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.

Risk of imported malaria infections in Zanzibar: a cross-sectional study.

BACKGROUND: Zanzibar has made substantial progress in malaria control with vector control, improved diagnosis, and artemisinin-based combination therapy. Parasite prevalence in the population has remained around 1% but imported infections from mainland Tanzania contribute to sustained local transmission. Understanding travel patterns between mainland Tanzania and Zanzibar, and the risk of malaria infection, may help to control malaria importation to Zanzibar. METHODS: A rolling cross-sectional survey linked to routine reactive case detection of malaria was carried out in Zanzibar between May 2017 and October 2018. Households of patients diagnosed with malaria at health facilities were surveyed and household members were tested for malaria using rapid diagnostic tests and a sub-sample by quantitative PCR (qPCR). Interviews elicited a detailed travel history of all household members who had travelled within the past two months, including trips within and outside of Zanzibar. We estimated the association of malaria infection with travel destinations in pre-defined malaria endemicity categories, trip duration, and other co-variates using logistic regression. RESULTS: Of 17,891 survey participants, 1177 (7%) reported a recent trip, of which 769 (65%) visited mainland Tanzania. Among travellers to mainland Tanzania with travel destination details and a qPCR result available, 241/378 (64%) reported traveling to districts with a 'high' malaria endemicity and for 12% the highest endemicity category was 'moderate'. Travelers to the mainland were more likely to be infected with malaria parasites (29%, 108/378) than those traveling within Zanzibar (8%, 16/206) or to other countries (6%, 2/17). Among travellers to mainland Tanzania, those visiting highly endemic districts had a higher odds of being qPCR-positive than those who travelled only to districts where malaria-endemicity was classified as low or very low (adjusted odd ratio = 7.0, 95% confidence interval: 1.9-25.5). Among travellers to the mainland, 110/378 (29%) never or only sometimes used a mosquito net during their travel. CONCLUSIONS: Strategies to reduce malaria importation to Zanzibar may benefit from identifying population groups traveling to highly endemic areas in mainland Tanzania. Targeted interventions to prevent and clear infections in these groups may be more feasible than attempting to screen and treat all travellers upon arrival in Zanzibar.

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.

Co-infection of the four major Plasmodium species: Effects on densities and gametocyte carriage.

BACKGROUND: Co-infection of the four major species of human malaria parasite Plasmodium falciparum (Pf), P. vivax (Pv), P. malariae (Pm), and P. ovale sp. (Po) is regularly observed, but there is limited understanding of between-species interactions. In particular, little is known about the effects of multiple Plasmodium species co-infections on gametocyte production. METHODS: We developed molecular assays for detecting asexual and gametocyte stages of Pf, Pv, Pm, and Po. This is the first description of molecular diagnostics for Pm and Po gametocytes. These assays were implemented in a unique epidemiological setting in Papua New Guinea with sympatric transmission of all four Plasmodium species permitting a comprehensive investigation of species interactions. FINDINGS: The observed frequency of Pf-Pv co-infection for asexual parasites (14.7%) was higher than expected from individual prevalence rates (23.8%Pf x 47.4%Pv = 11.3%). The observed frequency of co-infection with Pf and Pv gametocytes (4.6%) was higher than expected from individual prevalence rates (13.1%Pf x 28.2%Pv = 3.7%). The excess risk of co-infection was 1.38 (95% confidence interval (CI): 1.09, 1.67) for all parasites and 1.37 (95% CI: 0.95, 1.79) for gametocytes. This excess co-infection risk was partially attributable to malaria infections clustering in some villages. Pf-Pv-Pm triple infections were four times more frequent than expected by chance alone, which could not be fully explained by infections clustering in highly exposed individuals. The effect of co-infection on parasite density was analyzed by systematic comparison of all pairwise interactions. This revealed a significant 6.57-fold increase of Pm density when co-infected with Pf. Pm gametocytemia also increased with Pf co-infection. CONCLUSIONS: Heterogeneity in exposure to mosquitoes is a key epidemiological driver of Plasmodium co-infection. Among the four co-circulating parasites, Pm benefitted most from co-infection with other species. Beyond this, no general prevailing pattern of suppression or facilitation was identified in pairwise analysis of gametocytemia and parasitemia of the four species. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov, Trial ID: NCT02143934.

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.

Performance of highly sensitive and conventional rapid diagnostic tests for clinical and subclinical Plasmodium falciparum infections, and hrp2/3 deletion status in Burundi.

Rapid diagnostic tests (RDTs) are a key tool for the diagnosis of malaria infections among clinical and subclinical individuals. Low-density infections, and deletions of the P. falciparum hrp2/3 genes (encoding the HRP2 and HRP3 proteins detected by many RDTs) present challenges for RDT-based diagnosis. The novel Rapigen Biocredit three-band Plasmodium falciparum HRP2/LDH RDT was evaluated among 444 clinical and 468 subclinical individuals in a high transmission setting in Burundi. Results were compared to the AccessBio CareStart HRP2 RDT, and qPCR with a sensitivity of <0.3 parasites/µL blood. Sensitivity compared to qPCR among clinical patients for the Biocredit RDT was 79.9% (250/313, either of HRP2/LDH positive), compared to 73.2% (229/313) for CareStart (P = 0.048). Specificity of the Biocredit was 82.4% compared to 96.2% for CareStart. Among subclinical infections, sensitivity was 72.3% (162/224) compared to 58.5% (131/224) for CareStart (P = 0.003), and reached 88.3% (53/60) in children <15 years. Specificity was 84.4% for the Biocredit and 93.4% for the CareStart RDT. No (0/362) hrp2 and 2/366 hrp3 deletions were observed. In conclusion, the novel RDT showed improved sensitivity for the diagnosis of P. falciparum.

Malaria infection prevalence and sensitivity of reactive case detection in Zanzibar.

BACKGROUND: Reactive case detection (RCD) is a commonly used strategy for malaria surveillance and response in elimination settings. Many approaches to RCD assume detectable infections are clustered within and around homes of passively detected cases (index households), which has been evaluated in a number of settings with disparate results. METHODS: Household questionnaires and diagnostic testing were conducted following RCD investigations in Zanzibar, Tanzania, including the index household and up to 9 additional neighboring households. RESULTS: Of 12,487 participants tested by malaria rapid diagnostic test (RDT), 3·2% of those residing in index households and 0·4% of those residing in non-index households tested positive (OR = 8·4; 95%CI: 5·7, 12·5). Of 6,281 participants tested by quantitative polymerase chain reaction (qPCR), 8·4% of those residing in index households and 1·3% of those residing in non-index households tested positive (OR = 7·1; 95%CI: 6·1, 10·9). Within households of index cases defined as imported, odds of qPCR-positivity amongst members reporting recent travel were 1·4 times higher than among those without travel history (95%CI: 0·2, 4·4). Amongst non-index households, odds of qPCR-detectable infection were no different between households located within 50 m of the index household as compared with those located farther away (OR = 0·8, 95%CI: 0·5, 1·4). Sensitivity of RDT to detect qPCR-detectable infections was 34% (95%CI: 26·4, 42·3). CONCLUSIONS: Malaria prevalence in index households in Zanzibar is much higher than in non-index households, in which prevalence is very low. Travelers represent a high-risk population. Low sensitivity of RDTs due to a high prevalence of low-density infections results in an RCD system missing a large proportion of the parasite reservoir.

Molecular methods for tracking residual Plasmodium falciparum transmission in a close-to-elimination setting in Zanzibar.

BACKGROUND: Molecular detection of low-density Plasmodium falciparum infections is essential for surveillance studies conducted to inform malaria control strategies in close-to-elimination settings. Molecular monitoring of residual malaria infections usually requires a large study size, therefore sampling and diagnostic processes need to be economical and optimized for high-throughput. A method comparison was undertaken to identify the most efficient diagnostic procedure for processing large collections of community samples with optimal test sensitivity, simplicity, and minimal costs. METHODS: In a reactive case detection study conducted on Zanzibar, parasitaemia of 4590 individuals of all ages was investigated by a highly sensitive quantitative (q) PCR that targets multiple var gene copies per parasite genome. To reduce cost, a first round of positivity screening was performed on pools of dried blood spots from five individuals. Ten cycles of a pre-PCR were performed directly on the filter paper punches, followed by qPCR. In a second round, samples of positive pools were individually analysed by pre-PCR and qPCR. RESULTS: Prevalence in household members and neighbors of index cases was 1.7% (78/4590) with a geometric mean parasite density of 58 parasites/µl blood. Using qPCR as gold standard, diagnostic sensitivity of rapid diagnostic tests (RDTs) was 37% (29/78). Infections positive by qPCR but negative by RDT had mean densities of 15 parasites/µl blood. CONCLUSION: The approach of pre-screening reactive case detection samples in pools of five was ideal for a low prevalence setting such as in Zanzibar. Performing direct PCR on filter paper punches saves substantial time and justifies the higher cost for a polymerase suitable for amplifying DNA directly from whole blood. Molecular monitoring in community samples provided a more accurate picture of infection prevalence, as it identified a potential reservoir of infection that was largely missed by RDT. The developed qPCR-based methodology for screening large sample sets represents primarily a research tool that should inform the design of malaria elimination strategies. It may also prove beneficial for diagnostic tasks in surveillance-response activities.

Diagnostic Performance of Conventional and Ultrasensitive Rapid Diagnostic Tests for Malaria in Febrile Outpatients in Tanzania.

BACKGROUND: A novel ultrasensitive malaria rapid diagnostic test (us-RDT) has been developed for improved active Plasmodium falciparum infection detection. The usefulness of this us-RDT in clinical diagnosis and fever management has not been evaluated. METHODS: Diagnostic performance of us-RDT was compared retrospectively to that of conventional RDT (co-RDT) in 3000 children and 515 adults presenting with fever to Tanzanian outpatient clinics. The parasite density was measured by an ultrasensitive qPCR (us-qPCR), and the HRP2 concentration was measured by an enzyme-linked immunosorbent assay. RESULTS: us-RDT identified few additional P. falciparum-positive patients as compared to co-RDT (276 vs 265 parasite-positive patients detected), with only a marginally greater sensitivity (75% vs 73%), using us-qPCR as the gold standard (357 parasite-positive patients detected). The specificity of both RDTs was >99%. Five of 11 additional patients testing positive by us-RDT had negative results by us-qPCR. The HRP2 concentration was above the limit of detection for co-RDT (>3653 pg of HRP2 per mL of blood) in almost all infections (99% [236 of 239]) with a parasite density >100 parasites per µL of blood. At parasite densities <100 parasites/µL, the HRP2 concentration was above the limits of detection of us-RDT (>793 pg/mL) and co-RDT in 29 (25%) and 24 (20%) of 118 patients, respectively. CONCLUSION: There is neither an advantage nor a risk of using us-RDT, rather than co-RDT, for clinical malaria diagnosis. In febrile patients, only a small proportion of infections are characterized by a parasite density or an HRP2 concentration in the range where use of us-RDT would confer a meaningful advantage over co-RDT.