Longevity of Genotype-Specific Immune Responses to Plasmodium falciparum Merozoite Surface Protein 1 in Kenyan Children from Regions of Different Malaria Transmission Intensity

Naturally acquired immunity to Plasmodium falciparum presents a changing landscape as malaria control programs and vaccine initiatives are implemented. Determining which immunologic indicators remain surrogates of past infection, as opposed to mediators of protection, led us to compare stability of immune responses across regions with divergent malaria transmission intensities. A repeat cross-sectional study of Kenyan children from a malaria-holoendemic area and an epidemic-prone area was used to examine longitudinal antibody and interferon-gamma (IFN-gamma) responses to the 3D7 and FVO variants of merozoite surface protein 1 (MSP1). Antibodies to MSP1 were common in both study populations and did not significantly wane over a 21-month time period. IFN-gamma responses were less frequent and rapidly disappeared in children after a prolonged period of no malaria transmission. Antibody and IFN-gamma responses rarely correlated with each other; however, MSP1-specific IFN-gamma response correlated with lack of concurrent P. falciparum parasitemia of the same genotype, though only statistically significantly in the malaria-holoendemic region (odds ratio = 0.31, 95% confidence interval = 0.12-0.84). This study affirms that antimalarial antibodies are informative for evaluation of history of malaria exposure within individuals, whereas cell-mediated immunity, though short lived under natural exposure conditions, might provide an assessment of recent infection and protection from parasitemia

A deep sequencing tool for partitioning clearance rates following antimalarial treatment in polyclonal infections

BACKGROUND AND OBJECTIVES: Current tools struggle to detect drug-resistant malaria parasites when infections contain multiple parasite clones, which is the norm in high transmission settings in Africa. Our aim was to develop and apply an approach for detecting resistance that overcomes the challenges of polyclonal infections without requiring a genetic marker for resistance. METHODOLOGY: Clinical samples from patients treated with artemisinin combination therapy were collected from Tanzania and Cambodia. By deeply sequencing a hypervariable locus, we quantified the relative abundance of parasite subpopulations (defined by haplotypes of that locus) within infections and revealed evolutionary dynamics during treatment. Slow clearance is a phenotypic, clinical marker of artemisinin resistance; we analyzed variation in clearance rates within infections by fitting parasite clearance curves to subpopulation data. RESULTS: In Tanzania, we found substantial variation in clearance rates within individual patients. Some parasite subpopulations cleared as slowly as resistant parasites observed in Cambodia. We evaluated possible explanations for these data, including resistance to drugs. Assuming slow clearance was a stable phenotype of subpopulations, simulations predicted that modest increases in their frequency could substantially increase time to cure. CONCLUSIONS AND IMPLICATIONS: By characterizing parasite subpopulations within patients, our method can detect rare, slow clearing parasites in vivo whose phenotypic effects would otherwise be masked. Since our approach can be applied to polyclonal infections even when the genetics underlying resistance are unknown, it could aid in monitoring the emergence of artemisinin resistance. Our application to Tanzanian samples uncovers rare subpopulations with worrying phenotypes for closer examination

Amplicon sequencing as a potential surveillance tool for complexity of infection and drug resistance markers in plasmodium falciparum asymptomatic infections

Background: Genotyping Plasmodium falciparum subpopulations in malaria infections is an important aspect of malaria molecular epidemiology to understand within-host diversity and the frequency of drug resistance markers. Methods: We characterized P. falciparum genetic diversity in asymptomatic infections and subsequent first febrile infections using amplicon sequencing (AmpSeq) of ama1 in Coastal Kenya. We also examined temporal changes in haplotype frequencies of mdr1, a drug-resistant marker. Results: We found >60% of the infections were polyclonal (complexity of infection [COI] >1) and there was a reduction in COI over time. Asymptomatic infections had a significantly higher mean COI than febrile infections based on ama1 sequences (2.7 [95% confidence interval {CI}, 2.65–2.77] vs 2.22 [95% CI, 2.17–2.29], respectively). Moreover, an analysis of 30 paired asymptomatic and first febrile infections revealed that many first febrile infections (91%) were due to the presence of new ama1 haplotypes. The mdr1-YY haplotype, associated with chloroquine and amodiaquine resistance, decreased over time, while the NY (wild type) and the NF (modulates response to lumefantrine) haplotypes increased. Conclusions: This study emphasizes the utility of AmpSeq in characterizing parasite diversity as it can determine relative proportions of clones and detect minority clones. The usefulness of AmpSeq in antimalarial drug resistance surveillance is also highlighted

Real-time PCR detection of mixed Plasmodium ovale curtisi and wallikeri infections in human and mosquito hosts

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/μL (95% CI 0.4–1.6) for Poc and 4.5 plasmid copies/μL (95% CI 2.7–18) for Pow, or 0.1 and 0.8 parasites/μL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/μL (roughly 200 parasites/μL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/μL (<1 parasite/μL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated

Seroepidemiology and risk factors for SARS-CoV-2 infection among household members of food processing and farm workers in North Carolina

Background Racial and ethnic minorities have borne a disproportionate burden from COVID-19. Certain essential occupations, including food processing and farm work, employ large numbers of Hispanic migrant workers and have been shown to carry an especially high risk of infection. Methods We conducted an observational cohort study measuring the seroprevalence of SARS-CoV-2 and assessing risk factors for seropositivity among North Carolina food processing and farm workers and members of their households. Participants completed questionnaires and we collected blood samples and used an enzyme-linked immunosorbent assay to assess SARS-CoV-2 seropositivity. Univariate and multivariate analyses were carried out to identify risk factors associated with seropositivity, using generalized estimating equations to account for household clustering. Findings Among the 218 participants, 94.5% were Hispanic, and SARS-CoV-2 seropositivity was 50.0%. Most seropositive individuals did not report a history of illness compatible with COVID-19. Attending church, having a prior history of COVID-19, having a seropositive household member, and speaking Spanish as one's primary language were associated with SARS-CoV-2 seropositivity, while preventive behaviors were not. Interpretation These findings underscore the substantial burden of COVID-19 among a population of mostly Hispanic essential workers and their households in rural North Carolina. Our study contributes to a large body of evidence showing that Hispanic Americans have suffered a disproportionate COVID-19 burden. We also highlight the epidemiologic importance of viral transmission within the household

SARS-CoV-2 seroprevalence and risk factors among meat packing, produce processing, and farm workers

Meat packing, produce processing, and farm workers are known to have an elevated risk of COVID-19, but occupational risk factors in this population are unclear. We performed an observational cohort study of meat packing, produce processing, and farm workers in North Carolina in fall 2020. Blood, saliva, and nasal turbinate samples were collected to assess for SARS-CoV-2 seropositivity. Risk factors for SARS-CoV-2 seropositivity were investigated using chi-square tests, two-sample t-tests, and adjusted risk ratio analyses. Among 118 enrolled workers, the baseline SARS-CoV-2 seroprevalence was 50.0%. Meat packing plant workers had the highest SARS-CoV-2 seroprevalence (64.6%), followed by farm workers (45.0%) and produce processing workers (10.0%), despite similar sociodemographic characteristics. Compared to SARS-CoV-2 seronegative workers, seropositive workers were more likely to work in loud environments that necessitated yelling to communicate (RR: 1.83, 95% CI: 1.25–2.69), work in cold environments (RR: 1.58, 95% CI: 1.12–2.24), or continue working despite developing symptoms at work (RR: 1.63, 95% CI: 1.14–2.32). After adjusting for age and working despite symptoms, high occupational noise levels were associated with a 1.72 times higher risk of SARS-CoV-2 seropositivity (95% CI: 1.16–2.55). Half of food processing workers showed evidence of past SARS-CoV-2 infection, a prevalence five times higher than most of the United States population at the time of the study. Work environments with loud ambient noise may pose elevated risks for SARS-CoV-2 transmission. Our findings also highlight the disproportionate burden of COVID-19 among underserved and economically disadvantaged Latinx communities in the United States

Low Complexity of Infection Is Associated With Molecular Persistence of Plasmodium falciparum in Kenya and Tanzania

Background Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs) is a threat to malaria elimination. ACT-resistance in Asia raises concerns for emergence of resistance in Africa. While most data show high efficacy of ACT regimens in Africa, there have been reports describing declining efficacy, as measured by both clinical failure and prolonged parasite clearance times. Methods Three hundred children aged 2–10 years with uncomplicated P. falciparum infection were enrolled in Kenya and Tanzania after receiving treatment with artemether-lumefantrine. Blood samples were taken at 0, 24, 48, and 72 h, and weekly thereafter until 28 days post-treatment. Parasite and host genetics were assessed, as well as clinical, behavioral, and environmental characteristics, and host anti-malarial serologic response. Results While there was a broad range of clearance rates at both sites, 85% and 96% of Kenyan and Tanzanian samples, respectively, were qPCR-positive but microscopy-negative at 72 h post-treatment. A greater complexity of infection (COI) was negatively associated with qPCR-detectable parasitemia at 72 h (OR: 0.70, 95% CI: 0.53–0.94), and a greater baseline parasitemia was marginally associated with qPCR-detectable parasitemia (1,000 parasites/uL change, OR: 1.02, 95% CI: 1.01–1.03). Demographic, serological, and host genotyping characteristics showed no association with qPCR-detectable parasitemia at 72 h. Parasite haplotype-specific clearance slopes were grouped around the mean with no association detected between specific haplotypes and slower clearance rates. Conclusions Identifying risk factors for slow clearing P. falciparum infections, such as COI, are essential for ongoing surveillance of ACT treatment failure in Kenya, Tanzania, and more broadly in sub-Saharan Africa

A deep sequencing approach to estimate Plasmodium falciparum complexity of infection (COI) and explore apical membrane antigen 1 diversity

Abstract Background Humans living in regions with high falciparum malaria transmission intensity harbour multi-strain infections comprised of several genetically distinct malaria haplotypes. The number of distinct malaria parasite haplotypes identified from an infected human host at a given time is referred to as the complexity of infection (COI). In this study, an amplicon-based deep sequencing method targeting the Plasmodium falciparum apical membrane antigen 1 (pfama1) was utilized to (1) investigate the relationship between P. falciparum prevalence and COI, (2) to explore the population genetic structure of P. falciparum parasites from malaria asymptomatic individuals participating in the 2007 Demographic and Health Survey (DHS) in the Democratic Republic of Congo (DRC), and (3) to explore selection pressures on geospatially divergent parasite populations by comparing AMA1 amino acid frequencies in the DRC and Mali. Results A total of 900 P. falciparum infections across 11 DRC provinces were examined. Deep sequencing of both individuals, for COI analysis, and pools of individuals, to examine population structure, identified 77 unique pfama1 haplotypes. The majority of individual infections (64.5%) contained polyclonal (COI > 1) malaria infections based on the presence of genetically distinct pfama1 haplotypes. A minimal correlation between COI and malaria prevalence as determined by sensitive real-time PCR was identified. Population genetic analyses revealed extensive haplotype diversity, the vast majority of which was shared across the sites. AMA1 amino acid frequencies were similar between parasite populations in the DRC and Mali. Conclusions Amplicon-based deep sequencing is a useful tool for the detection of multi-strain infections that can aid in the understanding of antigen heterogeneity of potential malaria vaccine candidates, population genetics of malaria parasites, and factors that influence complex, polyclonal malaria infections. While AMA1 and other diverse markers under balancing selection may perform well for understanding COI, they may offer little geographic or temporal discrimination between parasite populations

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Additional file 7. Comparison of demographic factors between sample sequenced individually (n=79) and pooled population cluster samples (N=821) with analysable sequence reads

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Additional file 2. Pfama1 heminested primer sequences and Multiplex identifier (MID) sequences