Quantifying within-host diversity of H5N1 influenza viruses in humans and poultry in Cambodia.

Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift, and half of the variants detected within-host are never detected on the H5N1 virus phylogeny. However, we do detect a subset of mutations linked to human receptor binding and replication (PB2 E627K, HA A150V, and HA Q238L) that arose in multiple, independent humans. PB2 E627K and HA A150V were also enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adaptive. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection, many of which are detected at >5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses.

Co-circulation of Influenza A H5, H7, and H9 Viruses and Co-infected Poultry in Live Bird Markets, Cambodia.

Longitudinal surveillance of 2 live bird markets in Cambodia revealed year-round, high co-circulation of H5, H7, and H9 influenza viruses. We detected influenza A viruses in 51.3% of ducks and 39.6% of chickens, and co-infections, mainly by H5 and H9 viruses, in 0.8% of ducks and 4.5% of chickens.

Seroprevalence and Transmission of Human Influenza A(H5N1) Virus before and after Virus Reassortment, Cambodia, 2006-2014.

Thirty-five human influenza A(H5N1) cases were reported in Cambodia during 2013-2014 after emergence of a clade 1.1.2 reassortant virus. We tested 881 villagers and found 2 cases of pauci- or asymptomatic infection. Seroprevalence after emergence of the reassortant strain (0.2%) was lower than the aggregate seroprevalence of 1.3% reported in earlier studies.

Environmental contamination and risk factors for transmission of highly pathogenic avian influenza A(H5N1) to humans, Cambodia, 2006-2010.

BACKGROUND: Highly pathogenic avian influenza A (H5N1) virus has been of public health concern since 2003. Probable risk factors for A(H5N1) transmission to human have been demonstrated in several studies or epidemiological reports. However, transmission patterns may differ according to demographic characteristics of the population and local practices. This article aggregates these data from three studies with data collected in the previous surveys in 2006 and 2007 to further examine the risks factors associated with presence of anti-A(H5) antibodies among villagers residing within outbreak areas. METHODS: We aggregated 5-year data (2006-2010) from serology survey and matched case-control studies in Cambodia to further examine the risks factors associated with A(H5N1) infection among villagers in the outbreak areas. RESULTS: Serotesting among villagers detected 35 (1.5 % [0-2.6]) positive cases suggesting recent exposure to A(H5N1) virus. Practices associated with A(H5N1) infection among all ages were: having poultry cage or nesting area under or adjacent to the house (OR: 6.7 [1.6-28.3]; p = 0.010) and transporting poultry to market (OR: 17.6 [1.6-193.7]; p = 0.019). Practices found as risk factors for the infection among age under 20 years were swimming/bathing in ponds also accessed by domestic poultry (OR: 4.6 [1.1-19.1]; p = 0.038). Association with consuming wild birds reached borderline significance (p = 0.066). CONCLUSION: Our results suggest that swimming/bathing in contaminated pond water and close contact with poultry may present a risk of A(H5N1) transmission to human.

Identification of molecular markers associated with alteration of receptor-binding specificity in a novel genotype of highly pathogenic avian influenza A(H5N1) viruses detected in Cambodia in 2013.

Human infections with influenza A(H5N1) virus in Cambodia increased sharply during 2013. Molecular characterization of viruses detected in clinical specimens from human cases revealed the presence of mutations associated with the alteration of receptor-binding specificity (K189R, Q222L) and respiratory droplet transmission in ferrets (N220K with Q222L). Discovery of quasispecies at position 222 (Q/L), in addition to the absence of the mutations in poultry/environmental samples, suggested that the mutations occurred during human infection and did not transmit further.

Acute lower respiratory infections in ≥ 5 year -old hospitalized patients in Cambodia, a low-income tropical country: clinical characteristics and pathogenic etiology.

BACKGROUND: Few data exist on viral and bacterial etiology of acute lower respiratory infections (ALRI) in ≥ 5 year -old persons in the tropics. METHODS: We conducted active surveillance of community-acquired ALRI in two hospitals in Cambodia, a low-income tropical country. Patients were tested for acid-fast bacilli (AFB) by direct sputum examination, other bacteria by blood and/or sputum cultures, and respiratory viruses using molecular techniques on nasopharyngeal/throat swabs. Pulmonologists reviewed clinical/laboratory data and interpreted chest X-rays (CXR) to confirm ALRI. RESULTS: Between April 2007 - December 2009, 1,904 patients aged ≥5 years were admitted with acute pneumonia (50.4%), lung sequelae-associated ALRI (24.3%), isolated pleural effusions (8.9%) or normal CXR-related ALRI (17.1%); 61 (3.2%) died during hospitalization. The two former diagnoses were predominantly due to bacterial etiologies while viral detection was more frequent in the two latter diagnoses. AFB-positive accounted for 25.6% of acute pneumonia. Of the positive cultures (16.8%), abscess-prone Gram-negative bacteria (39.6%) and Haemophilus influenzae (38.0%) were most frequent, followed by Streptococcus pneumoniae (17.7%). Of the identified viruses, the three most common viruses included rhinoviruses (49.5%), respiratory syncytial virus (17.7%) and influenza viruses (12.1%) regardless of the diagnostic groups. Wheezing was associated with viral identification (31.9% vs. 13.8%, p < 0.001) independent of age and time-to-admission. CONCLUSIONS: High frequency of H. influenzae and S. pneumoniae infections support the need for introduction of the respective vaccines in the national immunization program. Tuberculosis was frequent in patients with acute pneumonia, requiring further investigation. The relationship between respiratory viruses and wheezing merits further studies.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

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

Human respiratory syncytial virus (HRSV) is the leading cause of hospitalization of children aged <5 years due to respiratory illness in industrialized countries, and pneumonia is the leading cause of mortality among children aged <5 years worldwide. Although HRSV was first identified in 1956, a preventative vaccine has yet to be developed. Here we report the results of the first study to investigate the circulation and genetic diversity of HRSV in Cambodia among an all-ages population over 5 consecutive years. The incidences of HRSV infection among all-ages outpatient and hospitalized populations were equivalent, at 9.5% and 8.2%, respectively. Infection was most prevalent among children aged <5 years, with bronchiolitis being the most frequently observed clinical syndrome in the same age group. Circulation of HRSV was seasonal, typically coinciding with the rainy season between July and November annually. Strains belonging to HRSV groups A and B were detected with equivalent frequencies; however, we observed a potentially biennial shift in the predominant circulating HRSV genotype. The majority of HRSV group B strains belonged to the recently described BA genotype, with the exception of 10 strains classified as belonging to a novel HRSV group B genotype, SAB4, first reported here.

Transmission experiments support clade-level differences in the transmission and pathogenicity of Cambodian influenza A/H5N1 viruses.

Influenza A/H5N1 has circulated in Asia since 2003 and is now enzootic in many countries in that region. In Cambodia, the virus has circulated since 2004 and has intermittently infected humans. During this period, we have noted differences in the rate of infections in humans, potentially associated with the circulation of different viral clades. In particular, a reassortant clade 1.1.2 virus emerged in early 2013 and was associated with a dramatic increase in infections of humans (34 cases) until it was replaced by a clade 2.3.2.1c virus in early 2014. In contrast, only one infection of a human has been reported in the 6 years since the clade 2.3.2.1c virus became the dominant circulating virus. We selected three viruses to represent the main viral clades that have circulated in Cambodia (clade 1.1.2, clade 1.1.2 reassortant, and clade 2.3.2.1c), and we conducted experiments to assess the virulence and transmissibility of these viruses in avian (chicken, duck) and mammalian (ferret) models. Our results suggest that the clade 2.3.2.1c virus is more "avian-like," with high virulence in both ducks and chickens, but there is no evidence of aerosol transmission of the virus from ducks to ferrets. In contrast, the two clade 1 viruses were less virulent in experimentally infected and contact ducks. However, evidence of chicken-to-ferret aerosol transmission was observed for both clade 1 viruses. The transmission experiments provide insights into clade-level differences that might explain the variation in A/H5N1 infections of humans observed in Cambodia and other settings.

Circulation and characterization of seasonal influenza viruses in Cambodia, 2012-2015.

BACKGROUND: Influenza virus circulation is monitored through the Cambodian influenza-like illness (ILI) sentinel surveillance system and isolates are characterized by the National Influenza Centre (NIC). Seasonal influenza circulation has previously been characterized by year-round activity and a peak during the rainy season (June-November). OBJECTIVES: We documented the circulation of seasonal influenza in Cambodia for 2012-2015 and investigated genetic, antigenic, and antiviral resistance characteristics of influenza isolates. PATIENTS/METHODS: Respiratory samples were collected from patients presenting with influenza-like illness (ILI) at 11 hospitals throughout Cambodia. First-line screening was conducted by the National Institute of Public Health and the Armed Forces Research Institute of Medical Sciences. Confirmation of testing and genetic, antigenic and antiviral resistance characterization was conducted by Institute Pasteur in Cambodia, the NIC. Additional virus characterization was conducted by the WHO Collaborating Centre for Reference and Research on Influenza (Melbourne, Australia). RESULTS: Between 2012 and 2015, 1,238 influenza-positive samples were submitted to the NIC. Influenza A(H3N2) (55.3%) was the dominant subtype, followed by influenza B (30.9%; predominantly B/Yamagata-lineage) and A(H1N1)pdm09 (13.9%). Circulation of influenza viruses began earlier in 2014 and 2015 than previously described, coincident with the emergence of A(H3N2) clades 3C.2a and 3C.3a, respectively. There was high diversity in the antigenicity of A(H3N2) viruses, and to a smaller extent influenza B viruses, during this period, with some mismatches with the northern and southern hemisphere vaccine formulations. All isolates tested were susceptible to the influenza antiviral drugs oseltamivir and zanamivir. CONCLUSIONS: Seasonal and year-round co-circulation of multiple influenza types/subtypes were detected in Cambodia during 2012-2015.

Intense circulation of A/H5N1 and other avian influenza viruses in Cambodian live-bird markets with serological evidence of sub-clinical human infections.

Surveillance for avian influenza viruses (AIVs) in poultry and environmental samples was conducted in four live-bird markets in Cambodia from January through November 2013. Through real-time RT-PCR testing, AIVs were detected in 45% of 1048 samples collected throughout the year. Detection rates ranged from 32% and 18% in duck and chicken swabs, respectively, to 75% in carcass wash water samples. Influenza A/H5N1 virus was detected in 79% of samples positive for influenza A virus and 35% of all samples collected. Sequence analysis of full-length haemagglutinin (HA) and neuraminidase (NA) genes from A/H5N1 viruses, and full-genome analysis of six representative isolates, revealed that the clade 1.1.2 reassortant virus associated with Cambodian human cases during 2013 was the only A/H5N1 virus detected during the year. However, multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of HA and NA genes revealed co-circulation of at least nine low pathogenic AIVs from HA1, HA2, HA3, HA4, HA6, HA7, HA9, HA10 and HA11 subtypes. Four repeated serological surveys were conducted throughout the year in a cohort of 125 poultry workers. Serological testing found an overall prevalence of 4.5% and 1.8% for antibodies to A/H5N1 and A/H9N2, respectively. Seroconversion rates of 3.7 and 0.9 cases per 1000 person-months participation were detected for A/H5N1 and A/H9N2, respectively. Peak AIV circulation was associated with the Lunar New Year festival. Knowledge of periods of increased circulation of avian influenza in markets should inform intervention measures such as market cleaning and closures to reduce risk of human infections and emergence of novel AIVs.

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

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.

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus.

BACKGROUND: Despite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny. STUDY DESIGN: We document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09. RESULTS: Complete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID50 of 10(4)/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia. CONCLUSIONS: It is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities.

Safety, potential efficacy, and pharmacokinetics of specific polyclonal immunoglobulin F(ab')2 fragments against avian influenza A (H5N1) in healthy volunteers: a single-centre, randomised, double-blind, placebo-controlled, phase 1 study.

BACKGROUND: Human infection with the avian influenza A H5N1 virus results in disease with a high fatality rate, against which antiviral treatments have limited efficacy. We aimed to investigate the safety, pharmacokinetics, and therapeutic potential of specific polyclonal immunoglobulin equine F(ab')2 fragments raised against influenza A/Vietnam/1194/2004 virus (H5N1 subtype) in healthy volunteers. METHODS: We did a randomised, double-blind, placebo-controlled, single-centre phase 1 study. In stage 1 (one infusion) and stage 2 (five infusions) of the trial, we randomly assigned healthy male volunteers to receive once-daily intravenous infusions of 0·85 U/kg body weight of F(ab')2 or once-daily saline placebo. Randomisation was done centrally, with one block of four patients and one block for substitutes (three actives, one placebo) in stage 1, and two blocks of six patients (five actives and one placebo) and the same block for substitutes in stage 2. The primary objective was assessment of the clinical and laboratory safety of F(ab')2, which was monitored for 22 days in the group that received one dose (assessments on days 0-2, 4, 8, 15, and 22) and 33 days in the group that received five doses (days 0-6, 8, 10, 12, 19, 26, and 33). A final post-study safety assessment was done at 120 days. We also assessed pharmacokinetic outcomes, and assayed haemagglutination and seroneutralisation activity. Analysis was done according to intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT02295813. FINDINGS: We enrolled 16 healthy Asian men between Sept 28 and Dec 28, 2012, and randomly assigned 13 to one or five doses of F(ab')2 and three to placebo. F(ab')2 was well tolerated, and no deaths or serious adverse events occurred. Three patients had mild adverse events (one each of blepharospasm, sinusitis, and pyrexia). The pyrexia (38°C) was regarded as probably related to the infusion, and resolved after 37 min. Our laboratory assessments of blood and urine samples and physical examinations of heart rate, electrocardiogram readings, and weight showed no clinically significant safety issues. Mean peak plasma concentrations were 19·3 µg/mL (SD 3·5) with the one dose schedule and 23·0 µg/mL (4·5) with the five-dose schedule. F(ab')2 were still detectable in plasma on average up to 5 days after five doses. Haemagglutination inhibition was only increased after the third dose, but in-vitro seroneutralisation activity was transiently increased after each of the five doses to concentrations regarded as clinically beneficial in infected patients. INTERPRETATION: F(ab')2 showed good safety, tolerability, and therapeutic potential for managing of H5N1 exposed patients. FUNDING: Fab'entech.

Specific polyclonal F(ab')2 neutralize a large panel of highly pathogenic avian influenza A viruses (H5N1) and control infection in mice.

AIM: There is still no specific therapy for infection with the highly pathogenic avian influenza A virus (HPAI) H5N1, which caused 39 human cases with a 64% fatality rate in 2013. MATERIALS & METHODS: We prepared highly purified specific equine polyclonal immunoglobulin fragments (F(ab')2) against H5N1 and tested them for efficacy in vitro and with different administration schedules in H5N1-challenged BALB/c mice. RESULTS: in vitro, F(ab')2 neutralized 21 different H5N1 strains from different areas, representative of 11 different clades and sub-clades and 9 years of evolution of the virus. In vivo mouse experiments identified that the most efficient administration protocol consists of five consecutive daily injections after infection; 10 mg/kg giving a 60% increase in survival. CONCLUSION: These data demonstrate the ability of anti-H5N1 F(ab')2 to markedly reduce the mortality and morbidity associated with infection of mice with HPAI H5N1 virus, and their potential for human therapy.

Epidemiological and virological characteristics of influenza viruses circulating in Cambodia from 2009 to 2011.

BACKGROUND: The Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011. METHODOLOGY/PRINCIPAL FINDINGS: Sentinel and study sites collected nasopharyngeal specimens for diagnostic detection, virus isolation, antigenic characterization, sequencing and antiviral susceptibility analysis from patients who fulfilled case definitions for influenza-like illness, acute lower respiratory infections and event-based surveillance. Each year in Cambodia, influenza viruses were detected mainly from June to November, during the rainy season. Antigenic analysis show that A/H1N1pdm09 isolates belonged to the A/California/7/2009-like group. Circulating A/H3N2 strains were A/Brisbane/10/2007-like in 2009 before drifting to A/Perth/16/2009-like in 2010 and 2011. The Cambodian influenza B isolates from 2009 to 2011 all belonged to the B/Victoria lineage represented by the vaccine strains B/Brisbane/60/2008 and B/Malaysia/2506/2004. Sequences of the M2 gene obtained from representative 2009-2011 A/H3N2 and A/H1N1pdm09 strains all contained the S31N mutation associated with adamantanes resistance except for one A/H1N1pdm09 strain isolated in 2011 that lacked this mutation. No reduction in the susceptibility to neuraminidase inhibitors was observed among the influenza viruses circulating from 2009 to 2011. Phylogenetic analysis revealed that A/H3N2 strains clustered each year to a distinct group while most A/H1N1pdm09 isolates belonged to the S203T clade. CONCLUSIONS/SIGNIFICANCE: In Cambodia, from 2009 to 2011, influenza activity occurred throughout the year with peak seasonality during the rainy season from June to November. Seasonal influenza epidemics were due to multiple genetically distinct viruses, even though all of the isolates were antigenically similar to the reference vaccine strains. The drug susceptibility profile of Cambodian influenza strains revealed that neuraminidase inhibitors would be the drug of choice for influenza treatment and chemoprophylaxis in Cambodia, as adamantanes are no longer expected to be effective.

Genetic variability of human metapneumovirus amongst an all ages population in Cambodia between 2007 and 2009.

First identified in 2001, human metapneumovirus (HMPV) is a novel pathogen and causative agent of acute respiratory tract infection. Re-infection with HMPV is common, and currently there is no available vaccine against HMPV infection. Two genotypes of HMPV have been identified, A and B, both of which can be divided further into at least two distinct sub-genotypes. Here we report the results of the first study to investigate the genetic variability of HMPV strains circulating within Cambodia. The overall incidence of HMPV infection amongst an all-ages population of patients hospitalised with ALRI in Cambodia during 3 consecutive years, between 2007 and 2009, was 1.7%. The incidence of HMPV infection was highest amongst children less than 5 years of age, with pneumonia or bronchopneumonia the most frequent clinical diagnoses across all age groups. The incidence of HMPV infection varied annually. As anticipated, genetic diversity was low amongst the conserved F gene sequences but very high amongst G gene sequences, some strains sharing as little as 56.3% and 34.2% homology at the nucleotide and amino acid levels, respectively. Simultaneous co-circulation of strains belonging to the HMPV sub-genotypes B1, B2 and lineage A2b, amongst patients recruited at 2 geographically distinct provincial hospitals, was detected. Sub-genotype B2 strains were responsible for the majority of the infections detected, and a significant (p=0.013) association between infection with lineage A2b strains and disease severity was observed.

Human bocavirus amongst an all-ages population hospitalised with acute lower respiratory infections in Cambodia.

BACKGROUND: Human bocavirus (HBoV) is a novel parvovirus that is associated with respiratory and gastrointestinal tract disease. OBJECTIVES: To investigate the prevalence and genetic diversity of HBoV amongst hospitalized patients with acute lower respiratory infection (ALRI) in Cambodia. STUDY DESIGN: Samples were collected from 2773 patients of all ages hospitalised with symptoms of ALRI between 2007 and 2009. All samples were screened by multiplex RT-PCR/PCR for 18 respiratory viruses. All samples positive for HBoV were sequenced and included in this study. RESULTS: Of the samples tested, 43 (1·5%) were positive for HBoV. The incidence of HBoV did not vary between the consecutive seasons investigated, and HBoV infections were detected year-round. The incidence of HBoV infection was highest in patients aged < 2 years, with pneumonia or bronchopneumonia the most common clinical diagnosis, regardless of age. A total of 19 patients (44%) were co-infected with HBoV and an additional respiratory pathogen. All isolates were classified as HBoV type 1 (HBoV-1). High conservation between Cambodian NP1 and V1V2 gene sequences was observed. CONCLUSIONS: Human bocavirus infection can result in serious illness, however is frequently detected in the context of viral co-infection. Specific studies are required to further understand the true pathogenesis of HBoV in the context of severe respiratory illness.

Acute lower respiratory infections on lung sequelae in Cambodia, a neglected disease in highly tuberculosis-endemic country.

BACKGROUND: Little is known about post-infectious pulmonary sequelae in countries like Cambodia where tuberculosis is hyper-endemic and childhood pulmonary infections are highly frequent. We describe the characteristics of hospitalized Cambodian patients presenting with community-acquired acute lower respiratory infections (ALRI) on post-infectious pulmonary sequelae (ALRIPS). METHODS: Between 2007 and 2010, inpatients ≥15 years with ALRI were prospectively recruited. Clinical, biological, radiological and microbiological data were collected. Chest radiographs were re-interpreted by experts to compare patients with ALRIPS, on previously healthy lungs (ALRIHL) and active pulmonary tuberculosis (TB). Patients without chest radiograph abnormality or with abnormality suggestive as other chronic respiratory diseases were excluded from this analysis. RESULTS: Among the 2351 inpatients with community-acquired ALRI, 1800 were eligible: 426 (18%) ALRIPS, 878 (37%) ALRIHL and 496 (21%) TB. ALRIPS patients had less frequent fever than other ALRI (p < 0.001) and more productive cough than ALRIHL (p < 0.001). Streptococcus pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa accounted for 83% of ALRIPS group positive cultures. H. influenzae and P. aeruginosa were significantly associated with ALRIPS compared with ALRIHL. Treatment was appropriate in 58% of ALRIPS patients. Finally, 79% of ALRIPS were not recognized by local clinicians. In-hospital mortality was low (1%) but probably underestimated in the ALRIPS group. CONCLUSION: ALRIPS remains often misdiagnosed as TB with inappropriate treatment in low-income countries. Better-targeted training programs would help reduce the morbidity burden and financial costs.

Serologic evidence of human influenza virus infections in swine populations, Cambodia.

BACKGROUND: This study was conducted from 2006 to 2010 and investigated the seroprevalence of influenza A viruses in Cambodian pigs, including human H1N1, H3N2, 2009 pandemic H1N1 (A(H1N1)pdm09), and highly pathogenic avian H5N1 influenza A viruses. METHODS: A total of 1147 sera obtained from pigs in Cambodia were tested by haemagglutination inhibition (HI) assays for antibody to human influenza A viruses along with both HI and microneutralization (MN) tests to assess immunological responses to H5N1 virus. The results were compared by year, age, and province. RESULTS: Antibodies against a human influenza A virus were detected in 14·9% of samples. A(H1N1)pdm09 virus were dominant over the study period (23·1%), followed by those to human H1N1 (17·3%) and H3N2 subtypes (9·9%). No pigs were serologically positive for avian H5 influenza viruses. The seroprevalence of human H1N1 and H3N2 influenza viruses peaked in 2008, while that of A(H1N1)pdm09 reached a peak in 2010. No significant differences in seroprevalence to human influenza subtypes were observed in different age groups. CONCLUSIONS: Cambodian pigs were exposed to human strains of influenza A viruses either prior to or during this study. The implications of these high prevalence rates imply human-to-swine influenza virus transmission in Cambodia. Although pigs are mostly raised in small non-commercial farms, our preliminary results provide evidence of sustained human influenza virus circulation in pig populations in Cambodia.