Veasna Duong

Asymptomatic humans transmit dengue virus to mosquitoes

Significance Our work provides evidence that people who are infected with dengue virus without developing detectable clinical symptoms or prior to the onset of symptoms are infectious to mosquitoes. At a given level of viremia, symptom-free people were markedly more infectious to mosquitoes than clinically symptomatic patients. Our results fundamentally change the current paradigm for dengue epidemiology and control, based on detection of dengue virus-infected cases with apparent illness. Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.

Clinical and Virological Factors Influencing the Performance of a NS1 Antigen-Capture Assay and Potential Use as a Marker of Dengue Disease Severity

Background Detection of dengue NS1 antigen in acute infection has been proposed for early diagnosis of dengue disease. The aim of this study was to evaluate the clinical and virological factors influencing the performance of the Platelia NS1 Ag kit (BioRad) and to assess the potential use of NS1 antigen and dengue viral loads as markers of dengue disease severity. Methodology/Principal Findings Blood specimens were collected from patients hospitalized at the Kampong Cham hospital during the 2006 and 2007 dengue epidemics in Cambodia. Dengue infection was confirmed in 243/339 symptomatic patients and in 17 asymptomatic individuals out of 214 household members tested. Overall sensitivity and specificity of Platelia NS1 Ag kit were 57.5% and 100% respectively. NS1 Ag assay combined with IgM antibody capture ELISA significantly increased the sensitivity for dengue diagnosis. NS1 Ag positivity rate was found significantly higher in DF than in DHF/DSS, in primary than in secondary infections, in patients with a high viremia (>5 log/mL) and in patients infected with DENV-1. In asymptomatic individuals, the NS1 Ag capture sensitivity tends to be lower than that in symptomatic patients. Milder disease severity was observed independently in patients with RNA copy number >5 log10 cDNA equivalents/mL or in high level of NS1 antigen ratio or in DENV-1 infection. Conclusions Overall sensitivity of NS1 Ag detection kit varied widely across the various forms of dengue infection or disease. Sensitivity was highest in patients sampled during the first 3 days after onset of fever, in patients with primary infection, DENV-1 infection, with high level of viremia and in DF rather than DHF/DSS. In asymptomatic patients, RT-PCR assay has proved to be more sensitive than NS1 antigen detection. The NS1 antigen level correlated significantly with viremia and a low NS1 antigen ratio was associated with more severe disease.

Dengue incidence in urban and rural Cambodia: results from population-based active fever surveillance, 2006-2008.

Background Dengue vaccines are now in late-stage development, and evaluation and robust estimates of dengue disease burden are needed to facilitate further development and introduction. In Cambodia, the national dengue case-definition only allows reporting of children less than 16 years of age, and little is known about dengue burden in rural areas and among older persons. To estimate the true burden of dengue in the largest province of Cambodia, Kampong Cham, we conducted community-based active dengue fever surveillance among the 0-to-19–year age group in rural villages and urban areas during 2006–2008. Methods and Findings Active surveillance for febrile illness was conducted in 32 villages and 10 urban areas by mothers trained to use digital thermometers combined with weekly home visits to identify persons with fever. An investigation team visited families with febrile persons to obtain informed consent for participation in the follow-up study, which included collection of personal data and blood specimens. Dengue-related febrile illness was defined using molecular and serological testing of paired acute and convalescent blood samples. Over the three years of surveillance, 6,121 fever episodes were identified with 736 laboratory-confirmed dengue virus (DENV) infections for incidences of 13.4–57.8/1,000 person-seasons. Average incidence was highest among children less than 7 years of age (41.1/1,000 person-seasons) and lowest among the 16-to-19–year age group (11.3/1,000 person-seasons). The distribution of dengue was highly focal, with incidence rates in villages and urban areas ranging from 1.5–211.5/1,000 person-seasons (median 36.5). During a DENV-3 outbreak in 2007, rural areas were affected more than urban areas (incidence 71 vs. 17/1,000 person-seasons, p<0.001). Conclusion The large-scale active surveillance study for dengue fever in Cambodia found a higher disease incidence than reported to the national surveillance system, particularly in preschool children and that disease incidence was high in both rural and urban areas. It also confirmed the previously observed focal nature of dengue virus transmission.

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.

Emergence and Transmission of Arbovirus Evolutionary Intermediates with Epidemic Potential

The high replication and mutation rates of RNA viruses can result in the emergence of new epidemic variants. Thus, the ability to follow host-specific evolutionary trajectories of viruses is essential to predict and prevent epidemics. By studying the spatial and temporal evolution of chikungunya virus during natural transmission between mosquitoes and mammals, we have identified viral evolutionary intermediates prior to emergence. Analysis of virus populations at anatomical barriers revealed that the mosquito midgut and salivary gland pose population bottlenecks. By focusing on virus subpopulations in the saliva of multiple mosquito strains, we recapitulated the emergence of a recent epidemic strain of chikungunya and identified E1 glycoprotein mutations with potential to emerge in the future. These mutations confer fitness advantages in mosquito and mammalian hosts by altering virion stability and fusogenic activity. Thus, virus evolutionary trajectories can be predicted and studied in the short term before new variants displace currently circulating strains.

Genome-Wide Expression Profiling Deciphers Host Responses Altered during Dengue Shock Syndrome and Reveals the Role of Innate Immunity in Severe Dengue

Background Deciphering host responses contributing to dengue shock syndrome (DSS), the life-threatening form of acute viral dengue infections, is required to improve both the differential prognosis and the treatments provided to DSS patients, a challenge for clinicians. Methodology/Principal Findings Based on a prospective study, we analyzed the genome-wide expression profiles of whole blood cells from 48 matched Cambodian children: 19 progressed to DSS while 16 and 13 presented respectively classical dengue fever (DF) or dengue hemorrhagic fever grades I/II (DHF). Using multi-way analysis of variance (ANOVA) and adjustment of p-values to control the False Discovery Rate (FDR<10%), we identified a signature of 2959 genes differentiating DSS patients from both DF and DHF, and showed a strong association of this DSS-gene signature with the dengue disease phenotype. Using a combined approach to analyse the molecular patterns associated with the DSS-gene signature, we provide an integrative overview of the transcriptional responses altered in DSS children. In particular, we show that the transcriptome of DSS children blood cells is characterized by a decreased abundance of transcripts related to T and NK lymphocyte responses and by an increased abundance of anti-inflammatory and repair/remodeling transcripts. We also show that unexpected pro-inflammatory gene patterns at the interface between innate immunity, inflammation and host lipid metabolism, known to play pathogenic roles in acute and chronic inflammatory diseases associated with systemic vascular dysfunction, are transcriptionnally active in the blood cells of DSS children. Conclusions/Significance We provide a global while non exhaustive overview of the molecular mechanisms altered in of DSS children and suggest how they may interact to lead to final vascular homeostasis breakdown. We suggest that some mechanisms identified should be considered putative therapeutic targets or biomarkers of progression to DSS.

Use of a multiplex PCR/RT-PCR approach to assess the viral causes of influenza-like illnesses in Cambodia during three consecutive dry seasons.

Acute respiratory infections are a major cause of mortality and morbidity worldwide. Using multiplex PCR/RT‐PCR methods for the detection of 18 respiratory viruses, the circulation of those viruses during 3 consecutive dry seasons in Cambodia was described. Among 234 patients who presented with influenza‐like illness, 35.5% were positive for at least one virus. Rhinoviruses (43.4%), parainfluenza (31.3%) viruses and coronaviruses (21.7%) were the most frequently detected viruses. Influenza A virus, parainfluenza virus 4 and SARS‐coronavirus were not detected during the study period. Ninety apparently healthy individuals were included as controls and 10% of these samples tested positive for one or more respiratory viruses. No significant differences were observed in frequency and in virus copy numbers for rhinovirus detection between symptomatic and asymptomatic groups. This study raises questions about the significance of the detection of some respiratory viruses, especially using highly sensitive methods, given their presence in apparently healthy individuals. The link between the presence of the virus and the origin of the illness is therefore unclear. J. Med. Virol. 82:1762–1772, 2010.

Clinical and virological study of dengue cases and the members of their households: the multinational DENFRAME Project.

Background Dengue has emerged as the most important vector-borne viral disease in tropical areas. Evaluations of the burden and severity of dengue disease have been hindered by the frequent lack of laboratory confirmation and strong selection bias toward more severe cases. Methodology A multinational, prospective clinical study was carried out in South-East Asia (SEA) and Latin America (LA), to ascertain the proportion of inapparent dengue infections in households of febrile dengue cases, and to compare clinical data and biological markers from subjects with various dengue disease patterns. Dengue infection was laboratory-confirmed during the acute phase, by virus isolation and detection of the genome. The four participating reference laboratories used standardized methods. Principal Findings Among 215 febrile dengue subjects—114 in SEA and 101 in LA—28 (13.0%) were diagnosed with severe dengue (from SEA only) using the WHO definition. Household investigations were carried out for 177 febrile subjects. Among household members at the time of the first home visit, 39 acute dengue infections were detected of which 29 were inapparent. A further 62 dengue cases were classified at early convalescent phase. Therefore, 101 dengue infections were found among the 408 household members. Adding these together with the 177 Dengue Index Cases, the overall proportion of dengue infections among the study participants was estimated at 47.5% (278/585; 95% CI 43.5–51.6). Lymphocyte counts and detection of the NS1 antigen differed significantly between inapparent and symptomatic dengue subjects; among inapparent cases lymphocyte counts were normal and only 20% were positive for NS1 antigen. Primary dengue infection and a specific dengue virus serotype were not associated with symptomatic dengue infection. Conclusion Household investigation demonstrated a high proportion of household members positive for dengue infection, including a number of inapparent cases, the frequency of which was higher in SEA than in LA.

Under-recognition and reporting of dengue in Cambodia: a capture–recapture analysis of the National Dengue Surveillance System

Robust disease burden estimates are important for decision-making concerning introduction of new vaccines. Dengue is a major public health problem in the tropics but robust disease burden estimates are lacking. We conducted a two-sample, capture-recapture study in the largest province in Cambodia to determine disease under-recognition to the National Dengue Surveillance System (NDSS). During 2006-2008, community-based active surveillance for acute febrile illness was conducted in 0- to 19-year-olds in rural and urban areas combined with testing for dengue virus infection. Of 14 354 individuals under active surveillance (22 498 person-seasons), the annual incidence ranged from 13·4 to 57·8/1000 person-seasons. During the same period, NDSS incidence rates ranged from 1·1/1000 to 5·7/1000, which was 3·9- to 29·0-fold lower than found in the capture-recapture study. In hospitalized cases, the rate of under-recognition was 1·1- to 2·4-fold. This study shows the substantial degree of under-recognition/reporting of dengue and that reported hospitalized cases are not a good surrogate for estimating dengue disease burden.

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.