Discovering disease-causing pathogens in resource-scarce Southeast Asia using a global metagenomic pathogen monitoring system.

SignificanceMetagenomic pathogen sequencing offers an unbiased approach to characterizing febrile illness. In resource-scarce settings with high biodiversity, it is critical to identify disease-causing pathogens in order to understand burden and to prioritize efforts for control. Here, metagenomic next-generation sequencing (mNGS) characterization of the pathogen landscape in Cambodia revealed diverse vector-borne and zoonotic pathogens irrespective of age and gender as risk factors. Identification of key pathogens led to changes in national program surveillance. This study is a "real world" example of the use of mNGS surveillance of febrile individuals, executed in-country, to identify outbreaks of vector-borne, zoonotic, and other emerging pathogens in a resource-scarce setting.

National dengue surveillance, Cambodia 2002-2020.

Global dengue incidence has increased dramatically over the past few decades from approximately 500 000 reported cases in 2000 to over 5 million in 2019. This trend has been attributed to population growth in endemic areas, rapid unplanned urbanization, increasing global connectivity, and climate change expanding the geographic range of the Aedes spp. mosquito, among other factors. Reporting dengue surveillance data is key to understanding the scale of the problem, identifying important changes in the landscape of disease, and developing policies for clinical management, vector control and vaccine rollout. However, surveillance practices are not standardized, and data may be difficult to interpret particularly in low- and middle-income countries with fragmented health-care systems. The latest national dengue surveillance data for Cambodia was published in 2010. Since its publication, the country experienced marked changes in health policies, population demographics, climate and urbanization. How these changes affected dengue control remains unknown. In this article, we summarize two decades of policy changes, published literature, country statistics, and dengue case data collected by the Cambodia National Dengue Control Programme to: (i) identify important changes in the disease landscape; and (ii) derive lessons to inform future surveillance and disease control strategies. We report that while dengue case morbidity and mortality rates in Cambodia fell between 2002 and 2020, dengue incidence doubled and age at infection increased. Future national surveillance, disease prevention and treatment, and vector control policies will have to account for these changes to optimize disease control.

Le taux d'incidence de la dengue dans le monde a considérablement augmenté au cours des dernières décennies, passant d'environ 500 000 cas notifiés en 2000 à plus de 5 millions en 2019. Cette tendance est attribuée à la croissance démographique dans les zones d'endémie, à l'urbanisation rapide non planifiée, au développement de la connectivité à l'échelle internationale, ainsi qu'au changement climatique, qui agrandit le territoire géographique du moustique Aedes spp., entre autres. La communication des données de surveillance de la dengue est essentielle pour comprendre l'étendue du problème, identifier les principales variations de contexte entourant la maladie et mettre au point des politiques pour la prise en charge clinique, la lutte contre les vecteurs et le déploiement des vaccins. Les pratiques en matière de surveillance ne sont toutefois pas standardisées et les données peuvent être difficiles à interpréter, surtout dans les pays à revenu faible et intermédiaire où les systèmes de soins de santé sont fragmentés. Les données de surveillance les plus récentes concernant la dengue au Cambodge ont été publiées en 2010. Depuis leur publication, le pays a subi de profondes mutations au niveau des politiques de santé, de l'évolution démographique, du climat et de l'urbanisation. L'impact de ces mutations sur la lutte contre la dengue reste à établir. Dans le présent article, nous résumons deux décennies d'amendements politiques, de documentation, de statistiques nationales et d'informations collectées sur les cas par le programme cambodgien de lutte contre la dengue afin de: (i) définir les changements importants survenus dans le contexte entourant la maladie; mais aussi (ii) tirer des leçons en vue d'élaborer, à l'avenir, des stratégies de surveillance et de lutte contre la maladie. Nous signalons qu'en dépit d'une baisse des taux de morbidité et de mortalité liés aux cas de dengue entre 2002 et 2020 au Cambodge, son incidence a doublé et l'âge des patients au moment de l'infection a augmenté. Les futures politiques nationales de surveillance, de prévention et de traitement de la dengue, mais aussi de lutte contre ses vecteurs, devront tenir compte de ces changements de façon à mieux maîtriser la maladie.

La incidencia del dengue a nivel mundial ha aumentado considerablemente en las últimas décadas, desde aproximadamente 500 000 casos notificados en el año 2000 a más de 5 millones en 2019. Esta tendencia se ha atribuido al crecimiento de la población en zonas endémicas, a una urbanización rápida y no planificada, al aumento de la conectividad a nivel mundial y al cambio climático, que está permitiendo una distribución geográfica más amplia del mosquito Aedes spp., entre otros factores. Para comprender la magnitud del problema resulta clave la notificación de datos sobre vigilancia del dengue, la identificación de cambios importantes dentro del escenario de la enfermedad, la creación de políticas enfocadas a la gestión clínica, así como el control de vectores y la implantación de la vacuna. Sin embargo, las prácticas sobre vigilancia no están estandarizadas y es posible que sea difícil interpretar los datos, especialmente en países con ingresos medios y bajos, que cuentan con sistemas fragmentados de atención sanitaria. Los datos nacionales más recientes sobre vigilancia del dengue en Camboya se publicaron en 2010. Desde su publicación, el país experimentó cambios significativos en las políticas sanitarias, la demografía de la población, el clima y la urbanización. Aún no se sabe cómo afectaron dichos cambios al control del dengue. En el presente artículo, resumimos dos décadas de cambios políticos, de bibliografía publicada, de datos estadísticos a nivel nacional y datos sobre casos de dengue recopilados por el programa nacional de control de dengue en Camboya, con el fin de: (i) identificar cambios importantes en el escenario de la enfermedad; y (ii) extraer conclusiones para orientar futuras estrategias sobre vigilancia y control de la enfermedad. Informamos de que, aunque las tasas de morbilidad y mortalidad de los casos de dengue en Camboya descendieron entre 2002 y 2020, la incidencia del dengue se duplicó y la edad de infección aumentó. Las futuras políticas nacionales sobre vigilancia, prevención y tratamiento de la enfermedad y control de vectores deberán tener en cuenta estos cambios para optimizar el control de la enfermedad.

Development of Inapparent Dengue Associated With Increased Antibody Levels to Aedes aegypti Salivary Proteins: A Longitudinal Dengue Cohort in Cambodia.

BACKGROUND: We established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins. METHODS: Children aged 2-9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing. RESULTS: From 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05-2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12-2.41]; P = .01). CONCLUSIONS: High levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children. CLINICAL TRIALS REGISTRATION: NCT03534245.

Dengue in Cambodia 2002-2020: Cases, Characteristics and Capture by National Surveillance.

Objective: Data from 19 years of national dengue surveillance in Cambodia (2002-2020) were analyzed to describe trends in dengue case characteristics and incidence. Methods: Generalized additive models were fitted to dengue case incidence and characteristics (mean age, case phenotype, fatality) over time. Dengue incidence in a pediatric cohort study (2018-2020) was compared to national data during the same period to evaluate disease under-estimation by national surveillance. Findings: During 2002-2020, there were 353,270 cases of dengue (average age-adjusted incidence 1.75 cases/1,000 persons/year) recorded in Cambodia, with an estimated 2.1-fold increase in case incidence between 2002 and 2020 (slope = 0.0058, SE = 0.0021, p = 0.006). Mean age of infected individuals increased from 5.8 years in 2002 to 9.1 years in 2020 (slope = 0.18, SE = 0.088, p <0.001); case fatality rates decreased from 1.77% in 2002 to 0.10% in 2020 (slope = -0.16, SE = 0.0050, p <0.001). When compared to cohort data, national data under-estimated clinically apparent dengue case incidence by 5.0-fold (95% CI 0.2 - 26.5), and overall dengue case incidence (both apparent and inapparent cases) by 33.6-fold (range: 18.7- 53.6). Conclusion: Dengue incidence in Cambodia is increasing and disease is shifting to older pediatric populations. National surveillance continues to under-estimate case numbers. Future interventions should account for disease under-estimation and shifting demographics for scaling and to target appropriate age groups.

Determinants of exposure to Aedes mosquitoes: A comprehensive geospatial analysis in peri-urban Cambodia.

Aedes mosquitoes are some of the most important and globally expansive vectors of disease. Public health efforts are largely focused on prevention of human-vector contact. A range of entomological indices are used to measure risk of disease, though with conflicting results (i.e. larval or adult abundance does not always predict risk of disease). There is a growing interest in the development and use of biomarkers for exposure to mosquito saliva, including for Aedes spp, as a proxy for disease risk. In this study, we conduct a comprehensive geostatistical analysis of exposure to Aedes mosquito bites among a pediatric cohort in a peri­urban setting endemic to dengue, Zika, and chikungunya viruses. We use demographic, household, and environmental variables (the flooding index (NFI), land type, and proximity to a river) in a Bayesian geostatistical model to predict areas of exposure to Aedes aegypti bites. We found that hotspots of exposure to Ae. aegypti salivary gland extract (SGE) were relatively small (< 500 m and sometimes < 250 m) and stable across the two-year study period. Age was negatively associated with antibody responses to Ae. aegypti SGE. Those living in agricultural settings had lower antibody responses than those living in urban settings, whereas those living near recent surface water accumulation were more likely to have higher antibody responses. Finally, we incorporated measures of larval and adult density in our geostatistical models and found that they did not show associations with antibody responses to Ae. aegypti SGE after controlling for other covariates in the model. Our results indicate that targeted house- or neighborhood-focused interventions may be appropriate for vector control in this setting. Further, demographic and environmental factors more capably predicted exposure to Ae. aegypti mosquitoes than commonly used entomological indices.

Immunity to non-dengue flaviviruses impacts dengue virus IgG ELISA specificity in Cambodia.

Seroprevalence studies are the gold standard for disease surveillance, and serology was used to determine eligibility for the first licensed dengue vaccine. However, expanding flavivirus endemicity, co-circulation, and vaccination complicate serology results. Among 713 healthy Cambodian children, a commonly used indirect dengue virus IgG ELISA (PanBio) had a lower specificity than previously reported (94% vs. 100%). Of those with false positive PanBio results, 46% had detectable neutralizing antibodies against other flaviviruses, with the highest frequency against West Nile virus (WNV). Immunity to non-dengue flaviviruses can impact dengue surveillance and potentially pre-vaccine screening efforts.

Reduced impact of pyrimethamine drug pressure on Plasmodium malariae dihydrofolate reductase gene.

Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax; observed in three Cambodian samples).

Case Report: Cambodian National Malaria Surveillance Program Detection of Plasmodium knowlesi.

Despite recent success in reducing the regional incidence of Plasmodium falciparum malaria, cases of zoonotic malaria are on the rise in Southeast Asia. The Cambodian National Malaria Surveillance Program has previously relied on rapid diagnostic tests and blood smear microscopy with confirmatory polymerase chain reaction (PCR) testing in a subset of cases to further distinguish P. falciparum, P. malariae, P. ovale, and P. vivax species. Here, metagenomic next-generation sequencing identified P. knowlesi mono-infection in six Cambodian patients initially diagnosed with P. malariae by blood smear microscopy in February-May 2020. These findings of recent human infections with P. knowlesi in Cambodia led to the incorporation of P. knowlesi-specific PCR diagnostics to national malaria surveillance efforts.

Evaluation of cutaneous immune response in a controlled human in vivo model of mosquito bites.

Mosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito 'bite', eliciting immune responses that shape the establishment of infection and pathogenesis. Here we assess the cutaneous innate and adaptive immune responses to controlled Aedes aegypti feedings in humans living in Aedes-endemic areas. In this single-arm, cross-sectional interventional study (trial registration #NCT04350905), we enroll 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We perform 3-mm skin biopsies at baseline as well as 30 min, 4 h, and 48 h after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints are measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. The results reveal induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2 macrophages. As the immune reaction progresses T cell priming and regulatory pathways are upregulated along with a shift to Th2-driven responses and CD8+ T cell activation. Stimulation of participants' bitten skin cells with Aedes aegypti salivary gland extract results in reduced pro-inflammatory cytokine production. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites and can be leveraged to inform and develop novel therapeutics and vector-targeted vaccine candidates to interfere with vector-mediated disease.

The PAGODAS protocol: pediatric assessment group of dengue and Aedes saliva protocol to investigate vector-borne determinants of Aedes-transmitted arboviral infections in Cambodia.

BACKGROUND: Mosquito-borne arboviruses, like dengue virus, continue to cause significant global morbidity and mortality, particularly in Southeast Asia. When the infectious mosquitoes probe into human skin for a blood meal, they deposit saliva containing a myriad of pharmacologically active compounds, some of which alter the immune response and influence host receptivity to infection, and consequently, the establishment of the virus. Previous reports have highlighted the complexity of mosquito vector-derived factors and immunity in the success of infection. Cumulative evidence from animal models and limited data from humans have identified various vector-derived components, including salivary components, that are co-delivered with the pathogen and play an important role in the dissemination of infection. Much about the roles and effects of these vector-derived factors remain to be discovered. METHODS/DESIGN: We describe a longitudinal, pagoda (community)-based pediatric cohort study to evaluate the burden of dengue virus infection and document the immune responses to salivary proteins of Aedes aegypti, the mosquito vector of dengue, Zika, and chikungunya viruses. The study includes community-based seroprevalence assessments in the peri-urban town of Chbar Mon in Kampong Speu Province, Cambodia. The study aims to recruit 771 children between the ages of 2 and 9 years for a three year period of longitudinal follow-up, including twice per year (rainy and dry season) serosurveillance for dengue seroconversion and Ae. aegypti salivary gland homogenate antibody intensity determinations by ELISA assays. Diagnostic tests for acute dengue, Zika and chikungunya viral infections will be performed by RT-PCR. DISCUSSION: This study will serve as a foundation for further understanding of mosquito saliva immunity and its impact on Aedes-transmitted arboviral diseases endemic to Cambodia. TRIAL REGISTRATION: NCT03534245 registered on 23 May 2018.