Search

Summary: Background: Encephalitis is a worldwide public health issue, with a substantially high burden among children in southeast Asia. We aimed to determine the causes of encephalitis in children admitted to hospitals across the Greater Mekong region by implementing a comprehensive state-of-the-art diagnostic procedure harmonised across all centres, and identifying clinical characteristics related to patients’ conditions. Methods: In this multicentre, observational, prospective study of childhood encephalitis, four referral hospitals in Cambodia, Vietnam, Laos, and Myanmar recruited children (aged 28 days to 16 years) who presented with altered mental status lasting more than 24 h and two of the following minor criteria: fever (within the 72 h before or after presentation), one or more generalised or partial seizures (excluding febrile seizures), a new-onset focal neurological deficit, cerebrospinal fluid (CSF) white blood cell count of 5 per mL or higher, or brain imaging (CT or MRI) suggestive of lesions of encephalitis. Comprehensive diagnostic procedures were harmonised across all centres, with first-line testing was done on samples taken at inclusion and results delivered within 24 h of inclusion for main treatable causes of disease and second-line testing was done thereafter for mostly non-treatable causes. An independent expert medical panel reviewed the charts and attribution of causes of all the included children. Using multivariate analyses, we assessed risk factors associated with unfavourable outcomes (ie, severe neurological sequelae and death) at discharge using data from baseline and day 2 after inclusion. This study is registered with ClinicalTrials.gov, NCT04089436, and is now complete. Findings: Between July 28, 2014, and Dec 31, 2017, 664 children with encephalitis were enrolled. Median age was 4·3 years (1·8–8·8), 295 (44%) children were female, and 369 (56%) were male. A confirmed or probable cause of encephalitis was identified in 425 (64%) patients: 216 (33%) of 664 cases were due to Japanese encephalitis virus, 27 (4%) were due to dengue virus, 26 (4%) were due to influenza virus, 24 (4%) were due to herpes simplex virus 1, 18 (3%) were due to Mycobacterium tuberculosis, 17 (3%) were due to Streptococcus pneumoniae, 17 (3%) were due to enterovirus A71, 74 (9%) were due to other pathogens, and six (1%) were due to autoimmune encephalitis. Diagnosis was made within 24 h of admission to hospital for 83 (13%) of 664 children. 119 (18%) children had treatable conditions and 276 (42%) had conditions that could have been preventable by vaccination. At time of discharge, 153 (23%) of 664 children had severe neurological sequelae and 83 (13%) had died. In multivariate analyses, risk factors for unfavourable outcome were diagnosis of M tuberculosis infection upon admission (odds ratio 3·23 [95% CI 1·04–10·03]), coma on day 2 (2·90 [1·78–4·72]), supplementary oxygen requirement (1·89 [1·25–2·86]), and more than 1 week duration between symptom onset and admission to hospital (3·03 [1·68–5·48]). At 1 year after inclusion, of 432 children who were discharged alive from hospital with follow-up data, 24 (5%) had died, 129 (30%) had neurological sequelae, and 279 (65%) had completely recovered. Interpretation: In southeast Asia, most causes of childhood encephalitis are either preventable or treatable, with Japanese encephalitis virus being the most common cause. We provide crucial information that could guide public health policy to improve diagnostic, vaccination, and early therapeutic guidelines on childhood encephalitis in the Greater Mekong region. Funding: Institut Pasteur, Institut Pasteur International Network, Fondation Merieux, Aviesan Sud, INSERM, Wellcome Trust, Institut de Recherche pour le Développement (IRD), and Fondation Total.

Abstract Background In many endemic areas, Plasmodium vivax malaria is predominantly a disease of young adults and children. International recommendations for radical cure recommend fixed target doses of 0.25 or 0.5 mg/kg/day of primaquine for 14 days in glucose-6-phosphate dehydrogenase normal patients of all ages. However, for many anti-malarial drugs, including primaquine, there is evidence that children have lower exposures than adults for the same weight-adjusted dose. The aim of the study was to develop 14-day weight-based and age-based primaquine regimens against high-frequency relapsing tropical P. vivax. Methods The recommended adult target dose of 0.5 mg/kg/day (30 mg in a 60 kg patient) is highly efficacious against tropical P. vivax and was assumed to produce optimal drug exposure. Primaquine doses were calculated using allometric scaling to derive a weight-based primaquine regimen over a weight range from 5 to 100 kg. Growth curves were constructed from an anthropometric database of 53,467 individuals from the Greater Mekong Subregion (GMS) to define weight-for-age relationships. The median age associated with each weight was used to derive an age-based dosing regimen from the weight-based regimen. Results The proposed weight-based regimen has 5 dosing bands: (i) 5–7 kg, 5 mg, resulting in 0.71–1.0 mg/kg/day; (ii) 8–16 kg, 7.5 mg, 0.47–0.94 mg/kg/day; (iii) 17–40 kg, 15 mg, 0.38–0.88 mg/kg/day; (iv) 41–80 kg, 30 mg, 0.37–0.73 mg/kg/day; and (v) 81–100 kg, 45 mg, 0.45–0.56 mg/kg/day. The corresponding age-based regimen had 4 dosing bands: 6–11 months, 5 mg, 0.43–1.0 mg/kg/day; (ii) 1–5 years, 7.5 mg, 0.35–1.25 mg/kg/day; (iii) 6–14 years, 15 mg, 0.30–1.36 mg/kg/day; and (iv) ≥ 15 years, 30 mg, 0.35–1.07 mg/kg/day. Conclusion The proposed weight-based regimen showed less variability around the primaquine dose within each dosing band compared to the age-based regimen and is preferred. Increased dose accuracy could be achieved by additional dosing bands for both regimens. The age-based regimen might not be applicable to regions outside the GMS, which must be based on local anthropometric data. Pharmacokinetic data in small children are needed urgently to inform the proposed regimens.

Abstract Background In 2017, New Caledonia experienced an outbreak of severe dengue causing high hospital burden (4379 cases, 416 hospital admissions, 15 deaths). We decided to build a local operational model predictive of dengue severity, which was needed to ease the healthcare circuit. Methods We retrospectively analyzed clinical and biological parameters associated with severe dengue in the cohort of patients hospitalized at the Territorial Hospital between January and July 2017 with confirmed dengue, in order to elaborate a comprehensive patient’s score. Patients were compared in univariate and multivariate analyses. Predictive models for severity were built using a descending step-wise method. Results Out of 383 included patients, 130 (34%) developed severe dengue and 13 (3.4%) died. Major risk factors identified in univariate analysis were: age, comorbidities, presence of at least one alert sign, platelets count 10 N, and previous dengue infection. Severity was not influenced by the infecting dengue serotype nor by previous Zika infection. Two models to predict dengue severity were built according to sex. Best models for females and males had respectively a median Area Under the Curve = 0.80 and 0.88, a sensitivity = 84.5 and 84.5%, a specificity = 78.6 and 95.5%, a positive predictive value = 63.3 and 92.9%, a negative predictive value = 92.8 and 91.3%. Models were secondarily validated on 130 patients hospitalized for dengue in 2018. Conclusion We built robust and efficient models to calculate a bedside score able to predict dengue severity in our setting. We propose the spreadsheet for dengue severity score calculations to health practitioners facing dengue outbreaks of enhanced severity in order to improve patients’ medical management and hospitalization flow.

Compared to the previous 2013–2014 outbreak, dengue 2016–2017 outbreak in New Caledonia was characterized by an increased number of severe forms associated with hepatic presentations. In this study, we assessed the virological factors associated with this enhanced severity. Whole-genome sequences were retrieved from dengue virus (DENV)-1 strains collected in 2013–2014 and from severe and non-severe patients in 2016–2017. Fitness, hepatic tropism and cytopathogenicity of DENV 2016–2017 strains were compared to those of 2013–2014 strains using replication kinetics in the human hepatic cell line HuH7. Whole-genome sequencing identified four amino acid substitutions specific to 2016–2017 strains and absent from 2013–2014 strains. Three of these mutations occurred in predicted T cell epitopes, among which one was also a B cell epitope. Strains retrieved from severe forms did not exhibit specific genetic features. DENV strains from 2016–2017 exhibited a trend towards reduced replicative fitness and cytopathogenicity in vitro compared to strains from 2013–2014. Overall, the 2016–2017 dengue outbreak in New Caledonia was associated with a viral genetic evolution which had limited impact on DENV hepatic tropism and cytopathogenicity. These mutations, however, may have modified DENV strains antigenicity, altering the anti-DENV immune response in some patients, in turn favoring the development of severe forms.Trial registration: ClinicalTrials.gov identifier: NCT04615364.

Abstract Background Mosquito vectors cause a significant human public health burden through the transmission of pathogens. Due to the expansion of international travel and trade, the dispersal of these mosquito vectors and the pathogens they carry is on the rise. Entomological surveillance is therefore required which relies on accurate mosquito species identification. This study aimed to optimize the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for mosquito identification. Methods Aedes aegypti of the Bora-Bora strain and 11 field-sampled mosquito species were used in this study. Analyses were performed to study the impact of the trapping duration on mosquito identification with MALDI-TOF MS. The best preservation methods to use for short, medium and long-term preservation before MALDI-TOF MS analysis were also assessed. In addition, the number of specimens per species required for MALDI-TOF MS database creation was determined. The first MALDI-TOF database of New Caledonian mosquitoes was assembled and the optimal threshold for mosquito species identification according to the sensitivity and specificity of this technique was determined. Results This study showed that the identification scores decreased as the trapping duration increased. High identification scores were obtained for mosquitoes preserved on silica gel and cotton at room temperature and those frozen at − 20 °C, even after two months of preservation. In addition, the results showed that the scores increased according to the number of main spectrum patterns (MSPs) used until they reached a plateau at 5 MSPs for Ae. aegypti. Mosquitoes (n = 67) belonging to 11 species were used to create the MALDI-TOF reference database. During blind test analysis, 96% of mosquitoes tested (n = 224) were correctly identified. Finally, based on MALDI-TOF MS sensitivity and specificity, the threshold value of 1.8 was retained for a secure identification score. Conclusions MALDI-TOF MS allows accurate species identification with high sensitivity and specificity and is a promising tool in public health for mosquito vector surveillance.

In Cambodia, dengue outbreaks occur each rainy season (May–October) but vary in magnitude. Using national surveillance data, we designed a tool that can predict 90% of the variance in peak magnitude by April, when typically

We investigated dengue virus (DENV) and asymptomatic DENV infections in rural villages of Kampong Cham Province, Cambodia, during 2012 and 2013. We conducted perifocal investigations in and around households for 149 DENV index cases identified through hospital and village surveillance. We tested participants 0.5–30 years of age by using nonstructural 1 rapid tests and confirmed DENV infections using quantitative reverse transcription PCR or nonstructural 1–capture ELISA. We used multivariable Poisson regressions to explore links between participants’ DENV infection status and household characteristics. Of 7,960 study participants, 346 (4.4%) were infected with DENV, among whom 302 (87.3%) were

With over one million cases worldwide annually and a high fatality in symptomatic forms, human leptospirosis is a growing public health concern for the most vulnerable populations, especially in the context of global warming and unplanned urbanization. Although the Asia-Pacific region is particularly affected, accurate epidemiological data are often lacking. We conducted an eleven-year retrospective laboratory-based epidemiological survey of human leptospirosis in New Caledonia. From 2006 to 2016, 904 cases were laboratory-confirmed, including 29 fatalities, corresponding to an average annual incidence of 30.6/100,000 and a case fatality rate of 3.2%. Over the period, there was a major shift from indirect serological diagnosis by MAT to direct diagnosis by real-time PCR, a more specific and sensitive test when performed early in the course of the disease. The systematic implementation of genotyping informed on the variety of the infective strains involved, with a predominance of serogroups Icterohaemorrhagiae and Pyrogenes. The epidemiological pattern showed a marked seasonality with an annual peak in March-April. Interestingly, the seasonal peak in children of school age was significantly earlier and corresponded to school holidays, suggesting that attending school from February on could protect children from environment-borne leptospirosis.

Abstract Zika virus (ZIKV) is a Flavivirus that is transmitted to humans by Aedes mosquitoes. ZIKV is divided into two phylogenetic lineages, African and Asian. In the Asian lineage, Pacific and American clades have been linked to the recent worldwide outbreak of ZIKV. The aim of this study was to measure the vector competence of Aedes aegypti for seven ZIKV strains belonging to both lineages. We demonstrate that Ae. aegypti from New Caledonia (NC), South Pacific region, is a low-competence vector for Asian ZIKV (37% transmission efficiency after 9 days post-infection) compared to recent ZIKV isolates from African (10% transmission efficiency) and Asian lineages (

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.

Although antibiotics are too often used inappropriately in Cambodia, published data on antimicrobial resistance in this country are scarce. Epidemic dissemination and the transfer of resistance genes to other bacterial species put the population at risk. The aim of this study was to evaluate the frequency and characteristics of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) isolated in consecutive samples tested at Institut Pasteur du Cambodge over a 4-year period (2012–2015). Antimicrobial susceptibility testing was performed by disk diffusion on agar technique and the results were read automatically using an OSIRIS system. The Etest was used to determine minimum inhibitory concentrations (MIC) for some resistance phenotypes. The strain most commonly identified was Escherichia coli (63.9%). The proportion of ESBL-E increased gradually over the study period, from 23.8% to 38.4%. ESBL was detected in 42.7% of the E. coli strains and 33.7% of all Klebsiella pneumoniae isolated. The proportion of ESBL-producing E. coli increased significantly from 28.9% in 2012 to 48.2% in 2015, while the increase for K. pneumoniae remained non-significant. Multidrug resistance was high in this Cambodian series, with some strains displaying resistance to all antibiotics available in the country. There is currently no established system for the surveillance of antimicrobial resistance in Cambodia. Collecting samples from clinical settings throughout the country is critical to assess the impact of antimicrobial drug use in patients in Cambodia and in the Mekong Region.

Objectives: In this study, we document the clinical characteristics and investigated risk factors for uncomplicated and severe forms of EV-A71 disease in Cambodian children. Methods: From March to July 2014 inclusive, all patients with suspicion of EV-A71 infection presenting to Kantha Bopha Hospitals in Phnom Penh and Siem Reap and confirmed by the Virology Unit at the Institut Pasteur du Cambodge were prospectively enrolled in this study. Throat swabs, rectal swabs and serum samples were collected from all consecutive patients with suspected EV-A71 infection. In addition, CSF was also collected from patients with suspected EV-A71 associated encephalitis. A total of 122 patients (29 with uncomplicated disease and 93 with severe disease) with confirmed EV-A71 infection with all available demographic and clinical data for clinical classification and further analysis were included in the study. Results: In this prospective EV-A71 study in Cambodia, we confirmed the previously reported association of male gender and absence of mouth or skin lesions with severe disease. We also highlighted the strong association of neutrophils in blood, but also in CSF in patients with pulmonary oedema. More importantly, we identified new putative nutrition-related risk factors for severe disease. Conclusions: EV-A71 is an important cause of encephalitis in the Asia-Pacific region. Further studies to determine the risk factors associated with severe EV-A71 disease are needed.

We describe a retrospective study on circulation of Zika virus in Cambodia during 2007–2016 among patients with dengue-like symptoms and Aedes aegypti mosquitoes. Our findings suggest that Zika virus in Cambodia belongs to the Asia genotype, is endemic, has low prevalence, and has had low-level impact on public health.

Objectives: This study aimed to investigate proteinuria occurring during dengue disease in children and assess if measurement of this parameter can help physicians in the clinical management of patients. Methods: Proteinuria was assessed by dipstick and quantified by urine protein:creatinine ratio (UPCR) in samples from patients hospitalized with a confirmed dengue infection and in healthy controls. Results: The dipstick tested positive in 42.9% of the patients presenting at hospital with dengue versus 20.0% in healthy controls. UPCR increased during the critical phase of the disease; peaking one week after fever onset then decreasing as the patients recovered. Patients with warnings signs or severe dengue were more likely to present with proteinuria detected by UPCR at the time of hospital admission compared to patients without warning signs. The sensitivity of this marker, however, was limited as only 16.1% of the patients with warning signs had proteinuria. Conclusions: Urine dipstick and UPCR do not seem to be very valuable for the triage of the patients at the time of the initial consultation but the observation of a decrease of the UPCR during the course of the illness appears to indicate an evolution towards recovery.

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.

Acute meningoencephalitis (AME) is associated with considerable morbidity and mortality in children in developing countries. Clinical specimens were collected from children presenting with AME at two Cambodian paediatric hospitals to determine the major aetiologies associated with AME in the country. Cerebrospinal fluid (CSF) and blood samples were screened by molecular and cell culture methods for a range of pathogens previously associated with AME in the region. CSF and serum (acute and convalescent) were screened for antibodies to arboviruses such as Japanese encephalitis virus (JEV), dengue virus (DENV), and chikungunya virus (CHIKV). From July 2010 through December 2013, 1160 children (one month to 15 years of age) presenting with AME to two major paediatric hospitals were enroled into the study. Pathogens associated with AME were identified using molecular diagnostics, cell culture and serology. According to a diagnostic algorithm, a confirmed or highly probable aetiologic agent was detected in 35.0% (n=406) of AME cases, with a further 9.2% (total: 44.2%, n=513) aetiologies defined as suspected. JEV (24.4%, n=283) was the most commonly identified pathogen followed by Orientia tsutsugamushi (4.7%, n=55), DENV (4.6%, n=53), enteroviruses (3.5%, n=41), CHIKV (2.0%, n=23) and Streptococcus pneumoniae (1.6%, n=19). The majority of aetiologies identified for paediatric AME in Cambodia were vaccine preventable and/or treatable with appropriate antimicrobials.Emerging Microbes & Infections (2017) 6, e35; doi:10.1038/emi.2017.15; published online 24 May 2017

The Pacific region, also referred to as Oceania, is a geographically widespread region populated by people of diverse cultures and ethnicities. Indigenous people in the region (Melanesians, Polynesians, Micronesians, Papuans, and Indigenous Australians) are over-represented on national, regional, and global scales for the burden of infectious and non-communicable diseases. Although social and environmental factors such as poverty, education, and access to health-care are assumed to be major drivers of this disease burden, there is also developing evidence that genetic and microbiotic factors should also be considered. To date, studies investigating genetic and/or microbiotic links with vulnerabilities to infectious and non-communicable diseases have mostly focused on populations in Europe, Asia, and USA, with uncertain associations for other populations such as indigenous communities in Oceania. Recent developments in personalized medicine have shown that identifying ethnicity-linked genetic vulnerabilities can be important for medical management. Although our understanding of the impacts of the gut microbiome on health is still in the early stages, it is likely that equivalent vulnerabilities will also be identified through the interaction between gut microbiome composition and function with pathogens and the host immune system. As rapid economic, dietary, and cultural changes occur throughout Oceania it becomes increasingly important that further research is conducted within indigenous populations to address the double burden of high rates of infectious diseases and rapidly rising non-communicable diseases so that comprehensive development goals can be planned. In this article, we review the current knowledge on the impact of nutrition, genetics, and the gut microbiome on infectious diseases in indigenous people of the Pacific region.

Dengue is a national priority disease in Cambodia. The Cambodian National Dengue Surveillance System is based on passive surveillance of dengue-like inpatients reported by public hospitals and on a sentinel, pediatric hospital-based active surveillance system. This system works well to assess trends but the sensitivity of the early warning and time-lag to usefully inform hospitals can be improved. During The ECOnomic development, ECOsystem MOdifications, and emerging infectious diseases Risk Evaluation (ECOMORE) project's knowledge translation platforms, Cambodian hospital staff requested an early warning tool to prepare for major outbreaks. Our objective was therefore to find adapted tools to improve the early warning system and preparedness. Dengue data was provided by the National Dengue Control Program (NDCP) and are routinely obtained through passive surveillance. The data were analyzed at the provincial level for eight Cambodian provinces during 2008-2015. The R surveillance package was used for the analysis. We evaluated the effectiveness of Bayesian algorithms to detect outbreaks using count data series, comparing the current count to an expected distribution obtained from observations of past years. The analyses bore on 78,759 patients with dengue-like syndromes. The algorithm maximizing sensitivity and specificity for the detection of major dengue outbreaks was selected in each province. The overall sensitivity and specificity were 73% and 97%, respectively, for the detection of significant outbreaks during 2008-2015. Depending on the province, sensitivity and specificity ranged from 50% to 100% and 75% to 100%, respectively. The final algorithm meets clinicians' and decisionmakers' needs, is cost-free and is easy to implement at the provincial level.

Pre-exposure rabies prophylaxis (PrEP) is recommended for people at frequent or increased risk of professional exposure to lyssavirus (including rabies virus). PrEP provides protection against unrecognized exposure. After the primary vaccination, one’s immune response against rabies may decline over time. We aimed to evaluate the immune response to rabies in individuals immunized for occupational reasons before and after a booster dose of the rabies vaccine. With this aim, we retrospectively documented factors associated with an inadequate response in individuals vaccinated for occupational purposes. Our findings analyzed data from 498 vaccinated individuals and found that 17.2% of participants had an inadequate antibody titration documented after their primary vaccination without the booster, while inadequate response after an additional booster of the vaccine was evidenced in 0.5% of tested participants. This study showed that a single booster dose of vaccine after PrEP conferred a high and long-term immune response in nearly all individuals except for rare, low responders. A systematic rabies booster after primary vaccination may result in alleviating the monitoring strategy of post-PrEP antibody titers among exposed professionals.

Abstract 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.

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.Emerging Microbes & Infections (2016) 5, e70; doi:10.1038/emi.2016.69; published online 20 July 2016

Human enterovirus 71 (EV-A71) causes hand, foot and mouth disease (HFMD). EV-A71 circulates in many countries and has caused large epidemics, especially in the Asia-Pacific region, since 1997. In April 2012, an undiagnosed fatal disease with neurological involvement and respiratory distress occurred in young children admitted to the Kantha Bopha Children’s Hospital in Phnom Penh, Cambodia. Most died within a day of hospital admission, causing public panic and international concern. In this study, we describe the enterovirus (EV) genotypes that were isolated during the outbreak in 2012 and the following year. From June 2012 to November 2013, 312 specimens were collected from hospitalized and ambulatory patients and tested by generic EV and specific EV-A71 reverse transcription PCR. EV-A71 was detected in 208 clinical specimens while other EVs were found in 32 patients. The VP1 gene and/or the complete genome were generated. Our phylogenetic sequencing analysis demonstrated that 80 EV-A71 strains belonged to the C4a subgenotype and 3 EV-A71 strains belonged to the B5 genotype. Furthermore, some lineages of EV-A71 were found to have appeared in Cambodia following separate introductions from neighboring countries. Nineteen EV A (CV-A6 and CV-A16), 9 EV B (EV-B83, CV-B3, CV-B2, CV-A9, E-31, E-2 and EV-B80) and 4 EV C (EV-C116, EV-C96, CV-A20 and Vaccine-related PV-3) strains were also detected. We found no molecular markers of disease severity. We report here that EV-A71 genotype C4 was the main etiological agent of a large outbreak of HFMD and particularly of severe forms associated with central nervous system infections. The role played by other EVs in the epidemic could not be clearly established.

Enterovirus 71 is reported to have emerged in Cambodia in 2012; at least 54 children with severe encephalitis died during that outbreak. We used serum samples collected during 2000–2011 to show that the virus had been widespread in the country for at least a decade before the 2012 outbreak.

Japanese encephalitis is mainly considered a rural disease, but there is growing evidence of a peri-urban and urban transmission in several countries, including Cambodia. We, therefore, compared the epidemiologic dynamic of Japanese encephalitis between a rural and a peri-urban setting in Cambodia. We monitored two cohorts of 15 pigs and determined the force of infection-rate at which seronegative pigs become positive-in two study farms located in a peri-urban and rural area, respectively. We also studied the mosquito abundance and diversity in proximity of the pigs, as well as the host densities in both areas. All the pigs seroconverted before the age of 6 months. The force of infection was 0.061 per day (95% confidence interval = 0.034-0.098) in the peri-urban cohort and 0.069 per day (95% confidence interval = 0.047-0.099) in the rural cohort. Several differences in the epidemiologic dynamic of Japanese encephalitis between both study sites were highlighted. The later virus amplification in the rural cohort may be linked to the later waning of maternal antibodies, but also to the higher pig density in direct proximity of the studied pigs, which could have led to a dilution of mosquito bites at the farm level. The force of infection was almost identical in both the peri-urban and the rural farms studied, which shifts the classic epidemiologic cycle of the virus. This study is a first step in improving our understanding of Japanese encephalitis virus ecology in different environments with distinct landscapes, human and animal densities.

Estimates of leptospirosis morbidity identified Oceania as the region with highest burden. Besides Australia and New Zealand, Oceania is home of Pacific Island Countries and Territories, most of which are developing countries facing a number of challenges. Their archipelago geography notably affects health infrastructure and access to healthcare. Although human leptospirosis was formerly identified in Vanuatu, there is a lack of knowledge of this disease in the country. We aimed to identify leptospirosis in outpatients visiting the hospital.We conducted a clinical study to investigate leptospirosis as a cause of non-malarial acute febrile illness in Vanuatu. A total 161 outpatients visiting the outpatient clinics at Port Vila Central Hospital for internal medicine were recruited over 20 month. We showed that leptospirosis significantly affects humans in Vanuatu: 12 cases were confirmed by real-time PCR on acute blood samples (n = 5) or by high serology titers evidencing a recent infection (MAT titer ≥800 or ELISA≥18 Units, n = 7). A high rate of positive serology was also evidenced, by MAT (100

Remote sensing can contribute to early warning for diseases with environmental drivers, such as flooding for leptospirosis. In this study we assessed whether and which remotely-sensed flooding indicator could be used in Cambodia to study any disease for which flooding has already been identified as an important driver, using leptospirosis as a case study. The performance of six potential flooding indicators was assessed by ground truthing. The Modified Normalized Difference Water Index (MNDWI) was used to estimate the Risk Ratio (RR) of being infected by leptospirosis when exposed to floods it detected, in particular during the rainy season. Chi-square tests were also calculated. Another variable-the time elapsed since the first flooding of the year-was created using MNDWI values and was also included as explanatory variable in a generalized linear model (GLM) and in a boosted regression tree model (BRT) of leptospirosis infections, along with other explanatory variables. Interestingly, MNDWI thresholds for both detecting water and predicting the risk of leptospirosis seroconversion were independently evaluated at -0.3. Value of MNDWI greater than -0.3 was significantly related to leptospirosis infection (RR = 1.61 [1.10-1.52]; χ2 = 5.64, p-value = 0.02, especially during the rainy season (RR = 2.03 [1.25-3.28]; χ2 = 8.15, p-value = 0.004). Time since the first flooding of the year was a significant risk factor in our GLM model (p-value = 0.042). These results suggest that MNDWI may be useful as a risk indicator in an early warning remote sensing tool for flood-driven diseases like leptospirosis in South East Asia.

Despite the increased use of vaccination in several Asian countries, Japanese Encephalitis (JE) remains the most important cause of viral encephalitis in Asia in humans with an estimated 68,000 cases annually. Considered a rural disease occurring mainly in paddy-field dominated landscapes where pigs are amplifying hosts, JE may nevertheless circulate in a wider range of environment given the diversity of its potential hosts and vectors. The main objective of this study was to assess the intensity of JE transmission to pigs in a peri-urban environment in the outskirt of Phnom Penh, Cambodia. We estimated the force of JE infection in two cohorts of 15 sentinel pigs by fitting a generalised linear model on seroprevalence monitoring data observed during two four-month periods in 2014. Our results provide evidence for intensive circulation of JE virus in a periurban area near Phnom Penh, the capital and most populated city of Cambodia. Understanding JE virus transmission in different environments is important for planning JE virus control in the long term and is also an interesting model to study the complexity of vector-borne diseases. Collecting quantitative data such as the force of infection will help calibrate epidemiological model that can be used to better understand complex vector-borne disease epidemiological cycles.

Several diseases and vulnerabilities associated with genetic or microbial factors are more frequent among populations of Oceanian, Non-European, Non-Asian descent (ONENA). ONENA are specific and have long been isolated geographically. To our knowledge, there are no published official, quantitative, aggregated data on the populations impacted by these excess vulnerabilities in Oceania. We searched official census reports for updated estimates of the total population for each of the Pacific Island Countries and Territories (including Australia) and the US State of Hawaii, privileging local official statistical or censual sources. We multiplied the most recent total population estimate by the cumulative percentage of the ONENA population as determined in official reports. Including Australia and the US State of Hawaii, Oceania counts 27 countries and territories, populated in 2016 by approximately 41 M inhabitants (17 M not counting Australia) among which approximately 12.5 M (11.6 M not counting Australia) consider themselves of entire or partial ONENA ancestry. Specific genetic and microbiome traits of ONENA may be unique and need further investigation to adjust risk estimates, risk prevention, diagnostic and therapeutic strategies, to the benefit of populations in the Pacific and beyond.

Arboviruses are viruses transmitted to humans by the bite of infected mosquito vectors. Over the last decade, arbovirus circulation has increasingly been detected in New Caledonia (NC), a French island territory located in the subtropical Pacific region. Reliable epidemiological, entomological, virological and climate data have been collected in NC over the last decade. Here, we describe these data and how they inform arboviruses’ epidemiological profile. We pinpoint areas which remain to be investigated to fully understand the peculiar epidemiological profile of arbovirus circulation in NC. Further, we discuss the advantages of conducting studies on arboviruses dynamics in NC. Overall, we show that conclusions drawn from observations conducted in NC may inform epidemiological risk assessments elsewhere and may be vital to guide surveillance and response, both in New Caledonia and beyond.

Chikungunya virus (CHIKV), probably Asian genotype, was first detected in Cambodia in 1961. Despite no evidence of acute or recent CHIKV infections since 2000, real-time reverse transcription PCR of serum collected in 2011 detected CHIKV, East Central South African genotype. Spatiotemporal patterns and phylogenetic clustering indicate that the virus probably originated in Thailand.

Leptospirosis is an emerging but neglected public health challenge in the Asia/Pacific Region with an annual incidence estimated at 10-100 per 100,000 population. No accurate data, however, are available for at-risk rural Cambodian communities.We conducted anonymous, unlinked testing for IgM antibodies to Leptospira spp. on paired sera of Cambodian patients

The East/Central/South African genotype of Chikungunya virus with the E1-A226V mutation emerged in 2011 in Cambodia and spread in 2012. An outbreak of 190 cases was documented in Trapeang Roka, a rural village. We surveyed 425 village residents within 3-4 weeks after the outbreak, and determined the sensitivity and specificity of case definitions and factors associated with infection by CHIKV. Self-reported clinical presentation consisted mostly of fever, rash and arthralgia. The presence of all three clinical signs or symptoms was identified as the most sensitive (67%) and specific (84%) self-reported diagnostic clinical indicator compared to biological confirmation by MAC-ELISA or RT-PCR used as a reference. Having an indoor occupation was associated with lower odds of infection compared with people who remained at home (adjOR 0.32, 95%CI 0.12-0.82). In contrast with findings from outbreaks in other settings, persons aged above 40 years were less at risk of CHIKV infection, likely reflecting immune protection acquired when Chikungunya circulated in Cambodia before the Khmer Rouge regime in 1975. In view of the very particular history of Cambodia, our epidemiological data from Trapeang Roka are the first to support the persistence of CHIKV antibodies over a period of 40 years.

BACKGROUND:Dengue laboratory diagnosis is essentially based on detection of the virus, its components or antibodies directed against the virus in blood samples. Blood, however, may be difficult to draw in some patients, especially in children, and sampling during outbreak investigations or epidemiological studies may face logistical challenges or limited compliance to invasive procedures from subjects. The aim of this study was to assess the possibility of using saliva and urine samples instead of blood for dengue diagnosis. METHODOLOGY/PRINCIPAL FINDINGS:Serial plasma, urine and saliva samples were collected at several time-points between the day of admission to hospital until three months after the onset of fever in children with confirmed dengue disease. Quantitative RT-PCR, NS1 antigen capture and ELISA serology for anti-DENV antibody (IgG, IgM and IgA) detection were performed in parallel on the three body fluids. RT-PCR and NS1 tests demonstrated an overall sensitivity of 85.4%/63.4%, 41.6%/14.5% and 39%/28.3%, in plasma, urine and saliva specimens, respectively. When urine and saliva samples were collected at the same time-points and tested concurrently, the diagnostic sensitivity of RNA and NS1 detection assays was 69.1% and 34.4%, respectively. IgG/IgA detection assays had an overall sensitivity of 54.4%/37.4%, 38.5%/26.8% and 52.9%/28.6% in plasma, urine and saliva specimens, respectively. IgM were detected in 38.1% and 36% of the plasma and saliva samples but never in urine. CONCLUSIONS:Although the performances of the different diagnostic methods were not as good in saliva and urine as in plasma specimens, the results obtained by qRT-PCR and by anti-DENV antibody ELISA could well justify the use of these two body fluids to detect dengue infection in situations when the collection of blood specimens is not possible.

The aim of this review is to guide clinicians in the practical management of patients suffering from rabies encephalomyelitis. This condition is eminently preventable by modern post-exposure vaccination, but is virtually always fatal in unvaccinated people. In the absence of any proven effective antiviral or other treatment, palliative care is an imperative to minimise suffering. Suspicion of rabies encephalomyelitis depends on recognising the classic symptomatology and eliciting a history of exposure to a possibly rabid mammal. Potentially treatable differential diagnoses must be eliminated, notably other infective encephalopathies. Laboratory confirmation of suspected rabies is not usually possible in many endemic areas, but is essential for public health surveillance. In a disease as agonising and terrifying as rabies encephalomyelitis, alleviation of distressing symptoms is the primary concern and overriding responsibility of medical staff. Calm, quiet conditions should be created, allowing relatives to communicate with the dying patient in safety and privacy. Palliative management must address thirst and dehydration, fever, anxiety, fear, restlessness, agitation, seizures, hypersecretion, and pain. As the infection progresses, coma and respiratory, cardiovascular, neurological, endocrine, or gastrointestinal complications will eventually ensue. When the facilities exist, the possibility of intensive care may arise, but although some patients may survive, they will be left with severe neurological sequelae. Recovery from rabies is extremely rare, and heroic measures with intensive care should be considered only in patients who have been previously vaccinated, develop rabies antibody within the first week of illness, or were infected by an American bat rabies virus. However, in most cases, clinicians must have the courage to offer compassionate palliation whenever the diagnosis of rabies encephalomyelitis is inescapable.

Following almost 30 years of relative silence, chikungunya fever reemerged in Kenya in 2004. It subsequently spread to the islands of the Indian Ocean, reaching Southeast Asia in 2006. The virus was first detected in Cambodia in 2011 and a large outbreak occurred in the village of Trapeang Roka Kampong Speu Province in March 2012, in which 44% of the villagers had a recent infection biologically confirmed. The epidemic curve was constructed from the number of biologically-confirmed CHIKV cases per day determined from the date of fever onset, which was self-reported during a data collection campaign conducted in the village after the outbreak. All individuals participating in the campaign had infections confirmed by laboratory analysis, allowing for the identification of asymptomatic cases and those with an unreported date of fever onset. We develop a stochastic model explicitly including such cases, all of whom do not appear on the epidemic curve. We estimate the basic reproduction number of the outbreak to be 6.46 (95% C.I. [6.24, 6.78]). We show that this estimate is particularly sensitive to changes in the biting rate and mosquito longevity. Our model also indicates that the infection was more widespread within the population on the reported epidemic start date. We show that the exclusion of asymptomatic cases and cases with undocumented onset dates can lead to an underestimation of the reproduction number which, in turn, could negatively impact control strategies implemented by public health authorities. We highlight the need for properly documenting newly emerging pathogens in immunologically naive populations and the importance of identifying the route of disease introduction.

BackgroundThe Cambodian National Influenza Center (NIC) monitored and characterized circulating influenza strains from 2009 to 2011.Methodology/principal findingsSentinel 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/significanceIn 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.

Community-acquired pneumonia (CAP) is one of the most important causes of morbidity and mortality worldwide. Etiological data for Cambodia is scarce. We aimed to describe the main etiological agents causing CAP, and their resistance patterns in Cambodia and the greater Mekong region.A review of bacterial etiologies of CAP and antimicrobial resistance in Cambodia and neighboring countries was conducted via: (1) a systematic review of published literature in all NCBI databases using Pubmed, Google scholar, EMBASE, the World Health Organization and the Cambodian Ministry of Health libraries; (2) a review of unpublished data from Cambodia provided by national and international stakeholders working at different tiers of the healthcare system.Twenty three articles and five data sources reported etiologies for 5919 CAP patients diagnosed between May 1995 and December 2012, including 1421 (24.0%), 3571 (60.3%) and 927 (15.7%) from Cambodia, Thailand and Vietnam, respectively. Streptococcus pneumoniae and Haemophilus influenzae were the most common pathogens ranking among the five most prevalent in 12 and 10 studies, respectively. Gram-negative bacteria such as Burkholderia pseudomallei and Klebsiella pneumoniae were also frequently diagnosed, particularly in bacteremic CAP in Thai adults and Cambodian children. In Thailand and Vietnam, Mycoplasma pneumoniae and Chlamydia pneumoniae were frequently identified in settings using indirect laboratory testing.Based on this analysis, CAP data in Cambodia seems to present etiological and resistance profiles comparable to those of neighboring countries. Findings have been shared with the national authorities upon the revision of the national therapeutic guidelines and were disseminated using a specially created website.

Diverse aetiologies of viral and bacterial encephalitis are widely recognized as significant yet neglected public health issues in the Mekong region. A robust analysis of the corresponding health burden is lacking. We retrieved 75 articles on encephalitis in the region published in English or in French from 1965 through 2011. Review of available data demonstrated that they are sparse and often derived from hospital-based studies with significant recruitment bias. Almost half (35 of 75) of articles were on Japanese encephalitis virus (JEV) alone or associated with dengue. In the Western Pacific region the WHO reported 30,000-50,000 annual JEV cases (15,000 deaths) between 1966 and 1996 and 4,633 cases (200 deaths) in 2008, a decline likely related to the introduction of JEV vaccination in China, Vietnam, or Thailand since the 1980s. Data on dengue, scrub typhus and rabies encephalitis, among other aetiologies, are also reviewed and discussed. Countries of the Mekong region are undergoing profound demographic, economic and ecological change. As the epidemiological aspects of Japanese encephalitis (JE) are transformed by vaccination in some countries, highly integrated expert collaborative research and objective data are needed to identify and prioritize the human health, animal health and economic burden due to JE and other pathogens associated with encephalitides.

After the 2006–2007 epidemic wave of Rift Valley fever (RVF) in East Africa and its circulation in the Comoros, laboratory case-finding of RVF was conducted in Mayotte from September 2007 through May 2008. Ten recent human RVF cases were detected, which confirms the indigenous transmission of RFV virus in Mayotte.

The French Institute for Public Health Surveillance monitors health events of potential international importance occurring worldwide to provide timely warning to French health authorities. We reviewed the nature and place of occurrence of the last 200 events. From an individual country’s perspective, the need for multiple sources is emphasized.

A patient with suspected malaria was hospitalized successively in 2 hospitals, first in Dakar, Senegal, then in Rennes, France, where tests diagnosed Crimean-Congo hemorrhagic fever. An international incident management group was set up in France and Senegal, which traced 181 contacts and analyzed 50 samples from 3 countries. No secondary cases were identified clinically.