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Human Sentinel Surveillance of Influenza and Other Respiratory Viral Pathogens in Border Areas of Western Cambodia.

Timmermans A, Melendrez MC, Se Y, Chuang I, Samon N, Uthaimongkol N, Klungthong C, Manasatienkij W, Thaisomboonsuk B, Tyner SD, Rith S, Horm VS, Jarman RG, Bethell D, Chanarat N, Pavlin J, Wongstitwilairoong T, Saingam P, El BS, Fukuda MM, Touch S, Sovann L, Fernandez S, Buchy P, Chanthap L, Saunders D - PLoS ONE (2016)

Bottom Line: Most genes showed signatures of purifying selection.A single specimen of EV71 was found.Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, US Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

ABSTRACT
Little is known about circulation of influenza and other respiratory viruses in remote populations along the Thai-Cambodia border in western Cambodia. We screened 586 outpatients (median age 5, range 1-77) presenting with influenza-like-illness (ILI) at 4 sentinel sites in western Cambodia between May 2010 and December 2012. Real-time reverse transcriptase (rRT) PCR for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ILI-specimens negative for influenza were cultured, followed by rRT-PCR for enterovirus and rhinovirus (EV/RV) and EV71. Influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from June to November. Isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in Cambodia. Influenza vaccination coverage was low (<20%). Western Cambodian H1N1(2009) isolate genomes were more closely related to 10 earlier Cambodia isolates (94.4% genome conservation) than to 13 Thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the Thai references. Most genes showed signatures of purifying selection. Viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus A4, A6, A8, A9, A12, B3, B4 and echovirus E6 and E9 using nested RT-PCR methods. A single specimen of EV71 was found. Despite proximity to Thailand, influenza epidemiology of these western Cambodian isolates followed patterns observed elsewhere in Cambodia, continuing to support current vaccine and treatment recommendations from the Cambodian National Influenza Center. Amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

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Laboratory testing flowchart for influenza-like-illness (ILI).
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pone.0152529.g002: Laboratory testing flowchart for influenza-like-illness (ILI).

Mentions: All specimens tested negative by influenza PCR were cultured for isolation of respiratory virus and detection by cytopathic effect (CPE) and/or with either immunofluorescence assay (IFA), or direct fluorescence assay (DFA). A subset of 164 culture-negative specimens (collected between May 2010 and April 2012), where we found a higher proportion (5.6%) of non-polio enteroviruses in children less than 5 years old as compared with previous studies (1%) in Cambodia [2], were tested for enterovirus and rhinovirus by two separate nested RT-PCR methods adapted from Coiras et al., 2004 and Singh et al., 2002 [29,30], one for simultaneous detection of pan-enteroviruses and rhinoviruses, and the other specific for enterovirus 71 (EV71). PCR-products positive for enterovirus or rhinovirus were sequenced for nucleotide analysis (Fig 2; S3 Table).


Human Sentinel Surveillance of Influenza and Other Respiratory Viral Pathogens in Border Areas of Western Cambodia.

Timmermans A, Melendrez MC, Se Y, Chuang I, Samon N, Uthaimongkol N, Klungthong C, Manasatienkij W, Thaisomboonsuk B, Tyner SD, Rith S, Horm VS, Jarman RG, Bethell D, Chanarat N, Pavlin J, Wongstitwilairoong T, Saingam P, El BS, Fukuda MM, Touch S, Sovann L, Fernandez S, Buchy P, Chanthap L, Saunders D - PLoS ONE (2016)

Laboratory testing flowchart for influenza-like-illness (ILI).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814059&req=5

pone.0152529.g002: Laboratory testing flowchart for influenza-like-illness (ILI).
Mentions: All specimens tested negative by influenza PCR were cultured for isolation of respiratory virus and detection by cytopathic effect (CPE) and/or with either immunofluorescence assay (IFA), or direct fluorescence assay (DFA). A subset of 164 culture-negative specimens (collected between May 2010 and April 2012), where we found a higher proportion (5.6%) of non-polio enteroviruses in children less than 5 years old as compared with previous studies (1%) in Cambodia [2], were tested for enterovirus and rhinovirus by two separate nested RT-PCR methods adapted from Coiras et al., 2004 and Singh et al., 2002 [29,30], one for simultaneous detection of pan-enteroviruses and rhinoviruses, and the other specific for enterovirus 71 (EV71). PCR-products positive for enterovirus or rhinovirus were sequenced for nucleotide analysis (Fig 2; S3 Table).

Bottom Line: Most genes showed signatures of purifying selection.A single specimen of EV71 was found.Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, US Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand.

ABSTRACT
Little is known about circulation of influenza and other respiratory viruses in remote populations along the Thai-Cambodia border in western Cambodia. We screened 586 outpatients (median age 5, range 1-77) presenting with influenza-like-illness (ILI) at 4 sentinel sites in western Cambodia between May 2010 and December 2012. Real-time reverse transcriptase (rRT) PCR for influenza was performed on combined nasal and throat specimens followed by viral culture, antigenic analysis, antiviral susceptibility testing and full genome sequencing for phylogenetic analysis. ILI-specimens negative for influenza were cultured, followed by rRT-PCR for enterovirus and rhinovirus (EV/RV) and EV71. Influenza was found in 168 cases (29%) and occurred almost exclusively in the rainy season from June to November. Isolated influenza strains had close antigenic and phylogenetic relationships, matching vaccine and circulating strains found elsewhere in Cambodia. Influenza vaccination coverage was low (<20%). Western Cambodian H1N1(2009) isolate genomes were more closely related to 10 earlier Cambodia isolates (94.4% genome conservation) than to 13 Thai isolates (75.9% genome conservation), despite sharing the majority of the amino acid changes with the Thai references. Most genes showed signatures of purifying selection. Viral culture detected only adenovirus (5.7%) and parainfluenza virus (3.8%), while non-polio enteroviruses (10.3%) were detected among 164 culture-negative samples including coxsackievirus A4, A6, A8, A9, A12, B3, B4 and echovirus E6 and E9 using nested RT-PCR methods. A single specimen of EV71 was found. Despite proximity to Thailand, influenza epidemiology of these western Cambodian isolates followed patterns observed elsewhere in Cambodia, continuing to support current vaccine and treatment recommendations from the Cambodian National Influenza Center. Amino acid mutations at non-epitope sites, particularly hemagglutinin genes, require further investigation in light of an increasingly important role of permissive mutations in influenza virus evolution. Further research about the burden of adenovirus and non-polio enteroviruses as etiologic agents in acute respiratory infections in Cambodia is also needed.

Show MeSH
Related in: MedlinePlus