A digital microscope for the diagnosis of Plasmodium falciparum and Plasmodium vivax, including P. falciparum with hrp2/hrp3 deletion.

Sensitive and accurate malaria diagnosis is required for case management to accelerate control efforts. Diagnosis is particularly challenging where multiple Plasmodium species are endemic, and where P. falciparum hrp2/3 deletions are frequent. The Noul miLab is a fully automated portable digital microscope that prepares a blood film from a droplet of blood, followed by staining and detection of parasites by an algorithm. Infected red blood cells are displayed on the screen of the instrument. Time-to-result is approximately 20 minutes, with less than two minutes hands-on time. We evaluated the miLab among 659 suspected malaria patients in Gondar, Ethiopia, where P. falciparum and P. vivax are endemic, and the frequency of hrp2/3 deletions is high, and 991 patients in Ghana, where P. falciparum transmission is intense. Across both countries combined, the sensitivity of the miLab for P. falciparum was 94.3% at densities >200 parasites/µL by qPCR, and 83% at densities >20 parasites/µL. The miLab was more sensitive than local microscopy, and comparable to RDT. In Ethiopia, the miLab diagnosed 51/52 (98.1%) of P. falciparum infections with hrp2 deletion at densities >20 parasites/µL. Specificity of the miLab was 94.0%. For P. vivax diagnosis in Ethiopia, the sensitivity of the miLab was 97.0% at densities >200 parasites/µL (RDT: 76.8%, microscopy: 67.0%), 93.9% at densities >20 parasites/µL, and specificity was 97.6%. In Ethiopia, where P. falciparum and P. vivax were frequent, the miLab assigned the wrong species to 15/195 mono-infections at densities >20 parasites/µL by qPCR, and identified only 5/18 mixed-species infections correctly. In conclusion, the miLab was more sensitive than microscopy and thus is a valuable addition to the toolkit for malaria diagnosis, particularly for areas with high frequencies of hrp2/3 deletions.

Using a mobile nanopore sequencing lab for end-to-end genomic surveillance of Plasmodium falciparum: A feasibility study.

Genomic epidemiology holds promise for malaria control and elimination efforts, for example by informing on Plasmodium falciparum genetic diversity and prevalence of mutations conferring anti-malarial drug resistance. Limited sequencing infrastructure in many malaria-endemic areas prevents the rapid generation of genomic data. To address these issues, we developed and validated assays for P. falciparum nanopore sequencing in endemic sites using a mobile laboratory, targeting key antimalarial drug resistance markers and microhaplotypes. Using two multiplexed PCR reactions, we amplified six highly polymorphic microhaplotypes and ten drug resistance markers. We developed a bioinformatics workflow that allows genotyping of polyclonal malaria infections, including minority clones. We validated the panels on mock dried blood spot (DBS) and rapid diagnostic test (RDT) samples and archived DBS, demonstrating even, high read coverage across amplicons (range: 580x to 3,212x median coverage), high haplotype calling accuracy, and the ability to explore within-sample diversity of polyclonal infections. We field-tested the feasibility of rapid genotyping in Zanzibar in close collaboration with the local malaria elimination program using DBS and routinely collected RDTs as sample inputs. Our assay identified haplotypes known to confer resistance to known antimalarials in the dhfr, dhps and mdr1 genes, but no evidence of artemisinin partial resistance. Most infections (60%) were polyclonal, with high microhaplotype diversity (median HE = 0.94). In conclusion, our assays generated actionable data within a few days, and we identified current challenges for implementing nanopore sequencing in endemic countries to accelerate malaria control and elimination.

Plasmodium falciparum transmission in the highlands of Ethiopia is driven by closely related and clonal parasites.

Malaria cases are frequently recorded in the Ethiopian highlands even at altitudes above 2000 m. The epidemiology of malaria in the Ethiopian highlands, and, in particular, the role of importation by human migration from the highly endemic lowlands is not well understood. We sequenced 187 Plasmodium falciparum samples from two sites in the Ethiopian highlands, Gondar (n = 159) and Ziway (n = 28), using a multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug resistance loci. Here, we characterize the parasite population structure and genetic relatedness. We identify moderate parasite diversity (mean HE : 0.54) and low infection complexity (74.9% monoclonal). A significant percentage of infections share microhaplotypes, even across transmission seasons and sites, indicating persistent local transmission. We identify multiple clusters of clonal or near-clonal infections, highlighting high genetic relatedness. Only 6.3% of individuals diagnosed with P. falciparum reported recent travel. Yet, in clonal or near-clonal clusters, infections of travellers were frequently observed first in time, suggesting that parasites may have been imported and then transmitted locally. 31.1% of infections are pfhrp2-deleted and 84.4% pfhrp3-deleted, and 28.7% have pfhrp2/3 double deletions. Parasites with pfhrp2/3 deletions and wild-type parasites are genetically distinct. Mutations associated with resistance to sulphadoxine-pyrimethamine or suggested to reduce sensitivity to lumefantrine are observed at near-fixation. In conclusion, genomic data corroborate local transmission and the importance of intensified control in the Ethiopian highlands.

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.

Is qPCR always the most sensitive method for malaria diagnostic quality surveillance?

In many studies to evaluate the quality of malaria diagnosis, microscopy or rapid diagnostic tests (RDT) are compared to PCR. Depending on the method for sample collection and storage (whole blood or dried blood spot), volume of blood used for extraction, volume of DNA used as PCR template, and choice of PCR target (single vs. multi-copy gene), the limit of detection (LOD) of PCR might not exceed the LOD of expert microscopy or RDT. One should not assume that PCR always detects the highest number of infections.

Bridging the Gap from Molecular Surveillance to Programmatic Decisions for Malaria Control and Elimination.

An increasing number of molecular and genomic assays are available to study malaria parasite populations. However, so far they have played a marginal role in informing policy and programmatic decision-making. Currently, molecular data are mainly used for monitoring drug efficacy against Plasmodium falciparum; assessing molecular markers of drug and insecticide resistance; and assessing P. falciparum histidine-rich protein 2 and 3 genes (Pfhrp2/3) deletion. We argue that additional use cases for molecular routine surveillance could be implemented in the near future, especially in transmission settings approaching elimination. These would include using quantitative polymerase chain reaction to monitor the prevalence of sub-patent infections in asymptomatic carriers, monitoring parasite genetic diversity as transmission intensity is changing, using genomic data to determine the origin of imported infections and characterize transmission chains in settings with very low malaria transmission, and using serology to monitor recent and past exposures in low-transmission settings. Molecular surveillance could inform control programs on adapting novel strategies, such as reactive case detection or focal mass drug administration, and help evaluate the impact of interventions currently in place. To better integrate molecular and genomic data into control program decision-making, engagement of national malaria control experts is crucial. Local laboratory capacity needs to be strengthened, shortening the time from sample collection to data availability. Here, we discuss opportunities and challenges of the use of molecular and genomic data for supporting malaria control and elimination efforts, as well as the avenues to link molecular and genomic data with gold standard epidemiological measurements through mathematical modeling.

Asymptomatic and submicroscopic malaria infections in sugar cane and rice development areas of Ethiopia.

BACKGROUND: Water resource development projects, such as dams and irrigation schemes, have a positive impact on food security and poverty reduction. However, such projects could increase prevalence of vector borne disease, such as malaria. This study investigate the impact of different agroecosystems and prevalence of malaria infection in Southwest Ethiopia. METHODS: Two cross-sectional surveys were conducted in the dry and wet seasons in irrigated and non-irrigated clusters of Arjo sugarcane and Gambella rice development areas of Ethiopia in 2019. A total of 4464 and 2176 study participants from 1449 households in Arjo and 546 households in Gambella enrolled in the study and blood samples were collected, respectively. All blood samples were microscopically examined and a subset of microscopy negative blood samples (n = 2244) were analysed by qPCR. Mixed effect logistic regression and generalized estimating equation were used to determine microscopic and submicroscopic malaria infection and the associated risk factors, respectively. RESULTS: Prevalence by microscopy was 2.0% (88/4464) in Arjo and 6.1% (133/2176) in Gambella. In Gambella, prevalence was significantly higher in irrigated clusters (10.4% vs 3.6%) than in non-irrigated clusters (p < 0.001), but no difference was found in Arjo (2.0% vs 2.0%; p = 0.993). On the other hand, of the 1713 and 531 samples analysed by qPCR from Arjo and Gambella the presence of submicroscopic infection was 1.2% and 12.8%, respectively. Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale were identified by qPCR in both sites. Irrigation was a risk factor for submicroscopic infection in both Arjo and Gambella. Irrigation, being a migrant worker, outdoor job, < 6 months length of stay in the area were risk factors for microscopic infection in Gambella. Moreover, school-age children and length of stay in the area for 1-3 years were significant predictors for submicroscopic malaria in Gambella. However, no ITN utilization was a predictor for both submicroscopic and microscopic infection in Arjo. Season was also a risk factor for microscopic infection in Arjo. CONCLUSION: The study highlighted the potential importance of different irrigation practices impacting on submicroscopic malaria transmission. Moreover, microscopic and submicroscopic infections coupled with population movement may contribute to residual malaria transmission and could hinder malaria control and elimination programmes in the country. Therefore, strengthening malaria surveillance and control by using highly sensitive diagnostic tools to detect low-density parasites, screening migrant workers upon arrival and departure, ensuring adequate coverage and proper utilization of vector control tools, and health education for at-risk groups residing or working in such development corridors is needed.

Welcome to the next generation of Malaria Rapid Diagnostic Tests: Comparative Analysis of NxTek Eliminate Malaria P.f, Biocredit Malaria Ag Pf, and SD Bioline Malaria Ag Pf for Plasmodium falciparum Diagnosis in Ghana.

Background: Accurate diagnosis and timely treatment are crucial in combating malaria. Methods: We evaluated the diagnostic performance of three Rapid Diagnostic Tests (RDTs) in diagnosing febrile patients, namely: Abbott NxTek Eliminate Malaria Ag Pf (detecting HRP2), Rapigen Biocredit Malaria Ag Pf (detecting HRP2 and LDH on separate bands), and SD Bioline Malaria Ag Pf (detecting HRP2). Results were compared to qPCR. Results: Among 449 clinical patients, 45.7% (205/449) tested positive by qPCR for P. falciparum with a mean parasite density of 12.5parasites/µL. The sensitivity of the Biocredit RDT was 52.2% (107/205), NxTek RDT was 49.3% (101/205), and Bioline RDT was 40.5% (83/205). When samples with parasite densities lower than 20 parasites/uL were excluded (n=116), a sensitivity of 88.8% (79/89, NxTek), 89.9% (80/89, Biocredit), and 78.7% (70/89, Bioline) was obtained. All three RDTs demonstrated specificity above 95%. The limits of detection was 84 parasites/µL (NxTek), 56 parasites/µL (Biocredit, considering either HRP2 or LDH), and 331 parasites/µL (Bioline). None of the three qPCR-confirmed P. falciparum positive samples, identified solely through the LDH target, carried hrp2/3 deletions. Conclusion: The Biocredit and NxTek RDTs demonstrated comparable diagnostic efficacies and both RDTs performed better than Bioline RDT.

Bangladesh in the era of malaria elimination.

Bangladesh has dramatically reduced malaria by 93% from 2008 to 2020. The strategy has been district-wise, phased elimination; however, the last districts targeted for elimination include remote, forested regions which present several challenges for prevention, detection, and treatment of malaria. These districts border Myanmar which harbors Plasmodium falciparum malaria parasites resistant to artemisinins, key drugs used in artemisinin-based combination therapies (ACTs) that have been vital for control programs. Challenges in monitoring emergence of artemisinin resistance (AR), tracking parasite reservoirs, changes in vector behavior and responses to insecticides, as well as other environmental and host factors (including the migration of Forcibly Displaced Myanmar Nationals; FDMNs) may pose added hazards in the final phase of eliminating malaria in Bangladesh.

Multiplexed ddPCR-amplicon sequencing reveals isolated Plasmodium falciparum populations amenable to local elimination in Zanzibar, Tanzania.

Zanzibar has made significant progress toward malaria elimination, but recent stagnation requires novel approaches. We developed a highly multiplexed droplet digital PCR (ddPCR)-based amplicon sequencing method targeting 35 microhaplotypes and drug-resistance loci, and successfully sequenced 290 samples from five districts covering both main islands. Here, we elucidate fine-scale Plasmodium falciparum population structure and infer relatedness and connectivity of infections using an identity-by-descent (IBD) approach. Despite high genetic diversity, we observe pronounced fine-scale spatial and temporal parasite genetic structure. Clusters of near-clonal infections on Pemba indicate persistent local transmission with limited parasite importation, presenting an opportunity for local elimination efforts. Furthermore, we observe an admixed parasite population on Unguja and detect a substantial fraction (2.9%) of significantly related infection pairs between Zanzibar and the mainland, suggesting recent importation. Our study provides a high-resolution view of parasite genetic structure across the Zanzibar archipelago and provides actionable insights for prioritizing malaria elimination efforts.

Genetic diversity and molecular evolution of Plasmodium vivax Duffy Binding Protein and Merozoite Surface Protein-1 in northwestern Thailand.

The local diversity and population structure of malaria parasites vary across different regions of the world, reflecting variations in transmission intensity, host immunity, and vector species. This study aimed to use amplicon sequencing to investigate the genotypic patterns and population structure of P. vivax isolates from a highly endemic province of Thailand in recent years. Amplicon deep sequencing was performed on 70 samples for the 42-kDa region of pvmsp1 and domain II of pvdbp. Unique haplotypes were identified and a network constructed to illustrate genetic relatedness in northwestern Thailand. Based on this dataset of 70 samples collected between 2015 and 2021, 16 and 40 unique haplotypes were identified in pvdbpII and pvmsp142kDa, respectively. Nucleotide diversity was higher in pvmsp142kDa than in pvdbpII (π = 0.027 and 0.012), as was haplotype diversity (Hd = 0.962 and 0.849). pvmsp142kDa also showed a higher recombination rate and higher levels of genetic differentiation (Fst) in northwestern Thailand versus other regions (0.2761-0.4881). These data together suggested that the genetic diversity of P. vivax in northwestern Thailand at these two studied loci evolved under a balancing selection, most likely host immunity. The lower genetic diversity of pvdbpII may reflect its stronger functional constrain. In addition, despite the balancing selection, a decrease in genetic diversity was observed. Hd of pvdbpII decreased from 0.874 in 2015-2016 to 0.778 in 2018-2021; π of pvmsp142kDa decreased from 0.030 to 0.022 over the same period. Thus, the control activities must have had a strong impact on the parasite population size. The findings from this study provide an understanding of P. vivax population structure and the evolutionary force on vaccine candidates. They also established a new baseline for tracking future changes in P. vivax diversity in the most malarious area of Thailand.

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.

Accuracy of diagnosis among clinical malaria patients: comparing microscopy, RDT and a highly sensitive quantitative PCR looking at the implications for submicroscopic infections.

BACKGROUND: The World Health Organization recommends parasitological confirmation of all suspected malaria cases by microscopy or rapid diagnostic tests (RDTs) before treatment. These conventional tools are widely used for point-of-care diagnosis in spite of their poor sensitivity at low parasite density. Previous studies in Ghana have compared microscopy and RDT using standard 18S rRNA PCR as reference with varying outcomes. However, how these conventional tools compare with ultrasensitive varATS qPCR has not been studied. This study, therefore, sought to investigate the clinical performance of microscopy and RDT assuming highly sensitive varATS qPCR as gold standard. METHODS: 1040 suspected malaria patients were recruited from two primary health care centers in the Ashanti Region of Ghana and tested for malaria by microscopy, RDT, and varATS qPCR. The sensitivity, specificity, and predictive values were assessed using varATS qPCR as gold standard. RESULTS: Parasite prevalence was 17.5%, 24.5%, and 42.1% by microscopy, RDT, and varATS qPCR respectively. Using varATS qPCR as the standard, RDT was more sensitive (55.7% vs 39.3%), equally specific (98.2% vs 98.3%), and reported higher positive (95.7% vs 94.5%) and negative predictive values (75.3% vs 69.0%) than microscopy. Consequently, RDT recorded better diagnostic agreement (kappa = 0.571) with varATS qPCR than microscopy (kappa = 0.409) for clinical detection of malaria. CONCLUSIONS: RDT outperformed microscopy for the diagnosis of Plasmodium falciparum malaria in the study. However, both tests missed over 40% of infections that were detected by varATS qPCR. Novel tools are needed to ensure prompt diagnosis of all clinical malaria cases.

Asymptomatic and submicroscopic malaria infections in sugar cane and rice development areas of Ethiopia.

Background: Water resource development projects such as dams and irrigation schemes have a positive impact on food security and poverty reduction but might result in increased prevalence of malaria. Methods: Two cross-sectional surveys were conducted in the dry and wet seasons in irrigated and non-irrigated clusters of Arjo sugarcane and Gambella rice development areas of Ethiopia in 2019. A total of 4464 and 2176 blood samples were collected from Arjo and Gambella. A subset of 2244 microscopy negative blood samples were analyzed by PCR. Results: Prevalence by microscopy was 2.0% (88/4464) in Arjo and 6.1% (133/2176) in Gambella. In Gambella, prevalence was significantly higher in irrigated clusters (10.4% vs 3.6%) than in non-irrigated clusters (p < 0.001), but no difference was found in Arjo (2.0% vs 2.0%; p = 0.993). Level of education was an individual risk factors associated with infection in Arjo [AOR: 3.2; 95%CI (1.27-8.16)] and in Gambella [AOR: 1.7; 95%CI (1.06-2.82)]. While duration of stay in the area for < 6 months [AOR: 4.7; 95%CI (1.84-12.15)] and being a migrant worker [AOR: 4.7; 95%CI (3.01-7.17)] were risk factors in Gambella. Season [AOR: 15.9; 95%CI (6.01-42.04)], no ITN utilization [AOR: 22.3; 95%CI (7.74-64.34)] were risk factors in Arjo, and irrigation [AOR: 2.4; 95%CI (1.45-4.07)] and family size [AOR: 2.3; 95%CI (1.30-4.09)] risk factors in Gambella. Of the 1713 and 531 randomly selected smear negative samples from Arjo and Gambella and analyzed by PCR the presence of Plasmodium infection was 1.2% and 12.8%, respectively. P. falciparum, P. vivax, and P. ovale were identified by PCR in both sites. Conclusion: Strengthening malaria surveillance and control in project development areas and proper health education for at-risk groups residing or working in such development corridors is needed.

Regional and socio-economic disparity in use of insecticide-treated nets to prevent malaria among pregnant women in Kenya.

BACKGROUND: Insecticide-treated net (ITN) use is among the most recommended strategies to prevent malaria in pregnancy. We analysed the regional and socio-economic patterns of ITN use among pregnant women in Kenya using data from the 2003, 2008 and 2014 Kenyan Demographic and Health Surveys (KDHSs). METHODS: Inequality was assessed using four dimensions: economic status, education, place of residence and region. Both relative and absolute summary measures were applied. In addition, simple and complex summary measures, i.e. difference, population attributable fraction, population attributable risk and ratio were considered based on the number of subgroups in each variable. RESULTS: There was overt inequality in the use of ITNs among pregnant women, with greater use among the better-off group in 2003 and 2014. Greater ITN use was also observed among pregnant women with a higher level of education. Pregnant women from urban settings tended to use ITNs (slept under a net the night before the survey) more than their rural counterparts in the 2003 KDHS. There were significant regional variations across the three surveys in all inequality summary measures, except ratio in the 2014 survey. CONCLUSIONS: Significant inequality in ITN use among pregnant women was observed at a macro scale.

The Impact of Emerging Plasmodium knowlesi on Accurate Diagnosis by Light Microscopy: A Systematic Review and Modeling Analysis.

The five major Plasmodium spp. that cause human malaria appear similar under light microscopy, which raises the possibility that misdiagnosis could routinely occur in clinical settings. Assessing the extent of misdiagnosis is of particular importance for monitoring P. knowlesi, which cocirculates with the other Plasmodium spp. We performed a systematic review and meta-analysis of studies comparing the performance of microscopy and polymerase chain reaction (PCR) for diagnosing malaria in settings with co-circulation of the five Plasmodium spp. We assessed the extent to which co-circulation of Plasmodium parasites affects diagnostic outcomes. We fit a Bayesian hierarchical latent class model to estimate variation in microscopy sensitivity and specificity measured against PCR as the gold standard. Mean sensitivity of microscopy was low, yet highly variable across Plasmodium spp., ranging from 65.7% (95% confidence interval: 48.1-80.3%) for P. falciparum to 0.525% (95% confidence interval 0.0210-3.11%) for P. ovale. Observed PCR prevalence was positively correlated with estimated microscopic sensitivity and negatively correlated with estimated microscopic specificity, though the strength of the associations varied by species. Our analysis suggests that cocirculation of Plasmodium spp. undermines the accuracy of microscopy. Sensitivity was considerably lower for P. knowlesi, P. malariae, and P. ovale. The negative association between specificity and prevalence imply that less frequently encountered species may be misdiagnosed as more frequently encountered species. Together, these results suggest that the burden of P. knowlesi, P. malariae, and P. ovale may be underappreciated in a clinical setting.

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.

High-throughput Plasmodium falciparum hrp2 and hrp3 gene deletion typing by digital PCR to monitor malaria rapid diagnostic test efficacy.

Most rapid diagnostic tests for Plasmodium falciparum malaria target the Histidine-Rich Proteins 2 and 3 (HRP2 and HRP3). Deletions of the hrp2 and hrp3 genes result in false-negative tests and are a threat for malaria control. A novel assay for molecular surveillance of hrp2/hrp3 deletions was developed based on droplet digital PCR (ddPCR). The assay quantifies hrp2, hrp3, and a control gene with very high accuracy. The theoretical limit of detection was 0.33 parasites/µl. The deletion was reliably detected in mixed infections with wild-type and hrp2-deleted parasites at a density of >100 parasites/reaction. For a side-by-side comparison with the conventional nested PCR (nPCR) assay, 248 samples were screened in triplicate by ddPCR and nPCR. No deletions were observed by ddPCR, while by nPCR hrp2 deletion was observed in 8% of samples. The ddPCR assay was applied to screen 830 samples from Kenya, Zanzibar/Tanzania, Ghana, Ethiopia, Brazil, and Ecuador. Pronounced differences in the prevalence of deletions were observed among sites, with more hrp3 than hrp2 deletions. In conclusion, the novel ddPCR assay minimizes the risk of false-negative results (i.e., hrp2 deletion observed when the sample is wild type), increases sensitivity, and greatly reduces the number of reactions that need to be run.

Tenfold difference in DNA recovery rate: systematic comparison of whole blood vs. dried blood spot sample collection for malaria molecular surveillance.

BACKGROUND: Molecular and genomic surveillance is becoming increasingly used to track malaria control and elimination efforts. Blood samples can be collected as whole blood and stored at - 20 °C until DNA extraction, or as dried blood spots (DBS), circumventing the need for a cold chain. Despite the wide use of either method, systematic comparisons of how the method of blood sample preservation affects the limit of detection (LOD) of molecular diagnosis and the proportion of DNA recovered for downstream applications are lacking. METHODS: Extractions based on spin columns, magnetic beads, Tween-Chelex, and direct PCR without prior extraction were compared for whole blood and dried blood spots (DBS) using dilution series of Plasmodium falciparum culture samples. Extracted DNA was quantified by qPCR and droplet digital PCR (ddPCR). RESULTS: DNA recovery was 5- to 10-fold higher for whole blood compared to DBS, resulting in a 2- to 3-fold lower LOD for both extraction methods compared to DBS. For whole blood, a magnetic bead-based method resulted in a DNA recovery rate of 88-98% when extracting from whole blood compared to 17-33% for a spin-column based method. For extractions from DBS, the magnetic bead-based method resulted in 8-20% DNA recovery, while the spin-column based method resulted in only 2% DNA recovery. The Tween-Chelex method was superior to other methods with 15-21% DNA recovery, and even more sensitive than extractions from whole blood samples. The direct PCR method was found to have the lowest LOD overall for both, whole blood and DBS. CONCLUSIONS: Pronounced differences in LOD and DNA yield need to be considered when comparing prevalence estimates based on molecular methods and when selecting sampling protocols for other molecular surveillance applications.