Bayesian modeling of post-vaccination serological data suggests that yearly vaccination of dog aged <2 years old is efficient to stop rabies circulation in Cambodia.

Rabies control remains challenging in low and middle-income countries, mostly due to lack of financial resources, rapid turnover of dog populations and poor accessibility to dogs. Rabies is endemic in Cambodia, where no national rabies vaccination program is implemented. The objective of this study was to assess the short and long-term vaccination-induced immunity in Cambodian dogs under field conditions, and to propose optimized vaccination strategies. A cohort of 351 dogs was followed at regular time points following primary vaccination only (PV) or PV plus single booster (BV). Fluorescent antibody virus neutralization test (FAVNT) was implemented to determine the neutralizing antibody titer against rabies and an individual titer ≥0·5 IU/mL indicated protection. Bayesian modeling was used to evaluate the individual duration of protection against rabies and the efficacy of two different vaccination strategies. Overall, 61% of dogs had a protective immunity one year after PV. In dogs receiving a BV, this protective immunity remained for up to one year after the BV in 95% of dogs. According to the best Bayesian model, a PV conferred a protective immunity in 82% of dogs (95% CI: 75-91%) for a mean duration of 4.7 years, and BV induced a lifelong protective immunity. Annual PV of dogs less than one year old and systematic BV solely of dogs vaccinated the year before would allow to achieve the 70% World Health Organization recommended threshold to control rabies circulation in a dog population in three to five years of implementation depending on dog population dynamics. This vaccination strategy would save up to about a third of vaccine doses, reducing cost and time efforts of mass dog vaccination campaigns. These results can contribute to optimize rabies control measures in Cambodia moving towards the global goal of ending human death from dog-mediated rabies by 2030.

Challenges of rabies surveillance in Madagascar based on a mixed method survey amongst veterinary health officers.

Introduction: In Madagascar, rabies is endemic and a notifiable disease. The objective of this mixed study was to understand the challenges faced by the Veterinary Health Officers (VHOs) in the current rabies surveillance system in Madagascar. Methods: A survey was conducted from mid-April to the end of July 2021 during which all officially-listed VHOs (N = 150) were contacted by phone at least twice. Participants, representative of the 22 regions of Madagascar, were interviewed by phone based on a semi-structured questionnaire on (1) their knowledge of rabies epidemiology in their area of activities, (2) the occurrence of human and animal rabies and the species affected in the region where they work, (3) the factors that might influence rabies surveillance depending on (a) their activities and area of operation, (b) the socio-cultural aspects of local communities, and (c) the general organization of animal rabies surveillance. Results: The majority (80/90) of VHOs declared having been informed of at least one suspected or confirmed case of human and/or animal rabies in their area of activity during their work as VHOs: at least once a month for 11 of them, at least once a year for 40 and with undetermined frequency for 29. Several obstacles hinder the surveillance of rabies resulting in under-reporting. The lack of funds to access suspect animals, collect, pack and ship samples in compliance with biosecurity measures and the cold chain, was mentioned as a major obstacle to surveillance. The second barrier is socio-cultural: in many large coastal regions, dogs are taboo and VHOs fear rejection by the community if they treat dogs. Discussion and conclusion: While the general population requires information on the rabies situation in Madagascar and on how to proceed in the event of a bite, veterinarians and decision-makers need to be fully aware of an evidence-based approach to rabies surveillance, prevention and control. Communication between the human and animal health sectors should be improved. Politicians need to be persuaded of the importance of funding to eliminate rabies in Madagascar. The adoption, in early 2023, of a national strategic plan for rabies control is a first step in this direction.

An Entomological Investigation during a Recent Rift Valley Fever Epizootic/Epidemic Reveals New Aspects of the Vectorial Transmission of the Virus in Madagascar.

A Rift Valley fever (RVF) outbreak occurred in at least five regions of Madagascar in 2021. The aim of this study was to provide an overview of the richness, abundance, ecology, and trophic preferences of mosquitoes in the Mananjary district and to investigate the distribution of mosquitoes that were RT-PCR-positive for RVFV. Three localities were prospected from 26 April to 4 May 2021, using light traps, BG-Sentinel traps baited with an artificial human odor, Muirhead-Thomson pit traps, and indoor pyrethroid spray catches. A total of 2806 mosquitoes belonging to at least 26 species were collected. Of 512 monospecific pools of mosquitoes tested with real-time RT-PCR, RVFV was detected in 37 pools representing 10 mosquito species. The RVFV-positive species were as follows: Aedes albopictus, Ae. argenteopunctatus, Anopheles coustani, An. gambiae s.l., An. mascarensis, An. squamosus/cydippis, Culex antennatus, Cx. decens, Cx. Tritaeniorhynchus, and Uranotaenia spp. Of the 450 tested engorged females, 78.7% had taken a blood meal on humans, 92.9% on cattle, and 71.6% had taken mixed (human-cattle) blood meals. This investigation suggests the potential role of mosquitoes in RVFV transmission within this epizootic/epidemic context and that the human populations at the three study sites were highly exposed to mosquitoes. Therefore, the use of impregnated mosquito nets as an appropriate prevention method is recommended.

The Re-Emergence of Rift Valley Fever in Mananjary District, Madagascar in 2021: A Call for Action.

An epizootic of rift valley fever (RVF) was suspected on 21 February 2021 in various districts of Madagascar, with a lab confirmation on 1 April 2021. A cross-sectional survey aiming to detect cases of RVF in humans and to study the circulation of rift valley fever virus (RVFV) in animals was conducted from 22 April to 4 May 2021 in the district of Mananjary. Blood samples from cattle and humans were tested using serological and molecular techniques. In cattle, the circulation of RVFV was confirmed between 5 February and 4 May 2021. The positivity rates of anti-RVFV IgG and IgM were 60% and 40%, respectively. In humans, the circulation of RVFV was observed from 1 April to 5 May 2021. The positivity rate of RVFV was estimated to be 11.7% by combining the results of the molecular and serological approaches. Of the 103 individuals who agreed to participate in the survey, 3 were determined to be positive by RT-PCR, and 10 had anti-RVFV IgM. Among them, one was positive for both. Given that previous studies have reported the circulation of RVFV during inter-epidemic periods and the occurrence of outbreaks due to imported RVFV in Madagascar, our findings suggest the importance of strengthening RVF surveillance from a "One Health" perspective by conducting syndromic and risk-based surveillance at the national and regional levels.

Multiple SARS-CoV-2 immunizations of an unvaccinated population lead to complex immunity. A T cell reactivity study of blood donors in Antananarivo.

BACKGROUND: Madagascar has undergone multiple and robust COVID-19 waves. The resulting immune background developed by its poorly vaccinated population has however not been described. METHODS: In this study, serological analysis and specific T cell response descriptions were used to describe the history of exposures of the capital's blood donors to SARS-CoV-2 and its VOCs. Samples were collected early 2022, and pools of multiple immunogenic peptides of SARS-CoV-2 were used in an IFN-γ secretion ELISPOT assay to characterize the specific T-cell immunity developed against these potential epitopes. RESULTS: Multiple epidemic waves have led to 92.1% of donors having detectable antibodies, and 94.8% having developed T-cells against SARS-CoV-2. Heterogeneous reactivities to different strain-derived peptides suggested multiple immunological backgrounds in the population including 16.1% of individuals exposed at least once to a unique strain, 27.1% to two strains, 28.5% to three strains, and 23.1% to four distinct strains. CONCLUSIONS: Cross-reactivity increased with multiple exposures but did not decrease the risk of re-infection. These results describe the extremely complex immunological background developed following multiple natural immunizations.

Neutralization of African enterovirus A71 genogroups by antibodies to canonical genogroups.

Enterovirus 71 (EV-A71) is a major public health problem, causing a range of illnesses from hand-foot-and-mouth disease to severe neurological manifestations. EV-A71 strains have been phylogenetically classified into eight genogroups (A to H), based on their capsid-coding genomic region. Genogroups B and C have caused large outbreaks worldwide and represent the two canonical circulating EV-A71 subtypes. Little is known about the antigenic diversity of new genogroups as compared to the canonical ones. Here, we compared the antigenic features of EV-A71 strains that belong to the canonical B and C genogroups and to genogroups E and F, which circulate in Africa. Analysis of the peptide sequences of EV-A71 strains belonging to different genogroups revealed a high level of conservation of the capsid residues involved in known linear and conformational neutralization antigenic sites. Using a published crystal structure of the EV-A71 capsid as a model, we found that most of the residues that are seemingly specific to some genogroups were mapped outside known antigenic sites or external loops. These observations suggest a cross-neutralization activity of anti-genogroup B or C antibodies against strains of genogroups E and F. Neutralization assays were performed with diverse rabbit and mouse anti-EV-A71 sera, anti-EV-A71 human standards and a monoclonal neutralizing antibody. All the batches of antibodies that were tested successfully neutralized all available isolates, indicating an overall broad cross-neutralization between the canonical genogroups B and C and genogroups E and F. A panel constituted of more than 80 individual human serum samples from Cambodia with neutralizing antibodies against EV-A71 subgenogroup C4 showed quite similar cross-neutralization activities between isolates of genogroups C4, E and F. Our results thus indicate that the genetic drift underlying the separation of EV-A71 strains into genogroups A, B, C, E and F does not correlate with the emergence of antigenically distinct variants.

Detection, characterization, and phylogenetic analysis of a near-whole genome sequence of a novel astrovirus in an endemic Malagasy fruit bat, Rousettus madagascariensis.

Bats (order: Chiroptera ) are known to host a diverse range of viruses, some of which present a public health risk. Thorough viral surveillance is therefore essential to predict and potentially mitigate zoonotic spillover. Astroviruses (family: Astroviridae ) are an understudied group of viruses with a growing amount of indirect evidence for zoonotic transfer. Astroviruses have been detected in bats with significant prevalence and diversity, suggesting that bats may act as important astrovirus hosts. Most astrovirus surveillance in wild bat hosts has, to date, been restricted to single-gene PCR detection and concomitant Sanger sequencing; additionally, many bat species and many geographic regions have not yet been surveyed for astroviruses at all. Here, we use metagenomic Next Generation Sequencing (mNGS) to detect astroviruses in three species of Madagascar fruit bats, Eidolon dupreanum, Pteropus rufus, and Rousettus madagascariensis . We detect numerous partial sequences from all three species and one near-full length astrovirus sequence from Rousettus madagascariensis , which we use to characterize the evolutionary history of astroviruses both within bats and the broader mammalian clade, Mamastrovirus . Taken together, applications of mNGS implicate bats as important astrovirus hosts and demonstrate novel patterns of bat astrovirus evolutionary history, particularly in the Southwest Indian Ocean region.

Longitudinal comparison of bacterial pathogen seropositivity among wet market vendors in the Lao People's Democratic Republic.

Wild animal trade for human consumption is a global issue, involving complex interactions between economics, culture, food security and conservation. Whilst being a biodiversity issue, it is also a major public health concern, with recent epidemics and pandemics of zoonotic pathogens linked to interactions with wildlife. At three time points, between March 2017 and June 2018, a longitudinal sero-survey of 150 market vendors from three wet markets in Laos (selling vegetables, domestic animal meat and/or wildlife meat) was conducted to determine if vendors had been differentially exposed to three endemic bacterial pathogens - Orientia tsutsugamushi, Rickettsia typhi, and Leptospira spp. A total of 367 serum samples were tested by IgG enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA, for scrub typhus group (STG) and typhus group (TG) only). Among vendors, 32.7% were IgG-positive for at least one pathogen, 13.3% sero-converted during the study. Multi-season occupancy modelling for STG indicated a significantly higher prevalence of STG IgG in vegetable vendors (27.3%) and wildlife vendors (28.4%) than in domestic animal meat vendors (6.9 %, p=0.05), and higher in Phonsavanh market (OR=9.6, p=0.03) compared to Lak Sao and Salavan markets. Estimated mean incidence was 57 cases per 10,000 per 7.5-month period. For TG, vendor age had a significant effect on prevalence (OR=1.04, p=0.006), estimated mean incidence was 64 cases per 10,000 per season (7.5-month period). Despite individuals selling domestic meat having a higher prevalence of Leptospira infections than those that did not (11.6% versus 4.5%), the difference was not significant. Whilst this study has a number of limitations, including vendors changing what food types they sold and no investigation of exposure outside of markets, the finding that the risk of exposure of vendors to zoonotic pathogens may be associated with types of food sold for human consumption warrants further investigation.

Comprehensive Characterization of Viral Diversity of Female Mosquitoes in Madagascar.

The diversity and circulation of arboviruses are not much studied in Madagascar. The fact is that arboviral emergences are rarely detected. The existing surveillance system primarily relies on serological detection and records only a few human infections annually. The city of Mahajanga, however, experienced a confirmed dengue fever epidemic in 2020 and 2021. This study aimed to characterize and analyze the virome of mosquitoes collected in Mahajanga, near patients with dengue-like syndromes to detect known and unknown viruses as well as investigate the factors contributing to the relative low circulation of arboviruses in the area. A total of 4280 mosquitoes representing at least 12 species from the Aedes, Anopheles, and Culex genera were collected during the dry and the rainy seasons from three sites, following an urbanization gradient. The virome analysis of 2192 female mosquitoes identified a diverse range of viral families and genera and revealed different patterns that are signatures of the influence of the mosquito genus or the season of collection on the composition and abundance of the virome. Despite the absence of known human or veterinary arboviruses, the identification and characterization of viral families, genera, and species in the mosquito virome contribute to our understanding of viral ecology and diversity within mosquito populations in Madagascar. This study serves as a foundation for ongoing surveillance efforts and provides a basis for the development of preventive strategies against various mosquito-borne viral diseases, including known arboviruses.

Uncovering the endemic circulation of rabies in Cambodia.

In epidemiology, endemicity characterizes sustained pathogen circulation in a geographical area, which involves a circulation that is not being maintained by external introductions. Because it could potentially shape the design of public health interventions, there is an interest in fully uncovering the endemic pattern of a disease. Here, we use a phylogeographic approach to investigate the endemic signature of rabies virus (RABV) circulation in Cambodia. Cambodia is located in one of the most affected regions by rabies in the world, but RABV circulation between and within Southeast Asian countries remains understudied. Our analyses are based on a new comprehensive data set of 199 RABV genomes collected between 2014 and 2017 as well as previously published Southeast Asian RABV sequences. We show that most Cambodian sequences belong to a distinct clade that has been circulating almost exclusively in Cambodia. Our results thus point towards rabies circulation in Cambodia that does not rely on external introductions. We further characterize within-Cambodia RABV circulation by estimating lineage dispersal metrics that appear to be similar to other settings, and by performing landscape phylogeographic analyses to investigate environmental factors impacting the dispersal dynamic of viral lineages. The latter analyses do not lead to the identification of environmental variables that would be associated with the heterogeneity of viral lineage dispersal velocities, which calls for a better understanding of local dog ecology and further investigations of the potential drivers of RABV spread in the region. Overall, our study illustrates how phylogeographic investigations can be performed to assess and characterize viral endemicity in a context of relatively limited data.

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.

Excess mortality associated with the COVID-19 pandemic during the 2020 and 2021 waves in Antananarivo, Madagascar.

INTRODUCTION: COVID-19-associated mortality remains difficult to estimate in sub-Saharan Africa because of the lack of comprehensive systems of death registration. Based on death registers referring to the capital city of Madagascar, we sought to estimate the excess mortality during the COVID-19 pandemic and calculate the loss of life expectancy. METHODS: Death records between 2016 and 2021 were used to estimate weekly excess mortality during the pandemic period. To infer its synchrony with circulation of SARS-CoV-2, a cross-wavelet analysis was performed. Life expectancy loss due to the COVID-19 pandemic was calculated by projecting mortality rates using the Lee and Carter model and extrapolating the prepandemic trends (1990-2019). Differences in life expectancy at birth were disaggregated by cause of death. RESULTS: Peaks of excess mortality in 2020-21 were associated with waves of COVID-19. Estimates of all-cause excess mortality were 38.5 and 64.9 per 100 000 inhabitants in 2020 and 2021, respectively, with excess mortality reaching ≥50% over 6 weeks. In 2021, we quantified a drop of 0.8 and 1.0 years in the life expectancy for men and women, respectively attributable to increased risks of death beyond the age of 60 years. CONCLUSION: We observed high excess mortality during the pandemic period, in particular around the peaks of SARS-CoV-2 circulation in Antananarivo. Our study highlights the need to implement death registration systems in low-income countries to document true toll of a pandemic.

Characterization of soluble TLR2 and CD14 levels during acute dengue virus infection.

Dengue virus infection results in a broad spectrum of diseases ranging from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Hitherto, there is no consensus biomarker for the prediction of severe dengue disease in patients. Yet, early identification of patients who progress to severe dengue is pivotal for better clinical management. We have recently reported that an increased frequency of classical (CD14 ++CD16-) monocytes with sustained high TLR2 expression in acutely infected dengue patients correlates with severe dengue development. Here, we hypothesized that the relatively lower TLR2 and CD14 expression in mild dengue patients is due to the shedding of their soluble forms (sTLR2 and sCD14) and that these could be used as indicators of disease progression. Therefore, using commercial sandwich ELISAs, we evaluated the release of sTLR2 and sCD14 by peripheral blood mononuclear cells (PBMCs) in response to in vitro dengue virus (DENV) infection and assessed their levels in acute-phase plasma of 109 dengue patients. We show that while both sTLR2 and sCD14 are released by PBMCs in response to DENV infection in vitro, their co-circulation in an acute phase of the disease is not always apparent. In fact, sTLR2 was found only in 20% of patients irrespective of disease status. In contrast, sCD14 levels were detected in all patients and were significantly elevated in DF patients when compared to DHF patients and age-matched healthy donors. Altogether, our results suggest that sCD14 may help in identifying patients at risk of severe dengue at hospital admittance.

Side-by-side Comparative Study of the Immunogenicity of the Intramuscular and Intradermal Rabies Post-exposure Prophylaxis Regimens in a Cohort of Suspected Rabies Virus Exposed Individuals.

All World Health Organization (WHO) pre-qualified rabies vaccines for humans are inactivated tissue culture rabies virus formulations produced for intramuscular (IM) administration. Due to costs and vaccine shortage, dose-saving intradermal (ID) administration of rabies post-exposure prophylaxis (PEP) is encouraged by WHO. This study compared the immunogenicity of the ID 2-site, 3-visit Institut Pasteur Cambodge (IPC) PEP regimen to the IM 1-site, 4-visit 4-dose Essen regimen using Verorab vaccine (Sanofi). The development of neutralizing antibodies (nAbs) and T cell response was assessed in 210 patients with a category II or III animal exposure in a rabies-endemic country. At day 28, all participants developed nAbs (≥0.5 IU/mL), irrespective of PEP scheme, age, or administration of rabies immunoglobulin. T cell response and nAb titers were similar for both PEP schemes. This study demonstrated that the 1-week ID IPC regimen is as effective as the 2-week IM 4-dose Essen regimen in inducing an anti-rabies immune response under real-life PEP.

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.

Review on Main Arboviruses Circulating on French Guiana, An Ultra-Peripheric European Region in South America.

French Guiana (FG), a French overseas territory in South America, is susceptible to tropical diseases, including arboviruses. The tropical climate supports the proliferation and establishment of vectors, making it difficult to control transmission. In the last ten years, FG has experienced large outbreaks of imported arboviruses such as Chikungunya and Zika, as well as endemic arboviruses such as dengue, Yellow fever, and Oropouche virus. Epidemiological surveillance is challenging due to the differing distributions and behaviors of vectors. This article aims to summarize the current knowledge of these arboviruses in FG and discuss the challenges of arbovirus emergence and reemergence. Effective control measures are hampered by the nonspecific clinical presentation of these diseases, as well as the Aedes aegypti mosquito's resistance to insecticides. Despite the high seroprevalence of certain viruses, the possibility of new epidemics cannot be ruled out. Therefore, active epidemiological surveillance is needed to identify potential outbreaks, and an adequate sentinel surveillance system and broad virological diagnostic panel are being developed in FG to improve disease management.

Investigation of Dengue Infection in Asymptomatic Individuals during a Recent Outbreak in La Réunion.

The number of dengue cases has increased dramatically over the past 20 years and is an important concern, particularly as the trends toward urbanization continue. While the majority of dengue cases are thought to be asymptomatic, it is unknown to what extent these contribute to transmission. A better understanding of their importance would help to guide control efforts. In 2019, a dengue outbreak in La Reunion resulted in more than 18,000 confirmed cases. Between October 2019 and August 2020, 19 clusters were investigated in the south, west, and east of the island, enabling the recruitment of 605 participants from 368 households within a 200 m radius of the home of the index cases (ICs). No active asymptomatic infections confirmed by RT-PCR were detected. Only 15% were possible asymptomatic dengue infections detected by the presence of anti-dengue IgM antibodies. Only 5.3% of the participants had a recent dengue infection confirmed by RT-PCR. Although the resurgence of dengue in La Réunion is very recent (2016), the rate of anti-dengue IgG positivity, a marker of past infections, was already high at 43% in this study. Dengue transmission was focal in time and space, as most cases were detected within a 100-m radius of the ICs, and within a time interval of less than 7 days between infections detected in a same cluster. No particular demographic or socio-cultural characteristics were associated with dengue infections. On the other hand, environmental risk factors such as type of housing or presence of rubbish in the streets were associated with dengue infections.

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.

Ribosomal RNA (rRNA) sequences from 33 globally distributed mosquito species for improved metagenomics and species identification.

Total RNA sequencing (RNA-seq) is an important tool in the study of mosquitoes and the RNA viruses they vector as it allows assessment of both host and viral RNA in specimens. However, there are two main constraints. First, as with many other species, abundant mosquito ribosomal RNA (rRNA) serves as the predominant template from which sequences are generated, meaning that the desired host and viral templates are sequenced far less. Second, mosquito specimens captured in the field must be correctly identified, in some cases to the sub-species level. Here, we generate mosquito rRNA datasets which will substantially mitigate both of these problems. We describe a strategy to assemble novel rRNA sequences from mosquito specimens and produce an unprecedented dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African Republic, Madagascar, and French Guiana). These sequences will allow both physical and computational removal of rRNA from specimens during RNA-seq protocols. We also assess the utility of rRNA sequences for molecular taxonomy and compare phylogenies constructed using rRNA sequences versus those created using the gold standard for molecular species identification of specimens-the mitochondrial cytochrome c oxidase I (COI) gene. We find that rRNA- and COI-derived phylogenetic trees are incongruent and that 28S and concatenated 28S+18S rRNA phylogenies reflect evolutionary relationships that are more aligned with contemporary mosquito systematics. This significant expansion to the current rRNA reference library for mosquitoes will improve mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA depletion protocols for a broader range of species and streamlining species identification by rRNA sequence and phylogenetics.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.