acute respiratory tract infection ( arti ) is one of the leading causes of morbidity and mortality in children and adults worldwide . the mortality rate is higher in children under five years of age , the elderly , and immunocompromised individuals ( 1 , 2 ) . influenza a virus ( ifv - a ) , influenza b virus ( ifv - b ) , respiratory syncytial virus ( rsv ) , parainfluenza viruses ( pivs ) , adenovirus ( adv ) , and rhinovirus ( rv ) are the most frequently reported viruses associated with artis in the general population . however , their distribution varies by season , geographic region , and age group ( 3 , 4 ) . the clinical presentations of different pathogens , either viral or bacterial , are similar and therefore the accurate etiologic diagnosis of artis relies entirely on laboratory investigations . early detection of related causative agents is crucial for providing an appropriate treatment regimen , decreasing the use of unnecessary antibiotics , limiting the spread of infection , and shortening the hospitalization duration ( 2 , 3 , 5 ) . the conventional methods , such as viral cell cultures and antigen detection tests ( such as enzyme immunoassays and direct fluorescent antibody tests ) , are effective and often complementary , but they have some limitations . although the cell culture technique is considered to be the gold standard for virus detection , the process is laborious and time - consuming , and it is almost impossible to obtain results during the acute phase of the disease . antigen - detection tests can provide more rapid results , but are less sensitive and/or specific compared to cell culture techniques ( 5 , 6 ) . molecular diagnostic methods allow the identification of a wide range of viral and bacterial pathogens within hours , with excellent sensitivity and specificity . multiplex real - time polymerase chain reaction ( pcr ) assays provide simultaneous amplification of several viruses or bacteria in a single reaction and make it easy to detect the common causative agents of artis ( 5 , 7 ) . in addition , these tests are able to detect emerging respiratory viruses , such as human metapneumovirus ( hmpv ) , human coronavirus ( hcov ) nl63 , hcov hku1 and human bocavirus ( hbov ) , which are difficult to grow in cell cultures ( 3 , 8 - 10 ) . the aim of this cross - sectional study was to determine the prevalence and seasonal distribution of causative viral agents over a one - year period in children and adults who were living in istanbul and had a pre - diagnosis of arti . a total of 845 nasopharyngeal swab specimens were obtained from patients with a clinical pre - diagnosis of arti between september 1 , 2014 , and august 31 , 2015 . the specimens were transferred from healthcare institutions in istanbul to gelisim medical laboratories , and analyzed directly or stored at -80c until tested . the samples were analyzed for respiratory viruses and bacteria by using the respifinder smart 22 assay ( pathofinder bv , the netherlands ) . each sample was simultaneously tested for the following 22 pathogens : ifv - a , ifv - a ( h1n1)pdm09 , ifv - b , piv-1 , piv-2 , piv-3 , piv-4 , rsv - a , rsv - b , hmpv , rhinovirus / enterovirus ( rv / ev ) , hbov , adv , hcov nl63 , hcov hku1 , hcov 229e , hcov oc43 , bordetella pertussis ( bp ) , chlamydophila pneumoniae ( cp ) , legionella pneumophila ( lp ) , and mycoplasma pneumoniae ( mp ) . first , viral and bacterial nucleic acids ( dna or rna ) were extracted according to the kit protocol . next , amplification , detection , and data analysis were performed with the rotor - gene 6000 real - time pcr system ( qiagen , germany ) according to the manufacturer s instructions ( 11 , 12 ) . the statistical analysis was performed using statistical package for the social sciences ( spss inc . , chi - square or fisher s exact tests were used for comparisons between groups in terms of categorical variables , wherever appropriate . a total of 845 nasopharyngeal swab specimens from 309 children and 536 adults with arti were analyzed . the median age was 29 years ( range 0 - 91 years ) ; 444 ( 52.5% ) of the patients were male and 401 ( 47.5% ) were female . in 233 ( 27.6% ) of the specimens , no pathogen was detected , while 612 ( 72.4% ) of the specimens were positive for one or more pathogens ( table 1 ) . the positive detection rate in children ( 232/309 , 75.1% ) was found to be higher than in adults ( 380/536 , 70.9% ) , but the difference was not statistically significant ( p = 0.190 ) . similarly , the positive detection rate in male patients ( 326/444 , 73.4% ) was higher than in female patients ( 286/401 , 71.3% ) , but the difference was not statistically significant ( p = 0.495 ) . overall , 902 pathogens were detected ; of these , 821 ( 91% ) were viruses and 81 ( 9% ) were bacteria . among the viruses , ifv - a ( n = 219 ) was the most commonly detected pathogen ( table 2 ) . seventy ( 32% ) of the ifv - a viruses were identified as the ( h1n1 ) pdm09 subtype . among the bacteria , 49 were identified as mp , 15 were bp , 11 were lp , and six were cp . the distribution of respiratory viruses in children ( aged 0 - 15 years ) is shown in table 2 . the detection rates of rsv a / b , adv , and pivs in children were significantly higher than in adults ( all p < 0.05 ) . in the 0 - 4-year - old subgroup , the most commonly detected viruses were rsv a / b ( n = 26 ) , ifv - a ( n = 23 ) , rv / ev ( n = 21 ) , adv ( n = 16 ) , ifv - b ( n = 14 ) , pivs ( n = 13 ) , hbov ( n = 11 ) , hcovs ( n = 7 ) , and hmpv ( n = 7 ) , respectively . in the 5 - 15-year - old subgroup , the most commonly detected viruses were ifv - a ( n = 37 ) , ifv - b ( n = 29 ) , rv / ev ( n = 28 ) , hbov ( n = 24 ) , rsv a / b ( n = 17 ) , adv ( n = 17 ) , pivs ( n=12 ) , hcovs ( n = 11 ) , and hmpv ( n = 4 ) , respectively . the distribution of respiratory viruses in adults ( > 15 years old ) is shown in table 2 . the detection rates of ifv - a and ifv - b in adults were significantly higher than in children ( all p < 0.05 ) . in the 16 - 64-year - old subgroup , the most commonly detected viruses were ifv - a ( n = 112 ) , ifv - b ( n = 81 ) , hbov ( n = 37 ) , rv / ev ( n = 36 ) , hcovs ( n = 24 ) , adv ( n = 17 ) , rsv a / b ( n = 16 ) , hmpv ( n = 13 ) , and pivs ( n = 9 ) , respectively . in the  65-year - old subgroup , the most commonly detected viruses were ifv - a ( n = 47 ) , ifv - b ( n = 33 ) , rv / ev ( n = 22 ) , hbov ( n = 19 ) , hmpv ( n = 12 ) , hcovs ( n = 9 ) , adv ( n = 6 ) , pivs ( n = 6 ) , and rsv a / b ( n = 5 ) , respectively . co - infection was seen in 215 specimens , with detection rates of 25.4% ( 215/845 ) of all specimens and 35.1% ( 215/612 ) of positive specimens . co - infection was more common in male patients ( 124/444 , 27.9% ) than in female patients ( 91/401 , 22.7% ) , but the difference was not statistically significant ( p = 0.081 ) . the co - infection rate in children ( 84/309 , 27.2% ) was found to be higher than in adults ( 131/536 , 24.4% ) , but the difference was not statistically significant ( p = 0.378 ) . the most frequently detected co - infection combinations were ifv - a / hbov ( n = 26 ) and ifv - a / ifv - b ( n = 20 ) , respectively . ifv - a / hbov co - infections ( n = 20 ) in adults and ifv - a / ifv - b co - infections ( n = 8) in children were the most common combinations . among the respiratory viruses , ifv - a ( 133/219 , 60.7% ) , rsv a / b ( 36/64 , 56.2% ) , and ifv - b ( 82/157 , 52.2% ) were more commonly detected as single pathogens , whereas adv ( 46/56 , 82.1% ) , hcovs ( 36/51,70.6% ) , hbov ( 59/91 , 64.8% ) , hmpv ( 23/36 , 63.9% ) , rv / ev ( 68/107 , 63.6% ) , and pivs ( 21/40 , 52.5% ) were predominantly detected in co - infections . among the bacteria , 17 mp , nine bp , seven lp , and five cp pathogens were detected as single pathogens , while 32 mp , six bp , four lp , and one cp pathogen were detected in co - infections . the monthly and seasonal distribution of respiratory viruses detected in 2014 - 2015 are shown in table 4 and figure 1 , respectively . ifv - a , rv / ev , rsv a / b , adv , and hcov were seen throughout the year , with different peak months . the total detection rate of respiratory viruses was found to be highest in march ( 197/821 , 24% ) and april ( 105/821 , 12.8% ) , and lowest in september ( 31/821 , 3.8% ) . seasonally , the total detection rates for respiratory viruses in spring , summer , autumn , and winter were 43.7% , 20.8% , 13.7% , and 21.8% , respectively . among the bacterial pathogens , mp was more frequently detected in summer ( 22/49 , 44.9% ) and winter ( 11/49 , 22.4% ) , lp was more frequent in winter ( 11/11 , 100% ) , and bp was more frequent in winter ( 6/15 , 40% ) . the co - infections were more frequently detected in spring ( 98/215 , 45.6% ) and winter ( 47/215 , 21.9% ) than in summer ( 40/215 , 18.6% ) and autumn ( 30/215 , 14% ) . a total of 845 nasopharyngeal swab specimens from 309 children and 536 adults with arti were analyzed . the median age was 29 years ( range 0 - 91 years ) ; 444 ( 52.5% ) of the patients were male and 401 ( 47.5% ) were female . in 233 ( 27.6% ) of the specimens , no pathogen was detected , while 612 ( 72.4% ) of the specimens were positive for one or more pathogens ( table 1 ) . the positive detection rate in children ( 232/309 , 75.1% ) was found to be higher than in adults ( 380/536 , 70.9% ) , but the difference was not statistically significant ( p = 0.190 ) . similarly , the positive detection rate in male patients ( 326/444 , 73.4% ) was higher than in female patients ( 286/401 , 71.3% ) , but the difference was not statistically significant ( p = 0.495 ) . overall , 902 pathogens were detected ; of these , 821 ( 91% ) were viruses and 81 ( 9% ) were bacteria . among the viruses , ifv - a ( n = 219 ) was the most commonly detected pathogen ( table 2 ) . seventy ( 32% ) of the ifv - a viruses were identified as the ( h1n1 ) pdm09 subtype . among the bacteria , 49 were identified as mp , 15 were bp , 11 were lp , and six were cp . the distribution of respiratory viruses in children ( aged 0 - 15 years ) is shown in table 2 . the detection rates of rsv a / b , adv , and pivs in children were significantly higher than in adults ( all p < 0.05 ) . in the 0 - 4-year - old subgroup , the most commonly detected viruses were rsv a / b ( n = 26 ) , ifv - a ( n = 23 ) , rv / ev ( n = 21 ) , adv ( n = 16 ) , ifv - b ( n = 14 ) , pivs ( n = 13 ) , hbov ( n = 11 ) , hcovs ( n = 7 ) , and hmpv ( n = 7 ) , respectively . in the 5 - 15-year - old subgroup , the most commonly detected viruses were ifv - a ( n = 37 ) , ifv - b ( n = 29 ) , rv / ev ( n = 28 ) , hbov ( n = 24 ) , rsv a / b ( n = 17 ) , adv ( n = 17 ) , pivs ( n=12 ) , hcovs ( n = 11 ) , and hmpv ( n = 4 ) , respectively . the distribution of respiratory viruses in adults ( > 15 years old ) is shown in table 2 . the detection rates of ifv - a and ifv - b in adults were significantly higher than in children ( all p < 0.05 ) . in the 16 - 64-year - old subgroup , the most commonly detected viruses were ifv - a ( n = 112 ) , ifv - b ( n = 81 ) , hbov ( n = 37 ) , rv / ev ( n = 36 ) , hcovs ( n = 24 ) , adv ( n = 17 ) , rsv a / b ( n = 16 ) , hmpv ( n = 13 ) , and pivs ( n = 9 ) , respectively . in the  65-year - old subgroup , the most commonly detected viruses were ifv - a ( n = 47 ) , ifv - b ( n = 33 ) , rv / ev ( n = 22 ) , hbov ( n = 19 ) , hmpv ( n = 12 ) , hcovs ( n = 9 ) , adv ( n = 6 ) , pivs ( n = 6 ) , and rsv a / b ( n = 5 ) , respectively . co - infection was seen in 215 specimens , with detection rates of 25.4% ( 215/845 ) of all specimens and 35.1% ( 215/612 ) of positive specimens . co - infection was more common in male patients ( 124/444 , 27.9% ) than in female patients ( 91/401 , 22.7% ) , but the difference was not statistically significant ( p = 0.081 ) . the co - infection rate in children ( 84/309 , 27.2% ) was found to be higher than in adults ( 131/536 , 24.4% ) , but the difference was not statistically significant ( p = 0.378 ) . the most frequently detected co - infection combinations were ifv - a / hbov ( n = 26 ) and ifv - a / ifv - b ( n = 20 ) , respectively . co - infections ( n = 20 ) in adults and ifv - a / ifv - b co - infections ( n = 8) in children were the most common combinations . among the respiratory viruses , ifv - a ( 133/219 , 60.7% ) , rsv a / b ( 36/64 , 56.2% ) , and ifv - b ( 82/157 , 52.2% ) were more commonly detected as single pathogens , whereas adv ( 46/56 , 82.1% ) , hcovs ( 36/51,70.6% ) , hbov ( 59/91 , 64.8% ) , hmpv ( 23/36 , 63.9% ) , rv / ev ( 68/107 , 63.6% ) , and pivs ( 21/40 , 52.5% ) were predominantly detected in co - infections . among the bacteria , 17 mp , nine bp , seven lp , and five cp pathogens were detected as single pathogens , while 32 mp , six bp , four lp , and one cp pathogen were detected in co - infections . the monthly and seasonal distribution of respiratory viruses detected in 2014 - 2015 are shown in table 4 and figure 1 , respectively . ifv - a , rv / ev , rsv a / b , adv , and hcov were seen throughout the year , with different peak months . the total detection rate of respiratory viruses was found to be highest in march ( 197/821 , 24% ) and april ( 105/821 , 12.8% ) , and lowest in september ( 31/821 , 3.8% ) . seasonally , the total detection rates for respiratory viruses in spring , summer , autumn , and winter were 43.7% , 20.8% , 13.7% , and 21.8% , respectively . among the bacterial pathogens , mp was more frequently detected in summer ( 22/49 , 44.9% ) and winter ( 11/49 , 22.4% ) , lp was more frequent in winter ( 11/11 , 100% ) , and bp was more frequent in winter ( 6/15 , 40% ) . the co - infections were more frequently detected in spring ( 98/215 , 45.6% ) and winter ( 47/215 , 21.9% ) than in summer ( 40/215 , 18.6% ) and autumn ( 30/215 , 14% ) . rapid and accurate identification of the viral agents of artis is critically important in order to initiate appropriate antiviral therapy and to prevent the overuse of antibiotics , nosocomial transmission , and lengthy hospital stays ( 7 , 13 ) . molecular techniques with higher sensitivity and rapidity play a critical role in the early identification of respiratory viral pathogens , particularly during epidemics ( 13 ) . however , the virus - detection rate varies depending on the specimen type , the method used , the working group , and when the study was performed ( 2 ) . in our study , nasopharyngeal swabs were tested for 18 respiratory viruses and four bacteria by using a multiplex real - time pcr assay , and at least one agent was identified in 72.4% of the samples . this high positive detection rate is similar to the rates reported in other studies , in which molecular methods were performed ( 13 - 18 ) . the results indicated that individuals in all age groups were susceptible to multiple respiratory viruses that simultaneously circulate in the community . moreover , despite no significant differences , the detection rate of pathogens in children ( 75.1% ) was slightly higher than in adults ( 70.9% ) ( table 1 ) . previous reports suggested that positive detection rates ( in the range of 30.9% - 96.1% ) for respiratory viruses in pediatric groups were higher than in adult groups ( 19 ) . viral artis have been suggested to occur more frequently in males ( 15 , 20 ) . our data also showed a slight male preponderance ( 53.4% ) among the positive cases , but the overall positive detection rate was not significantly different between male and female patients . among the respiratory viruses , ifvs are some of the most important genera due to their epidemic and pandemic potential in terms of public health . ifvs can have different clinical manifestations , from mild upper respiratory tract infections to severe pneumonia resulting in death ( 21 ) . in this study , ifv - a was the most commonly detected agent , both in children over 5 years of age and in adults ( table 2 ) . in addition , the detection rates of ifv - a and ifv - b in adults were found to be significantly higher than in children . ( 2 ) detected the most common agents as rv in the 0 - 4-year - old age group and ifv - b in the 5 - 50-year - old age group in iran . in another study conducted in the united states , hbov ( in the 0 - 4-year - old age group ) and rv ( in the 5 - 50-year - old age group ) were found to be the most prevalent agents ( 22 ) . ( 23 ) found the most common agent in adults in china to be ifv - a , which was similar to our findings . these results support that the prevalence of respiratory viruses changes based on geographic region and age group . rv and ev are both members of the enterovirus genus , and the kit used in this study was not designed for the identification of these two viruses at species level . rv , once thought to cause only the common cold in children and adults , is now considered to be a major cause of lower artis and asthmatic exacerbations ( 3 , 15 ) . in our study , rv / ev were the second most frequent agents following ifvs , comprising 13% of the detected viruses ( table 2 ) 24 ) found the most common agent to be ifvs and the second most common to be rv / ev . respiratory syncytial virus is known as the most common cause of bronchiolitis and pneumonia in infants and young children worldwide , and is divided into two subgroups , a and b , depending on the antigenic and genetic variety ( 3 ) . in this study , the detection rate of rsv a / b in children was significantly higher than in adults , and rsv a / b was the most commonly detected pathogen in the 0 - 4-year - old age group . our results confirmed the previous observations regarding the importance of rsv a / b in children under 5 years of age . in the current study , two or more pathogens were detected in 25.4% of all samples , with a higher rate in children and in male patients . in a systematic review , goka et al . ( 19 ) reported that co - infection rates ranged from 5% to 62% , and rsv has been found to be the most predominant co - infecting virus in many studies . 10 ) detected the most prevalent viral agents in co - infections to be ifvs and rv . in another study conducted in our country , hbov and hmpv were found to be the most common co - infecting viruses ( 20 ) . in this study , adv , hbov , hmpv , rv / ev , and hcovs were more frequently found in co - infections , and the most commonly detected co - infections were ifv - a / hbov and ifv - a / ifv - b ( table 3 ) . it was reported that the high co - infection prevalence of adv and hbov may have resulted from asymptomatic persistence , prolonged nasopharyngeal shedding , or a tendency to infect or colonize in the presence of other viruses ( 9 , 14 , 18 , 25 ) . ( 22 ) found that the detection of hbov was not associated with clinical symptoms in 54% of cases . on the other hand , although nasopharyngeal samples were suggested to reveal reliable results for detecting viral agents , the findings described above may indicate that pathogen detection in the nasopharynx may not accurately represent the situation in the lower respiratory tract ( 14 , 17 , 18 ) . moreover , the possibility of contamination during sample collection or analysis could not be excluded , and this should be kept in mind in clinical evaluations . assessment of viral load may be a better choice in the interpretation of positive co - infection results , but it has been reported that more studies would be required to clarify the potential value of quantitative test results ( 9 ) . in addition , in most studies , it has been suggested that the presence of more than one pathogen in a respiratory sample did not affect the clinical presentation of artis , but the relationship between co - infection and severity of disease remains debatable ( 4 , 13 - 15 , 19 ) . it is known that viral artis have a seasonal character , particularly in regions with temperate climates , and the peak periods may change from year to year . in many studies , respiratory viruses have been reported to be active during the cold seasons ( generally from november to march ) in the northern hemisphere ( 20 ) . in our study , interestingly , ifv - b was not detected in the autumn months or in december ; it began to appear in january ( table 4 , figure 1 ) . in contrast , ifv - a was seen throughout the year , with peak activity in march and april . rv / ev was most frequently seen in the autumn and winter months , rsv a / b and hmpv in the winter and spring months , hcovs in the spring months , adv in the spring and summer months , and hbov and pivs in the summer months . the differences in the peak periods , compared to previous reports , could be explained by the smaller number of cases in our study ( particularly for hcovs , pivs , and hmpv ) , regional differences , or annual variability , which has been demonstrated in other studies ( 13 , 20 , 25 ) . first , we were unable to obtain accurate inpatient or outpatient data of the cases and thus we could not present the distribution of respiratory viruses according to inpatient and outpatient groups . however , most of our contracted healthcare institutions consisted of local outpatient polyclinics in istanbul , and a high proportion of the samples may have been collected from outpatients and a minority from inpatients . second , although multiplex real - time pcr assays are reported to have excellent sensitivity and specificity , there is a possibility of false - positive or false - negative results . as mentioned above , it may be difficult to diagnose whether a positive nasopharyngeal swab shows the etiology or evidence of nasopharyngeal colonization . lastly , one year may be considered too short a time for an epidemiologic study , but we think that our preliminary data could provide useful findings for further investigations . in conclusion , ifv - a was found to be the most prevalent respiratory pathogen in all age groups except for children under 5 years of age , in whom rsv a / b was the most common agent . in our region , respiratory viruses were generally active in the early spring and winter , and the peak months for these agents were different from each other over a one - year period . early detection and monitoring of respiratory pathogens is essential in order to avoid the unnecessary use of antibiotics , to control the spread of infection , and to contribute to public health surveillance efforts , and the multiplex real - time pcr assay could be a suitable and effective method in this regard .
backgroundacute respiratory tract infection ( arti ) is one of the most common infections worldwide , causing significant morbidity and mortality.objectivesthis study was conducted to determine the prevalence and seasonal distribution of respiratory viruses in our region , in children and adults with a pre - diagnosis of arti.methodsa total of 845 nasopharyngeal swab specimens were analyzed with the respifinder smart 22 kit ( pathofinder bv , netherlands ) and the rotor - gene 6000 real - time pcr system.resultsat least one pathogen was detected in 612 ( 72.4% ) of the specimens . 
 overall , 902 pathogens were detected ; 821 ( 91% ) were viruses and 81 ( 9% ) were bacteria . 
 the most commonly detected pathogens were influenza a virus ( ifv - a ) ( n = 219 ) , influenza b virus ( ifv - b ) ( n=157 ) , rhinovirus / enterovirus ( n = 107 ) , human bocavirus ( hbov ) ( n = 91 ) , respiratory syncytial virus ( rsv ) a / b ( n = 64 ) , adenovirus ( n = 56 ) , human coronaviruses ( n = 51 ) , mycoplasma pneumoniae ( n = 49 ) , parainfluenza viruses ( n = 40 ) , human metapneumovirus ( n = 36 ) , bordetella pertussis ( n = 15 ) , legionella pneumophila ( n = 11 ) , and chlamydophila pneumoniae ( n = 6 ) , respectively . among the 215 ( 25.4% ) co - infected cases , ifv - a / hbov and ifv - a / ifv - b were the most common co - infections . 
 ifv - a was the most prevalent agent in all age groups except for children under 5 years of age , in whom rsv a / b was the most common pathogen . 
 approximately two thirds of the respiratory viruses were detected in early spring and winter , with peaks in january , march , and april.conclusionswith regard to the prevalence and seasonal distribution of respiratory viruses , our epidemiological data for the 2014 - 2015 season in istanbul showed a predominance of ifv - a infections with a peak activity in early spring . 
 enhanced surveillance and early detection of respiratory viral pathogens can be useful in the diagnosis , treatment , and prevention of artis , and for guiding the development of appropriate public health strategies .