Manabe et al. BMC Infectious Diseases (2016) 16:64
DOI 10.1186/s12879-016-1391-8

RESEARCH ARTICLE

Open Access

Chronological, geographical, and seasonal
trends of human cases of avian influenza A(H5N1) in Vietnam, 2003–2014: a spatial
analysisToshie Manabe1,2,3,4*, Kazue Yamaoka1, Toshiro Tango1,5, Nguyen Gia Binh6, Dao Xuan Co6, Nguyen Dang Tuan6,
Shinyu Izumi7, Jin Takasaki7, Ngo Quy Chau8 and Koichiro Kudo3,9Abstract
Background: Human cases of highly pathogenic avian influenza A (H5N1) virus infection continue to occur inSoutheast Asia. The objective of this study was to identify when and where human H5N1 cases have occurred in
Vietnam and how the situation has changed from the beginning of the H5N1 outbreaks in 2003 through 2014, toassist with implementing methods of targeted disease management.
Methods: We assessed the disease clustering and seasonal variation of human H5N1 cases in Vietnam to evaluate
the geographical and monthly timing trends. The clustering of H5N1 cases and associated mortality were examinedover three time periods: the outbreak period (2003–2005), the post-outbreak (2006–2009), and the recent period(2010–2014) using the flexibly shaped space-time scan statistic. The most likely cases to co-cluster and the elevatedrisks for incidence and mortality were assessed via calculation of the relative risk (RR). The H5N1 case seasonalvariation was analysed as the cyclic trend in incidence data using Roger’s statistical test.
Results: Between 2003 and 2005, H5N1 cases (RR: 2.15, p = 0.001) and mortality (RR: 2.49, p = 0.021) were significantly
clustered in northern Vietnam. After 2010, H5N1 cases tended to occur on the border with Cambodia in the south,
while H5N1 mortality clustered significantly in the Mekong delta area (RR: 6.62, p = 0.002). A significant seasonalvariation was observed (p < 0.001), with a higher incidence of morbidity in December through April.
Conclusions: These findings indicate that clinical preparedness for H5N1 in Vietnam needs to be strengthened insouthern Vietnam in December–April.
Keywords: Avian influenza, H5N1, Geographical trend, Spatial analysis, Disease clustering, Seasonal variation

Background
In Southeast Asia, including in Vietnam, human cases ofhighly pathogenic avian influenza A (H5N1) virus infection continue to occur and have been associated withhigh mortality rates [1]. The current increased numberof cases in Egypt [1, 2] should encourage clinicians andpolicy makers to enhance the clinical and public health* Correspondence: manabe@kklabo.gr.jp
1
Teikyo University, Graduate School of Public Health, 2-11-1 Kaga, Itabashi-ku,
Tokyo 173-8605, Japan2
Department of Hygiene and Public Health, Teikyo University School of
Medicine, Tokyo, Japan
Full list of author information is available at the end of the article

preparation in each country and region for H5N1 infection as well as for a potential future influenza pandemic.
Human cases of H5N1 infection often have a rapid
progression and cause high mortality [3–6]. Previous
studies indicate that early diagnosis and administrationof antiviral agents relate to patient survival [7–11]. In
many developing countries, the diagnosis using real-timereverse-transcriptase polymerase chain reaction (RTPCR), one of the most sensitive methods for detecting
influenza virus, is only possible in centralised locations
with well-trained staff. Additionally, limited medical resources, including antiviral drugs, often delay the initiation of treatment [12]. Furthermore, patients frequently© 2016 Manabe et al. 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, andreproduction 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.

Manabe et al. BMC Infectious Diseases (2016) 16:64

need to travel hundreds of kilometres to receive the appropriate medical services [13]. Education for residents
in high-risk areas is a good way to provide the appropriate knowledge for preventing H5N1 infection [14], but it
is difficult to provide these programs for all residents.
Without an in-depth analysis, human cases of H5N1 appear to occur sporadically. However, we hypothesized if
a spatial and temporal pattern of human H5N1 cases
can be discovered, more tailored clinical preparedness
plans will be possible, including preventive measures,the effective allocation of medical and human resources,
and the provision of education programs.
Direct or indirect contact with sick or dead domesticated poultry is a potential cause of H5N1 infection inhumans [15]. As a result, previous studies have examined the circulation and movement of poultry flocks
across nations and performed spatial and temporal
analyses of H5N1 outbreaks among birds in Vietnam[15–17]. One study examining eight countries inSoutheast Asia reaffirmed that human H5N1 casesand H5N1 outbreaks among poultry often co-occur
seasonally [18]. Understanding the spatial and temporal patterns of H5N1 outbreaks among poultry is
crucial for understanding the spatial and temporal
patterns of human H5N1 cases. However, a detailed
evaluation using spatial and temporal analyses of thetiming and location of human H5N1 cases in Vietnam
has not been reported.
The aims of the present study were to use space-time
analyses to evaluate when and where human H5N1 cases
have occurred in Vietnam and how the situation haschanged from the beginning of the H5N1 outbreaks in2003 to the present. The results may contribute toward
more directed and targeted interventions for preventing
and controlling the spread of H5N1 human infections.

Methods
A space-time analysis was conducted to evaluate thedisease clustering and mortality of human cases of
H5N1 infection as well as the seasonal variation ofthese cases in Vietnam. The seasonal variation was defined as a cyclic trend of incidence on a monthly basis[19]. The incidence and mortality data were collectedfrom case reports of H5N1 human infections by theWorld Health Organization (WHO) and the WHO
Western Pacific Region between December 2003 andDecember 2014 [20, 21].
Data on the location (province) of occurrence,
dates of disease onset and hospital admission, andoutcome (death or survival) of each human H5N1
case were collected.
Because a reporting system with detailed informationon H5N1 patients had not yet been established during
the 2003–2005 outbreak period, cases from this time

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were only included in our study if they were confirmed
by RT-PCR. These cases were used in the disease clustering analysis only if the name of the province where
the case occurred and the patient outcome were bothreported, and they were used for the seasonal variation
analysis only if the date of disease onset, hospitalization,
or death were reported clearly. In contrast, all of thecases from 2006 to 2014 were included in the present
study. Thus, the number of included cases from the
2003–2005 outbreak period used in analysing the seasonal variation and the disease clustering was 74 and 68
cases (36 fatal), respectively. According to the WHO,
however, the number of reported cases in Vietnam during this period was 93 (42 fatal). After the reporting
system was established in late 2005, detailed informationon all H5N1 cases in Vietnam were provided from theWHO. Thus, from 2003 to 2014, a total of 108 cases
were analysed for seasonal variation and 98 cases (60fatal) for disease clustering. A sub-analysis to determine
the sensitivity of our study was conducted with the datafrom 2006 through 2014 because the data from theseyears was complete for all reported H5N1 cases.
The population of each Vietnamese province wasobtained from a 2012 report by the General Statistics
Office of Vietnam [22] and used to analyse a standardised morality ratio (SMR) and a standardised incidence
ratio (SIR) of disease clustering [23]. The SMR and SIR
for each province in Vietnam were then plotted on amap. Geographical information, including the latitude
and longitude of each province and its neighbouringprovinces, was also collected for our disease mapping.
The collected cases were divided into three time periods: the outbreak period (2003–2005), the postoutbreak period (2006–2009), and the recent period(2010–2014). These time periods were chosen to evaluate possible changes over time as well as to evaluate
the effects of a strategic disease control project being
conducted in northern Vietnam that our group began
in 2010 [9, 14, 24, 25].
Statistical analyses

Disease clustering was detected using the flexibly shaped
space-time scan statistic (FleXScan) [23, 26–29]. The
FlexScan analysis was implemented with the restricted
likelihood ratio proposed by Tango [23, 29]. It was
shown that as the relative risk (RR) of the cluster becomes large via a Monte Carlo simulation, this method
is able to detect clusters of any shape reasonably well[23, 26]. We selected a flexible spatial scan as our scanning method. A Poisson model was used for spatial
analysis scanning for clustering. The p-values were
calculated using the original log likelihood ratios. To
characterise a detected spatial cluster, we estimated theRR, the SIR, and the SMR. The SIR and SMR are theManabe et al. BMC Infectious Diseases (2016) 16:64

observed number of cases divided by the expected frequency of incidence and mortality, respectively, within
the clusters. For the circulating spatial scan statistic, themaximum length of the geographical window was set to
20 of the nearest neighbours [23, 26]. The number of
replications for the Monte Carlo procedure was set to999. Seasonal variations in the monthly incidence of human H5N1 infection were evaluated using the numberof cases according to the month of disease onset or hospital admission. To evaluate if there was a seasonal variation in the incidence data, we used the cyclic trend
significance test proposed by Roger [30]. In this method,
the null hypothesis is that the incidences are equally
likely to be allocated in each of the months.
Data analyses were conducted using FleXScan version 3.1 [28] and SAS version 9.3 (SAS Institute Inc.,
Cary, North Carolina). The p-values were two-tailed,and p < 0.05 was considered significant.

Results
Human cases of avian influenza H5N1 infection in
Vietnam

Between December 2003 and December 2014, a total of
127 confirmed human H5N1 cases were reported to the
WHO from Vietnam, 64 of which were fatal [1]. The
highest numbers of cases were reported between 2003
and 2005 (Fig. 1). Although there were no cases reported
in 2006 or 2011, some cases were reported in 2014.
Seasonal trends of human H5N1 cases in Vietnam

To investigate the seasonal variation in human H5N1
cases, we evaluated the trends in the monthly timing ofthese cases. A significant seasonal variation was observed from the beginning of the H5N1 outbreak in2003 to the end of 2014, (p < 0.001), and from 2006 to
the end of 2014 (p < 0.001) (Fig. 2). Similarly, the seasonal variations within the outbreak period (2003–
2005) (p < 0.001) and the recent period (2010–2014)(p < 0.001) were significant, and although there was atrend toward seasonal variation within the postoutbreak period (2007–2009), this failed to reachstatistical significance (p = 0.059). Compared with theother months, H5N1 cases occurred at a higher

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incidence between December and April and a lower
incidence between August and November.
Geographical disease clustering of human H5N1 infection
incidence and mortality in Vietnam

Disease and mortality clustering of H5N1 cases wasshown in Fig. 3. From 2003 to 2014, the incidence(Fig. 3a) and mortality (Fig. 3b) of H5N1 cases were significantly clustered in the central area of northern
Vietnam and in the rural mountainous and coastal areasof northern Vietnam, respectively. During the H5N1
outbreak period (2003–2005), a cluster of H5N1 cases
was detected in the central area of northern Vietnam(Fig. 3c) and the H5N1 case mortality was clustered in
the central and coastal areas (Fig. 3d). In the postoutbreak period, the incidence of H5N1 cases (Fig. 3e)and H5N1 mortality (Fig. 3f ) spread to the agriculturalareas in northern Vietnam other than the central areasof Hanoi. From 2010 to 2014, there was no significant
clustering of H5N1 cases, only a trend toward cases being predominantly located in the areas that border
Cambodia in southern Vietnam (Fig. 3g). During thisperiod, a significant cluster of H5N1 mortality was detected near the border of Cambodia and the Mekong
delta in southern Vietnam (Fig. 3h).
We also performed a subgroup analysis using datafrom 2006 through 2014 because the data from the outbreak period (2003–2005) did not include all reported
cases, as described in the Methods section. The H5N1
cases (RR: 4.15, p = 0.001) and H5N1 mortality (RR:
4.55, p = 0.044) from this period were significantly clustered in northern Vietnam (Additional file 1).

Discussion
The present study revealed that H5N1 mortality in
Vietnam was significantly clustered in the northern partof Vietnam from 2003 through 2014, but during the recent period, H5N1 mortality has clustered in the southernpart of Vietnam. We also observed a significant seasonalvariation in the human H5N1 cases, with a higher disease
incidence in December through April.
Large outbreaks of human H5N1 cases occurred toward the end of 2003, simultaneous with large H5N1

Fig. 1 Observed human cases of H5N1 infection from December 2003 to December 2014

Manabe et al. BMC Infectious Diseases (2016) 16:64

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Fig. 2 Seasonal variation during the entire observational period (2003–2014) was tested by Roger’s statistic (p < 0.001). The p values for eachperiod were p < 0.001 in the outbreak period (2003–2005), p = 0.059 in the post-outbreak period (2006–2009), and p < 0.001 in the recent
period (2010–2014)

outbreaks among poultry throughout Asia [6, 31, 32].
Since then, a history of contact with infected poultry has
been reported for many of the human H5N1 cases andis believed to be one of the risk factors of human H5N1
infection [7, 15, 33–35]. Therefore, the seasonal variation in human H5N1 cases might correspond to thetime when migratory birds typically infect domesticatedpoultry with H5N1 [36]. The present study has shownthat human H5N1 cases in Vietnam are most common
from December to April (Fig. 2). This timing overlaps
with that of H5N1 infections in poultry, which typicallyoccur between January and March [18]. These results
indicate that the clinical preparedness and disease monitoring for H5N1 should be enhanced during these overlap months.
Temperature may also be a factor for H5N1 outbreaks
among poultry. A previous report indicated that avian
influenza virus replication increases at cooler temperatures [37], and colder weather may enable prolonged
viral survival in the secretions and faeces of infected
poultry [18, 38]. The temperature and humidity in
Vietnam between December and March are lower thanthose in the other months, especially in northern
Vietnam, which has a subtropical climate [39].
Temperature and humidity are known ecological risk
factors for seasonal influenza; therefore, it is likely thatthey similarly influence the occurrence of human H5N1
cases [40–43]. The seasonal variation in the number ofhuman H5N1 cases typically overlaps with the periodof seasonal influenza incidence.
Another possible reason for the seasonal variation of
H5N1 infection, possibly unique to Vietnam, may be theincreases in poultry population density and trafficking
due to the Tet holiday festival (the Lunar New Year of
Vietnam), which takes place in January or February ofeach year. During this period, the distribution, trading,and consumption of chickens increase dramatically because chicken is a traditional dish for the celebration of
Tet [44]. Thus, the risk of H5N1 human infection is
likely to increase dramatically around the Tet holiday[44]. A similar situation has been suggested for the

Chinese New Year festival [45]. The results of our study
point to the necessity of understanding the relevant
cultural factors to achieve clinical preparedness for avian
influenza infection. Although patterns on seasonal variations on each period were similar with the above reasons, the number of incidence of H5N1 human infection
has been decreasing. It may be caused by no evidence ofhuman-to-human spread [46].
In Vietnam, human H5N1 cases and mortality weresignificantly clustered in the coastal and mountainous
areas of northern Vietnam from 2003 through 2014
(Fig. 3a, b). At the beginning of the human H5N1 outbreaks between 2003 and 2005, H5N1 cases were reported both from the southern and northern parts ofVietnam [19]. However, significant clusters of H5N1
cases and mortality were detected in northern Vietnam
during this period (Fig. 3c, d). Since then, although thenumber of confirmed human H5N1 cases of has been
lower, human H5N1 cases have still occurred sporadically and continued to be present through 2014. In
2005, Vietnam conducted nationwide vaccination programs against poultry that were thought to contributeto the absence of widespread outbreaks in poultry[33]. It might affect to occurrence on human H5N1
cases throughout 2006 [17]. After 2007, significant
clusters of H5N1 cases and mortality were still observed in northern Vietnam, but they moved to morerural and mountainous areas. After 2010, no significant clusters of H5N1 cases were found, although
there was a trend toward cases being located near theborder with Cambodia in southern Vietnam. However,a significant cluster of H5N1 mortality was foundduring the recent period in the Mekong delta areas in
the south.
The outbreak of human H5N1 cases started in
December 2003, and cooler temperatures in northern
Vietnam may have led to more cases in that area thanin southern Vietnam, which has a tropical climate.
However, the movement of H5N1 mortality clusters
over time may also be strongly influenced by reporting
systems, medical techniques and resources, and/or

Manabe et al. BMC Infectious Diseases (2016) 16:64

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a

b

c

d

e

f

g

h

Fig. 3 Clustering of the incidence and mortality of human H5N1 in Vietnam from 2003 to 2014. Clustering of H5N1 cases (left panels) andmortality (right panels) in 2003–2014 a, b, 2003–2005 c, d, 2006–2009 e, f, and 2010–2014 g, h. Black areas represent the clusters of human H5N1
incidence (left panels) and mortality (right panels) due to H5N1 infection during each time period

Manabe et al. BMC Infectious Diseases (2016) 16:64

information availability, rather than climatic factors.
For example, in the beginning of the H5N1 outbreak,
H5N1 cases and mortality were primarily located inand around the central city of Hanoi. The reporting
system of Hanoi may be more efficient than those inrural areas, and the RT-PCR technique is more common in Hanoi. Additionally, Hanoi’s hospitals have themedical and human resources to provide advanced
medicine for treating patients with unknown serious
diseases, and patients with serious conditions are better able to access hospitals in the central city.
There were no significant clusters of H5N1 cases from2010 to 2014 (Fig. 3g), but a significant cluster of H5N1
mortality was detected during this time period in thearea bordering Cambodia and the Mekong delta area insouthern Vietnam (Fig. 3h). This cluster of mortality led
to a high RR (6.62) compared with those of other periods. Since 2011, the numbers of reported human
H5N1 cases and mortality in Cambodia have increased
dramatically [1]. The movement of humans and trade,including poultry, between Cambodia and Vietnam
might affect the occurrence of human H5N1 cases inareas close to the border of Cambodia [40]. This area isalso thought to have fewer medical and human resources
and less clinical information and support for treating
H5N1 patients. The other neighbour countries including
Thailand and Lao did not report H5N1 in human cases
since 2007, and China has been reporting continuously;
however, the number of reported cases was less than 2
in 2010–2014 [1]. The situation of neighbour countries
may not affect to those of Vietnam other than
Cambodia. Starting in 2010, our group has conducted an
H5N1 treatment network program, which includes
education for residents and medical providers as well as
clinical consultations for clinicians in the local hospitals
in northern Vietnam [9, 20–22]. This network may havehelped to reduce the mortality from human H5N1 in thenorth and could be one of the factors that led to thecluster of H5N1 mortality moving from northern tosouthern Vietnam.
One limitation of the present study is that the collected data are from the WHO’s official reports ofhuman H5N1 cases. It is possible that the number ofcases reported to the WHO is underestimated. Additionally, some of these reports lack detailed information.
Consistent monitoring activities on a local basis would
help with obtaining a more accurate evaluation of H5N1
human incidence and mortality. Another potential limitation of this study could be our use of the 2012
Vietnamese population throughout all the observational
years for our SIR and SMR analyses of disease clustering;
however, the mean population over these years was similar to the population during 2012 and it has not changed
drastically over this time period.

Page 6 of 8

Conclusion
In conclusion, the results of the present study provide
statistically significant evidence of H5N1 disease incidence and mortality clustering. These findings will contribute toward directed and targeted interventions,improving our preparedness and control activities for
H5N1 human infections. To reduce the disease severity
and number of deaths from H5N1 infection, clinical andpublic health preparedness for H5N1 in Vietnam should
be strengthened from December–April, particularly inthe areas bordering Cambodia and the Mekong deltaregion in southern Vietnam. Continuous and timely
disease monitoring are necessary for the detection of
H5N1 outbreaks and other novel influenza virus infections throughout Vietnam and may assist in worldwide
prevention of influenza infections.
Ethics approval and consent to participate

The ethical approval was waived by the ethics committeeof Teikyo University for this study using open-accessed
anonymized information.

Additional file
Additional file 1: Geographical disease clustering of incidence andmortality of human H5N1 infection in Vietnam between 2007 and2014. (JPG 113 kb)
Abbreviations
RT-PCR: real-time reverse-transcription polymerase chain reaction; RR: relative
risk; SMR: standardised morality ratio; SIR: standardised incidence ratio;
FleXScan: the flexibly shaped space-time scan statistic..
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived and designed the experiments: TM, KY, TT, and KK. Performedexperiments and analysed data: TM, KY, and TT. Interpreted study results: TM,
NGB, DXC, NDT, SI, JT, NQC, and KK. Wrote the manuscript: TM. All authors
have read and approved of the final manuscript.
Acknowledgements
The authors thank Tran Thuy Hanh, Dang Hung Minh, Vu Thi Tuong Van,
Pham Thi Phuong Thuy, Tran Thi Mai Phuong, and the medical providers in
the provincial hospitals for providing information on the local conditions fortreating H5N1 patients and for assisting with our research activities inVietnam.
Funding
This study was supported by the Japan Society for the Promotion of Science(JSPS) grant KAKENHI26293115.
Author details
1
Teikyo University, Graduate School of Public Health, 2-11-1 Kaga, Itabashi-ku,
Tokyo 173-8605, Japan. 2Department of Hygiene and Public Health, Teikyo
University School of Medicine, Tokyo, Japan. 3Waseda University, 1-21-1
Nishi-Waseda, Shinjuku-ku, Tokyo, Japan. 4University of Tsukuba, Graduate
School of Comprehensive Human Sciences, Ibaraki, Japan. 5Center for
Medical Statistics, Tokyo, Japan. 6Bach Mai Hospital, Intensive Care Unit, 78
Giai Phong, Dong Da, Hanoi, Vietnam. 7National Center for Global Health andMedicine, Division of Pulmonary Medicine, Tokyo, Japan. 8Department ofManabe et al. BMC Infectious Diseases (2016) 16:64

Pulmonary Medicine, Bach Mai Hospital, 78 Giai Phong, Dong Da, Hanoi,
Vietnam. 9Koto Hospital, 6-8-5 Ojima, Koto-ku, Tokyo, Japan.
Received: 22 September 2015 Accepted: 27 January 2016

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