key: cord-0764352-g13e5lvb
authors: Snoeck, Chantal J.; Ponghsavath, Valy; Luetteke, Nina; Kaufmann, Sabine; Sausy, Aurélie; Samountry, Bounthome; Jutavijittum, Prapan; Weber, Bernard; Muller, Claude P.
title: Etiology of viral respiratory infections in Northern Lao People's Democratic Republic
date: 2018-07-13
journal: J Med Virol
DOI: 10.1002/jmv.25237
sha: 7062ac6e3487d6e4054207964b1c6de6bddbfb51
doc_id: 764352
cord_uid: g13e5lvb

In Lao People's Democratic Republic (PDR), acute respiratory infections overburden the health care system, but viral etiology, genetic diversity, and seasonality, especially in light of the introduction of influenza vaccination in the country, are poorly understood. From August 2010 to April 2011, 309 outpatients were recruited at the Luang Prabang Provincial Hospital covering highland Lao communities. Nasopharyngeal swabs were screened for the presence of 13 respiratory viruses. At least one virus was detected in 69.6% and dual/triple viral infections in 12.9%/1.9% of the patients. Influenza A and B viruses combined were the most frequently detected pathogens, followed by human adenovirus and respiratory syncytial virus (RSV). The other viruses were detected in less than 10% of the patients. Phylogenetic analyses on a representative set of RSV strains revealed that, while otherwise very rare, the RSV‐B CB1/THB genotype cocirculated with other common genotypes. A single wave of influenza virus and RSV activity was observed during the rainy season, providing further support to influenza vaccination before the onset of the rains. This study provides recommendations for influenza vaccination that still needs optimization and highlights the need for revised guidelines for treatment and prevention of respiratory infections in Lao PDR, as well as for increased surveillance efforts.

The etiology of respiratory diseases is complex. Influenza A and B viruses (family Orthomyxoviridae, genus Influenzavirus A and B) are frequently detected at all ages, but a variety of pathogens, including viruses and bacteria, cause similar symptoms that cannot be differentiated without laboratory confirmation. In Lao PDR, the influenza sentinel network was initiated only in 2007 3 and other respiratory viruses are normally not diagnosed. 3, 4 In particular, respiratory syncytial virus (RSV; order Mononegavirales, family Pneumoviridae, genus Orthopneumovirus, and species human orthopneumovirus) is a leading cause of lower respiratory tract infections in infants and young children worldwide. 5 A proper understanding of the etiology, seasonal patterns, and genetic diversity of respiratory viruses in Lao PDR is warranted. Notably, the success of the influenza vaccination recently introduced in the country relies on recommendations for optimal timing with respect to the season with highest influenza transmission. This is still poorly understood in subtropical countries. Nasopharyngeal swabs from patients with acute respiratory infections at the Luang Prabang Provincial Hospital in Northern Laos, catering to highland communities in Northern Laos, were screened for 13 viruses.

Samples were collected at the Provincial Hospital in Luang Prabang, a town with a population of 56 000 located at an altitude of 700 m. The hospital provides primary care without particular specialization to the local population of the town and is a major referral center for the highland communities living throughout this mountainous province. [9] [10] [11] human metapneumovirus (hMPV), 12 human coronaviruses 229E (hCoV-229E) and OC43 (hCoV-OC43). 13 The same pathogens as well as influenza B virus, human adenovirus (hAdV), human bocavirus (hBoV), human rhinovirus (hRV), and human coronavirus HKU1 (hCoV-HKU1) were detected by Lab2 using the FTD respiratory pathogens 21 kit (Fast-track Diagnostics, Esch-sur-Alzette, Luxembourg). All samples were tested by the two laboratories to compare detection rates of the in-house PCRs against commercial tests and to broaden the range of pathogens detected. Results of conventional PCRs were visualized on an agarose gel, and positivity was confirmed by sequencing on an ABI 3130 Avant Capillary Sequencer (Applied Biosystems, Nieuwerkerk, The Netherlands) using the PCR primers also as sequencing primers. The ectodomain of the G-gene of RSV was also amplified as described before 14 and sequenced (GenBank accession numbers MG773863-MG773881).

Genetic distances and phylogenetic trees were calculated using maximum composite likelihood model with 500 bootstrap values in MEGA6. 15 Figure 1A ). hAdV (40 of 309, 12.9%) and RSV (31 of 309, 10.0%) were also frequently detected, while the other viruses were detected in less than 10% of the patients, and PIV4 was not detected at all (Table 1) .

When comparing the results obtained by the two laboratories, more samples were positive for RSV and PIV1-3 in Lab1 compared to Lab2, while more samples were positive for influenza A, hMPV, hCoV-OC43, and hCoV-229E in Lab2 (Table 1) . Discrepant results were observed only in weak positive samples. A statistical difference between the results obtained by the two laboratories was only found for hCoV-OC43 (P = 0.041; Table 1 ), suggesting that most in-house PCRs were performing well and can be more sustainably used in resource-limited countries compared to commercial PCRs.

Dual and triple infections were detected in 12.9% (40 of 309) and

1.9% (6 of 309) of the patients (Table 1) 

All viruses combined, the virological status (ie, positivity for ≥1 virus) of the patients was not significantly associated with age. However, patients with viral mixed infections were significantly younger than those with a single infection (median age, 19.5 vs 28.0 months; P = 0.015). PIV2, PIV3, hMPV, hCoV-OC43, hCoV-229E, hAdV, and hBoV were also exclusively found in children under 15 years old (Table 1) , whereas influenza viruses were detected in all age groups and RSV (n = 1), PIV1 (n = 2), hCoV-HKU1 (n = 2), and hRV (n = 2) were only sporadically found in the 15-to 50-year age group (Table 1) 

World Health Organization. WHO Country Cooperation Strategy for the Lao People's Democratic Republic

Epidemiological and virological characteristics of seasonal and pandemic influenza in Lao PDR

An early report from newly established laboratory-based influenza surveillance in Lao PDR. Influenza Other Respir Viruses

Respiratory virus infections in hospitalized children and adults in Lao PDR. Influenza Other Respir Viruses

Burden of respiratory syncytial virus infections in China: Systematic review and meta-analysis

Avian flu: multiple introductions of H5N1 in Nigeria

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement

Respiratory syncytial virus heterogeneity during an epidemic: analysis by limited nucleotide sequencing (SH gene) and restriction mapping (N gene)

Detection and identification of human parainfluenza viruses 1, 2, 3, and 4 in clinical samples of pediatric patients by multiplex reverse transcription-PCR

Simultaneous detection and identification of human parainfluenza viruses 1, 2, and 3 from clinical samples by multiplex PCR

Rapid detection of parainfluenza virus type 3 RNA in respiratory specimens: use of reverse transcription-PCR-enzyme immunoassay

Real-time reverse transcriptase PCR assay for detection of human metapneumoviruses from all known genetic lineages

Direct diagnosis of human respiratory coronaviruses 229E and OC43 by the polymerase chain reaction

Molecular epidemiology of respiratory syncytial virus infections among children with acute respiratory symptoms in a community over three seasons

Molecular Evolutionary Genetics Analysis version 6.0

Genetic variation in attachment glycoprotein genes of human respiratory syncytial virus subgroups a and B in children in recent five consecutive years

Molecular characterization of human respiratory syncytial virus, 2010-2011: identification of genotype ON1 and a new subgroup B genotype in Thailand

Sequence analysis of G glycoprotein of human respiratory syncytial virus subtype B strains isolated from children with acute respiratory infections in Beijing

Capacity building in response to pandemic influenza threats: Lao PDR case study

Causes of non-malarial fever in Laos: a prospective study

Association of RSV-A ON1 genotype with increased pediatric acute lower respiratory tract infection in Vietnam

Direct whole-genome deepsequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter-and intrahost evolution

A study of the genetic variability of human respiratory syncytial virus (HRSV) in Cambodia reveals the existence of a new HRSV group B genotype

Acute respiratory infections in hospitalized children in Vientiane, Lao PDR -the importance of respiratory syncytial virus

Lao People's Democratic Republic Launches Seasonal Flu Vaccination Program

Influenza seasonality and vaccination timing in tropical and subtropical areas of southern and south-eastern Asia

Influenza seasonality in the tropics and subtropics -when to vaccinate?

Influenza Vaccine Use in Low and Middle Income Countries in the Tropics and Subtropics. A systematic review

Influenza-like illness sentinel surveillance in Peru

Genomic loads and genotypes of respiratory syncytial virus: viral factors during lower respiratory tract infection in Chilean hospitalized infants

Characterization of hospital and community-acquired respiratory syncytial virus in children with severe lower respiratory tract infections in Ho Chi Minh City

Genetic variability of human respiratory syncytial virus A strains circulating in Ontario: a novel genotype with a 72 nucleotide G gene duplication

Genetic diversity and evolutionary insights of respiratory syncytial virus A ON1 genotype: global and local transmission dynamics

Antibiotic prescription behaviours in Lao People's Democratic Republic: a knowledge, attitude and practice survey

Etiology of viral respiratory infections in Northern Lao People's Democratic Republic

The authors thank the study volunteers for their participation and the staff from Luang Prabang Provincial Hospital for sample collection. They also thank S. Wolter and R. Sinner for their technical help. This study was supported by the Ministère des Affaires étrangères et européennes, Luxembourg and the Luxembourg Institute of Health through "PaReCIDS" project.

The authors declare that there are no competing conflicts of interest.

http://orcid.org/0000-0002-0000-1850