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.

Phase 1 trial to model primary, secondary, and tertiary dengue using a monovalent vaccine.

BACKGROUND: The four co-circulating and immunologically interactive dengue virus serotypes (DENV1-4) pose a unique challenge to vaccine design because sub-protective immunity can increase the risk of severe dengue disease. Existing dengue vaccines have lower efficacy in DENV seronegative individuals but higher efficacy in DENV exposed individuals. There is an urgent need to identify immunological measures that are strongly associated with protection against viral replication and disease following sequential exposure to distinct serotypes. METHODS/DESIGN: This is a phase 1 trial wherein healthy adults with neutralizing antibodies to zero (seronegative), one non-DENV3 (heterotypic), or more than one (polytypic) DENV serotype will be vaccinated with the live attenuated DENV3 monovalent vaccine rDEN3Δ30/31-7164. We will examine how pre-vaccine host immunity influences the safety and immunogenicity of DENV3 vaccination in a non-endemic population. We hypothesize that the vaccine will be safe and well tolerated, and all groups will have a significant increase in the DENV1-4 neutralizing antibody geometric mean titer between days 0 and 28. Compared to the seronegative group, the polytypic group will have lower mean peak vaccine viremia, due to protection conferred by prior DENV exposure, while the heterotypic group will have higher mean peak viremia, due to mild enhancement. Secondary and exploratory endpoints include characterizing serological, innate, and adaptive cell responses; evaluating proviral or antiviral contributions of DENV-infected cells; and immunologically profiling the transcriptome, surface proteins, and B and T cell receptor sequences and affinities of single cells in both peripheral blood and draining lymph nodes sampled via serial image-guided fine needle aspiration. DISCUSSION: This trial will compare the immune responses after primary, secondary, and tertiary DENV exposure in naturally infected humans living in non-endemic areas. By evaluating dengue vaccines in a new population and modeling the induction of cross-serotypic immunity, this work may inform vaccine evaluation and broaden potential target populations. TRIAL REGISTRATION: NCT05691530 registered on January 20, 2023.

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.

SARS-CoV-2 Cross-Reactivity in Prepandemic Serum from Rural Malaria-Infected Persons, Cambodia.

Inhabitants of the Greater Mekong Subregion in Cambodia are exposed to pathogens that might influence serologic cross-reactivity with severe acute respiratory syndrome coronavirus 2. A prepandemic serosurvey of 528 malaria-infected persons demonstrated higher-than-expected positivity of nonneutralizing IgG to spike and receptor-binding domain antigens. These findings could affect interpretation of large-scale serosurveys.

Safety and immunogenicity of a mosquito saliva peptide-based vaccine: a randomised, placebo-controlled, double-blind, phase 1 trial.

BACKGROUND: In animal models, immunity to mosquito salivary proteins protects animals against mosquito-borne disease. These findings provide a rationale to vaccinate against mosquito saliva instead of the pathogen itself. To our knowledge, no vector salivary protein-based vaccine has been tested for safety and immunogenicity in humans. We aimed to assess the safety and immunogenicity of Anopheles gambiae saliva vaccine (AGS-v), a peptide-based vaccine derived from four A gambiae salivary proteins, in humans. METHODS: In this randomised, placebo-controlled, double-blind, phase 1 trial, participants were enrolled at the National Institutes of Health Clinical Center in Bethesda, MD, USA. Participants were eligible if they were healthy adults, aged 18-50 years with no history of severe allergic reactions to mosquito bites. Participants were randomly assigned (1:1:1), using block randomisation and a computer-generated randomisation sequence, to treatment with either 200 nmol of AGS-v vaccine alone, 200 nmol of AGS-v with adjuvant (Montanide ISA 51), or sterile water as placebo. Participants and clinicians were masked to treatment assignment. Participants were given a subcutaneous injection of their allocated treatment at day 0 and day 21, followed by exposure to feeding by an uninfected Aedes aegypti mosquito at day 42 to assess subsequent risk to mosquito bites in a controlled setting. The primary endpoints were safety and immunogenicity at day 42 after the first immunisation. Participants who were given at least one dose of assigned treatment were assessed for the primary endpoints and analysis was by intention to treat. The trial was registered with ClinicalTrials.gov, NCT03055000, and is closed for accrual. FINDINGS: Between Feb 15 and Sept 10, 2017, we enrolled and randomly assigned 49 healthy adult participants to the adjuvanted vaccine (n=17), vaccine alone (n=16), or placebo group (n=16). Five participants did not complete the two-injection regimen with mosquito feeding at day 42, but were included in the safety analyses. No systemic safety concerns were identified; however, one participant in the adjuvanted vaccine group developed a grade 3 erythematous rash at the injection site. Pain, swelling, erythema, and itching were the most commonly reported local symptoms and were significantly increased in the adjuvanted vaccine group compared with both other treatment groups (nine [53%] of 17 participants in the adjuvanted vaccine group, two [13%] of 16 in the vaccine only group, and one [6%] of 16 in the placebo group; p=0·004). By day 42, participants who were given the adjuvanted vaccine had a significant increase in vaccine-specific total IgG antibodies compared with at baseline than did participants who were give vaccine only (absolute difference of log10-fold change of 0·64 [95% CI 0·39 to 0·89]; p=0·0002) and who were given placebo (0·62 [0·34 to 0·91]; p=0·0001). We saw a significant increase in IFN-γ production by peripheral blood mononuclear cells at day 42 in the adjuvanted vaccine group compared with in the placebo group (absolute difference of log10 ratio of vaccine peptide-stimulated vs negative control 0·17 [95% CI 0·061 to 0·27]; p=0·009) but we saw no difference between the IFN-γ production in the vaccine only group compared with the placebo group (0·022 [-0·072 to 0·116]; p=0·63). INTERPRETATION: AGS-v was well tolerated, and, when adjuvanted, immunogenic. These findings suggest that vector-targeted vaccine administration in humans is safe and could be a viable option for the increasing burden of vector-borne disease. FUNDING: Office of the Director and the Division of Intramural Research at the National Institute of Allergy and Infectious Diseases, and National Institutes of Health.

Material strategies and considerations for serologic testing of global infectious diseases.

The SARS-CoV-2 pandemic has brought to light multiple considerations when approaching infectious diseases on the global level. These range from diagnostic platforms, to therapeutics, and prevention agents. In this article, we focus on the engineering platforms and considerations when applying serologic assays to multiple geographic locations, climates with varying endemic virus repertoires, and different laboratory and clinical resource settings. Serologic assays detect antibodies that react against viral proteins, suggesting prior infection and correlative of an increased likelihood of immunity to future infection. As these assays are focused on the human immune response to a pathogen, and humans are variable, there are a number of important engineering steps to optimize assay performance, from sample collection, to assay execution and data analysis. Moving forward, a global approach to infectious disease detection and prevention is necessary to prevent the spread of future viruses with pandemic potential.

Mosquito Saliva: The Hope for a Universal Arbovirus Vaccine?

Arthropod-borne viruses (arboviruses) are taxonomically diverse causes of significant morbidity and mortality. In recent decades, important mosquito-borne viruses such as West Nile, chikungunya, dengue, and Zika have re-emerged and spread widely, in some cases pandemically, to cause serious public health emergencies. There are no licensed vaccines against most of these viruses, and vaccine development and use has been complicated by the number of different viruses to protect against, by subtype and strain variation, and by the inability to predict when and where outbreaks will occur. A new approach to preventing arboviral diseases is suggested by the observation that arthropod saliva facilitates transmission of pathogens, including leishmania parasites, Borrelia burgdorferi, and some arboviruses. Viruses carried within mosquito saliva may more easily initiate host infection by taking advantage of the host's innate and adaptive immune responses to saliva. This provides a rationale for creating vaccines against mosquito salivary proteins, rather than against only the virus proteins contained within the saliva. As proof of principle, immunization with sand fly salivary antigens to prevent leishmania infection has shown promising results in animal models. A similar approach using salivary proteins of important vector mosquitoes, such as Aedes aegypti, might protect against multiple mosquito-borne viral infections.

Piperaquine Population Pharmacokinetics and Cardiac Safety in Cambodia.

Despite the rising rates of resistance to dihydroartemisinin-piperaquine (DP), DP remains a first-line therapy for uncomplicated malaria in many parts of Cambodia. While DP is generally well tolerated as a 3-day DP (3DP) regimen, compressed 2-day DP (2DP) regimens were associated with treatment-limiting cardiac repolarization effects in a recent clinical trial. To better estimate the risks of piperaquine on QT interval prolongation, we pooled data from three randomized clinical trials conducted between 2010 and 2014 in northern Cambodia. A population pharmacokinetic model was developed to compare exposure-response relationships between the 2DP and 3DP regimens while accounting for differences in regimen and sample collection times between studies. A 2-compartment model with first-order absorption and elimination without covariates best fit the data. The linear slope-intercept model predicted a 0.05-ms QT prolongation per ng/ml of piperaquine (5 ms per 100 ng/ml) in this largely male population. Though the plasma half-life was similar in both regimens, peak and total piperaquine exposures were higher in those treated with the 2DP regimen. Furthermore, the correlation between the plasma piperaquine concentration and the QT interval prolongation was stronger in the population receiving the 2DP regimen. Neither the time since the previous meal nor the baseline serum magnesium or potassium levels had additive effects on QT interval prolongation. As electrocardiographic monitoring is often nonexistent in areas where malaria is endemic, 2DP regimens should be avoided and the 3DP regimen should be carefully considered in settings where viable alternative therapies exist. When DP is employed, the risk of cardiotoxicity can be mitigated by combining a 3-day regimen, enforcing a 3-h fast before and after administration, and avoiding the concomitant use of QT interval-prolonging medications. (This study used data from three clinical trials that are registered at ClinicalTrials.gov under identifiers NCT01280162, NCT01624337, and NCT01849640.).

Expression of complement and toll-like receptor pathway genes is associated with malaria severity in Mali: a pilot case control study.

BACKGROUND: The host response to infection by Plasmodium falciparum, the parasite most often responsible for severe malaria, ranges from asymptomatic parasitaemia to death. The clinical trajectory of malaria is influenced by host genetics and parasite load, but the factors determining why some infections produce uncomplicated malaria and some proceed to severe disease remain incompletely understood. METHODS: To identify molecular markers of severe falciparum malaria, human gene expression patterns were compared between children aged 6 months to 5 years with severe and uncomplicated malaria who were enrolled in a case-control study in Bandiagara, Mali. Microarrays were used to obtain expression data on severe cases and uncomplicated controls at the time of acute disease presentation (five uncomplicated and five severe), 1 week after presentation (three uncomplicated and three severe) and treatment initiation, and in the subsequent dry season (late convalescence, four uncomplicated and four severe). This is a pilot study for the first use of microarray technology in Mali. RESULTS: Complement and toll-like receptor (TLR) pathways were differentially expressed, with severe cases showing higher expression of the C1q, TLR2, TLR4, TLR8, and CR1 genes. Other genes previously associated with malaria pathogenesis, GZMB, FOS and HSPA6, were also higher among severe cases. TLR2, TLR4, TLR8, CR1, GZMB, FOS, and HSPA6 genes were expressed at lower levels in severe cases at late convalescence. CONCLUSIONS: Overexpression of genes previously associated with uncomplicated malaria was associated with severe disease. Low baseline expression of these genes may represent candidate markers for severe malaria. Despite the small sample size, results of this pilot study offer promising targets for follow-up analyses.

Randomized, double-blind, placebo-controlled clinical trial of a two-day regimen of dihydroartemisinin-piperaquine for malaria prevention halted for concern over prolonged corrected QT interval.

Dihydroartemisinin-piperaquine, the current first-line drug for uncomplicated malaria caused by Plasmodium falciparum and Plasmodium vivax in Cambodia, was previously shown to be of benefit as malaria chemoprophylaxis when administered as a monthly 3-day regimen. We sought to evaluate the protective efficacy of a compressed monthly 2-day treatment course in the Royal Cambodian Armed Forces. The safety and efficacy of a monthly 2-day dosing regimen of dihydroartemisinin-piperaquine were evaluated in a two-arm, randomized, double-blind, placebo-controlled cohort study with 2:1 treatment allocation. Healthy military volunteers in areas along the Thai-Cambodian border where there is a high risk of malaria were administered two consecutive daily doses of 180 mg dihydroartemisinin and 1,440 mg piperaquine within 30 min to 3 h of a meal once per month for a planned 4-month period with periodic electrocardiographic and pharmacokinetic assessment. The study was halted after only 6 weeks (69 of 231 projected volunteers enrolled) when four volunteers met a prespecified cardiac safety endpoint of QTcF (Fridericia's formula for correct QT interval) prolongation of >500 ms. The pharmacodynamic effect on the surface electrocardiogram (ECG) peaked approximately 4 h after piperaquine dosing and lasted 4 to 8 h. Unblinded review by the data safety monitoring board revealed mean QTcF prolongation of 46 ms over placebo at the maximum concentration of drug in serum (Cmax) on day 2. Given that dihydroartemisinin-piperaquine is one of the few remaining effective antimalarial agents in Cambodia, compressed 2-day treatment courses of dihydroartemisinin-piperaquine are best avoided until the clinical significance of these findings are more thoroughly evaluated. Because ECG monitoring is often unavailable in areas where malaria is endemic, repolarization risk could be mitigated by using conventional 3-day regimens, fasting, and avoidance of repeated dosing or coadministration with other QT-prolonging medications. (This study has been registered at ClinicalTrials.gov under registration no. NCT01624337.).

Antibodies to Aedes aegypti D7L salivary proteins as a new serological tool to estimate human exposure to Aedes mosquitoes.

Introduction: Aedes spp. are the most prolific mosquito vectors in the world. Found on every continent, they can effectively transmit various arboviruses, including the dengue virus which continues to cause outbreaks worldwide and is spreading into previously non-endemic areas. The lack of widely available dengue vaccines accentuates the importance of targeted vector control strategies to reduce the dengue burden. High-throughput tools to estimate human-mosquito contact and evaluate vector control interventions are lacking. We propose a novel serological tool that allows rapid screening of human cohorts for exposure to potentially infectious mosquitoes. Methods: We tested 563 serum samples from a longitudinal pediatric cohort study previously conducted in Cambodia. Children enrolled in the study were dengue-naive at baseline and were followed biannually for dengue incidence for two years. We used Western blotting and enzyme-linked immunosorbent assays to identify immunogenic Aedes aegypti salivary proteins and measure total anti-Ae. aegypti IgG. Results: We found a correlation (rs=0.86) between IgG responses against AeD7L1 and AeD7L2 recombinant proteins and those to whole salivary gland homogenate. We observed seasonal fluctuations of AeD7L1+2 IgG responses and no cross-reactivity with Culex quinquefasciatus and Anopheles dirus mosquitoes. The baseline median AeD7L1+2 IgG responses for young children were higher in those who developed asymptomatic versus symptomatic dengue. Discussion: The IgG response against AeD7L1+2 recombinant proteins is a highly sensitive and Aedes specific marker of human exposure to Aedes bites that can facilitate standardization of future serosurveys and epidemiological studies by its ability to provide a robust estimation of human-mosquito contact in a high-throughput fashion.

Case Report: Risk Factors Associated with Mortality in Adults with Burkholderia Pseudomallei Bacteremia: A Retrospective Case Series of Melioidosis in Cambodia.

In resource-scarce settings, melioidosis is associated with up to 80% mortality. Studies of melioidosis in Cambodia report primarily on pediatric populations with localized infection; however, literature describing Cambodian adults with severe melioidosis is lacking. We present a case series of 35 adults with sequence-confirmed Burkholderia pseudomallei bacteremia presenting to a provincial referral hospital in rural Cambodia. More than 90% of the patients had diabetes, an important risk factor for developing melioidosis. Inappropriate antimicrobial therapy was significantly associated with lower odds of survival. Improved diagnostic testing and greater access to first-line antibiotics for acute melioidosis treatment present potential targets for intervention to reduce mortality associated with this disease in resource-limited settings.

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Evaluation of parasite subpopulations and genetic diversity of the msp1, msp2 and glurp genes during and following artesunate monotherapy treatment of Plasmodium falciparum malaria in Western Cambodia.

BACKGROUND: Despite widespread coverage of the emergence of artemisinin resistance, relatively little is known about the parasite populations responsible. The use of PCR genotyping around the highly polymorphic Plasmodium falciparum msp1, msp2 and glurp genes has become well established both to describe variability in alleles within a population of parasites, as well as classify treatment outcome in cases of recurrent disease. The primary objective was to assess the emergence of minority parasite clones during seven days of artesunate (AS) treatment in a location with established artemisinin resistance. An additional objective was to investigate whether the classification of clinical outcomes remained valid when additional genotyping was performed. METHODS: Blood for parasite genotyping was collected from 143 adult patients presenting with uncomplicated falciparum malaria during a clinical trial of AS monotherapy in Western Cambodia. Nested allelic type-specific amplification of the genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) and the glutamate-rich protein (glurp) was performed at baseline, daily during seven days of treatment, and again at failure. Allelic variants were analysed with respect to the size of polymorphisms using Quantity One software to enable identification of polyclonal infections. RESULTS: Considerable variation of msp2 alleles but well-conserved msp1 and glurp were identified. At baseline, 31% of infections were polyclonal for one or more genes. Patients with recurrent malaria were significantly more likely to have polyclonal infections than patients without recurrence (seven of nine versus 36 of 127, p = 0.004). Emergence of minority alleles during treatment was detected in only one of twenty-three cases defined as being artemisinin resistant. Moreover, daily genotyping did not alter the final outcome classification in any recurrent cases. CONCLUSIONS: The parasites responsible for artemisinin-resistant malaria in a clinical trial in Western Cambodia comprise the dominant clones of acute malaria infections rather than minority clones emerging during treatment. Additional genotyping during therapy was not beneficial. Disproportionately high rates of polyclonal infections in cases of recurrence suggest complex infections lead to poor treatment outcomes. Current research objectives should be broadened to include identification and follow-up of recurrent polyclonal infections so as to define their role as potential agents of emerging resistance.

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.

Antibodies to Aedes aegypti D7L salivary proteins as a new serological tool to estimate human exposure to Aedes mosquitoes.

Introduction: Aedes spp. are the most prolific mosquito vectors in the world. Found on every continent, they can effectively transmit various arboviruses, including the dengue virus which continues to cause outbreaks worldwide and is spreading into previously non-endemic areas. The lack of widely available dengue vaccines accentuates the importance of targeted vector control strategies to reduce the dengue burden. High-throughput sensitive tools to estimate human-mosquito contact and evaluate vector control interventions are lacking. We propose a novel serological tool that allows rapid screening of large human cohorts for exposure to potentially infectious mosquitoes and effective targeting of vector control. Methods: We tested 563 serum samples from a longitudinal pediatric cohort study previously conducted in Cambodia. Children enrolled in the study were dengue-naïve at baseline and were followed biannually for dengue incidence for two years. We used Western blotting and enzyme-linked immunosorbent assays to identify the most immunogenic Aedes aegypti salivary proteins and measure total anti- Ae. Aegypti IgG. Results: We found a strong correlation (r s =0.86) between the combined IgG responses against AeD7L1 and AeD7L2 recombinant proteins and those to whole salivary gland homogenate. We observed seasonal fluctuations of AeD7L1+2 IgG responses, corresponding to Aedes spp. abundance in the region, and no cross-reactivity with Culex quinquefasciatus and Anopheles dirus mosquitoes. The baseline median AeD7L1+2 IgG responses for young children were higher in those who developed asymptomatic dengue versus those who developed symptomatic dengue. Conclusion: The IgG response against AeD7L1+2 recombinant proteins is a highly sensitive and Aedes specific marker of human exposure to Aedes bites that can facilitate standardization of future serosurveys and epidemiological studies by its ability to provide a robust estimation of human-mosquito contact in a high-throughput fashion.

Genomic epidemiology of SARS-CoV-2 in Cambodia, January 2020 to February 2021.

The first case of coronavirus disease 2019 (COVID-19) in Cambodia was confirmed on 27 January 2020 in a traveller from Wuhan. Cambodia subsequently implemented strict travel restrictions, and although intermittent cases were reported during the first year of the COVID-19 pandemic, no apparent widespread community transmission was detected. Investigating the routes of severe acute respiratory coronavirus 2 (SARS-CoV-2) introduction into the country was critical for evaluating the implementation of public health interventions and assessing the effectiveness of social control measures. Genomic sequencing technologies have enabled rapid detection and monitoring of emerging variants of SARS-CoV-2. Here, we detected 478 confirmed COVID-19 cases in Cambodia between 27 January 2020 and 14 February 2021, 81.3 per cent in imported cases. Among them, fifty-four SARS-CoV-2 genomes were sequenced and analysed along with representative global lineages. Despite the low number of confirmed cases, we found a high diversity of Cambodian viruses that belonged to at least seventeen distinct PANGO lineages. Phylogenetic inference of SARS-CoV-2 revealed that the genetic diversity of Cambodian viruses resulted from multiple independent introductions from diverse regions, predominantly, Eastern Asia, Europe, and Southeast Asia. Most cases were quickly isolated, limiting community spread, although there was an A.23.1 variant cluster in Phnom Penh in November 2020 that resulted in a small-scale local transmission. The overall low incidence of COVID-19 infections suggests that Cambodia's early containment strategies, including travel restrictions, aggressive testing and strict quarantine measures, were effective in preventing large community outbreaks of COVID-19.