Francis Kimani

Malaria in pregnancy remains a major driver of poor maternal and neonatal health outcomes in sub-Saharan Africa. For decades, intermittent preventive treatment in pregnancy (IPTp), with sulphadoxine-pyrimethamine (SP), has mitigated malaria-associated health risks, but concerns have been raised regarding accumulated Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations on the efficacy of SP. Western Kenya, including Busia County, is a high malaria transmission setting where molecular surveillance of dhfr and dhps mutations remains limited. This study assessed the prevalence and haplotype structure of dhfr and dhps mutations in P. falciparum isolates from Busia County, Kenya. A total of 66 samples of P. falciparum isolates collected from patients attending Matayos Sub-County Hospital between November 2024 and January 2025 were analysed. PCR amplification and Sanger sequencing targeted dhfr codons C50R, N51I, C59R, S108N/T, I164L, and dhps codons I431V, S436A/F, A437G, K540E, A581G, and A613S/T to determine mutation frequencies, haplotypes, and combined dhps and dhfr haplotype profiles. High frequencies of dhfr and dhps mutations were observed across the parasite isolates. The most common dhfr substitutions included N51I (85.2%) and C59R (75.4%), while S108N (32.8%) and S108T (19.7%) were detected at lower frequencies. Dhfr haplotypes identified included N51I + C59R, N51I + C59R + S108N, and a N51I + C59R + S108T + I164L variant. The I164L mutation was detected at a frequency of 18.0% and was observed exclusively on a non-canonical S108T background (19.7%). Dhps haplotypes were dominated by A437G (92.3%), K540E (40%) alone, and the A437G + K540E double mutant. Combined dhfr and dhps haplotype analyses revealed circulation of classical dhfr triple-mutant (N51I + C59R + S108N) backgrounds with dhps A437G. Quintuple haplotypes (dhfr N51I + C59R + S108T + I164L with dhps A437G) and rare complex haplotypes incorporating both I164L and K540E or I164L and S436F were also detected. These findings indicate the persistence and circulation of both canonical and non-canonical dhfr and dhps haplotypes in P. falciparum isolates from Busia County. This study highlights the need for continuous molecular and phenotypic surveillance to clarify the functional and epidemiological significance of parasites carrying S108T and I164L mutations, and to inform IPT policy.

The current study sought to re-evaluate malaria prevalence, susceptibility to artemisinin-based combination therapy (ACT), transmission patterns and the presence of malaria vectors in the Kikuyu area of the Kenyan Central highlands, a non-traditional/low risk malaria transmission zone where there have been anecdotal reports of emerging malaria infections. Sampling of adult mosquitoes was done indoors, while larvae were sampled outdoors in June 2019. The malaria clinical study was an open label non-randomized clinical trial where the efficacy of one ACT drug, was evaluated in two health facilities. Microscopy was used at the facility while nested 18 s rRNA subunit gene PCR amplification and MSP-1 and MSP-2 family alleles genotyping was done in the laboratory. Anti-malarial resistance gene markers Pfk13 and Pfmdr1 were profiled. Anopheles funestus mosquitoes were the predominant vectors at 76.35% of all larvae collections (N = 148). Only two non-blood fed, parasites negative adult mosquitoes were collected from houses sampled. Parasitological analysis of the 838 patients screened resulted in 41 positives whose treatment outcome was 100% Adequate Clinical and Parasitological Response (ACPR). From the 35 positive samples genotyped, 29 (82.9%) were polyclonal. The overall mean MOI was 2.8 (95% CI 2.36–3.35). The MOI for msp-1 and msp-2 genes, was 2.02 (95% CI 0.72–2.27) and 2.9 (95% CI 2.22–3.55), and parasite strains range of 1–3 and 1–7, respectively. Polyclonal variation in the two genes was at 76.4% and 70.3%, respectively. The Pfk13 gene revealed no single nucleotide polymorphisms (SNP) associated with suspected artemisinin resistance nor was there any pfmdr1 N86 mutant allele detected. The Plasmodium infections positivity rate observed in the study site was very low but significant. A proportion of participants who tested positive did not report recent history of travel. This observation together with the finding of competent known vectors can probably suggest that several of the cases could have been acquired and transmitted locally. The observed genetic diversity and polyclonal variations was on the contrary and suggest that these are imported cases. This however does not rule out a likely changing malaria transmission scenario in this zone, thus the need for further investigations.

Background Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. Methods We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1. Using qPCR analysis, we measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages. Results We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His, while DAGK contained K270R and K292R mutations. PI4Kβ had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites. Conclusions The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and their impact on drug responses require investigations using reverse genetics techniques in malaria parasites.

Background: Malaria mortality and morbidity in Kenya has all along been largely due to Plasmodium falciparum infections with no cases attributed to P.vivax. The epidemiology in northern Kenya counties is traditionally classified as zero to low-risk transmission zones. A recent upsurge in febrile illnesses was reported in the counties of Marsabit, Mandera and Turkana, in mid-2023 which required an emergency pandemic response by the county and national health teams. In addition to possible viral infection that had featured in these areas previously, malaria disease was also investigated as part of the response. An analysis determining the presence of Plasmodium infection and the species present in this rapid response are hereby reported. Methods: A total of 89 blood samples were collected from patients suspected of malaria in the Northern Kenya counties, after which they were analyzed. Positivity and speciation was performed using 18S rRNA and qPCR TaqMan probes with species specific primers Results: From 3 health facilities in Marsabit, Mandera and Turkana counties a total of 24, 50 and 15 dried blood spot samples were received respectively. There were 11, 40 and 15 Plasmodium positive samples in the three county health facilities respectively. This translated to a total positivity of 74.2%. Speciation analysis was done for the four common plasmodium species. This analysis recorded a total of 60 P. falciparum positive samples, 2 samples with a mixed infection of P. malariae and P. falciparum, 2 P.vivaxmono infection samples, and 2 P. vivax P. falciparum co-infected samples. No P.ovale was observed in any of the facilities. Three of the P.vivax infections were in Marsabit county while the remaining case was from Mandera County translating to an incidence rate of 12.5% (3/24)and 2%(1/50) respectively. No P.vivax infection was identified in Turkana county. Conclusion: The findings of this analysis show that the incidence of malaria from the response sites was quite high, at 74.2%. Although the burden of P. vivax in these counties is undetermined, the presence of the observed proportions in such a small sample size is an indicator of an underlying problem of cases that are rarely diagnosed in a clinical setting and may often be asymptomatic. The presence of Anopheles stephensi that was recently identified for the first time in Kenya in the same study region, implies the potential expansion of a highly competent vector here, which could dramatically enhance the transmission of P. vivax and reverse all the malaria control gains made over the years.

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ) are the essential long-acting partner drugs in the artemisinin-based combination therapies (ACTs) treatment regimens globally. The recent report on the emergence of artemisinin-resistant parasites portends an imminent failure of the partner drug in clearing the high residual parasite densities. Understanding the resistance mechanisms to partner drugs remains critical for tracking resistant parasites. Cysteine desulfurase IscS (nfs1), one of the proteins involved in the iron-sulfur (FeS) biogenesis pathway, has been implicated in mediating malaria parasite drug resistance. Methods: Using the rodent malaria parasites Plasmodium berghei ANKA in mice, we assessed whether the nfs1 gene is associated with LM, PQ, and AQ resistance. We first verified the stability of the LM, PQ, and AQ-resistant parasites in the standard 4-Day Suppressive Test. By means of PCR and sequencing analysis, we probed for single nucleotide polymorphisms (SNPs) in the nfs1 gene. Using qPCR, we then measured the expression of the nfs1 gene in resistant parasites relative to the drug-sensitive parent parasites. Results: Our analyses of nfs1 reveal a non-synonymous Gln142Arg mutation in the LM and PQ-resistant parasites. This mutation was not detected in the AQ-resistant parasites. The mRNA quantification of the nfs1 gene reveals differential expression in both LM and PQ-resistant parasites. Conversely, nfs1 expression remained unchanged in the AQ-resistant parasites. Conclusion: Our data suggest that LM and PQ selection pressure induces nonsynonymous mutation and differential expression of the nfs1 gene in Plasmodium berghei. Collectively, these findings provide a premise for investigating LM and PQ resistance mechanisms in both P. berghei and P. falciparum.

Introduction: Emerging infectious diseases are infections that have recently appeared within a population or those whose incidence or geographic range is rapidly increasing or threatens to increase in the near future. The current study sought to re-evaluate malaria prevalence, susceptibility to ACTs, transmission patterns and the presence of malaria vectors in the Kikuyu area of the Kenyan Central highlands, a non-traditional/ low risk malaria transmission zone where there have been anecdotal reports of malaria cases The potential role of climate factors was also evaluated. The aim of the study was to generate data to inform malaria treatment policy and practice in the study area and country. Methodology: Sampling of adult mosquitoes was carried indoors by manual aspiration and using CDC light traps while mosquito larvae were sampled outdoors using larval dippers and reared to adults in the laboratory. Mosquitoes were identified by morphology and subsequently using PCR and the presence of malaria parasites in field sampled adult mosquitoes investigated using ELISA. The malaria clinical study was an open label nonrandomized clinical trial where the efficacy of one artemisinin-based antimalarial combination drug, Artemether Lumefantrine (AL) was evaluated. Two health facilities Lusigeti and Gikambura were identified for the study. Microscopy was used to identify positive cases at the health facility and nested PCR amplification targeting subunit 18s rRNA gene used to confirm positivity in the lab. P. falciparum isolates were genotyped using nested-PCR of MSP-1 (block 2) and MSP-2 (block 3) family alleles to determine the multiplicity of the infections (MOI) and characterize any subsequent infection. Antimalarial resistance gene markers Pfk13 and Pfmdr1 were analyzed Climate data for the study area was obtained from Climate Engine (http://climateengine.org) and analyzed to understand long term trends. Results: A rich repertoire of mosquito vector species was identified from the area, with the Anopheles funestus group being the predominant vector species and comprising 76.35% of all collections. Only two adult mosquitoes which were non-blood fed and negative for malaria parasites were collected. Of the 838 patients screened, 471, with a slide positivity rate of 2.1% (10) were from Lusigeti and 421, with a slide positivity rate of 7.4% (31) were from Gikambura. Parasitological analysis of microscopy outcome of the 41 cases revealed 100% (95% CI 1.96) as Adequate Clinical and Parasitological Response (ACPR). There was probable delayed parasite clearance (parasites present on Day 3) in 3(7.3%) of the cases, and no severe adverse reaction was observed. Analysis of the Pfk13 gene in the positive P. falciparum cases from the study sites revealed no SNP associated with artemisinin resistance. The pfmdr1 86Y mutation was found in 0% (0/41) of the isolates while the N86 wild allele was detected in 100%(37/37). Analysis of long term climate data showed an increase of about 1.3ºC in both the mean minimum and maximum temperatures consistent with forecasts from other sources. Conclusion: The positivity rate observed in the study site was very low but the fact that 87% of participants who tested positive did not report recent history of travel from the area and the finding of highly competent known vectors of malaria suggest a changing malaria transmission scenario requiring further investigations. That circulating parasite strains showed full sensitivity to the available treatment option indicating the absence of antimalarial drug resistance which is a positive finding.

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ) are the essential long-acting partner drugs in the artemisinin-based combination therapies (ACTs) treatment regimens globally. The recent report on the emergence of artemisinin-resistant parasites portends an imminent failure of the partner drug in clearing the high residual parasite densities. Understanding the resistance mechanisms to partner drugs remains critical for tracking resistant parasites. Cysteine desulfurase IscS (nfs1), one of the proteins involved in the iron-sulfur (FeS) biogenesis pathway, has been implicated in mediating malaria parasite drug resistance. Methods: Using the rodent malaria parasites Plasmodium berghei ANKA in mice, we assessed whether the nfs1 gene is associated with LM, PQ, and AQ resistance. We first verified the stability of the LM, PQ, and AQ-resistant parasites in the standard 4-Day Suppressive Test. By means of PCR and sequencing analysis, we probed for single nucleotide polymorphisms (SNPs) in the nfs1 gene. Using qPCR, we then measured the expression of the nfs1 gene in resistant parasites relative to the drug-sensitive parent parasites. Results: Our analyses of nfs1 reveal a non-synonymous Gln142Arg mutation in the LM and PQ-resistant parasites. This mutation was not detected in the AQ-resistant parasites. The mRNA quantification of the nfs1 gene reveals differential expression in both LM and PQ-resistant parasites. Conversely, nfs1 expression remained unchanged in the AQ-resistant parasites. Conclusion: Our data suggest that LM and PQ selection pressure induces nonsynonymous mutation and differential expression of the nfs1 gene in Plasmodium berghei. Collectively, these findings provide a premise for investigating LM and PQ resistance mechanisms in both P. berghei and P. falciparum.

Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. Methods: We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1. Using qPCR analysis, we finally measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages. Results: We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His, while DAGK contained K270R and K292R mutations. PI4Kβ had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites. Conclusions: The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and the impact on drug responses require further investigations in malaria parasites.