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Abstract Background The World Health Organization 2022 malaria chemoprevention guidelines recommend providing a full course of antimalarial treatment at pre-defined intervals, regardless of malaria status to prevent illness among children resident in moderate to high perennial malaria transmission settings as perennial malaria chemoprevention (PMC) with sulfadoxine-pyrimethamine (SP). The dhps I431V mutation circulating in West Africa has unknown effect on SP protective efficacy. Methods This protocol is for a three-arm, parallel, double-blinded, placebo-controlled, randomised trial in Cameroon among children randomly assigned to one of three directly-observed treatment groups: (i) Group 1 (n = 450) receives daily artesunate (AS) placebo on days − 7 to -1, then active SP plus placebo amodiaquine (AQ) on day 0, and placebo AQ on days 1 and 2; (ii) Group 2 (n = 250) receives placebo AS on days − 7 to -1, then active SP and AQ on day 0, and active AQ on days 1 and 2; and (iii) Group 3 (n = 200) receives active AS on days − 7 to -1, then placebo SP on day 0 and placebo AQ on days 0 to 2. On days 0, 2, 5, 7, and thereafter weekly until day 28, children provide blood for thick smear slides. Dried blood spots are collected on the same days and weekly from day 28 to day 63 for quantitative polymerase chain reaction (qPCR) and genotype analyses. Discussion Our aim is to quantify the chemopreventive efficacy of SP, and SP plus AQ, and measure the effect of the parasite genotypes associated with SP resistance on parasite clearance and protection from infection when exposed to SP chemoprevention. We will report unblinded results including: (i) time-to-parasite clearance among SP and SP plus AQ recipients who were positive on day 0 by qPCR and followed to day 63; (ii) mean duration of SP and SP plus AQ protection against infection, and (iii) mean duration of symptom-free status among SP and SP plus AQ recipients who were parasite free on day 0 by qPCR. Our study is designed to compare the 28-day follow-up of the new WHO malaria chemoprevention efficacy study protocol with extended follow-up to day 63. Trial registration ClinicalTrials.gov NCT06173206; 15/12/2023.

Objectives Malaria and curable sexually transmitted and reproductive tract infections (STIs/RTIs) are associated with adverse pregnancy outcomes. This study reports the prevalence and risk factors of curable STIs/RTIs, STI/RTI co-infection and STI/RTI and malaria co-infection among HIV-negative pregnant women at their first antenatal care visit in Kenya, Malawi and Tanzania.Methods HIV-negative pregnant women of all gravidae (n=4680) were screened for syphilis with point-of-care tests and treated if positive. Separately, women provided blood samples (n=4569) for rapid plasma reagin (RPR) testing; positive cases were confirmation by Treponema pallidum particle agglutination (TPPA). Women also provided dried blood spots for batch testing of malaria by retrospective polymerase chain reaction (PCR (n=4226) methods. A randomly selected subgroup of women provided vaginal swabs for chlamydia, gonorrhoea and trichomoniasis testing by retrospective PCR batch testing (n=1431), and bacterial vaginosis diagnosis by Nugent scoring (n=1402).Results Malaria prevalence was 14.6% (95% CI 13.6 to 15.7), 45.9% (43.4 to 48.4) of women were positive for at least one curable STI/RTI and 6.7% (5.5 to 8.1) were co-infected with malaria and a curable STI/RTI. Prevalence of individual STIs/RTIs ranged from 28.5% (26.2 to 30.9) for bacterial vaginosis to 14.5% (12.7 to 16.4) for trichomoniasis, 13.8% (12.1 to 15.7) for chlamydia, 2.7% (1.9 to 3.6) for gonorrhoea and 1.7% (1.4 to 2.2) for RPR/TPPA-confirmed syphilis. The prevalence of STI/RTI co-infection was 10.1% (8.7 to 11.8). Paucigravidae, at highest risk of malaria, were also at greater risk of having chlamydia, gonorrhoea and bacterial vaginosis than multigravidae.Conclusions Of women infected with malaria, 49.0% also had a curable STI/RTI and one in five women with at least one STI/RTI were co-infected with more than one STI/RTI. Current antenatal interventions that address malaria and curable STIs/RTIs remain suboptimal. New approaches to preventing and managing these infections in pregnancy are urgently needed.Trial registration number NCT03208179.

Objective: Intermittent Preventive Treatment of schoolchildren (IPTsc) is recommended by WHO as a strategy to protect against malaria; to explore whether IPTsc with dihydroartemisinin-piperaquine (DP) or artesunate-amodiaquine (ASAQ) cause a selection of molecular markers in Plasmodium falciparum genes associated with resistance in children in seven schools in Tanga region, Tanzania. Methods: SNPs in P. falciparum genes Pfmdr1, Pfexo, Pfkelch13, and Pfcrt and copy number variations in Pfplasmepsin-2 and Pfmdr1 were assessed in samples collected at 12 months (visit 4, n=74) and 20 months (visit 6, n=364) after initiation of IPTsc and compared with the baseline prevalence (n=379). Results: The prevalence of Pfmdr1 N86 and Pfexo 415G was >99% and 0%, respectively without any temporal differences observed. The prevalence of Pfmdr1 184F changed significantly from baseline (52.2%) to visit 6 (64.6%) (χ2=6.11, P=0.013), but no differences were observed between the treatment arms (χ2=0.05, P=0.98). Finally, only minor differences in the amplification of Pfmdr1 were observed; from 10.2% at baseline to 16.7% at visit 6 (χ2=0.98, P=0.32). Conclusions: The IPTsc strategy does not seem to pose a risk for the selection of markers associated with DP or ASAQ resistance. Continuously and timely surveillance of markers of antimalarial drug resistance is recommended.

Objectives: Partial artemisinin resistance, mediated by Plasmodium falciparum K13 (PfK13) mutations, has been confirmed in certain areas of East Africa that are historically associated with high-level antimalarial resistance. The Democratic Republic of Congo (DRC) borders these areas in the East. This study aimed to determine the prevalence of resistance markers in six National Malaria Control Program surveillance sites; Boende, Kabondo, Kapolowe, Kimpese, Mikalayi, and Rutshuru. Methods: The single nucleotide polymorphisms (SNPs) in P. falciparum genes PfK13, Pfdhfr, Pfdhps, Pfmdr1, and Pfcrt were assessed using targeted next-generation sequencing of isolates collected at enrollment in therapeutic efficacy studies. Results: PfK13 SNPs were detected in two samples: in Kabondo (R561H) and in Rutshuru (P441L), both areas near Uganda and Rwanda. The Pfdhps ISGEGA haplotype, associated with reduced sulfadoxine-pyrimethamine chemoprevention efficacy, ranged from 0.8% in Mikalayi (central DRC) to 42.2% in Rutshuru (East DRC). Conclusions: R561H and P441L observed in eastern DRC are a concern, as they are associated with delayed artemisinin-based combination therapies-clearance and candidate marker of resistance, respectively. This is consistent with previous observations of shared drug resistance profiles in parasites of that region with bordering areas of Rwanda and Uganda. The likely circulation of parasites has important implications for the ongoing surveillance of partial artemisinin-resistant P. falciparum and for future efforts to mitigate its dispersal.

BackgroundRecently revised WHO guidelines on malaria chemoprevention have opened the door to more tailored implementation. Countries face choices on whether to replace old drugs, target additional age groups, and adapt delivery schedules according to local drug resistance levels and malaria transmission patterns. Regular routine assessment of protective efficacy of chemoprevention is key. Here, we apply a novel modelling approach to aid the design and analysis of chemoprevention trials and generate measures of protection that can be applied across a range of transmission settings.Methods and findingsWe developed a model of genotype-specific drug protection, which accounts for underlying risk of infection and circulating genotypes. Using a Bayesian framework, we fitted the model to multiple simulated scenarios to explore variations in study design, setting, and participant characteristics. We find that a placebo or control group with no drug protection is valuable but not always feasible. An alternative approach is a single-arm trial with an extended follow-up (>42 days), which allows measurement of the underlying infection risk after drug protection wanes, as long as transmission is relatively constant. We show that the currently recommended 28-day follow-up in a single-arm trial results in low precision of estimated 30-day chemoprevention efficacy and low power in determining genotype differences of 12 days in the duration of protection (power = 1.4%). Extending follow-up to 42 days increased precision and power (71.5%) in settings with constant transmission over this time period. However, in settings of unstable transmission, protective efficacy in a single-arm trial was overestimated by 24.3% if recruitment occurred during increasing transmission and underestimated by 15.8% when recruitment occurred during declining transmission. Protective efficacy was estimated with greater precision in high transmission settings, and power to detect differences by resistance genotype was lower in scenarios where the resistant genotype was either rare or too common.ConclusionsThese findings have important implications for the current guidelines on chemoprevention efficacy studies and will be valuable for informing where these studies should be optimally placed. The results underscore the need for a comparator group in seasonal settings and provide evidence that the extension of follow-up in single-arm trials improves the accuracy of measures of protective efficacy in settings with more stable transmission. Extension of follow-up may pose logistical challenges to trial feasibility and associated costs. However, these studies may not need to be repeated multiple times, as the estimates of drug protection against different genotypes can be applied to different settings by adjusting for transmission intensity and frequency of resistance.

Objectives: Malaria and sexually transmitted and reproductive tract infections (STIs/RTIs) are highly prevalent in sub-Saharan Africa and associated with poor pregnancy outcomes. We investigated the individual and combined effects of malaria and curable STIs/RTIs on fetal growth in Kenya, Tanzania, and Malawi. Methods: This study was nested within a randomized trial comparing monthly intermittent preventive treatment for malaria in pregnancy with sulfadoxine-pyrimethamine vs dihydroartemisinin-piperaquine, alone or combined with azithromycin. Fetal weight gain was assessed by serial prenatal ultrasound. Malaria was assessed monthly, and Treponema pallidum, Neisseria gonorrhoeae, Trichomonas vaginalis, Chlamydia trachomatis, and bacterial vaginosis at enrollment and in the third trimester. The effect of malaria and STIs/RTIs on fetal weight/birthweight Z-scores was evaluated using mixed-effects linear regression. Results: In total, 1435 pregnant women had fetal/birth weight assessed 3950 times. Compared to women without malaria or STIs/RTIs (n = 399), malaria-only (n = 267), STIs/RTIs only (n = 410) or both (n = 353) were associated with reduced fetal growth (adjusted mean difference in fetal/birth weight Z-score [95% confidence interval]: malaria = -0.18 [-0.31,-0.04], P = 0.01; STIs/RTIs = -0.14 [-0.26,-0.03], P = 0.01; both = -0.20 [-0.33,-0.07], P = 0.003). Paucigravidae experienced the greatest impact. Conclusion: Malaria and STIs/RTIs are associated with poor fetal growth especially among paucigravidae women with dual infections. Integrated antenatal interventions are needed to reduce the burden of both malaria and STIs/RTIs.

Objectives: This study aimed to investigate the evolution of Plasmodium falciparum antimalarial drug resistance markers by comparing the pre- and post-adoption of artemisinin-based combination therapies (ACTs) in Yaounde, Cameroon. Methods: The molecular characterization of known antimalarial drug resistance markers (Pfcrt, Pfmdr1, Pfdhfr, Pfdhps, and Pfk13) in P. falciparum-positive samples collected in 2014 and 2019-2020 was achieved using nested polymerase chain reaction, followed by targeted amplicon deep sequencing on the Illumina MiSeq platform. Data derived were compared with those published during the pre-ACT adoption period from 2004 to 2006. Results: A high prevalence of Pfmdr1 184F, Pfdhfr 51I/59R/108N, and Pfdhps 437G mutant alleles was observed during the post-ACT adoption period. The Pfcrt 76T and Pfmdr1 86Y mutant alleles significantly declined between 2004 and 2020 (P

The pathogenesis of severe Plasmodium falciparum malaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family of var genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for var gene profiling and classification of predicted binding phenotypes in clinical P. falciparum isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for var gene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wichers et al., 2021, on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate the var transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of var genes in malaria pathogenesis. We applied this new method to investigate changes in var gene expression through early transition of parasite isolates to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictable var gene switching and convergence towards var2csa were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.

Abstract Background Malaria is a major public health issue with substantial risks among vulnerable populations. Currently, the World Health Organization (WHO) recommends SP-IPTp in the second and third trimesters. However, the efficacy of SP-IPTp is threatened by the emergence of sulfadoxine-pyrimethamine resistant malaria parasites due to single nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. This study aimed to assess the current prevalence of Pfdhfr/Pfdhps mutations in P. falciparum isolates collected from individuals residing in Ile-Ife, Nigeria, and also present maps of the prevalence of Pfdhps 431V and 581G within Nigeria and surrounding countries. Methods Between October 2020 and April 2021, samples were collected as dried blood spots among 188 participants who showed malaria positivity with a histidine-rich-protein-based rapid diagnostic test (RDT). Nested PCR assays were used to confirm falciparum in the samples with RDT positivity, and to amplify fragments of the Pfdhfr/Pfdhps genes followed by targeted amplicon sequencing. Published data since 2007 on the prevalence of the Pfdhps genotypes in Nigeria and the neighbouring countries were used to produce maps to show the distribution of the mutant genotypes. Results Only 74 and 61 samples were successfully amplified for the Pfdhfr and Pfdhps genes, respectively. At codons resulting in N51I, C59R, and S108N, Pfdhfr carried mutant alleles of 97.3% (72/74), 97.3% (72/74) and 98.6% (73/74), respectively. The Pfdhps gene carried mutations at codons resulting in amino acid changes at 431–436-437–540-581–613; I431V [45.9%, (28/61)], A581G [31.1% (19/61)] and A613S [49.2% (30/61)]. Constructed haplotypes were mainly the triple Pfdhfr mutant 51I-59R-108N (95.9%), and the most common haplotypes observed for the Pfdhps gene were the ISGKAA (32.8%), ISGKGS (8.2%), VAGKAA (14.8%), VAGKAS (9.8%) and VAGKGS (14.8%). In the context of the previously published data, a high prevalence of 431V/581G mutations was found in the study population. It seems quite evident that the Pfdhps 431V, 581G and 613S often co-occur as Pfdhps-VAGKGS haplotype. Conclusion This study showed that the prevalence of VAGKGS haplotype seems to be increasing in prevalence. If this is similar in effect to the emergence of 581G in East Africa, the efficacy of SP-IPTp in the presence of these novel Pfdhps mutants should be re-assessed.

Abstract Background Artemisinin-based combination therapy (ACT) is the first-line treatment for uncomplicated malaria in Ghana. Artemisinin (ART) tolerance in Plasmodium falciparum has arisen in Southeast Asia and recently, in parts of East Africa. This is ascribed to the survival of ring-stage parasites post treatment. The present study sought to assess and characterize correlates of potential ART tolerance based on post-treatment parasite clearance, ex vivo and in vitro drug sensitivity, and molecular markers of drug resistance in P. falciparum isolates from children with uncomplicated malaria in Ghana. Methods Six months to fourteen years old children presenting with acute uncomplicated malaria (n = 115) were enrolled in two hospitals and a Health Centre in Ghana’s Greater Accra region and treated with artemether-lumefantrine (AL) according to body weight. Pre- and post-treatment parasitaemia (day 0 and day 3) was confirmed by microscopy. The ex vivo ring-stage survival assay (RSA) was used to detect percent ring survival while the 72 h SYBR Green I assay was used to measure the 50% inhibition concentration (IC50s) of ART and its derivatives and partner drugs. Genetic markers of drug tolerance /resistance were evaluated using selective whole genome sequencing. Results Of the total of 115 participants, 85 were successfully followed up on day 3 post-treatment and 2/85 (2.4%) had parasitaemia. The IC50 values of ART, artesunate (AS), artemether (AM), dihydroartemisinin (DHA), amodiaquine (AQ), and lumefantrine (LUM) were not indicative of drug tolerance. However, 7/90 (7.8%) pre-treatment isolates had > 10% ring survival rates against DHA. Of the four isolates (2 RSA positive and 2 RSA negative) with high genomic coverage, P. falciparum (Pf) kelch 13 K188* and Pfcoronin V424I mutations were only present in the two RSA positive isolates with > 10% ring survival rates. Conclusions The observed low proportion of participants with day-3 post-treatment parasitaemia is consistent with rapid ART clearance. However, the increased rates of survival observed in the ex vivo RSA against DHA, maybe a pointer of an early start of ART tolerance. Furthermore, the role of two novel mutations in PfK13 and Pfcoronin genes, harboured by the two RSA positive isolates that had high ring survival in the present study, remains to be elucidated.

Global efforts to control Aedes mosquito-transmitted pathogens still rely heavily on insecticides. However, available information on vector resistance is mainly restricted to mosquito populations located in residential and public areas, whereas commercial settings, such as hotels are overlooked. This may obscure the real magnitude of the insecticide resistance problem and lead to ineffective vector control and resistance management. We investigated the profile of insecticide susceptibility of Aedes aegypti mosquitoes occurring at selected hotel compounds on Zanzibar Island. At least 100 adults Ae. aegypti females from larvae collected at four hotel compounds were exposed to papers impregnated with discriminant concentrations of DDT (4%), permethrin (0.75%), 0.05 deltamethrin (0.05%), propoxur (0.1%) and bendiocarb (0.1%) to determine their susceptibility profile. Allele-specific qPCR and sequencing analysis were applied to determine the possible association between observed resistance and presence of single nucleotide polymorphisms (SNPs) in the voltage-gated sodium channel gene (VGSC) linked to DDT/pyrethroid cross-resistance. Additionally, we explored the possible involvement of Glutathione-S-Transferase gene (GSTe2) mutations for the observed resistance profile. In vivo resistance bioassay indicated that Ae. aegypti at studied sites were highly resistant to DDT, mortality rate ranged from 26.3% to 55.3% and, moderately resistant to deltamethrin with a mortality rate between 79% to and 100%. However, genotyping of kdr mutations affecting the voltage-gated sodium channel only showed a low frequency of the V1016G mutation (n = 5; 0.97%). Moreover, for GSTe2, seven non-synonymous SNPs were detected (L111S, C115F, P117S, E132A, I150V, E178A and A198E) across two distinct haplotypes, but none of these were significantly associated with the observed resistance to DDT. Our findings suggest that cross-resistance to DDT/deltamethrin at hotel compounds in Zanzibar is not primarily mediated by mutations in VGSC. Moreover, the role of identified GSTe2 mutations in the resistance against DDT remains inconclusive. We encourage further studies to investigate the role of other potential insecticide resistance markers.