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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 peri‑urban 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.

BackgroundWe 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.MethodsChildren 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.ResultsFrom 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).ConclusionsHigh 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 registrationNCT03534245.

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.

Introduction The global incidence of reported dengue has increased by tenfold over the last two decades, from 505 430 cases in 2000 to 5.2 million in 2019. (1) To contain [...], 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 considerablement augmente au cours des dernieres decennies, passant d'environ 500 000 cas notifies en 2000 a plus de 5 millions en 2019. Cette tendance est attribuee a la croissance demographique dans les zones d'endemie, a l'urbanisation rapide non planifiee, au developpement de la connectivite a l'echelle internationale, ainsi qu'au changement climatique, qui agrandit le territoire geographique du moustique Aedes spp, entre autres. La communication des donnees de surveillance de la dengue est essentielle pour comprendre l'etendue du probleme, 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 deploiement des vaccins. Les pratiques en matiere de surveillance ne sont toutefois pas standardisees et les donnees peuvent etre difficiles a interpreter, surtout dans les pays a revenu faible et intermediaire ou les systemes de soins de sante sont fragmentes. Les donnees de surveillance les plus recentes concernant la dengue au Cambodge ont ete publiees en 2010. Depuis leur publication, le pays a subi de profondes mutations au niveau des politiques de sante, de l'evolution demographique, du climat et de l'urbanisation. L'impact de ces mutations sur la lutte contre la dengue reste a etablir. Dans le present article, nous resumons deux decennies d'amendements politiques, de documentation, de statistiques nationales et d'informations collectees sur les cas par le programme cambodgien de lutte contre la dengue afin de: (i) definir les changements importants survenus dans le contexte entourant la maladie; mais aussi (ii) tirer des lecons en vue d'elaborer, a l'avenir, des strategies de surveillance et de lutte contre la maladie. Nous signalons qu'en depit d'une baisse des taux de morbidite et de mortalite lies aux cas de dengue entre 2002 et 2020 au Cambodge, son incidence a double et l'age des patients au moment de l'infection a augmente. Les futures politiques nationales de surveillance, de prevention et de traitement de la dengue, mais aussi de lutte contre ses vecteurs, devront tenir compte de ces changements de facon a mieux maitriser la maladie. La incidencia del dengue a nivel mundial ha aumentado considerablemente en las ultimas decadas, desde aproximadamente 500 000 casos notificados en el ano 2000 a mas de 5 millones en 2019. Esta tendencia se ha atribuido al crecimiento de la poblacion en zonas endemicas, a una urbanizacion rapida y no planificada, al aumento de la conectividad a nivel mundial y al cambio climatico, que esta permitiendo una distribucion geografica mas amplia del mosquito Aedes spp, entre otros factores. Para comprender la magnitud del problema resulta clave la notificacion de datos sobre vigilancia del dengue, la identificacion de cambios importantes dentro del escenario de la enfermedad, la creacion de politicas enfocadas a la gestion clinica, asi como el control de vectores y la implantacion de la vacuna. Sin embargo, las practicas sobre vigilancia no estan estandarizadas y es posible que sea dificil interpretar los datos, especialmente en paises con ingresos medios y bajos, que cuentan con sistemas fragmentados de atencion sanitaria. Los datos nacionales mas recientes sobre vigilancia del dengue en Camboya se publicaron en 2010. Desde su publicacion, el pais experimento cambios significativos en las politicas sanitarias, la demografia de la poblacion, el clima y la urbanizacion. Aun no se sabe como afectaron dichos cambios al control del dengue. En el presente articulo, resumimos dos decadas de cambios politicos, de bibliografia publicada, de datos estadisticos 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 duplico y la edad de infeccion aumento. Las futuras politicas nacionales sobre vigilancia, prevencion y tratamiento de la enfermedad y control de vectores deberan tener en cuenta estos cambios para optimizar el control de la enfermedad. [phrase omitted]

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.

BackgroundMosquito-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/designWe 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.DiscussionThis 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 registrationNCT03534245 registered on 23 May 2018.

Background: Seroprevalence studies are the standard for disease surveillance, and serology determined eligibility for the first dengue vaccine. Expanding flavivirus co-circulation and vaccination complicate testing. We evaluate the accuracy of a common dengue virus serological assay, examine immunity to non-dengue flaviviruses as a contributor to decreased performance, and assess whether alternative cut points may improve assay performance., Methods: Children (n = 770) aged 2-9 years in Kampong Speu, Cambodia were enrolled in a prospective longitudinal study, and PanBio indirect dengue virus immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) was performed. Plaque reduction neutralization tests (PRNTs) using dengue viruses were performed on a subset to assess the accuracy of the IgG ELISA, and PRNTs with Zika, Japanese encephalitis, and West Nile viruses evaluated immunity to non-dengue flaviviruses. Receiver operating curve analysis identified an alternative cut point to improve IgG ELISA accuracy., Results: The dengue IgG ELISA had a lower specificity than previously reported (58% vs 93%-100%). Of those with false-positive IgG results, 46% had detectable neutralizing antibodies against other flaviviruses including 14% against West Nile virus. A higher IgG cut point improved the test accuracy in this population., Conclusions: Physicians and public health authorities should be alert for West Nile in Cambodia. Immunity to non-dengue flaviviruses can impact dengue surveillance., Clinical Trials Registration: NCT03534245., Competing Interests: Potential conflicts of interest. J. E. M. is now a Clinical Franchise Leader of Influenza in Vaccines Research and Development at Sanofi Pasteur. The work reported in this manuscript was conducted prior to her switching positions. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The incidence of dengue virus disease has increased globally across the past half-century, with highest number of cases ever reported in 2019 and again in 2023. We analyzed climatological, epidemiological, and phylogenomic data to investigate drivers of two decades of dengue in Cambodia, an understudied endemic setting. Using epidemiological models fit to a 19-y dataset, we first demonstrate that climate-driven transmission alone is insufficient to explain three epidemics across the time series. We then use wavelet decomposition to highlight enhanced annual and multiannual synchronicity in dengue cycles between provinces in epidemic years, suggesting a role for climate in homogenizing dynamics across space and time. Assuming reported cases correspond to symptomatic secondary infections, we next use an age-structured catalytic model to estimate a declining force of infection for dengue through time, which elevates the mean age of reported cases in Cambodia. Reported cases in >70-y-old individuals in the 2019 epidemic are best explained when also allowing for waning multitypic immunity and repeat symptomatic infections in older patients. We support this work with phylogenetic analysis of 192 dengue virus (DENV) genomes that we sequenced between 2019 and 2022, which document emergence of DENV-2 Cosmopolitan Genotype-II into Cambodia. This lineage demonstrates phylogenetic homogeneity across wide geographic areas, consistent with invasion behavior and in contrast to high phylogenetic diversity exhibited by endemic DENV-1. Finally, we simulate an age-structured, mechanistic model of dengue dynamics to demonstrate how expansion of an antigenically distinct lineage that evades preexisting multitypic immunity effectively reproduces the older-age infections witnessed in our data., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., Competing Interests: Conflict of Interest: The authors declare no conflict of interest.

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.