Influenza B associated paediatric acute respiratory infection hospitalization in central vietnam

Abstract Background Influenza B is one of the major etiologies for acute respiratory infections (ARI) among children worldwide; however, its clinical‐epidemiological information is limited. We aimed to investigate the hospitalization incidence and clinical‐epidemiological characteristics of influenza B‐associated paediatric ARIs in central Vietnam. Methods We collected clinical‐epidemiological information and nasopharyngeal swabs from ARI children hospitalized at Khanh Hoa General Hospital, Nha Trang, Vietnam from February 2007 through June 2013. Nasopharyngeal samples were screened for 13 respiratory viruses using Multiplex‐PCRs. Influenza B‐confirmed cases were genotyped by Haemagglutinin gene sequencing. We analyzed the clinical‐epidemiological characteristics of influenza B Lineages (Victoria/Yamagata) and WHO Groups. Results In the pre‐A/H1N1pdm09 period, influenza B‐associated ARI hospitalization incidence among children under five was low, ranging between 14.7 and 80.7 per 100 000 population. The incidence increased to between 51.4 and 330 in the post‐A/H1N1pdm09. Influenza B ARI cases were slightly older with milder symptoms. Both Victoria and Yamagata lineages were detected before the A/H1N1pdm09 outbreak; however, Victoria lineage became predominant in 2010‐2013 (84% Victoria vs 16% Yamagata). Victoria and Yamagata lineages did not differ in demographic and clinical characteristics. In Victoria lineage, Group1 ARI cases were clinically more severe compared to Group5, presenting a greater proportion of wheeze, tachypnea, and lower respiratory tract infection. Conclusions The current results highlight the increased incidence of influenza B‐related ARI hospitalization among children in central Vietnam in the post‐A/H1N1pdm09 era. Furthermore, the difference in clinical severity between Victoria lineage Group1 and 5 implies the importance of influenza B genetic variation on clinical presentation.


| INTRODUC TI ON
Influenza viruses belong to the family Orthomyxoviridae, which possess a segmented negative-stranded RNA genome. 1,2 Among three influenza types, namely, type-A, B and C, influenza A and B often cause seasonal acute respiratory infections (ARI) epidemics and impose a high socioeconomic burden, particularly among children and the elderly population. 3,4 In temperate climate regions, peaks of influenza-associated ARIs may appear in early autumn through winter, 5 whereas year-round circulation with no apparent seasonal trend may be seen in tropical climate regions. [6][7][8][9] Influenza B differs from type-A in terms of genetic composition and its ability to infect only humans and seals. 10,11 Two surface glycoproteins, Haemagglutinin (HA) and Neuraminidase (NA), play pivotal roles during pathogenesis. 12 Unlike influenza A, influenza B does not possess multiple subtypes. Instead, influenza B obtains its genetic variation mainly through genetic drift, including nucleotide substitutions, insertions, and deletions, which explains the slower molecular evolutionary rate and smaller capacity to cause seasonal ARI outbreaks compared to influenza A. 13,14 Genetic reassortment of influenza B genetic components was previously documented 15 ; however, its frequency is not as high as that of influenza A due to its limited animal reservoir.
Despite the clinical importance of influenza B among ARI cases, majority of previous literature focused on influenza A. Therefore, clinical and molecular epidemiological information on influenza B is relatively limited worldwide.
The first influenza B strain B/Lee/40 was isolated in 1940. 16 In the 1970s, influenza B diverged into two genetically and antigenically distinct lineages, Victoria-like (B/Victoria/2/87) and Yamagata-like (B/Yamagata/16/88) lineages. 17,18 Since then, the two lineages have been co-circulating in many regions of the world. 19 Previous studies from Cambodia, China, India, and Taiwan have reported the increase of influenza B-associated ARI cases in the following seasons after the emergence of the pandemic A/ H1N1pdm09 strain. 4,17,18,20,21 Furthermore, some studies presented the circulation of Victoria lineage as a dominant type in the post-A/H1N1pdm09 period, 17,18,[20][21][22][23] whereas others showed cocirculation of both lineages. 3,4 Regardless of a few epidemiological studies on influenza B lineage-specific clinical presentation, 21,23 the clinical aspect of two genetically distinct lineages has not been clearly understood up to date.
In this study, we investigated the incidence and clinicalepidemiological characteristics of paediatric hospitalized influenza B ARI cases in Vietnam.

| Study site, period, and case enrollment
Population-based prospective paediatric ARI surveillance in Khanh Hoa province, Nha Trang, Vietnam was established in February 2007. 24 All children from 16 communes admitted to the paediatric ward of Khanh Hoa General Hospital (KHGH), which is the only hospital in the region, due to ARI symptoms (cough and/or difficulty breathing) from February 2007 to June 2013 were enrolled in the current study. Nha Trang city in south central region of Vietnam has a hot and dry season or tropical climate with a short rainy or wet season from September to December. The detailed characterization of the target population was described in the previous study. 24 Written informed consent from the parents or guardians of all the enrolled ARI children were obtained. Nasopharyngeal (NP) swab, clinical-epidemiological information, chest radiograph, and laboratory test data were collected from each enrollee. Currently, vaccination against influenza type A and B is not included in the nationwide immunization program in Vietnam. Its availability in our study site is limited and not commonly used among children.

| Clinical information categorization
Lower respiratory tract infection (LRTI) was defined based on the modified World Health Organization (WHO) Integrated Management of Childhood Illnesses (IMCI) algorithms. 25 The presence of tachypnea (respiratory rate >60/min for children ≦1 month, >50/min for 2-11 months and >40/min for 12-59 months of age) was categorized as mild LRTIs. Furthermore, children with a general danger sign (the situation in which children were either unable to drink, under convulsion, or lethargy), chest-wall indrawing, or stridor were categorized as severe LRTIs. Radiologically-confirmed pneumonia was defined as the presence of either substantial alveolar consolidation or pleural effusion in chest X-ray result following the standardized interpretation method established by WHO Vaccine Trial Investigators Group. 26 ARI cases with abnormal shadow but neither substantial alveolar consolidation nor pleural effusion were categorized into chest X-ray abnormality or other lower respiratory infections. 26    Adenovirus, and Bocavirus as previously described. 24 For the cur-

| Yearly incidences of paediatric ARI hospitalizations, LRTIs, and influenza (A/B) ARIs
confirmed that there was statistical evidence of an increase in influenza B-associated paediatric ARI hospitalization in "Post-A/

| Demographic and clinical characteristics comparison between influenza B and non-influenza B ARIs
With regard to the demographic and clinical characterization of overall paediatric ARI hospitalization cases enrolled in the current study (n = 4,429), 2,602 were male (58.8%), and the median age (in months) was 16.6 (IQR: 8.6-27.3). Other demographic information, including socioeconomic status, medical history, as well as detailed clinical information, was summarized in Table 2.
The demographic and clinical characteristics between influenza B (n = 133) and non-influenza B ARI groups (n = 4296) were compared ( Table 2). The male proportion was higher in the non-influenza B ARI group (50.4%, influenza B vs 59.0%, non-influenza B, P = 0.046).
Regarding the median age (in month), the influenza B ARI group was older (22.9, influenza B vs 16.5, non-influenza B, P < 0.001), and the greater proportion of ARI children were 3 years or older in the influenza B ARI group. Furthermore, family smoking was more frequent in the influenza B ARI group (P = 0.003).
With respect to the clinical information, the respiratory rate (per min) was faster in the non-influenza B ARI group (32.0, influenza B vs 33.0, non-influenza B, P = 0.012) ( Table 2). Furthermore, ARI children with wheeze (P = 0.005) and breathing difficulty (P = 0.022) were more common in the non-influenza B ARI group. The proportion of paediatric ARI hospitalizations with chest X-ray abnormal findings was also greater in the non-influenza B ARI group ( Furthermore, in the comparison between influenza A and B, influenza B-associated ARI hospitalizations were slightly older with different age distribution pattern (P = 0.002) (Table S2). Parental smoking was more common in influenza B ARI group (P = 0.011).
Regarding the clinical information, influenza A group was associated with higher body temperature (P = 0.003) as well as frequent presence of chest X-ray abnormality (P < 0.001).   (Table S3). On the contrary, the overall number of Yamagata lineage-related ARI hospitalizations was limited throughout the investigation period in both pre-and post-A/ H1N1pdm09 periods.

| Demographic and clinical characteristics comparison between Victoria and Yamagata lineages
In the demographic characteristics comparison between influenza B      Table S3.

| Demographic and clinical characteristics comparison among WHO Groups of Victoria and Yamagata lineages
Firstly, demographic information among WHO Groups (1, 4, and 5) of Victoria lineage was compared (Table 4). There were no significant differences in sex distribution and median age among the three groups. Daycare attendance was more common in Group 5 com-  (Table S4). With regard to demographic characterization, no statistically significant differences were detected between Groups 2 and 3. Both Group 2-and Group 3-associated ARI hospitalizations were commonly observed among children younger than 2 years. The results of a comparison in clinical manifestations did not reveal any significant differences.  Interestingly, we also observed unusual split of RSV seasonal peak in 2010 as we previously described. 37 Further studies would be necessary to understand the effect of A/H1N1pdm09 on respiratory virus circulation trend.

| D ISCUSS I ON
Among all the paediatric ARI hospitalization cases enrolled in the current study, 133 were influenza B positive ( Table 2). The median age in influenza B ARI group was older than non-influenza B ARIs, and influenza B group presented the higher proportion of family smoking, which is known to be one of the major risk factors for influenza morbidity and mortality. 38 We also have to take the lineage-specific sero-epidemiological information into account to gain a better understanding of influenza B lineage circulation pattern. A study from Taiwan suggested switching of dominantly circulating lineage may occur every 2-3 years. 18 In the future study, we will continue monitoring the lineage-specific circulation trend of influenza B strains in order to investigate the lineagespecific viral transmissibility based on the viral genetic variation.
Regarding the demographic information of influenza B lineages, median age was slightly older in Victoria lineage (Table 3), which contradicted the reports from China and Slovenia that presented Victoria lineage ARIs were younger than Yamagata lineage. 21,45 The difference in age of infection has been controversial due to the differences in inclusion criteria among studies. Furthermore, clinical aspect of influenza B lineages is poorly understood. In the current study, the clinical data did not present a significant difference between lineages (Table 3), which was in line with previous reports from China, France, Serbia, and Slovenia. 41,[45][46][47][48] Although the body temperature was slightly higher in Yamagata lineage ARIs, it was probably due to the difference in age distribution. In our study, the hospitalization duration did not differ between lineages, which con-

ACK N OWLED G EM ENTS
We would like to express our sincere gratitude to the medical doctors, nurses, and laboratory technicians at Khanh Hoa General Hospital and staffs in Khanh Hoa Health Service for their kind support on clinical sample collection and ARI clinical data management.
We would like to express our special appreciation for administrative staff and professors in Leading Program at Nagasaki University, Graduate School of Biomedical Sciences for their kind support during the research. This study was conducted in part at the Joint Usage/Research Center on Tropical Disease, Institute of Tropical Medicine, Nagasaki University, Japan.

CO N FLI C T O F I NTE R E S T
The authors have declared that no competing interests exist.