Genetic and Antigenic Characterization of an Influenza A(H3N2) Outbreak in Cambodia and the Greater Mekong Subregion during the COVID-19 Pandemic, 2020.

Introduction of non-pharmaceutical interventions to control COVID-19 in early 2020 coincided with a global decrease in active influenza circulation. However, between July and November 2020, an influenza A(H3N2) epidemic occurred in Cambodia and in other neighboring countries in the Greater Mekong Subregion in Southeast Asia. We characterized the genetic and antigenic evolution of A(H3N2) in Cambodia and found that the 2020 epidemic comprised genetically and antigenically similar viruses of Clade3C2a1b/131K/94N, but they were distinct from the WHO recommended influenza A(H3N2) vaccine virus components for 2020-2021 Northern Hemisphere season. Phylogenetic analysis revealed multiple virus migration events between Cambodia and bordering countries, with Laos PDR and Vietnam also reporting similar A(H3N2) epidemics immediately following the Cambodia outbreak: however, there was limited circulation of these viruses elsewhere globally. In February 2021, a virus from the Cambodian outbreak was recommended by WHO as the prototype virus for inclusion in the 2021-2022 Northern Hemisphere influenza vaccine. IMPORTANCE The 2019 coronavirus disease (COVID-19) pandemic has significantly altered the circulation patterns of respiratory diseases worldwide and disrupted continued surveillance in many countries. Introduction of control measures in early 2020 against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection has resulted in a remarkable reduction in the circulation of many respiratory diseases. Influenza activity has remained at historically low levels globally since March 2020, even when increased influenza testing was performed in some countries. Maintenance of the influenza surveillance system in Cambodia in 2020 allowed for the detection and response to an influenza A(H3N2) outbreak in late 2020, resulting in the inclusion of this virus in the 2021-2022 Northern Hemisphere influenza vaccine.

Human Infection with Avian Influenza A(H9N2) Virus, Cambodia, February 2021.

In February 2021, routine sentinel surveillance for influenza-like illness in Cambodia detected a human avian influenza A(H9N2) virus infection. Investigations identified no recent H9N2 virus infections in 43 close contacts. One chicken sample from the infected child's house was positive for H9N2 virus and genetically similar to the human virus.

Human Outbreak of Trichinellosis Caused by Trichinella papuae Nematodes, Central Kampong Thom Province, Cambodia.

In September 2017, a severe trichinellosis outbreak occurred in Cambodia after persons consumed raw wild pig meat; 33 persons were infected and 8 died. We collected and analyzed the medical records for 25 patients. Clinical signs and symptoms included myalgia, facial or peripheral edema, asthenia, and fever. We observed increased levels of creatine phosphokinase and aspartate aminotransferase-, as well as eosinophilia. Histopathologic examination of muscle biopsy specimens showed nonencapsulated Trichinella larvae. A Trichinella excretory/secretory antigen ELISA identified Trichinella IgM and IgG. Biopsy samples were digested and larvae were isolated and counted. PCR for the 5S rDNA intergenic spacer region and a multiplex PCR, followed by sequencing identified the parasite as Trichinella papuae. This species was identified in Papua New Guinea during 1999 and in several outbreaks in humans in Thailand. Thus, we identified T. papuae nematodes in humans in Cambodia.

Detecting Spread of Avian Influenza A(H7N9) Virus Beyond China.

During February 2013-March 2015, a total of 602 human cases of low pathogenic avian influenza A(H7N9) were reported; no autochthonous cases were reported outside mainland China. In contrast, since highly pathogenic avian influenza A(H5N1) reemerged during 2003 in China, 784 human cases in 16 countries and poultry outbreaks in 53 countries have been reported. Whether the absence of reported A(H7N9) outside mainland China represents lack of spread or lack of detection remains unclear. We compared epidemiologic and virologic features of A(H5N1) and A(H7N9) and used human and animal influenza surveillance data collected during April 2013-May 2014 from 4 Southeast Asia countries to assess the likelihood that A(H7N9) would have gone undetected during 2014. Surveillance in Vietnam and Cambodia detected human A(H5N1) cases; no A(H7N9) cases were detected in humans or poultry in Southeast Asia. Although we cannot rule out the possible spread of A(H7N9), substantial spread causing severe disease in humans is unlikely.

Genomic epidemiology of SARS-CoV-2 in Cambodia, January 2020 to February 2021.

The first case of coronavirus disease 2019 (COVID-19) in Cambodia was confirmed on 27 January 2020 in a traveller from Wuhan. Cambodia subsequently implemented strict travel restrictions, and although intermittent cases were reported during the first year of the COVID-19 pandemic, no apparent widespread community transmission was detected. Investigating the routes of severe acute respiratory coronavirus 2 (SARS-CoV-2) introduction into the country was critical for evaluating the implementation of public health interventions and assessing the effectiveness of social control measures. Genomic sequencing technologies have enabled rapid detection and monitoring of emerging variants of SARS-CoV-2. Here, we detected 478 confirmed COVID-19 cases in Cambodia between 27 January 2020 and 14 February 2021, 81.3 per cent in imported cases. Among them, fifty-four SARS-CoV-2 genomes were sequenced and analysed along with representative global lineages. Despite the low number of confirmed cases, we found a high diversity of Cambodian viruses that belonged to at least seventeen distinct PANGO lineages. Phylogenetic inference of SARS-CoV-2 revealed that the genetic diversity of Cambodian viruses resulted from multiple independent introductions from diverse regions, predominantly, Eastern Asia, Europe, and Southeast Asia. Most cases were quickly isolated, limiting community spread, although there was an A.23.1 variant cluster in Phnom Penh in November 2020 that resulted in a small-scale local transmission. The overall low incidence of COVID-19 infections suggests that Cambodia's early containment strategies, including travel restrictions, aggressive testing and strict quarantine measures, were effective in preventing large community outbreaks of COVID-19.

Epidemiological characteristics, clinical presentation and diagnosis at point-of-care during the first wave of the H1N1 influenza pandemic in Cambodia.

We conducted clinic-based surveillance for influenza virus among cases with acute febrile illness at 9 medical clinics in south-central Cambodia during 2006-2009. Patients greater than or equal to 24 months old presenting with acute fever (> 38 degrees C) were enrolled. In late July 2009, the study identified its first case of pandemic H1N1 (pH1N1) influenza virus infection. The prevalence of pH1N1 infections increased rapidly during August and September and by October, pH1N1 infections had peaked replacing H3N2 as the dominant subtype. The incidence of pH1N1 subsequently decreased, with only one case identified in late December. From late July through December 2009, 42.4% of all influenza cases were caused by pH1N1. Except for headache, less frequently reported among pH1N1-infected patients, patients infected with the pH1N1 reported symptoms (eg, cough, diarrhea, vomiting and nausea) similar to seasonal H3N2 and B virus infections. Among children 6 to 12 years old, there was a higher number of hospitalizations campared to other age groups. Identification of influenza virus types A and B using the QuickVue rapid diagnostic test was found to be equally sensitive for pH1N1 (50.4%), H3N2 (51.7%) and influenza B (53.9%) viruses, although the sensitivity was low among all subtypes. The pH1N1 virus rapidly became the dominant virus subtype in 2009 in Cambodia, but no symptoms consistently distinguished the pandemic strain from other influenza virus subtypes. The QuickVue test was as sensitive for detecting pH1N1 viral as well as other circulating seasonal influenza viruses.

Rapid Generation of In-House Serological Assays Is Comparable to Commercial Kits Critical for Early Response to Pandemics: A Case With SARS-CoV-2.

Introduction: Accurate and sensitive measurement of antibodies is critical to assess the prevalence of infection, especially asymptomatic infection, and to analyze the immune response to vaccination during outbreaks and pandemics. A broad variety of commercial and in-house serological assays are available to cater to different laboratory requirements; however direct comparison is necessary to understand utility. Materials and Methods: We investigate the performance of six serological methods against SARS-CoV-2 to determine the antibody profile of 250 serum samples, including 234 RT-PCR-confirmed SARS-CoV-2 cases, the majority with asymptomatic presentation (87.2%) at 1-51 days post laboratory diagnosis. First, we compare to the performance of two in-house antibody assays: (i) an in-house IgG ELISA, utilizing UV-inactivated virus, and (ii) a live-virus neutralization assay (PRNT) using the same Cambodian isolate as the ELISA. In-house assays are then compared to standardized commercial anti-SARS-CoV-2 electrochemiluminescence immunoassays (Elecsys ECLIAs, Roche Diagnostics; targeting anti-N and anti-S antibodies) along with a flow cytometry based assay (FACS) that measures IgM and IgG against spike (S) protein and a multiplex microsphere-based immunoassay (MIA) determining the antibodies against various spike and nucleoprotein (N) antigens of SARS-CoV-2 and other coronaviruses (SARS-CoV-1, MERS-CoV, hCoVs 229E, NL63, HKU1). Results: Overall, specificity of assays was 100%, except for the anti-S IgM flow cytometry based assay (96.2%), and the in-house IgG ELISA (94.2%). Sensitivity ranged from 97.3% for the anti-S ECLIA down to 76.3% for the anti-S IgG flow cytometry based assay. PRNT and in-house IgG ELISA performed similarly well when compared to the commercial ECLIA: sensitivity of ELISA and PRNT was 94.7 and 91.1%, respectively, compared to S- and N-targeting ECLIA with 97.3 and 96.8%, respectively. The MIA revealed cross-reactivity of antibodies from SARS-CoV-2-infected patients to the nucleocapsid of SARS-CoV-1, and the spike S1 domain of HKU1. Conclusion: In-house serological assays, especially ELISA and PRNT, perform similarly to commercial assays, a critical factor in pandemic response. Selection of suitable immunoassays should be made based on available resources and diagnostic needs.

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.

Disability from Japanese encephalitis in Cambodia and Viet Nam.

A cohort of Japanese encephalitis (JE) survivors in Cambodia and Viet Nam were assessed at least 4 months after hospital discharge in order to understand the extent of disability after JE. We used a simple assessment tool which focuses on the impact on daily life. In total, 64 disability assessments were conducted: 38 in Cambodia and 26 in Viet Nam. In Cambodia, 4 (11%) children had severe sequelae, suggesting the children would likely be dependent, 15 (39%) had moderate sequelae and 17 (45%) had mild sequelae. In Viet Nam, two (8%) persons had severe sequelae, five (19%) had moderate sequelae and eight (31%) had mild sequelae. In many JE-endemic areas there are no multi-disciplinary teams with sophisticated equipment to assess patients after JE disease. This assessment tool can assist with patient management and generate data to support the need for programmes to prevent disease and improve outcomes for survivors.

The Pandemic Experience in Southeast Asia: Interface Between SARS-CoV-2, Malaria, and Dengue.

Southeast Asia (SEA) emerged relatively unscathed from the first year of the global SARS-CoV-2 pandemic, but as of July 2021 the region is experiencing a surge in case numbers primarily driven by Alpha (B.1.1.7) and subsequently the more transmissible Delta (B.1.617.2) variants. While initial disease burden was mitigated by swift government responses, favorable cultural and societal factors, the more recent rise in cases suggests an under-appreciation of prior prevalence and over-appreciation of possible cross-protective immunity from exposure to endemic viruses, and highlights the effects of vaccine rollout at varying tempos and of variable efficacy. This burgeoning crisis is further complicated by co-existence of malaria and dengue in the region, with implications of serological cross-reactivity on interpretation of SARS-CoV-2 assays and competing resource demands impacting efforts to contain both endemic and pandemic disease.

Evaluation of an influenza-like illness case definition in the diagnosis of influenza among patients with acute febrile illness in Cambodia.

BACKGROUND: Influenza-like illness (ILI) is often defined as fever (>38.0°C) with cough or sore throat. In this study, we tested the sensitivity, specificity, and positive and negative predictive values of this case definition in a Cambodia patient population. METHODS: Passive clinic-based surveillance was established at nine healthcare centers to identify the causes of acute undifferentiated fever in patients aged two years and older seeking treatment. Fever was defined as tympanic membrane temperature >38°C lasting more than 24 hours and less than 10 days. Influenza virus infections were identified by polymerase chain reaction. RESULTS: From July 2008 to December 2008, 2,639 patients were enrolled. From 884 (33%) patients positive for influenza, 652 presented with ILI and 232 acute fever patients presented without ILI. Analysis by age group identified no significant differences between influenza positive patients from the two groups. Positive predictive values (PPVs) varied during the course of the influenza season and among age groups. CONCLUSION: The ILI case definition can be used to identify a significant percentage of patients with influenza infection during the influenza season in Cambodia, assisting healthcare providers in its diagnosis and treatment. However, testing samples based on the criteria of fever alone increased our case detection by 34%.

Rapid metagenomic characterization of a case of imported COVID-19 in Cambodia.

Rapid production and publication of pathogen genome sequences during emerging disease outbreaks provide crucial public health information. In resource-limited settings, especially near an outbreak epicenter, conventional deep sequencing or bioinformatics are often challenging. Here we successfully used metagenomic next generation sequencing on an iSeq100 Illumina platform paired with an open-source bioinformatics pipeline to quickly characterize Cambodia's first case of COVID-2019.

Cholera in selected countries in Asia.

INTRODUCTION: Although the current pandemic of cholera originated in Asia, reports of cholera cases and outbreaks in the region are sparse. To provide a sub-regional assessment of cholera in South and Southeast Asia, we collated published and unpublished data from existing surveillance systems from Bangladesh, Cambodia, India, Malaysia, Nepal, Pakistan, Philippines, Thailand and Vietnam. METHODS: Data from existing country surveillance systems on diarrhea, acute watery diarrhea, suspected cholera and/or confirmed cholera in nine selected Asian countries (Bangladesh, Cambodia, India, Malaysia, Nepal, Pakistan, Philippines, Thailand and Vietnam) from 2011 to 2015 (or 2016, when available) were collated. We reviewed annual cholera reports from WHO and searched PubMed and/or ProMED to complement data, where information is not completely available. RESULTS: From 2011 to 2016, confirmed cholera cases were identified in at least one year of the 5- or 6-year period in the countries included. Surveillance for cholera exists in most countries, but cases are not always reported. India reported the most number of confirmed cases with a mean of 5964 cases annually. The mean number of cases per year in the Philippines, Pakistan, Bangladesh, Malaysia, Nepal and Thailand were 760, 592, 285, 264, 148 and 88, respectively. Cambodia and Vietnam reported 51 and 3 confirmed cholera cases in 2011, with no subsequent reported cases. DISCUSSION AND CONCLUSION: We present consolidated results of available surveillance in nine Asian countries and supplemented these with publication searches. There is paucity of readily accessible data on cholera in these countries. We highlight the continuing existence of the disease even in areas with improved sanitation and access to safe drinking water. Continued vigilance and improved surveillance in countries should be strongly encouraged.

Epidemiology and burden of disease from Japanese encephalitis in Cambodia: results from two years of sentinel surveillance.

OBJECTIVES: To describe the results from two years of Japanese encephalitis (JE) sentinel surveillance in Cambodia. METHODS: Sentinel site surveillance for JE in children aged 15 years and under was implemented in Cambodia in mid-2006. It was integrated into the routine meningoencephalitis surveillance system. Six hospitals were selected as sentinel sites. Epidemiological information and diagnostic specimens were collected from each patient presenting with meningoencephalitis. Cerebrospinal fluid and sera were tested for presence of immunoglobulin M antibodies against JE and dengue viruses by an ELISA. Surveillance data from 2006 to 2008 were analysed. RESULTS: Of 586 patients presenting with meningoencephalitis, 110 (19%) were confirmed to have JE. The percentage of confirmed JE cases at individual sentinel sites ranged from 13% to 35% of all meningoencephalitis cases. Mean age was 6.2 years, with 95% of JE cases in children aged 12 years and under. Cases occurred year-round in both 12-month reporting periods. CONCLUSIONS: JE is an important cause of meningoencephalitis in Cambodian children. As JE is a vaccine-preventable disease, an immunization programme could result in a considerable reduction in morbidity and mortality from JE among children in Cambodia.

Descriptive assessment of rabies post-exposure prophylaxis procurement, distribution, monitoring, and reporting in four Asian countries: Bangladesh, Bhutan, Cambodia, and Sri Lanka, 2017-2018.

BACKGROUND: There are approximately 35,000 human deaths from rabies in Asia annually. Rabies can be prevented through timely post-exposure prophylaxis (PEP) consisting of wound washing, rabies vaccine, and in some cases, rabies immunoglobulin (RIG). However, access to rabies PEP often remains limited to urban areas and is cost-prohibitive. There is little information on procurement, distribution, monitoring, and reporting of rabies PEP. METHODS: We interviewed key informants in the public sector from various levels in Bangladesh, Bhutan, Cambodia, and Sri Lanka between March 2017 and May 2018 using a descriptive assessment tool to obtain information on procurement, distribution, monitoring, and reporting of rabies PEP. These four countries in Asia were chosen to showcase a range of rabies PEP systems. National rabies focal points were interviewed in each country and focal points helped identify additional key informants at lower levels. RESULTS: A total of 22 key informants were interviewed at various levels (central level to health facility level) including national rabies focal points in each country. Each country has a unique system for managing rabies PEP procurement, distribution, monitoring, and reporting. There are varying levels of PEP access for those with potential rabies exposures. Rabies PEP is available in select health facilities throughout the country in Bangladesh, Bhutan, and Sri Lanka. In Cambodia, rabies PEP is limited to two urban centers. The availability of RIG in all four countries is limited. In these four countries, most aspects of the rabies PEP distribution system operate independently of systems for other vaccines. However, in Bhutan, rabies PEP and Expanded Programme on Immunization (EPI) vaccines share cold chain space in some locations at the lowest level. All countries have a monitoring system in place, but there is limited reporting of data, particularly to the central level. CONCLUSION: Systems to procure, deliver, monitor, and report on rabies PEP are variable across countries. Sharing information on practices more widely among countries can help programs to increase access to this life-saving treatment.

Data quality and timeliness of outbreak reporting system among countries in Greater Mekong subregion: Challenges for international data sharing.

Cross-border disease transmission is a key challenge for prevention and control of outbreaks. Variation in surveillance structure and national guidelines used in different countries can affect their data quality and the timeliness of outbreak reports. This study aimed to evaluate timeliness and data quality of national outbreak reporting for four countries in the Mekong Basin Disease Surveillance network (MBDS). Data on disease outbreaks occurring from 2010 to 2015 were obtained from the national disease surveillance reports of Cambodia, Lao PDR, Myanmar, and Vietnam. Data included total cases, geographical information, and dates at different timeline milestones in the outbreak detection process. Nine diseases or syndromes with public health importance were selected for the analysis including: dengue, food poisoning & diarrhea, severe diarrhea, diphtheria, measles, H5N1 influenza, H1N1 influenza, rabies, and pertussis. Overall, 2,087 outbreaks were reported from the four countries. The number of outbreaks and number of cases per outbreak varied across countries and diseases, depending in part on the outbreak definition used in each country. Dates on index onset, report, and response were >95% complete in all countries, while laboratory confirmation dates were 10%-100% incomplete in most countries. Inconsistent and out of range date data were observed in 1%-5% of records. The overall timeliness of outbreak report, response, and public communication was within 1-15 days, depending on countries and diseases. Diarrhea and severe diarrhea outbreaks showed the most rapid time to report and response, whereas diseases such as rabies, pertussis and diphtheria required a longer time to report and respond. The hierarchical structure of the reporting system, data collection method, and country's resources could affect the data quality and timeliness of the national outbreak reporting system. Differences in data quality and timeliness of outbreak reporting system among member countries should be considered when planning data sharing strategies within a regional network.

Influenza A/H5N1 virus infection in humans in Cambodia.

BACKGROUND: Between January 2005 and April 2006, six patients of influenza A/H5N1 virus infection were reported in Cambodia, all with fatal outcome. OBJECTIVES: We describe the virological findings of these six H5N1 patients in association with clinical and epidemiologic findings. STUDY DESIGN: Broncho-alveolar lavage, nasopharyngeal, throat and rectal swabs and sera were cultured for virus isolation and viral load quantified in clinical specimens by real-time RT-PCR. We compared sequences obtained from different body sites within the same patient to detect viral quasi-species. RESULTS: H5N1 virus strains isolated in Cambodia belong to genotype Z, clade 1 viruses. H5N1 viruses were isolated from serum and rectal swab specimens in two patients. The haemagglutinin gene sequences of the virus in different body sites did not differ. Amino acid substitutions known to be associated with a change in virus binding were not observed. CONCLUSION: The high frequency of virus isolation from serum and faecal swabs highlights that H5N1 is likely to be a disseminated infection in humans and this has implications for antiviral treatment, biosafety in clinical laboratories and on risks for nosocomial and human-to-human transmission. There were no tissue-specific adaptive mutations in the HA gene from viruses isolated from different organs.

Complete Genome Sequences of Five Zika Virus Isolates.

Zika virus is an emerging human pathogen of great concern due to putative links to microcephaly and Guillain-Barre syndrome. Here, we report the complete genomes, including the 5' and 3' untranslated regions, of five Zika virus isolates, one from the Asian lineage and four from the African lineage.

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.