Amplicon deep sequencing of five highly polymorphic markers of Plasmodium falciparum reveals high parasite genetic diversity and moderate population structure in Ethiopia.

BACKGROUND: Plasmodium falciparum genetic diversity can add information on transmission intensity and can be used to track control and elimination interventions. METHODS: Dried blood spots (DBS) were collected from patients who were recruited for a P. falciparum malaria therapeutic efficacy trial in three malaria endemic sites in Ethiopia from October to December 2015, and November to December 2019. qPCR-confirmed infections were subject to amplicon sequencing of polymorphic markers ama1-D3, csp, cpp, cpmp, msp7. Genetic diversity, the proportion of multiclonal infections, multiplicity of infection, and population structure were analysed. RESULTS: Among 198 samples selected for sequencing, data was obtained for 181 samples. Mean MOI was 1.38 (95% CI 1.24-1.53) and 17% (31/181) of infections were polyclonal. Mean He across all markers was 0.730. Population structure was moderate; populations from Metema and Metehara 2015 were very similar to each other, but distinct from Wondogent 2015 and Metehara 2019. CONCLUSION: The high level of parasite genetic diversity and moderate population structure in this study suggests frequent gene flow of parasites among sites. The results obtained can be used as a baseline for additional parasite genetic diversity and structure studies, aiding in the formulation of appropriate control strategies in Ethiopia.

Heterogeneity in prevalence of subclinical Plasmodium falciparum and Plasmodium vivax infections but no parasite genomic clustering in the Chittagong Hill Tracts, Bangladesh.

BACKGROUND: Malaria remains endemic in Bangladesh, with the majority of cases occurring in forested, mountainous region in the Chittagong Hill Tracts (CHT). This area is home to Bengali and diverse groups of indigenous people (Pahari) residing largely in mono-ethnic villages. METHODS: 1002 individuals of the 9 most prominent Pahari and the Bengali population were randomly selected and screened by RDT and qPCR. Parasites were genotyped by msp2 and deep sequencing of 5 amplicons (ama1-D3, cpmp, cpp, csp, and msp7) for Plasmodium falciparum (n = 20), and by microsatellite (MS) typing of ten loci and amplicon sequencing of msp1 for Plasmodium vivax (n = 21). Population structure was analysed using STRUCTURE software. Identity-by-state (IBS) was calculated as a measure of parasite relatedness and used to generate relatedness networks. RESULTS: The prevalence of P. falciparum and P. vivax infection was 0.7% by RDT (P. falciparum 6/1002; P. vivax 0/1002, mixed: 1/1002) and 4% by qPCR (P. falciparum 21/1002; P. vivax 16/1002, mixed: 5/1002). Infections were highly clustered, with 64% (27/42) of infections occurring in only two Pahari groups, the Khumi and Mro. Diversity was high; expected heterozygosity was 0.93 for P. falciparum and 0.81 for P. vivax. 85.7% (18/21) of P. vivax and 25% (5/20) of P. falciparum infections were polyclonal. No population structure was evident for either species, suggesting high transmission and gene flow among Pahari groups. CONCLUSIONS: High subclinical infection prevalence and genetic diversity mirror ongoing transmission. Control activities should be specifically directed to Pahari groups at greatest risk.

Altitude, not potential larval habitat availability, explains pronounced variation in Plasmodium falciparum infection prevalence in the western Kenya highlands.

Progress in malaria control has stalled over the recent years. Knowledge on main drivers of transmission explaining small-scale variation in prevalence can inform targeted control measures. We collected finger-prick blood samples from 3061 individuals irrespective of clinical symptoms in 20 clusters in Busia in western Kenya and screened for Plasmodium falciparum parasites using qPCR and microscopy. Clusters spanned an altitude range of 207 meters (1077-1284 m). We mapped potential mosquito larval habitats and determined their number within 250 m of a household and distances to households using ArcMap. Across all clusters, P. falciparum parasites were detected in 49.8% (1524/3061) of individuals by qPCR and 19.5% (596/3061) by microscopy. Across the clusters, prevalence ranged from 26% to 70% by qPCR. Three to 34 larval habitats per cluster and 0-17 habitats within a 250m radius around households were observed. Using a generalized linear mixed effect model (GLMM), a 5% decrease in the odds of getting infected per each 10m increase in altitude was observed, while the number of larval habitats and their proximity to households were not statistically significant predictors for prevalence. Kitchen located indoors, open eaves, a lower level of education of the household head, older age, and being male were significantly associated with higher prevalence. Pronounced variation in prevalence at small scales was observed and needs to be taken into account for malaria surveillance and control. Potential larval habitat frequency had no direct impact on prevalence.

qPCR in a suitcase for rapid Plasmodium falciparum and Plasmodium vivax surveillance in Ethiopia.

Many Plasmodium spp. infections, both in clinical and asymptomatic patients, are below the limit of detection of light microscopy or rapid diagnostic test (RDT). Molecular diagnosis by qPCR can be valuable for surveillance, but is often hampered by absence of laboratory capacity in endemic countries. To overcome this limitation, we optimized and tested a mobile qPCR laboratory for molecular diagnosis in Ziway, Ethiopia, where transmission intensity is low. Protocols were optimized to achieve high throughput and minimize costs and weight for easy transport. 899 samples from febrile patients and 1021 samples from asymptomatic individuals were screened by local microscopy, RDT, and qPCR within a period of six weeks. 34/52 clinical Plasmodium falciparum infections were missed by microscopy and RDT. Only 4 asymptomatic infections were detected. No hrp2 deletions were observed among 25 samples typed, but 19/24 samples carried hrp3 deletions. The majority (25/41) of Plasmodium vivax infections (1371 samples screened) were found among asymptomatic individuals. All asymptomatic P. vivax infections were negative by microscopy and RDT. In conclusion, the mobile laboratory described here can identify hidden parasite reservoirs within a short period of time, and thus inform malaria control activities.

Spatial and genetic clustering of Plasmodium falciparum and Plasmodium vivax infections in a low-transmission area of Ethiopia.

The distribution of malaria infections is heterogeneous in space and time, especially in low transmission settings. Understanding this clustering may allow identification and targeting of pockets of transmission. In Adama district, Ethiopia, Plasmodium falciparum and P. vivax malaria patients and controls were examined, together with household members and immediate neighbors. Rapid diagnostic test and quantitative PCR (qPCR) were used for the detection of infections that were genetically characterized by a panel of microsatellite loci for P. falciparum (26) and P. vivax (11), respectively. Individuals living in households of clinical P. falciparum patients were more likely to have qPCR detected P. falciparum infections (22.0%, 9/41) compared to individuals in control households (8.7%, 37/426; odds ratio, 2.9; 95% confidence interval, 1.3-6.4; P = .007). Genetically related P. falciparum, but not P. vivax infections showed strong clustering within households. Genotyping revealed a marked temporal cluster of P. falciparum infections, almost exclusively comprised of clinical cases. These findings uncover previously unappreciated transmission dynamics and support a rational approach to reactive case detection strategies for P. falciparum in Ethiopia.

Interactions between Parasitic Infections and the Human Gut Microbiome in Odisha, India.

Soil-transmitted helminth (STH) infections and malaria are parasitic diseases with enormous global health burdens. Research has demonstrated a relationship between each of these parasites and the gut microbiome, suggesting that the gut microbiota may be implicated in governing host susceptibility to diverse pathogens, and perhaps even coinfection by different pathogens, through similar microbiome-influenced pathways. Here, we have derived a first microbiome community profile associated with STH infections in Odisha, India, and tested the hypothesis that the gut microbiome can modulate host susceptibility to multiple parasite infections through the same pathways. This study revealed several bacterial taxa negatively associated with specific STH infections, including Lactobacillus and Lachnospiracaea. Our results also suggest that relative abundance of Lactobacillus is driven by the STH infection status more so than by the Plasmodium infection status. This study contributes to efforts to understand the effects of the microbiome on host susceptibility to parasitic infections in endemic communities.