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Background There is growing recognition of metagenomic next-generation sequencing (mNGS) as a valuable diagnostic tool capable of providing unbiased pathogen detection, but data on performance in low-resource settings remains scant. Here, we use mNGS of nasopharyngeal (NP) swabs taken from subjects in Cambodia to identify potential pathogens causing acute febrile illness. Methods Febrile subjects aged 2 months to 65 years were enrolled in a cross-sectional study conducted across 4 tertiary hospitals in Cambodia. NP swabs were collected at hospital presentation. Depending on reported symptom constellations, sera was also taken in a subset of subjects for comparison of mNGS results. RNA was isolated from biosamples, converted to cDNA libraries, and sequenced on a NextSeq2000 (Illumina). Raw sequence reads were stripped for host reads and aligned to NCBI nucleotide and protein databases using a cloud-based bioinformatics platform (CZID). Results NP swabs were collected from 97 subjects between April 2020 and June 2021. Subjects were predominantly male (53.6%) and young (median age 3 years [IQR 1-25]). Pathogens were identified in 42 (43.2%) NP swabs; of these, 26 (61.9%) were respiratory viruses including 9 rhinovirus, 7 coronavirus (1 SARS-CoV-2), and 5 respirovirus cases. Co-infection was identified in 3 subjects with coronavirus and respirovirus (N=2) and coronavirus and rhinovirus (N=1). Of subjects with paired sera and NP samples (N=61), 18 (29.5%) had positive NP swabs but negative sera, 7 (11.5%) had negative NP swabs but positive sera, 12 (19.7%) had positive NP swabs and sera, and 24 (39.3%) had negative NP swabs and sera. Pathogen hits correlated in NP swabs and sera in 10 of 12 subjects, including six subjects with chikungunya. Conclusion mNGS can be successfully implemented in low-resource settings to identify emerging pathogens and common respiratory pathogens, including co-infecting pathogens, from NP swabs of febrile patients. mNGS may also be able to detect chikungunya from NP swab alone, raising the possibility of non-invasive diagnostics for infections associated with high viremic states. Disclosures All Authors: No reported disclosures.

Information on causative diarrheal pathogens and their associated antimicrobial susceptibility remains limited for Cambodia. This study describes antimicrobial resistance patterns for Shigella and nontyphoidal Salmonella isolates collected in Cambodia over a 5-year period. Multidrug resistance was shown in 98% of Shigella isolates, with 70%, 11%, and 29% of isolates being resistant to fluoroquinolones, azithromycin, and cephalosporin, respectively. As many as 11% of Shigella isolates were resistant to nearly all oral and parenteral drugs typically used for shigellosis, demonstrating extreme drug resistance phenotypes. Although a vast majority of nontyphoidal Salmonella isolates remained susceptible to cephalosporins (99%) and macrolides (98%), decreased susceptibility to ciprofloxacin was found in 67% of isolates, which is notably higher than previous reports. In conclusion, increasing antimicrobial resistance of Shigella and nontyphoidal Salmonella is a major concern for selecting empirical treatment of acute infectious diarrhea in Cambodia. Treatment practices should be updated and follow local antimicrobial resistance data for the identified pathogens.

Information on causative diarrheal pathogens and their associated antimicrobial susceptibility remains limited for Cambodia. This study describes antimicrobial resistance patterns for Shigella and nontyphoidal Salmonella isolates collected in Cambodia over a 5-year period. Multidrug resistance was shown in 98% of Shigella isolates, with 70%, 11%, and 29% of isolates being resistant to fluoroquinolones, azithromycin, and cephalosporin, respectively. As many as 11% of Shigella isolates were resistant to nearly all oral and parenteral drugs typically used for shigellosis, demonstrating extreme drug resistance phenotypes. Although a vast majority of nontyphoidal Salmonella isolates remained susceptible to cephalosporins (99%) and macrolides (98%), decreased susceptibility to ciprofloxacin was found in 67% of isolates, which is notably higher than previous reports. In conclusion, increasing antimicrobial resistance of Shigella and nontyphoidal Salmonella is a major concern for selecting empirical treatment of acute infectious diarrhea in Cambodia. Treatment practices should be updated and follow local antimicrobial resistance data for the identified pathogens.

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.

BACKGROUND: Global influenza virus circulation decreased during the COVID-19 pandemic, possibly due to widespread community mitigation measures. Cambodia eased some COVID-19 mitigation measures in June and July 2020. On 20 August a cluster of respiratory illnesses occurred among residents of a pagoda, including people who tested positive for influenza A but none who were positive for SARS-CoV-2. METHODS: A response team was deployed on 25 August 2020. People with influenza-like illness (ILI) were asked questions regarding demographics, illness, personal prevention measures, and residential arrangements. Respiratory swabs were tested for influenza and SARS-Cov-2 by real-time reverse transcription PCR, and viruses were sequenced. Sentinel surveillance data were analyzed to assess recent trends in influenza circulation in the community. RESULTS: Influenza A (H3N2) viruses were identified during sentinel surveillance in Cambodia in July 2020 prior to the reported pagoda outbreak. Among the 362 pagoda residents, 73 (20.2%) ILI cases were identified and 40 were tested, where 33/40 (82.5%) confirmed positive for influenza A (H3N2). All 40 were negative for SARS-CoV-2. Among the 73 residents with ILI, none were vaccinated against influenza, 47 (64%) clustered in 3/8 sleeping quarters, 20 (27%) reported often wearing a mask, 27 (36%) reported often washing hands, and 11 (15%) reported practicing social distancing. All viruses clustered within clade 3c2.A1 close to strains circulating in Australia in 2020. CONCLUSIONS: Circulation of influenza viruses began in the community following the relaxation of national COVID-19 mitigation measures, and prior to the outbreak in a pagoda with limited social distancing. Continued surveillance and influenza vaccination are required to limit the impact of influenza globally.

We regret that this article is behind a paywall., Safe and efficacious pan-genotypic direct-acting antiviral (DAA) regimens, such as sofosbuvir and daclatasvir (SOF+DCV)facilitate simplified models of care for hepatitis C virus (HCV). However, in Cambodia access to HCV testing and treatment has typically been low. In response, Médecins Sans Frontières(MSF) implemented a HCV testing and treatment pilot project in Phnom Penh, Cambodia in 2016. This project provides the first real-world evidence of SOF+DCV effectiveness across a large patient cohort using a simplified care model in Cambodia.Patients treated with SOF+DCV from September 2016 to June 2019 were included in the analysis. Medical standard operational procedures (SOPs) were simplified significantly across the study period. Treatment effectiveness was assessed by sustained viral response at 12 weeks post-treatment (SVR12) according to a modified intention to treat methodology. Treatment safety was assessed by clinical outcome and occurrence of serious and non-serious adverse events (S/AE). Of 9158 patients, median age was 57 years and 39.6% were male. At baseline assessment, 27.2% of patients had compensated cirrhosis and 2.9% had decompensated cirrhosis. Genotype 6 was predominant (53.0%). Among patients analysed according to modified intention to treat (n=8525), treatment effectiveness was high, with 97.2% of patients achieving SVR12. Occurrence of SAE was low (0.7%). Treatment effectiveness and safety was not affected by the iterative simplification to treatment modality. In conclusion, in this large treatment cohort in Phnom Penh, Cambodia, the SOF+DCV regimen showed high rates of treatment effectiveness and safety across patient sub-groups and during progressive simplification.

Introduction: The burden of influenza in Cambodia is not well known, but it would be useful for understanding the impact of seasonal epidemics and pandemics and to design appropriate policies for influenza prevention and control. The severe acute respiratory infection (SARI) surveillance system in Cambodia was used to estimate the national burden of SARI hospitalizations in Cambodia. Methods: We estimated age-specific influenza-associated SARI hospitalization rates in three sentinel sites in Svay Rieng, Siem Reap and Kampong Cham provinces. We used influenza-associated SARI surveillance data for one year to estimate the numerator and hospital admission surveys to estimate the population denominator for each site. A national influenza-associated SARI hospitalization rate was calculated using the pooled influenza-associated SARI hospitalizations for all sites as a numerator and the pooled catchment population of all sites as denominator. National influenza-associated SARI case counts were estimated by applying hospitalization rates to the national population. Results: The national annual rates of influenza-associated hospitalizations per 100 000 population was highest for the two youngest age groups at 323 for < 1 year and 196 for 1–4 years. We estimated 7547 influenza-associated hospitalizations for Cambodia with almost half of these represented by children younger than 5 years. Discussion: We present national estimates of influenza-associated SARI hospitalization rates for Cambodia based on sentinel surveillance data from three sites. The results of this study indicate that the highest burden of severe influenza infection is borne by the younger age groups. These findings can be used to guide future strategies to reduce influenza morbidity.

Background Understanding the burden of influenza‐associated severe acute respiratory infection (SARI) is important for setting national influenza surveillance and vaccine priorities. Estimating influenza‐associated SARI rates requires hospital‐based surveillance data and a population‐based denominator, which can be challenging to determine. Objectives We present an application of the World Health Organization's recently developed manual (WHO Manual) including hospital admission survey (HAS) methods for estimating the burden of influenza‐associated SARI, with lessons learned to help others calculate similar estimates. Methods Using an existing SARI surveillance platform in Cambodia, we counted influenza‐associated SARI cases during 2015 at one sentinel surveillance site in Svay Rieng Province. We applied WHO Manual‐derived methods to count respiratory hospitalizations at all hospitals within the catchment area, where 95% of the sentinel site case‐patients resided. We used HAS methods to adjust the district‐level population denominator for the sentinel site and calculated the incidence rate of influenza‐associated SARI by dividing the number of influenza‐positive SARI infections by the adjusted population denominator and multiplying by 100 000. We extrapolated the rate to the provincial population to derive a case count for 2015. We evaluated data sources, detailed steps of implementation, and identified lessons learned. Results We estimated an adjusted influenza‐associated 2015 SARI rate of 13.5/100 000 persons for the catchment area of Svay Rieng Hospital and 77 influenza‐associated SARI cases in Svay Rieng Province after extrapolation. Conclusions Methods detailed in the WHO Manual and operationalized successfully in Cambodia can be used in other settings to estimate rates of influenza‐associated SARI.

Objective: To establish seasonal and alert thresholds and transmission intensity categories for influenza to provide timely triggers for preventive measures or upscaling control measures in Cambodia. Methods: Using Cambodia's influenza-like illness (ILI) and laboratory-confirmed influenza surveillance data from 2009 to 2015, three parameters were assessed to monitor influenza activity: the proportion of ILI patients among all outpatients, proportion of ILI samples positive for influenza and the product of the two. With these parameters, four threshold levels (seasonal, moderate, high and alert) were established and transmission intensity was categorized based on a World Health Organization alignment method. Parameters were compared against their respective thresholds. Results: Distinct seasonality was observed using the two parameters that incorporated laboratory data. Thresholds established using the composite parameter, combining syndromic and laboratory data, had the least number of false alarms in declaring season onset and were most useful in monitoring intensity. Unlike in temperate regions, the syndromic parameter was less useful in monitoring influenza activity or for setting thresholds. Conclusion: Influenza thresholds based on appropriate parameters have the potential to provide timely triggers for public health measures in a tropical country where monitoring and assessing influenza activity has been challenging. Based on these findings, the Ministry of Health plans to raise general awareness regarding influenza among the medical community and the general public. Our findings have important implications for countries in the tropics/subtropics and in resource-limited settings, and categorized transmission intensity can be used to assess severity of potential pandemic influenza as well as seasonal influenza.

International audience; Background: Influenza virus circulation is monitored through the Cambodian in- fluenza-like illness (ILI) sentinel surveillance system and isolates are characterized by the National Influenza Centre (NIC). Seasonal influenza circulation has previ- ously 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 characteris- tics of influenza isolates.Patients/Methods: Respiratory samples were collected from patients presenting with influenza-like illness (ILI) at 11 hospitals throughout Cambodia. First-line screen- ing was conducted by the National Institute of Public Health and the Armed Forces Research Institute of Medical Sciences. Confirmation of testing and genetic, anti- genic 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 in- fluenza 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 de- scribed, coincident with the emergence of A(H3N2) clades 3C.2a and 3C.3a, respec- tively. 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 north- ern and southern hemisphere vaccine formulations. All isolates tested were suscepti- ble to the influenza antiviral drugs oseltamivir and zanamivir.Conclusions: Seasonal and year-round co-circulation of multiple influenza types/sub- types were detected in Cambodia during 2012-2015.

Objectives: Following the launch of the Global Antimicrobial Resistance Surveillance System (GLASS), antimicrobial resistance (AMR) rates in many countries remain poorly described. This review provides an overview of published AMR data from Cambodia in the context of recently initiated national human and food-animal surveillance. Methods: PubMed and the Cochrane Database of Systematic Reviews were searched for articles published from 2000 to 2018, which reported antimicrobial susceptibility testing (AST) data for GLASS specific organisms isolated from Cambodia. Articles were screened using strict inclusion/exclusion criteria. AST data was extracted, with medians and ranges of resistance rates calculated for specific bug-drug combinations. Results: Twenty-four papers were included for final analysis, with 20 describing isolates from human populations. Escherichia coli was the most commonly described organism, with median resistance rates from human isolates of 92.8% (n = 6 articles), 46.4% (n = 4), 55.4% (n = 8), and 46.4% (n = 5) to ampicillin, 3rd generation cephalosporins, fluoroquinolones, and gentamicin respectively. Conclusions: Whilst resistance rates are high for several GLASS organisms, there were insufficient data to draw robust conclusions about the AMR situation in Cambodia. The recently implemented national AMR surveillance systems will begin to address this data gap. Keywords: Antimicrobial resistance, Cambodia, Bacteria, Review, One Health

International audience; In a 10-year incidence survey of breast cancer in the City of Boston, 14 cases in American Chinese women were observed, while approximately 13 were expected on the basis of age-specific incidence in the white population. In Hawaii, on the other hand, while breast cancer incidence rates for ethnic Chinese are approximately three times as high as those of indigenous Chinese populations, data indicate that they are still 35% lower than those of whites in Hawaii or on the US mainland. We have compared estrogen concentrations in the urine of Chinese women in the Orient, Honolulu, and Boston. Levels of estrone (E1) and estradiol (E2) increased greatly from the low to the high risk group. The increase was evident among both the younger (15-19 years) and the older (20-24 years) women, in both follicular and luteal phase specimens. The crude unweighted average increase from Oriental to continental US Chinese was about 38% and 34% for E1 and E2, respectively. On the other hand, levels of E3 decreased from the low-risk to the high-risk group, but the decrease was modest (16%) and not always regular. There were no systematic or significant differences in the frequency of ovulation in the three groups of women. These results are compatible with the hypothesis that high levels of estrone and estradiol are important to breast carcinogenesis. Whether E3 has some additional "protective" effect or is irrelevant is unclear. The data do not support the hypothesis that total urine estrogens (E1 + E2 + E3) are predictive of population groups at high risk for breast cancer and they appear inconsistent with the hypothesis that frequency of ovulation (or anovulation) is an important aspect of breast carcinogenesis.

International audience; Rabies causes 60,000 deaths worldwide annually. Rabies post-exposure prophylaxis is highly effective but often geographically and financially beyond reach in endemic developing countries. We conducted a retrospective study on clinical outcome at ≥6 months in 3318 Cambodians who received intradermal Vero cell vaccine post-exposure prophylaxis after a bite by a rabid or sick-looking but untested dog in 2003-2014. An external expert panel examined verbal autopsy reports to identify rabies deaths. 1739 (93.65%) persons bitten by rabid- and 1066 (72.96%) bitten by sick-looking but untested dogs were traced and 513 were lost to follow-up. Among the former, 1591 (91.49%) and 129 (7.42%) patients referred for 4+ and 3 post-exposure prophylaxis sessions, respectively. Three persons died of probable rabies so that the overall percentage of survival was 99.83% (95% exact confidence interval: 99.49-99.96%) in post-exposure prophylaxis recipients bitten by confirmed rabid dogs. No significant difference was found in survival among patients who received 3 vs. 4+ sessions (with or without rabies immunoglobin). The power of the study, however, was limited. The current four sessions/one month intradermal regimen can be reduced to a three sessions/one week at no detectable added risk to patients, with the limitation of study power at 49%. A clinical follow-up system should be adopted by rabies prevention centers, especially to monitor implementation of an abridged course. The Institut Pasteur in Cambodia regimen will improve vaccine equity by treating 33% more patients with available doses, reduce direct cost of vaccination, transportation and other indirect costs to vaccinees.

Helicobacter fennelliae (H. fennelliae) is associated with human gastroenteritis; however, H. fennelliae was isolated and confirmed by phenotypic and genotypic identification from a non-diarrheal child stool sample in Cambodia. Antimicrobial susceptibility testing demonstrated that this isolate had a high minimal inhibitory concentration against macrolides and quinolones, which are first-line antibiotic treatment choices for Campylobacter infections. Consequently, macrolides and quinolones were likewise expected to be ineffective against Campylobacter-like organisms such as H. fennelliae. This isolate warranted further genetic characterization to better understand associated antibiotic resistance mechanisms. Resistant pathogens from asymptomatic diarrheal cases are likely underestimated, and as such colonized individuals may spread resistant organisms to local community members and the environment.

International audience; Postexposure prophylaxis (PEP) prevents human rabies and is accessible in Cambodia principally in Phnom Penh, the capital. Timely, affordable access to PEP is a challenge for the mainly rural population. We aimed to identify districts independently associated with PEP noncompletion to position frontline vaccination centers. We analyzed the 2009–2013 database at the Rabies Prevention Center at the Institut Pasteur du Cambodge, Phnom Penh. Logistic regressions identified nongeographic determinants of PEP noncompletion as well as the districts that were independently associated with noncompletion after adjustment for these determinants. The influence of distance by road was estimated using a boosted regression-trees model. We computed a population attributable fraction (rabies index (RI)) for each district and developed a map of this RI distribution. A cartographic analysis based on the statistic developed by Getis and Ord identified clusters of high-RI districts. Factors independently associated with noncompletion were pa-tients' district of residence, male sex, age 15–49 years, initial visit during rice harvest, the dog's status (culled or disappeared), and a prescribed PEP protocol requiring more than 3 PEP sessions (4 or 5). Four clusters of high-RI districts were identified using this analytical strategy, which is applicable to many vaccination or other health services. Positioning frontline PEP centers in these districts could significantly widen access to timely and adequate PEP.