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In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What is the date of the event?

{'answer_start': [27], 'text': [' 2011 ']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What is the location of the event?

{'answer_start': [107], 'text': ['Sweden']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

How was the event first detected?

{'answer_start': [25356], 'text': ['large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, ']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

How many people were ill?

{'answer_start': [705], 'text': ['18,500 individuals']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What are the pathogens?

{'answer_start': [888], 'text': ['Cryptosporidium hominis oocysts ']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What is the concentration of the contaminant after analysis?

{'answer_start': [20456], 'text': ['150,000 oocysts/L and de- clined to 6200 oocysts/L']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What are the symptoms?

{'answer_start': [23325], 'text': ['fatigue, abdominal pain, upset stomach, and watery diarrhoea ']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What is the concentration of the pathogen?

{'answer_start': [20682], 'text': ['12,000 oocysts/L']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What is the event?

{'answer_start': [40], 'text': [' outbreak of cryptosporidiosis']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What are the initial steps of investigation?

{'answer_start': [495], 'text': ['a retrospective study based on phone calls to a health advice line ']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What is the duration of the event?

{'answer_start': [1086], 'text': ['The outbreak went unnoticed to authorities for several months.']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

From when until when the event happened

{'answer_start': [19603], 'text': ['Between 1 January and 1 July 2011']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What are the first steps of mitigation?

{'answer_start': [4495], 'text': ['A boil water notice (BWN)']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What did they do to mitigate the event?

{'answer_start': [4560], 'text': ['web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What type of samples were examined?

{'answer_start': [953], 'text': ['patient and sewage samples,']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What steps were taken to restore the problem?

{'answer_start': [33849], 'text': ['the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started.']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What was the age of the affected people?

{'answer_start': [22721], 'text': ['children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1%']}

Contamination Question Answering

In the winter and spring of 2011 a large outbreak of cryptosporidiosis occurred in Skellefte municipality, Sweden. This study summarizes the outbreak investigation in terms of outbreak size, duration, clinical characteristics, possible source(s) and the potential for earlier detection using calls to a health advice line. Methods: The investigation included two epidemiological questionnaires and microbial analysis of samples from patients, water and other environmental sources. In addition, a retrospective study based on phone calls to a health advice line was performed by comparing patterns of phone calls between different water distribution areas. Results: Our analyses showed that approximately 18,500 individuals were affected by a waterborne outbreak of cryptosporidiosis in Skellefte in 2011. This makes it the second largest outbreak of cryptosporidiosis in Europe to date. Cryptosporidium hominis oocysts of subtype IbA10G2 were found in patient and sewage samples, but not in raw water or in drinking water, and the initial contamination source could not be determined. The outbreak went unnoticed to authorities for several months. The analysis of the calls to the health advice line provides strong indications early in the outbreak that it was linked to a particular water treatment plant. Conclusions: We conclude that an earlier detection of the outbreak by linking calls to a health advice line to water distribution areas could have limited the outbreak substantially. Keywords: Early outbreak detection, Cryptosporidiosis, Syndromic surveillance, Cryptosporidium hominis Background The protozoan parasite Cryptosporidium is a major cause of gastroenteritis in humans worldwide [1]. At least 29 valid species of Cryptosporidium have been identified [2] and the two most common species infecting humans are Crypto- sporidium parvum and Cryptosporidium hominis [3]. Cryptosporidium hominis has been the cause of several large waterborne outbreaks. The largest took place in 1993 in Milwaukee, USA, where more than 400,000 people were infected [4]. Cryptosporidiosis is mainly transmitted by the fecal-oral route, usually through oocyst-contaminated water or food, or through contact with infected humans or ani- mals. As few as 10 ingested oocysts can cause infection [5]. Asymptomatic carriage occurs [6, 7] while symptomatic in- fection is associated with diarrhoea, abdominal pain, nau- sea, vomiting and fever that usually resolve within 2 weeks. Symptoms occur a few days up to 2 weeks after ingestion of oocysts [5]. Severe life-threating diarrhoea may develop among immunocompromised patients [8]. Gastrointestinal- and joint symptoms can persist for several months after the initial infection with Cryptosporidium [9]. The public health impact of the parasite was recognised in Sweden in 2004 * Correspondence: par.bjelkmar@folkhalsomyndigheten.se 1Department of Monitoring and Evaluation, Public Health Agency of Sweden, 171 83 Solna, Sweden Full list of author information is available at the end of the article The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bjelkmar et al. BMC Public Health (2017) 17:328 DOI 10.1186/s12889-017-4233-8 when cryptosporidiosis became a notifiable disease and the parasite was also included in the World Health Organiza- tions Neglected Diseases Initiative in the same year [10]. In November and December 2010 a massive waterborne outbreak of C. hominis occurred in the city of stersund in Jmtland County, Sweden. Based on a retrospective cohort study, it was concluded that approximately 27,000 individ- uals were infected through the drinking water which made it the second biggest reported waterborne outbreak of cryptosporidiosis globally [11]. A couple of months later, in April 2011, drinking water from a municipal water treat- ment plant (WTP) in the neighboring county of Vsterbot- ten was suspected to be the source of a number of cases of cryptosporidiosis. A boil water notice (BWN) was therefore issued on 19 April and a web-based questionnaire was im- mediately created and published on the webpage of the mu- nicipality to collect epidemiological data. In order to complement the web-based questionnaire and better esti- mate the extent of the outbreak and to find its source, an additional study based on a postal questionnaire was per- formed in June 2011. The study was managed by the Vs- terbotten County Medical Office and the municipal environmental and health authorities in collaboration with the Public Health Agency of Sweden (at the time named Swedish Institute for Communicable Disease Control). Syndromic surveillance is defined as the real-time (or near real-time) collection, analysis, interpretation and dis- semination of health-related data [12]. As part of the on- going effort of development and evaluation of a syndromic surveillance system at the Public Health Agency, a retro- spective analysis of phone calls to a national health advice line from inhabitants living in Skellefte municipality dur- ing the time period of the outbreak was performed. No contemporaneous analysis of the phone calls was per- formed at the time of the outbreak. A new approach for early detection and improved situational awareness of local waterborne outbreaks was used where call patterns from individuals living in different drinking-water distribution areas were compared. The utility of syndromic surveillance systems for detecting and tracking local gastrointestinal outbreaks (GI) has been questioned [13] but systems based on data from health advice lines have been shown to be successful in a few cases [14, 15]. Globally, there are several examples of similar syndromic surveillance systems based on health advice lines [1618]. This study describes the outbreak investigation by sum- marizing the results from web-based and postal question- naires, human and environmental sampling and the analysis of phone calls to the health advice line. It outlines the extent and duration of the outbreak, risk factors and clinical characteristics of the infected persons, and discusses the potential for detecting the outbreak earlier. Figure 1 de- picts a time line indicating for which time periods the dif- ferent data sources were used in the analyses. Methods Study setting Skellefte is situated in Vsterbotten County geographic- ally located next to Jmtland County where the stersund outbreak occurred. The distance between Skellefte and stersund is almost 500 km. Skellefte is a municipality with a population of approximately 72,000 inhabitants. Twenty-eight water treatment plants are operating within the municipality. Two of these deliver water to the city of Skellefte; Slind WTP and Abborrverket WTP, where the latter delivers water to the majority of the inhabitants. All water treatment plants in the municipality use ground- water as the water source except Abborrverket which uses surface water obtained from the river Skelleftelven. Abborrverket WTP produces approximately 18,000 m3 of treated water daily to 44,000 of the 72,000 inhabitants in the municipality (31 March 2011). The normal water in- take to Abborrverket is located far out and deep in the river but due to icing during the winter months the intake is shifted to a more shallow position closer to shore where the ice can be removed more easily. Microbiological investigation Human samples Fecal samples from patients seeking healthcare for gastro- intestinal illness were analysed with standard techniques for enteric bacterial pathogens; polymerase chain reaction (PCR) for analysis of noro- and sapoviruses and micros- copy for analysis of Entamoeba spp. and Giardia intesti- nalis. Samples were only sporadically analysed for presence of Cryptosporidium oocysts up until 19 April 2011, when the current outbreak was first suspected. An intensification of testing for Cryptosporidium followed from that time until 1 July 2011 when the outbreak was considered over. Samples tested for Cryptosporidium were analysed using standard concentration technique followed by modified Ziehl-Neelsen staining [19]. A subset of posi- tive Cryptosporidium samples (n = 26) were sent to the Swedish Institute for Communicable Disease Control for species identification by PCR restriction fragment length polymorphism (RFLP) analysis of the rRNA gene [20, 21]. Subtypes were characterized by sequence analysis of the 60 kDa glycoprotein (gp60) gene [22, 23]. Environmental samples At the time of the outbreak Abborrverket WTP used floc- culation and sedimentation followed by sand filtering and chlorination for water treatment. This water treatment setup could be sufficient for removal of Cryptosporidium oocysts if the processes work optimally and the concentra- tion of oocysts is relatively low, but ultraviolet (UV) treat- ment is generally preferred as a disinfectant [24]. The winter intake was used from 19 November 2010 until 19 April 2011. A total of 38 samples were collected from the Bjelkmar et al. BMC Public Health (2017) 17:328 Page 2 of 10 drinking water system during a period of 5 months, 19 April to 15 September 2011. These samples included raw water from the river Skelleftelven, i.e. incoming water to Abborrverket WTP, treated water at Abborrverket WTP and samples taken from the distribution net. Twelve influent and twelve effluent wastewater samples were collected at the main sewage water treatment plant (SWTP) Tuvan. Moreover, in order to investigate possible causes of contamination of Skelleftelven and to trace sources of oocysts, 9 samples were collected from the wastewater and storm water systems and from other rele- vant locations. Water samples were analysed for Cryptosporidium oo- cysts according to ISO 15553:2006 [25] with filtration of water (101000 L), immunomagnetic separation (IMS) and immunofluorescence (IFL) microscopy. The slides with concentrated and purified material were identified by fluorescent-marked oocysts specific in size, shape, internal structure and DAPI-(4,6-diamidino-2-phenylindole)- stained nuclei. Wastewater samples were analyzed as water samples but without passing filters and in smaller volumes, 50100 mL for influent wastewater and 0.30.5 L for efflu- ent wastewater. Two sediment samples from the inside of the influent raw water pipe were also analysed as water sample but without filtration before IMS. DNA from one wastewater concentrate was analysed by sequence analysis of the gp60 gene as described for human samples [22, 23]. Epidemiological investigation Web-based questionnaire The same day as the BWN was issued, on 19 April 2011, a web-based questionnaire (Additional file 1) was created in order to immediately start collecting epidemiological data. The value of such a questionnaire was demonstrated in the preceding cryptosporidiosis outbreak in stersund [11] and those experiences were applied here as well. The questionnaire was made available to the public on the website of the municipality on the evening the same day, and was closed on 9 May 2011. The public was informed of the questionnaire by press releases and there were also links to it from key web pages such as the local newspaper and Vsterbotten County Council. The full data set was summarised after the outbreak was considered to be over. Visitors to the webpage who were residents of Skellefte municipality, both individuals with and without GI symp- toms, were asked to answer a set of questions regarding gastrointestinal illness in the family. A case attributed to the outbreak was defined as a person with residential ad- dress within Skellefte municipality with 3 loose stools per day for at least 1 day with onset between 1 April and 5 May 2011. Respondents with a date of symptom onset be- fore 1 April or after 5 May, persons who had travelled abroad 2 weeks prior to symptom onset, as well as individ- uals with a residential postal code outside Vsterbotten County were excluded from the analysis. Remaining re- spondents who did not fulfil the criteria of having 3 loose stools per day were considered non-cases. More detailed analyses of the data were not performed since the follow- up postal survey was conducted. Postal questionnaire A retrospective cohort study was performed in June 2011 by sending a questionnaire to a random sample of 1754 citizens in the municipality of Skellefte (Additional file 2, Additional file 3). The random sample was stratified by age (05 years, 615 years, 1665 years and 66 years or older) and gender. Questions were asked to find out about the start and magnitude of the outbreak, the source of the outbreak and risk factors for disease. The questionnaire contained questions on demographics, onset, duration and occurrence of symptoms indicating cryptosporidiosis, and water consumption as well as history of symptoms be- fore 1 January 2011. Caretakers were asked to answer for children <15 years of age. A case attributed to the out- break was defined as a person with 3 loose stools per day for at least 1 day with onset between 1 December 2010 and 31 May 2011. Statistical analysis of the postal questionnaire Each of the 1754 respondents were assigned a random number and a barcode on the questionnaire was used to identify each respondent. The postal codes were matched to the water distribution areas of the WTPs. In a stratified survey study, weights are used to calculate the number of individuals in the population represented by each individ- ual in the sample. Binary logistic regression was used to Fig. 1 Time line indicating for which time periods the different data sources were used in the analyses Bjelkmar et al. BMC Public Health (2017) 17:328 Page 3 of 10 find associated variables for the propensity of responding to the survey. Age, gender and water supply were used to calibrate the weights for non-response to adjust for unbal- ance between the sample and the population. The association between the binary outcome of case/ non-case and the exposure variables was analysed by binary logistic regression. Included in the model as covariates and exposure variables were gender, age (05 years, 615 years, 1665 years, and 66 years or older), gastric ulcer (yes, no), irritable bowel syndrome (yes, no), Crohns disease (yes, no), celiac disease (yes, no), lactose intolerance (yes, no), immunodeficiency disease (yes, no), average tap water con- sumption (<1 glass, 1 glass, 25 glasses, >5 glasses) and household water supply (Abborrverket, not Abborrverket or not from any WTP/own well). The results from the binary logistic regression were expressed as odds ratios (OR). All I do not know an- swers for binary questions were regarded as non- informative and were set as missing values prior to the analysis. Missing values for binary variables were then given a value (yes, no) using multiple imputation chain equations [26]. The chains contained all exposure vari- ables plus the outcome non-case/case [27]. Twenty data- sets with different imputed values for missing data were created and binary logistic regression results from each dataset were weighted together into one result using Rubins formula [28]. All analyses were performed in the statistical software R (version 3.3.2) using the packages survey (version 3.31.2), MICE (version 2.25) and the gen- eralized linear model function (glm) in the base R package stats. In all analyses a p-value less than 0.05 was used as a significant result and in case of estimated confidence in- tervals a confidence level of 95% was applied. Analysis of phone calls to a health advice line Healthcare Guide 1177 is a national Swedish telephone health advice line staffed by nurses. The service provides advice and information about urgent, but non-life- threatening, health problems. The medical record cre- ated for each consultation includes a structured data field, called the contact cause, that represents the most severe symptom as assessed by the nurse [29]. There are almost 200 contact causes in the services medical deci- sion support system but only a handful are related to GI problems. For the purpose of this study daily call counts on GI symptoms were retrospectively extracted from the service for inhabitants in Skellefte municipality from 1 August 2010 to 18 April 2011. The contact causes vomiting or nausea, diarrhoea and stomach pain were used since changes in contact patterns for these symptoms previously have been shown in outbreaks of cryptosporidiosis [14]. In addition, for each call, infor- mation on the postal code of the registered residence ad- dress of the patient was extracted. Postal codes were divided into two geographical regions; belonging to the distribution area of Abborrverket WPT or not, and the number of inhabitants in the corresponding regions were calculated. To compare the call patterns of GI-related symptoms between these two regions a previ- ously published outbreak detection algorithm [14] was used but with a minor modification. No analyses were performed for the period from the BWN and onwards since, as the in- formation of an ongoing outbreak becomes public, the con- tact pattern to the health advice line changes drastically and it is challenging to adjust for this in the analyses. The daily call count, Ct , i, for one contact cause or a sin- gle group of contact causes at day t for geographical re- gion i was classified as an outbreak signal if it exceeded a threshold Tt , i: Tt;i max L; V ; V Et;i L SDt;i; L 3; 5 f g Et;i pt;i Ni; SDt;i Ni pt;i q 1pt;i; pt;i Pni j1;ji P C;j 10 Pni j1;jiNj ; t7; t8; t9; t10; t11; t12; t13; t14 f g; 2; if t7; t14 f g 1; if t8; t9; t10; t11; t12; t13 f g where L is the threshold level for a weak and strong outbreak signal respectively, V is the threshold for a positive outbreak signal, Et , i is the expected number of calls and SDt , i is the standard deviation, both based on a binomial distribution, Niis the population size of geographical region i, pt , i is the probability of a single call per inhabitant per day, and ni is the number of geographical regions in the analysis. In the current study, two geographical regions were in- cluded: Abborrverket distribution area and not Abborrverket distribution area. It is important to note that calls from inhabitants of the geographical region under investigation are not included in the calculation of its threshold since that would increase the thresh- old if an outbreak in that region has been ongoing for more than 6 days. Compared to the previously published algorithm, the time period for the calcula- tion of pt , i has been modified. Here, calls for 8 days, t-7 to t-14, were included. Since the call patterns dif- fer between different weekdays, call counts for the weekdays matching the day for which the threshold is calculated, t-7 and t-14, has a weight of 2. The Bjelkmar et al. BMC Public Health (2017) 17:328 Page 4 of 10 motivation for this modification of the algorithm was primarily to reduce the risk of calculating pt , i based on small number of calls. Results Microbiological investigation Human samples Between 1 January and 1 July 2011, 145 laboratory con- firmed cases of domestic cryptosporidiosis were reported from Vsterbotten County. Only a handful were reported before 19 April, including one case on 15 April and two on 18 April. Genotyping identified C. hominis subtype IbA10G2 in samples from 24 confirmed cases, while no amplification product was obtained from the remaining two samples that were tested. No other gastrointestinal pathogens were found in a subset of the samples that were positive for Cryptosporidium. Environmental samples Cryptosporidium oocysts could not be detected in any of the 38 samples collected from the drinking water system. In influent and effluent wastewater samples from Tuvan SWTP oocysts were detected in 10 out of 24 samples. The concentration of oocysts in influent wastewater was highest on 22 April 2011 at 150,000 oocysts/L and de- clined to 6200 oocysts/L on 1 June 2011. From 27 June 2011 no oocysts were detected in influent wastewater. In effluent wastewater the concentration was highest on 8 June 2011 with a concentration of 12,000 oocysts/L. In subsequent samples the concentration varied between 1700 and 4200 oocysts/L and from 27 June 2011 the concentration was below the detection limit. Molecular investigation of one wastewater sample revealed C. hominis subtype IbA10G2. In the remaining 9 water and sediment samples collected at other places no oocysts were detected. Epidemiological investigation Web-based questionnaire The epidemiological curve based on the web-based questionnaire (Fig. 2) showed that the number of cases declined after a couple of days following the BWN and verified the hypothesis of an ongoing waterborne out- break. Importantly, it also indicated that the outbreak started well before 1 April. In total 12,358 individuals answered the questionnaire and 11,065 remained after exclusions. The results from the questionnaire were con- tinually monitored in order to provide information for decision making based on the extent of the outbreak, who were being affected and to follow up the effect of interventions. Moreover, it was used to inform the in- habitants about the progress of the outbreak and these reports were highly appreciated. Postal questionnaire In total, 1099 out of 1754 (63%) questionnaires were an- swered and returned for analysis. The survey showed that 26.4% of the respondents fulfilled the case defin- ition, i.e. self-reported diarrhoea (3 loose stools per day) between 1 December 2010 and 31 May 2011, which corresponds to an estimate of 18,449 cases (Table 1). The data from the survey also provided evidence that the outbreak started in January and ended by the end of May (Fig. 3). April was the peak month with 6969 cases. If the outbreak had been detected earlier and we assume that all cases from 1 February forward had remained healthy, the estimation is that the outbreak would have affected 2273 individuals, corresponding to approxi- mately 12% the current outbreak size. Only the variables age and water supply were identi- fied as risk factors for infection (Table 2). Divided into age groups, children up to 5 years were most affected, 37.2%, while the group of 66 years and above were least affected, 12.1% (Table 3). Among the different water supplies in Skellefte, water from Abborrverket WPT was the only supply that significantly correlated with an increased risk of infection (p < 0.001). Approximately 1 in 3 (32.7%) living in the distribution area of Abborrver- ket WPT had symptoms of cryptosporidiosis, compared to 16.2% of inhabitants living in other areas (Table 4). The most common symptoms, each present in more than 70% of the respondents that fulfilled the case defin- ition, were fatigue, abdominal pain, upset stomach, and watery diarrhoea (Table 5). Health advice line Starting on 30 December 2010, the retrospective analysis showed a sequence of 6 days of consecutive outbreak sig- nals regarding GI symptoms from individuals living in the distribution area of Abborrverket WTP (Fig. 4). Four of those were strong. A large number of outbreak signals for inhabitants living in the distribution area of Abborrverket WTP were evident during the following time period up until the BWN. In contrast, very few outbreak signals regarding GI symptoms were present for Abborrverket WPT distribu- tion area during the autumn and early winter of 2010. However, there was a small cluster of five outbreak signals between 20 November and 29 November 2010, but they were weak and not on consecutive days, hence the inter- pretation was that those outbreak signals were inconclu- sive. For the other geographical area in the analysis, individuals not living in the distribution area of Abborr- verket WTP, only one weak outbreak signal was present during the entire time-period under investigation - em- phasizing the abnormality of the identified outbreak signal pattern for the distribution area of Abborrverket WTP in the beginning of 2011. Similar results are obtained using Bjelkmar et al. BMC Public Health (2017) 17:328 Page 5 of 10 the original definition of the outbreak algorithm and with other groupings of ages and contact causes related to cryptosporidiosis (results not shown). When comparing the current outbreak signals based on geographical regions corresponding to the distribution of drinking water with outbreak signals based on (adult) GI calls from the entire Skellefte municipality with respect to the other municipalities within Vsterbotten County [14], the two patterns are similar and suggest the same time for outbreak detection: beginning of January 2011. Discussion The three cases of cryptosporidiosis in the middle of April together with other indications from informal sources re- garding large numbers of sick people, and higher-than- normal contacts regarding symptoms of gastrointestinal illness reported by the nurses staffing the regional health advice line, led the authorities to suspect an outbreak. However, our epidemiological investigation shows that the outbreak had already started, unnoticed to the authorities, in the beginning of January 2011. High norovirus activity together with only a handful of domestic cases of crypto- sporidiosis reported in Vsterbotten County between January and 19 April 2011 were contributing factors to the late detection of the outbreak. New routines are now in place in Vsterbotten County where analysis of Crypto- sporidium is performed on fecal samples if there are clus- ters of cases with gastrointestinal symptoms or other indications of an outbreak. If the outbreak had been detected in the beginning of the year by systematic monitoring of the telephone calls as described in this study, it is very likely that the outbreak would have ended during January. Two facts support this conclusion. First, once the outbreak was suspected the BWN was an effective intervention that substantially lim- ited illness within a few days. A similar delay in the Fig. 2 Epidemiological curve based on observed cases in the web questionnaire Table 1 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 Status N SE (N) 95% CI (N) N (%) Non-cases 51,618 1214 49,239 53,997 73.7 Cases 18,448 1191 16,114 20,782 26.3 N Number, SE standard error, CI confidence interval Based on postal questionnaire Fig. 3 Epidemiological curve of population estimates of number of cases from the postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 6 of 10 decrease of reported cases was seen in the stersund out- break [11] and it is explained by the time it takes to de- velop symptoms after ingestion of oocyst. Second, the outbreak signals from the described analyses of telephone calls would have given a strong indication during January that the outbreak was waterborne and which drinking water supply to suspect (Abborrverket WTP). We therefor argue that such an early outbreak detection followed by a timelier BWN in January 2011 would have limited the out- break substantially from approximately 18,500 cases down to 2300 cases if all who fell ill after 31 January had remained healthy. The potential of syndromic surveillance systems based on analysing telephone call patterns to Healthcare Guide 1177 for early event detection and situational awareness of local outbreaks has been shown previously [14]. In the current work the concept was taken a step further by comparing call patterns between water distribution areas that were based on groups of postal codes. The im- portance of this should not be underestimated. In the situation of an unknown waterborne outbreak, or other types of local outbreaks where the spread matches geographical areas used in the analysis, this procedure gives a more timely indication of the underlying cause and therefore substantially increases the chances of ef- fective countermeasures. Since water distribution areas are known, the approach can be used in systems for syn- dromic surveillance. There is always a tradeoff between sensitivity and spe- cificity in signal detection. In practical terms it is the in- stitution that is eligible to act on the signal that needs to find a reasonable protocol for signal evaluation and val- idation. To increase the sensitivity and hence the poten- tial of timely detection of local outbreaks, which usually are very short-lived in contrast to the outbreak under in- vestigation here, the outbreak algorithm used operates on a daily basis. This has the drawback of reduced speci- ficity, i.e. that more false positive outbreak signals are generated due to randomness, especially for geographical regions where the population size is small. Despite this, and even though the daily call counts are relatively low, the outbreak signal pattern shown in Fig. 4 is excep- tional and clearly indicates that individuals living in the distribution area of Abborrverket WTP report more GI symptoms compared to individuals living in other areas. Moreover, this outbreak signal pattern is similar if other groupings of age and contact causes related to symptoms of cryptosporidiosis are used. Although possible sources of contamination were in- vestigated and discussed no conclusive information could be found. Several samples from the drinking water system and the environment were analysed for Crypto- sporidium oocysts but none were detected - apart from the findings in wastewater samples. The most likely the- ory in our opinion is that the winter intake of water to Abborrverket WTP, which is more exposed to contamin- ation since it is located more shallowly in the river and closer to shore compared to the summer intake, was contaminated with Cryptosporidium oocysts from sew- age from one or several sources. However, data on weekly maximal turbidity and bacteriological counts (Escherichia coli, general coliform bacteria, enterococci and Clostridium perfringens) in raw water to Abborrver- ket WTP for the period October 2010 to March 2011 had been within normal levels so such a contamination, if present, was not detected in the routine testing at the WTP. The water intake was shifted to the summer position on the same day as the BWN was issued on 19 April 2011, which may explain why no Cryptosporidium oo- cysts were found in the water samples, since they were all taken after 19 April. It is worth noting that the out- break signals from the syndromic surveillance algorithm present in November 2010 coincide with the shift to the winter intake which might indicate a contamination at that point in time as well - although probably unrelated Table 2 Significant risk factors for infection based on postal questionnaire Odds ratio 95% CI P-value* Age 0-5 4.22 2.66 6.68 <0.001 6-15 2.28 1.42 3.68 0.001 16-65 3.08 1.96 4.83 <0.001 66- 1.00 Water source Not from any WTP/own well 1.00 Abborrverket WTP 2.30 1.49 3.56 <0.001 Not Abborrverket WTP 1.14 0.68 1.91 0.613 CI confidence interval, WTP water treatment plant *Fishers exact P - value Table 3 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into age groups Status Age N SE (N) 95% CI (N) N (%) Non-cases 0-5 2433 121 2195 2671 62.8 6-15 5542 208 5135 5950 75.3 16-65 30,728 1148 28,479 32,977 69.6 66- 12,914 326 12,276 13,553 87.9 Cases 0-5 1442 120 1207 1676 37.2 6-15 1819 202 1423 2215 24.7 16-65 13,402 1132 11,183 15,621 30.4 66- 1786 301 1196 2377 12.1 N number, SE standard error; CI confidence interval Based on postal questionnaire Bjelkmar et al. BMC Public Health (2017) 17:328 Page 7 of 10 to the current outbreak. The fact that Cryptosporidium oocysts were found in wastewater is in our opinion re- lated to the fact that a substantial part of the population connected to the municipal SWTP Tuvan was infected with C. hominis IbA10G2. Thus, although the initial cause of the outbreak remains unknown, it was most certainly caused by fecal contamination of human origin since C. hominis is almost exclusively human specific. Compared to the stersund outbreak [11] only age was the common risk factor. In contrast, the current study did not find statistical significance for any of the underlying diseases nor amount of water consumed. Symptom pro- files were almost identical between the outbreaks. The same Cryptosporidium gp60 subtype was found respon- sible for both the current and the stersund outbreak. It is in our opinion likely that the outbreaks were related since this subtype seldom is found in domestic cases in Sweden, in contrast to other European countries, and the time period between the outbreaks was short. However, similarity on gp60 is not conclusive evidence [30] and the question of whether two outbreaks were related is currently investigated by whole genome sequencing. Speculatively, one or a few infected individuals from the stersund outbreak brought the parasite to Skellefte and caused a second outbreak through spread of Cryptosporid- ium oocysts via sewage, into the river, finally ending up in the drinking water since the microbial barriers present in Abborrverket WPT at the time were insufficient to inacti- vate or remove the oocysts. After the outbreak was identified, the water distribution system was flushed to remove the contamination and work to improve the water treatment in Abborrverket WTP was started. Since the large outbreak in stersund only took place a few months earlier the municipality of Skellefte could utilize experience from the actions taken to stop the former outbreak. Even so, the BWN had to be kept in place for 5 months, compared to 3 months in stersund. This was partly due to the longer period of time it took to install an ultraviolet unit as an additional Table 4 Population estimates of cases and non-cases of Cryptosporidium-infection in Skellefte between December 2010 and May 2011 divided into water supply categories Status Category N SE (N) 95% CI (N) N (%) Non-cases Not Abborrverket WTP 22,649 642 21,392 23,906 83.8 Abborrverket WTP 28,696 1033 26,944 30,994 67.3 Cases Not Abborrverket WTP 4368 624 3145 5590 16.2 Abborrverket WTP 14,081 1019 12,084 16,078 32.7 N number, SE standard error, CI confidence interval Based on postal questionnaire Table 5 Clinical features of Cryptosporidium-infection cases in the municipality of Skellefte during December 2010 to May 2011 Proportion with symptom (%) 95% CI (%) Fatigue 78.5 72.4 84.7 Abdominal pain 73.3 66.8 79.8 Upset stomach 71.4 64.7 78.2 Diarrhoea Watery 70.2 63.4 77.0 Bloody 0.9 0.0 2.5 Nausea 63.5 56.1 70.9 Headache 46.9 39.3 54.5 Vomiting 35.8 28.6 43.1 Fever >38 C 36.5 28.2 42.8 Joint pain 27.4 20.4 34.4 Pain in eyes 14.6 9.1 20.1 CI confidence interval Based on postal questionnaire Fig. 4 Daily call counts from Skellefte municipality to Healthcare Guide 1177 regarding GI symptoms from 1 August 2010 until the day before the BWN on 19 April 2011. Inhabitants are divided into two groups; those living in the water distribution area of Abborrverket WPT (blue) and those who are not (red). Outbreak signals from the detection algorithm [14] are shown as blue (Abborrverket WTP) and red circles (not Abborrverket WTP) with weak (hollow circles) and strong outbreak signals (filled circles) along the lower horizontal and upper horizontal, respectively Bjelkmar et al. BMC Public Health (2017) 17:328 Page 8 of 10 microbiological barrier in Abborrverket WTP as well as a longer and more complex water distribution network that had to be flushed. As of November 2016, the municipality of Skellefte is in the process of rebuilding their infrastruc- ture for production of drinking water. This work had started before the outbreak of cryptosporidiosis. Conclusions Our investigation concludes that approximately 18,500 people in the municipality of Skellefte were infected by Cryptosporidium during the winter and spring of 2011 making it the second largest outbreak of cryptosporidiosis described in Europe to date. Cryptosporidium hominis subtype IbA10G2 was isolated from patient samples and wastewater. The epidemiological investigation strongly indicates that this outbreak was waterborne based on the vast number of cases, as well as the fact that the BWN were an effective countermeasure, and that people living in the water distribution area of one specific WTP were more likely to become ill. This conclusion is also strongly supported by the pattern of phone calls to the national health advice line Healthcare Guide 1177. We therefore firmly believe that the outbreak was waterborne and caused by C. hominis transmitted through the public water supplied by Abborrverket WTP even though no oocysts could be found in raw water or in drinking water. Moreover, our results show that the outbreak went unnoticed to the authorities for several months and that systematic monitoring of phone calls to the health advice line, as described in this study, could have limited the out- break to approximately 2300 cases compared to the current estimate of 18,500 cases. This new approach of linking health advice line calls to water distribution areas has been implemented in a system for syndromic surveillance deployed by the Public Health Agency of Sweden in 2016. Additional files Additional file 1: Web-based questionnaire. Translated web-based questionnaire. (PDF 162 kb) Additional file 2: Postal questionnaire for adults. Translated postal questionnaire for adults. (PDF 128 kb) Additional file 3: Postal questionnaire for children. Translated postal questionnaire for children. (PDF 128 kb) Abbreviations BWN: Boil water notice; GI: Gastrointestinal; IFL: Immunofluorescence; IMS: Immunomagnetic separation; OR: Odds ratio; PCR: Polymerase chain reaction; RFLP: Fragment length polymorphism; SWTP: Sewage water treatment plan; UV: Ultraviolet; WTP: Water treatment plant Acknowledgements The authors would like to thank Leah Martin at the Public Health Agency of Sweden for useful comments on the manuscript and Stefan Johansson of Skellefte municipality for the information on the drinking water infrastructure, production and testing. Funding The study was partly funded by the Swedish Agency for Contingency Planning through a research and development project named Event-based Surveillance System (ESS). The funding organisation was not involved in any part of the study. Availability of data and materials The datasets used are available from the corresponding author on request. Authors contributions PB conceived and performed the health advice line study and wrote the manuscript. AH performed water analysis and was part of the outbreak team. JB designed and made the statistical analyses of the epidemiology part of the manuscript. ML was part of the outbreak team. ML performed analysis on human samples, including typing and sub-typing and was part of the outbreak team. GA performed water analysis and was part of the outbreak team. SS was responsible for the local outbreak team and contributed to the study design and acquisition of data. JL conceived the study and performed epidemiological analysis. All authors contributed to the interpretation of data and made substantial contributions to the overall content of the manuscript, and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Consent for publication Not applicable. Ethics approval and consent to participate The study was approved by Stockholm ethical review board, reference number 2011/220-31/4. Written informed consent was not necessary due to outbreak investigation. However, a letter stating the art of the investigation was handed out to all participants. Here it was also stated that all personal identification issues were handled according to Swedish law. Furthermore, answers to questionnaires would be seen as written consent to participate in study. Collection of field samples The field samples were collected as part of an ongoing outbreak investigation, followed Swedish routines and legislation, and the sampling was performed in collaboration with local authorities and the drinking water producer. Requirement for permission was waived. Publishers Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

What is the source of contamination?

{'answer_start': [4338], 'text': ['municipal water treat- ment plant (WTP) ']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What type of samples were analyzed?

{'answer_start': [241], 'text': ['faecal specimens']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What symptoms did people report?

{'answer_start': [3085], 'text': ['diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What is the date of the event?

{'answer_start': [1172], 'text': ['2006']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What is the location of the event?

{'answer_start': [102], 'text': ['New Zealand']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What pathogen was connected to the outbreak?

{'answer_start': [1281], 'text': ['C. parvum']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What is the source that started the event?

{'answer_start': [3470], 'text': ['direct zoonotic transfer through contact with calves']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

How many people were ill?

{'answer_start': [2632], 'text': ['25/80 cases']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What is the attack rate?

{'answer_start': [2669], 'text': ['31%']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What are the pathogens?

{'answer_start': [174], 'text': ['Cryptosporidium parvum']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What are the symptoms?

{'answer_start': [769], 'text': ['abdominal discomfort, diarrhoea, and in some cases, vomiting']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What is the event?

{'answer_start': [1197], 'text': ['outbreak of gastrointestinal illness in a class of 96 veterinary students']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What type of samples were examined?

{'answer_start': [1371], 'text': ['water sample']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What is the source of contamination?

{'answer_start': [451], 'text': ['contact with calves ']}

Contamination Question Answering

The occurrence of a gastrointestinal illness among a class of 96 undergraduate veterinary students in New Zealand prompted laboratory and questionnaire-based investigations. Cryptosporidium parvum was the only enteropathogen identied in 4/7 faecal specimens analysed. The C. parvum isolates carried a rare IIa GP60 allele, indicating a point-source outbreak. The infection source could not be microbiologically traced, but the investigation suggested contact with calves during a practical class as the most likely exposure. The inferred median incubation period was 5 days (range 011 days), and the median illness duration was 56 days (range 223 days), corroborating previous observations in experimental cryptosporidiosis. Disease was self-limiting, characterized by abdominal discomfort, diarrhoea, and in some cases, vomiting. Originating from a rural area and having had previously handled ruminants were associated with a signicant risk reduction in males. All the three students who reported chronic use of steroid inhalers for treatment of asthma were cases. This case highlighted, once again, the potential hazard for explosive outbreaks of cryptosporidiosis. In 2006, we investigated an outbreak of gastrointestinal illness in a class of 96 veterinary students, in which C. parvum was identified as the sole enteropathogen in multiple faecal specimens. A 100-l water sample was collected from one of the drinking water taps on the same day of the investigation and filtered on-site using a commercial filter (FiltaMax, Idexx Laboratories, USA), at a ow rate of 2 l/min. A 200-ml sample was also collected in a sterile bottle, placed on ice and transported to an environmental microbiology laboratory for analysis for the presence of Escherichia coli bacteria. The same day, the dairy farm that provided the calves for the practical class was visited. The farm was situated about 5 km from LATU. Faecal specimens from nine calves aged >30 days were submitted on ice to IVABS Unfortunately, the calves actually used in the class had been sold and could not be traced, and other calves aged <1 month were not present at the day of the visit as the calving season had ended. Seven faecal specimens submitted by students were analysed for the presence of enteropathogens. The three specimens initially analysed at the regional diagnostic laboratory tested negative for Campylobacter and one tested positive for Cryptosporidium. All the seven specimens submitted to IVABS tested negative for rotavirus, Salmonella spp., norovirus and Giardia, and four tested positive for Cryptosporidium oocysts by immunouorescence. There were 25/80 cases among respondents to Q1 (31% attack rate), and 15/64 among respondents to Q2 (P=0.35). Nine out of 25 cases (36%) sought medical advice during the course of the illness. All 64 respondents to Q2 had attended the calf-handling practical (Table 2), and none of them responded as having drunk tap water at LATU during the same practical, arguing against a water-borne source of infection there (Table 2). Sixteen out of 25 cases (64%) reported diarrhoea and abdominal discomfort in Q1; six (24%) abdominal discomfort, diarrhoea, and vomiting; two (8%) abdominal discomfort only, and one (4%) vomiting only. Although a single negative water sample was of limited value to completely rule out a water-borne outbreak of cryptosporidiosis, Q2 indicated none of the respondents had drunk tap water during the practical class, leaving direct zoonotic transfer through contact with calves as the most likely route of transmission. An attack rate of 31% was estimated based on the responses to Q1.

What were the associated pathogens of concern?

{'answer_start': [2416], 'text': ['Cryptosporidium']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What is the initial cause of the event?

{'answer_start': [359], 'text': ['bathing water']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. Finnish Decree. Sosiaali- ja terveysministerin asetus yleisten uimarantojen uimaveden laatuvaatimuksista ja valvonnasta. [Decree of the Ministry of Social Affairs and Health concerning the quality requirements and surveillance of bathing water in public beaches]. Document no. 177/2008. Helsinki: Finlex database; 2008. Finnish. Available from: http://www.finlex.fi/ fi/laki/alkup/2008/20080177 4. Finnish Decree. Sosiaali- ja terveysministerin asetus pienten yleisten uimarantojen uimaveden laatuvaatimuksista ja valvonnasta. [Decree of the Ministry of Social Affairs and Health concerning the quality requirements and surveillance of bathing water in small public beaches]. Document no. 354/2008. Helsinki: Finlex database; 2008. Finnish. Available from: http://www.finlex.fi/fi/laki/alkup/2008/20080354 5. European Union. Directive 2006/7/EC of the European Parliament and of the Council 15 February 2006 concerning the management of bathing water quality and repealing Directive 76/160/EEC. Off J Eur Union. 2006;L64:37-51. Available from: http://eur-lex.europa.eu/legal-content/EN/ TXT/?uri=CELEX%3A32006L0007 6. Shuval H. Estimating the global burden of thalassogenic diseases: human infectious diseases caused by wastewater pollution of the marine environment.J Water Health. 2003;1(2):53-64.PMID: 15382734 7. Gibson KE. Viral pathogens in water: occurrence, public health impact, and available control strategies.Curr Opin Virol. 2014;4:50-7. DOI: 10.1016/j.coviro.2013.12.005 PMID: 24440908 8. Hoebe CJ, Vennema H, de Roda Husman AM, van Duynhoven YT. Norovirus outbreak among primary schoolchildren who had played in a recreational water fountain.J Infect Dis. 2004;189(4):699-705. DOI: 10.1086/381534 PMID: 14767824 9. Maunula L, Kalso S, Von Bonsdorff CH, Pnk A. Wading pool water contaminated with both noroviruses and astroviruses as the source of a gastroenteritis outbreak.Epidemiol Infect. 2004;132(4):737-43. DOI: 10.1017/S0950268804002249 PMID: 15310176 10. 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Enteric viruses in a large waterborne outbreak of acute gastroenteritis in Finland. Food Environ Virol. 2009;1(1):31-6. DOI: 10.1007/s12560-008-9004-3 15. Vinj J, Hamidjaja RA, Sobsey MD. Development and application of a capsid VP1 (region D) based reverse transcription PCR assay for genotyping of genogroup I and II noroviruses.J Virol Methods. 2004;116(2):109-17. DOI: 10.1016/j.jviromet.2003.11.001 PMID: 14738976 16. Humphries RM, Linscott AJ. Laboratory diagnosis of bacterial gastroenteritis.Clin Microbiol Rev. 2015;28(1):3-31. DOI: 10.1128/CMR.00073-14 PMID: 25567220 17. Jalava K, Rintala H, Ollgren J, Maunula L, Gomez-Alvarez V, Revez J, et al. Novel microbiological and spatial statistical methods to improve strength of epidemiological evidence in a community-wide waterborne outbreak. PLoS One. 2014;9(8):e104713. DOI: 10.1371/journal.pone.0104713 PMID: 25147923 18. Kauppinen A, Ikonen J, Pursiainen A, Pitknen T, Miettinen IT. Decontamination of a drinking water pipeline system contaminated with adenovirus and Escherichia coli utilizing peracetic acid and chlorine.J Water Health. 2012;10(3):406-18. DOI: 10.2166/wh.2012.003 PMID: 22960485 19. Kauppinen A, Martikainen K, Matikka V, Veijalainen A-M, Pitknen T, Heinonen-Tanski H, et al. Sand filters for removal of microbes and nutrients from wastewater during a one-year pilot study in a cold temperate climate. J Environ Manage. 2014;133:206-13. DOI: 10.1016/j.jenvman.2013.12.008 PMID: 24384282 8 www.eurosurveillance.org 20. International Organization for Standardization (ISO). ISO/TS 15216-1. Microbiology of food and animal feed - Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR - Part 1: Method for quantification. Geneva: ISO; 2013. Available from: http://www.iso.org/iso/ catalogue_detail.htm?csnumber=55382 21. International Organization for Standardization (ISO). ISO 9308- 2. Water quality -- Enumeration of Escherichia coli and coliform bacteria -- Part 2: Most probable number method. Geneva: ISO; 2012. Available from: http://www.iso.org/iso/iso_catalogue/ catalogue_tc/catalogue_detail.htm?csnumber=52246 22. International Organization for Standardization (ISO). ISO 7899- 2. Water quality -- Detection and enumeration of intestinal enterococci -- Part 2: Membrane filtration method. Geneva: ISO; 2000. Geneva, Switzerland. Available from: http://www. iso.org/iso/catalogue_detail.htm?csnumber=14854 23. Rnnqvist M, Rtt M, Tuominen P, Salo S, Maunula L. Swabs as a tool for monitoring the presence of norovirus on environmental surfaces in the food industry.J Food Prot. 2013;76(8):1421-8. DOI: 10.4315/0362-028X.JFP-12-371 PMID: 23905799 24. Jothikumar N, Cromeans TL, Hill VR, Lu X, Sobsey MD, Erdman DD. Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41.Appl Environ Microbiol. 2005;71(6):3131-6. 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DOI: 10.1002/jmv.21237 PMID: 18551613 30. Dufour AP, Evans O, Behymer TD, Cant R. Water ingestion during swimming activities in a pool: a pilot study.J Water Health. 2006;4(4):425-30.PMID: 17176813 31. Robilotti E, Deresinski S, Pinsky BA. Norovirus.Clin Microbiol Rev. 2015;28(1):134-64. DOI: 10.1128/CMR.00075-14 PMID: 25567225 32. Prez-Sautu U, Sano D, Guix S, Kasimir G, Pint RM, Bosch A. Human norovirus occurrence and diversity in the Llobregat river catchment, Spain.Environ Microbiol. 2012;14(2):494-502. DOI: 10.1111/j.1462-2920.2011.02642.x PMID: 22118046 33. Lysn M, Thorhagen M, Brytting M, Hjertqvist M, Andersson Y, Hedlund KO. Genetic diversity among food-borne and waterborne norovirus strains causing outbreaks in Sweden.J Clin Microbiol. 2009;47(8):2411-8. DOI: 10.1128/JCM.02168-08 PMID: 19494060 34. Vega E, Barclay L, Gregoricus N, Shirley SH, Lee D, Vinj J. 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In: Mandell G, Bennet J, Dolin R, editors. Principles and practice of infectious diseases. 7th ed. Philadelphia: Churchill Livingstone Elsevier. 2010;2027-33. 40. Jovanovi Galovi A, Bijelovi S, Miloevi V, Hrnjakovi Cvjetkovic I, Popovi M, Kovaevi G, et al. Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014. Euro Surveill. 2016;21(15):30196. DOI: 10.2807/1560-7917. ES.2016.21.15.30196 PMID: 27105473 41. Boehm AB, Ashbolt NJ, Colford JM, Dunbar LE, Fleming LE, Gold MA, et al. A sea change ahead for recreational water quality criteria. J Water Health. 2009;7(1):9-20. DOI: 10.2166/ wh.2009.122 PMID: 18957771 42. Fujioka RS, Solo-Gabriele HM, Byappanahalli MN, Kirs M. U.S. Recreational Water Quality Criteria: A Vision for the Future.Int J Environ Res Public Health. 2015;12(7):7752-76. DOI: 10.3390/ ijerph120707752 PMID: 26184253 43. Updyke EA, Wang Z, Sun S, Connell C, Kirs M, Wong M, et al. Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What is the date of the event?

{'answer_start': [165], 'text': ['July and August 2014']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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In: Mandell G, Bennet J, Dolin R, editors. Principles and practice of infectious diseases. 7th ed. Philadelphia: Churchill Livingstone Elsevier. 2010;2027-33. 40. Jovanovi Galovi A, Bijelovi S, Miloevi V, Hrnjakovi Cvjetkovic I, Popovi M, Kovaevi G, et al. Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014. Euro Surveill. 2016;21(15):30196. DOI: 10.2807/1560-7917. ES.2016.21.15.30196 PMID: 27105473 41. Boehm AB, Ashbolt NJ, Colford JM, Dunbar LE, Fleming LE, Gold MA, et al. A sea change ahead for recreational water quality criteria. J Water Health. 2009;7(1):9-20. DOI: 10.2166/ wh.2009.122 PMID: 18957771 42. Fujioka RS, Solo-Gabriele HM, Byappanahalli MN, Kirs M. U.S. Recreational Water Quality Criteria: A Vision for the Future.Int J Environ Res Public Health. 2015;12(7):7752-76. DOI: 10.3390/ ijerph120707752 PMID: 26184253 43. Updyke EA, Wang Z, Sun S, Connell C, Kirs M, Wong M, et al. Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What is the location of the event?

{'answer_start': [7453], 'text': ['Finland']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What pathogen was connected to the outbreak?

{'answer_start': [567], 'text': ['noroviruses']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. 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Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

How was the event first detected?

{'answer_start': [1513], 'text': ['via the media']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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Decontamination of a drinking water pipeline system contaminated with adenovirus and Escherichia coli utilizing peracetic acid and chlorine.J Water Health. 2012;10(3):406-18. DOI: 10.2166/wh.2012.003 PMID: 22960485 19. Kauppinen A, Martikainen K, Matikka V, Veijalainen A-M, Pitknen T, Heinonen-Tanski H, et al. Sand filters for removal of microbes and nutrients from wastewater during a one-year pilot study in a cold temperate climate. J Environ Manage. 2014;133:206-13. DOI: 10.1016/j.jenvman.2013.12.008 PMID: 24384282 8 www.eurosurveillance.org 20. International Organization for Standardization (ISO). ISO/TS 15216-1. Microbiology of food and animal feed - Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR - Part 1: Method for quantification. Geneva: ISO; 2013. Available from: http://www.iso.org/iso/ catalogue_detail.htm?csnumber=55382 21. International Organization for Standardization (ISO). ISO 9308- 2. Water quality -- Enumeration of Escherichia coli and coliform bacteria -- Part 2: Most probable number method. Geneva: ISO; 2012. Available from: http://www.iso.org/iso/iso_catalogue/ catalogue_tc/catalogue_detail.htm?csnumber=52246 22. International Organization for Standardization (ISO). ISO 7899- 2. Water quality -- Detection and enumeration of intestinal enterococci -- Part 2: Membrane filtration method. Geneva: ISO; 2000. Geneva, Switzerland. Available from: http://www. iso.org/iso/catalogue_detail.htm?csnumber=14854 23. Rnnqvist M, Rtt M, Tuominen P, Salo S, Maunula L. Swabs as a tool for monitoring the presence of norovirus on environmental surfaces in the food industry.J Food Prot. 2013;76(8):1421-8. DOI: 10.4315/0362-028X.JFP-12-371 PMID: 23905799 24. Jothikumar N, Cromeans TL, Hill VR, Lu X, Sobsey MD, Erdman DD. Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41.Appl Environ Microbiol. 2005;71(6):3131-6. 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In: Mandell G, Bennet J, Dolin R, editors. Principles and practice of infectious diseases. 7th ed. Philadelphia: Churchill Livingstone Elsevier. 2010;2027-33. 40. Jovanovi Galovi A, Bijelovi S, Miloevi V, Hrnjakovi Cvjetkovic I, Popovi M, Kovaevi G, et al. Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014. Euro Surveill. 2016;21(15):30196. DOI: 10.2807/1560-7917. ES.2016.21.15.30196 PMID: 27105473 41. Boehm AB, Ashbolt NJ, Colford JM, Dunbar LE, Fleming LE, Gold MA, et al. A sea change ahead for recreational water quality criteria. J Water Health. 2009;7(1):9-20. DOI: 10.2166/ wh.2009.122 PMID: 18957771 42. Fujioka RS, Solo-Gabriele HM, Byappanahalli MN, Kirs M. U.S. Recreational Water Quality Criteria: A Vision for the Future.Int J Environ Res Public Health. 2015;12(7):7752-76. DOI: 10.3390/ ijerph120707752 PMID: 26184253 43. Updyke EA, Wang Z, Sun S, Connell C, Kirs M, Wong M, et al. Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

How many people were ill?

{'answer_start': [468], 'text': ['1,453']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What are the pathogens?

{'answer_start': [955], 'text': ['faecal indicator bacteria']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What are the symptoms?

{'answer_start': [9786], 'text': ['vomiting, diarrhoea, stomach pain, and fever.']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What is the event?

{'answer_start': [33], 'text': ['outbreaks of gastroenteritis linked to bathing water']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. 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In: Mandell G, Bennet J, Dolin R, editors. Principles and practice of infectious diseases. 7th ed. Philadelphia: Churchill Livingstone Elsevier. 2010;2027-33. 40. Jovanovi Galovi A, Bijelovi S, Miloevi V, Hrnjakovi Cvjetkovic I, Popovi M, Kovaevi G, et al. Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014. Euro Surveill. 2016;21(15):30196. DOI: 10.2807/1560-7917. ES.2016.21.15.30196 PMID: 27105473 41. Boehm AB, Ashbolt NJ, Colford JM, Dunbar LE, Fleming LE, Gold MA, et al. A sea change ahead for recreational water quality criteria. J Water Health. 2009;7(1):9-20. DOI: 10.2166/ wh.2009.122 PMID: 18957771 42. Fujioka RS, Solo-Gabriele HM, Byappanahalli MN, Kirs M. U.S. Recreational Water Quality Criteria: A Vision for the Future.Int J Environ Res Public Health. 2015;12(7):7752-76. DOI: 10.3390/ ijerph120707752 PMID: 26184253 43. Updyke EA, Wang Z, Sun S, Connell C, Kirs M, Wong M, et al. Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What are the first steps of mitigation?

{'answer_start': [7952], 'text': ['Restrictions against bathing ']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What measures were taken to prevent the event?

{'answer_start': [1333], 'text': ['published guidelines for outbreak control']}

Contamination Question Answering

An increased number of suspected outbreaks of gastroenteritis linked to bathing water were reported to the Finnish food- and waterborne outbreak (FWO) registry in July and August 2014. The investigation reports were assessed by a national outbreak investigation panel. Eight confirmed outbreaks were identified among the 15 suspected outbreaks linked to bathing water that had been reported to the FWO registry. According to the outbreak investigation reports, 1,453 persons fell ill during these outbreaks. Epidemiological and microbiological data revealed noroviruses as the main causative agents. During the outbreaks, exceptionally warm weather had boosted the use of beaches. Six of eight outbreaks occurred at small lakes; for those, the investigation strongly suggested that the beach users were the source of contamination. In one of those eight outbreaks, an external source of contamination was identified and elevated levels of faecal indicator bacteria (FIB) were noted in water. In the remaining outbreaks, FIB analyses were insufficient to describe the hygienic quality of the water. Restrictions against bathing proved effective in controlling the outbreaks. In spring 2015, the National Institute for Health and Welfare(THL)and the National Supervisory Authority for Welfare and Health (Valvira) published guidelines for outbreak control to prevent bathing water outbreaks. In July 2014, THL received primary information on several suspected outbreaks linked to bathing water via the media, while no notifications were reported to the FWO registry. This resulted in direct contacts with the health authorities, and a reminder about notifying outbreaks related to bathing water was posted in a THL Infectious Disease Bulletin sent to the municipal health authorities. The message was also distributed to municipal environmental authorities by the National Supervisory Authority for Welfare and Health (Valvira). Following these reminders, several notifications were reported to the FWO registry. We identified outbreaks caused by bathing water from the FWO registry for 2014 and reviewed the epidemiological and microbio- logical data in order to assess and compile guidelines for outbreak control to prevent similar outbreaks in the future. Methods Epidemiological investigation We reviewed outbreak notifications and investigation reports from the FWO registry for 2014. Outbreaks with a suspected link to bathing water were included in this study. We evaluated the strength of association for waterborne outbreaks based on classification criteria (Table 1) modified from those presented by Tillett et al. [12] and on information collected from local investigation reports (i.e. time and place of swimming, number of ill persons, clinical and microbiological findings). Microbiological investigation Description of the laboratories and their roles Analyses of enteric virus were carried out in four laboratories. Clinical samples were analysed at the Helsinki University Hospital (HUSLAB) and/or at the Viral Infection Unit of the National Institute for Health and Welfare (THL). Water samples were analysed either at the Water and Health Unit of the National Institute for Health and Welfare (THL) or at the Department of Food Hygiene and Environmental Health, University of Helsinki (UH). Surface samples were analysed at the UH. Pathogenic bacteria, faecal indicator bacteria (FIB) and water temperature analyses were conducted in local clinical and/or environmental laboratories. Clinical samples Viruses were analysed in patients stools for seven out- breaks. At the HUSLAB laboratory, noroviruses were analysed according to Kanerva et al. [13]. For astrovi- ruses, viral RNA was extracted from a 10% suspension of the stool using MagNa Pure LC (Roche, Germany). After RT-PCR, the amplified DNA was detected by liquid hybridisation using an astrovirus-specific probe [14]. At the THL laboratory, norovirus RNAs were extracted using the RNeasy Mini Kit (Qiagen, Germany) and the polymerase/capsid gene junction was amplified as pre- viously described [14]. Genotyping analysis was done for several norovirus isolates at the THL laboratory. Viral RNA was amplified in polymerase region A using a one-step RT-PCR kit (Qiagen) according to Vinj et al. [15]. Sequences were analysed using Geneious soft- ware. NoroNet online software was used for genotyp- ing. For three outbreaks, stool specimens were tested for pathogenic bacteria (Campylobacter,Salmonella, Shigella and Yersinia) by routine methods [16]. Water samples At the THL laboratory, noroviruses and adenoviruses were concentrated from 0.52 L water samples as Table1 Classification criteria used for evaluating the strength of association for waterborne outbreaks, Finland, 2014 A: Same pathogen identified in patients and in the environment B: Water quality failure or other deviation in the quality of environment C: Association between illness and environment shown in analytical epidemiological investigation D: Descriptive epidemiological investigation suggests that the outbreak is related to the environment and excludes other obvious exposures Strong association: A + C or A + D or B + C. Probable association: B + D or C or A. Possible association: B or D. Criteria modified from Tillett et al. [12]. 3 www.eurosurveillance.org previously described [17] and using glass fibre pre- filters (Millipore). Viral nucleic acids were extracted and detected using RT-qPCR and qPCR methods, as previously described [18,19], with the exception of using Taqman Environmental Master Mix 2.0 (Life Technologies) in the adenovirus qPCR. At the UH laboratory, noroviruses and adenoviruses were concentrated by using membrane disks HA and Nanoceram to filter a total volume of 4.5 L of water. When necessary, a prefilter (Waterra) was used, oth- erwise the protocol was as described in Maunula et al. [14]. As a modification, Taqman primerprobe sets were applied as published in ISO/TS 152162 [20] for norovirus GI and GII. Mengovirus was added as a pro- cess control. MPN of E. coli and CFU of intestinal enterococci were analysed according to standards ISO 93082 and ISO 78992, respectively [21,22]. Surface samples In outbreak IV, 10 environmental swabs were taken from the toilet facilities (toilets for females, toilets for males and two latrines). Swabs taken from taps, door handles and toilet seats were analysed for noroviruses according to Rnnqvist et al. using nucleic acid detec- tion by RT-qPCR [23]. For adenovirus investigation, a primerprobe set from Jothikumar et al. was included [24]. Statistical analyses The statistical analyses were conducted using SPSS 22 software for Windows. The related samples Wilcoxon signed-rank test was used to test the significance of temperature and FIB analyses, while comparing the outbreak samples with frequent-monitoring samples collected during the summer. Differences were consid- ered significant if the p value was < 0.05. Results Review of the outbreak notifications and investigation reports In 2014, 15 outbreaks suspected to be caused by bath- ing water were reported to the FWO registry. We identi- fied eight outbreaks in which an association between bathing water and the illness could be confirmed based on classification criteria (Table 1). These out- breaks occurred on public beaches in different parts of Finland in July and August, 2014 (Table 2; Table 3). Six of eight confirmed outbreaks occurred at rather small lakes or ponds (< 141 ha) and eight of 13 beaches were categorised as large public beaches with more than 100 bathers per day (Table 2). According to the BWD classification criteria based on the last four bathing seasons, all these large public beaches were classified as excellent, except for one beach that was opened in 2012 and therefore did not have data for classification. Restrictions against bathing were set for each beach (Table 2). The length of these restrictions varied from 2 days to more than 3 weeks and for one beach, the advice against bathing was set for the rest of the bathing season. Seven of eight outbreaks occurred at inland lakes where no clear source of contamination was identified according to the bathing water profiles and/or outbreak investigation reports, although for five of these outbreaks at inland lakes, non-specific quality deviations were reported (Table 3). In the one Table2 Description of beaches with outbreaks linked to recreational water, Finland, summer 2014 (n = 13) Outbreak Type Size (ha) Category Estimated number of bathers/day EU BWD classification (2014)a Estimated outbreak start time Restriction against bathing I Lake 2,420 Small < 100 NA 26 July 16 August II Lake 2.9 Large 150500 Excellent 25 July 29 July21 August IIIb Lake 5.5141 2/6 small 4/6 large < 100 > 100 NA Excellent 2427 July 28 July12 August IV Lake 16.6 Large 1002,000 Excellent 24 July 31 July31 August (until the end of the bathing season) V Lake 9.7 Small < 100 NA 3 August 1522 August VI Lake 71.1 Large 150 Excellent 5 August 1121 August VII Sea 393,00,000 Small < 100 NA NK 1315 August and 19 August9 September VIII Lake/pond 0.8 Large 1,000 NAc 27 July 621 August EU BWD: European Unions Bathing Water Directive [5]; NA: not available; NK: not known. a Based on frequent monitoring during the last four bathing seasons [5]. b Combined results from six beaches. c New beach, no classification. 4 www.eurosurveillance.org coastal sea water outbreak, a wastewater overflow was identified as a potential source of contamination. According to the outbreak investigation reports, 1,453 persons fell ill in these outbreaks (Table 3). The most common symptoms were vomiting, diarrhoea, stomach pain, and fever. Information on the incubation period was available for four outbreaks, the median incuba- tion period ranging from 20 to 62 hours. The dura- tion of illness was reported for five outbreaks, with a median ranging from 19 to 60 hours. None of the patients required hospital care. Patient samples were collected in seven outbreaks and tested for gastrointestinal pathogenic viruses and bacteria. Several types of norovirus were identified, with norovirus GI.2 detected in three outbreaks (Table 3). In addition, norovirus GI.4, GII.2 and GII.4 were detected in patient samples. In one patient, astrovirus was identified. According to outbreak investigation reports, pathogenic bacteria were analyzed in three investigations (outbreaks III, IV and VIII). Campylobacter was found in one patient (outbreak III). Salmonella, Shigella or Yersinia spp. were not found in any of the specimens tested. Water samples were collected for noro- and adenovi- rus analyses in seven outbreaks, and noro- and/or adenoviruses were detected in the samples from three outbreaks (Table 3). In the remaining outbreak, these analyses were not requested by the municipal health protection authority. FIB were analysed from water in all outbreaks. In addition, water quality monitoring was carried out at every beach according to EU BWD and national regulations. Elevated levels of both FIB were found in two of the outbreaks (VII and VIII; Table 4), but only in outbreak VII did the number of E. coli exceed the limit for management actions, with maximum con- centrations of 1,100 and 190 CFU/100 mL for E. coli and enterococci, respectively. Elevated levels of ente- rococci were also noted in outbreak I. In the remain- ing outbreaks, the levels of FIB were low. Overall, no statistical difference in the levels of E. coli (p = 0.8) or enterococci (p = 0.086) were noted between the out- break samples (n = 14) and the frequent-monitoring samples (n = 42), excluding the samples from outbreak VII, where a clear contamination source was noted. At one outbreak (IV), 10 surface samples from the toi- let area were analysed, and norovirus GII was found on the tap of the womens toilet. Adenoviruses were not detected in the surface samples. Water temperature During the outbreak period, exceptionally warm weather raised the temperature of the bathing water by several degrees (Table 4). The average tempera- ture of the bathing water samples collected during the outbreaks was 24.3 1.3 C (n = 16), while the average temperature of other frequent-monitoring samples col- lected at these beaches in summer 2014 (2 June to 26 August) was 19.4 3.6 C (n = 47; p = 0.002). Table3 Strength of association for waterborne outbreaks, number of patients, virological findings and observed quality deviations, Finland, summer 2014 (n = 1,453 patients) Outbreak Strength of associationa No. of patients Viruses found in patients No. of virus findings per water samples tested Viruses found in water Observed quality deviation I Possible (D) 40 NA 0/1 ND Not observed II Probable (A + B) 85 Norovirus GI.2 2/4 Adenovirus, norovirus GI Untidy toilets IIIb Strong (B + C) 819b 1,093c Norovirus GI.2, GI.4, GII.2 0/3 ND Untidy toilets, defecation in water IV Strong (A + B + D) 185 Norovirus GII 0/1 ND Untidy toilets V Probable (A) 4 Norovirus GI.2 and GII.4 1/2 Norovirus GII Not observed VI Possible (B) 17 Norovirus (not typed) 0/2 ND Untidy toilets, used nappies in water VII Possible (B) 2 Norovirus GI NA NA Wastewater overflow VIII Possible (B) 27 Astrovirus 1/3 Adenovirus Faeces on the dock NA: not analysed; ND: not detected. a Letters refer to classification criteria detailed in Table 1. b Combined results from six beaches that were investigated in detail. c Total number from all 32 suspected beaches from which the local health authority received notifications of illness. 5 www.eurosurveillance.org Discussion In 2014, an increased number of suspected outbreaks linked to bathing water were reported to the Finnish FWO registry. Reminders about the need to notify outbreaks borne by bathing water were sent to the municipal authorities and probably triggered the fol- lowing notifications seeing as only one outbreak linked to bathing water had been reported during the period 2012 to 2013. In addition, the publicity around out- breaks in 2014 probably made the beach users more alert so that they reported their suspicions of bath- ing water-related sickness to the health authorities. Generally, it could be difficult to attribute individually reported gastroenteritis cases to a particular bathing activity and therefore these outbreaks may remain undocumented. Nearly 1,500 persons fell ill during the outbreaks linked to bathing water in 2014. Although the exact number of people visiting the beaches was not known, some municipal investigation reports estimated that hun- dreds to thousands of persons per day had been swim- ming at each beach during the outbreak period before restrictions against bathing were set. In the summer of 2014, the period of continuous hot weather in Finland, with temperatures of more than 25 C, was exception- ally long and lasted for 38 days [25]. Because of this heatwave, it is likely that more people than usual were visiting the beaches and spent more time in the water. A previous study noted a positive correlation between the number of days with temperatures over 25 C and the number of outbreaks per bathing season [26]. Some investigation reports also stated that the toilets at the beaches were untidy, rubbish bins were overloaded, and used nappies were floating in the water, indicating overcrowded conditions. In 2015, no outbreaks linked to bathing water were reported. This was probably due in part to the weather conditions, namely 3 days with temperatures over 25 C in July 2015, compared with 26 such days in July 2014. In Helsinki, the average tem- perature and precipitation in July differed considerably between 2015 and 2014 (16.2 C/76.1 mm vs 20 C/12.5 mm) [27]. Most of the beaches were small, suggesting that the volume of users exceeded the self-cleaning capacity of the beach. For example, the volume of the smallest lake (outbreak VIII) is 20,800 m3. In theory, if a single infected person excreted large numbers of noroviruses (up to 1011 genomic copies/g) [28], and if these viruses were evenly diluted in the total volume of the lake, 1 g of faeces would result in a virus concentration of nearly 5,000 genomic copies/L. Considering the low infectious dose of norovirus (as few as 18 virus particles) [29] and the average ingestion of water while swimming (37 mL and 16 mL for children and adults, respectively, per Table4 Levels of faecal indicator bacteria and water temperature in outbreak samples (n = 17) and frequent-monitoring samples (n = 47), Finland, summer 2014 Outbreak No. of analysed water samples Escherichia coli MPN/100 mL Intestinal enterococci CFU/100 mL Temperature C I Outbreak samples Monitoring samples 1 3 6 8 6 190 4 2 25.7 22.1 3.3 II Outbreak samples Monitoring samples 2 6 39 26 72 72 9 8 6 4 25.0 1.4 20.5 4.4 IIIa Outbreak samples Monitoring samples 5 18 14 10 19 4 3 3 15 22 25.2 0 19.0 3.8 IV Outbreak samples Monitoring samples 1 4 9 3 3 7 1 2 24.0 19.8 4.2 V Outbreak samples Monitoring samples 1 2 12 34 47 22 6 8 24.0 19.3 2.5 VI Outbreak samples Monitoring samples 2 4 4 1 1 0 3 2 1 1 23.0 0 17.5 3.7 VII Outbreak samples Monitoring samples 3 5 670 580 2 4 110 98 4 4 22.3 1b 20.2 3.5 VIII Outbreak samples Monitoring samples 2 5 130 120 17 5 48 46 8 7 23.9 1 18.1 2.2 CFU: colony-forming units; MPN: most probable number. a Combined results from the five beaches for which indicator bacteria were analysed. b Average from n = 2 samples. 6 www.eurosurveillance.org 45 min swimming session [30]), it is obvious that the bathing water at this particular beach would have the potential to cause a considerable number of infections. Norovirus was detected in ill persons in most of the out- breaks. The symptoms reported by municipal authori- ties fit the clinical picture of a norovirus illness [31]. In three outbreaks, norovirus GI.2 was identified. In addi- tion, also GI.4, GII.2 and GII.4 were detected in patient samples. The prevalence of GI in these outbreaks is consistent with the observation that GI genotypes are more frequently involved in food- or waterborne out- breaks than GII, which could imply that GI is more sta- ble in the environment [32,33]. Genotype GII.4 is the most common genotype causing infections in humans and is more likely to be associated with person-to-per- son transmission [34]. In two outbreaks, norovirus GI and GII were found in bathing water and in one outbreak, GII was determined in a swab taken from the tap of the toilet, but the num- ber of particles obtained was too small to allow typ- ing of these viruses. Therefore, an exact comparison between patient and water samples could not be car- ried out. In two outbreaks, adenovirus was found in water. Adenoviruses are commonly found in human wastewater and owing to their high stability in aqueous environments, they are recognised as good viral indi- cators of human sewage pollution [19,35,36]. Moreover, adenoviruses can spread via contaminated water and they have been linked to waterborne outbreaks [14,37,38]. Since adenoviruses most often result in subclinical disease, and symptomatic infections tend to be mild and self-resolving, most infections remain undocumented [39]. In the outbreaks of this study, no adenoviruses were identified in ill persons. In Finland, the hygienic quality of the bathing water is evaluated according to BWD and national regulations [3-5]. According to Finnish legislation, the minimum number of bathing water samples to be taken during a bathing season is three for small public beaches and four for large public beaches. The legislation con- tains rules how to monitor and manage bathing waters, indicates microbiological threshold values, regulates measures to be taken when bathing water fails to meet the quality and requires the dissemination of informa- tion about bathing water quality. In Finland, the concen- trations of FIB in bathing water are typically very low; 70% of the E. coli and 58% of the intestinal enterococci concentrations were < 10 CFU or MPN/100 mL in bathing water samples collected from all large public beaches (n = 302) during the seasons from 2013 to 2015 (data not shown). In this study, the microbiological thresh- old for management actions was exceeded only in one of eight outbreaks. For this outbreak, a clear external contamination source was identified as 2,0003,000 m3 of raw wastewater had overflowed near the bath- ing site. In the other outbreaks, the levels of FIB were low and the bathing water quality was classified as excellent according to the BWD criteria. The sources of contamination in these outbreaks were most probably the bathers and other beach users. This suggestion is supported by the observed pollution of the beach environment. The poor indicator value of FIB in these outbreaks raises questions about the current practices for assessing bathing water quality. This finding is consistent with a recent study showing high prevalence of adenovi- ruses (75%) in bathing water samples, which neverthe- less complied with the regulations for recreational use [40]. Moreover, Boehm et al. reviewed the lack of cor- relation between FIB and human pathogen concentra- tions and between FIB and human health, especially in recreational areas of non-point-source contamination [41]. It is also widely known that pathogenic microbes, especially enteric viruses, survive substantially bet- ter than the currently used FIB in water environments. Therefore, new candidates, such as Clostridium per- fringens, coliphages, Bacteroides and human enteric viruses as well as new genomic approaches, e.g. metagenomics, have been proposed for water quality assessment [41-43]. However, during the summer, the higher temperature of bathing water and the increased amount of ultraviolet light have a negative impact on microbe survival. In this study, noro- and adenoviruses in outbreak II were detected in the water on at least six days but fewer than 12 days. These relatively short contamination episodes may remain undetected with routine FIB sampling. In most of the outbreaks, the quality of bathing water was questioned only after people visiting the beaches fell ill, and restrictions against bathing were set for the beaches only then. The length of the restrictions was determined according to the results of water analyses and proved effective in con- trolling of the outbreaks. Investigation reports of outbreaks linked to bathing water were assessed by a panel that included experts from THL, Valvira and UH. By using agreed criteria, reports can be assessed more consistently over time [12]. When the same pathogen has been identified in patients and in the beach environment, results from the analytical epidemiological study point towards a certain source and water quality failures have been detected, outbreaks are often easy to categorise. More discussion in the panel will be needed on the relation between illness and the beach environment when pol- lution of the beach is mentioned but no obvious other exposures are described in outbreak reports. In this study, eight outbreaks were identified among the 15 outbreaks suspected to be caused by bathing water that were reported to the FWO registry. Four outbreaks were classified as having a strong or probable associa- tion with the beach environment, and four as having a possible association. Analytical epidemiological inves- tigations were lacking in all but one investigation, indi- cating that more training and practical experience in analytical epidemiology may be needed in the munici- pal outbreak investigation groups. 7 www.eurosurveillance.org Because of an increase in the number of bathing water outbreaks in the summer of 2014, THL and Valvira pub- lished guidelines for outbreak control in spring 2015 to prevent bathing water outbreaks. If, based on the labo- ratory or epidemiological findings, the water is consid- ered to be contaminated, visitors should be informed about a bathing prohibition or advice against bath- ing should be posted by means of the international symbols presented in the Commission Implementing Decision (2011/321/EU) [44]. To prevent outbreaks, rooms intended for washing and dressing as well as toilets at the beach should be kept clean, and soap, hand towels and toilet paper should be available. Visitors should be encouraged to wash their hands or use freshen-up towels. Nappies should not be changed and the babies bottoms should not be washed in the bathing water, and people with gastrointestinal illness should avoid swimming. In the case of an outbreak suspicion, municipal authorities should notify the FWO registry and an outbreak investigation, including epi- demiological and microbiological analyses, should be initiated. Acknowledgements Appreciation is given to the municipal health authorities for their investigations and assistance. We acknowledge the help of the personnel at the National Institute for Health and Welfare and the University of Helsinki. The research at THL was partly supported by the personal research grant to Ari Kauppinen from the Doctoral School of the University of Eastern Finland. The research at UH was partly supported by EU project Aquavalens (311846). Conflict of interest None declared. Authors contributions Ari Kauppinen, Haider Al-Hello, Outi Zacheus, Jaana Kilponen, Leena Maunula, Sari Huusko, Ilkka Miettinen, Soile Blomqvist and Ruska Rimhanen-Finne participated in the national outbreak evaluation panel and the design of the study. Ruska Rimhanen-Finne coordinated the national panel. Ari Kauppinen was responsible for performing the data analyses and virus analyses from water performed at THL. Haider Al-Hello, Soile Blomqvist and Maija Lappalainen were responsible for analysing viruses from patient samples. Leena Maunula was responsible for analysing viruses from the water and environmental samples performed at UH. Ari Kauppinen and Ruska Rimhanen-Finne drafted the manu- script. All authors were involved in the preparation and re- view of the manuscript and approved the final version. References 1. Zacheus O, Miettinen IT. Increased information on waterborne outbreaks through efficient notification system enforces actions towards safe drinking water.J Water Health. 2011;9(4):763-72. DOI: 10.2166/wh.2011.021 PMID: 22048435 2. Finnish Decree. Valtioneuvoston asetus elintarvikkeiden ja veden vlityksell levivien epidemioiden selvittmisest. [Government Decree concerning the follow-up and reporting of food- and waterborne outbreaks]. Document no. 1365/2011. Helsinki: Finlex database; 2011. Finnish. Available from: http:// www.finlex.fi/fi/laki/alkup/2011/20111365 3. 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In: Mandell G, Bennet J, Dolin R, editors. Principles and practice of infectious diseases. 7th ed. Philadelphia: Churchill Livingstone Elsevier. 2010;2027-33. 40. Jovanovi Galovi A, Bijelovi S, Miloevi V, Hrnjakovi Cvjetkovic I, Popovi M, Kovaevi G, et al. Testing for viral material in water of public bathing areas of the Danube during summer, Vojvodina, Serbia, 2014. Euro Surveill. 2016;21(15):30196. DOI: 10.2807/1560-7917. ES.2016.21.15.30196 PMID: 27105473 41. Boehm AB, Ashbolt NJ, Colford JM, Dunbar LE, Fleming LE, Gold MA, et al. A sea change ahead for recreational water quality criteria. J Water Health. 2009;7(1):9-20. DOI: 10.2166/ wh.2009.122 PMID: 18957771 42. Fujioka RS, Solo-Gabriele HM, Byappanahalli MN, Kirs M. U.S. Recreational Water Quality Criteria: A Vision for the Future.Int J Environ Res Public Health. 2015;12(7):7752-76. DOI: 10.3390/ ijerph120707752 PMID: 26184253 43. Updyke EA, Wang Z, Sun S, Connell C, Kirs M, Wong M, et al. Human enteric viruses--potential indicators for enhanced monitoring of recreational water quality. Virol Sin. 2015;30(5):344-53. DOI: 10.1007/s12250-015-3644-x PMID: 26494480 44. European Union. Commission Implementing Decision 2011/321/ EU of 27 May 2011 establishing, pursuant to Directive 2006/7/ EC of the European Parliament and of the Council, a symbol for information to the public on bathing water classification and any bathing prohibition or advice against bathing. Off J. Eur Union. 2011;L143:38-40. Available from: http://eur-lex.europa. eu/eli/dec_impl/2011/321/oj License and copyright This is an open-access article distributed under the terms of the Creative Commons Attribution (CC BY 4.0) Licence. You may share and adapt the material, but must give appropriate credit to the source, provide a link to the licence, and indi- cate if changes were made. This article is copyright of the authors, 2017.

What is the source of contamination?

{'answer_start': [799], 'text': ['beach users']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What is the initial cause of the event?

{'answer_start': [25017], 'text': ['contamination of surface water by human sewage']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. This work was supported by grants from the Research and Development Unit, Jmtland County Council, Sweden, and the Medical Faculty of Ume University, Ume, Sweden. Dr Widerstrm is the county medical officer at the Depart- ment of Communicable Diseases Control and Prevention, Coun- ty Council of Jmtland, Sweden, and senior infectious disease consultant at the Department of Infectious Diseases, stersund Hospital. His primary research interests include epidemiology of communicable diseases, especially healthcare-associated staphy- lococcal infections. References 1. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol. 2013;29:23751. http://dx.doi.org/10.1016/j.pt.2013.03.001 2. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev. 2013;26:11534. http://dx.doi.org/10.1128/CMR.00076-12 3. Insulander M, Silverlas C, Lebbad M, Karlsson L, Mattsson JG, Svenungsson B. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What is the date of the event?

{'answer_start': [3], 'text': ['November 2010']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What is the location of the event?

{'answer_start': [57], 'text': ['Sweden']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. This work was supported by grants from the Research and Development Unit, Jmtland County Council, Sweden, and the Medical Faculty of Ume University, Ume, Sweden. Dr Widerstrm is the county medical officer at the Depart- ment of Communicable Diseases Control and Prevention, Coun- ty Council of Jmtland, Sweden, and senior infectious disease consultant at the Department of Infectious Diseases, stersund Hospital. His primary research interests include epidemiology of communicable diseases, especially healthcare-associated staphy- lococcal infections. References 1. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol. 2013;29:23751. http://dx.doi.org/10.1016/j.pt.2013.03.001 2. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev. 2013;26:11534. http://dx.doi.org/10.1128/CMR.00076-12 3. Insulander M, Silverlas C, Lebbad M, Karlsson L, Mattsson JG, Svenungsson B. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

How was the event first detected?

{'answer_start': [6221], 'text': ['received reports from several employers that 10%-20% of employees had gastroenteritis.']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

How many people were ill?

{'answer_start': [18], 'text': ['27,000']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. This work was supported by grants from the Research and Development Unit, Jmtland County Council, Sweden, and the Medical Faculty of Ume University, Ume, Sweden. Dr Widerstrm is the county medical officer at the Depart- ment of Communicable Diseases Control and Prevention, Coun- ty Council of Jmtland, Sweden, and senior infectious disease consultant at the Department of Infectious Diseases, stersund Hospital. His primary research interests include epidemiology of communicable diseases, especially healthcare-associated staphy- lococcal infections. References 1. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol. 2013;29:23751. http://dx.doi.org/10.1016/j.pt.2013.03.001 2. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev. 2013;26:11534. http://dx.doi.org/10.1128/CMR.00076-12 3. Insulander M, Silverlas C, Lebbad M, Karlsson L, Mattsson JG, Svenungsson B. 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J Infect Dis. 2001;183:13739. http://dx.doi.org/10.1086/319862 36. Borad A, Ward H. Human immune responses in cryptosporidiosis. Fu- ture Microbiol. 2010;5:50719. http://dx.doi.org/10.2217/fmb.09.128 37. Howe AD, Forster S, Morton S, Marshall R, Osborn KS, Wright P, et al. Cryptosporidium oocysts in a water supply associated with a cryptosporidiosis outbreak. Emerg Infect Dis. 2002;8:61924. http://dx.doi.org/10.3201/eid0806.010271 38. Ongerth JE. The concentration of Cryptosporidium and Giardia in waterthe role and importance of recovery efficiency. Water Res. 2013;47:247988. http://dx.doi.org/10.1016/j.watres. 2013.02.015 39. Causer LM, Handzel T, Welch P, Carr M, Culp D, Lucht R, et al. An outbreak of Cryptosporidium hominis infection at an Illinois recreational waterpark. Epidemiol Infect. 2006;134:14756. http:// dx.doi.org/10.1017/S0950268805004619 40. Gallas-Lindemann C, Sotiriadou I, Plutzer J, Karanis P. Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What is the attack rate?

{'answer_start': [8170], 'text': ['50%']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What are the pathogens?

{'answer_start': [16630], 'text': ['Cryptosporidium oocysts']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. This work was supported by grants from the Research and Development Unit, Jmtland County Council, Sweden, and the Medical Faculty of Ume University, Ume, Sweden. Dr Widerstrm is the county medical officer at the Depart- ment of Communicable Diseases Control and Prevention, Coun- ty Council of Jmtland, Sweden, and senior infectious disease consultant at the Department of Infectious Diseases, stersund Hospital. His primary research interests include epidemiology of communicable diseases, especially healthcare-associated staphy- lococcal infections. References 1. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol. 2013;29:23751. http://dx.doi.org/10.1016/j.pt.2013.03.001 2. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev. 2013;26:11534. http://dx.doi.org/10.1128/CMR.00076-12 3. Insulander M, Silverlas C, Lebbad M, Karlsson L, Mattsson JG, Svenungsson B. 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Syndromic surveillance for local outbreak detec- tion and awareness: evaluating outbreak signals of acute gastroen- teritis in telephone triage, Web-based queries and over-the-counter pharmacy sales. Epidemiol Infect. 2014;142:30313. http://dx.doi. org/10.1017/S0950268813001088 20. Casemore DP. ACP Broadsheet 128: June 1991. Laboratory methods for diagnosing cryptosporidiosis. J Clin Pathol. 1991;44:44551. http://dx.doi.org/10.1136/jcp.44.6.445 21. Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W, et al. Identification of 5 types of Cryptosporidium parasites in chil- dren in Lima, Peru. J Infect Dis. 2001;183:4927. http://dx.doi. org/10.1086/318090 22. Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F. Sub- genotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol. 2003;41:27447. http://dx.doi.org/10.1128/JCM.41.6.2744-2747.2003 23. Loveridge P, Cooper D, Elliot AJ, Harris J, Gray J, Large S, et al. Vomiting calls to NHS Direct provide an early warning of norovirus outbreaks in hospitals. J Hosp Infect. 2010;74:38593. http://dx.doi. org/10.1016/j.jhin.2009.10.007 24. Keller M, Blench M, Tolentino H, Freifeld CC, Mandl KD, Mawudeku A, et al. Use of unstructured event-based reports for global infectious disease surveillance. Emerg Infect Dis. 2009;15:68995. http://dx.doi.org/10.3201/eid1505.081114 25. Public Health Laboratory Study Group. Cryptosporidiosis in England and Wales: prevalence and clinical and epidemiological features. BMJ. 1990;300:7747. http://dx.doi.org/10.1136/bmj.300.6727.774 26. Mason BW, Chalmers RM, Carnicer-Pont D, Casemore DP. A Cryptosporidium hominis outbreak in north-west Wales associated with low oocyst counts in treated drinking water. J Water Health. 2010;8:299310. http://dx.doi.org/10.2166/wh.2009.184 27. Yoder JS, Beach MJ. Cryptosporidium surveillance and risk factors in the United States. 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J Infect Dis. 2001;183:13739. http://dx.doi.org/10.1086/319862 36. Borad A, Ward H. Human immune responses in cryptosporidiosis. Fu- ture Microbiol. 2010;5:50719. http://dx.doi.org/10.2217/fmb.09.128 37. Howe AD, Forster S, Morton S, Marshall R, Osborn KS, Wright P, et al. Cryptosporidium oocysts in a water supply associated with a cryptosporidiosis outbreak. Emerg Infect Dis. 2002;8:61924. http://dx.doi.org/10.3201/eid0806.010271 38. Ongerth JE. The concentration of Cryptosporidium and Giardia in waterthe role and importance of recovery efficiency. Water Res. 2013;47:247988. http://dx.doi.org/10.1016/j.watres. 2013.02.015 39. Causer LM, Handzel T, Welch P, Carr M, Culp D, Lucht R, et al. An outbreak of Cryptosporidium hominis infection at an Illinois recreational waterpark. Epidemiol Infect. 2006;134:14756. http:// dx.doi.org/10.1017/S0950268805004619 40. Gallas-Lindemann C, Sotiriadou I, Plutzer J, Karanis P. Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What are the symptoms?

{'answer_start': [507], 'text': ['diarrhea']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. This work was supported by grants from the Research and Development Unit, Jmtland County Council, Sweden, and the Medical Faculty of Ume University, Ume, Sweden. Dr Widerstrm is the county medical officer at the Depart- ment of Communicable Diseases Control and Prevention, Coun- ty Council of Jmtland, Sweden, and senior infectious disease consultant at the Department of Infectious Diseases, stersund Hospital. His primary research interests include epidemiology of communicable diseases, especially healthcare-associated staphy- lococcal infections. References 1. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol. 2013;29:23751. http://dx.doi.org/10.1016/j.pt.2013.03.001 2. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev. 2013;26:11534. http://dx.doi.org/10.1128/CMR.00076-12 3. Insulander M, Silverlas C, Lebbad M, Karlsson L, Mattsson JG, Svenungsson B. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What is the event?

{'answer_start': [95], 'text': ['outbreak of cryptosporidiosis']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. This work was supported by grants from the Research and Development Unit, Jmtland County Council, Sweden, and the Medical Faculty of Ume University, Ume, Sweden. Dr Widerstrm is the county medical officer at the Depart- ment of Communicable Diseases Control and Prevention, Coun- ty Council of Jmtland, Sweden, and senior infectious disease consultant at the Department of Infectious Diseases, stersund Hospital. His primary research interests include epidemiology of communicable diseases, especially healthcare-associated staphy- lococcal infections. References 1. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol. 2013;29:23751. http://dx.doi.org/10.1016/j.pt.2013.03.001 2. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev. 2013;26:11534. http://dx.doi.org/10.1128/CMR.00076-12 3. Insulander M, Silverlas C, Lebbad M, Karlsson L, Mattsson JG, Svenungsson B. 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J Infect Dis. 2001;183:13739. http://dx.doi.org/10.1086/319862 36. Borad A, Ward H. Human immune responses in cryptosporidiosis. Fu- ture Microbiol. 2010;5:50719. http://dx.doi.org/10.2217/fmb.09.128 37. Howe AD, Forster S, Morton S, Marshall R, Osborn KS, Wright P, et al. Cryptosporidium oocysts in a water supply associated with a cryptosporidiosis outbreak. Emerg Infect Dis. 2002;8:61924. http://dx.doi.org/10.3201/eid0806.010271 38. Ongerth JE. The concentration of Cryptosporidium and Giardia in waterthe role and importance of recovery efficiency. Water Res. 2013;47:247988. http://dx.doi.org/10.1016/j.watres. 2013.02.015 39. Causer LM, Handzel T, Welch P, Carr M, Culp D, Lucht R, et al. An outbreak of Cryptosporidium hominis infection at an Illinois recreational waterpark. Epidemiol Infect. 2006;134:14756. http:// dx.doi.org/10.1017/S0950268805004619 40. Gallas-Lindemann C, Sotiriadou I, Plutzer J, Karanis P. Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What are the initial steps of investigation?

{'answer_start': [7149], 'text': ['questionnaire']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. This work was supported by grants from the Research and Development Unit, Jmtland County Council, Sweden, and the Medical Faculty of Ume University, Ume, Sweden. Dr Widerstrm is the county medical officer at the Depart- ment of Communicable Diseases Control and Prevention, Coun- ty Council of Jmtland, Sweden, and senior infectious disease consultant at the Department of Infectious Diseases, stersund Hospital. His primary research interests include epidemiology of communicable diseases, especially healthcare-associated staphy- lococcal infections. References 1. Chalmers RM, Katzer F. Looking for Cryptosporidium: the application of advances in detection and diagnosis. Trends Parasitol. 2013;29:23751. http://dx.doi.org/10.1016/j.pt.2013.03.001 2. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium pathogenicity and virulence. Clin Microbiol Rev. 2013;26:11534. http://dx.doi.org/10.1128/CMR.00076-12 3. Insulander M, Silverlas C, Lebbad M, Karlsson L, Mattsson JG, Svenungsson B. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What are the first steps of mitigation?

{'answer_start': [6771], 'text': ['boil-water advisory']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What measures were taken to prevent the event?

{'answer_start': [930], 'text': ['optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What was the age of the affected people?

{'answer_start': [12942], 'text': ['44 years ']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What is the source of contamination?

{'answer_start': [24923], 'text': ['Water Supply']}

Contamination Question Answering

In November 2010, 27,000 (45%) inhabitants of stersund, Sweden, were affected by a waterborne outbreak of cryptosporidiosis. The outbreak was characterized by a rapid onset and high attack rate, especially among young and middle-aged persons. Young age, number of infected family members, amount of water consumed daily, and gluten intolerance were identified as risk factors for acquiring cryptosporidiosis. Also, chronic intestinal disease and young age were significantly associated with prolonged diarrhea. Identification of Cryptosporidium hominis subtype IbA10G2 in human and environmental samples and consistently low numbers of oocysts in drinking water confirmed insufficient reduction of parasites by the municipal water treatment plant. The current outbreak shows that use of inadequate microbial barriers at water treatment plants can have serious consequences for public health. This risk can be minimized by optimizing control of raw water quality and employing multiple barriers that remove or inactivate all groups of pathogens. Protozoan parasites of the genus Cryptosporidium can cause gastrointestinal illness in humans and animals (1). Twenty-six species and >60 genotypes have been identified (2). C. parvum and C. hominis are the most prevalent species that infect humans (1,3). Cryptosporidiosis is transmitted mainly by the fecal-oral route, usually through oocyst-contaminated water or food or by direct contact with an infected person or animal (2). Infectivity is dose de- pendent and certain subtypes are apparently more virulent, requiring only a few oocysts to establish infection (1,4). In healthy persons, gastrointestinal symptoms usually resolve spontaneously within 12 weeks, although asymptomatic carriage can occur (2). Nonetheless, in immunocompromised patients, severe life-threatening watery diarrhea can develop (2). Information is limited regarding the long-term effects of Cryptosporidium infection (3,5,6). The global incidence of cryptosporidiosis is largely unknown, although the disease was recently identified as one of the major causes of moderate to severe diarrhea in children <5 years of age in low-income countries (7). In Sweden, cryptosporidiosis has been a notifiable disease since 2004, and 150 cases (1.7/100,000 population/year) were reported annually until 2009. However, cryptosporid- iosis is probably underreported, mainly because sampling from patients with gastrointestinal symptoms and requests for diagnostic tests are insufficient (3,8). Because of some inherent characteristics of the patho- gen, Cryptosporidium infection has critical public health implications for drinking water and recreational waters. The oocysts are excreted in large numbers in feces, can survive for months in the environment (5), and are resis- tant to the concentrations of chlorine commonly used to treat drinking water (9). The first reported outbreak of wa- terborne human cryptosporidiosis occurred in the United States in 1984 (10), and since then, numerous outbreaks involving up to hundreds of persons have been identified in several parts of the world (11,12). However, only a few very large outbreaks have been documented (1315); the most extensive occurred in 1993 in Milwaukee, Wis- consin, USA, in which 400,000 persons were infected with Cryptosporidium oocysts by drinking water from a Large Outbreak of Cryptosporidium hominis Infection Transmitted through the Public Water Supply, Sweden Micael Widerstrm, Caroline Schnning, Mikael Lilja, Marianne Lebbad, Thomas Ljung, Grel Allestam, Martin Ferm, Britta Bjrkholm, Anette Hansen, Jari Hiltula, Jonas Lngmark, Margareta Lfdahl, Maria Omberg, Christina Reuterwall, Eva Samuelsson, Katarina Widgren, Anders Wallensten, and Johan Lindh Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 581 Author affiliations: Ume University, Ume, Sweden (M. Widerstrm, M. Lilja, M. Ferm, C. Reuterwall, E. Samuelsson); Jmtland County Council, stersund, Sweden (M. Widerstrm, M. Omberg); Public Health Agency of Sweden, Solna, Sweden (C. Schnning, M. Leb- bad, G. Allestam, B. Bjrkholm, A. Hansen, J. Lngmark, M. Lf- dahl, K. Widgren, A. Wallensten, J. Lindh); Mid Sweden University, stersund (T. Ljung); stersund Municipality, stersund (J. Hitula); and Karolinska Institutet, Stockholm (J. Lindh) DOI: http://dx.doi.org/10.3201/eid2004.121415 RESEARCH water treatment plant (WTP) (14). Cryptosporidium spp. are the predominant protozoan parasites causing water- borne outbreaks worldwide (11). In 2012, an increase in Cryptosporidium infections, particularly by C. hominis IbA10G2, was reported in Europe (16). In Sweden, only 1 drinking water outbreak involving Cryptosporidium has been recognized (Y. Andersson, pers. comm.), and a C. parvum outbreak associated with fecal contamination of a public swimming pool occurred in 2002 and affected 1,000 persons (17). A study of Cryptospo- ridium species and subtypes isolated from samples from 194 patients in Sweden during 20062008 identified 111 C. parvum infections and 65 C. hominis infections. Most pa- tients with C. hominis infection had been infected abroad, and only 3 were considered to have sporadic domestic in- fections (3). A recent investigation of Cryptosporidium in raw water from 7 large WTPs in Sweden (not including the WTP of interest in the present study) identified 23 (11.5%) of 200 positive samples containing 130 oocysts/10 L, al- though neither species nor subtypes were analyzed (18). The city of stersund is located in central Sweden and has a population of 60,000. The major WTP in stersund (WTP-) draws surface water from nearby Lake Storsjn and supplies drinking water to 51,000 of the citys inhab- itants. At the time of the onset of the outbreak reported here, the purification process at WTP included preozonation, flocculation, and sedimentation, followed by rapid sand filtering and chloramination. WTP- is situated 4 km upstream from the major wastewater treatment plant (WWTP-) to ensure that the drinking water intake will not be affected by the wastewater outlet (Figure 1). In late November 2010, the County Medical Office in stersund received reports from several employers that 10%-20% of employees had gastroenteritis. The office advised that patients with acute gastroenteritis be tested for bacterial, viral, and protozoan pathogens. Among 20 patients from whom samples were obtained, 14 cases of cryptosporidiosis were detected on November 26. The local health advice line received numerous calls from persons with gastroenteritis, most of whom lived within the municipality (19). These facts indicated that the outbreak could be traced to the drinking water, and thus a boil-water advisory was issued for the municipality on November 26. This study describes the outbreak investigation and outlines the extent of the outbreak, clinical characteristics of persons infected, and risk factors for acquiring cryptosporidiosis. Methods Epidemiologic Investigation Electronic Survey To estimate the extent of the outbreak, the municipality published a questionnaire on its website during November 27- December 13, 2010. Persons in stersund who 582 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 1. Map of Lake Storsjn, showing water currents (arrows) and locations of wastewater treatment plant, water treatment plant, and contaminating stream during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. C. hominis Infection Transmitted through Water Supply had gastrointestinal symptoms were encouraged to provide information about day of onset, home address, and recent food intake. Written Questionnaire Two months after the outbreak began, we conducted a retrospective cohort study, which included a random sample of 1,524 persons living in stersund, to assess the extent of the outbreak, clinical characteristics of infected persons, and risk factors for acquiring cryptosporidiosis. We estimated the proportion infected among the population of stersund with a 3% margin of error (95% CI) by assuming a 50% attack rate and a 70% response rate when calculating the sample size. The patient questionnaire con- tained items on demographic characteristics, onset and oc- currence of possible symptoms of cryptosporidiosis, water consumption, underlying diseases, and whether the WTP- supplied water to the persons workplace. Residential WTP supply was ascertained through population registers. Parents or guardians were asked to respond for children <15 years of age. A case-patient was defined as a person who lived in stersund in mid-January 2011 and had had 3 ep- isodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010, and before January 31, 2011. The study was approved by the Research Ethics Committee of the Faculty of Medicine, Ume University, Ume, Sweden. Microbiological Investigation Human Samples From November 1, 2010, through January 31, 2011, fecal samples from inhabitants of stersund who had acute gastroenteritis were tested for various pathogens. Cryptosporidium oocysts were analyzed by standard concentration techniques and modified Ziehl-Neelsen staining (20); enteric bacterial pathogens by standard methods; noroviruses and sapoviruses by PCR; and Entamoeba spp. and Giardia duodenalis by conventional light microscopy. Environmental Samples During the outbreak, 163 samples of drinking water, raw water, and wastewater were collected to trace the source and monitor the presence of oocysts. Most water samples were collected at or near WTP- and at WWTP- . However, as the outbreak spread to nearby regions, sampling was also conducted at 14 other WTPs and 6 additional WWTPs. The municipality identified 4 differ- ent streams with high counts of Escherichia coli that may have contaminated the raw water, and samples from those streams were analyzed for Cryptosporidium. Also, as part of a then-ongoing national survey regarding presence of parasites in wastewater, 7 preoutbreak samples were collected at WWTP-. The methods used are described in the online Technical Appendix (wwwnc.cdc.gov/EID/ article/20/4/12-1415-Techapp1.pdf). Molecular Analysis/Typing In a subset of fecal samples, Cryptosporidium species were determined by PCR restriction fragment-length poly- morphism analysis of the 18S rRNA gene (21). Species were further characterized by sequence analysis of the 60- kDa glycoprotein (gp60) gene (22). Oocysts in wastewater and stream water samples were isolated from the contaminating debris by immunomagnetic separation (IMS), and DNA was extracted (online Techni- cal Appendix). DNA was also extracted from oocysts that had been obtained from 1 raw water sample and 1 drinking water sample by use of Envirochek filters (Pall Life Science, Ann Arbor, MI, USA) followed by IMS. Microscope slides containing 113 oocysts from 4 raw water samples and 4 drinking water samples were sent to the Cryptosporidium Reference Unit, Swansea, United Kingdom (online Techni- cal Appendix), where molecular analyses were performed. Statistical Analysis We conducted statistical analyses to test associations between risk factors and duration of diarrhea after con- trolling for age, sex, and residence in the area served by WTP-. Student t test was used to analyze differences in attack rate and relapse rate. Relationships between risk fac- tors and clinical cryptosporidiosis as the outcome variable were investigated by logistic regression. For dichotomous predictors, odds ratios were used to measure associations between clinical cryptosporidiosis and risk factors. Because of overdispersion in the data, negative binomial regression was applied to model the duration of infection in accor- dance with the case definition. Age and number of glasses of water consumed per day were evaluated as continuous variables. All statistical analyses were performed by using SPSS software version 19 (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant. Results Epidemiologic Investigation Electronic Survey Gastrointestinal symptoms were reported by 10,653 persons over a period of 2.5 weeks, confirming the large outbreak in the city and contamination of the drinking water (Figure 2). The number of cases of gastrointestinal illness increased from mid-November and peaked on No- vember 29, three days after the boil-water advisory was is- sued. Thereafter, the number of new cases reported per day rapidly declined. Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 583 RESEARCH Written Questionnaire Questionnaires were distributed by mail to 1,524 addressees; 10 persons had moved, and 6 were unable to respond. Of the remaining 1,508, a total of 1,044 (69.2%) responded: 481 men (46.1%) and 563 women (53.9%) (median age 44 years Diarrhea[range 098 years])(Table 1). The response rate was highest for women 6069 years of age (90.0%) and lowest for men 2029 years (43.8%), and 45.2% (95% CI 42.1%48.3%) of all the responders met the case definition criteria. When the rate of 45.2% was applied to the total population of stersund (59,500), results indicated that 27,000 (95% CI 25,04928,738) inhabitants contracted clinical cryptosporidiosis during the survey period. The attack rate decreased with age (p<0.0001; Table 1, Figure 3), was highest (58.0%) for persons 2029 years of age and lowest (26.1%) for per- sons >69 years of age (Table 1), and was similar for men and women. The attack rate was 52.2% for respondents who lived and worked in areas served by the WTP- but only 12.8% for inhabitants of stersund who neither lived nor worked in areas served by that plant (p<0.0001; data not shown). The most common symptoms among case- patients were episodes of diarrhea >3 times daily (89.0%), watery diarrhea (84.3%), abdominal cramps (78.8%), fa- tigue (73.1%), nausea (63.9%), and headache (57.1%) (Table 2). Diarrhea lasted a median of 4 days (range 151 days). Duration of diarrhea decreased significantly with age (p<0.0001; Table 3, Figure 3), as did the incidence of 584 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Figure 2. Epidemiologic curve of data from the electronic survey (10,653 participants; light gray) and written questionnaire (434 participants; dark gray) showing number of patients with suspected cases by date of onset of illness during Cryptosporidium infection outbreak, stersund, Sweden, 20102011. Table 1. Distribution of survey respondents and attack rate in Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y No. respondents (%) Attack rate, % All Female Male All Women Men p value 09 115 (67.3) 58 (67.4) 57 (67.1) 50.9 42.6 58.9 0.09 1019 117 (66.5) 58 (61.1) 59 (72.8) 47.2 55.6 38.5 0.08 2029 103 (48.8) 57 (53.8) 46 (43.8) 58.0 58.2 57.8 0.97 3039 110 (55.8) 58 (60.4) 52 (51.5) 52.8 51.9 53.8 0.84 4049 150 (66.7) 71 (70.3) 79 (63.7) 55.0 52.9 57.0 0.62 5059 145 (79.2) 85 (84.2) 60 (73.2) 42.1 45.1 37.9 0.40 6069 148 (89.2) 81 (90.0) 67 (88.2) 35.3 41.3 27.6 0.10 >69 156 (87.2) 95 (88.8) 61 (84.7) 26.1 24.4 28.8 0.57 Total 1,044 (69.2) 563 (72.0) 481 (66.3) 45.2 45.1 45.4 0.94 C. hominis Infection Transmitted through Water Supply fever, headache, nausea, vomiting, and fatigue (data not shown). Recurrence of diarrhea after >2 days of normal stools (defined as a relapse) was reported in 49.1% of the cases, and >1 relapse occurred significantly more often among women than men (p = 0.016; Table 4). Higher con- sumption of water and gluten intolerance were significant risks for Cryptosporidium infection (Table 3). Chronic intestinal disease (defined as inflammatory bowel dis- ease [IBD], lactose intolerance, or gluten intolerance) and young age were significantly associated with more days with diarrhea (Table 3). Microbiological Investigation Human Samples A total of 186 laboratory-confirmed cases of cryp- tosporidiosis related to the outbreak were reported to the national surveillance system: 149 in Jmtland County and 37 in other counties. Genotyping identified C. hominis sub- type IbA10G2 in 37 samples. A representative sequence has been deposited into GenBank under accession no. KF574041. Analyses showed that the 149 Cryptospori- dum-positive samples from Jmtland County were negative for other gastrointestinal pathogens. Environmental Samples Cryptosporidium oocysts were found in drinking water and raw water samples collected at the WTP- on November 27 and in all samples of WTP- drink- ing water, water from the distribution network, and raw water from Lake Storsjn over the next 2 months (Table 5). The highest number of oocysts in drinking water (1.4 presumptive oocysts/10 L) was detected on December 12, 2010 (online Technical Appendix Figure 1. During the outbreak, the average oocyst density in drinking wa- ter was 0.32/10 L in WTP- samples and 0.20/10 L in samples from the distribution network. Densities in raw water samples were generally higher: 0.23.1 oocysts/ 10 L. In WWTP- wastewater, the pre-outbreak low den- sity (<200 oocysts/10 L), had increased to 1,800/10 L on November 16, was highest at 270,000/10 L on Novem- ber 29, and then gradually declined to preoutbreak levels from December 31 onward (online Technical Appendix Figure 2). Oocysts were detected in 4 of 22 raw water samples from other municipalities near Lake Storsjn but in only 1 drinking water sample from a WTP (online Technical Appendix Table). All samples of untreated wastewater, most samples of treated wastewater (11/18), and samples from recipient water bodies (6/9) contained oocysts. Two of the 4 investigated streams connected to Lake Storsjn Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 585 Figure 3. Percentage of ill persons (A) and mean duration of symptoms fulfilling the case definition (B), stratified by age group during Cryptosporidium infection outbreak, stersund, Sweden, 20102011 . Error bars represent 1 SE. Table 2. Clinical characteristics of surveyed case-patients and noncase-patients in Cryptosporidium infection outbreak, stersund, Sweden, 2010-2011 Symptom No. positive answers/total no. respondents (%)* All respondents, N = 972 Case-patients, n = 434 Noncase-patients, n = 538 Diarrhea, >3 stools/d 382/967 (39.5) 382/429 (89.0) 0/538 (0) Watery diarrhea 343/945 (36.3) 343/407 (84.3) 0/538 (0) Abdominal cramps 382/952 (40.1) 328/416 (78.8) 54/536 (10.1) Fatigue 342/921 (37.1) 302/413 (73.1) 40/508 (7.9) Nausea 301/931 (32.3) 253/396 (63.9) 48/535 (9.0) Headache 267/920 (29.0) 232/406 (57.1) 35/514 (6.8) Fever >38.0C 128/909 (14.1) 121/393 (30.8) 7/516 (1.4) Muscle or joint aches 95/875 (10.9) 80/366 (21.9) 15/509 (2.9) Vomiting 89/894 (10.0) 76/357 (21.3) 13/537 (2.4) Eye pain 81/877 (9.2) 67/367 (18.3) 14/510 (2.7) Bloody diarrhea 16/883 (1.8) 15/345 (4.3) 1/538 (0.2) *Respondents who answered yes (case-patients) compared with those who answered no (noncase-patients) about whether they had experienced >3 episodes of diarrhea daily and/or watery diarrhea with onset after November 1, 2010. Results on the basis of answers from 972 of 1,044 respondents. RESEARCH contained oocysts (Table 5). The stream closest to WTP- (Figure 1) had densities of 1,300 and 5,000 oocysts/10 L on November 30 and December 2, respectively; this finding could be explained by wastewater leaking from an apart- ment building into the storm water system, which was re- paired on December 3. Isolated DNA from 1 concentrate of raw water, sep- arated from other particulate matter by IMS, was suc- cessfully amplified at the 18S rRNA gene locus, and C. hominis was determined by restriction fragment length polymorphism and sequence analysis. Subtyping was not possible because amplification of the gp60 gene failed. Also, despite repeated attempts, we were unable to amplify any DNA sequences from oocysts detected in raw water and drinking water by microscopy and removed from mi- croscope slides. C. hominis IbA10G2 was identified in 2 samples from the stream closest to WTP-, in 5 from untreated wastewa- ter at WWTP-, and in 4 from other WWTPs in Jmtland County. No other Cryptosporidium species or subtypes were detected in any of the analyzed samples. Discussion We describe a confirmed outbreak of Cryptosporidium infection affecting at least 27,000 inhabitants of stersund, Sweden, which represents the largest known outbreak in Europe and the second largest worldwide after the Milwau- kee outbreak. The etiologic agent was detected in drinking water, repeatedly over >2 months. Although Cryptosporid- ium spp. are occasionally found in untreated surface water, to our knowledge, this is the first time this pathogen has been detected in drinking water in Sweden. Three factors facilitated detection of the outbreak. First, before the outbreak was recognized, alert staff at the county laboratory suspected oocysts in wet smears of unstained, concentrated fecal specimens and subsequently confirmed the presence of Cryptosporidium spp. by modi- fied Ziehl-Neelsen staining, even though this analysis had not been specifically requested. Second, data from the lo- cal health advice line indicated that most persons with gas- troenteritis resided within the city limits, which proved to be crucial for the decision to issue a boil-water advisory. Third, the electronic survey was a valuable tool for daily monitoring of the epidemic curve and evaluating the effect of the boil-water advisory. Previous research has demon- strated the benefits of event-based surveillance data and website questionnaires in early detection and monitoring of an outbreak (23,24). The distribution of symptoms among case-patients with cryptosporidiosis in this study is comparable to ob- servations from other studies (6,17,25), except regarding muscle or joint aches, which were reported less frequently in stersund. Moreover, the median duration of diarrhea, the level of attack rates in different age groups, and recur- rence rate of diarrhea correspond to findings in other out- breaks (6,14). We identified young age, amount of water consumed, and number of infected family members as risk factors, which agrees with results from other studies (26,27). Also, gluten intolerance remained a risk factor after we controlled for age, sex, and residence in the WTP area, but this analysis was based on information from only 17 persons and hence should be interpreted with caution. The mechanism by which gluten intolerance might constitute a risk factor for cryptosporidiosis is unknown. Duration of diarrhea was significantly associated with young age and chronic intestinal disease. Exacerbation of IBD in cryptosporidiosis patients has been documented (28), and Cryptosporidium-induced loss of intestinal barrier func- tion has been suggested to mimic changes seen in IBD (29). Additional studies are needed to clarify any long- term effects of Cryptosporidium infection and are being undertaken in relation to the current outbreak. Molecular typing identified C. hominis IbA10G2 in both human and environmental samples. This early iden- tification of nonlivestock-associated Cryptosporidium 586 Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 Table 3. Risk factors for cryptosporidiosis and duration of infection in Cryptosporidium infection outbreak, stersund, Sweden, 2010 2011* Risk factor Infection Duration, p value Adjusted OR (95% CI) p value Age, continuous 0.99 (0.980.99) <0.0001 <0.0001 Chronic intestinal disease 1.86 (0.952.63) 0.08 <0.01 Chronic underlying disease# 1.15 (0.731.8) 0.55 0.59 Gluten intolerance 4.06 (1.2413.29) 0.02 0.05 Lactose intolerance 1.40 (0.792.46) 0.25 <0.01 No. additional family members with cryptosporidiosis 1.99 (1.702.33) <0.0001 NA No. glasses of water consumed daily 1.07 (1.031.11) <0.0001 0.07 No. persons in household 0.98 (0.871.07) 0.54 NA Peptic ulcer or medication 1.26 (0.722.22) 0.42 0.43 Smoking 1.01 (0.581.75) 0.98 0.40 *OR, odds ratio, adjusted for age, sex, and residence in the water treatment plant area; NA, not applicable. Participants with watery diarrhea and/or >3 episodes of diarrhea daily were defined as having cryptosporidiosis. Duration (i.e., time fulfilling the case definition). Defined as inflammatory bowel disease, lactose intolerance, or gluten intolerance. #Defined as cancer, rheumatic disease, cardiac failure, asthma, chronic obstructive pulmonary disease, or diabetes. C. hominis Infection Transmitted through Water Supply isolates facilitated the outbreak investigation by indicating that the cause was contamination of surface water by human sewage rather than contamination from an animal source (4,30). C. hominis IbA10G2 is reported to be highly virulent; is excreted in high numbers in feces (1,31,32); and is the most commonly identified subtype in waterborne cryptosporidiosis outbreaks, including that in Milwaukee (3,30,33,34). These characteristics, along with occurrence of the outbreak in a population that may have been par- ticularly susceptible because of limited previous exposure, contributed to the high attack rate (35,36). Although the infectious dose for Cryptosporidium in- fection is low, the oocyst densities in the stersund drink- ing water (maximum 1/10 L) cannot readily explain the high attack rate, even if the low recovery rate is taken into account. Densities may have been higher at the onset of the outbreak because of a surge of oocysts in the inlet before sampling, and secondary household transmission could have contributed to some of the cases. However, similar low numbers of oocysts have been detected in drinking wa- ter samples in other outbreaks (26,37). The level of recov- ery efficiency of the methods used in the outbreak required analysis of at least 100 L of water to identify the low level of Cryptosporidium contamination, which agrees with find- ings reported by other investigators (26). Recovery studies were not performed during the acute phase of the stersund outbreak, which underscores the uncertainty of extrapolating the numbers of oocysts de- tected in raw and drinking water to the actual density of oocysts (38). Moreover, no reliable assays to test viability and infectivity of oocysts are available (1). Other limita- tions of the present study include potential response bias in the electronic survey and the mailed questionnaire (39). Moreover, we could not assess the contribution of second- ary transmission to the attack rate or ascertain the number of subclinical cases by serologic testing. Several possible factors could explain Cryptospo- ridium contamination of the drinking water. In the rou- tine bacteriologic analysis performed weekly at WTP-, E. coli densities were 10 times greater than the average level on 3 occasions a few weeks before the outbreak (H. Dahlsten, pers. comm.), which implies abnormally high fecal contamination of the source water. Furthermore, Cryptosporidium oocysts were detected repeatedly in both raw and drinking water for months after the outbreak peaked, which illustrates the environmental persistence of oocysts and/or continuing contamination. Survival of the oocysts in Lake Storsjn was probably prolonged be- cause the outbreak occurred in winter when the lake was covered with ice. The municipality of stersund made Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 587 Table 5. Presence of Cryptosporidium oocysts in environmental samples collected in stersund, Sweden, November 27, 2010 March 22, 2011* Sample type No. samples No. positive samples Analyzed volume, L Presumptive no. oocysts, minmax/10 L Confirmed no. oocysts, minmax/10 L Time span for positive samples Raw water 18 10 100 0.23.1 0.10.7 2010 Nov 272011 Feb 9 Drinking water, WTP- 7 7 8001,500 0.0471.4 0.021.3 2010 Nov 272011 Jan 20 Drinking water, distribution network 9 9 8001,400 0.0630.36 0.050.05 2010 Nov 292011 Jan 31 Wastewater, untreated 21 13 0.05 200270,000 160,000 2010 Nov 292011 Feb 17 Wastewater, treated 15 14 0.250.3 3021,000 3010,000 2010 Dec 12011 Jan 24 Recipient (Lake Storsjn) 14 8 910 221 118 2010 Nov 292011 Mar 22 Connected streams 8 5 210 1,3005,000 9503,500 2010 Nov 30Dec 14 Other 10 2 1017 13 13 2010 Nov 302011 Jan 17 Total 102 68 0.047270,000 0.02160,000 2010 Nov 272011 Mar 22 *Min, minimum; max, maximum; WTP-, water treatment plantstersund. Details are available in Technical Appendix Figures 1 and 2, wwwnc.cdc.gov/EID/article/20/4/12-1415-Techapp1.pdf. These samples consisted of 30-mL aliquots from every 5060 m3 of wastewater produced over 24 h. Not possible to determine the lowest density by microscopy because of substantial background material in the concentrated water sample. Samples from sources, such as swimming pools, water used to flush the distribution network, and sediment from fire hydrants. Table 4. Distribution of respondents and relapse of diarrhea among surveyed case-patients in the Cryptosporidium infection outbreak, stersund, Sweden, 20102011 Age group, y All relapses, % 1 Relapse, % >1 Relapse, % Female Male p value Women Men p value 09 68.5 50.0 43.8 0.66 22.7 21.9 0.94 1019 48.9 20.7 50.0 0.04 20.7 10.0 0.30 2029 40.4 22.6 19.2 0.76 22.6 15.4 0.50 3039 47.3 25.9 32.1 0.63 29.6 7.1 0.03 4049 51.3 27.8 36.4 0.42 25.0 13.6 0.21 5059 47.4 22.2 23.8 0.89 25.0 23.8 0.92 6069 47.8 22.6 20.0 0.85 29.0 20.0 0.52 >69 35.3 15.0 35.7 0.20 15.0 7.1 0.50 Total 49.1 25.4 33.5 0.07 24.1 15.0 0.016 RESEARCH considerable efforts to trace the sources of Cryptosporid- ium contamination, and tentatively identified 2 streams, 1 of which was located closer to (upstream of) the raw water intake (Figure 1) and had higher densities of oo- cysts. However, we could not establish whether the initial input of oocysts to Lake Storsjn and the raw water intake had actually come from these streams, or whether it re- sulted from the outbreak itself. Perhaps these 2 streams contributed to a transmission cycle in which infectious persons were shedding oocysts into leaking wastewater that reached the raw water intake. Because only C. homi- nis IbA10G2 was identified in environmental samples, we suggest that the outbreak was caused by a single common source of contamination, although this hypothesis could not be definitively demonstrated. Failure of the WTP- and onset of the outbreak has sev- eral plausible explations. To our knowledge, no posttreatment contamination or extensive failures in the treatment processes occurred, and routine tests of the drinking water showed no increased levels of fecal indicator bacteria. The WTP- had 2 microbiological barriers (ozonation and chloramination) as recommended by the drinking water regulations in Sweden for surface waterworks, but these barriers were simply inad- equate to remove or inactivate the Cryptosporidium oocysts in the raw water. The long-term solution to reduce infective parasites in stersund was to install a UV water disinfection system, which was done after the outbreak in December 2010. In addition, pipes were repeatedly flushed, and and further sampling was conducted to verify that no potentially viable oocysts remained in the distribution network. Previous research has suggested that analysis of Cryp- tosporidium in wastewater can aid in early detection of an outbreak (40). In stersund, the number of Cryptospo- ridium oocysts in influent wastewater increased slightly 10 days before the boil-water advisory (1,800 oocysts/10 L), which indeed implies that monitoring the level of oocysts in influent wastewater might facilitate early detection of an ongoing outbreak, although the cost of such an approach would render it impractical. Six months after the outbreak in stersund, another waterborne outbreak of C. hominis IbA10G2 infection occurred in the city of Skellefte, 450 km northeast of stersund, possibly because persons from that city had visited stersund during the outbreak there and had sub- sequently spread Cryptosporidium oocysts on their return to Skellefte. In Sweden, recommendations to prevent out- breaks of parasites include identifying and limiting sources of contamination of raw water in combination with sam- pling (100-L volumes). The awareness of parasites as a probable cause of waterborne outbreaks has increased tre- mendously in this country since these outbreaks, and many WTPs have evaluated the efficiency of their current barri- ers, for example, by quantitative microbial risk assessment. This study has documented the largest outbreak of waterborne cryptosporidiosis in Europe, affecting 27,000 persons. C. hominis subtype 1bA10G2 was identified in clinical samples and in wastewater. Low levels of oocysts were repeatedly detected in drinking water for >2 months. Our results emphasize the value of assessing microbial risks in raw water and using multiple barriers in WTPs to substantially reduce or inactivate all groups of microorgan- isms, including parasites such as Cryptosporidium spp. Acknowledgments We thank Joyce Eriksson, Tomas Nilsson, Jessica Ns, and Lill Welinder for their excellent technical assistance. We also thank Johan Wistrm for invaluable intellectual comments. 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Prevalence and distribution of Cryptosporidium and Giardia in wastewater and the surface, drinking and ground waters in the Lower Rhine, Germany. Epidemiol Infect. 2013;141:921. http://dx.doi. org/10.1017/S0950268812002026 Address for correspondence: Micael Widerstrm, Department of Clinical MicrobiologyClinical Bacteriology, Ume University, Ume 90185, Sweden; email: micael.widerstrom@jll.se Emerging Infectious Diseases www.cdc.gov/eid Vol. 20, No. 4, April 2014 589

What was the alert of the event?

{'answer_start': [6571], 'text': ['received numerous calls from persons with gastroenteritis']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What type of samples were analyzed?

{'answer_start': [1236], 'text': ['stool samples']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What did the local authorities investigate?

{'answer_start': [1203], 'text': ['microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What were the first steps of investigation?

{'answer_start': [519], 'text': ['case ascertainment and descriptive epidemiology']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What is the date of the event?

{'answer_start': [14], 'text': ['July 2006']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What is the location of the event?

{'answer_start': [142], 'text': ['Taranto, Apulia']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

How was the event first detected?

{'answer_start': [25], 'text': ['an unusually high number of patients with acute diarrhoea were reported']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

How many people were ill?

{'answer_start': [6325], 'text': ['166 cases']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What are the pathogens?

{'answer_start': [4700], 'text': ['norovirus and 11 (25%) for rotavirus']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What are the symptoms?

{'answer_start': [6268], 'text': ['fever >= 38C, headache, vomit, abdominal pain, nausea']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What is the event?

{'answer_start': [38], 'text': ['high number of patients with acute diarrhoea ']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What is the duration of the event?

{'answer_start': [422], 'text': ['between July and October 2006']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What are the first steps of mitigation?

{'answer_start': [7230], 'text': ['extra chlorination treatment']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What type of samples were examined?

{'answer_start': [1326], 'text': ['environmental samples']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What was the age of the affected people?

{'answer_start': [2425], 'text': ['mean age was 25']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What is the source of contamination?

{'answer_start': [7155], 'text': ['drinkable tap water contaminated with (at least) rota- and noroviruses']}

Contamination Question Answering

At the end of July 2006, an unusually high number of patients with acute diarrhoea were reported by the accident and emergency departments in Taranto, Apulia. Subsequently, a field investigation was conducted jointly by the Apulia Regional Epidemiological Observatory and the Regional Reference Laboratory in Bari, and the Epidemiological Department of Taranto Local Health Unit. The outbreak investigation carried out between July and October 2006, involving hospitals in the whole province of Taranto, included a case ascertainment and descriptive epidemiology. A case was defined as a patient with diarrhoea (at least three loose or liquid stools in a day) and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea [1,2]. Five out of six hospitals in the province of Taranto provided information on patients with acute gastroenteritis. Data were collected retrospectively for the period between May and July and prospectively for August and September 2006. In addition, the special medical facilities set up for tourists in the summer season (June-August) in the province of Taranto were also asked to report cases. The outbreak investigation included microbiological investigation of stool samples of hospitalized patients. It also included microbiological investigation of environmental samples (including tap water, sea water and shellfish) [1-4]. G Case control study performed between 1 August and 15 September 2006 in order to identify the possible sources of infection. From 1 May to 30 September 2006, a total of 2,860 patients with gastroenteritis symptoms were either admitted to hospital or seen by the hospitals outpatient accident and emergency units. This significantly exceeded the number reported in the same period in 2005, when a total of 586 patients with gastroenteritis were treated by the same hospitals. The epidemic curve is shown in Figure 1. Figure 1. Number of patients with gastroenteritis seeking hospital care, by week. Taranto province, 1 May-30 September, 2005 and 2006 The first peak in incidence was observed at the end of June (26 week of the year), followed by a second peak at the end of July (weeks 29 and 30). The number of patients with gastroenteritis seeking hospital care decreased in the following weeks. By mid-September, the number of cases per week was similar to that seen in the same period of 2005. Patients mean age was 25 years; 19% of the cases were under 5 years of age, 16% were 5 to 15 years old, and 65% were above 15 years of age. Incubation time was not calculated because it was not possible to determine the exact time of exposure. Incidence by town of residence was highest in the city of Taranto (9.5 cases per 1,000 inhabitants) (Figure 2). http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (2 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Figure 2. Number of patients with gastroenteritis seeking hospital care, by town of residency per 1,000 inhabitants. Taranto province, 1 May-30 September 2006 Data collected by the tourist medical facilities in Taranto province showed a total of 361 cases of acute gastroenteritis, significantly more than a year before. Hence the same trend was observed as in the case of hospital data. Microbiological analysis A total of 70 stool samples from patients affected by the outbreak were collected and analysed. Results by age group are reported in Table 1. Table 1. Stool samples collected from patients and tested by the Regional Reference Laboratory (U.O.C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Age Number of samples Rotavirus () Norovirus (*) < 15 51 32 (62%) 19 (37%) >= 15 7 1 (14%) 4 (57%) Unknown 12 1 (8%) 5 (41%) Total 70 34 (48%) 28 (40%) () Nested PCR in VP7 region (*) Nested PCR in the polymerase gene Stool samples were also examined with respect to gastrointestinal bacteria and parasites. No samples examined were positive for the entire range of pathogens tested. Further genotyping of the samples is currently being done. Environmental samples, systematically collected for microbiological analyses, were tap water from the water distribution system across the whole area affected by the outbreak, sea water and shellfish. The water samples were collected at the local waterworks, from major water pipelines and wells, and from tap water in pubs. No faecal indicator bacteria and endotoxins were detected in the environmental samples of tap water collected in Taranto city. Of 44 samples tested, four (9%) were positive for norovirus and 11 (25%) for rotavirus (Table 2). The tests were performed using molecular techniques. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (3 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 Table 2. Drinkable tap water samples collected and tested Regional Reference Laboratory (U.O. C. Igiene, Azienda Ospedaliera Policlinico), Bari, August September 2006 Date Number of samples Norovirus Rotavirus Both 31.07.2006 2 1 1 0 2.08.2006 3 0 2 0 8-9.08.2006 3 0 0 0 12.09.2006 4 1 1 0 14.09.2006 2 0 0 0 18-19.09.2006 8 1 3 1 12.10.2006 6 1 2 0 19.10.2006 13 0 2 0 27.10.2006 3 0 0 0 Total 44 4 (9%) 11 (25%) 1 (2%) Molecular profiles of rotavirus and norovirus identified in some tap water samples were the same as the ones found in some patients stool samples. Sequence analysis showed the new norovirus strain GGII.4 2006a and rotavirus genotype G9. The laboratory investigations, however, are still ongoing and more results are expected in the future. Of 12 sea water samples tested, four (33%) were positive for norovirus and one (8,3%) for rotavirus. No shell fish samples were positive for bacteria or viruses. Case control study A case control study was performed in order to find an association between the occurrence of gastroenteritis and the exposure to one or more risk factors. A case was defined as a patient with at least 3 loose or liquid stools in a day and one or more of the following symptoms: fever >= 38C, headache, vomit, abdominal pain, nausea. 166 cases were selected among patients treated at the accident and emergency departments of the hospitals in Taranto province, in the period between 1 August and 15 September 2006. The control group consisted of 146 non-hospitalised healthy individuals who during the study period were resident in the same area as the case patients. Cases and controls were age-matched. A standard questionnaire was used for the interview. Risk factors which were shown to be significantly associated with the onset of acute diarrhoea/ gastroenteritis were the use of tap water (OR= 2; 95% CI: 1,23-3,36), and the use of water of uncertain origin in the 72 hours before the onset of the symptoms (OR= 3,9; 95% CI: 1,41- 10,54). The epidemiological investigation and the laboratory tests showed that the possible source of infection was the drinkable tap water contaminated with (at least) rota- and noroviruses. An extra chlorination treatment for household water supplies was therefore performed starting from the 34th week of the year in order to stop a possible contamination of the water. Systematic technical and microbiological investigations of the pipelines and wells of the water distribution system did not reveal the source of contamination even though technical problems at the local chlorination treatment facilities could not have been excluded. http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (4 di 5)19/05/2008 10.16.21 http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 To date, the outbreak of viral gastroenteritis described in this paper is probably the largest one associated with drinking tap water in Italy. Acknowledgements A. Mincuzzi, T. Battista, R. Mongelli, M.T. Balducci, C. Ladalardo, A. Fusco, S. Tafuri, A. Panebianco, F. Fortunato, A. Arbore, L. Lamarina (Department of Biomedical Science and Human Oncology, Hygiene Section, University of Bari - Apulia Regional Epidemiological Observatory); A. Pesare, S. Minerba, G. Grassi, M. Rollo, D. Zuppiroli (Taranto Local Health Unit); F. Portincasa, P. Montemurro, G. Vitucci (Apulia Water Distribution Company Ente Acquedotto Pugliese) References: 1. Chironna M, Prato R. Lopalco PL, Germinario C, Sallustio A, Barbuti S, Quarto M. Norovirus GI e GII in corso di epidemia di gastroenterite acuta associata al consumo di molluschi bivalvi in Puglia. Rapporti ISTISAN 2003; 3/C5: 39. 2. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in South Italy. BMC Infectious Diseases, 2004; 4: 37. 3. Liang JL, Dziuban EJ, Craun GF, Hill V, Moore MR, Gelting RJ, Calderon RL, Beach MJ, Roy SL. Surveillance for waterborne disease and outbreaks associated with drinking water and water not intended for drinking, United States, 2003-2004. In: Surveillance Summaries, MMWR, 2006; 55: 31-58. 4. Boccia D, Tozzi AE, Cotter B, Rizzo C, Russo T, Buttinelli G, Caprioli A, Marziano ML, Ruggeri FM. Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy. Emerging Infection Diseases, 2002; 8(6):563-568. back to top Back to Table of Contents Next To top | Recommend this page Disclamer:The opinions expressed by authors contributing to Eurosurveillance do not necessarily reflect the opinions of the European Centre for Disease Prevention and Control (ECDC) or the Editorial team or the institutions with which the authors are affiliated. Neither the ECDC nor any person acting on behalf of the ECDC is responsible for the use which might be made of the information in this journal. Eurosurveillance [ISSN] - 2008 All rights reserved http://www.eurosurveillance.org/ViewArticle.aspx?PublicationType=W&Volume=12&Issue=16&OrderNumber=1 (5 di 5)19/05/2008 10.16.21 View publication stats View publication stats

What were the associated pathogens of concern?

{'answer_start': [5479], 'text': ['rotavirus and norovirus ']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What symptoms did the people had?

{'answer_start': [5856], 'text': ['chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%).']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What type of samples were analyzed?

{'answer_start': [838], 'text': ['stool samples']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What were the first steps of investigation?

{'answer_start': [249], 'text': ['a message on a social media website asked racers to report any symptoms by email']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What is the initial cause of the event?

{'answer_start': [1083], 'text': ['human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risk']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What is the date of the event?

{'answer_start': [104], 'text': ['summer of 2015']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What is the location of the event?

{'answer_start': [89], 'text': [' France']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What pathogen was connected to the outbreak?

{'answer_start': [6129], 'text': ['Shigella sonnei)']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

How was the event first detected?

{'answer_start': [1370], 'text': ['a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

How many people were ill?

{'answer_start': [597], 'text': ['1,264 adults ']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What is the attack rate?

{'answer_start': [4819], 'text': ['attack rate: 16%']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What are the pathogens?

{'answer_start': [889], 'text': ['norovirus genogroup I and genotype 2']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What is the concentration of the contaminant after analysis?

{'answer_start': [6421], 'text': ['counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL)']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What are the symptoms?

{'answer_start': [5450], 'text': ['vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and feve']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What is the event?

{'answer_start': [6889], 'text': ['A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What are the initial steps of investigation?

{'answer_start': [397], 'text': ['a retrospective cross-sectional study was conducted through an interactive questionnaire']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What are the first steps of mitigation?

{'answer_start': [4440], 'text': ['provided recommendations on measures to prevent secondary spread during a press conference ']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What was the outbreak investigation?

{'answer_start': [120], 'text': ['An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What measures were taken to prevent the event?

{'answer_start': [1202], 'text': ['Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What was the age of the affected people?

{'answer_start': [5348], 'text': ['The mean age of adult respondents was 33 years (range: 18-61 years)']}

Contamination Question Answering

An acute gastroenteritis (AG) outbreak occurred among participants in an obstacle race in France in the summer of 2015. An investigation in two phases was conducted to identify the source of infection and document the extent of the outbreak. First, a message on a social media website asked racers to report any symptoms by email to the Regional Health Agency of Provence-Alpes-Cte dAzur. Second, a retrospective cross-sectional study was conducted through an interactive questionnaire for all participants, followed by an analytical study of potential risks factors. Of 8,229 persons registered, 1,264 adults reported AG resolved within 48 hours. Of adults who reported AG, 866 met the case definition. Age group, departure time and ingestion of mud were associated with AG. Twenty stool specimens tested negative for bacteria. All four stool samples tested for viruses were positive for norovirus genogroup I and genotype 2. No indicator bac- teria for faecal contamination were found in drinking water but muddy water of ponds tested positive. The outbreak was possibly caused by human-to-human transmission of a norovirus introduced by one or more persons and transmitted through contaminated mud. Risks related to similar races should be assessed and recommendations be proposed to raise awareness among health authorities and organisers. On 22 June 2015 at 16:00, a hospital near Nice alerted the Regional Health Agency of Provence-Alpes-Cte dAzur (ARS PACA) of 22 patients with AG after participation in an obstacle race, in the south of France. On a social media website, many other participants reported also suffering from similar symptoms. An investigation was immediately conducted in order to identify the source of infection and document the extent of the outbreak. Methods The ARS PACA, responsible for the outbreak inves- tigation and implementation of control measures, requested support of the Regional Office of the French Public Health Agency (Cire Sud). Epidemiological investigation The investigation of the outbreak was conducted in two phases. The first phase started immediately, upon receipt of the outbreak alert, on 22 June 2015. ARS PACA and Cire Sud sent a message on a social media website informing the participants of the obstacle race that an investigation was conducted and asked them to report any recent or current gastrointestinal illness by email to a dedicated address of ARS. This information was also relayed by the organisers of the race and the local press. The second phase, aimed at collecting additional information, was conducted retrospectively through an interactive application, Voozanoo (a secure web-based platform for hosting personal health data) including sections on socio-demographic characteristics, presence of symptoms or not, and sections on potential exposures (catering, means of contact with mud). This short questionnaire (available from the authors upon request) was intended for all participants of the obstacle race, whether or not they had clinical signs of AG. On 30 June, the company that had organised the event sent the Internet link for this survey by email in a news- letter to all the participants of the obstacle race. Cross-sectional descriptive studies were conducted in two phases, and were followed by an analytical study of the potential risks factors (in the second phase). A case of AG was defined as a racer in the obstacle race that took place on 20 June, with self-reported gastrointestinal illness (vomiting and/or diarrhoea) associated or not with other symptoms within eight days from the race. A secondary case of AG was defined as a person who did not participate in the obstacle race as racer, with gastrointestinal illness following at least 24 hours after a contact with a case of AG among racers. We recommended all laboratories to test stool specimens from the racers for Salmonella, Shigella, Campylobacter and Yersinia, and to send stool specimens to the National Reference Laboratory (NRL) for Enteric viruses in Dijon in order to test for norovirus, sapovirus, rotavirus, adenovirus and astrovirus. These samples were tested by a laboratory in Lyon. Water samples were tested for Campylobacter and norovirus by the Laboratory for Hydrology of the French Agency for Food, Environmental and Occupational Health & Safety (ANSES,) in Nancy. The University Laboratory of Pharmacy in Lyon tested the specimens for species of Naegleria [10]. We provided recommendations on measures to prevent secondary spread during a press conference organised on 22 June, through a weekly report of the Cire Sud, on the ARS PACA website, and we posted prevention messages on a social media website. the outbreak investiga- tion was published on the social media website. Among the respondents, 1,300 AG cases were reported: 1,264 adults (attack rate: 16%; 1,264/7,804) and 36 secondary cases (not included in this analysis). Phase 2 was conducted from 30 June to 27 July and a total of 748 questionnaires were completed (for 745 adults and 3 children). Among the participants who filled out the questionnaire, more than half (404/748; 54%) did it the day the newsletter came out and one third (247/748; 33%) the day after. There were 375 AG cases and 373 non cases. Secondary cases reported by respondents rose to a total of 177 cases, not included in this analysis. The mean age of adult respondents was 33 years (range: 18-61 years). The most common symptoms besides vomiting and/or diarrhoea, were abdominal cramps (369/866; 43%) and fever (354/866; 41%). Those symptoms were over or being spontaneously resolved in 48 hours. In phase 2, 69% of participants notified being ill (513/745) and among them, 73% met the case definition (375/513); of these, 67% (251/375) presented fever and 43% (161/375) other digestive symptoms. The other symptoms were non-specific, such as chills (215/375; 57%), headaches (101/375; 27%), asthenia (322/375; 86%), and muscle ache (262/375; 70%). Microbiological investigations We obtained the results of 20 stool samples. Results of bacteriological testing were negative for all samples, except one (positive for Shigella sonnei). Among the four specimens sent to the NRL for enteric viruses, all were positive for norovirus genogroup I and genotype 2 (GI.2). Of the environmental water specimens (n=5) taken from five ponds on 23 June, all were found contaminated by aerobic microorganisms at 36 C (with counts ranging from 86 to 3,200,000 UFC/100 mL) and 22 C (with counts ranging from 2,500 to 1,400,000 UFC/100 mL), indicating bacterial contamination. All specimens were negative for Salmonella, Campylobacter, Enterovirus, V. cholerae and V. parahemolyticus. All environmental samples were positive for Naegleria spp. (with cells ranging from 400 to 280,000/L), but the pathogenic species N. fowleri was not detected. Norovirus geno- group I and II were not detected. A large AG outbreak occurred among the participants of obstacle race in the department of Alpes-Maritimes, France, on 20 June 2015. Of 7,804 adult participants, 1,264 were ill and 866 met the case definition of AG. This outbreak occurred from 20 to 25 June and the epidemiological curves during two different phases of the outbreak investigation were characteristic of a point-source outbreak. The epidemiological investigation enabled us to identify mud ingestion as the main risk factor of developing AG; no other source of infection was identified. Several arguments support a common source outbreak of human origin related to the ingestion of contaminated mud.

What is the source of contamination?

{'answer_start': [7469], 'text': ['common source outbreak of human origin related to the ingestion of contaminated mud']}

Contamination Question Answering

On Monday 15 January 2007, a municipality in Denmark received the first of several complaints from citizens who reported severe diarrhoea and vomiting over the weekend. Over the same period, the drinking water in many houses was reported to be discoloured and of unusual smell and taste. The local authorities immediately prohibited any use of the water - except for toilet flushing - in the entire area supplied with untreated drinking water from the local waterworks, which involved 5,802 citizens and a number of companies (Figure 1). The citizens in the area were warned by the police and through radio broadcasts. To reveal the nature and the geographical spread of the suspected water contamination, water samples for microbiological and chemical analyses were systematically collected from the water distribution system across the whole area supplied from the local waterworks. The water samples were collected at the waterworks itself, from major water pipelines and wells, and from tap water in private houses and companies. High concentrations of faecal indicator bacteria (primarily presumptive coliform counts and�Escherichia coli) and endotoxins in the water samples indicated a massive faecal contamination of a part of the water distribution system, while other parts of the distribution system appeared not to be affected. On the basis of the geographical distribution of indicator bacteria, and the technical information about directions of the water flow in the different sections of the water distribution system, the area suspected to be contaminated was systematically reduced step by step during the next days. Based on the analyses of 530 water samples collected at 200 different sites, the area finally considered to be affected by the water contamination was defined on 26 January 2007 (indicated with a red line in Figure 1). This area comprised 177 households with 450 residents and several companies, among which six dealt with food. Restrictions on water use were maintained in this area, while the water was released for normal use in the area that was not considered to be contaminated. Gastrointestinal illness A line-list of patients with gastrointestinal illness associated with the water contamination was established on the basis of notifications from general practitioners, enquiries made to the medical health officer, patients seeking advice from the emergency medical service, and patients contacting an ad hoc telephone �hotline� established by the local authorities. Additional information was collected during a house-to-house questionnaire survey conducted on 16 January among 20 households in the most severely affected street. Stool samples were examined at the Department of Bacteriology, Mycology and Parasitology, and the Department of Virology, Statens Serum Institut, Copenhagen. A case of acute gastroenteritis was defined as a person with diarrhoea, vomiting and/or abdominal pain/cramps with fever. To verify whether reported cases met the case definition, and to confirm the geographical extent of gastrointestinal illness associated with the water contamination, patients on the line-list were contacted by telephone and/or postal questionnaires. By the end of February 2007, 140 cases had been registered: 110 were residents of the area that was judged to be contaminated on the basis of the environmental investigations, 12 were shoppers or employees at the food companies in the area, and 18 affected people came from outside the contaminated area. The epidemic curve for cases among residents in the contaminated area with known date of illness onset is shown in Figure 2. No new cases in the contaminated area were registered after 24 January. Cases were largely confined to the contaminated area. A total of 24% of the residents of the contaminated area were registered with gastrointestinal illness, compared with 0.3% in the other sections of the waterworks� supply area (relative risk 73; 95% CI 44-127). From the most severely affected street in the contaminated area, 43% of residents were reported to have fallen ill. Four patients were temporarily admitted to hospital. Microbiological results By the end of February 2007, stool samples from 139 patients affected by the outbreak (including 99 patients who met the case-definition criteria) had been examined with respect to gastrointestinal bacteria, viruses and parasites. Among these, 77 patients (43 cases) had one or more samples that tested positive, including 23 patients with 2-5 different pathogenic gastrointestinal organisms (Table). Not all samples were tested for the entire range of pathogens identified. Further microbiological testing and genotyping of the samples are being undertaken. Technical assessment and intervention Technical and microbiological investigations of the water indicated that the most probable cause of the contamination was the combination of a technical and a human error at a local sewage treatment facility, which allowed at least 27 m3�of partially filtered waste water to enter into the drinking water system in the period between 12 and 14 January 2007. The two pipelines were separated, and the exact circumstances of the incident that allowed the backflow of the sewage water remain to be revealed. The conclusion of the technical investigations was supported by the large variety of gastrointestinal pathogens found in the stool samples, which corroborated that the contamination was due to backflow of grossly contaminated sewage water rather than, for example, surface water or sewage from only a few households. The sewage treatment plant receives sewage from a population of approximately 40,000, as well as industrial enterprises, food production establishments and a hospital. Flushing of the area�s distribution system was initiated immediately and sustained for several weeks. As faecal indicator bacteria were still found in the drinking water after two weeks of sustained flushing, the distribution system was subsequently disinfected by chlorination on 10 and 11 February. After another two days of flushing, the drinking water was released for normal use, but recommended for drinking still only after boiling. By 12 March, the boiling restrictions were lifted for the majority of households, since by then the environmental water samples from the distribution system had fulfilled the quality criteria for untreated drinking water as defined by the Danish Ministry of Environment [3]. Commentary The handling of the outbreak called for interdisciplinary cooperation and the epidemiological investigations supported the technical and water-microbiological analyses. Geographical information systems were used to define the contaminated area, and the 18 episodes of illness reported outside of the contaminated area (Figure 1) probably represent sporadic illness or could be associated with the outbreak in a way that was not investigated, for example by secondary transmission. Although the drinking water supply in Denmark is primarily from untreated ground water, disease cases are rarely registered in connection with incidents of contamination [1,2]. The outbreak described was unusual, partly because of the high morbidity among the exposed citizens, and partly because of the extraordinary complexity of positive microbiological findings; a recent review of disease outbreaks caused by contaminated drinking water in the United States from 2003 to 2004 reported that in only two out of 25 outbreaks with known aetiology more than one microorganism was detected [4]. The consequences were considerable for the affected families, but also for the food companies, which were not allowed to resume production until the middle of February.

What type of samples were analyzed?

{'answer_start': [706], 'text': ['water samples']}

Contamination Question Answering

On Monday 15 January 2007, a municipality in Denmark received the first of several complaints from citizens who reported severe diarrhoea and vomiting over the weekend. Over the same period, the drinking water in many houses was reported to be discoloured and of unusual smell and taste. The local authorities immediately prohibited any use of the water - except for toilet flushing - in the entire area supplied with untreated drinking water from the local waterworks, which involved 5,802 citizens and a number of companies (Figure 1). The citizens in the area were warned by the police and through radio broadcasts. To reveal the nature and the geographical spread of the suspected water contamination, water samples for microbiological and chemical analyses were systematically collected from the water distribution system across the whole area supplied from the local waterworks. The water samples were collected at the waterworks itself, from major water pipelines and wells, and from tap water in private houses and companies. High concentrations of faecal indicator bacteria (primarily presumptive coliform counts and�Escherichia coli) and endotoxins in the water samples indicated a massive faecal contamination of a part of the water distribution system, while other parts of the distribution system appeared not to be affected. On the basis of the geographical distribution of indicator bacteria, and the technical information about directions of the water flow in the different sections of the water distribution system, the area suspected to be contaminated was systematically reduced step by step during the next days. Based on the analyses of 530 water samples collected at 200 different sites, the area finally considered to be affected by the water contamination was defined on 26 January 2007 (indicated with a red line in Figure 1). This area comprised 177 households with 450 residents and several companies, among which six dealt with food. Restrictions on water use were maintained in this area, while the water was released for normal use in the area that was not considered to be contaminated. Gastrointestinal illness A line-list of patients with gastrointestinal illness associated with the water contamination was established on the basis of notifications from general practitioners, enquiries made to the medical health officer, patients seeking advice from the emergency medical service, and patients contacting an ad hoc telephone �hotline� established by the local authorities. Additional information was collected during a house-to-house questionnaire survey conducted on 16 January among 20 households in the most severely affected street. Stool samples were examined at the Department of Bacteriology, Mycology and Parasitology, and the Department of Virology, Statens Serum Institut, Copenhagen. A case of acute gastroenteritis was defined as a person with diarrhoea, vomiting and/or abdominal pain/cramps with fever. To verify whether reported cases met the case definition, and to confirm the geographical extent of gastrointestinal illness associated with the water contamination, patients on the line-list were contacted by telephone and/or postal questionnaires. By the end of February 2007, 140 cases had been registered: 110 were residents of the area that was judged to be contaminated on the basis of the environmental investigations, 12 were shoppers or employees at the food companies in the area, and 18 affected people came from outside the contaminated area. The epidemic curve for cases among residents in the contaminated area with known date of illness onset is shown in Figure 2. No new cases in the contaminated area were registered after 24 January. Cases were largely confined to the contaminated area. A total of 24% of the residents of the contaminated area were registered with gastrointestinal illness, compared with 0.3% in the other sections of the waterworks� supply area (relative risk 73; 95% CI 44-127). From the most severely affected street in the contaminated area, 43% of residents were reported to have fallen ill. Four patients were temporarily admitted to hospital. Microbiological results By the end of February 2007, stool samples from 139 patients affected by the outbreak (including 99 patients who met the case-definition criteria) had been examined with respect to gastrointestinal bacteria, viruses and parasites. Among these, 77 patients (43 cases) had one or more samples that tested positive, including 23 patients with 2-5 different pathogenic gastrointestinal organisms (Table). Not all samples were tested for the entire range of pathogens identified. Further microbiological testing and genotyping of the samples are being undertaken. Technical assessment and intervention Technical and microbiological investigations of the water indicated that the most probable cause of the contamination was the combination of a technical and a human error at a local sewage treatment facility, which allowed at least 27 m3�of partially filtered waste water to enter into the drinking water system in the period between 12 and 14 January 2007. The two pipelines were separated, and the exact circumstances of the incident that allowed the backflow of the sewage water remain to be revealed. The conclusion of the technical investigations was supported by the large variety of gastrointestinal pathogens found in the stool samples, which corroborated that the contamination was due to backflow of grossly contaminated sewage water rather than, for example, surface water or sewage from only a few households. The sewage treatment plant receives sewage from a population of approximately 40,000, as well as industrial enterprises, food production establishments and a hospital. Flushing of the area�s distribution system was initiated immediately and sustained for several weeks. As faecal indicator bacteria were still found in the drinking water after two weeks of sustained flushing, the distribution system was subsequently disinfected by chlorination on 10 and 11 February. After another two days of flushing, the drinking water was released for normal use, but recommended for drinking still only after boiling. By 12 March, the boiling restrictions were lifted for the majority of households, since by then the environmental water samples from the distribution system had fulfilled the quality criteria for untreated drinking water as defined by the Danish Ministry of Environment [3]. Commentary The handling of the outbreak called for interdisciplinary cooperation and the epidemiological investigations supported the technical and water-microbiological analyses. Geographical information systems were used to define the contaminated area, and the 18 episodes of illness reported outside of the contaminated area (Figure 1) probably represent sporadic illness or could be associated with the outbreak in a way that was not investigated, for example by secondary transmission. Although the drinking water supply in Denmark is primarily from untreated ground water, disease cases are rarely registered in connection with incidents of contamination [1,2]. The outbreak described was unusual, partly because of the high morbidity among the exposed citizens, and partly because of the extraordinary complexity of positive microbiological findings; a recent review of disease outbreaks caused by contaminated drinking water in the United States from 2003 to 2004 reported that in only two out of 25 outbreaks with known aetiology more than one microorganism was detected [4]. The consequences were considerable for the affected families, but also for the food companies, which were not allowed to resume production until the middle of February.

What caused the event?

{'answer_start': [4886], 'text': ['combination of a technical and a human error at a local sewage treatment facility,']}

Contamination Question Answering

On Monday 15 January 2007, a municipality in Denmark received the first of several complaints from citizens who reported severe diarrhoea and vomiting over the weekend. Over the same period, the drinking water in many houses was reported to be discoloured and of unusual smell and taste. The local authorities immediately prohibited any use of the water - except for toilet flushing - in the entire area supplied with untreated drinking water from the local waterworks, which involved 5,802 citizens and a number of companies (Figure 1). The citizens in the area were warned by the police and through radio broadcasts. To reveal the nature and the geographical spread of the suspected water contamination, water samples for microbiological and chemical analyses were systematically collected from the water distribution system across the whole area supplied from the local waterworks. The water samples were collected at the waterworks itself, from major water pipelines and wells, and from tap water in private houses and companies. High concentrations of faecal indicator bacteria (primarily presumptive coliform counts and�Escherichia coli) and endotoxins in the water samples indicated a massive faecal contamination of a part of the water distribution system, while other parts of the distribution system appeared not to be affected. On the basis of the geographical distribution of indicator bacteria, and the technical information about directions of the water flow in the different sections of the water distribution system, the area suspected to be contaminated was systematically reduced step by step during the next days. Based on the analyses of 530 water samples collected at 200 different sites, the area finally considered to be affected by the water contamination was defined on 26 January 2007 (indicated with a red line in Figure 1). This area comprised 177 households with 450 residents and several companies, among which six dealt with food. Restrictions on water use were maintained in this area, while the water was released for normal use in the area that was not considered to be contaminated. Gastrointestinal illness A line-list of patients with gastrointestinal illness associated with the water contamination was established on the basis of notifications from general practitioners, enquiries made to the medical health officer, patients seeking advice from the emergency medical service, and patients contacting an ad hoc telephone �hotline� established by the local authorities. Additional information was collected during a house-to-house questionnaire survey conducted on 16 January among 20 households in the most severely affected street. Stool samples were examined at the Department of Bacteriology, Mycology and Parasitology, and the Department of Virology, Statens Serum Institut, Copenhagen. A case of acute gastroenteritis was defined as a person with diarrhoea, vomiting and/or abdominal pain/cramps with fever. To verify whether reported cases met the case definition, and to confirm the geographical extent of gastrointestinal illness associated with the water contamination, patients on the line-list were contacted by telephone and/or postal questionnaires. By the end of February 2007, 140 cases had been registered: 110 were residents of the area that was judged to be contaminated on the basis of the environmental investigations, 12 were shoppers or employees at the food companies in the area, and 18 affected people came from outside the contaminated area. The epidemic curve for cases among residents in the contaminated area with known date of illness onset is shown in Figure 2. No new cases in the contaminated area were registered after 24 January. Cases were largely confined to the contaminated area. A total of 24% of the residents of the contaminated area were registered with gastrointestinal illness, compared with 0.3% in the other sections of the waterworks� supply area (relative risk 73; 95% CI 44-127). From the most severely affected street in the contaminated area, 43% of residents were reported to have fallen ill. Four patients were temporarily admitted to hospital. Microbiological results By the end of February 2007, stool samples from 139 patients affected by the outbreak (including 99 patients who met the case-definition criteria) had been examined with respect to gastrointestinal bacteria, viruses and parasites. Among these, 77 patients (43 cases) had one or more samples that tested positive, including 23 patients with 2-5 different pathogenic gastrointestinal organisms (Table). Not all samples were tested for the entire range of pathogens identified. Further microbiological testing and genotyping of the samples are being undertaken. Technical assessment and intervention Technical and microbiological investigations of the water indicated that the most probable cause of the contamination was the combination of a technical and a human error at a local sewage treatment facility, which allowed at least 27 m3�of partially filtered waste water to enter into the drinking water system in the period between 12 and 14 January 2007. The two pipelines were separated, and the exact circumstances of the incident that allowed the backflow of the sewage water remain to be revealed. The conclusion of the technical investigations was supported by the large variety of gastrointestinal pathogens found in the stool samples, which corroborated that the contamination was due to backflow of grossly contaminated sewage water rather than, for example, surface water or sewage from only a few households. The sewage treatment plant receives sewage from a population of approximately 40,000, as well as industrial enterprises, food production establishments and a hospital. Flushing of the area�s distribution system was initiated immediately and sustained for several weeks. As faecal indicator bacteria were still found in the drinking water after two weeks of sustained flushing, the distribution system was subsequently disinfected by chlorination on 10 and 11 February. After another two days of flushing, the drinking water was released for normal use, but recommended for drinking still only after boiling. By 12 March, the boiling restrictions were lifted for the majority of households, since by then the environmental water samples from the distribution system had fulfilled the quality criteria for untreated drinking water as defined by the Danish Ministry of Environment [3]. Commentary The handling of the outbreak called for interdisciplinary cooperation and the epidemiological investigations supported the technical and water-microbiological analyses. Geographical information systems were used to define the contaminated area, and the 18 episodes of illness reported outside of the contaminated area (Figure 1) probably represent sporadic illness or could be associated with the outbreak in a way that was not investigated, for example by secondary transmission. Although the drinking water supply in Denmark is primarily from untreated ground water, disease cases are rarely registered in connection with incidents of contamination [1,2]. The outbreak described was unusual, partly because of the high morbidity among the exposed citizens, and partly because of the extraordinary complexity of positive microbiological findings; a recent review of disease outbreaks caused by contaminated drinking water in the United States from 2003 to 2004 reported that in only two out of 25 outbreaks with known aetiology more than one microorganism was detected [4]. The consequences were considerable for the affected families, but also for the food companies, which were not allowed to resume production until the middle of February.

With what symptoms they were presented?

{'answer_start': [2892], 'text': ['diarrhoea, vomiting and/or abdominal pain/cramps with fever.']}

Contamination Question Answering

On Monday 15 January 2007, a municipality in Denmark received the first of several complaints from citizens who reported severe diarrhoea and vomiting over the weekend. Over the same period, the drinking water in many houses was reported to be discoloured and of unusual smell and taste. The local authorities immediately prohibited any use of the water - except for toilet flushing - in the entire area supplied with untreated drinking water from the local waterworks, which involved 5,802 citizens and a number of companies (Figure 1). The citizens in the area were warned by the police and through radio broadcasts. To reveal the nature and the geographical spread of the suspected water contamination, water samples for microbiological and chemical analyses were systematically collected from the water distribution system across the whole area supplied from the local waterworks. The water samples were collected at the waterworks itself, from major water pipelines and wells, and from tap water in private houses and companies. High concentrations of faecal indicator bacteria (primarily presumptive coliform counts and�Escherichia coli) and endotoxins in the water samples indicated a massive faecal contamination of a part of the water distribution system, while other parts of the distribution system appeared not to be affected. On the basis of the geographical distribution of indicator bacteria, and the technical information about directions of the water flow in the different sections of the water distribution system, the area suspected to be contaminated was systematically reduced step by step during the next days. Based on the analyses of 530 water samples collected at 200 different sites, the area finally considered to be affected by the water contamination was defined on 26 January 2007 (indicated with a red line in Figure 1). This area comprised 177 households with 450 residents and several companies, among which six dealt with food. Restrictions on water use were maintained in this area, while the water was released for normal use in the area that was not considered to be contaminated. Gastrointestinal illness A line-list of patients with gastrointestinal illness associated with the water contamination was established on the basis of notifications from general practitioners, enquiries made to the medical health officer, patients seeking advice from the emergency medical service, and patients contacting an ad hoc telephone �hotline� established by the local authorities. Additional information was collected during a house-to-house questionnaire survey conducted on 16 January among 20 households in the most severely affected street. Stool samples were examined at the Department of Bacteriology, Mycology and Parasitology, and the Department of Virology, Statens Serum Institut, Copenhagen. A case of acute gastroenteritis was defined as a person with diarrhoea, vomiting and/or abdominal pain/cramps with fever. To verify whether reported cases met the case definition, and to confirm the geographical extent of gastrointestinal illness associated with the water contamination, patients on the line-list were contacted by telephone and/or postal questionnaires. By the end of February 2007, 140 cases had been registered: 110 were residents of the area that was judged to be contaminated on the basis of the environmental investigations, 12 were shoppers or employees at the food companies in the area, and 18 affected people came from outside the contaminated area. The epidemic curve for cases among residents in the contaminated area with known date of illness onset is shown in Figure 2. No new cases in the contaminated area were registered after 24 January. Cases were largely confined to the contaminated area. A total of 24% of the residents of the contaminated area were registered with gastrointestinal illness, compared with 0.3% in the other sections of the waterworks� supply area (relative risk 73; 95% CI 44-127). From the most severely affected street in the contaminated area, 43% of residents were reported to have fallen ill. Four patients were temporarily admitted to hospital. Microbiological results By the end of February 2007, stool samples from 139 patients affected by the outbreak (including 99 patients who met the case-definition criteria) had been examined with respect to gastrointestinal bacteria, viruses and parasites. Among these, 77 patients (43 cases) had one or more samples that tested positive, including 23 patients with 2-5 different pathogenic gastrointestinal organisms (Table). Not all samples were tested for the entire range of pathogens identified. Further microbiological testing and genotyping of the samples are being undertaken. Technical assessment and intervention Technical and microbiological investigations of the water indicated that the most probable cause of the contamination was the combination of a technical and a human error at a local sewage treatment facility, which allowed at least 27 m3�of partially filtered waste water to enter into the drinking water system in the period between 12 and 14 January 2007. The two pipelines were separated, and the exact circumstances of the incident that allowed the backflow of the sewage water remain to be revealed. The conclusion of the technical investigations was supported by the large variety of gastrointestinal pathogens found in the stool samples, which corroborated that the contamination was due to backflow of grossly contaminated sewage water rather than, for example, surface water or sewage from only a few households. The sewage treatment plant receives sewage from a population of approximately 40,000, as well as industrial enterprises, food production establishments and a hospital. Flushing of the area�s distribution system was initiated immediately and sustained for several weeks. As faecal indicator bacteria were still found in the drinking water after two weeks of sustained flushing, the distribution system was subsequently disinfected by chlorination on 10 and 11 February. After another two days of flushing, the drinking water was released for normal use, but recommended for drinking still only after boiling. By 12 March, the boiling restrictions were lifted for the majority of households, since by then the environmental water samples from the distribution system had fulfilled the quality criteria for untreated drinking water as defined by the Danish Ministry of Environment [3]. Commentary The handling of the outbreak called for interdisciplinary cooperation and the epidemiological investigations supported the technical and water-microbiological analyses. Geographical information systems were used to define the contaminated area, and the 18 episodes of illness reported outside of the contaminated area (Figure 1) probably represent sporadic illness or could be associated with the outbreak in a way that was not investigated, for example by secondary transmission. Although the drinking water supply in Denmark is primarily from untreated ground water, disease cases are rarely registered in connection with incidents of contamination [1,2]. The outbreak described was unusual, partly because of the high morbidity among the exposed citizens, and partly because of the extraordinary complexity of positive microbiological findings; a recent review of disease outbreaks caused by contaminated drinking water in the United States from 2003 to 2004 reported that in only two out of 25 outbreaks with known aetiology more than one microorganism was detected [4]. The consequences were considerable for the affected families, but also for the food companies, which were not allowed to resume production until the middle of February.

What did the local authorities investigate?

{'answer_start': [6645], 'text': ['Geographical information systems were used to define the contaminated area']}

Contamination Question Answering

On Monday 15 January 2007, a municipality in Denmark received the first of several complaints from citizens who reported severe diarrhoea and vomiting over the weekend. Over the same period, the drinking water in many houses was reported to be discoloured and of unusual smell and taste. The local authorities immediately prohibited any use of the water - except for toilet flushing - in the entire area supplied with untreated drinking water from the local waterworks, which involved 5,802 citizens and a number of companies (Figure 1). The citizens in the area were warned by the police and through radio broadcasts. To reveal the nature and the geographical spread of the suspected water contamination, water samples for microbiological and chemical analyses were systematically collected from the water distribution system across the whole area supplied from the local waterworks. The water samples were collected at the waterworks itself, from major water pipelines and wells, and from tap water in private houses and companies. High concentrations of faecal indicator bacteria (primarily presumptive coliform counts and�Escherichia coli) and endotoxins in the water samples indicated a massive faecal contamination of a part of the water distribution system, while other parts of the distribution system appeared not to be affected. On the basis of the geographical distribution of indicator bacteria, and the technical information about directions of the water flow in the different sections of the water distribution system, the area suspected to be contaminated was systematically reduced step by step during the next days. Based on the analyses of 530 water samples collected at 200 different sites, the area finally considered to be affected by the water contamination was defined on 26 January 2007 (indicated with a red line in Figure 1). This area comprised 177 households with 450 residents and several companies, among which six dealt with food. Restrictions on water use were maintained in this area, while the water was released for normal use in the area that was not considered to be contaminated. Gastrointestinal illness A line-list of patients with gastrointestinal illness associated with the water contamination was established on the basis of notifications from general practitioners, enquiries made to the medical health officer, patients seeking advice from the emergency medical service, and patients contacting an ad hoc telephone �hotline� established by the local authorities. Additional information was collected during a house-to-house questionnaire survey conducted on 16 January among 20 households in the most severely affected street. Stool samples were examined at the Department of Bacteriology, Mycology and Parasitology, and the Department of Virology, Statens Serum Institut, Copenhagen. A case of acute gastroenteritis was defined as a person with diarrhoea, vomiting and/or abdominal pain/cramps with fever. To verify whether reported cases met the case definition, and to confirm the geographical extent of gastrointestinal illness associated with the water contamination, patients on the line-list were contacted by telephone and/or postal questionnaires. By the end of February 2007, 140 cases had been registered: 110 were residents of the area that was judged to be contaminated on the basis of the environmental investigations, 12 were shoppers or employees at the food companies in the area, and 18 affected people came from outside the contaminated area. The epidemic curve for cases among residents in the contaminated area with known date of illness onset is shown in Figure 2. No new cases in the contaminated area were registered after 24 January. Cases were largely confined to the contaminated area. A total of 24% of the residents of the contaminated area were registered with gastrointestinal illness, compared with 0.3% in the other sections of the waterworks� supply area (relative risk 73; 95% CI 44-127). From the most severely affected street in the contaminated area, 43% of residents were reported to have fallen ill. Four patients were temporarily admitted to hospital. Microbiological results By the end of February 2007, stool samples from 139 patients affected by the outbreak (including 99 patients who met the case-definition criteria) had been examined with respect to gastrointestinal bacteria, viruses and parasites. Among these, 77 patients (43 cases) had one or more samples that tested positive, including 23 patients with 2-5 different pathogenic gastrointestinal organisms (Table). Not all samples were tested for the entire range of pathogens identified. Further microbiological testing and genotyping of the samples are being undertaken. Technical assessment and intervention Technical and microbiological investigations of the water indicated that the most probable cause of the contamination was the combination of a technical and a human error at a local sewage treatment facility, which allowed at least 27 m3�of partially filtered waste water to enter into the drinking water system in the period between 12 and 14 January 2007. The two pipelines were separated, and the exact circumstances of the incident that allowed the backflow of the sewage water remain to be revealed. The conclusion of the technical investigations was supported by the large variety of gastrointestinal pathogens found in the stool samples, which corroborated that the contamination was due to backflow of grossly contaminated sewage water rather than, for example, surface water or sewage from only a few households. The sewage treatment plant receives sewage from a population of approximately 40,000, as well as industrial enterprises, food production establishments and a hospital. Flushing of the area�s distribution system was initiated immediately and sustained for several weeks. As faecal indicator bacteria were still found in the drinking water after two weeks of sustained flushing, the distribution system was subsequently disinfected by chlorination on 10 and 11 February. After another two days of flushing, the drinking water was released for normal use, but recommended for drinking still only after boiling. By 12 March, the boiling restrictions were lifted for the majority of households, since by then the environmental water samples from the distribution system had fulfilled the quality criteria for untreated drinking water as defined by the Danish Ministry of Environment [3]. Commentary The handling of the outbreak called for interdisciplinary cooperation and the epidemiological investigations supported the technical and water-microbiological analyses. Geographical information systems were used to define the contaminated area, and the 18 episodes of illness reported outside of the contaminated area (Figure 1) probably represent sporadic illness or could be associated with the outbreak in a way that was not investigated, for example by secondary transmission. Although the drinking water supply in Denmark is primarily from untreated ground water, disease cases are rarely registered in connection with incidents of contamination [1,2]. The outbreak described was unusual, partly because of the high morbidity among the exposed citizens, and partly because of the extraordinary complexity of positive microbiological findings; a recent review of disease outbreaks caused by contaminated drinking water in the United States from 2003 to 2004 reported that in only two out of 25 outbreaks with known aetiology more than one microorganism was detected [4]. The consequences were considerable for the affected families, but also for the food companies, which were not allowed to resume production until the middle of February.

What is the date of the event?

{'answer_start': [13], 'text': ['January 2007']}