Asymptomatic Dengue Virus Infections, Cambodia, 2012-2013.

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 <15 years of age and 225 (65.0%) were <9 years of age. We identified 26 (7.5%) participants with strictly asymptomatic DENV infection at diagnosis and during follow-up. We linked symptomatic DENV infection status to familial relationships with index cases. During the 2-year study, we saw fewer asymptomatic DENV infections than expected based on the literature.

Low Circulation of Zika Virus, Cambodia, 2007-2016.

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.

Evaluation of the performances of six commercial kits designed for dengue NS1 and anti-dengue IgM, IgG and IgA detection in urine and saliva clinical specimens.

BACKGROUND: Rapid diagnostic tests (RDTs) have been commercialized in order to help physicians in dengue diagnosis. Until recently, only blood samples were used for those tests but it has been shown in several studies that urine and saliva can also be employed for dengue diagnosis. RDTs for the detection of NS1 antigen and anti-dengue IgG, IgM and IgA in urine and saliva specimens have thus been developed by Standard Diagnostics. The aim of this study was to evaluate the performances these new commercial assays. METHODS: Two panels of clinical specimens were used: one for the evaluation of the NS1-detection devices and the second for the evaluation of the antibody-detection kits. Each panel consisted of urine and saliva specimens collected sequentially from 86 patients with a confirmed dengue infection. A total of 291 saliva and 440 urine samples were included in the NS1-evaluation panel and 530 saliva and 528 urine specimens constituted the antibody-evaluation panel. All samples were tested in parallel by in-house ELISAs and by the commercial RDTs. RESULTS: The RDTs demonstrated an overall sensitivity of 15.5 %/27.9 %/10.7 % for NS1/IgG/IgA detection in urine samples and 20.4 %/ 34.8 %/11 %/6.2 % for NS1/IgG/IgM/IgA detection in saliva samples. Compared to the in-house NS1 ELISA, the results obtained with the NS1 RDT demonstrated a good correlation with urine samples (kappa coefficient: 0.88) but not with saliva specimens (kappa coefficient: 0.28). RDTs designed for antibody detection in saliva and urine were extremely specific (100 %), but less sensitive than the in-house ELISAs (i.e., reduction of the overall sensitivity by 12.2 % for the RDT designed for IgG detection in urine and by 23.7 % for the RDT detecting anti-DENV IgM in saliva). IgM were not detected in urine, either by RDT or ELISA. CONCLUSIONS: Although the RDTs evaluated here offer an apparently attractive approach for dengue diagnosis, this study suggests that these new commercial kits would require further improvement to increase the sensitivity.

Reemergence of Chikungunya virus in Cambodia.

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.

National dengue surveillance in Cambodia 1980-2008: epidemiological and virological trends and the impact of vector control.

OBJECTIVE: Dengue has been reportable in Cambodia since 1980. Virological surveillance began in 2000 and sentinel surveillance was established at six hospitals in 2001. Currently, national surveillance comprises passive and active data collection and reporting on hospitalized children aged 0-15 years. This report summarizes surveillance data collected since 1980. METHODS: Crude data for 1980-2001 are presented, while data from 2002-2008 are used to describe disease trends and the effect of vector control interventions. Trends in dengue incidence were analysed using the Prais-Winsten generalized linear regression model for time series. FINDINGS: During 1980-2001, epidemics occurred in cycles of 3-4 years, with the cycles subsequently becoming less prominent. For 2002-2008 data, linear regression analysis detected no significant trend in the annual reported age-adjusted incidence of dengue (incidence range: 0.7-3.0 per 1000 population). The incidence declined in 2.7% of the 185 districts studied, was unchanged in 86.2% and increased in 9.6%. The age-specific incidence was highest in infants aged < 1 year and children aged 4-6 years. The incidence was higher during rainy seasons. All four dengue virus (DENV) serotypes were permanently in circulation, though the predominant serotype has alternated between DENV-3 and DENV-2 since 2000. Although larvicide has been distributed in 94 districts since 2002, logistic regression analysis showed no association between the intervention and dengue incidence. CONCLUSION: The dengue burden remained high among young children in Cambodia, which reflects intense transmission. The national vector control programme appeared to have little impact on disease incidence.

Highly pathogenic avian influenza virus (H5N1) outbreak in captive wild birds and cats, Cambodia.

From December 2003 through January 2004, the Phnom Tamao Wildlife Rescue Centre, Cambodia, was affected by the highly pathogenic influenza virus (H5N1). Birds from 26 species died. Influenza virus subtype H5N1 was detected in 6 of 7 species tested. Cats from 5 of 7 species were probably infected; none died.

An algorithm applied to national surveillance data for the early detection of major dengue outbreaks in Cambodia.

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.

A 1-week intradermal dose-sparing regimen for rabies post-exposure prophylaxis (RESIST-2): an observational cohort study.

BACKGROUND: The international health authorities are backing an effort to eliminate canine-mediated rabies in humans by 2030. This effort will require improving access to adequate and timely rabies post-exposure prophylaxis as compliance is low with WHO-recommended regimens (given in four to five visits over 1 month). Access could be substantially improved by an abridged regimen to reduce doses, direct and indirect costs, and improve vaccine equity by better sharing of available vaccine. We aimed to compare rabies virus neutralising antibody titres before and after the fourth visit to determine whether that session was needed or the current regimen could be abridged. METHODS: In this observational cohort study, we measured rabies virus neutralising antibody titres using rapid fluorescent focus inhibition tests in 116 people bitten by dogs with laboratory-confirmed rabies and 20 control individuals. Percentages of circulating plasmablasts were determined by flow cytometry. All individuals had been referred to the rabies prevention clinic at Institut Pasteur in Cambodia and received two intradermal injections of post-exposure prophylaxis on days 0, 3, 7, and 28 (Thai Red Cross regimen) with or without equine rabies immunoglobulin, as per 2010 WHO recommendations. FINDINGS: All individuals had rabies virus neutralising antibody titres considered protective (≥0·5 IU/mL) and plasmablast activation on day 28 before the last injection. The median rabies virus neutralising antibody concentration in the group of individuals bitten by rabies virus-positive dogs was 1·08 IU/mL (IQR 0·37-3·09) on day 7, 26·86 (22·68-49·50) on day 28, and 26·74 (11·78-49·06) on day 42. No significant differences were observed in titres between days 28 and 42, after titres reached a plateau. These titres were reached notwithstanding equine rabies immunoglobulin use, age, sex, nutrition status as indicated by upper-arm circumference in children or BMI in adults, or dog infection status. Titres or plasmablast percentages did not increase between the day of the last injection and 2 weeks later. All patients were alive 1 year after post-exposure prophylaxis. INTERPRETATION: The fourth vaccine session on day 28 provides no additional benefit. Rabies post-exposure prophylaxis can be abridged to a two-dose, three-session, 1 week regimen to improve post-exposure prophylaxis coverage and equity at no risk to patients. FUNDING: Institut Pasteur.

Comparison of the dynamics of Japanese encephalitis virus circulation in sentinel pigs between a rural and a peri-urban setting in Cambodia.

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.

Broad and long-lasting immune protection against various Chikungunya genotypes demonstrated by participants in a cross-sectional study in a Cambodian rural community.

Chikungunya virus (CHIKV) is an alphavirus circulating worldwide. Its presence in Asia has been reported since the 1950s, constituting the Asian genotype. Since 2005, strains from the Eastern, Central, and Southern African (ECSA) genotype have caused several outbreaks across Asia. Viruses from the ECSA genotype were also detected in Cambodia in late 2011 and led to an outbreak in a rural community in 2012. A former investigation from 2012 found a higher risk of infection in people younger than 40 years, suggesting a pre-existing herd immunity in the older Cambodian population due to infection with an Asian genotype. In 2016, we collected serum from equivalent numbers of individuals born before 1975 and born after 1980 that were also part of the 2012 study. We analyzed the 154 serum samples from 2016 for neutralization against the Cambodian ECSA isolate and three strains belonging to the Asian genotype. This experiment revealed that 22.5% (18/80) of the younger study participants had no CHIKV antibodies, whereas 5.4% (4/74) of the older population remained naive. Study participants infected during the ECSA outbreak had twofold neutralizing titers against the ECSA and the most ancient Asian genotype virus (Thailand 1958) compared to the other two Asian genotype viruses. The neutralization data also support the older population's exposure to an Asian genotype virus during the 1960s. The observed cross-reactivity confirms that the investigated CHIKV strains belong to a single serotype despite the emergence of novel ECSA genotype viruses and supports the importance of the development of a Chikungunya vaccine.

Proteinuria during dengue fever in children.

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.

Laboratory diagnostics in dog-mediated rabies: an overview of performance and a proposed strategy for various settings.

The diagnosis of dog-mediated rabies in humans and animals has greatly benefited from technical advances in the laboratory setting. Approaches to diagnosis now include the detection of rabies virus (RABV), RABV RNA, or RABV antigens. These assays are important tools in the current efforts aimed at the global elimination of dog-mediated rabies. The assays available for use in laboratories are reviewed herein, as well as their strengths and weaknesses, which vary with the types of sample analyzed. Depending on the setting, however, the public health objectives and use of RABV diagnosis in the field will also vary. In non-endemic settings, the detection of all introduced or emergent animal or human cases justifies exhaustive testing. In dog RABV-endemic settings, such as rural areas of developing countries where most cases occur, the availability of or access to testing may be severely constrained. Thus, these issues are also discussed along with a proposed strategy to prioritize testing while access to rabies testing in the resource-poor, highly endemic setting is improved. As the epidemiological situation of rabies in a country evolves, the strategy should shift from that of an endemic setting to one more suitable for a decreased rabies incidence following the implementation of efficient control measures and when nearing the target of dog-mediated rabies elimination.

Evidence of two distinct phylogenetic lineages of dog rabies virus circulating in Cambodia.

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.

Intensive Circulation of Japanese Encephalitis Virus in Peri-urban Sentinel Pigs near Phnom Penh, Cambodia.

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.

Rabies Vaccine and Rabies Immunoglobulin in Cambodia: Use and Obstacles to Use.

BACKGROUND: Authorities have pledged to eliminate canine rabies by 2020 in Cambodia, a country with a very high rabies burden. Logistic and financial access to timely and adequate postexposure prophylaxis (PEP) is essential for preventing rabies in humans. METHODS: We undertook a survey of the few identified sites where PEP rabies vaccination and rabies immunoglobulin (RIG) are available in Cambodia. We examined the Rabies Prevention Center at Institut Pasteur du Cambodge (rpc@ipc) database and rpc@ipc order forms for 2012 to assess vaccine and RIG use. We conducted a rapid internet survey of centers that provide rabies vaccine and RIG in Cambodia, other than rpc@ipc. RESULTS: The cost of a full course of intramuscular or intradermal PEP in Cambodia, with and without RIG, was also estimated. Rabies vaccination is free of charge in one foundation hospital and is accessible for a fee at Institut Pasteur du Cambodge (IPC), some institutions, and some Cambodian private clinics. In 2012, 27,500 rabies vaccine doses (0.5 mL) and 591 equine RIG doses were used to provide intradermal PEP to 20,610 persons at rpc@ipc following animal bites. Outside of rpc@ipc, an estimated total of 53,400 vaccine doses and 200 RIG doses were used in Cambodia in 2012. The wholesale cost of full rabies PEP was estimated at 50% to 100% of a Cambodian farmer's monthly wage. CONCLUSIONS: Local populations and travelers cannot be sure to locally access adequate and timely PEP due to high costs and low access to RIG. Travelers to high-endemic areas such as Cambodia are strongly encouraged to undergo pre-exposure vaccination or seek expert advice, as per World Health Organization (WHO) recommendations. State-subsidized, pre-positioned stocks of human vaccine and RIG in bite management centers would extend the rabies prevention centers network. Support from Institut Pasteur du Cambodge for staff training, cold chain, and quality control would contribute to reducing the risk of rabies deaths in Cambodia.

Value of Routine Dengue Diagnostic Tests in Urine and Saliva Specimens.

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.

Etiology of encephalitis syndrome among hospitalized children and adults in Takeo, Cambodia, 1999-2000.

Whether or not Japanese encephalitis virus (JEV) is an important causative agent of acute encephalitis in Cambodia remains unclear. This study was carried out to determine the cause of encephalitis syndrome among children and adults admitted to Takeo Provincial Hospital from October 1999 to September 2000. Ninety-nine cases were included in the study: 52 pediatric cases (12 were fatal) and 47 adult cases (10 were fatal). A causative agent such as human herpesvirus (HHV-3 or HHV-4), Cryptococcus neoformans, or Mycobacterium tuberculosis had been identified in 8 of the 11 adults who had human immunodeficiency virus type 1 (HIV-1). An infectious agent was identified in 35 (40%) of 88 HIV-1-seronegative patients (60% of the causes remains unidentified). These comprised 11 bacterial infections, 1 fungal infection, and 23 viral infections. The viral infections were 1 fatal HHV-4 infection, 5 dengue virus infections (2 fatal), 1 coinfection with flavivirus and alphavirus, and 16 presumptive infections JEV (no virus detected), one case of which was fatal. Infection with JEV, the principal cause identified in the 99 encephalitis syndromes, concerned 16 (31%) of 52 children.

Complex dynamic of dengue virus serotypes 2 and 3 in Cambodia following series of climate disasters.

The Dengue National Control Program was established in Cambodia in 2000 and has reported between 10,000 and 40,000 dengue cases per year with a case fatality rate ranging from 0.7 to 1.7. In this study 39 DENV-2 and 57 DENV-3 viruses isolated from patients between 2000 and 2008 were fully sequenced. Five DENV2 and four DENV3 distinct lineages with different dynamics were identified. Each lineage was characterized by the presence of specific mutations with no evidence of recombination. In both DENV-2 and DENV-3 the lineages present prior to 2003 were replaced after that date by unrelated lineages. After 2003, DENV-2 lineages D2-3 and D2-4 cocirculated until 2007 when they were almost completely replaced by a lineage D2-5 which emerged from D2-3 Conversely, all DENV-3 lineages remained, diversified and cocirculated with novel lineages emerging. Years 2006 and 2007 were marked by a high prevalence of DENV-3 and 2007 with a large dengue outbreak and a high proportion of patients with severe disease. Selective sweeps in DENV-1 and DENV-2 were linked to immunological escape to a predominately DENV-3-driven immunological response. The complex dynamic of dengue in Cambodia in the last ten years has been associated with a combination of stochastic climatic events, cocirculation, coevolution, adaptation to different vector populations, and with the human population immunological landscape.

Genetic diversity and lineage dynamic of dengue virus serotype 1 (DENV-1) in Cambodia.

In Cambodia, dengue virus (DENV) was first isolated in 1963 and has become endemic with peak epidemic during raining season. Since 2000, the Dengue National Control Program has reported from 10,000 to 40,000 cases per year with fatality rates ranging from 0.7 to 1.7. All four dengue serotypes are found circulating in Cambodia with alternative predominance of serotypes DENV-2 and DENV-3. The DENV-1 represents from 5% to 20% of all circulating viruses, depending upon the year. In this work, 79 clinical strains of DENV-1 were isolated between 2000 and 2009 and their genome fully sequenced. Four distinct lineages with different dynamics were identified. The main evolutionary drive was negative selective pressure but each lineage was characterized by the presence of specific mutations acquired through evolution. Coexistence, extinction and replacement of lineages occurred over the 10-year period. Lineages 1, 2 and 3 were all detected since 2000-2002 and disappeared in 2003, 2004-2005 and 2007, respectively. Lineages 1 and 2 displayed different dynamics. Lineage 1 was very diverse whereas lineage 2 was very homogeneous. Lineage 4 which derived from lineage 3 in 2003 remained the only one at the end of the sampling period in 2008-2009 owing to a selective sweep. The lineages dynamic of DENV-1 viruses and consequences for molecular epidemiology are discussed.

Field evaluation and impact on clinical management of a rapid diagnostic kit that detects dengue NS1, IgM and IgG.

BACKGROUND: Dengue diagnosis is complex and until recently only specialized laboratories were able to definitively confirm dengue infection. Rapid tests are now available commercially making biological diagnosis possible in the field. The aim of this study was to evaluate a combined dengue rapid test for the detection of NS1 and IgM/IgG antibodies. The evaluation was made prospectively in the field conditions and included the study of the impact of its use as a point-of-care test for case management as well as retrospectively against a panel of well-characterized samples in a reference laboratory. METHODOLOGY/PRINCIPAL FINDINGS: During the prospective study, 157 patients hospitalized for a suspicion of dengue were enrolled. In the hospital laboratories, the overall sensitivity, specificity, PPV and NPV of the NS1/IgM/IgG combination tests were 85.7%, 83.9%, 95.6% and 59.1% respectively, whereas they were 94,4%, 90.0%, 97.5% and 77.1% respectively in the national reference laboratory at Institut Pasteur in Cambodia. These results demonstrate that optimal performances require adequate training and quality assurance. The retrospective study showed that the sensitivity of the combined kit did not vary significantly between the serotypes and was not affected by the immune status or by the interval of time between onset of fever and sample collection. The analysis of the medical records indicates that the physicians did not take into consideration the results obtained with the rapid test including for care management and use of antibiotic therapy. CONCLUSIONS: In the context of our prospective field study, we demonstrated that if the SD Bioline Dengue Duo kit is correctly used, a positive result highly suggests a dengue case but a negative result doesn't rule out a dengue infection. Nevertheless, Cambodian pediatricians in their daily practice relied on their clinical diagnosis and thus the false negative results obtained did not directly impact on the clinical management.