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International audience; Aedes-transmitted diseases, especially dengue, are increasing throughout the world and the main preventive methods include vector control and the avoidance of mosquito bites. A simple Premise Condition Index (PCI) categorizing shade, house, and yard conditions was previously developed to help prioritize households or geographical areas where resources are limited. However, evidence about the accuracy of the PCI is mixed. The current study aimed to contribute to a better understanding of the relevance by collecting data from 2,400 premises at four time points over 1 year in Kampong Cham, Cambodia. Regression models were then used to identify associations between PCI and Aedes adult female mosquitoes and pupae. In addition, receiver operating characteristic curves were used to measure the ability of PCI to identify premises in the top quartile of mosquito abundance. The density of adult Aedes females was positively associated with PCI at the household (ratio of means = 1.16 per point on the PCI scale) and cluster level (ratio of means = 1.54). However, the number of Aedes pupae was negatively associated with PCI at the household level (rate ratio = 0.74) and did not have a statistically significant association at the cluster level. Receiver operating characteristic curves suggest the PCI score had "rather low accuracy" (area under the ROC curve = 0.52 and 0.54) at identifying top-quartile premises in terms of adult female Aedes and pupae, respectively. These results suggest that caution is warranted in the programmatic use of PCI in areas of similar geography and mosquito abundance.

Background Artesunate-amodiaquine is a potential therapy for uncomplicated malaria in Cambodia. Methods Between September 2016 and January 2017, artesunate-amodiaquine efficacy and safety were evaluated in a prospective, open-label, single-arm observational study at health centers in Mondulkiri, Pursat, and Siem Reap Provinces, Cambodia. Adults and children with microscopically confirmed Plasmodium falciparum malaria received oral artesunate-amodiaquine once daily for 3 days plus single-dose primaquine, with follow-up on days 7, 14, 21, and 28. The primary outcome was day-28 polymerase chain reaction (PCR)-adjusted adequate clinical and parasitological response (ACPR). An amodiaquine parasite survival assay (AQSA) was developed and applied to whole genome sequencing results to evaluate potential amodiaquine resistance molecular markers. Results In 63 patients, day-28 PCR-adjusted ACPR was 81.0% (95% confidence interval [CI], 68.9–88.7). Day 3 parasite positivity rate was 44.4% (28/63; 95% CI, 31.9–57.5). All 63 isolates had the K13(C580Y) marker for artemisinin resistance; 79.4% (50/63) had Pfpm2 amplification. The AQSA resistance phenotype (≥45% parasite survival) was expressed in 36.5% (23/63) of isolates and was significantly associated with treatment failure (P = .0020). Pfmdr1 mutant haplotypes were N86/184F/D1246, and Pfcrt was CVIET or CVIDT at positions 72–76. Additional Pfcrt mutations were not associated with amodiaquine resistance, but the G353V mutant allele was associated with ACPR compared to Pfmdr1 haplotypes harboring F1068L or S784L/R945P mutations (P = .030 and P = .0004, respectively). Conclusions For uncomplicated falciparum malaria in Cambodia, artesunate-amodiaquine had inadequate efficacy owing to amodiaquine-resistant P. falciparum. Amodiaquine resistance was not associated with previously identified molecular markers., Artesunate-amodiaquine has inadequate efficacy in Cambodian patients with uncomplicated falciparum malaria. Amodiaquine resistance is present in Cambodia and not associated with molecular markers reported in Africa and South America.

Background Cambodia is the epicentre of the emergence of Plasmodium falciparum drug resistance. Much less is known regarding the drug susceptibility of the co-endemic Plasmodium vivax. Only in vitro drug assays can determine the parasite’s intrinsic susceptibility, but these are challenging to implement for P. vivax and rarely performed. Objectives To evaluate the evolution of Cambodian P. vivax susceptibility to antimalarial drugs and determine their association with putative markers of drug resistance. Methods In vitro response to three drugs used in the past decade in Cambodia was measured for 52 clinical isolates from Eastern Cambodia collected between 2015 and 2018 and the sequence and copy number variation of their pvmdr1 and pvcrt genes were analysed. pvmdr1 polymorphism was also determined for an additional 250 isolates collected in Eastern Cambodia between 2014 and 2019. Results Among the 52 cryopreserved isolates tested, all were susceptible to the three drugs, with overall median IC50s of 16.1 nM (IQR 11.4–22.3) chloroquine, 3.4 nM (IQR 2.1–5.0) mefloquine and 4.6 nM (IQR 2.7–7.0) piperaquine. A significant increase in chloroquine and piperaquine susceptibility was observed between 2015 and 2018, unrelated to polymorphisms in pvcrt and pvmdr1. Susceptibility to mefloquine was significantly lower in parasites with a single mutation in pvmdr1 compared with isolates with multiple mutations. The proportion of parasites with this single mutation genotype increased between 2014 and 2019. Conclusions P. vivax with decreased susceptibility to mefloquine is associated with the introduction of mefloquine-based treatment during 2017–18.

Knowledge of the origin and reservoir of the coronavirus responsible for the ongoing COVID-19 pandemic is still fragmentary. To date, the closest relatives to SARS-CoV-2 have been detected in Rhinolophus bats sampled in the Yunnan province, China. Here we describe the identification of SARS-CoV-2 related coronaviruses in two Rhinolophus shameli bats sampled in Cambodia in 2010. Metagenomic sequencing identifies nearly identical viruses sharing 92.6% nucleotide identity with SARS-CoV-2. Most genomic regions are closely related to SARS-CoV-2, with the exception of a region of the spike, which is not compatible with human ACE2-mediated entry. The discovery of these viruses in a bat species not found in China indicates that SARS-CoV-2 related viruses have a much wider geographic distribution than previously reported, and suggests that Southeast Asia represents a key area to consider for future surveillance for coronaviruses., In this study, Delaune et al., isolate and characterise a SARS-CoV-2-related coronavirus from two bats sampled in Cambodia. Their findings suggest that the geographic distribution of SARS-CoV-2-related viruses is wider than previously reported.

Background Rapid elimination of Plasmodium falciparum malaria in Cambodia is a goal with both national and international significance. Transmission of malaria in Cambodia is limited to forest environments, and the main population at risk consists of forest-goers who rely on forest products for income or sustenance. The ideal interventions to eliminate malaria from this population are unknown. Methods In two forested regions of Cambodia, forest-goers were trained to become forest malaria workers (FMWs). In one region, FMWs performed mass screening and treatment, focal screening and treatment, and passive case detection inside the forest. In the other region, FMWs played an observational role for the first year, to inform the choice of intervention for the second year. In both forests, FMWs collected blood samples and questionnaire data from all forest-goers they encountered. Mosquito collections were performed in each forest. Results Malaria prevalence by PCR was high in the forest, with 2.3–5.0% positive for P. falciparum and 14.6–25.0% positive for Plasmodium vivax among forest-goers in each study site. In vectors, malaria prevalence ranged from 2.1% to 9.6%, but no P. falciparum was observed. Results showed poor performance of mass screening and treatment, with sensitivity of rapid diagnostic tests equal to 9.1% (95% CI 1.1%, 29.2%) for P. falciparum and 4.4% (95% CI 1.6%, 9.2%) for P. vivax. Malaria infections were observed in all demographics and throughout the studied forests, with no clear risk factors emerging. Conclusions Malaria prevalence remains high among Cambodian forest-goers, but performance of rapid diagnostic tests is poor. More adapted strategies to this population, such as intermittent preventive treatment of forest goers, should be considered.

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

International audience; Proposed interventions for eliminating drug-resistant Plasmodium falciparum malaria include the targeting of asymptomatic carriers through screening and treatment. We report on the diagnostic performance of the recently developed ultrasensitive rapid diagnostic test (uRDT) compared with screening with conventional RDTs (cRDT) and polymerase chain reaction (PCR) under field conditions in Cambodia in a total of 2,729 individuals. The P. falciparum positivity by quantitative PCR (qPCR) was 3.8% (26/678) in those screened during active case detection and 0.5% (10/2,051) in the cross-sectional survey. Compared with qPCR, the sensitivity of the uRDTs was 53.8% (95% CI: 33.4-73.4%) when used in active case detection and 60.0% (95% CI: 26.2-87.8%) in the cross-sectional survey. The uRDTs did not show a significant improvement in diagnostic performance over cRDTs when used for active case detection and for a malaria prevalence survey in the context of this low-transmission setting.

International audience; In January 2017, an estimated 3,700 (93%) of 4,000 Khaki Campbell ducks (Anas platyrhynchos domesticus) died in Kampong Thom Province, Cambodia. We detected low pathogenicity avian influenza A(H7N3) virus and anatid herpesvirus 1 (duck plague) in the affected flock; however, the exact cause of the mortality event remains unclear.

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.

International audience; 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.

International audience; 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.

Highly pathogenic avian influenza virus (AIV) has been endemic in Cambodia since 2004, and is a major agricultural and public health concern. Cambodia is a tropical, resource poor, lower-middle inc...

International audience; Low pathogenic A(H9N2) subtype avian influenza viruses (AIVs) were originally detected in Cambodian poultry in 2013, and now circulate endemically. We sequenced and characterised 64 A(H9N2) AIVs detected in Cambodian poultry (chickens and ducks) from January 2015 to May 2016. All A(H9) viruses collected in 2015 and 2016 belonged to a new BJ/94like h9-4.2.5 sub-lineage that emerged in the region during or after 2013, and was distinct to previously detected Cambodian viruses. Overall, there was a reduction of genetic diversity of H9N2 since 2013, however two genotypes were detected in circulation, P and V, with extensive reassortment between the viruses. Phylogenetic analysis showed a close relationship between A(H9N2) AIVs detected in Cambodian and Vietnamese poultry, highlighting cross-border trade/movement of live, domestic poultry between the countries. Wild birds may also play a role in A(H9N2) transmission in the region. Some genes of the Cambodian isolates frequently clustered with zoonotic A(H7N9), A(H9N2) and A(H10N8) viruses, suggesting a common ecology. Molecular analysis showed 100% of viruses contained the hemagglutinin (HA) Q226L substitution, which favours mammalian receptor type binding. All viruses were susceptible to the neuraminidase inhibitor antivirals; however, 41% contained the matrix (M2) S31N substitution associated with resistance to adamantanes. Overall, Cambodian A(H9N2) viruses possessed factors known to increase zoonotic potential, and therefore their evolution should be continually monitored.

Background The World Health Organization (WHO) proposed guidelines on dengue clinical classification in 1997 and more recently in 2009 for the clinical management of patients. The WHO 1997 classification defines three categories of dengue infection according to severity: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). Alternative WHO 2009 guidelines provide a cross-sectional classification aiming to discriminate dengue fever from dengue with warning signs (DWWS) and severe dengue (SD). The primary objective of this study was to perform a comparison of two dengue classifications. The secondary objective was to describe the changes of hematological and biochemical parameters occurring in patients presenting with different degrees of severity during the course of the disease, since progression to more severe clinical forms is unpredictable. Methodology/Principal findings We performed a prospective, monocentric, cross-sectional study of hospitalized children in Cambodia, aged from 2 to 15 years old with severe and non-severe dengue. We enrolled 243 patients with acute dengue-like illness: 71.2% were dengue infections confirmed using quantitative reverse transcription PCR or NS1 antigen capture ELISA, of which 87.2% and 9.0% of DF cases were respectively classified DWWS and SD, and 35.9% of DHF were designated SD using an adapted WHO 2009 classification for SD case definition. Systematic use of ultrasound at patient admission was crucial for detecting plasma leakage. No difference was observed in the concentration of secreted NS1 protein between different dengue severity groups. Lipid profiles were different between DWWS and SD at admission, characterized by a decrease in total cholesterol, HDL cholesterol, and LDL cholesterol, in SD. Conclusions/Significance Our results show discrepancies between the two classifications, including misclassification of severe dengue cases as mild cases by the WHO 1997 classification. Using an adapted WHO 2009 classification, SD more precisely defines the group of patients requiring careful clinical care at a given time during hospitalization., Author summary Dengue is a viral disease that results in various health conditions, ranging from strictly asymptomatic infections to life-threatening severe dengue. Here we have compared the two World Health Organization (WHO) dengue classification schemes from 1997 and 2009, which define different categories of dengue fever and have been at the root of much debate in the dengue research community. We enrolled a Cambodian pediatric cohort of hospitalized dengue-confirmed patients, with clinical and biological follow-up. Our findings demonstrate that (i) the WHO 1997 classification is a longitudinal dengue-case classification which uses strict prerequisite clinical and/or biological signs to move from one level of severity to another, and (ii) the WHO 2009 classification, being a transversal dengue-case classifier, is more flexible and when adapted with systematic use of ultrasound is more accurate in identifying severe dengue case for timely and appropriate clinical management of hospitalized patients. Our laboratory results found that lipid profiles exhibited changes and differences between different dengue severity groups at hospital.

International audience; Active surveillance in high-risk sites in Cambodia has iden- tified multiple low-pathogenicity influenza A(H7) viruses, mainly in ducks. None fall within the A/Anhui/1/2013(H7N9) lineage; however, some A(H7) viruses from 2018 show temporal and phylogenetic similarity to the H7N4 virus that caused a nonfatal infection in Jiangsu Province, China, in December 2017.

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.

Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses., Author summary H5N1 avian influenza viruses can cross species barriers and cause severe disease in humans. H5N1 viruses currently cannot replicate and transmit efficiently among humans, but animal infection studies and modeling experiments have suggested that human adaptation may require only a few mutations. However, data from natural spillover infection has been limited, posing a challenge for risk assessment. Here, we analyze a unique dataset of deep sequence data from H5N1 virus-infected humans and domestic ducks in Cambodia. We find that well-known markers of human receptor binding and replication arise in multiple, independent humans. We also find that 3 mutations detected within-host are enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adapting. However, we also show that within-host evolution in both humans and ducks are shaped heavily by purifying selection and genetic drift, and that a large fraction of within-host variation is never detected on the H5N1 phylogeny. Taken together, our data show that H5N1 viruses do generate human-adapting mutations during natural infection. However, short infection times, purifying selection, and genetic drift may severely limit how much H5N1 viruses can evolve during the course of a single infection.

International audience; 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.

Erratum in : Corrections. [Lancet Infect Dis. 2017]Comment in : Evidence of a hard selective sweep for artemisinin resistant Plasmodium falciparum. [Lancet Infect Dis. 2017]; International audience; BACKGROUND:Evidence suggests that the PfKelch13 mutations that confer artemisinin resistance in falciparum malaria have multiple independent origins across the Greater Mekong subregion, which has motivated a regional malaria elimination agenda. We aimed to use molecular genotyping to assess antimalarial drug resistance selection and spread in the Greater Mekong subregion.METHODS:In this observational study, we tested Plasmodium falciparum isolates from Myanmar, northeastern Thailand, southern Laos, and western Cambodia for PfKelch13 mutations and for Pfplasmepsin2 gene amplification (indicating piperaquine resistance). We collected blood spots from patients with microscopy or rapid test confirmed uncomplicated falciparum malaria. We used microsatellite genotyping to assess genetic relatedness.FINDINGS:As part of studies on the epidemiology of artemisinin-resistant malaria between Jan 1, 2008, and Dec 31, 2015, we collected 434 isolates. In 2014-15, a single long PfKelch13 C580Y haplotype (-50 to +31·5 kb) lineage, which emerged in western Cambodia in 2008, was detected in 65 of 88 isolates from northeastern Thailand, 86 of 111 isolates from southern Laos, and 14 of 14 isolates from western Cambodia, signifying a hard transnational selective sweep. Pfplasmepsin2 amplification occurred only within this lineage, and by 2015 these closely related parasites were found in ten of the 14 isolates from Cambodia and 15 of 15 isolates from northeastern Thailand. C580Y mutated parasites from Myanmar had a different genetic origin.INTERPRETATION:Our results suggest that the dominant artemisinin-resistant P falciparum C580Y lineage probably arose in western Cambodia and then spread to Thailand and Laos, outcompeting other parasites and acquiring piperaquine resistance. The emergence and spread of fit artemisinin-resistant P falciparum parasite lineages, which then acquire partner drug resistance across the Greater Mekong subregion, threatens regional malaria control and elimination goals. Elimination of falciparum malaria from this region should be accelerated while available antimalarial drugs still remain effective.

International audience; Rabies circulates intensely in Cambodia, mainly affecting rural populations. We conducted a prospective study to estimate the baseline incidence of potentially infective dog bites in rural villages of Siem Reap province, Cambodia. The study was conducted in a convenience sample of 844 families totaling 1779 persons in four villages. The study collected data in a total of 802.3 person-years. Trained village health workers (VHW) exhaustively documented consecutive dog bites at the end of each month. Between May 15th and November 15th, 2011, a total of 40 attacks (43 bites; 1.07 bites per attack) were notified by 39 persons (50% female; one suffered two distinct incidents) to VHW. The all-age attack rate for bites over this 6-month period was 2.3% (CI95%: 1.7-3.1%), with a global incidence rate estimated at 4.84 bites/100 person-years (CI95%: 3.5-6.6). The mean age in bite victims was 20.8±18.9years (median 12.5; interquartile range 6-36; range 1-63). The dog was identified in 39 (97.5%) of cases, being the household dog in 9 (22.5%) of cases. Bites were classified as severe (WHO Category III-broken skin with bleeding) in 33 (82.5%) of cases with a severe dog bites incidence estimated at 4/100 person-years (CI95%: 2.8-5.6). The bites involved the hand or face in 1 (2.5%) case each (both Category III). In 20 incidents (50%), only rice was applied to the wounds. There were no suspected or confirmed human rabies deaths during the study period but one dog died after biting (2 others were lost to follow-up and 14 were put down by their owner). Our study documented an extremely high incidence of dog bites in of rural Cambodian adults and children. Adapted control policies for canine vaccination are urgently needed.

International audience; BACKGROUND:The diagnosis of tuberculosis in human immunodeficiency virus (HIV)-infected children is challenging. We assessed the performance of alternative specimen collection methods for tuberculosis diagnosis in HIV-infected children using Xpert MTB/RIF (Xpert).METHODS:HIV-infected children aged ≤13 years with suspected intrathoracic tuberculosis were enrolled in 8 hospitals in Burkina Faso, Cambodia, Cameroon, and Vietnam. Gastric aspirates were taken for children aged

International audience; Background:In the frame of elimination strategies of Plasmodium falciparum (Pf), active case detection has been recommended as complementary approach to the existing passive case detection programs. We trialed a polymerase chain reaction (PCR)-based active detection strategy targeting asymptomatic individuals, named proactive case detection (PACD), with the aim of assessing its feasibility, the extra yield of Pf infections, and the at-risk population for Pf carriage status.Methods:A pilot of PACD was conducted in 3 villages in Chey Saen district (Preah Vihear province, Cambodia), from December 2015 to March 2016. Voluntary screening and treatment, following health promotion sensitization, was used as mobilization strategy.Results:A total of 2802 persons were tested, representing 54% of the population. PACD (n = 30) and the respective reactive case detection (RACD) (n = 3) identified 33 Pf carriers, approximately twice as many as the Pf infections (n = 17) diagnosed in passive case detection and respective RACD, by health centers and village malaria workers using PCR, in the same villages/period. Final positivity rate was 1.07% (30/2802). People spending nighttime in forests and plantations were found to be at increased risk for Pf infection (odds ratio [OR], 3.4 [95% CI, 1.6-7.2], P = .002 and OR, 2.3 [95% CI, 1.1-4.9], P = .03, respectively).Conclusions:We demonstrated the usefulness of the PACD component in identifying Pf asymptomatic carriers. Social mobilization and promotion led to good attendance of specific risk groups, identified to be, in the Cambodian context, individuals spending nighttime in forest and plantations.

International audience; Background:Eliminating falciparum malaria in Cambodia is a top priority, requiring the implementation of novel tools and strategies to interrupt its transmission. To date, few data are available regarding the contributions to malaria transmission of symptomatic and asymptomatic carriers.Methods:Direct-membrane and skin feeding assays (DMFAs, SFAs) were performed, using Anopheles minimus and Anopheles dirus, to determine infectivity of symptomatic falciparum-infected patients and malaria asymptomatic carriers; a subset of the latter were followed up for 2 months to assess their transmission potential.Results:By microscopy and real-time polymerase chain reaction, Plasmodium falciparum gametocyte prevalence rates were, respectively, 19.3% (n = 21/109) and 44% (n = 47/109) on day (D) 0 and 17.9% (n = 5/28) and 89.3% (n = 25/28) in recrudescent patients (Drec) (RT-PCR Drec vs D0 P = .002). Falciparum malaria patient infectivity was low on D0 (6.2%; n = 3/48) and in Drec (8.3%; n = 1/12). Direct-membrane feeding assays and SFAs gave similar results. None of the falciparum (n = 0/19) and 3 of 28 Plasmodium vivax asymptomatic carriers were infectious to mosquitoes, including those that were followed up for 2 months. Overall, P. falciparum gametocytemias were low except in a few symptomatic carriers.Conclusions:Only symptomatic falciparum malaria patients were infectious to mosquito vectors at baseline and recrudescence, highlighting the need to detect promptly and treat effectively P. falciparum patients.

Background Malaria elimination needs a concentration of activities towards identification of residual transmission foci and intensification of efforts to eliminate the last few infections, located in so-called ‘malaria hotspots’. Previous work on characterizing malaria transmission hotspots has mainly focused on falciparum malaria and especially on symptomatic cases, while the malaria reservoir is expected to be mainly concentrated in the asymptomatic human population when transmission is low. For Plasmodium vivax, there has been less effort in identifying transmission hotspots. The main aim of this study was to uncover micro-epidemiological mechanisms of clustering of malaria infections at a sub-village level, based on geographical or behavioural features. Methods A cross-sectional survey was performed in three villages within the highest malaria endemic province of Cambodia. The survey took place in the dry season, when the malaria reservoir is expected to be low and residing in the asymptomatic part of the population. Village and field locations of households were georeferenced, blood samples were taken from as many residents as possible and a short questionnaire probing for individual risk factors was taken. Asymptomatic malaria carriers were detected by PCR, and geographical clustering analysis (SaTScan) as well as risk factor analysis were performed. Results A total of 1540 out of 1792 (86%) individuals were sampled. Plasmodial DNA was detected in 129 individuals (8.4%). P. vivax was most prevalent (5.5%) followed by Plasmodium malariae (2.1%) and Plasmodium falciparum (1.6%). Mixed infection occurred in 12 individuals. In two out of three villages geographical clustering of high and low malaria infection risk was clearly present. Cluster location and risk factors associated with the infection differed between the parasite species. Age was an important risk factor for the combined Plasmodium infections, while watching television at evenings was associated with increased odds of P. vivax infections [OR (CI): 1.86 (0.95–3.64)] and bed net use was associated with reduced odds of P. falciparum infections [OR (CI): 0.25 (0.077–0.80)]. Conclusions Clusters of malaria carriers were malaria species specific and often located remotely, outside village centres. As such, at micro-epidemiological level, malaria is not a single disease. Further unravelling the micro-epidemiology of malaria can enable programme managers to define the interventions likely to contribute to halt transmission in a particular hotspot location. Electronic supplementary material The online version of this article (10.1186/s12936-017-2169-1) contains supplementary material, which is available to authorized users.

International audience

International audience; Reactive case detection around falciparum malaria cases in Cambodia presents a low output. We improved it by including individuals occupationally coexposed with index case patients and using polymerase chain reaction-based diagnosis. The positivity rate increased from 0.16% to 3.9%.

International audience; 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.

International audience; In Cambodia, highly pathogenic avian influenza A(H5N1) subtype viruses circulate endemically causing poultry outbreaks and zoonotic human cases. To investigate the genomic diversity and development of endemicity of the predominantly circulating clade 2.3.2.1c A (H5N1) viruses, we characterised 68 AIVs detected in poultry, the environment and from a single human A(H5N1) case from January 2014 to December 2016. Full genomes were generated for 42 A(H5N1) viruses. Phylogenetic analysis shows that five clade 2.3.2.1c genotypes, designated KH1 to KH5, were circulating in Cambodia during this period. The genotypes arose through multiple reassortment events with the neuraminidase (NA) and internal genes belonging to H5N1 clade 2.3.2.1a, clade 2.3.2.1b or A(H9N2) lineages. Phylogenies suggest that the Cambodian AIVs were derived from viruses circulating between Cambodian and Vietnamese poultry. Molecular analyses show that these viruses contained the hemagglutinin (HA) gene substitutions D94N, S133A, S155N, T156A, T188I and K189R known to increase binding to the human-type α2,6-linked sialic acid receptors. Two A (H5N1) viruses displayed the M2 gene S31N or A30T substitutions indicative of adamantane resistance, however, susceptibility testing towards neuraminidase inhibitors (oseltamivir, zanamivir, lananmivir and peramivir) of a subset of thirty clade 2.3.2.1c viruses showed susceptibility to all four drugs. This study shows that A(H5N1) viruses continue to reassort with other A(H5N1) and A(H9N2) viruses that are endemic in the region, highlighting the risk of introduction and emergence of novel A(H5N1) genotypes in Cambodia.

Background The presence of artemisinin-resistant malaria parasites was confirmed in western Cambodia in 2009. In 2013, mutations in the propeller domain of the kelch protein K13 was found to be associated with artemisinin resistance. A cross-sectional study was conducted to determine the prevalence of Day-3 parasitaemia, estimate the frequency of k13 molecular marker and assess their relationship in the context of operational research. Methods Blood smears and filter paper blood spots were collected from febrile patients in Kravanh District, Pursat Province. The blood smears were examined by microscopy, and blood spots by a k13 mutation assay. Results Data from 92 patients were analysed. Only one was positive for Day-3 parasitaemia. Results of the k13 assay were interpretable for 76 of the 92 samples. The findings were: wild type: 9 (12%), C580Y: 64 (84%), Y493H: 3 (4%). Therefore, despite the high prevalence of k13 mutants (67/76: 88%), only 1 of the 92 patients remained blood smear positive for Plasmodium falciparum on Day-3. Conclusions These preliminary findings suggest good potency of artemisinin despite the dominance of k13 mutation in Kravanh, but the result is not necessarily representative of the western part of Cambodia. Further investigation should be made to determine if k13 marker remains useful as a tool for tracking artemisinin resistance and predicting the trend of the efficacy of artemisinin combination therapy once the mutant alleles have been well established in the population.

International audience; 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.

South-East Asia is a hot spot for emerging zoonotic diseases, and bats have been recognized as hosts for a large number of zoonotic viruses such as Severe Acute Respiratory Syndrome (SARS), responsible for acute respiratory syndrome outbreaks. Thus, it is important to expand our knowledge of the presence of viruses in bats which could represent a risk to humans. Coronaviruses (CoVs) have been reported in bat species from Thailand, China, Indonesia, Taiwan and the Philippines. However no such work was conducted in Cambodia or Lao PDR. Between 2010 and 2013, 1965 bats were therefore sampled at interfaces with human populations in these two countries. They were tested for the presence of coronavirus by consensus reverse transcription-PCR assay. A total of 93 samples (4.7%) from 17 genera of bats tested positive. Sequence analysis revealed the presence of potentially 37 and 56 coronavirus belonging to alpha-coronavirus (αCoV) and beta-CoV (βCoV), respectively. The βCoVs group is known to include some coronaviruses highly pathogenic to human, such as SARS-CoV and MERS-CoV. All coronavirus sequences generated from frugivorous bats (family Pteropodidae) (n = 55) clustered with other bat βCoVs of lineage D, whereas one coronavirus from Pipistrellus coromandra fell in the lineage C of βCoVs which also includes the MERS-CoV. αCoVs were all detected in various genera of insectivorous bats and clustered with diverse bat αCoV sequences previously published. A closely related strain of PEDV, responsible for severe diarrhea in pigs (PEDV-CoV), was detected in 2 Myotis bats. We highlighted the presence and the high diversity of coronaviruses circulating in bats from Cambodia and Lao PDR. Three new bat genera and species were newly identified as host of coronaviruses, namely Macroglossus sp., Megaerops niphanae and Myotis horsfieldii, Graphical abstract Image 1, Highlights • Coronaviruses detected in bats from Lao PDR and Cambodia. • High diversity of αCoVs and βCoVs circulating in bats in Cambodia and Lao PDR. • One strain of βCoV, a new member of the MERS-CoV sister-clade, detected from Pipistrellus coromandra. • A αCoV strain genetically related to PEDV-CoV, detected from Myotis horsfieldii. • CoVs detected for the first time in Megaerops niphanae, Myotis horsfieldii and Macroglossus sp.

Astroviruses are known to infect humans and a wide range of animal species, and can cause gastroenteritis in their hosts. Recent studies have reported astroviruses in bats in Europe and in several locations in China. We sampled 1876 bats from 17 genera at 45 sites from 14 and 13 provinces in Cambodia and Lao PDR respectively, and tested them for astroviruses. Our study revealed a high diversity of astroviruses among various Yangochiroptera and Yinpterochiroptera bats. Evidence for varying degrees of host restriction for astroviruses in bats was found. Furthermore, additional Pteropodid hosts were detected. The astroviruses formed distinct phylogenetic clusters within the genus Mamastrovirus, most closely related to other known bat astroviruses. The astrovirus sequences were found to be highly saturated indicating that phylogenetic relationships should be interpreted carefully. An astrovirus clustering in a group with other viruses from diverse hosts, including from ungulates and porcupines, was found in a Rousettus bat. These findings suggest that diverse astroviruses can be found in many species of mammals, including bats., Highlights • Diverse astroviruses detected in bats in Lao PDR and Cambodia • High polymorphism of astroviruses found in insectivorous and frugivorous bats • High level of genome saturation and hypermutation potential evidenced in astrovirus • Detection of additional astrovirus bat hosts, i.e. nectar bats from the genus Eonycteris • Discovery of a new species of astrovirus in fruit bats (Rousettus sp.), Graphical abstract. Image 1

International audience; 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.

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., Author Summary Japanese Encephalitis Virus (JEV) is the most important cause of viral encephalitis in Asia in humans with an estimated 68,000 cases annually. The disease is considered a mainly rural one because it occurs mainly in rural areas dominated by paddy fields where the main mosquito species vector of JEV breed. However, other mosquito species, breeding in urban areas, and a large range of animal hosts can play a role in the transmission of JEV, and JEV could therefore be transmitted in peri-urban and urban areas. Our results show an intensive circulation of JEV in sentinel pigs in a peri-urban area of Phnom Penh Cambodia at two different periods of the year. It shows the potential for JEV to circulate in a large range of landscapes and suggest that JEV control should not be limited to rural areas and that JEV may have the potential to emerge and be and be maintained in new areas.

Western Cambodia is recognized as the epicenter of Plasmodium falciparum multidrug resistance. Recent reports of the efficacy of dihydroartemisinin (DHA)-piperaquine (PP), the latest of the artemisinin-based combination therapies (ACTs) recommended by the WHO, have prompted further investigations. The clinical efficacy of dihydroartemisinin-piperaquine in uncomplicated falciparum malaria was assessed in western and eastern Cambodia over 42 days. Day 7 plasma piperaquine concentrations were measured and day 0 isolates tested for in vitro susceptibilities to piperaquine and mefloquine, polymorphisms in the K13 gene, and the copy number of the Pfmdr-1 gene. A total of 425 patients were recruited in 2011 to 2013. The proportion of patients with recrudescent infections was significantly higher in western (15.4%) than in eastern (2.5%) Cambodia ( P −3 ). Day 7 plasma PP concentrations and median 50% inhibitory concentrations (IC 50 ) of PP were independent of treatment outcomes, in contrast to median mefloquine IC 50 , which were found to be lower for isolates from patients with recrudescent infections (18.7 versus 39.7 nM; P = 0.005). The most significant risk factor associated with DHA-PP treatment failure was infection by parasites carrying the K13 mutant allele (odds ratio [OR], 17.5; 95% confidence interval [CI], 1 to 308; P = 0.04). Our data show evidence of P. falciparum resistance to PP in western Cambodia, an area of widespread artemisinin resistance. New therapeutic strategies, such as the use of triple ACTs, are urgently needed and must be tested. (This study has been registered at the Australian New Zealand Clinical Trials Registry under registration no. ACTRN12614000344695.)

International audience; 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.

International audience; Accurate information regarding malaria prevalence at national level is required to design and assess malaria control/elimination efforts. Although many comparisons of microscopy and polymerase chain reaction (PCR)-based methods have been conducted, there is little published literature covering such comparisons in southeast Asia especially at the national level. Both microscopic examination and PCR detection were performed on blood films and dried blood spots samples collected from 8,067 individuals enrolled in a nationwide, stratified, multistage, cluster sampling malaria prevalence survey conducted in Cambodia in 2007. The overall malaria prevalence and prevalence rates of Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae infections estimated by microscopy (N = 8,067) were 2.74% (95% confidence interval [CI]: 2.39-3.12%), 1.81% (95% CI: 1.53-2.13%), 1.14% (95% CI: 0.92-1.40%), and 0.01% (95% CI: 0.003-0.07%), respectively. The overall malaria prevalence based on PCR detection (N = 7,718) was almost 2.5-fold higher (6.31%, 95% CI: 5.76-6.89%, P < 0.00001). This difference was significantly more pronounced for P. falciparum (4.40%, 95% CI: 3.95-4.90%, P < 0.00001) compared with P. vivax (1.89%, 95% CI: 1.60-2.22%, P < 0.001) and P. malariae infections (0.22%, 95% CI: 0.13-0.35%, P < 0.0001). The significant proportion of microscopy-negative but PCR-positive individuals (289/7,491, 3.85%) suggest microscopic examination frequently underestimated malaria infections and that active case detection based on microscopy may miss a significant reservoir of infection, especially in low-transmission settings.

Background Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008–2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential. Methods To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010–2011 from 16 health centres in malaria endemics areas in Cambodia. Results Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance. Conclusions The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1370-y) contains supplementary material, which is available to authorized users.

International audience; BACKGROUND: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.METHOD:We conducted anonymous, unlinked testing for IgM antibodies to Leptospira spp. on paired sera of Cambodian patients

International audience; Artemisinin-based combination therapies (ACTs) are the cornerstone of current strategies for fighting malaria. Over the last decade, ACTs have played a major role in decreasing malaria burden. However, this progress is being jeopardized by the emergence of artemisinin-resistant Plasmodium falciparum parasites. Artemisinin resistance was first detected in western Cambodia in 2008 and has since been observed in neighboring countries in southeast Asia. The problem of antimalarial drug resistance has recently worsened in Cambodia, with reports of parasites resistant to piperaquine, the latest generation of partner drug used in combination with dihydroartemisinin, leading to worrying rates of clinical treatment failure. The monitoring and the comprehension of both types of resistance are crucial to prevent the spread of multidrug-resistant parasites outside southeast Asia, and particularly to Africa, where the public health consequences would be catastrophic. To this end, new tools are required for studying the biological and molecular mechanisms underlying resistance to antimalarial drugs and for monitoring the geographic distribution of the resistant parasites. In this review, we detail the major advances in our understanding of resistance to artemisinin and piperaquine and define the challenges that the malaria community will have to face in the coming years.

International audience; This first extensive retrospective study of the molecular epidemiology of dog rabies in Cambodia included 149 rabies virus (RABV) entire nucleoprotein sequences obtained from 1998-2011. The sequences were analyzed in conjunction with RABVs from other Asian countries. Phylogenetic reconstruction confirmed the South-East Asian phylogenetic clade comprising viruses from Cambodia, Vietnam, Thailand, Laos and Myanmar. The present study represents the first attempt to classify the phylogenetic lineages inside this clade, resulting in the confirmation that all the Cambodian viruses belonged to the South-East Asian (SEA) clade. Three distinct phylogenetic lineages in the region were established with the majority of viruses from Cambodia closely related to viruses from Thailand, Laos and Vietnam, forming the geographically widespread phylogenetic lineage SEA1. A South-East Asian lineage SEA2 comprised two viruses from Cambodia was identified, which shared a common ancestor with RABVs originating from Laos. Viruses from Myanmar formed separate phylogenetic lineages within the major SEA clade. Bayesian molecular clock analysis suggested that the time to most recent common ancestor (TMRCA) of all Cambodian RABVs dated to around 1950. The TMRCA of the Cambodian SEA1 lineage was around 1964 and that of the SEA2 lineage was around 1953. The results identified three phylogenetically distinct and geographically separated lineages inside the earlier identified major SEA clade, covering at least five countries in the region. A greater understanding of the molecular epidemiology of rabies in South-East Asia is an important step to monitor progress on the efforts to control canine rabies in the region.

International audience; To determine the origin of influenza A virus (H5N1) epizootics in Cambodia, we used maximum-likelihood and Bayesian methods to analyze the genetic sequences of subtype H5N1 strains from Cambodia and neighboring areas. Poultry movements, rather than repeated reintroduction of subtype H5N1 viruses by wild birds, appear to explain virus circulation and perpetuation.

International audience; BACKGROUND:The number of human deaths due to rabies is currently underestimated to be 55,000 deaths per year. Biological diagnostic methods for confirmation of rabies remain limited, because testing on postmortem cerebral samples is the reference method, and in many countries, sampling brain tissue is rarely practiced. There is a need for a reliable method based on a simple collection of nonneural specimens.METHODS:A new reverse-transcription, heminested polymerase chain reaction (RT-hnPCR) protocol was standardized at 3 participating centers in Cambodia, Madagascar, and France. Fifty-one patients from Cambodia, Madagascar, Senegal, and France were prospectively enrolled in the study; 43 (84%) were ultimately confirmed as having rabies. A total of 425 samples were collected from these patients during hospitalization. We studied the accuracy of the diagnosis by comparing the results obtained with use of biological fluid specimens (saliva and urine) and skin biopsy specimens with the results obtained with use of the standard rabies diagnostic procedure performed with a postmortem brain biopsy specimen.RESULTS:The data obtained indicate a high specificity (100%) of RT-hnPCR and a higher sensitivity (>/=98%) when the RT-hnPCR was performed with skin biopsy specimens than when the test was performed with fluid specimens, irrespective of the time of collection (i.e., 1 day after the onset of symptoms or just after death). Also, a sensitivity of 100% was obtained with the saliva sample when we analyzed at least 3 successive samples per patient.CONCLUSIONS:Skin biopsy specimens should be systematically collected in cases of encephalitis of unknown origin. These samples should be tested by RT-hnPCR immediately to confirm rabies; if the technique is not readily available locally, the samples should be tested retrospectively for epidemiological purposes.

International audience; The Cambodian National Strategic Plan for Elimination of Malaria aims to move step by step toward elimination of malaria across Cambodia with an initial focus on Plasmodium falciparum malaria before achieving elimination of all forms of malaria, including Plasmodium vivax in 2025. The emergence of artemisinin-resistant P. falciparum in western Cambodia over the last decade has drawn global attention to support the ultimate goal of P. falciparum elimination, whereas the control of P. vivax lags much behind, making the 2025 target gradually less achievable unless greater attention is given to P. vivax elimination in the country. The following review presents in detail the past and current situation regarding P. vivax malaria, activities of the National Malaria Control Program, and interventional measures applied. Constraints and obstacles that can jeopardize our efforts to eliminate this parasite species are discussed.

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., Author Summary Dengue is the most important arthropod-borne disease affecting humans and represents a huge public health burden in affected countries. Symptoms are often non-specific hence the need for an early, sensitive and specific diagnosis of dengue for appropriate management as well as for early epidemic detection. Currently, almost all laboratory diagnostic methods require a blood specimen that may be sometimes be difficult or inconvenient to obtain. In this study, we assessed the possibility to use saliva and urine samples as alternatives to blood specimens in dengue diagnosis. We demonstrated that the performances of the different diagnostic methods (RT-PCR, NS1 antigen detection and anti-DENV IgM/IgG/IgA ELISAs) were in general not as good in saliva and urine as in plasma, but that the use of these body fluids obtained by non-invasive methods could be of value in certain circumstances such as outbreak investigations or in young children (once they are old enough to comply to instructions), in addition to the situations when blood cannot be easily collected (e.g., lack of phlebotomist, refusal of the procedure, etc.).

International audience; To study biological factors related to protection against HIV-1 infection in Cambodia, we recruited 48 partners of HIV-1-infected patients who remained uninfected (exposed uninfected individuals, EUs) despite unprotected sexual intercourse for more than 1 year and 49 unexposed controls (UCs). HIV-1-specific antibodies (IgA anti-gp41 and IgG anti-CD4-gp120 complex), T-cell responses, and cellular factors that may be involved in protection (peripheral blood mononuclear cell [PBMC] resistance to HIV-1 infection and beta-chemokine production) were evaluated. Anti-HIV-1 antibodies were higher in EUs than those in UCs (P = 0.01 and P = 0.04 for anti-gp41 and anti-CD4-gp120, respectively). We observed a decreased susceptibility to a primary Cambodian isolate, HIV-1KH019, in EU PBMCs as compared with UC PBMCs (P = 0.03). A weak T-cell response to one pool of HIV-1 Gag peptides was found by ELISpot in 1 of 19 EUs. Whereas T-cell specific immunity was not associated to protection, our results suggest that HIV-specific humoral immunity and reduced cell susceptibility to infection may contribute to protection against HIV-1 infection in Cambodian EUs.

International audience; In Cambodia, 1,303 bats of 16 species were tested for lyssavirus. No lyssavirus nucleocapsid was detected in 1,283 brains tested by immunofluorescence assay. Antibodies against lyssaviruses were detected by enzyme-linked immunosorbent assay in 144 (14.7%) of 981 serum samples. Thirty of 187 serum samples contained neutralizing antibodies against different lyssaviruses.

Background Epidemiological surveillance is a key activity in malaria control and elimination in low-transmission and pre-elimination settings. Hence, sensitive tools for estimating malaria force of infection are crucial. Serological markers might provide additional information in estimating force of infection in low-endemic areas along with classical parasite detection methods. Serological markers can be used to estimate recent, past or present malaria exposure, depending on the used markers and their half-life. Methods An assay based on 14 Plasmodium-specific peptides, one peptide specific for Anopheles gambiae saliva protein and five Plasmodium-specific recombinant proteins was developed for the MAGPIX system, assessed for its performance, and applied on blood spots from 2000 individuals collected in the Ratanakiri Province, Cambodia. Results A significant correlation for the use of 1000 and 2000 beads/antigen/well as well as for the monoplex versus multiplex assay was observed for all antigens (p

The authors are indebted to Wallis Annenberg whose generosity made this study possible, and to John Moores for his generous gift. We are grateful to Francoise Barré-Sinoussi for her help, advice, and support over the years. We thank Eric Nerrienet, Claire Rekacewicz, Daniel Scott, Gianfranco Pancino, Jean-Louis Sarthou, and Charles Mayaud for helpful discussions and advice as we began the study, and Jean-Louis Sarthou for his long time support of the Harvard team’s collaboration with the Institut Pasteur du Cambodge. We thank the CAMELIA study team, including the site nurses, doctors, and health workers, and Vincent Deubel and the staff at the Institut Pasteur du Cambodge. We thank Larry Fox, Rod Hoff, Jane Bupp, and Brigitte Bazin for their support and helpful discussions from the beginning of CAMELIA onward and the staff of the Cambodian Health Committee, especially Sam Sophan, for his contributions to and support of the work. We thank Leslie Kalish of Children’s Hospital Boston and the Harvard Catalyst Biostatistical Consulting program (NIH UL1 TR001102) for expert statistical advice and help as we started the analysis. We also thank Jerry Sadoff for his early support and Judy Lieberman for helpful comments on the manuscript. Finally, we are deeply indebted to the patients who took part in this study, who generously offered their participation to help the scientific community find solutions to AIDS and TB; International audience; OBJECTIVE: To investigate the impact of tuberculosis-associated immune reconstitution inflammatory syndrome (TB-IRIS) upon immunological recovery and the T-cell compartment after initiation of TB and antiretroviral therapy (ART).DESIGN AND METHODS: We prospectively evaluated T-cell immunophenotypes by flow cytometry and cytokines by Luminex assays in a subset (n = 154) of highly immunosuppressed HIV-infected patients with TB from the Cambodian Early versus Late Introduction of Antiretrovirals randomized clinical trial. We compared findings from patients who developed TB-IRIS with findings from patients who did not develop TB-IRIS. Data were evaluated with mixed-effect linear regression, Kaplan-Meier estimates, and Wilcoxon rank-sum tests, and q-values were calculated to control for multiple comparisons.RESULTS: Development of TB-IRIS was associated with significantly greater pre-ART frequencies of HLA-DRCD45ROCD4, CCR5CD4, OX40CD4, and Fas effector memory CD8 T cells, and significantly elevated levels of plasma interleukin (IL)-6, IL-1β, IL-8, and IL-10, and viral load. Post-ART initiation, effector memory CD4 and Fas effector memory CD4 T-cell frequencies significantly expanded, and central memory CD4 T-cell frequencies significantly contracted in patients who experienced TB-IRIS. By week 34 post-TB treatment initiation, effector memory/central memory CD4 T-cell ratios were markedly higher in TB-IRIS versus non-TB-IRIS patients.CONCLUSIONS: A distinct pattern of pre-ART T-cell and cytokine markers appear to poise the immune response of certain patients to develop TB-IRIS. Experience of TB-IRIS is then associated with long-term remodeling of the CD4 T-cell memory compartment towards an effector memory-dominated phenotype. We speculate that these pre and post-ART TB-IRIS-associated immune parameters may contribute to superior immune control of TB/HIV co-infection and better clinical outcome.

International audience; Two cases of Plasmodium knowlesi infection in humans were identified in Cambodia by 3 molecular detection assays and sequencing. This finding confirms the widespread distribution of P. knowlesi malaria in humans in Southeast Asia. Further wide-scale studies are required to assess the public health relevance of this zoonotic malaria parasite.