key: cord-0961700-a4ograau authors: Snoeck, Chantal J.; Evdokimov, Konstantin; Xaydalasouk, Kinnaly; Mongkhoune, Sodaly; Sausy, Aurélie; Vilivong, Keoudomphone; Pauly, Maude; Hübschen, Judith M.; Billamay, Somxay; Muller, Claude P.; Black, Antony P. title: Epidemiology of acute respiratory viral infections in children in Vientiane, Lao People's Democratic Republic date: 2021-05-12 journal: J Med Virol DOI: 10.1002/jmv.27004 sha: 9cc0e83ff8a74604cdc48260d9b90437d7262bff doc_id: 961700 cord_uid: a4ograau Respiratory infections are one of the most frequent reasons for medical consultations in children. In low resource settings such as in Lao People's Democratic Republic, knowledge gaps and the dearth of laboratory capacity to support differential diagnosis may contribute to antibiotic overuse. We studied the etiology, temporal trends, and genetic diversity of viral respiratory infections in children to provide evidence for prevention and treatment guidelines. From September 2014 to October 2015, throat swabs and nasopharyngeal aspirates from 445 children under 10 years old with symptoms of acute respiratory infection were collected at the Children Hospital in Vientiane. Rapid antigen tests were performed for influenza A and B and respiratory syncytial virus. Real‐time reverse‐transcription polymerase chain reactions (RT‐PCRs) were performed to detect 16 viruses. Influenza infections were detected with a higher sensitivity using PCR than with the rapid antigen test. By RT‐PCR screening, at least one pathogen could be identified for 71.7% of cases. Human rhinoviruses were most frequently detected (29.9%), followed by influenza A and B viruses combined (15.9%). We identify and discuss the seasonality of some of the infections. Altogether these data provide a detailed characterization of respiratory pathogens in Lao children and we provide recommendations for vaccination and further studies. In children, respiratory tract infections are one of the most common reasons for seeking medical care. 1 In Lao People's Democratic Republic (PDR), consultations for influenza-like illness (ILI) represented 10% of outpatient consultations in Vientiane between 2008 and 2010. 2 The incidence of ILI was estimated at 10.7 episodes per 100 person years in the Vientiane metropolitan area between 2015 and 2016, with the highest rates in children below 15 years old. 3 The etiology of respiratory tract infections is very diverse. A high proportion of viral infections present with mild symptoms, 4,5 which may worsen due to bacteria superinfections. 6 Measures to mitigate bacteria coinfections or gaps in differential diagnosis often lead to empirical antibiotic treatment. Antibiotic prescription levels in primary care settings in low and middle-income countries are often high, 7 and are influenced by staff training level. However, innate and adaptive immune responses to pathogenic microorganisms are influenced by commensal bacteria, which are present in site-specific communities on mucosal surfaces. 8 Resident non-pathogenic bacteria of the airways augment the immunity against viral infections. 9 Therefore, empirical antibiotic treatment of respiratory infections without a laboratory confirmation may disrupt the balance necessary for effective virus clearance. In addition, unnecessary antibiotic administration increases the risk of emergence and spread of antibiotic resistance. In Lao PDR, knowledge gaps in respiratory tract infection diagnosis and management exist. In a previous study, nearly 30% of the medical doctors questioned thought that unnecessary antibiotic treatment was harmless. 10 In addition, weak laboratory capacity to substantiate differential diagnosis can lead to unnecessary antibiotic prescription, often driven by patient demand or antibiotic availability rather than necessity. 10 A surge of patients with ILI was reported between May and July 2008 in Vientiane but this did not coincide with increased transmission of influenza cases. 11 Previous studies in the country were confined to a limited number of pathogens, mainly influenza viruses, with almost half of all patients left without etiological diagnosis. 2, 11, 12 This, combined with the age-specific pathogen susceptibility, warrants a broadening of respiratory surveillance programs. This study therefore aimed at investigating the etiology of respiratory infections in children, a population with higher susceptibility and disease severity. The study aimed at including a larger panel of viral agents than previous studies, to provide better evidence on disease etiology to local medical doctors. It aimed at investigating seasonal trends of virus circulation and to genetically characterize influenza A virus, respiratory syncytial virus, and human metapneumovirus, often responsible for more severe disease outcomes in young children. The study was approved by the Lao National Ethics Committee for Health Research (No. 027/NIOPH/NECHR). Clinical samples and data were collected after obtaining informed consent from the patient's parents or legal guardians. The inclusion criteria were: age below 10-year old, presenting to the Children Hospital in Vientiane with symptoms of acute respiratory infection; informed consent obtained. Throat swabs and nasopharyngeal aspirates were collected by trained hospital personnel. A questionnaire capturing demographic and clinical data was administered by health care personnel. Complete glycoprotein (G) sequences of RSV strains and partial fusion (F) and glycoprotein (G) sequences of hMPV were amplified using previously published 16, 17 and newly designed primer pairs. PCR product purification and sequencing were performed as described before. 18 The nucleotide substitution model that best fitted the data was determined with MEGA6. 19 Genetic distances and phylogenetic analyses were then calculated with the best model using the Neighbor-joining method and 500 bootstrap replicates in MEGA6. McNemar's test with Yates correction for continuity was used for comparing detection rates between swab types or between RT-PCR and rapid tests. Mann-Whitney rank-sum tests were used to assess the effect of age on overall positivity rate (≥1 virus detected), on the detection of single or mixed infections, on the positivity rate for each virus, on antibiotic prescription, and to assess the effect of delay between onset of symptoms and sampling on the outcome of rapid tests. A one-tailed t test was applied to assess the effect of influenza C t values on rapid test outcomes. Odds ratios were calculated to describe the risk associated with positivity for each viral species and hospital admission. Odds ratios were also calculated to evaluate antibiotic prescription with regard to in-or outpatient treatment. Based on the sample collection date, cases were classified as occurring during the rainy or dry seasons. In Lao PDR, the rainy season, typically taking place between mid-October and mid-May of the following year, is marked by frequent and heavy rain with high humidity. Rainy seasons alternate with dry seasons characterized by low humidity and high temperatures that occurs from mid-May to mid-October. 20 Z-scores were calculated to assess the seasonality of virus occurrence in the dry or rainy season. From September 2014 to August 2015, 445 children (median age 18.6 months; Table 1 ) were recruited at the Children's Hospital in Vientiane. Patients presented with fever (n = 390) and/or cough (n = 418) and/or nasal congestion (n = 403; Table 1 ). The majority (80.9%) were outpatients while 19.1% (85 of 445) were admitted to the hospital. Throat swabs and nasopharyngeal aspirates were collected from all patients (n = 445). Overall, at least one virus was detected by RT-PCR in 71.7% (319 of 445; Table 1 ) of the patients when combining results from both types of swabs. hRV was the most frequently detected pathogen (133 of 445, 29.9%) followed by influenza A virus (54 of 445, 12.1%; Figure 1 ). All other viruses were detected at lower levels (2.7%-8.1%) and PIV2, PIV4, and the 4 hCoVs were detected only sporadically; in less than 2% of the patients ( Figure 1 ). Although detection rates were the same or higher in nasopharyngeal aspirates compared to throat swabs for most pathogens (10 of 13), a statistically significant difference was only observed for RSV (p = .039; Figure 1B ). Mixed infections were detected in 13.5% (60 of 445; Table 1) Figure S1 ). The genetic similarity between Lao strains ranged from 98.2% to 100% over the complete G gene sequence. All RSV-B sequences clustered within the BA9 genotype and were distinct from BA9 strains that were circulating in the country in 2010. 21 The THB/CB1 genotype previously identified in Lao PDR in 2010 21 was not observed in this study. hMPV was detected almost all year round without any temporal variation (p = .738). hMPV strains were typed as genotype A2b In Lao PDR, limited disease surveillance data prompted us to investigate and characterize a wide range of respiratory pathogens. Here we show that respiratory viral infections occur throughout the year in Lao PDR, with hRV being the most frequently detected virus (29.9% overall and 75.0% of co-infections; Figure 1 ). High frequencies of hRV infections were also reported in other studies carried out in Lao PDR, regardless of the population investigated. 3, 11, 12, 22 Two studies found that hRV was the most frequently detected virus in all age groups in patients with ILI or acute lower respiratory infections. 11, 12 hRV was also the second most frequent virus reported, after RSV, in children with severe respiratory acute infection. 22 Although other studies carried out elsewhere have shown that hRV can be found in 10%-33% of asymptomatic children [23] [24] [25] and has typically been associated with upper respiratory tract infections, its implication in more severe diseases such as bronchiolitis, pneumonia, or asthma exacerbation has also been suggested. 26 Co-infections tend to increase illness duration: hospital stay for children with hRV-RSV co-infections has been shown to be significantly longer than children infected with RSV or hRV alone. 27 In addition, hRV-C may be more virulent than hRV-A and -B. Our study also confirms the seasonality of RSV circulation in Lao PDR, with increased incidence in the rainy season. 12, 21, 22 However, we did not detect RSV cases outside September 2014 and 2015. This contrasts with previous studies suggesting that RSV is present yearround with a surge of cases during the rainy season. 22 Younger children had an increased chance of being positive for RSV and had an increased risk of hospital admission, again highlighting the contribution of RSV to respiratory disease severity in young children. hMPV, discovered only two decades ago, shares clinical and epidemiological characteristics with RSV. 34 It has been shown that most children are infected with hMPV before 5 years of age. hMPV infections lead to a wide range of clinical symptoms including frequent bronchiolitis and pneumonia, with a peak of hospitalization for children aged 6-12 months. 35 However, no association with age or hospital admission was observed in our study. Five main genotypes have been described so far, often co-circulating. Indeed, our study provides the first molecular characterization of hMPV in Lao PDR and shows co-circulation of 3 genotypes. The predominant genotype has been shown to change over time and is influenced by genotypespecific levels of immunity in the community, virus evolution and escape 36,37 and differential replication advantage rather than a difference in virulence. 38 An In our study, we combined results from both nasopharyngeal aspirates and throat swabs. This approach allowed for improved sensitivity for identifying the etiological agents of respiratory infections in a pediatric population. Others have also found that multiple sampling in an adult population with lower respiratory tract manifestations revealed an improved sensitivity for virus detection. 40 Nasopharyngeal aspirates provided a significantly increased sensitivity for detection only for RSV. This contrasts with a comparison of nasal, throat or nasopharyngeal swabs for the detection of RSV in hospitalized children in Lao PDR that showed no statistical difference between all three sampling materials. 41 However, viral loads in throat swabs in that study were lower than in nasal or nasopharyngeal swabs, 41 | 4753 Easier access to information such as through the Internet or training likely contributes to improved knowledge. With an infection characterization rate of more than 70%, our data provide a detailed overview of the dynamics of respiratory pathogens in the study population. This information will be useful to raise awareness among local medical practitioners and may provide guidance in planning vaccination campaigns in the country. Given the importance of influenza A/B infections, seasonal influenza vaccination for young children in Lao PDR could help decrease the annual burden. However, other childhood vaccinations may have a higher priority. 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