genotype with Increased Pediatric Acute Lower Respiratory Tract Infection in Vietnam
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KeisukeYoshihara,, Minh Nhat Le,, Michiko Okamoto, Anita Carolle AkpeedjeWadagni, Hien Anh Nguyen, MichikoToizumi,, Enga Pham, Motoi Suzuki, Ai Thi Thuy Nguyen, Hitoshi Oshitani, Koya Ariyoshi,, Hiroyuki Moriuchi,, Masahiro Hashizume,,Duc Anh Dang & Lay-MyintYoshida
prospective pediatric ARI surveillance at Khanh Hoa General Hospital, Central Vietnam from January
Human Respiratory Syncytial Virus (RSV) is a pneumovirus under the family of paramyxoviridae with a negative-sense single stranded RNA genome1. RSV is widely known as one of the most common respiratory viral pathogens for lower respiratory tract infection (LRTI) among infants and young children worldwide2.
RSV-related Acute Respiratory Infection (ARI) morbidity causes huge public health concerns particularly among children less than 5 with more than 60 million LRTI episodes annually in developing nations3. Previously, our population-based ARI surveillance in Central Vietnam reported that RSV was one of the main viral pathogens among hospitalized ARI children less than 2 years of age4. In fact, virtually all the children are exposed to RSV at some point before 2 years of age5. Furthermore, recurrence of RSV infections is common and particularly cause life-threatening LRTI in children less than 6 months6. Younger age, lower socioeconomic status, shorter gestational age, prematurity, low birthweight, lack of breastfeeding and family smoking have been previously reviewed as risk-factors for developing severe RSV-related LRTIs79.
Department of Virology, Tohoku Graduate School of Biomedical Sciences, Department Department
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Year (Jan
2010Dec
2012)
Incidence per 100,000
population (95% CI)
Total enrolled ARI cases (n=1854) n=542 3976.2 (3654.44317.9) n=513 3763.5 (3450.24096.7) n=799 5861.6 (5473.16269.2) Total LRTI cases (n=398) n=99 726.3 (590.7883.5) n=53 388.8 (291.4508.3) n=246 1804.7 (1587.92042.4) RSV positive ARI cases (n=426) n=135 990.4 (831.01171.2) n=80 586.9 (465.6729.9) n=211 1547.9 (1347.41769.5) RSV subgroup (A/B)
RSV-A single infection (n=253) n=63 462.2 (355.3591.0) n=32 234.8 (160.6331.3) n=158 1159.1 (986.31353.3) RSV-B single infection (n=77) n=38 278.8 (197.4382.4) n=25 183.4 (118.7270.6) n=14 102.7 (56.2172.3) RSV-A and B co-detection (n=16) n=3 22.0 (4.564.3) n=2 14.7 (1.853.0) n=11 80.7 (40.3144.4)
Table 1. Yearly incidence data for ARIs, LRTIs, RSV and RSV subgroups (A/B) in Nha Trang, Khanh Hoa province during January 2010December 2012.
RSV is classied into two antigenically and genetically distinct subgroups (A/B)10. Each subgroup is further categorized into genotypes based on the nucleotide sequence variation within the 2nd hypervariable region of heavily glycosylated G-glycoprotein (G-protein). There are 12 genotypes for RSV-A (GA1-7, SAA1, NA1-2 and ON1-2) and 20 genotypes for RSV-B (GB1-4, SAB1-4, URU1-2 and BA1-10)1113. Primary function of the G-protein is associated with viral attachment to the cell receptors and acts as an immunologic peptide that induces neutralizing antibody1416. The relevance of distinct RSV subgroups with clinical manifestations has been controversial. For instance, the studies from the U.S. and Argentina previously demonstrated that RSV-A was most likely associated with clinical severity17,18; however, a study from Brazil presented the opposite result19, while the other studies showed no signicant dierence2022. Furthermore, the studies from Canada and the U.S. implicated that RSV-A GA2 or RSV-B GB3 genotype may be linked to clinical severity20,23. To gain a better understanding of the association of RSV subgroup and genotype with clinical outcome, it is essential to further expand RSV molecular epidemiological surveillance worldwide.
In 2010, ON1 genotype, a new variant of RSV-A, was initially detected in Ontario, Canada by Eshaghi et al.24.
As a unique genetic characteristic, ON1 possesses 72-nucleotide tandem duplication within the G-protein 2nd hypervariable region. Since the rst discovery of ON1 in Canada24, it has been reported in countries around the globe11,2531, including South East Asian countries such as Philippines, Malaysia and Thailand3234. Furthermore, the reports from Cyprus, Germany, Italy, Kenya and Philippine indicated that the emergence of ON1 has rapidly replaced the previously predominant NA1 genotype2931,3436. Although the clinical aspects of ON1 genotype were investigated in the previous reports from Cyprus, Germany and Italy2931, the clinical relevance and pathogenicity of newly emerged ON1 genotype remain unclear.
There is still limited information regarding the molecular and clinical epidemiological characteristics of RSV particularly in South East Asian nations including Vietnam. Therefore in this study, we investigated the annual incidence of RSV-related pediatric ARI hospitalization, circulation dynamics of RSV subgroups (A/B) and geno-types, and the clinical signicance of RSV-A ON1 genotype among the hospitalized pediatric ARI cases in Central Vietnam.
Results
A total of 1854 hospitalized acute respiratory infection (ARI) cases were enrolled into our population-based pediatric ARI surveillance at Khanh Hoa General Hospital (KHGH) during the three years study period. Chest X-ray result were available for 1796 (96.9%) of enrolled ARI cases. Annual numbers of pediatric ARI cases enrolled were 542 in 2010, 513 in 2011 and 799 in 2012 (Table1). Based on the 2010 population census data, the pediatric ARI hospitalization incidence rates were 3976.2 cases per 100,000 children under 5 per year (95% CI: 3654.44317.9) in 2010, 3763.5 (95% CI: 3450.2 4096.7) in 2011 and 5861.6 (95% CI: 5473.16269.2) in 2012 respectively. Result of the respiratory virus screening by multiplex PCR assays revealed that viruses were detected in 66.2% of the enrolled ARI cases, in which RSV (n = 426, 23.0%), Inuenza (overall, 12.0%: type-A, 7.5% and type-B, 4.5%) and Rhinovirus (25.1%) were the leading respiratory viruses detected (data not shown).
RSV-related hospitalized ARI cases in Central Vietnam presented a clear seasonal circulation pattern with peaks in hot and dry seasons, which extended from July to September (Fig.1). The RSV-related ARI hospitalization incidence rates varied yearly: 990.4 (95% CI: 831.0 1171.2) in 2010, 586.9 (95% CI: 465.6729.9) in 2011 and 1547.9 (95% CI: 1347.41769.5) in 2012 season respectively. The highest RSV-related ARI hospitalization incidence recorded in 2012 corresponded to the highest LRTI incidence in the same season, accounting for 1804.7 (95% CI: 1587.92042.4) (Table1).
Demographic characteristics of the RSV ARI cases (n = 426) were compared with non-RSV ARI cases (n = 1428) (Table2). Both RSV and non-RSV ARI cases were more common among males (59.6%, RSV vs 56.0%, non-RSV, p = 0.179). RSV-positive ARI cases were signicantly younger (median age in month) (12, RSV vs 17, non-RSV, p <0.001) and more commonly seen in the rst year of life (51.2%, RSV vs 37.0%, non-RSV cases, p < 0.001). Daycare
January 2010December 2010
Number of
ARI cases
(n)
January 2011December 2011
Number of
ARI cases
(n)
January 2012December 2012
Number of
ARI cases
(n)
Overall cases
(n)
Incidence per 100,000
population (95% CI)
Incidence per 100,000
population (95% CI)
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Figure 1. Seasonal trend of RSV related pediatric ARI hospitalizations in Nha Trang, Khanh Hoa province during January 2010December 2012. Each box corresponds to the cumulative pediatric ARI cases in each month during the study period from January 2010 to December 2012. RED-lled boxes correspond to theRSV subgroup-A ARI cases, and BLUE-lled boxes are for the RSV subgroup-B ARI cases. RSV subgroup unclassied ARI cases were added on top of the BLUE-lled boxes as GREEN-lled boxes. Lower respiratory tract infection (LRTI) in each month was indicated as the BLUE solid line. *ARI is abbreviation for Acute
Respiratory Infection. *LRTI is abbreviation for lower respiratory tract infection.
attendance was less common among the RSV-positive ARI cases (32.2%, RSV vs 38.7%, non-RSV, p=0.015), and antibiotic usage prior to hospital admission was less frequent among the RSV-positive ARI cases (39.7% vs 44.3%, p = 0.042). Furthermore, the presence of underlying medical condition was commonly seen in non-RSV ARI cases (31.2%, RSV vs 37.9%, non-RSV, p=0.012).
In terms of the clinical presentation comparison, wheezing (52.8%, RSV vs 45.9%, non-RSV, p = 0.012), tachypnea (31.0%, RSV vs 21.1%, non-RSV, p < 0.001), crackle (21.6%, RSV vs 16.3%, non-RSV, p = 0.012) and chest-wall indrawing (9.9%, RSV vs 6.8%, non-RSV, p = 0.035) were signicantly more common among the RSV-positive ARI cases (Table2). Furthermore, the association of RSV-positive ARI cases with greater clinical severity remained signicant even aer controlling for the demographic confounding factors in the multivariate regression analysis (Table S1).
RSV subgrouping and genotyping were performed for all the RSV-positive conrmed ARI samples (n= 426). We were able to classify the subgroup (A/B) in 346 RSV-positive ARI cases: 253 RSV-A and 77 RSV-B respectively (Table1). We found that 16 samples were co-infected with both RSV subgroup A and B. Throughout the study period from January 2010 to December 2012, RSV-A was the major subgroup identied. RSV-A related ARI hospitalization incidences rates (per 100,000) were 462.2 (in 2010), 234.8 (in 2011) and 1159.1 (in 2012) respectively. The RSV-B ARI hospitalization rates were 278.8 (in 2010), 183.4 (in 2011) and 102.7 (in 2012) respectively. Notably, the total number and proportion of RSV-A related ARI hospitalization dramatically increased in 2012 season (Fig.1), which was statistically signi-cant compared to the previous two seasons (p<0.001) (Table3).
Genotyping was performed for all the RSV subgroup (A/B) conrmed ARI samples by sequencing and phylogenetic analysis of the G-protein 2nd hypervariable region (Figs S1 and 2). All the RSV-A conrmed ARI samples from both 2010 and 2011 seasons were NA1 genotype, while in 2012, RSV-A ON1 emerged and immediately became the predominant RSV-A genotype. On the other hand, the proportion of NA1 among the RSV subgroup A conrmed ARI samples decreased to 22%. Overall, RSV-A phylogenetic tree presented distinct genetic clustering of the ARI hospitalization cases in 20102011 seasons from 2012 season. RSV-A ARI cases from 20102011 seasons were bundled into NA1 whereas RSV-A from 2012 season formed distinct cluster within ON1 genotype (Fig. S1). In fact, NA1 genotype was further divided into genetically distinct clades (1 or 2). Eight RSV-A conrmed ARI samples were not able to be categorized into genotype (Table3).
With respect to the RSV-B genotype circulation pattern, the phylogenetic tree revealed that BA9, BA10 and BA-C had been circulating during the three years study period (Fig. S2). Overall, there was no noticeable geno-type shi in RSV subgroup B during the study period (Table3).
To investigate the clinical impact of RSV-A ON1 genotype, we compared the demographic and clinical characteristics of RSV-A ON1 with NA1 genotype (Table4). A total of 123 RSV-A ON1 and 138 NA1 genotype conrmed ARI cases were included in the statistical analysis.
The overall demographic characteristics were similar between ON1 and NA1 ARI cases. ON1 ARI cases was younger (median age in month) (11, ON1 vs 13, NA1, p= 0.020) with slightly more NA1 ARI cases in the older age groups (Table4). Daycare attendance was signicantly higher among the NA1 ARI cases (24.4%, ON1 vs
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Total pediatric ARI hospitalization cases during Jan 2010Dec 2012 (n= 1854) RSV positive ARI group (n=426) Non-RSV ARI group (n=1428)p-value#
Demographic information
Male sex (%) 254 (59.6%) 799 (56.0%) 0.179 Median age (in month) 12 (IQR: 521) 17 (IQR: 929) <0.001***
Age group (%)
012month 218 (51.2%) 528 (37.0%) <0.001***
1324month 131 (30.8%) 442 (31.0%)
2536month 52 (12.2%) 187 (13.1%)
3748month 17 (4.0%) 94 (6.6%)
4960month 2 (0.5%) (4.3%)
>60month 6 (1.4%) 116 (8.1%)
Socioeconomic status
Daycare attendance (%) 137 (32.2%) 552 (38.7%) 0.015* History
Antibiotic used prior to hospitalization (%) 169 (39.7%) 633 (44.3%) 0.042* Underlying medical condition (%) 133 (31.2%) 541 (37.9%) 0.012* Clinical information Total number (%)/Mean (95% CI) Total number (%)/Mean (95% CI)
Vital sign (s)
Mean respiratory rate (per min) 37.2 (95% CI: 36.338.1) 35.7 (95% CI: 35.236.1) 0.003** Mean body temperature (C.) 37.9 (95% CI: 37.838.0) 37.8 (95% CI: 37.837.9) 0.045* SpO2 (90%) 12 (2.8%) 40 (2.8%) 0.986 Respiratory symptom and sign (s)
Wheeze (%) 225 (52.8%) 655 (45.9%) 0.012* Tachypnea (%) 132 (31.0%) 301 (21.1%) <0.001*** Difficulty breathing (%) 51 (12.0%) 157 (11.0%) 0.575 Crackle (%) 92 (21.6%) 233 (16.3%) 0.012*
Cough (%) 426 (100%) 1423 (99.7%) 0.221 Chest-wall indrawing (%) 42 (9.9%) 97 (6.8%) 0.035*
LRTI and chest X-ray result
LRTI (%) 92 (21.6%) 307 (21.5%) 0.966
Mild LRTI (%) 43 (10.1%) 165 (11.6%) 0.402 Severe LRTI (%) 49 (11.5%) 142 (9.9%) 0.353 Abnormal chest X-ray (%) 62 (14.6%) 210 (14.7%) 0.856 Radiologically-conrmed pneumonia (%) 55 (12.9%) 182 (12.8%) 0.991 Treatment and outcome (s)
Mean onset to hospitalization (in day) 2.4 (95% CI: 2.22.5) 2.4 (95% CI: 2.22.5) 0.831 Mean hospitalization duration (in day) 5.2 (95% CI: 4.95.4) 4.9 (95% CI: 4.75.1) 0.132 Antibiotic used (%) 426 (100%) 1428 (100%) 1.000 Steroid used (%) 213 (50.0%) 694 (48.6%) 0.612
Table 2. Demographic and clinical characteristics comparison between RSV and Non-RSV pediatric ARI cases. #All the statistically signicant p-values were indicated in bold font. As the index for the statistically signicant values: * were used for p-value<0.05, ** were for p-value<0.01 and *** were for p-value0.001.
IQR is abbreviation for Interquartile Range (1st and 3rd), and 95% CI is abbreviation for 95% Condence Interval. LRTI is abbreviation for lower respiratory tract infection and based on the WHO denition of clinical pneumonia45. Severe lower respiratory tract infection (LRTI) was dened as presence of either danger sign, stridor or chest-wall indrawing.
39.1%, NA1, p= 0.011). Furthermore, the ON1 ARI cases were more frequently associated with underlying medical condition (43.1%, ON1 vs 21.7%, NA1, p<0.001).
Regarding the respiratory clinical signs and symptoms, occurrence of respiratory sign and symptoms such as wheeze (81.3%, ON1 vs 33.3%, NA1, p< 0.001), tachypnea (50.4%, ON1 vs 25.4%, NA1, p< 0.001) and difficulty in breathing (18.7%, ON1 vs 6.5%, NA1, p = 0.004) were signicantly more common among ON1 ARI cases (Table4). In addition, ARI cases with LRTI (34.2%, ON1 vs 15.2%, NA1, p< 0.001) and radiologically-conrmed pneumonia (19.5%, ON1 vs 8.7%, NA1, p= 0.011) were more commonly seen in ON1 ARI cases. Furthermore, ON1 ARI cases tended to be admitted to the hospital signicantly earlier, since they had a shorter mean period from disease onset to the hospital admission (in day) (1.7, ON1 vs 2.7, NA1, p< 0.001). On the other hand, mean duration of hospitalization (in day) between ON1 and NA1 ARI cases did not dier signicantly (5.3, ON1 vs 5.1, NA1, p=0.329).
Total number (%)/Median (IQR) Total number (%)/Median (IQR)
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Pediatric ARI cases admitted to KHGH# (Jan 2010Dec 2012)
Year of sample collection 2010 (JanDec) 2011 (JanDec) 2012 (JanDec)
Total ARI cases (n=1854) (n=542) (n=513) (n=799) RSV positive ARI cases (n=426) n=135 (24.9%) n=80 (15.6%) n=211 (26.4%)
RSV subgroup A 63 (46.7%) 32 (40.0%) 158 (74.9%)
RSV subgroup B 38 (28.2%) 25 (31.3%) 14 (6.6%) RSV-A and B mixed-infection 3 (2.2%) 2 (2.5%) 11 (5.2%) p-value p=0.711 p<0.001
(Not classied) 31 (23.0%) 21 (26.3%) 28 (13.3%)
RSV-A genotype (n=269) (n=66) (n=34) (n=169)
NA1 genotype 66 (100%) 34 (100%) 38 (22.5%)
ON1 genotype 0 0 123 (72.8%) (Not classied) 0 0 8 (4.7%) RSV-B genotype (n=93) (n=41) (n=27) (n=25)
BA9 genotype 31 (75.6%) 17 (63.0%) 9 (36.0%)
BA10 genotype 6 (14.6%) 6 (22.2%) 0BA-C genotype 1 (2.4%) 3 (11.1%) 8 (32.0%) (Not classied) 3 (7.3%) 1 (3.7%) 8 (32.0%) Total RSV-A&B genotype conrmed n= 107 (in 2010) n= 61 (in 2011) n= 194 (in 2012)
Table 3. Yearly prevalence of RSV subgroups (A/B) and genotypes in Nha Trang, Khanh Hoa province during January 2010December 2012. #KHGH is abbreviation for Khanh Hoa General Hospital in Nha Trang city, Khanh Hoa province. For the statistical tests for RSV subgroups (A/B) proportion comparison among sampling years, two-tailed Fishers exact tests were performed. Statistically signicant p-value was indicatedin bold font. p-value was for RSV subgroups (A/B) proportion comparison between year of 2010 and 2011.
p-value was for RSV subgroups (A/B) proportion comparison among 2010, 2011 and 2012.
The respiratory clinical signs and symptoms that presented signicant dierence in proportion between ON1 and NA1 ARI cases in Table4 were further proceeded to the multivariate regression analysis. Multivariate analysis with log-binominal regression was performed to estimate the relative risks (RR) (Table5). Sex, age, antibiotic-use, daycare attendance, viral co-infection and underlying medical condition were adjusted for estimating adjusted Relative Risk (Adj RR).
Result of the multivariate regression analysis illustrated the signicant association of ON1 ARI cases with greater clinical severity even aer controlling for the demographic confounding factors. For instance, ON1 ARI cases had greater risk of wheezing (Adj RR: 2.21 (95% CI: 1.722.86)), tachypnea (Adj RR: 1.83 (95% CI: 1.302.57)) and difficulty in breathing (Adj RR: 2.46 (95% CI: 1.125.39)). Furthermore, RSV-A ON1 had signicantly greater risk for severe LRTI (Adj RR: 2.42 (95% CI: 1.125.25)) and radiologically-conrmed pneumonia (Adj RR: 1.97 (95% CI: 1.043.74)). On the other hand, relative risk for SpO2 (90%) and mild LRTI became no longer signicant in the multivariate analysis: SpO2 (90%) (Adj RR: 3.18 (95% CI: 0.7114.29)) and mild LRTI (Adj RR: 1.94 (95% CI: 0.904.19)) respectively.
Discussion
Our study illustrated that RSV played a major clinical role among pediatric ARI cases in Central Vietnam. The result illustrated that RSV-related pediatric ARI hospitalization incidences were high during hot and dry season (July through September), timing of which was similar to previous ndings from Cambodia, Thailand, Vietnam4,37,38. However correlation between climatic parameters (such as temperature and relative humidity) and RSV incidences is not clearly understood in tropical climate regions like Vietnam39. Future studies are required to clarify RSV seasonality in tropical countries.
The majority of RSV-related ARI cases were detected among children less than 2 years of age (Table2), which was consistent with the previous nding5. According to the RSV subgroup specic circulation dynamics in our study site, RSV-A and B had been co-circulating during the three years study period from January 2010 to December 2012. RSV subgroup A was predominant throughout the study period, which was similar to previous studies in other South East Asian Countries3234. In 2012 season, the RSV-A related ARI hospitalization incidence increased remarkably (1159.1 cases per 100,000) compared to the previous two seasons, which corresponded to the highest LRTI incidence recorded in the same season (1804.7 cases per 100,000) (Table1).
Since the initial discovery of RSV-A ON1 genotype in Ontario, Canada in 201024, ON1 have been reported in numbers of European and Asian nations primarily during 20102012 season11,2536,40. This study is the rst report regarding the emergence of ON1 in Vietnam. In our study site, NA1 was circulating as the only RSV-A genotype in 2010 and 2011 seasons (Table3). However, ON1 emerged in mid-2012 and became the predominant genotype in 2012 season. The prevalence of ON1 among RSV-A reached nearly 73% in 2012 season whereas proportion of NA1 decreased down to about 23% from 100% in both 2010 and 2011 season (Table3). ON1 genotype emerged and was detected as major genotype during 20112012 seasons in countries such as Cyprus, Italy, Germany and Philippine2931,3436. However, further studies are required to monitor whether ON1 persisted as the predominant genotype aer its emergence in these countries. On the other hand, studies from Canada, China, Thailand
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RSV subgroup A positive pediatric ARI cases during Jan 2010Dec 2012 (n= 269) RSV-A ON1 ARI group (n=123) RSV-A NA1 ARI group (n=138)p-value#
Demographic information
Male sex (%) 76 (61.8%) 79 (57.3%) 0.456 Median age (in month) 11 (IQR: 318) 13 (IQR: 623) 0.020*
Age group (%)
012month 68 (55.3%) 68 (49.3%) 0.198 1324month 41 (33.3%) 40 (29.0%)2536month 12 (9.8%) 22 (15.9%)3748month 1 (0.8%) 6 (4.4%)4960month 0 0> 60month 1 (0.8%) 2 (1.5%)
Socioeconomic status
Daycare attendance (%) 30 (24.4%) 54 (39.1%) 0.011* History
Antibiotic used prior to hospitalization (%) 55 (44.7%) 53 (38.4%) 0.832 Underlying medical condition (%) 53 (43.1%) 30 (21.7%) < 0.001***
Respiratory virus co-infection (%) 27 (22.0%) 34 (24.6%) 0.609 Clinical information Total number (%)/Mean (95% CI) Total number (%)/Mean (95% CI)
Vital sign (s)
Mean respiratory rate (per min) 38.8 (95% CI: 36.940.8) 35.9 (95% CI: 34.437.5) 0.021* Mean body temperature (C.) 38.0 (95% CI: 37.938.2) 37.9 (95% CI: 37.838.0) 0.261 SpO2 (90%) 9 (7.3%) 2 (1.5%) 0.028*
Respiratory symptom and sign (s)
Wheeze (%) 100 (81.3%) 46 (33.3%) <0.001*** Tachypnea (%) 62 (50.4%) 35 (25.4%) <0.001*** Difficulty breathing (%) 23 (18.7%) 9 (6.5%) 0.004** Crackle (%) 46 (37.4%) 25 (18.1%) <0.001*** Cough (%) 123 (100%) 138 (100%) 1.000 Chest-wall indrawing (%) 19 (15.5%) 12 (8.7%) 0.092 LRTI and chest X-ray result
LRTI(%) 42 (34.2%) 21 (15.2%) <0.001***
Mild LRTI (%) 19 (15.5%) 9 (6.5%) 0.027*
Severe LRTI(%) 23 (18.7%) 12 (8.7%) 0.028* Abnormal chest X-ray (%) 28 (22.8%) 13 (9.4%) 0.003** Radiologically-conrmed pneumonia (%) 24 (19.5%) 12 (8.7%) 0.011* Treatment and outcome (s)
Mean onset to hospitalization (in day) 1.7 (95% CI: 1.51.9) 2.7 (95% CI: 2.33.0) <0.001*** Mean hospitalization duration (in day) 5.3 (95% CI: 4.95.8) 5.1 (95% CI: 4.75.4) 0.329 Antibiotic used (%) 123 (100%) 138 (100%) 1.000 Steroid used (%) 69 (56.1%) 64 (46.4%) 0.117
Table 4. Demographic and clinical characteristics comparison between RSV-A ON1 and NA1 genotype pediatric ARI cases. #All the statistically signicant p-values were indicated in bold font. As the index forthe statistically signicant values: * were used for p-value<0.05, ** were for p-value<0.01 and *** were for p-value0.001. IQR is abbreviation for Interquartile Range (1st and 3rd), and 95% CI is abbreviation for 95% Condence Interval. LRTI is abbreviation for lower respiratory tract infection and based on the WHO denition of clinical pneumonia45. Severe lower respiratory tract infection (LRTI) was dened as presence of either danger sign, stridor or chest-wall indrawing.
and Malaysia reported its emergence but did not nd ON1 genotype as the most prevalent type in their rep ort11,24,32,33,40.
Regardless of the numbers of molecular epidemiological surveillances on RSV describing the emergence of ON1 genotype during the last couple of years, the clinical and pathological signicance of RSV-A ON1 and its 72-nucleotide tandem duplication within the G-protein 2nd hypervariable region has not been clearly understood. Since our current pediatric ARI surveillance possessed a relatively large sample size in both RSV-A genotype ON1 (n = 123) and NA1 (n = 138) ARI cases, we were able to evaluated the demographic and clinical characteristics of ON1 ARI cases, in comparison with NA1 ARI cases (Table4).
Total number (%)/Median (IQR) Total number (%)/Median (IQR)
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Clinical manifestation (s)
Vital sign
SpO2 (90%) RSV-A ON1 ARI
group (n=123) 5.05 1.1122.92 3.18 0.7114.29 RSV-A NA1 ARI
group (n=138)# (ref.)
Clinical symptom and sign (s)
Wheeze 2.44 1.903.13 2.21 1.722.86 Tachypnea 1.99 1.422.78 1.83 1.302.57 Difficulty breathing 2.87 1.385.96 2.46 1.125.39 Crackle 2.06 1.353.15 1.96 1.283.00 Chest-wall indrawing 1.78 0.903.51 1.95 0.884.31 LRTI and chest X-ray result
LRTI 2.24 1.413.57 2.26 1.373.72
Mild LRTI 2.37 1.115.04 1.94 0.904.19 Severe LRTI 2.15 1.124.14 2.42 1.125.25 Abnormal chest X-ray 2.42 1.324.46 2.14 1.134.04 Radiologically-conrmed pneumonia 2.25 1.184.30 1.98 1.013.87
Table 5. Multivariate log-binomial regression analysis of clinical severity comparison between RSV-A ON1 and NA1 genotype ARI cases. #In the log-binomial regression analysis, RSV-A NA1 ARI group (n=138) was used as the reference group. 95% CI is abbreviation for 95% Condence Interval. In the multivariate log-binomial regression, variables including sex, age, antibiotic use prior to hospitalization, daycare attendance, viral co-infection and underlying medical condition were adjusted for estimating adjusted Relative Risk (Adj RR) and 95% Condence Interval (CI). LRTI is abbreviation for lower respiratory tract infection and based on the WHO denition of clinical pneumonia45. Severe lower respiratory tract infection (LRTI) was dened as presence of either danger sign, stridor or chest-wall indrawing. All the statistically signicant values were indicated in bold font.
With respect to the demographic characteristics, ON1 ARI cases were seen in slightly younger (median age in month) (11, ON1 vs 13, NA1, p= 0.020), which may explain the lower prevalence of daycare attendance in the ON1 ARI cases (24.4%, ON1 vs 39.1%, NA1, p = 0.011). One study from Italy, Pierangeli et al. also found that ON1 cases were seen in slightly younger age group30, while other studies did not nd signicant dierences31,36. The discrepancies in age distribution may have occurred due to dierences in study design, method for case enrollment criteria, herd-immunity against RSV (subgroups and genotypes) and circulating RSV genotypes prior to ON1 emergence in each study.
With respect to the clinical characteristics, the multivariate analysis using log-binomial regression revealed that ON1 ARI cases were associated with increased risk of respiratory clinical signs/symptoms and severity compared to NA1 ARI cases. For instance, risk of wheezing was 2.21 (95% CI: 1.722.86) times, LRTI was 2.26 (95% CI: 1.373.72) times, and chest X-ray abnormality was 2.14 (95% CI: 1.134.04) times greater among ON1 ARI cases compared to NA1 ARI cases (Table5). Furthermore, signicantly shorter mean period from disease onset to the hospital admission (in day) was seen in ON1 ARI cases (1.7, ON1 vs 2.7, NA1, p < 0.001). Although the detailed biological mechanism has not been clearly understood, the G-protein 72-nucleotide tandem duplication of ON1 might have crucial biological role by enhancing the efficiency for viral attachment to the cell receptors or faster viral replication capacity during pathogenesis. Further studies are necessary to clarify the biological significance of the 72-nucleotide insertion in the G-protein.
In contrast to the our major nding of ON1s association with clinical severity, other RSV surveillances from Cyprus, Germany and Italy did not nd any remarkable clinical impact of ON1 genotype29,31,36,41. The clinical
impact of a newly emerged virus may depend on the herd-immunity in the community, pre-circulating viruses and genotypes in respective study area. Recently, two studies have described the molecular evolutionary characteristics of globally circulating RSV-A NA1 and ON1 genotype13,42. Further genetic and antigenic analysis on the Central Vietnam RSV-A genotypes will give us insight into possible underlying mechanisms of the association between RSV ON1 and clinical severity.
As considerable limitations in the current study, we were not able to categorize RSV subgroups (A/B) or geno-types in about 20% of RSV conrmed ARI samples (Table3). This may have been due to the fact that we used the RNA extracted directly from NP samples , which may have contained low viral copies. Previous study described that higher RSV viral load was associated with clinical severity21; however, RSV viral load data was not available in the current study. Furthermore, we did not take into account the co-infection with respiratory bacterial pathogens. In fact, it has been previously reported that RSV may increase the Streptococcus pneumoniae bacterial load which was associated with increased risk of radiologically-conrmed pneumonia43,44. It will be important to further investigate the underlying biological mechanism, interaction with nasopharyngeal bacteria that may leads to the clinical severity of RSV-A ON1.
RSV (subgroup/
genotype) Unadjusted RR 95% CI Adjusted RR 95% CI
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Conclusion
In conclusion, our current study highlights the clinical importance of RSV among the pediatric ARI cases in Central Vietnam. The emergence of RSV-A ON1 was associated with increased ARI hospitalization incidence. Furthermore, the ON1 ARI cases were associated with greater risk of LRTI, radiologically-conrmed pneumonia compared to the previously predominant NA1 genotype. Further molecular and clinical epidemiological studies on RSV-A ON1 genotype circulating across the world would be important for better understanding of RSV-A ON1s clinical signicance which may have impact on future vaccine development.
Materials and Methods
A population-based prospective pediatric ARI surveillance was established at Khanh Hoa province, Nha Trang, Central Vietnam in 2007. Khanh Hoa General Hospital (KHGH) is the provincial hospital and the only hospital in Nha Trang city. In Vietnam, all children less than 6 years of age are covered by a free government health insurance. Transportation system in Nha Trang city is considerably good as children living in the study area can reach KHGH within one hour. Therefore, access to medical care is relatively good in the study area. All children from the catchment area admitted to KHGH presenting with cough and/or difficulty breathing were recorded as ARI cases and enrolled in the current study. Written informed consents were obtained from the parents or guardians of the pediatric ARI cases to enroll in the study. Clinical-epidemiological information, chest radiographs result (Chest X-ray), laboratory test data and nasopharyngeal (NP) swab samples were collected from all the participants. The catchment area covered 198,729 individuals living in 42,770 households from 16 communities with 13,631 children less than 5 years of age. Detailed methods and characteristics of the study population have been described previously4.
This study was approved by the institutional ethical review boards of National Institute of Hygiene and Epidemiology (NIHE), Vietnam, and Institute of Tropical Medicine, Nagasaki University, Japan. The study was conducted in accordance with the approved guidelines.
Pediatric ARI cases enrolled to the ARI surveillance in Nha Trang, Central Vietnam during the period of January 2010December 2012 were selected and utilized for this study.
Clinical categories were dened using modied World Health Organization (WHO) Integrated Management of Childhood Illnesses (IMCI) algorithms45. The presence of tachypnea (Respiratory Rate >60/min for children 1month, >50/min for 211 months and >40/min for 1259 months) were categorized as mild LRTIs. Furthermore, children with general danger signs (situation in which children were either unable to drink, under convulsion or lethargy), chest-wall indrawing or stridor were categorized as severe LRTIs. Radiologically-conrmed pneumonia was dened as substantial alveolar consolidation or pleural eusion in chest X-ray result following the standardized interpretation method established by WHO Vaccine Trial Investigators Group46. Cases with abnormal shadow but not substantial alveolar consolidation or pleural eusion were considered as abnormal chest X-ray or other lower respiratory infection46.
Viral nucleic acids were extracted from patients NP swab samples using QIA viral RNA Minikit (QIAGEN Inc., Valencia, CA) following the manufacturers manual. Four Multiplex -PCR assays were performed for screening 13 respiratory viral pathogens including RSV, Inuenza-A and B, Human Metapneumovirus, Parainuenzavirus 14, Human Coronaviruses (229E, OC43), Adenovirus and Bocavirus. The detailed protocol of respiratory viruses screening was previously described4.
RSV-positive conrmed samples were further screened for subgroup (A/B) and genotype by amplifying and sequencing the 2nd hypervariable region of G-protein as previously described1,47. BigDye Terminator ver.3.1 (Applied Biosystem, Foster City, CA, USA) was utilized for the sequencing reaction, and nucleotide sequence analysis was performed with 3730 DNA Analyzer (Applied Biosystem, Foster City, CA, USA). Multiple nucleotide sequences were aligned and edited with ClustalW ver.1.8. Phylogenetic analysis was executed using the Neighbor-Joining method with bootstrap value of 1000 replicates for testing statistical signicance of the tree topology using MEGA ver.5.2.2.
For the categorical variables, either two-tailed Pearson Chi-squared or Fishers exact tests were performed to test the statistical dierence in proportion between two independent groups. For the numerical variables, two-sample t-test was performed for mean value comparison, and Mann-Whitney U test was used for median comparison. In the multivariate analysis, Generalized Linear Model with log-binomial regression was applied to estimate adjusted Relative Risk (Adj RR) and 95% Condence Interval (CI). To control demographic confounding variables in the multivariate regression analysis, both forward-selection step and biologically plausible approaches were taken into account. All the statistical analysis was performed using STATA ver.12.1 (StataCorp LP, College Station, TX, USA). P-values less than 0.05 were considered to be statistically signicant.
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Acknowledgements
We would like to thank for the medical doctors, nurses and laboratory technicians at Khanh Hoa General Hospital for their technical support on clinical data and sample collection, processing and management. We would also like to acknowledge the stas in Khanh Hoa Health Service for clinical-epidemiological data entry and necessary administrative support for the study. This study was supported by the Japan Initiative for Global Research Network on Infectious Diseases (JGRID) (10008012), Japan Agency for Medical Research and development (AMED) and Grants-in-Aid for Scientic Research (Japan Society for the Promotion of Science).
K.A., D.A.D. and L.M.Y. designed the study. H.A.N., M.T., E.P. and A.T.T.N. performed data collection and management. K.Y., M.N.L., A.C.A.W. and H.A.N. designed and performed the sample testing. K.Y., M.N.L., M.O., A.C.A.W., H.O. and L.M.Y. analyzed and interpreted the data. K.Y., M.S., H.M., M.H. and L.M.Y. wrote and edited the manuscript. All authors reviewed the manuscript and accepted the nal version of manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: The authors declare no competing nancial interests.
How to cite this article: Yoshihara, K. et al. Association of RSV-A ON1 genotype with Increased Pediatric Acute Lower Respiratory Tract Infection in Vietnam. Sci. Rep. 6, 27856; doi: 10.1038/srep27856 (2016).
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Copyright Nature Publishing Group Jun 2016
Abstract
Since the initial discovery of RSV-A ON1 in Canada in 2010, ON1 has been reported worldwide, yet information regarding its clinical impact and severity has been controversial. To investigate the clinical relevance of RSV-A ON1,acute respiratory infection (ARI) cases enrolled to our population-based prospective pediatric ARI surveillance at Khanh Hoa General Hospital, Central Vietnam from January 2010 through December 2012 were studied. Clinical-epidemiological information and nasopharyngeal samples were collected. Multiplex PCR assays were performed for screening 13 respiratory viruses. RSV-positive samples were further tested for subgroups (A/B) and genotypes information by sequencing the G-glycoprotein 2nd hypervariable region. Statistical analysis was performed to evaluate the clinical-epidemiological characteristics of RSV-A ON1. A total of 1854 ARI cases were enrolled and 426 (23.0%) of them were RSV-positive. During the study period, RSV-A and B had been co-circulating. NA1 was the predominant RSV-A genotype until the appearance of ON1 in 2012. RSV-related ARI hospitalization incidence significantly increased after the emergence of ON1. Moreover, multivariate analysis revealed that risk of lower respiratory tract infection was 2.26 (95% CI: 1.37-3.72) times, and radiologically-confirmed pneumonia was 1.98 (95% CI: 1.01-3.87) times greater in ON1 compared to NA1 cases. Our result suggested that ON1 ARI cases were clinically more severe than NA1.
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