Application of a new highly multiplexed amplicon sequencing tool to evaluate Plasmodium falciparum antimalarial resistance and relatedness in individual and pooled samples from Dschang, Cameroon.

Background: Resistance to antimalarial drugs remains a major obstacle to malaria elimination. Multiplexed, targeted amplicon sequencing is being adopted for surveilling resistance and dissecting the genetics of complex malaria infections. Moreover, genotyping of parasites and detection of molecular markers drug resistance in resource-limited regions requires open-source protocols for processing samples, using accessible reagents, and rapid methods for processing numerous samples including pooled sequencing. Methods: P lasmodium f alciparum Streamlined Multiplex Antimalarial Resistance and Relatedness Testing (Pf-SMARRT) is a PCR-based amplicon panel consisting of 15 amplicons targeting antimalarial resistance mutations and 9 amplicons targeting hypervariable regions. This assay uses oligonucleotide primers in two pools and a non-proprietary library and barcoding approach. Results: We evaluated Pf-SMARRT using control mocked dried blood spots (DBS) at varying levels of parasitemia and a mixture of 3D7 and Dd2 strains at known frequencies, showing the ability to genotype at low parasite density and recall within-sample allele frequencies. We then piloted Pf-SMARRT to genotype 100 parasite isolates collected from uncomplicated malaria cases at three health facilities in Dschang, Western Cameroon. Antimalarial resistance genotyping showed high levels of sulfadoxine-pyrimethamine resistance mutations, including 31% prevalence of the DHPS A613S mutation. No K13 candidate or validated artemisinin partial resistance mutations were detected, but one low-level non-synonymous change was observed. Pf-SMARRT's hypervariable targets, used to assess complexity of infections and parasite diversity and relatedness, showed similar levels and patterns compared to molecular inversion probe (MIP) sequencing. While there was strong concordance of antimalarial resistance mutations between individual samples and pools, low-frequency variants in the pooled samples were often missed. Conclusion: Overall, Pf-SMARRT is a robust tool for assessing parasite relatedness and antimalarial drug resistance markers from both individual and pooled samples. Control samples support that accurate genotyping as low as 1 parasite per microliter is routinely possible.

Population genomics of Plasmodium ovale species in sub-Saharan Africa.

Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) are relapsing malaria parasites endemic to Africa and Asia that were previously thought to represent a single species. Amid increasing detection of ovale malaria in sub-Saharan Africa, we performed a population genomic study of both species across the continent. We conducted whole-genome sequencing of 25 isolates from Central and East Africa and analyzed them alongside 20 previously published African genomes. Isolates were predominantly monoclonal (43/45), with their genetic similarity aligning with geography. Pow showed lower average nucleotide diversity (1.8×10-4) across the genome compared to Poc (3.0×10-4) (p < 0.0001). Signatures of selective sweeps involving the dihydrofolate reductase gene were found in both species, as were signs of balancing selection at the merozoite surface protein 1 gene. Differences in the nucleotide diversity of Poc and Pow may reflect unique demographic history, even as similar selective forces facilitate their resilience to malaria control interventions.

Prevalence of mutations associated with artemisinin partial resistance and sulfadoxine-pyrimethamine resistance in 13 regions in Tanzania in 2021: a cross-sectional survey.

BACKGROUND: The emergence of the artemisinin partial resistance (ART-R) mutation in the Plasmodium falciparum kelch13 gene (k13), Arg561His, in Rwanda and the regional presence of polymorphisms affecting sulfadoxine-pyrimethamine have raised concern in neighbouring Tanzania. The goal of this study was to assess the status of antimalarial resistance in Tanzania, with a focus on the border with Rwanda, to understand the distribution of the Arg561His mutation, partner drug resistance, and resistance to chemoprevention drugs. METHODS: In this cross-sectional survey, capillary dried blood spots were collected from malaria positive asymptomatic individuals in the community and symptomatic individuals in health facilities aged 6 months and older, in 13 regions of mainland Tanzania from Jan 31 to June 26, 2021. Exclusion criteria included residence of the areas other than the target sites, presenting to the health facility for care and treatment of conditions other than malaria, and not providing informed consent. Samples were assessed for antimalarial resistance polymorphisms and genetic relatedness using molecular inversion probes targeting P falciparum and short-read whole-genome sequencing. The primary outcome was the prevalence of molecular markers of antimalarial resistance at the region level, as well as at the district level in Kagera, a region in the northwest of the country at the border with Rwanda. FINDINGS: 6855 (88·1%) of 7782 capillary dried blood spot samples collected were successfully genotyped. The overall prevalence of k13 Arg561His in Kagera was 7·7% (90% CI 6·0-9·4; 50 of 649), with the highest prevalence in the districts near the Rwandan border (22·8% [31 of 136] in Karagwe, 14·4% [17 of 118]) in Kyerwa, and 1·4% [two of 144] in Ngara). k13 Arg561His was uncommon in the other regions. Haplotype analysis suggested that some of these parasites are related to isolates collected in Rwanda in 2015, supporting regional spread of Arg561His. However, a novel k13 Arg561His haplotype was observed, potentially indicating a second origin in the region. Other validated k13 resistance polymorphisms (one Arg622Ile and two Ala675Val isolates) were also identified. A region of prevalent dihydrofolate reductase Ile164Leu mutation, associated with sulfadoxine-pyrimethamine resistance, was also identified in Kagera (15·2% [12·6-17·8%]; 80 of 526). The mutant crt Lys76Thr mutation, associated with chloroquine and amodiaquine resistance, was uncommon, occurring only in 75 of 2861 genotyped isolates, whereases the wild-type mdr1 Asn86Tyr allele, associated with reduced sensitivity to lumefantrine, was found in 99·7% (3819 of 3830) of samples countrywide. INTERPRETATION: These findings show that the k13 Arg561His mutation is common in northwest Tanzania and that multiple emergences of ART-R, similar as to what was seen in southeast Asia, have occurred. Mutations associated with high levels of sulfadoxine-pyrimethamine resistance are common. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Understanding how multiple emergences interact with drivers of regional spread is essential for combating ART-R in Africa. FUNDING: This study was funded by the Bill & Melinda Gates Foundation and the National Institutes of Health.

High-Throughput Genotyping of Plasmodium vivax in the Peruvian Amazon via Molecular Inversion Probes.

Plasmodium vivax transmission occurs throughout the tropics and is an emerging threat in areas of Plasmodium falciparum decline, causing relapse infections that complicate treatment and control. Targeted sequencing for P. falciparum has been widely deployed to detect population structure and the geographic spread of antimalarial and diagnostic resistance. However, there are fewer such tools for P. vivax . Leveraging global variation data, we designed four molecular inversion probe (MIP) genotyping panels targeting geographically differentiating SNPs, neutral SNPs, putative antimalarial resistance genes, and vaccine candidate genes. We deployed these MIP panels on 866 infections from the Peruvian Amazon and identified transmission networks with clonality (IBD>0.99), copy number variation in Pvdbp and multiple Pvrbps , fixation of putative antimalarial resistance, and balancing selection in 13 vaccine candidate genes. Our MIP panels are the broadest genotyping panel currently available and are poised for successful deployment in other regions of P. vivax transmission.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission. Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018. Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors. Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania, and continued local transmission. To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo District on the coastal mainland from 2016-2018. Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Our data support importation as a main source of genetic diversity and contribution to the parasite population on Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive for malaria reemergence due to susceptible hosts and competent vectors.

Country wide surveillance reveals prevalent artemisinin partial resistance mutations with evidence for multiple origins and expansion of high level sulfadoxine-pyrimethamine resistance mutations in northwest Tanzania.

Background: Emergence of artemisinin partial resistance (ART-R) in Plasmodium falciparum is a growing threat to the efficacy of artemisinin combination therapies (ACT) and the efforts for malaria elimination. The emergence of Plasmodium falciparum Kelch13 (K13) R561H in Rwanda raised concern about the impact in neighboring Tanzania. In addition, regional concern over resistance affecting sulfadoxine-pyrimethamine (SP), which is used for chemoprevention strategies, is high. Methods: To enhance longitudinal monitoring, the Molecular Surveillance of Malaria in Tanzania (MSMT) project was launched in 2020 with the goal of assessing and mapping antimalarial resistance. Community and clinic samples were assessed for resistance polymorphisms using a molecular inversion probe platform. Findings: Genotyping of 6,278 samples collected countrywide in 2021 revealed a focus of K13 561H mutants in northwestern Tanzania (Kagera) with prevalence of 7.7% (50/649). A small number of 561H mutants (about 1%) were found as far as 800 km away in Tabora, Manyara, and Njombe. Genomic analysis suggests some of these parasites are highly related to isolates collected in Rwanda in 2015, supporting regional spread of 561H. However, a novel haplotype was also observed, likely indicating a second origin in the region. Other validated resistance polymorphisms (622I and 675V) were also identified. A focus of high sulfadoxine-pyrimethamine drug resistance was also identified in Kagera with a prevalence of dihydrofolate reductase 164L of 15% (80/526). Interpretation: These findings demonstrate the K13 561H mutation is entrenched in the region and that multiple origins of ART-R, similar as to what was seen in Southeast Asia, have occurred. Mutations associated with high levels of SP resistance are increasing. These results raise concerns about the long-term efficacy of artemisinin and chemoprevention antimalarials in the region. Funding: This study was funded by the Bill and Melinda Gates Foundation and the National Institutes of Health.

Background malaria incidence and parasitemia during the three-dose RTS,S/AS01 vaccination series do not reduce magnitude of antibody response nor efficacy against the first case of malaria.

BACKGROUND: RTS,S/AS01 has been recommended by WHO for widespread implementation in medium to high malaria transmission settings. Previous analyses have noted lower vaccine efficacies in higher transmission settings, possibly due to the more rapid development of naturally acquired immunity in the control group. METHODS: To investigate a reduced immune response to vaccination as a potential mechanism behind lower efficacy in high transmission areas, we examine initial vaccine antibody (anti-CSP IgG) response and vaccine efficacy against the first case of malaria (to exclude the effect of naturally acquired immunity) using data from three study areas (Kintampo, Ghana; Lilongwe, Malawi; Lambaréné, Gabon) from the 2009-2014 phase III trial (NCT00866619). Our key exposures are parasitemia during the vaccination series and background malaria incidence. We calculate vaccine efficacy (one minus hazard ratio) using a cox-proportional hazards model and allowing for the time-varying effect of RTS,S/AS01. RESULTS: We find that antibody responses to the primary three-dose vaccination series were higher in Ghana than in Malawi and Gabon, but that neither antibody levels nor vaccine efficacy against the first case of malaria varied by background incidence or parasitemia during the primary vaccination series. CONCLUSIONS: We find that vaccine efficacy is unrelated to infections during vaccination. Contributing to a conflicting literature, our results suggest that vaccine efficacy is also unrelated to infections before vaccination, meaning that control-group immunity is likely a major reason for lower efficacy in high transmission settings, not reduced immune responses to RTS,S/AS01. This may be reassuring for implementation in high transmission settings, though further studies are needed.

Evolution of Partial Resistance to Artemisinins in Malaria Parasites in Uganda.

BACKGROUND: Partial resistance of Plasmodium falciparum to the artemisinin component of artemisinin-based combination therapies, the most important malaria drugs, emerged in Southeast Asia and now threatens East Africa. Partial resistance, which manifests as delayed clearance after therapy, is mediated principally by mutations in the kelch protein K13 (PfK13). Limited longitudinal data are available on the emergence and spread of artemisinin resistance in Africa. METHODS: We performed annual surveillance among patients who presented with uncomplicated malaria at 10 to 16 sites across Uganda from 2016 through 2022. We sequenced the gene encoding kelch 13 (pfk13) and analyzed relatedness using molecular methods. We assessed malaria metrics longitudinally in eight Ugandan districts from 2014 through 2021. RESULTS: By 2021-2022, the prevalence of parasites with validated or candidate resistance markers reached more than 20% in 11 of the 16 districts where surveillance was conducted. The PfK13 469Y and 675V mutations were seen in far northern Uganda in 2016-2017 and increased and spread thereafter, reaching a combined prevalence of 10 to 54% across much of northern Uganda, with spread to other regions. The 469F mutation reached a prevalence of 38 to 40% in one district in southwestern Uganda in 2021-2022. The 561H mutation, previously described in Rwanda, was first seen in southwestern Uganda in 2021, reaching a prevalence of 23% by 2022. The 441L mutation reached a prevalence of 12 to 23% in three districts in western Uganda in 2022. Genetic analysis indicated local emergence of mutant parasites independent of those in Southeast Asia. The emergence of resistance was observed predominantly in areas where effective malaria control had been discontinued or transmission was unstable. CONCLUSIONS: Data from Uganda showed the emergence of partial resistance to artemisinins in multiple geographic locations, with increasing prevalence and regional spread over time. (Funded by the National Institutes of Health.).

Malaria transmission-blocking vaccines Pfs230D1-EPA and Pfs25-EPA in Alhydrogel in healthy Malian adults; a phase 1, randomised, controlled trial.

BACKGROUND: Malaria transmission-blocking vaccines target mosquito-stage parasites and will support elimination programmes. Gamete vaccine Pfs230D1-EPA/Alhydrogel induced superior activity to zygote vaccine Pfs25-EPA/Alhydrogel in malaria-naive US adults. Here, we compared these vaccines in malaria-experienced Malians. METHODS: We did a pilot safety study then double-blind, block-randomised, comparator-controlled main-phase trial in malaria-intense Bancoumana, Mali. 18-50-year-old healthy non-pregnant, non-breastfeeding consenting adult residents were randomly assigned (1:1:1:1) to receive four doses at months 0, 1, 4·5, and 16·5 of either 47 µg Pfs25, 40 µg Pfs230D1 or comparator (Twinrix or Menactra)-all co-administered with normal saline for blinding-or 47 µg Pfs25 plus 40 µg Pfs230D1 co-administered. We documented safety and tolerability (primary endpoint in the as-treated populations) and immunogenicity (secondary endpoint in the as-treated populations: ELISA, standard-membrane-feeding assay, and mosquito direct skin feed assay). This trial is registered at ClinicalTrials.gov, NCT02334462. FINDINGS: Between March 19, and June 2, 2015, we screened 471 individuals. Of 225 enrolled for the pilot and main cohorts, we randomly assigned 25 participants to pilot safety cohort groups of five (20%) to receive a two-dose series of Pfs25-EPA/Alhydrogel (16 µg), Pfs230D1-EPA/Alhydrogel (15 µg) or comparator, followed by Pfs25-EPA/Alhydrogel (16 µg) plus Pfs230D1-EPA/Alhydrogel (15 µg) or comparator plus saline. For the main cohort, we enrolled 200 participants between May 11 and June 2, 2015, to receive a four-dose series of 47 µg Pfs25-EPA/Alhydrogel plus saline (n=50 [25%]; Pfs25), 40 µg Pfs230D1-EPA/Alhydrogel plus saline (n=49 [25%]; Pfs230D1), 47 µg Pfs25-EPA/Alhydrogel plus 40 µg Pfs230D1-EPA/Alhydrogel (n=50 [25%]; Pfs25 plus Pfs230D1), or comparator (Twinrix or Menactra) plus saline (n=51 [25%]). Vaccinations were well tolerated in the pilot safety and main phases. Most vaccinees became seropositive after two Pfs230D1 or three Pfs25 doses; peak titres increased with each dose thereafter (Pfs230D1 geometric mean: 77·8 [95% CI 56·9-106·3], 146·4 [108·3-198·0], and 410·2 [301·6-558·0]; Pfs25 geometric mean 177·7 [130·3-242·4] and 315·7 [209·9-474·6]). Functional activity (mean peak transmission-reducing activity) appeared for Pfs230D1 (74·5% [66·6-82·5]) and Pfs25 plus Pfs230D1 (68·6% [57·3-79·8]), after the third dose and after the fourth dose (88·9% [81·7-96·2] for Pfs230D1 and 85·0% [78·4-91·5] Pfs25 plus Pfs230D1) but not for Pfs25 (58·2% [49·1-67·3] after the third dose and 58·2% [48·5-67·9] after the fourth dose). Pfs230D1 transmission-reducing activity (73·7% [64·1-83·3]) persisted 10 weeks after the fourth dose. Transmission-reducing activity of 80% was estimated at 1659 ELISA units for Pfs25, 218 for Pfs230D1, and 223 for Pfs230D1 plus Pfs25. After 3850 direct skin feed assays, 35 participants (12 Pfs25, eight Pfs230D1, five Pfs25 plus Pfs230D1, and ten comparator) had transmitted parasites at least once. The proportion of positive assays in vaccine groups (Pfs25 33 [3%] of 982 [-0·013 to 0·014], Pfs230D1 22 [2%] of 954 [-0·005 to 0·027], and combination 11 [1%] of 940 [-0·024 to 0·002]) did not differ from that of the comparator (22 [2%] of 974), nor did Pfs230D1 and combination groups differ (-0·024 to 0·001). INTERPRETATION: Pfs230D1 but not Pfs25 vaccine induces durable serum functional activity in Malian adults. Direct skin feed assays detect parasite transmission to mosquitoes but increased event rates are needed to assess vaccine effectiveness. FUNDING: Intramural Research Program of the National Institute of Allergy and Infectious Diseases and US National Institutes of Health.

An optimized GATK4 pipeline for Plasmodium falciparum whole genome sequencing variant calling and analysis.

BACKGROUND: Accurate variant calls from whole genome sequencing (WGS) of Plasmodium falciparum infections are crucial in malaria population genomics. Here a falciparum variant calling pipeline based on GATK version 4 (GATK4) was optimized and applied to 6626 public Illumina WGS samples. METHODS: Control WGS and accurate PacBio assemblies of 10 laboratory strains were leveraged to optimize parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs. From these controls, a high-quality training dataset was generated to recalibrate the raw variant data. RESULTS: On current high-quality samples (read length = 250 bp, insert size = 405-524 bp), the optimized pipeline shows improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P < 0.001) and previous variant calling with GATK version 3 (GATK3, 70.3 ± 3.0% for SNPs and 59.7 ± 5.8% for indels, adjusted P < 0.001). Its sensitivity on simulated mixed infection samples (80.8 ± 6.1% for SNPs and 78.3 ± 5.1% for indels) was again improved relative to default GATK4 (68.8 ± 6.0% for SNPs and 38.9 ± 0.7% for indels, adjusted, adjusted P < 0.001). Precision was high and comparable across all pipelines on each type of data tested. The resulting combination of high-quality SNPs and indels increases the resolution of local population population structure detection in sub-Saharan Africa. Finally, increasing ploidy improves the detection of drug resistance mutations and estimation of complexity of infection. CONCLUSIONS: Overall, this study provides an optimized falciparum GATK4 pipeline resource for variant calling which should help improve genomic studies of malaria.

Malaria Transmission Intensity and Parasitemia during the Three-Dose RTS,S/AS01 Vaccination Series do not Reduce Magnitude of Antibody Response nor Efficacy Against the First Case of Malaria.

Background: RTS,S/AS01 has been recommended by WHO for widespread implementation in medium to high malaria transmission settings. Previous analyses have noted lower vaccine efficacies in higher transmission settings, possibly due to the more rapid development of naturally acquired immunity in the control group. Methods: To investigate a reduced immune response to vaccination as a potential mechanism behind lower efficacy in high transmission areas, we examine initial vaccine antibody (anti-CSP IgG) response and vaccine efficacy against the first case of malaria to exclude the delayed malaria effect using data from three study areas (Kintampo, Ghana; Lilongwe, Malawi; Lambaréné, Gabon) from the 2009-2014 phase III trial (NCT00866619). Our key exposures are parasitemia during the vaccination series and malaria transmission intensity. We calculate vaccine efficacy (one minus hazard ratio) using a cox-proportional hazards model and allowing for the time-varying effect of RTS,S/AS01. Results: We find that antibody responses to the primary three-dose vaccination series were higher in Ghana than in Malawi and Gabon, but that neither antibody levels nor vaccine efficacy against the first case of malaria varied by transmission intensity or parasitemia during the primary vaccination series. Conclusions: We find that vaccine efficacy is unrelated to infections during vaccination. Contributing to a conflicting literature, our results suggest that vaccine efficacy is also unrelated to infections before vaccination, meaning that delayed malaria is likely the main reason for lower efficacy in high transmission settings, not reduced immune responses. This may be reassuring for implementation in high transmission settings, though further studies are needed.

Characterization of a novel Plasmodium falciparum merozoite surface antigen and potential vaccine target.

Introduction: Detailed analyses of genetic diversity, antigenic variability, protein localization and immunological responses are vital for the prioritization of novel malaria vaccine candidates. Comprehensive approaches to determine the most appropriate antigen variants needed to provide broad protection are challenging and consequently rarely undertaken. Methods: Here, we characterized PF3D7_1136200, which we named Asparagine-Rich Merozoite Antigen (ARMA) based on the analysis of its sequence, localization and immunogenicity. We analyzed IgG and IgM responses against the common variants of ARMA in independent prospective cohort studies in Burkina Faso (N = 228), Kenya (N = 252) and Mali (N = 195) using a custom microarray, Div-KILCHIP. Results: We found a marked population structure between parasites from Africa and Asia. African isolates shared 34 common haplotypes, including a dominant pair although the overall selection pressure was directional (Tajima's D = -2.57; Fu and Li's F = -9.69; P < 0.02). ARMA was localized to the merozoite surface, IgG antibodies induced Fc-mediated degranulation of natural killer cells and strongly inhibited parasite growth in vitro. We found profound serological diversity, but IgG and IgM responses were highly correlated and a hierarchical clustering analysis identified only three major serogroups. Protective IgG and IgM antibodies appeared to target both cross-reactive and distinct epitopes across variants. However, combinations of IgG and IgM antibodies against selected variants were associated with complete protection against clinical episodes of malaria. Discussion: Our systematic strategy exploits genomic data to deduce the handful of antigen variants with the strongest potential to induce broad protection and may be broadly applicable to other complex pathogens for which effective vaccines remain elusive.

An Optimized GATK4 Pipeline for Plasmodium falciparum Whole Genome Sequencing Variant Calling and Analysis.

Background Accurate variant calls from whole genome sequencing (WGS) of Plasmodium falciparum infections are crucial in malaria population genomics. Here we optimized a falciparum variant calling pipeline based on GATK version 4 (GATK4) and applied it to 6,626 public Illumina WGS samples. Methods We optimized parameters that control the heterozygosity, local assembly region size, ploidy, mapping and base quality in both GATK HaplotypeCaller and GenotypeGVCFs leveraging control WGS and accurate PacBio assemblies of 10 laboratory strains. From these controls we generated a high-quality training dataset to recalibrate the raw variant data. Results On current high-quality samples (read length = 250bp, insert size = 405 - 524 bp ), we show improved sensitivity (86.6 ± 1.7% for SNPs and 82.2 ± 5.9% for indels) compared to the default GATK4 pipeline (77.7 ± 1.3% for SNPs; and 73.1 ± 5.1% for indels, adjusted P < 0.001) and previous variant calling with GATK version 3 (GATK3, 70.3 ± 3.0% for SNPs and 59.7 ± 5.8% for indels, adjusted P < 0.001). The sensitivity of our pipeline on simulated mixed infection samples (80.8 ± 6.1% for SNPs and 78.3 ± 5.1% for indels) was again improved relative to default GATK4 (68.8 ± 6.0% for SNPs and 38.9 ± 0.7% for indels, adjusted P < 0.001). Precision was high and comparable across all pipelines on each type of data tested. We further show that using the combination of high-quality SNPs and indels increases the resolution of local population population structure detection in sub-Saharan Africa. We finally demonstrate that increasing ploidy improves the detection of drug resistance mutations and estimation of complexity of infection. Conclusions Overall, we provide an optimized GATK4 pipeline and resource for falciparum variant calling which should help improve genomic studies of malaria.

The prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine among pregnant women at first antenatal clinic attendance and delivery in the forest-savannah area of Ghana.

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is used to prevent malaria and associated unfavorable maternal and foetal outcomes in pregnancy in moderate to high malaria transmission areas. Effectiveness of IPTp-SP is, however, threatened by mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to pyrimethamine and sulfadoxine, respectively. This study determined the prevalence of molecular markers of SP resistance among pregnant women in a high malaria transmission area in the forest-savannah area of Ghana. Genomic DNA was extracted from 286 P. falciparum-positive dried blood spots obtained from pregnant women aged ≥18 years (255 at first Antenatal Care (ANC) clinic visit and 31 at delivery from 2017 to 2019) using Chelex 100. Mutations in Pfdhfr and Pfdhps genes were detected using molecular inversion probes and next generation sequencing. In the Pfdhfr gene, single nucleotide polymorphisms (SNPs) were detected in 83.1% (157/189), 92.0% (173/188) and 91.0% (171/188) at codons 51, 59, and 108 respectively in samples collected at first ANC visit, while SNPs were detected in 96.6 (28/29), 96.6% (28/29) and 96.8% (30/31) in isolates collected at delivery. The Pfdhfr triple mutant N51I, C59R and S108N (IRN) was carried by 80.5% (128/159) and 96.5% (28/29) of the typed isolates collected at ANC visit and at delivery respectively. In the Pfdhps gene, SNPs were detected in 0.6% (1/174), 76.2% (138/181), 33.2% (60/181), 1.2% (2/174), 0% (0/183), and 16.6% (27/173) at codons 431, 436, 437, 540, 581 and 613 respectively in samples collected at ANC, and 0% (0/25), 72% (18/25), 40% (10/25), 3.6% (1/25), 0% (0/29) and 7.4% (2/27) in samples collected at delivery. Quadruple mutant Pfdhfr N51I, C59R, and S108N + Pfdhps A437G (IRN-GK) was present in 25.8% (33/128) and 34.8% (8/23) of isolates at ANC and at delivery respectively. Quintuple mutant alleles Pfdhfr N51I, C59R, and S108N + Pfdhps A437G and K540E (IRN-GE) were detected in 0.8% (1/128) and 4.4% (1/23) of samples collected at ANC and at delivery respectively. No mutations were identified at Pfdhfr codons 16 or 164 or Pfdhps 581. There is a high prevalence of Pfdhfr triple mutant P. falciparum infections among pregnant women in the study area. However, prevalence of the combined Pfdhfr/Pfdhps quadruple and quintuple mutants IRN-GK and IRN-GE respectively prior to commencement of IPTp-SP were low, and no Pfdhps A581G mutant was detected, indicating that SP is still likely to be efficacious for IPTp-SP in the forest-savannah area in the middle belt of Ghana.

Malaria Transmission Intensity Likely Modifies RTS, S/AS01 Efficacy Due to a Rebound Effect in Ghana, Malawi, and Gabon.

BACKGROUND: RTS,S/AS01 is the first malaria vaccine to be approved and recommended for widespread implementation by the World Health Organization (WHO). Trials reported lower vaccine efficacies in higher-incidence sites, potentially due to a "rebound" in malaria cases in vaccinated children. When naturally acquired protection in the control group rises and vaccine protection in the vaccinated wanes concurrently, malaria incidence can become greater in the vaccinated than in the control group, resulting in negative vaccine efficacies. METHODS: Using data from the 2009-2014 phase III trial (NCT00866619) in Lilongwe, Malawi; Kintampo, Ghana; and Lambaréné, Gabon, we evaluate this hypothesis by estimating malaria incidence in each vaccine group over time and in varying transmission settings. After estimating transmission intensities using ecological variables, we fit models with 3-way interactions between vaccination, time, and transmission intensity. RESULTS: Over time, incidence decreased in the control group and increased in the vaccine group. Three-dose efficacy in the lowest-transmission-intensity group (0.25 cases per person-year [CPPY]) decreased from 88.2% to 15.0% over 4.5 years, compared with 81.6% to -27.7% in the highest-transmission-intensity group (3 CPPY). CONCLUSIONS: These findings suggest that interventions, including the fourth RTS,S dose, that protect vaccinated individuals during the potential rebound period should be implemented for high-transmission settings.

Mutation in the Plasmodium falciparum BTB/POZ Domain of K13 Protein Confers Artemisinin Resistance.

Partial artemisinin resistance, defined in patients as a delayed parasite clearance following artemisinin-based treatment, is conferred by non-synonymous mutations in the Kelch beta-propeller domain of the Plasmodium falciparum k13 (pfk13) gene. Here, we carried out in vitro selection over a 1-year period on a West African P. falciparum strain isolated from Kolle (Mali) under a dose-escalating artemisinin regimen. After 18 cycles of sequential drug pressure, the selected parasites exhibited enhanced survival to dihydroartemisinin in the ring-stage survival assay (RSA0-3h = 9.2%). Sanger and whole-genome sequence analyses identified the PfK13 P413A mutation, localized in the BTB/POZ domain, upstream of the propeller domain. This mutation was sufficient to confer in vitro artemisinin resistance when introduced into the PfK13 coding sequence of the parasite strain Dd2 by CRISPR/Cas9 gene editing. These results together with structural studies of the protein demonstrate that the propeller domain is not the sole in vitro mediator of PfK13-mediated artemisinin resistance, and highlight the importance of monitoring for mutations throughout PfK13.

Safety and efficacy of a three-dose regimen of Plasmodium falciparum sporozoite vaccine in adults during an intense malaria transmission season in Mali: a randomised, controlled phase 1 trial.

BACKGROUND: WHO recently approved a partially effective vaccine that reduces clinical malaria in children, but increased vaccine activity is required to pursue malaria elimination. A phase 1 clinical trial was done in Mali, west Africa, to assess the safety, immunogenicity, and protective efficacy of a three-dose regimen of Plasmodium falciparum sporozoite (PfSPZ) Vaccine (a metabolically active, non-replicating, whole malaria sporozoite vaccine) against homologous controlled human malaria infection (CHMI) and natural P falciparum infection. METHODS: We recruited healthy non-pregnant adults aged 18-50 years in Donéguébougou, Mali, and surrounding villages (Banambani, Toubana, Torodo, Sirababougou, Zorokoro) for an open-label, dose-escalation pilot study and, thereafter, a randomised, double-blind, placebo-controlled main trial. Pilot study participants were enrolled on an as-available basis to one group of CHMI infectivity controls and three staggered vaccine groups receiving: one dose of 4·5 × 105, one dose of 9 × 105, or three doses of 1·8 × 106 PfSPZ via direct venous inoculation at approximately 8 week intervals, followed by homologous CHMI 5 weeks later with infectious PfSPZ by direct venous inoculation (PfSPZ Challenge). Main cohort participants were stratified by village and randomly assigned (1:1) to receive three doses of 1·8 × 106 PfSPZ or normal saline at 1, 13, and 19 week intervals using permuted block design by the study statistician. The primary outcome was safety and tolerability of at least one vaccine dose; the secondary outcome was vaccine efficacy against homologous PfSPZ CHMI (pilot study) or against naturally transmitted P falciparum infection (main study) measured by thick blood smear. Combined artesunate and amodiaquine was administered to eliminate pre-existing parasitaemia. Outcomes were analysed by modified intention to treat (mITT; including all participants who received at least one dose of investigational product; safety and vaccine efficacy) and per protocol (vaccine efficacy). This trial is registered with ClinicalTrials.gov, number NCT02627456. FINDINGS: Between Dec 20, 2015, and April 30, 2016, we enrolled 56 participants into the pilot study (five received the 4·5 × 105 dose, five received 9 × 105, 30 received 1·8 × 106, 15 were CHMI controls, and one withdrew before vaccination) and 120 participants into the main study cohort with 60 participants assigned PfSPZ Vaccine and 60 placebo in the main study. Adverse events and laboratory abnormalities post-vaccination in all dosing groups were few, mainly mild, and did not differ significantly between vaccine groups (all p>0·05). Unexpected severe transaminitis occured in four participants: one participant in pilot phase that received 1·8 × 106 PfSPZ Vaccine, one participant in main phase that received 1·8 × 106 PfSPZ Vaccine, and two participants in the main phase placebo group. During PfSPZ CHMI, approximately 5 weeks after the third dose of 1·8 × 106 PfSPZ, none of 29 vaccinees and one of 15 controls became positive on thick blood smear; subsequent post-hoc PCR analysis for submicroscopic blood stage infections detected P falciparum parasites in none of the 29 vaccine recipients and eight of 15 controls during CHMI. In the main trial, 32 (58%) of 55 vaccine recipients and 42 (78%) of 54 controls became positive on thick blood smear during 24-week surveillance after vaccination. Vaccine efficacy (1-hazard ratio) was 0·51 per protocol (95% CI 0·20-0·70; log-rank p=0·0042) and 0·39 by mITT (0·04-0·62; p=0·033); vaccine efficacy (1-risk ratio) was 0·24 per-protocol (0·02-0·41; p=0·031) and 0·22 mITT (0·01-0·39; p=0·041). INTERPRETATION: A three-dose regimen of PfSPZ Vaccine was safe, well tolerated, and conferred 51% vaccine efficacy against intense natural P falciparum transmission, similar to 52% vaccine efficacy reported for a five-dose regimen in a previous trial. FUNDING: US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Sanaria. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Safety and immunogenicity of Pfs25H-EPA/Alhydrogel, a transmission-blocking vaccine against Plasmodium falciparum: a randomised, double-blind, comparator-controlled, dose-escalation study in healthy Malian adults.

BACKGROUND: Pfs25H-EPA is a protein-protein conjugate transmission-blocking vaccine against Plasmodium falciparum that is safe and induces functional antibodies in malaria-naive individuals. In this field trial, we assessed Pfs25H-EPA/Alhydrogel for safety and functional immunogenicity in Malian adults. METHODS: This double-blind, randomised, comparator-controlled, dose-escalation trial in Bancoumana, Mali, was done in two staggered phases, an initial pilot safety assessment and a subsequent main phase. Healthy village residents aged 18-45 years were eligible if they had normal laboratory results (including HIV, hepatitis B, hepatitis C tests) and had not received a previous malaria vaccine or recent immunosuppressive drugs, vaccines, or blood products. Participants in the pilot safety cohort and the main cohort were assigned (1:1) by block randomisation to a study vaccine group. Participants in the pilot safety cohort received two doses of Pfs25H-EPA/Alhydrogel 16 µg or Euvax B (comparator vaccine), and participants in the main cohort received Pfs25H-EPA/Alhydrogel 47 µg or comparator vaccine (Euvax B for the first, second, and third vaccinations and Menactra for the fourth vaccination). Participants and investigators were masked to group assignment, and randomisation codes in sealed envelopes held by a site pharmacist. Vials with study drug for injection were covered by opaque tape and labelled with a study identification number. Group assignments were unmasked at final study visit. The primary outcomes were safety and tolerability for all vaccinees. The secondary outcome measure was immunogenicity 14 days after vaccination in the per-protocol population, as confirmed by the presence of antibodies against Pfs25H measured by ELISA IgG and antibody functionality assessed by standard membrane feeding assays and by direct skin feeding assays. This trial is registered with ClinicalTrials.gov, number NCT01867463. FINDINGS: Between May 15, and Jun 16, 2013, 230 individuals were screened for eligibility. 20 individuals were enrolled in the pilot safety cohort; ten participants were assigned to receive Pfs25H-EPA/Alhydrogel 16 µg, and ten participants were assigned to receive comparator vaccine. 100 individuals were enrolled in the main cohort; 50 participants were assigned to receive Pfs25H-EPA/Alhydrogel 47 µg, and 50 participants were assigned to receive comparator vaccine. Compared with comparator vaccinees, Pfs25H vaccinees had more solicited adverse events (137 events vs 86 events; p=0·022) and treatment-related adverse events (191 events vs 126 events, p=0·034), but the number of other adverse events did not differ between study vaccine groups (792 vs 683). Pfs25H antibody titres increased with each dose, with a peak geometric mean of 422·3 ELISA units (95% CI 290-615) after the fourth dose, but decreased relatively rapidly thereafter, with a half-life of 42 days for anti-Pfs25H and 59 days for anti-EPA (median ratio of titres at day 600 to peak, 0·19 for anti-Pfs25H vs 0·29 for anti-EPA; p=0·009). Serum transmission-reducing activity was greater for Pfs25H than for comparator vaccine after the fourth vaccine dose (p<0·001) but not after the third dose (p=0·09). Repeated direct skin feeds were well tolerated, but the number of participants who infected at least one mosquito did not differ between Pfs25H and comparator vaccinees after the fourth dose (p=1, conditional exact). INTERPRETATION: Pfs25H-EPA/Alhydrogel was well tolerated and induced significant serum activity by standard membrane feeding assays but transmission blocking activity was not confirmed by weekly direct skin feed. This activity required four doses, and titres decreased rapidly after the fourth dose. Alternative antigens or combinations should be assessed to improve activity. FUNDING: Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Multiple Phenotypic and Genotypic Artemisinin Sensitivity Evaluation of Malian Plasmodium falciparum Isolates.

We assessed the ex vivo/in vitro sensitivity of 54 Malian Plasmodium falciparum isolates to artemisinin for the monitoring of drug resistance in this area. The artemisinin sensitivity of parasites was evaluated using 1) the ex vivo and in vitro parasite recrudescence detection after treatment of the ring stage with 1-200 nM artemisinin for 48 hours and 2) the in vitro parasite recrudescence kinetics assay over 7 days after 6-hour treatment of the ring stage with 700 nM dihydroartemisinin (DHA). In addition, as recommended by the World Health Organization for artemisinin resistance characterization, the ring-stage survival assay (RSA0-3 h) was performed and the parasite isolates were sequenced at the kelch 13 propeller locus. No clinical and molecular evidence of artemisinin resistance was observed. However, these isolates present different phenotypic profiles in response to artemisinin treatments. Despite all RSA0-3 h values less than 1.5%, six out of 46 (13.0%) isolates tested ex vivo and four out of six (66.7%) isolates tested in vitro were able to multiply after 48-hour treatments with 100 nM artemisinin. Moreover, five out of eight isolates tested showed faster parasite recovery after DHA treatment in kinetic assays. The presence of such phenotypes needs to be taken into account in the assessment of the efficacy of artemisinins in Mali. The assays presented here appear as valuable tools for the monitoring of artemisinin sensitivity in the field and thus could help to evaluate the risk of emergence of artemisinin resistance in Africa.