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Principal Investigator: Christina Yek
Organization: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
Fiscal Year: 2024
Award: $1,364,178
Funding agency: National Institute of Allergy and Infectious Diseases
A DIVERSE PATHOGEN LANDSCAPE IS RESPONSIBLE FOR ACUTE FEBRILE ILLNESS IN CAMBODIA.
In 2019, we implemented an in-country pathogen short-read sequencing pipeline for evaluation of clinical samples from febrile patients aged 6 months to 65 years of age in peri-urban Cambodia (NIH 19-I-N109). As of 2023, mNGS analysis of over 3000 patients identified vector-borne pathogens as the largest clinical category responsible for febrile disease (an interim analysis of the first 500 patients was published in PNAS in 2022). Dengue virus (DENV) was the most abundant although Plasmodium vivax, undetectable on rapid tests used for elimination efforts, was also often identified. Metagenomic sequencing of clinical samples enabled identification of chikungunya virus that presaged a larger national outbreak, leading to vector control interventions and the addition of chikungunya PCR to the national surveillance system. Interestingly, underappreciated vector-borne and zoonotic pathogens such as Plasmodium knowlesi, Rickettsia spp., scrub typhus, leptospirosis, and co-infecting HIV were also detected. Among febrile persons with positive clinical cultures, untargeted sequencing approaches identified a diverse range of bacterial pathogens, among which the three most common were E coli, Acinetobacter baumannii, and Burkholderia pseudomallei. In FY24, partial and whole genome data generated through this approach enabled pathogen characterization including identification of genetic markers of antimicrobial resistance, recognition of disease outbreaks, and descriptions of pathogen evolution and phylogeography. We demonstrated that untargeted sequencing approaches can be used to identify and monitor the breadth of pathogens leading to acute febrile illness, and in turn inform development of diagnostic algorithms and tailored solutions.
A NOVEL DENV2 GENOTYPE DROVE THE UNPRECEDENTED 2019 DENGUE EPIDEMIC IN CAMBODIA WITH GEOGRAPHICALLY DISPERSED TRANSMISSION PATTERNS IN COMPARISON TO ENDEMIC DENV1.
To better understand the 2019 dengue epidemic, we employed phylogeographic analyses of 192 georeferenced DENV genomes (Brook et al, PNAS 2024). We discovered that a novel introduction of DENV2 Cosmopolitan genotype, a strain phylogenetically distant from any previously published sequence data from Cambodia, as likely responsible for the historic epidemic. Further, DENV2 transmission patterns were more intensely local than DENV-1, suggesting DENV1 as the endemic serotype and DENV-2 as an introduction. Older children infected with DENV2, but with likely pre-existing DENV-1 immunity, were more likely to share a transmission chain at a given spatial distance, suggesting those with secondary infections were sicker and more likely to stay at home (and infect others). This novel DENV2 genotype finding raises important questions about viral evolutionary expansion and associated clinical pathogenesis.
IDENTIFICATION OF BIOMARKERS OF EXPOSURE TO AEDES AEGYPTI MOSQUITO.
After confirming a sufficient burden of dengue circulating in our pediatric cohort, we investigated semi-annual sero-reactivity as total IgG to Ae. aegypti salivary gland homogenate (SGH), which was notably higher in the younger participants and during wet season, which is not unexpected. When we surveyed our cohort for reactivity to Ae. aegypti SGH via ELISA and Western blot, we identified five immunodominant proteins by molecular weight. We produced these selected targets, expressed in HEK293 cells, as recombinant proteins and tested them in sera from our cohort and NIH Clinical Center healthy volunteers (Chea et al, Front Immunol 2024). As expected, we see reactivity in likely-exposed Cambodian sera but not NIH Clinical Center nave sera, suggesting that these proteins may serve as markers of differential exposure to Ae. aegypti. The most robust marker of exposure is a combined D7L1 and D7L2 and also tracks along with disease risk showing that those with higher levels of these proteins tend to have asymptomatic dengue infection as previously shown with Ae. aegypti salivary antibody levels as well.
HIGH LEVELS OF CROSS-REACTIVE IMMUNITY RAISED AGAINST ENDEMIC PATHOGENS COMPLICATE DISEASE DETECTION AND CLINICAL PRESENTATION IN CAMBODIA.
Numerous arboviruses, including CHIKV, ZIKV, DENV, and JEV, co-circulate in Cambodia and are often difficult to distinguish based on clinical presentation alone. In FY24, we tested the ability of a commercial NS1 antigen-based dengue assay to accurately distinguish between DENV, ZIKV, JEV, and WNV neutralizing immunity (Odio et al, JID 2024) in a pediatric cohort of 713 children aged two to nine years. We found lower assay specificity than previously reported (94% vs. 100%). We hypothesize that cross-reactive immunity raised to infection with other flaviviruses, and potentially other viruses and/or pathogens, may lead to assay false positivity. We next performed neutralization assays for DENV and ZIKV on the subset of baseline-DENV-naive children over 3 years of follow-up and found a low prevalence and incidence of ZIKV (3.3%) versus DENV (40.4%). We are continuing to analyze the impacts of these infections on one another, including in the development of antibody-dependent enhancement.
In two mixed adult and pediatric cohorts sampled adventitiously prior to the SARS-CoV-2 pandemic (total N=821), we explored humoral immunity to SARS-CoV-2 and found 7-14% seroprevalence that could not be explained by immunity against seasonal betacoronaviruses (Manning et al, EID 2022) or Plasmodium falciparum (Fathi et al, manuscript in submission). In FY24, we performed surrogate virus neutralization tests against a panel of 2 human and 8 animal sarbecoviruses in a subset of SARS-CoV-2 (SCV2) reactive Cambodian sera and found that 14/30 (46.7%) of samples demonstrating neutralizing activity against other SCV2-related betacoronaviruses including several previously isolated among bats and pangolins in Cambodia and the greater Mekong subregion. We hypothesize that previously unappreciated exposure to animal sarbecoviruses may have generated cross-reactive immunity against SCV2 prior to the pandemic. Next, we are designing an observational cohort study to sample Cambodians living and/or working in proximity to known animal reservoirs of potentially zoonotic viruses (henipaviruses, betacoronaviruses, and/or alphainfluenzaviruses) with a goal to use adaptive immune signatures to describe spillover rates and risk and develop immune-based therapies.
A PILOT STUDY OF BACTERIAL GENOMICS AS PART OF ANTIMICROBIAL RESISTANCE SURVEILLANCE IN CAMBODIA.
Integration of genomics into the national AMR surveillance network of bacteremic patients in 2021-2022 demonstrated a high burden of Burkholderia pseudomallei infections and allowed for identification of community spread of drug-resistant typhoidal Salmonella and E. coli strains outbreaks, and nosocomial outbreaks of Acinetobacter spp (Yek et al, SSRN 2024). This pilot study represented the first nationwide survey of antimicrobial resistance genomic markers associated with human disease in Cambodia, and informed the application of PCR-based AMR gene detection as part of national AMR surveillance.
Terms: <0-11 years old><2019 novel corona virus><2019 novel coronavirus><2019-nCoV><21+ years old><7S Gamma Globulin><A baumanni><A baumannii><A. baumanni><A. baumannii><A.baumannii><AIDS Virus><Abdominal Typhus><Acinetobacter><Acinetobacter baumanni><Acinetobacter baumannii><Acquired Immune Deficiency Syndrome Virus><Acquired Immunodeficiency Syndrome Virus><Active Follow-up><Acute><Adult><Adult Human><Aedes><Age Years><Agriculture><Animal Husbandries><Animal Husbandry><Animals><Antibodies><Antibody-Dependent Enhancement><Antigens><Antimicrobial Resistance><Arboviral><Arboviruses><Arthropod-Borne Viruses><Assay><Aves><Avian><B pseudomallei><B. pseudomallei><Bats><Bioassay><Biological Assay><Biology><Birds><Breakbone Fever Virus><Burkholderia pseudomallei><CHIKV><COVID crisis><COVID epidemic><COVID pandemic><COVID-19 crisis><COVID-19 epidemic><COVID-19 era><COVID-19 global health crisis><COVID-19 global pandemic><COVID-19 health crisis><COVID-19 immunity><COVID-19 pandemic><COVID-19 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