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Background: Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised., Methods: We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug 31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility., Findings: We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts., Interpretation: The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp., Funding: French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Timely molecular surveillance of Plasmodium falciparum kelch 13 (k13) gene mutations is essential for monitoring the emergence and stemming the spread of artemisinin resistance. Widespread artemisinin resistance, as observed in Southeast Asia, would reverse significant gains that have been made against the malaria burden in Africa. The purpose of this study was to assess the prevalence of k13 polymorphisms in western Kenya and Ethiopia at sites representing varying transmission intensities between 2018 and 2022., Methods: Dried blood spot samples collected through ongoing passive surveillance and malaria epidemiological studies, respectively, were investigated. The k13 gene was genotyped in P. falciparum isolates with high parasitaemia: 775 isolates from four sites in western Kenya (Homa Bay, Kakamega, Kisii, and Kombewa) and 319 isolates from five sites across Ethiopia (Arjo, Awash, Gambella, Dire Dawa, and Semera). DNA sequence variation and neutrality were analysed within each study site where mutant alleles were detected., Results: Sixteen Kelch13 haplotypes were detected in this study. Prevalence of nonsynonymous k13 mutations was low in both western Kenya (25/783, 3.19%) and Ethiopia (5/319, 1.57%) across the study period. Two WHO-validated mutations were detected: A675V in three isolates from Kenya and R622I in four isolates from Ethiopia. Seventeen samples from Kenya carried synonymous mutations (2.17%). No synonymous mutations were detected in Ethiopia. Genetic variation analyses and tests of neutrality further suggest an excess of low frequency polymorphisms in each study site. Fu and Li's F test statistic in Semera was 0.48 (P > 0.05), suggesting potential population selection of R622I, which appeared at a relatively high frequency (3/22, 13.04%)., Conclusions: This study presents an updated report on the low frequency of k13 mutations in western Kenya and Ethiopia. The WHO-validated R622I mutation, which has previously only been reported along the north-west border of Ethiopia, appeared in four isolates collected from eastern Ethiopia. The rapid expansion of R622I across Ethiopia signals the need for enhanced monitoring of the spread of drug-resistant P. falciparum parasites in East Africa. Although ACT remains currently efficacious in the study areas, continued surveillance is necessary to detect early indicators of artemisinin partial resistance., (© 2024. The Author(s).)

Background: Currently, chemotherapy stands out as the major malaria intervention strategy, however, anti-malarial resistance may hamper global elimination programs. Artemisinin-based combination therapy (ACT) stands as the drug of choice for the treatment of Plasmodium falciparum malaria. Plasmodium falciparum kelch13 gene mutations are associated with artemisinin resistance. Thus, this study was aimed at evaluating the circulation of P. falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of ACT deployment., Methods: Participants suspected to have malaria were recruited. Plasmodium falciparum was confirmed using the microscopy method. Malaria-positive patients were treated with artemether-lumefantrine (AL). Blood from participants who tested positive for parasites after day 3 was kept on filter papers. DNA was extracted using chelex-suspension method. A nested polymerase chain reaction (PCR) was conducted and the second-round products were sequenced using the Sanger method. Sequenced products were analysed using DNAsp 5.10.01 software and then blasted on the NCBI for k13 propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST). To assess the selection pressure in P. falciparum parasite population, Tajima' D statistic and Fu & Li's D test in DnaSP software 5.10.01 was used., Results: Out of 275 enrolled participants, 231 completed the follow-up schedule. 13 (5.6%) had parasites on day 28 hence characterized for recrudescence. Out of the 13 samples suspected of recrudescence, 5 (38%) samples were positively amplified as P. falciparum, with polymorphisms in the k13-propeller gene detected. Polymorphisms detected in this study includes R539T, N458T, R561H, N431S and A671V, respectively. The sequences have been deposited in NCBI with bio-project number PRJNA885380 and accession numbers SAMN31087434, SAMN31087433, SAMN31087432, SAMN31087431 and SAMN31087430 respectively., Conclusions: WHO validated polymorphisms in the k13-propeller gene previously reported to be associated with ACT resistance were not detected in the P. falciparum isolates from Kisii County, Kenya. However, some previously reported un-validated k13 resistant single nucleotide polymorphisms were reported in this study but with limited occurrences. The study has also reported new SNPs. More studies need to be carried out in the entire country to understand the association of reported mutations if any, with ACT resistance., (© 2023. The Author(s).)

Background: Dihydroartemisinin-piperaquine (DHA-PPQ) is an alternative first-line antimalarial to artemether-lumefantrine in Kenya. However, recent reports on the emergence of PPQ resistance in Southeast Asia threaten its continued use in Kenya and Africa. In line with the policy on continued deployment of DHA-PPQ, it is imperative to monitor the susceptibility of Kenyan parasites to PPQ and other antimalarials., Methods: Parasite isolates collected between 2008 and 2021 from individuals with naturally acquired P. falciparum infections presenting with uncomplicated malaria were tested for in vitro susceptibility to piperaquine, dihydroartemisinin, lumefantrine, artemether, and chloroquine using the malaria SYBR Green I method. A subset of the 2019-2021 samples was further tested for ex vivo susceptibility to PPQ using piperaquine survival assay (PSA). Each isolate was also characterized for mutations associated with antimalarial resistance in Pfcrt, Pfmdr1, Pfpm2/3, Pfdhfr, and Pfdhps genes using real-time PCR and Agena MassARRAY platform. Associations between phenotype and genotype were also determined., Results: The PPQ median IC 50 interquartile range (IQR) remained stable during the study period, 32.70 nM (IQR 20.2-45.6) in 2008 and 27.30 nM (IQR 6.9-52.8) in 2021 (P=0.1615). The median ex vivo piperaquine survival rate (IQR) was 0% (0-5.27) at 95% CI. Five isolates had a PSA survival rate of ≥10%, consistent with the range of PPQ-resistant parasites, though they lacked polymorphisms in Pfmdr1 and Plasmepsin genes. Lumefantrine and artemether median IC 50 s rose significantly to 62.40 nM (IQR 26.9-100.8) (P = 0.0201); 7.00 nM (IQR 2.4-13.4) (P = 0.0021) in 2021 from 26.30 nM (IQR 5.1-64.3); and 2.70 nM (IQR 1.3-10.4) in 2008, respectively. Conversely, chloroquine median IC 50 s decreased significantly to 10.30 nM (IQR 7.2-20.9) in 2021 from 15.30 nM (IQR 7.6-30.4) in 2008, coinciding with a decline in the prevalence of Pfcrt 76T allele over time (P = 0.0357). The proportions of piperaquine-resistant markers including Pfpm2/3 and Pfmdr1 did not vary significantly. A significant association was observed between PPQ IC 50 and Pfcrt K76T allele (P=0.0026)., Conclusions: Circulating Kenyan parasites have remained sensitive to PPQ and other antimalarials, though the response to artemether (ART) and lumefantrine (LM) is declining. This study forms a baseline for continued surveillance of current antimalarials for timely detection of resistance., (© 2022. The Author(s).)

Background: Rapid Diagnostic Tests (RDTs) have become the cornerstone for the management of malaria in many endemic settings, but their use is constrained for several reasons: (i) persistent malaria antigen (histidine-rich protein 2; HRP2) leading to false positive test results; (ii) hrp2 deletions leading to false negative PfHRP2 results; and (iii) limited sensitivity with a detection threshold of around 100 parasites/μl blood (pLDH- and HRP2-based) leading to false negative tests. Microscopy is still the gold standard for malaria diagnosis, and allows for species determination and quantitation, but requires trained microscopists, maintained microscopes and has detection limit issues. Consequently, there is a pressing need to develop and evaluate more sensitive and accurate diagnostic tests. To address this need we have developed a direct on blood mini PCR-NALFIA test that combines the benefits of molecular biology with low infrastructural requirements and extensive training., Methods: This is a Phase 3 diagnostic evaluation in 5 African countries. Study sites (Sudan, Ethiopia, Burkina, Kenya and Namibia) were selected to ensure wide geographical coverage of Africa and to address various malaria epidemiological contexts ranging from high transmission to near elimination settings with different clinical scenarios and diagnostic challenges. Study participants will be enrolled at the study health facilities after obtaining written informed consent. Diagnostic accuracy will be assessed following the WHO/TDR guidelines for the evaluation of diagnostics and reported according to STARD principles. Due to the lack of a 100% specific and sensitive standard diagnostic test for malaria, the sensitivity and specificity of the new test will be compared to the available diagnostic practices in place at the selected sites and to quantitative PCR as the reference test., Discussion: This phase 3 study is designed to validate the clinical performance and feasibility of implementing a new diagnostic tool for the detection of malaria in real clinical settings. If successful, the proposed technology will improve the diagnosis of malaria. Enrolment started in November 2022 (Kenya) with assessment of long term outcome to be completed by 2023 at all recruitment sites., Trial Registration: Pan African Clinical Trial Registry (www.pactr.org) PACTR202202766889963 on 01/02/2022 and ISCRTN (www.isrctn.com/) ISRCTN13334317 on 22/02/2022., Competing Interests: The authors have declared that no competing interests exist.

Background: High levels of genetic diversity are common characteristics of Plasmodium falciparum parasite populations in high malaria transmission regions. There has been a decline in malaria transmission intensity over 12 years of surveillance in the community in Kilifi, Kenya. This study sought to investigate whether there was a corresponding reduction in P. falciparum genetic diversity, using msp2 as a genetic marker., Methods: Blood samples were obtained from children (< 15 years) enrolled into a cohort with active weekly surveillance between 2007 and 2018 in Kilifi, Kenya. Asymptomatic infections were defined during the annual cross-sectional blood survey and the first-febrile malaria episode was detected during the weekly follow-up. Parasite DNA was extracted and successfully genotyped using allele-specific nested polymerase chain reactions for msp2 and capillary electrophoresis fragment analysis., Results: Based on cross-sectional surveys conducted in 2007-2018, there was a significant reduction in malaria prevalence (16.2-5.5%: P-value < 0.001), however msp2 genetic diversity remained high. A high heterozygosity index (He) (> 0.95) was observed in both asymptomatic infections and febrile malaria over time. About 281 (68.5%) asymptomatic infections were polyclonal (> 2 variants per infection) compared to 46 (56%) polyclonal first-febrile infections. There was significant difference in complexity of infection (COI) between asymptomatic 2.3 [95% confidence interval (CI) 2.2-2.5] and febrile infections 2.0 (95% CI 1.7-2.3) (P = 0.016). Majority of asymptomatic infections (44.2%) carried mixed alleles (i.e., both FC27 and IC/3D7), while FC27 alleles were more frequent (53.3%) among the first-febrile infections., Conclusions: Plasmodium falciparum infections in Kilifi are still highly diverse and polyclonal, despite the reduction in malaria transmission in the community., (© 2022. The Author(s).)

Severe falciparum malaria is a major cause of preventable child mortality in sub-Saharan Africa. Plasma concentrations of P. falciparum Histidine-Rich Protein 2 (PfHRP2) have diagnostic and prognostic value in severe malaria. We investigate the potential use of plasma PfHRP2 and the sequestration index (the ratio of PfHRP2 to parasite density) as quantitative traits for case-only genetic association studies of severe malaria. Data from 2198 Kenyan children diagnosed with severe malaria, genotyped for 14 major candidate genes, show that polymorphisms in four major red cell genes that lead to hemoglobin S, O blood group, α-thalassemia, and the Dantu blood group, are associated with substantially lower admission plasma PfHRP2 concentrations, consistent with protective effects against extensive parasitized erythrocyte sequestration. In contrast the known protective ATP2B4 polymorphism is associated with higher plasma PfHRP2 concentrations, lower parasite densities and a higher sequestration index. We provide testable hypotheses for the mechanism of protection of ATP2B4., (© 2022. The Author(s).)

Molecular surveillance of Plasmodium falciparum parasites is important to track emerging and new mutations and trends in established mutations and should serve as an early warning system for antimalarial resistance. Dried blood spots were obtained from a Plasmodium falciparum malaria survey in school children conducted across eight counties in western Kenya in 2019. Real-time PCR identified 500 P. falciparum-positive samples that were amplified at five drug resistance loci for targeted amplicon deep sequencing (TADS). The absence of important kelch 13 mutations was similar to previous findings in Kenya pre-2019, and low-frequency mutations were observed in codons 569 and 578. The chloroquine resistance transporter gene codons 76 and 145 were wild type, indicating that the parasites were chloroquine and piperaquine sensitive, respectively. The multidrug resistance gene 1 haplotypes based on codons 86, 184, and 199 were predominantly present in mixed infections with haplotypes NYT and NFT, driven by the absence of chloroquine pressure and the use of lumefantrine, respectively. The sulfadoxine-pyrimethamine resistance profile was a "superresistant" combination of triple mutations in both Pfdhfr (51I 59R 108N) and Pfdhps (436H 437G 540E), rendering sulfadoxine-pyrimethamine ineffective. TADS highlighted the low-frequency variants, allowing the early identification of new mutations, Pfmdr1 codon 199S and Pfdhfr codon 85I and emerging 164L mutations. The added value of TADS is its accuracy in identifying mixed-genotype infections and for high-throughput monitoring of antimalarial resistance markers.

Background: Leading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities., Methods: Dry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1-4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparum-infected blood samples., Results: Nine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low nucleotide diversity (0.04×10-2) and low mutation frequencies (1.3% to 7.7%). Chulaimbo had the highest frequency (15.4%) of mutated sites followed by Kisii (6.7%) and Homa Bay (5.1%). Neutrality tests of Pfs25 variations showed significant negative values of Tajima's D (-2.15, p<0.01) and Fu's F (-10.91, p<0.001) statistics tests. Three loci pairs (123, 372), (364, 428) and (390, 394) were detected to be under linkage disequilibrium and none had history of recombination. These results suggested that purifying selection and inbreeding might be the drivers of the observed variation in Pfs25., Conclusion: Given the low level of nucleotide diversity, it is unlikely that a Pfs25 antigen-based vaccine would be affected by antigenic variations. However, continued monitoring of Pfs25 immunogenic domain 3 for possible variants that might impact vaccine antibody binding is warranted., Competing Interests: The authors have declared that no competing interests exist.

Histidine-rich protein 2 (HRP2)-based rapid diagnostic tests detect Plasmodium falciparum malaria and are used throughout sub-Saharan Africa. However, deletions in the pfhrp2 and related pfhrp3 (pfhrp2/3) genes threaten use of these tests. Therapeutic efficacy studies (TESs) enroll persons with symptomatic P. falciparum infection. We screened TES samples collected during 2016-2018 in Ethiopia, Kenya, Rwanda, and Madagascar for HRP2/3, pan-Plasmodium lactate dehydrogenase, and pan-Plasmodium aldolase antigen levels and selected samples with low levels of HRP2/3 for pfhrp2/3 genotyping. We observed deletion of pfhrp3 in samples from all countries except Kenya. Single-gene deletions in pfhrp2 were observed in 1.4% (95% CI 0.2%-4.8%) of Ethiopia samples and in 0.6% (95% CI 0.2%-1.6%) of Madagascar samples, and dual pfhrp2/3 deletions were noted in 2.0% (95% CI 0.4%-5.9%) of Ethiopia samples. Although this study was not powered for precise prevalence estimates, evaluating TES samples revealed a low prevalence of pfhrp2/3 deletions in most sites.

Background: In Plasmodium falciparum the monoallelic expression of var virulence genes is regulated through epigenetic mechanisms. A study in the Gambia showed that an increase in var genes commonly expressed in patients with severe malaria is associated with fever and high blood lactate. A strong association was demonstrated between the upregulation of PfSir2A and group B var genes. A subsequent study in Kenya extended this association to show a link between elevated expression of PfSir2A and overall var transcript levels. We investigate here the link between heat shock and/or lactate levels on sirtuin and var gene expression levels in vitro., Methods: In vitro experiments were conducted using laboratory and recently-laboratory-adapted Kenyan isolates of P. falciparum. To investigate a potential cause-and-effect relationship between host stress factors and parasite gene expression, qPCR was used to measure the expression of sirtuins and var genes after highly synchronous cultured parasites had been exposed to 2 h or 6 h of heat shock at 40 °C or elevated lactate., Results: Heat shock was shown to increase the expression ofPfSir2B in the trophozoites, whereas exposure to lactate was not. After the ring stages were exposed to heat shock and lactate, there was no alteration in the expression of sirtuins and severe-disease-associated upsA and upsB var genes. The association between high blood lactate and sirtuin/var gene expression that was previously observed in vivo appears to be coincidental rather than causative., Conclusions: This study demonstrates that heat stress in a laboratory and recently-laboratory-adapted isolates of P. falciparum results in a small increase in PfSir2B transcripts in the trophozoite stages only. This finding adds to our understanding of how patient factors can influence the outcome of Plasmodium falciparum infections., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Plasmodium falciparum parasites have evolved genetic adaptations to overcome immune responses mounted by diverse Anopheles vectors hindering malaria control efforts. Plasmodium falciparum surface protein Pfs47 is critical in the parasite's survival by manipulating the vector's immune system hence a promising target for blocking transmission in the mosquito. This study aimed to examine the genetic diversity, haplotype distribution, and population structure of Pfs47 and its implications on malaria infections in endemic lowlands in Western Kenya. Cross-sectional mass blood screening was conducted in malaria endemic regions in the lowlands of Western Kenya: Homa Bay, Kombewa, and Chulaimbo. Dried blood spots and slide smears were simultaneously collected in 2018 and 2019. DNA was extracted using Chelex method from microscopic Plasmodium falciparum positive samples and used to genotype Pfs47 using polymerase chain reaction (PCR) and DNA sequencing. Thirteen observed haplotypes of the Pfs47 gene were circulating in Western Kenya. Population-wise, haplotype diversity ranged from 0.69 to 0.77 and the nucleotide diversity 0.10 to 0.12 across all sites. All the study sites displayed negative Tajima's D values although not significant. However, the negative and significant Fu's Fs statistical values were observed across all the study sites, suggesting population expansion or positive selection. Overall genetic differentiation index was not significant (FST = -0.00891, P > 0.05) among parasite populations. All Nm values revealed a considerable gene flow in these populations. These results could have important implications for the persistence of high levels of malaria transmission and should be considered when designing potential targeted control interventions., Competing Interests: All authors declare that they have no conflict of interest.

The spread of drug resistance to antimalarial treatments poses a serious public health risk globally. To combat this risk, molecular surveillance of drug resistance is imperative. We report the prevalence of mutations in the Plasmodium falciparum kelch 13 propeller domain associated with partial artemisinin resistance, which we determined by using Sanger sequencing samples from patients enrolled in therapeutic efficacy studies from 9 sub-Saharan countries during 2014-2018. Of the 2,865 samples successfully sequenced before treatment (day of enrollment) and on the day of treatment failure, 29 (1.0%) samples contained 11 unique nonsynonymous mutations and 83 (2.9%) samples contained 27 unique synonymous mutations. Two samples from Kenya contained the S522C mutation, which has been associated with delayed parasite clearance; however, no samples contained validated or candidate artemisinin-resistance mutations.

Cryptosporidium felis is the major etiologic agent of cryptosporidiosis in felines and has been reported in numerous human cryptosporidiosis cases. Sequence analysis of the 60-kDa glycoprotein ( gp60 ) gene has been developed for subtyping C. felis recently. In this study, 66 C. felis isolates from the United States, Jamaica, Peru, Portugal, Slovakia, Nigeria, Ethiopia, Kenya, China, India and Australia were subtyped using the newly established tool. Forty-four specimens yielded gp60 sequences, generating 23 subtypes clustered in 4 subtype families (XIXa, XIXc, XIXd and XIXe) with high bootstrap support in a phylogenetic analysis of sequence data. Among them, XIXa showed high genetic diversity at the nucleotide level, with the formation of 18 subtypes from both cats and humans with different geographic distribution. In contrast, all 11 XIXd isolates derived from humans from various countries had identical sequences. Results of this study improve our understanding of the genetic diversity, host specificity and transmission dynamics of C. felis .

Background: Sulfadoxine-pyrimethamine (SP) is the only anti-malarial drug formulation approved for intermittent preventive treatment in pregnancy (IPTp). However, mutations in the Plasmodium falciparum dhfr (Pfdhfr) and dhps (Pfdhps) genes confer resistance to pyrimethamine and sulfadoxine, respectively. Here, the frequencies of SP resistance-associated mutations from 2005 to 2018 were compared in samples from Kenyan children with malaria residing in a holoendemic transmission region., Methods: Partial sequences of the Pfdhfr and Pfdhps genes were amplified and sequenced from samples collected in 2005 (n = 81), 2010 (n = 95), 2017 (n = 43), and 2018 (n = 55). The frequency of known mutations conferring resistance to pyrimethamine and sulfadoxine were estimated and compared. Since artemisinin-based combination therapy (ACT) is the current first-line treatment for malaria, the presence of mutations in the propeller domain of P. falciparum kelch13 gene (Pfk13) linked to ACT-delayed parasite clearance was studied in the 2017/18 samples., Results: Among other changes, the point mutation of Pfdhps S436H increased in frequency from undetectable in 2005 to 28% in 2017/18. Triple Pfdhfr mutant allele (CIRNI) increased in frequency from 84% in 2005 to 95% in 2017/18, while the frequency of Pfdhfr double mutant alleles declined (allele CICNI from 29% in 2005 to 6% in 2017/18, and CNRNI from 9% in 2005 to undetectable in 2010 and 2017/18). Thus, a multilocus Pfdhfr/Pfdhps genotype with six mutations (HGEAA/CIRNI), including Pfdhps S436H, increased in frequency from 2010 to 2017/18. Although none of the mutations associated with ACT-delayed parasite clearance was observed, the Pfk13 mutation A578S, the most widespread Pfk13 SNP found in Africa, was detected in low frequency (2.04%)., Conclusions: There were changes in SP resistance mutant allele frequencies, including an increase in the Pfdhps S436H. Although these patterns seem consistent with directional selection due to drug pressure, there is a lack of information to determine the actual cause of such changes. These results suggest incorporating molecular surveillance of Pfdhfr/Pfdhps mutations in the context of SP efficacy studies for intermittent preventive treatment in pregnancy (IPTp).

Background: Prevention and treatment of malaria during pregnancy is crucial in dealing with maternal mortality and adverse fetal outcomes. The World Health Organization recommendation to treat all pregnant women with sulfadoxine-pyrimethamine (SP) through antenatal care structures was implemented in Kenya in the year 1998, but concerns about its effectiveness in preventing malaria in pregnancy has arisen due to the spread of SP resistant parasites. This study aimed to determine the prevalence of SP resistance markers in Plasmodium falciparum parasites isolated from pregnant women seeking antenatal care at Msambweni County Referral Hospital, located in coastal Kenya, between the year 2013 and 2015., Methods: This hospital-based study included 106 malaria positive whole blood samples for analysis of SP resistance markers within the Pfdhfr gene (codons 51, 59 and 108) and Pfdhps gene (codons 437 and 540). The venous blood collected from all pregnant women was tested for malaria via light microscopy, then the malaria positive samples were separated into plasma and red cells and stored in a - 86° freezer for further studies. Archived red blood cells were processed for molecular characterization of SP resistance markers within the Pfdhfr and Pfdhps genes using real time PCR platform and Sanger sequencing., Results: All samples had at least one mutation in the genes associated with drug resistance; polymorphism prevalence of Pfdhfr51I, 59R and 108N was at 88.7%, 78.3% and 93.4%, respectively, while Pfdhps polymorphism accounted for 94.3% and 91.5% at 437G and 540E, respectively. Quintuple mutations (at all the five codons) conferring total SP resistance had the highest prevalence of 85.8%. Quadruple mutations were observed at a frequency of 10.4%, and 24.5% had a mixed outcome of both wildtype and mutant genotypes in the genes of interest., Conclusion: The data suggest a high prevalence of P. falciparum genetic variations conferring resistance to SP among pregnant women, which may explain reduced efficacy of IPTp treatment in Kenya. There is need for extensive SP resistance profiling in Kenya to inform IPTp drug choices for successful malaria prevention during pregnancy.

Background: Many health facilities in malaria endemic countries are dependent on Rapid diagnostic tests (RDTs) for diagnosis and some National Health Service (NHS) hospitals without expert microscopists rely on them for diagnosis out of hours. The emergence of P. falciparum lacking the gene encoding histidine-rich protein 2 and 3 (HRP2 and HRP3) and escaping RDT detection threatens progress in malaria control and elimination. Currently, confirmation of RDT negative due to the deletion of pfhrp2 and pfhrp3, which encodes a cross-reactive protein isoform, requires a series of PCR assays. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of the deletions with certainty., Methods: We developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers., Results: The qPCR assay demonstrated diagnostic sensitivity of 100% (n = 19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n = 31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 deletions. In addition, the assay estimates P. falciparum parasite density and accurately detects pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers., Interpretation: The new qPCR is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control., Competing Interests: Declaration of Competing Interest JBP reports non-financial support in the form of in-kind donation of laboratory testing and reagents from Abbott Laboratories for studies of viral hepatitis and financial support from the World Health Organization. Other authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Resistance to the mainstay antimalarial drugs is a major concern in the control of malaria. Delayed Plasmodium falciparum parasite clearance has been associated with Single Nucleotide Polymorphisms (SNPs) in the kelch propeller region (K13). However, SNPs in the Pf-adaptor protein complex 2 mu subunit (Pfap2-mu), Pfcrt and Pfmdr1 are possible markers associated with multi-drug resistance. Here, we explored the prevalence of SNPs in the K13, Pfap2-mu, Pfcrt, and Pfmdr1 in 94 dried blood spot field isolates collected from children aged below 12 years infected with P. falciparum during a cross-sectional study. The samples were collected in 2015 during the peak malaria transmission season in the Nyando region of Western Kenya before treatment with Artemether-Lumefantrine, the first-line artemisinin-based combination therapy (ACT) in Kenya. However, 47 of the 94 samples had recurrent parasitemia and were interrogated for the presence of the SNPs in K13 and Pfap2-mu. We used PCR amplification and sequencing to evaluate specific regions of K13 (codons 432-702), Pfap2-mu (codons 1-350), Pfmdr1 (codons 86, 1034-1246), and Pfcrt (codons 72-76) gene(s). The majority of parasites harbored the wild type K13 sequence. However, we found a unique non-synonymous W611S change. In silico studies on the impact of the W611S predicted structural changes in the overall topology of the K13 protein. Of the 47 samples analyzed for SNPs in the Pfap2-mu gene, 14 (29%) had S160 N/T mutation. The CVIET haplotype associated with CQ resistance in the Pfcrt yielded a 7.44% (7/94), while CVMNK haplotype was at 92.56%. Mutations in the Pfmdr1 region were detected only in three samples (3/94; 3.19%) at codon D1246Y. Our data suggest that parasites in the western part of Kenya harbor the wildtype strains. However, the detection of the unique SNP in K13 and Pfap2-mu linked with ACT delayed parasite clearance may suggest slow filtering of ACT-resistant parasites., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Community-based active case detection of malaria parasites with conventional rapid diagnostic tests (cRDTs) is a strategy used most commonly in low-transmission settings. We estimated the sensitivity of this approach in a high-transmission setting in Western Kenya. We tested 3,547 members of 912 households identified in 2013-2014 by index children with (case) and without (control) cRDT-positive malaria. All were tested for Plasmodium falciparum with both a cRDT targeting histidine-rich protein 2 and with an ultrasensitive real-time polymerase chain reaction (PCR). We computed cRDT sensitivity against PCR as the referent, compared prevalence between participant types, and estimated cRDT detectability as a function of PCR-estimated parasite density. Parasite prevalence was 22.9% by cRDTs and 61.5% by PCR. Compared with children aged < 5 years or adults aged > 15 years, geometric mean parasite densities (95% CI) were highest in school-age children aged 5-15 years (8.4 p/uL; 6.6-10.6). The overall sensitivity of cRDT was 36%; among asymptomatic household members, cRDT sensitivity was 25.5% and lowest in adults aged > 15 years (15.8%). When modeled as a function of parasite density, relative to school-age children, the probability of cRDT positivity was reduced in both children aged < 5 years (odds ratio [OR] 0.48; 95% CI: 0.34-0.69) and in adults aged > 15 years (OR: 0.35; 95% CI: 0.27-0.47). An HRP2-detecting cRDT had poor sensitivity for active P. falciparum case detection in asymptomatic community members, and sensitivity was lowest in highly prevalent low-density infections and in adults. Future studies can model the incremental effects of high-sensitivity rapid diagnostic tests and the impacts on transmission.

Theileria parva is a tick-transmitted, apicomplexan protozoan found in buffalo (Syncerus caffer) and cattle in eastern, central and southern Africa. The parasite causes a fatal, lymphoproliferative disease in susceptible cattle. Previous studies have shown that the parasites in buffalo comprise a more heterogeneous population than those in cattle, which has led to the concept that the population of parasites circulating in cattle represents a restricted subpopulation of those in buffalo. The present study was undertaken to identify if and where this restriction may occur in cattle naturally infected with parasites from buffalo, by sequencing the T. parva p67 antigen gene from eight buffalo and 12 acutely infected cattle from the same endemic site in Kenya. From 103 sequences, we detected 44 different alleles. Nine alleles were found in both cattle and buffalo, and 17 and 18 found only in the cattle and buffalo populations respectively. Nucleotide and amino acid sequence analyses revealed a similar level of diversity of parasites in both hosts. Principal coordinates and phylogenetic tree analyses did not reveal any clustering associated with the host animals, and the number and degree of mixed T. parva infections was similar in the respective populations. The results suggest that any restriction in the ability of T. parva from buffalo to survive and be transmitted from cattle occurs after entry into and initial transformation of bovine lymphocytes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objective: Cotrimoxazole prevents opportunistic infections including falciparum malaria in HIV-infected individuals but there are concerns of cross-resistance to other antifolate drugs such as sulphadoxine-pyrimethamine (SP). In this study, we investigated the prevalence of antifolate-resistance mutations in Plasmodium falciparum that are associated with SP resistance in HIV-infected individuals on antiretroviral treatment randomized to discontinue (STOP-CTX), or continue (CTX) cotrimoxazole in Western Kenya., Design: Samples were obtained from an unblinded, non-inferiority randomized controlled trial where participants were recruited on a rolling basis for the first six months of the study, then followed-up for 12 months with samples collected at enrollment, quarterly, and during sick visits., Method: Plasmodium DNA was extracted from blood specimens. Initial screening to determine the presence of Plasmodium spp. was performed by quantitative reverse transcriptase real-time PCR, followed by genotyping for the presence of SP-resistance associated mutations by Sanger sequencing., Results: The prevalence of mutant haplotypes associated with SP-resistant parasites in pfdhfr (51I/59R/108N) and pfdhps (437G/540E) genes were significantly higher (P = 0.0006 and P = 0.027, respectively) in STOP-CTX compared to CTX arm. The prevalence of quintuple haplotype (51I/59R/108N/437G/540E) was 51.8% in STOP-CTX vs. 6.3% (P = 0.0007) in CTX arm. There was a steady increase in mutant haplotypes in both genes in STOP-CTX arm overtime through the study period, reaching statistical significance (P < 0.0001)., Conclusion: The frequencies of mutations in pfdhfr and pfdhps genes were higher in STOP-CTX arm compared to CTX arm, suggesting cotrimoxazole effectively controls and selects against SP-resistant parasites., Trial Registration: ClinicalTrials.gov NCT01425073., Competing Interests: The authors have declared that no competing interests exist.

Plasmodium falciparum histidine-rich proteins 2 (PfHRP2) based RDTs are advocated in falciparum malaria-endemic regions, particularly when quality microscopy is not available. However, diversity and any deletion in the pfhrp2 and pfhrp3 genes can affect the performance of PfHRP2-based RDTs. A total of 400 samples collected from uncomplicated malaria cases from Kenya were investigated for the amino acid repeat profiles in exon 2 of pfhrp2 and pfhrp3 genes. In addition, PfHRP2 levels were measured in 96 individuals with uncomplicated malaria. We observed a unique distribution pattern of amino acid repeats both in the PfHRP2 and PfHRP3. 228 PfHRP2 and 124 PfHRP3 different amino acid sequences were identified. Of this, 214 (94%) PfHRP2 and 81 (65%) PfHRP3 amino acid sequences occurred only once. Thirty-nine new PfHRP2 and 20 new PfHRP3 amino acid repeat types were identified. PfHRP2 levels were not correlated with parasitemia or the number of PfHRP2 repeat types. This study shows the variability of PfHRP2, PfHRP3 and PfHRP2 concentration among uncomplicated malaria cases. These findings will be useful to understand the performance of PfHRP2-based RDTs in Kenya.

The development of artemisinin (ART)-resistant parasites in Southeast Asia (SEA) threatens malaria control globally. Mutations in the Kelch 13 (K13)-propeller domain have been useful in identifying ART resistance in SEA. ART combination therapy (ACT) remains highly efficacious in the treatment of uncomplicated malaria in Sub-Saharan Africa (SSA). However, it is crucial that the efficacy of ACT is closely monitored. Toward this effort, this study profiled the prevalence of K13 nonsynonymous mutations in different malaria ecological zones of Kenya and in different time periods, before (pre) and after (post) the introduction of ACT as the first-line treatment of malaria. Nineteen nonsynonymous mutations were present in the pre-ACT samples ( N = 64) compared with 22 in the post-ACT samples ( N = 251). Eight of these mutations were present in both pre- and post-ACT parasites. Interestingly, seven of the shared single-nucleotide polymorphisms were at higher frequencies in the pre-ACT than the post-ACT parasites. The A578S mutation reported in SSA and the V568G mutation reported in SEA were found in both pre- and post-ACT parasites, with their frequencies declining post-ACT. D584Y and R539K mutations were found only in post-ACT parasites; changes in these codons have also been reported in SEA with different amino acids. The N585K mutation described for the first time in this study was present only in post-ACT parasites, and it was the most prevalent mutation at a frequency of 5.2%. This study showed the type, prevalence, and frequency of K13 mutations that varied based on the malaria ecological zones and also between the pre- and post-ACT time periods.

Deletions of the Plasmodium falciparum hrp2 and hrp3 genes can affect the performance of HRP2-based malaria rapid diagnostic tests (RDTs). Such deletions have been reported from South America, India and Eritrea. Whether these parasites are widespread in East Africa is unknown. A total of 274 samples from asymptomatic children in Mbita, western Kenya, and 61 genomic  data from Kilifi, eastern Kenya, were available for analysis. PCR-confirmed samples were investigated for the presence of pfhrp2 and pfhrp3 genes. In samples with evidence of deletion, parasite presence was confirmed by amplifying three independent genes. We failed to amplify pfhrp2 from 25 of 131 (19.1%) PCR-confirmed samples. Of these, only 8 (10%) samples were microscopic positive and were classified as pfhrp2-deleted. Eight microscopically-confirmed pfhrp2-deleted samples with intact pfhrp3 locus were positive by HRP2-based RDT. In addition, one PCR-confirmed infection showed a deletion at the pfhrp3 locus. One genomic sample lacked pfhrp2 and one lacked pfhrp3. No sample harbored parasites lacking both genes. Parasites lacking pfhrp2 are present in Kenya, but may be detectable by HRP-based RDT at higher parasitaemia, possibly due to the presence of intact pfhrp3. These findings warrant further systematic study to establish prevalence and diagnostic significance.

Background: Single low-dose primaquine (PQ) reduces Plasmodium falciparum infectivity before it impacts gametocyte density. Here, we examined the effect of PQ on gametocyte sex ratio as a possible explanation for this early sterilizing effect., Methods: Quantitative reverse-transcription polymerase chain reaction assays were developed to quantify female gametocytes (targeting Pfs25 messenger RNA [mRNA]) and male gametocytes (targeting Pf3D7&#95;1469900 mRNA) in 2 randomized trials in Kenya and Mali, comparing dihydroartemisinin-piperaquine (DP) alone to DP with PQ. Gametocyte sex ratio was examined in relation to time since treatment and infectivity to mosquitoes., Results: In Kenya, the median proportion of male gametocytes was 0.33 at baseline. Seven days after treatment, gametocyte density was significantly reduced in the DP-PQ arm relative to the DP arm (females: 0.05% [interquartile range {IQR}, 0.0-0.7%] of baseline; males: 3.4% [IQR, 0.4%-32.9%] of baseline; P < .001). Twenty-four hours after treatment, gametocyte sex ratio became male-biased and was not significantly different between the DP and DP-PQ groups. In Mali, there was no significant difference in sex ratio between the DP and DP-PQ groups (>0.125 mg/kg) 48 hours after treatment, and gametocyte sex ratio was not associated with mosquito infection rates., Conclusions: The early sterilizing effects of PQ may not be explained by the preferential clearance of male gametocytes and may be due to an effect on gametocyte fitness., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America.)

Background: The Plasmodium falciparum multidrug resistance 1 (PfMDR1), P. falciparum Ca 2+ -ATPase (PfATP6) and Kelch-13 propeller domain (PfK13) loci are molecular markers of parasite susceptibility to anti-malarial drugs. Their frequency distributions were determined in the isolates collected from children with severe malaria originating from three African countries., Methods: Samples from 287 children with severe malaria [(Gabon: n = 114); (Ghana: n = 89); (Kenya: n = 84)] were genotyped for pfmdr1, pfatp6 and pfk13 loci by DNA sequencing and assessing pfmdr1 copy number variation (CNV) by real-time PCR., Results: Pfmdr1-N86Y mutation was detected in 48, 10 and 10% in Lambaréné, Kumasi and Kisumu, respectively. At codon 184, the prevalence of the mutation was 73% in Lambaréné, 63% in Kumasi and 49% Kisumu. The S1034C and N1042D variants were absent at all three sites, while the frequency of the D1246Y mutation was 1, 3 and 13% in Lambaréné, Kumasi and Kisumu, respectively. Isolates with two pfmdr1 gene copy number predominantly harboured the N86Y wild-type allele and were mostly found in Kumasi (10%) (P < 0.0001). Among the main pfmdr1 haplotypes (NFD, NYD and YFD), NYD was associated with highest parasitaemia (P = 0.04). At the pfatp6 locus, H243Y and A623E mutations were observed at very low frequency at all three sites. The prevalence of the pfatp6 E431K variant was 6, 18 and 17% in Lambaréné, Kumasi and Kisumu, respectively. The L263E and S769N mutations were absent in all isolates. The pfk13 variants associated with artemisinin resistance in Southeast Asia were not observed. Eleven novel substitutions in the pfk13 locus occurring at low frequency were observed., Conclusions: Artemisinins are still highly efficacious in large malaria-endemic regions though declining efficacy has occurred in Southeast Asia. The return of chloroquine-sensitive strains following the removal of drug pressure is observed. However, selection of wild-type alleles in the multidrug-resistance gene and the increased gene copy number is associated with reduced lumefantrine sensitivity. This study indicates a need to constantly monitor drug resistance to artemisinin in field isolates from malaria-endemic countries.

Background: Anti-malarial drugs are the major focus in the prevention and treatment of malaria. Artemisinin-based combination therapy (ACT) is the WHO recommended first-line treatment for Plasmodium falciparum malaria across the endemic world. Also ACT is increasingly relied upon in treating Plasmodium vivax malaria where chloroquine is failing. The emergence of artemisinin drug-resistant parasites is a serious threat faced by global malaria control programmes. Therefore, the success of treatment and intervention strategies is highly pegged on understanding the genetic basis of resistance., Methods: Here, resistance in P. falciparum was generated in vitro for artemisinin to produce levels above clinically relevant concentrations in vivo, and the molecular haplotypes investigated. Genomic DNA was extracted using the QIAamp mini DNA kit. DNA sequences of Pfk13, Pfcrt and Pfmdr1 genes were amplified by PCR and the amplicons were successfully sequenced. Single nucleotide polymorphisms were traced by standard bidirectional sequencing and reading the transcripts against wild-type sequences in Codon code Aligner Version 5.1 and NCBI blast., Results: Exposure of parasite strains D6 and W2 to artemisinin resulted in a decrease in parasite susceptibility to artemisinin (W2 and D6) and lumefantrine (D6 only). The parasites exhibited elevated IC50s to multiple artemisinins, with >twofold resistance to artemisinin; however, the resistance index obtained with standard methods was noticeably less than expected for parasite lines recovered from 50 µg/ml 48 h drug pressure. The change in parasite susceptibility was associated with Pfmdr-185K mutation, a mutation never reported before. The Pfcrt-CVMNK genotype (Pfcrt codons 72-76) was retained and notably, the study did not detect any polymorphisms reported to reduce P. falciparum susceptibility in vivo in the coding sequences of the Pfk13 gene., Discussion: This data demonstrate that P. falciparum has the capacity to develop resistance to artemisinin derivatives in vitro and that this phenotype is achieved by mutations in Pfmdr1, the genetic changes that are also underpinning lumefantrine resistance. This finding is of practical importance, because artemisinin drugs in Kenya are used in combination with lumefantrine for the treatment of malaria., Conclusion: Artemisinin resistance phenotype as has been shown in this work, is a decrease in parasites susceptibility to artemisinin derivatives together with the parasite's ability to recover from drug-induced dormancy after exposure to drug dosage above the in vivo clinical concentrations. The study surmises that Pfmdr1 may play a role in the anti-malarial activity of artemisinin.

Introduction: Globally Cryptosporidium and Giardia species are the most common non-bacterial causes of diarrhoea in children and HIV infected individuals, yet data on their role in paediatric diarrhoea in Kenya remains scant. This study investigated the occurrence of Cryptosporidium species, genotypes and subtypes in children, both hospitalized and living in an informal settlement in Nairobi., Methods: This was a prospective cross-sectional study in which faecal specimen positive for Cryptosporidium spp. by microscopy from HIV infected and uninfected children aged five years and below presenting with diarrhoea at selected outpatient clinics in Mukuru informal settlements, or admitted to the paediatric ward at the Mbagathi District Hospital were characterized. The analysis was done by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 18srRNA gene for species identification and PCR-sequencing of the 60 kDa glycoprotein (GP60) gene for subtyping., Results: C. hominis was the most common species of Cryptosporidium identified in125/151(82.8%) of the children. Other species identified were C. parvum 18/151(11.9%), while C. felis and C. meleagridis were identified in 4 and 2 children, respectively. Wide genetic variation was observed within C. hominis, with identification of 5 subtype families; Ia, Ib, Id, Ie and If and 21 subtypes. Only subtype family IIc was identified within C. parvum. There was no association between species and HIV status or patient type., Conclusion: C. hominis is the most common species associated with diarrhoea in the study population. There was high genetic variability in the C. hominis isolates with 22 different subtypes identified, whereas genetic diversity was low within C. parvum with only one subtype family IIc identified.

Background: Immunoglobulin G antibodies (Abs) to Plasmodium falciparum antigens have been associated with naturally acquired immunity to symptomatic malaria., Methods: We probed protein microarrays covering 824 unique P. falciparum protein features with plasma from residents of a community in Kenya monitored for 12 weeks for (re)infection and symptomatic malaria after administration of antimalarial drugs. P. falciparum proteins recognized by Abs from 88 children (aged 1-14 years) and 86 adults (aged ≥ 18 years), measured at the beginning of the observation period, were ranked by Ab signal intensity., Results: Abs from immune adults reacted with a total 163 of 824 P. falciparum proteins. Children gradually acquired Abs to the full repertoire of antigens recognized by adults. Abs to some antigens showed high seroconversion rates, reaching maximal levels early in childhood, whereas others did not reach adult levels until adolescence. No correlation between Ab signal intensity and time to (re)infection was observed. In contrast, Ab levels to 106 antigens were significantly higher in children who were protected from symptomatic malaria compared with those who were not. Abs to antigens predictive of protection included P. falciparum erythrocyte membrane protein 1, merozoite surface protein (MSP) 10, MSP2, liver-stage antigen 3, PF70, MSP7, and Plasmodium helical interspersed subtelomeric domain protein., Conclusions: Protein microarrays may be useful in the search for malaria antigens associated with protective immunity., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Plasmodium falciparum resistance to chloroquine (CQ) denied healthcare providers access to a cheap and effective anti-malarial drug. Resistance has been proven to be due to point mutations on the parasite's pfcrt gene, particularly on codon 76, resulting in an amino acid change from lysine to threonine. This study sought to determine the prevalence of the pfcrt K76T mutation 13 years after CQ cessation in Msambweni, Kenya., Methods: Finger-prick whole blood was collected on 3MM Whatman(®) filter paper from 99 falciparum malaria patients. Parasite DNA was extracted via the Chelex method from individual blood spots and used as template in nested PCR amplification of pfcrt. Apo1 restriction enzyme was used to digest the amplified DNA to identify the samples as wild type or sensitive at codon 76. Prevalence figures of the mutant pfcrt 76T gene were calculated by dividing the number of samples bearing the mutant gene with the total number of samples multiplied by 100 %. Chi square tests were used to test the significance of the findings against previous prevalence figures., Results: Out of 99 clinical samples collected in 2013, prevalence of the mutant pfcrt 76T gene stood at 41 %., Conclusion: The results indicate a significant [χ(2) test, P ≤ 0.05 (2006 vs 2013)] reversal to sensitivity by the P. falciparum population in the study site compared to the situation reported in 2006 at the same study site. This could primarily be driven by diminished use of CQ in the study area in line with the official policy. Studies to establish prevalence of the pfcrt 76T gene could be expanded countrywide to establish the CQ sensitivity status and predict a date when CQ may be re-introduced as part of malaria chemotherapy.

The prevalence of a genetic polymorphism(s) at codon 268 in the cytochrome b gene, which is associated with failure of atovaquone-proguanil treatment, was analyzed in 227 Plasmodium falciparum parasites from western Kenya. The prevalence of the wild-type allele was 63%, and that of the Y268S (denoting a Y-to-S change at position 268) mutant allele was 2%. There were no pure Y268C or Y268N mutant alleles, only mixtures of a mutant allele(s) with the wild type. There was a correlation between parasite 50% inhibitory concentration (IC50) and parasite genetic polymorphism; mutant alleles had higher IC50s than the wild type., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

We looked for mutations in the Plasmodium falciparum K13 propeller gene of an artemisinin-resistant parasite on islands in Lake Victoria, Kenya, where transmission in 2012-2013 was high. The 4 new types of nonsynonymous, and 5 of synonymous, mutations we detected among 539 samples analyzed provide clues to understanding artemisinin-resistant parasites.

The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem)., (Copyright © 2015, Ochola-Oyier et al.)

Background: The efficacy of artemisinin-based combination therapy (ACT) for Plasmodium falciparum malaria may be threatened by parasites with reduced responsiveness to artemisinins. Among 298 ACT-treated children from Mbita, Kenya, submicroscopic persistence of P. falciparum on day 3 posttreatment was associated with subsequent microscopically detected parasitemia at days 28 or 42., Methods: DNA sequences of resistance-associated parasite loci pfcrt, pfmdr1, pfubp1, and pfap2mu were determined in the Mbita cohort before treatment, on days 2 and 3 after initiation of treatment, and on the day of treatment failure., Results: Parasites surviving ACT on day 2 or day 3 posttreatment were significantly more likely than the baseline population to carry the wild-type haplotypes of pfcrt (CVMNK at codons 72-76; P < .001) and pfmdr1 (NFD at codons 86, 184, 1246; P < .001). In contrast, variant alleles of the novel candidate resistance genes pfap2mu (S160N/T; P = .006) and pfubp-1 (E1528D; P < .001) were significantly more prevalent posttreatment. No genetic similarities were found to artemisinin-tolerant parasites recently described in Cambodia., Conclusions: Among treated children in western Kenya, certain P. falciparum genotypes defined at pfcrt, pfmdr1, pfap2mu, and pfubp1 more often survive ACT at the submicroscopic level, and contribute to onward transmission and subsequent patent recrudescence., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Doxycycline is widely used for malaria prophylaxis by international travelers. However, there is limited information on doxycycline efficacy in Kenya, and genetic polymorphisms associated with reduced efficacy are not well defined. In vitro doxycycline susceptibility profiles for 96 Plasmodium falciparum field isolates from Kenya were determined. Genetic polymorphisms were assessed in P. falciparum metabolite drug transporter (Pfmdt) and P. falciparum GTPase tetQ (PftetQ) genes. Copy number variation of the gene and the number of KYNNNN amino acid motif repeats within the protein encoded by PftetQ were determined. Reduced in vitro susceptibility to doxycycline was defined by 50% inhibitory concentrations (IC50s) of ≥35,000 nM. The odds ratio (OR) of having 2 PfTetQ KYNNNN amino acid repeats in isolates with IC50s of >35,000 nM relative to those with IC50s of <35,000 nM is 15 (95% confidence interval [CI], 3.0 to 74.3; P value of <0.0002). Isolates with 1 copy of the Pfmdt gene had a median IC50 of 6,971 nM, whereas those with a Pfmdt copy number of >1 had a median IC50 of 9,912 nM (P = 0.0245). Isolates with 1 copy of PftetQ had a median IC50 of 6,370 nM, whereas isolates with a PftetQ copy number of >1 had a median IC50 of 3,422 nM (P < 0.0007). Isolates with 2 PfTetQ KYNNNN motif repeats had a median IC50 of 26,165 nM, whereas isolates with 3 PfTetQ KYNNNN repeats had a median IC50 of 3,352 nM (P = 0.0023). PfTetQ sequence polymorphism is associated with a reduced doxycycline susceptibility phenotype in Kenyan isolates and is a potential marker for susceptibility testing., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: The primary target of the human immune response to the malaria parasite Plasmodium falciparum, P. falciparum erythrocyte membrane protein 1 (PfEMP1), is encoded by the members of the hyper-diverse var gene family. The parasite exhibits antigenic variation via mutually exclusive expression (switching) of the ~60 var genes within its genome. It is thought that different variants exhibit different host endothelial binding preferences that in turn result in different manifestations of disease., Results: Var sequences comprise ancient sequence fragments, termed homology blocks (HBs), that recombine at exceedingly high rates. We use HBs to define distinct var types within a local population. We then reanalyze a dataset that contains clinical and var expression data to investigate whether the HBs allow for a description of sequence diversity corresponding to biological function, such that it improves our ability to predict disease phenotype from parasite genetics. We find that even a generic set of HBs, which are defined for a small number of non-local parasites: capture the majority of local sequence diversity; improve our ability to predict disease severity from parasite genetics; and reveal a previously hypothesized yet previously unobserved parasite genetic basis for two forms of severe disease. We find that the expression rates of some HBs correlate more strongly with severe disease phenotypes than the expression rates of classic var DBLα tag types, and principal components of HB expression rate profiles further improve genotype-phenotype models. More specifically, within the large Kenyan dataset that is the focus of this study, we observe that HB expression differs significantly for severe versus mild disease, and for rosetting versus impaired consciousness associated severe disease. The analysis of a second much smaller dataset from Mali suggests that these HB-phenotype associations are consistent across geographically distant populations, since we find evidence suggesting that the same HB-phenotype associations characterize this population as well., Conclusions: The distinction between rosetting versus impaired consciousness associated var genes has not been described previously, and it could have important implications for monitoring, intervention and diagnosis. Moreover, our results have the potential to illuminate the molecular mechanisms underlying the complex spectrum of severe disease phenotypes associated with malaria--an important objective given that only about 1% of P. falciparum infections result in severe disease.

Background: Informing and evaluating malaria control efforts relies on knowledge of local transmission dynamics. Serological and molecular tools have demonstrated great sensitivity to quantify transmission intensity in low endemic settings where the sensitivity of traditional methods is limited. Filter paper blood spots are commonly used a source of both DNA and antibodies. To enhance the operational practicability of malaria surveys, a method is presented for combined DNA extraction and antibody elution., Methods: Filter paper blood spots were collected as part of a large cross-sectional survey in the Kenyan highlands. DNA was extracted using a saponin/chelex method. The eluate of the first wash during the DNA extraction process was used for antibody detection and compared with previously validated antibody elution procedures. Antibody elution efficiency was assessed by total IgG ELISA for malaria antigens apical membrane antigen-1 (AMA-1) and merozoite-surface protein-1 (MSP-142). The sensitivity of nested 18S rRNA and cytochrome b PCR assays and the impact of doubling filter paper material for PCR sensitivity were determined. The distribution of cell material and antibodies throughout filter paper blood spots were examined using luminescent and fluorescent reporter assays., Results: Antibody levels measured after the combined antibody/DNA extraction technique were strongly correlated to those measured after standard antibody elution (p < 0.0001). Antibody levels for both AMA-1 and MSP-142 were generally slightly lower (11.3-21.4%) but age-seroprevalence patterns were indistinguishable. The proportion of parasite positive samples ranged from 12.9% to 19.2% in the different PCR assays. Despite strong agreement between outcomes of different PCR assays, none of the assays detected all parasite-positive individuals. For all assays doubling filter paper material for DNA extraction increased sensitivity. The concentration of cell and antibody material was not homogenously distributed throughout blood spots., Conclusion: Combined DNA extraction and antibody elution is an operationally attractive approach for high throughput assessment of cumulative malaria exposure and current infection prevalence in endemic settings. Estimates of antibody prevalence are unaffected by the combined extraction and elution procedure. The choice of target gene and the amount and source of filter paper material for DNA extraction can have a marked impact on PCR sensitivity.

Background: The use of amodiaquine in prophylaxis is associated with serious toxicity, resulting from its metabolic conversion into a reactive quinone-imine metabolite by the hepatic cytochrome P450. To circumvent this toxicity, several amodiaquine analogues that lack the potential to form a quinone-imine derivative, while retaining antimalarial activity, have been designed. Isoquine is one of these promising molecules that has already reached Phase I clinical trials in humans., Methods: We analysed the in vitro activity of isoquine against 62 Plasmodium falciparum isolates collected in Kenya and the association of this activity with polymorphisms in pfcrt and pfmdr1 genes., Results: The median concentration of isoquine that inhibited 50% of parasite growth (IC50) was 9 nM, compared with 56 nM chloroquine, 8 nM amodiaquine, 10 nM desethylamodiaquine, 69 nM lumefantrine and 1 nM dihydroartemisinin. Isoquine activity was correlated with polymorphisms in pfcrt at codon 76, but not in pfmdr1 at codon 86., Conclusions: The high activity of isoquine against field isolates, including chloroquine-resistant isolates, with IC50 <10 nM, warrants its further development as an antimalarial.

Chloroquine (CQ) drug was withdrawn in 1998 as a first-line treatment of uncomplicated malaria in Kenya. This was in response to resistance to the drug in Plasmodium falciparum malaria parasite. Investigations were conducted to determine prevalence of CQ resistance genotypes in the parasites in Tiwi, a malaria endemic town in Kenya, before and about a decade after the withdrawal of the drug. Blood samples were collected and spotted on filter papers in 1999 and 2008 from 75 and 77 out-patients respectively with uncomplicated malaria. The sampling was conducted using finger pricking technique. DNA was extracted from individual spots in the papers and screened for the presence of P. falciparum chloroquine resistance transporter (Pfcrt) and multi drug resistance (Pfmdr-1) markers using nested PCR. Nature of mutations (haplotypes) in the Pfcrt and Pfmdr-1 markers in the samples were confirmed using dot blot hybridization technique. Changes in pattern of CQ resistance in the parasite samples in 1999 and 2008 were assessed by Chi Square test. There was a significant (P<0.05) reduction in CQ resistant genotypes of the parasite between 1999 and 2008. Pfmdr and Pfcrt CQ resistant genotypes in 2008 reduced to 54.10 and 63.64% respectively, from 75.39 and 88.0% respectively in 1999. This reduction was accompanied by emergence of Pfcrt specific CQ sensitive (IEK) and intermediate/partially CQ resistant (MET) haplotypes. Results suggest significant reversal of the phenotype of the parasite from chloroquine resistant to wild/sensitive type. The novel haplotypes indicates transitional phase of the parasite to the wild type. Current prevalence of chloroquine resistant genotype is definitely above the threshold for efficacious re-introduction of chloroquine for treatment of malaria in Tiwi., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Plasmodium falciparum is the major cause of malaria globally and is transmitted by mosquitoes. During parasitic development, P. falciparum-infected erythrocytes (P. falciparum-IEs) express multiple polymorphic proteins known as variant surface antigens (VSAs), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1). VSA-specific antibodies are associated with protection from symptomatic and severe malaria. However, the importance of the different VSA targets of immunity to malaria remains unclear, which has impeded an understanding of malaria immunity and vaccine development. In this study, we developed assays using transgenic P. falciparum with modified PfEMP1 expression to quantify serum antibodies to VSAs among individuals exposed to malaria. We found that the majority of the human antibody response to the IE targets PfEMP1. Furthermore, our longitudinal studies showed that individuals with PfEMP1-specific antibodies had a significantly reduced risk of developing symptomatic malaria, whereas antibodies to other surface antigens were not associated with protective immunity. Using assays that measure antibody-mediated phagocytosis of IEs, an important mechanism in parasite clearance, we identified PfEMP1 as the major target of these functional antibodies. Taken together, these data demonstrate that PfEMP1 is a key target of humoral immunity. These findings advance our understanding of the targets and mediators of human immunity to malaria and have major implications for malaria vaccine development.

Background: Resistance to sulphadoxine-pyrimethamine (SP) in Plasmodium falciparum parasites is associated with mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes and has spread worldwide. SP remains the recommended drug for intermittent preventive treatment for malaria in pregnancy (IPTp) and information on population prevalence of the SP resistance molecular markers in pregnant women is limited., Methods: Temporal trends of SP resistance molecular markers were investigated in 489 parasite samples collected from pregnant women at delivery from three different observational studies between 1996 and 2009 in Kenya, where SP was adopted for both IPTp and case treatment policies in 1998. Using real-time polymerase chain reaction, pyrosequencing and direct sequencing, 10 single-nucleotide polymorphisms (SNPs) of SP resistance molecular markers were assayed., Results: The prevalence of quintuple mutant (dhfr N51I/C59R/S108N and dhps A437G/K540E combined genotype) increased from 7% in the first study (1996-2000) to 88% in the third study (2008-2009). When further stratified by sample collection year and adoption of IPTp policy, the prevalence of the quintuple mutant increased from 2.4% in 1998 to 44.4% three years after IPTp policy adoption, seemingly in parallel with the increase in percentage of SP use in pregnancy. However, in the 1996-2000 study, more mutations in the combined dhfr/dhps genotype were associated with SP use during pregnancy only in univariable analysis and no associations were detected in the 2002-2008 and 2008-2009 studies. In addition, in the 2008-2009 study, 5.3% of the parasite samples carried the dhps triple mutant (A437G/K540E/A581G). There were no differences in the prevalence of SP mutant genotypes between the parasite samples from HIV + and HIV- women over time and between paired peripheral and placental samples., Conclusions: There was a significant increase in dhfr/dhps quintuple mutant and the emergence of new genotype containing dhps 581 in the parasites from pregnant women in western Kenya over 13 years. IPTp adoption and SP use in pregnancy only played a minor role in the increased drug-resistant parasites in the pregnant women over time. Most likely, other major factors, such as the high prevalence of resistant parasites selected by the use of SP for case management in large non-pregnant population, might have contributed to the temporally increased prevalence of SP resistant parasites in pregnant women. Further investigations are needed to determine the linkage between SP drug resistance markers and efficacy of IPTp-SP.

Background: Understanding the origin and spread of mutations associated with drug resistance, especially in the context of combination therapy, will help guide strategies to halt and prevent the emergence of resistance. Unfortunately, studies have assessed these complex processes when resistance is already highly prevalent. Even further, information on the evolutionary dynamics leading to multidrug-resistant parasites is scattered and limited to areas with low or seasonal malaria transmission. This study describes the dynamics of strong selection for mutations conferring resistance against sulphadoxine-pyrimethamine (SP), a combination therapy, in western Kenya between 1992 and 1999, just before SP became first-line therapy (1999). Importantly, the study is based on longitudinal data, which allows for a comprehensive analysis that contrasts with previous cross-sectional studies carried out in other endemic regions., Methods: This study used 236 blood samples collected between 1992 and 1999 in the Asembo Bay area of Kenya. Pyrosequencing was used to determine the alleles of dihydrofolate reductase (dhfr) and dihydropterote synthase (dhps) genes. Microsatellite alleles spanning 138 kb around dhfr and dhps, as well as, neutral markers spanning approximately 100 kb on chromosomes 2 and 3 were characterized., Results: By 1992, the South-Asian dhfr triple mutant was already spreading, albeit in low frequency, in this holoendemic Kenyan population, prior to the use of SP as a first-line therapy. Additionally, dhfr triple mutant alleles that originated independently from the predominant Southeast Asian lineage were present in the sample set. Likewise, dhps double mutants were already present as early as 1992. There is evidence for soft selective sweeps of two dhfr mutant alleles and the possible emergence of a selective sweep of double mutant dhps alleles between 1992 and 1997. The longitudinal structure of the dataset allowed estimation of selection pressures on various dhfr and dhps mutants relative to each other based on a theoretical model tailored to P. falciparum. The data indicate that drug selection acted differently on the resistant alleles of dhfr and dhps, as evidenced by fitness differences. Thus a combination drug therapy such as SP, by itself, does not appear to select for "multidrug"-resistant parasites in areas with high recombination rate., Conclusions: The complexity of these observations emphasizes the importance of population-based studies to evaluate the effects of strong drug selection on Plasmodium falciparum populations.

Ferroquine (FQ), a chloroquine (CQ) analog, is being developed to treat persons with Plasmodium falciparum malaria. In 146 P. falciparum field isolates from western Kenya, we measured 50% inhibitory concentrations (IC(50); nM) of CQ and FQ by a SYBR Green I in vitro assay. Reference clones included W2 (CQ resistant) and D6 (CQ sensitive). Mutation analysis was done for P. falciparum CQ-resistance transporter gene (Pfcrt K76T). Median IC(50) values for FQ were lower than CQ for field isolates and the W2 clone (both P < 0.05). The Pfcrt mutation (76T), which was detected in > 80% of isolates, conferred higher CQ IC(50) values (P < 0.05) and modestly lower FQ IC(50) values (P < 0.05), versus Pfcrt wild type (K76). FQ is more potent than CQ against CQ-resistant P. falciparum field isolates and the W2 clone, and is less affected by Pfcrt 76T. These findings support the notion that FQ could be useful in treating persons with P. falciparum malaria.

Despite the clear public health benefit of insecticide-treated bednets (ITNs), the impact of malaria transmission-reduction by vector control on the spread of drug resistance is not well understood. In the present study, the effect of sustained transmission reduction by ITNs on the prevalence of Plasmodium falciparum gene mutations associated with resistance to the antimalarial drugs sulfadoxine-pyrimethamine (SP) and chloroquine (CQ) in children under the age of five years was investigated during an ITN trial in Asembo area, western Kenya. During the ITN trial, the national first line antimalarial treatment changed from CQ to SP. Smear-positive samples collected from cross sectional surveys prior to ITN introduction (baseline, n = 250) and five years post-ITN intervention (year 5 survey, n = 242) were genotyped for single nucleotide polymorphisms (SNPs) at dhfr-51, 59, 108, 164 and dhps-437, 540 (SP resistance), and pfcrt-76 and pfmdr1-86 (CQ resistance). The association between the drug resistance mutations and epidemiological variables was evaluated. There were significant increases in the prevalence of SP dhps mutations and the dhfr/dhps quintuple mutant, and a significant reduction in the proportion of mixed infections detected at dhfr-51, 59 and dhps-437, 540 SNPs from baseline to the year 5 survey. There was no change in the high prevalence of pfcrt-76 and pfmdr1-86 mutations. Multivariable regression analysis further showed that current antifolate use and year of survey were significantly associated with more SP drug resistance mutations. These results suggest that increased antifolate drug use due to drug policy change likely led to the high prevalence of SP mutations 5 years post-ITN intervention and reduced transmission had no apparent effect on the existing high prevalence of CQ mutations. There is no evidence from the current study that sustained transmission reduction by ITNs reduces the prevalence of genes associated with malaria drug resistance.

Plasmodium merozoites attach to and invade red blood cells (RBCs) during the erythrocytic cycle. The invasion process requires recognition of RBC surface receptors by proteins of the Plasmodium Duffy binding like erythrocyte binding like (DBL-EBP) family. Clones and isolates of Plasmodium falciparum have varying abilities to utilize different RBC receptors, and multiple distinct pathways so far identified depend on glycophorins A, B, C, and as yet unidentified receptors. At present, five members of the DBL-EBP family have been identified in the P. falciparum genome, based on gene structure and amino acid sequence homology. The cardinal features of this family consist of conserved 5' and 3' cysteine-rich regions (regions II and VI, respectively) whose cysteine residues are highly conserved along with the majority of aromatic amino acids. In contrast to the single DBL-EBP family member in Plasmodium vivax, in P. falciparum all DBL-EBP family members have a duplication of the conserved 5' cysteine-rich region denoted as the F1 and F2 domains. These cysteine-rich regions are considered crucial in recognition of erythrocyte receptors and it has been shown that several bind to glycophorins on the erythrocyte surface. Several studies, on both field isolates and laboratory strains have uncovered a relatively high degree of sequence polymorphism in the DBP-EBL genes. This study is now extended to include field isolates collected from sites within Kenya. DNA isolated from blood samples of infected patients was utilized to amplify the region I sequence of ebl-1 gene in order to investigate polymorphism in the region immediately adjacent to the 5' cysteine-rich domains, and to determine the prevalence of an insertion mutant that effectively knocks out the gene., (Copyright 2009 Elsevier B.V. All rights reserved.)

We have developed two loop-mediated isothermal amplification (LAMP) assays for the detection of Theileria parva, the causative agent of East Coast fever (ECF), an economically important cattle disease in eastern, central and southern Africa. These assays target the polymorphic immunodominant molecule (PIM) and p150 LAMP genes. The primer set for each gene target consists of six primers, and each set recognises eight distinct regions on the target gene to give highly specific detection of T. parva. The detection limit of each primer set is 1fg, which is equivalent to one copy of the PIM and p150 T. parva genes. These PIM and p150 LAMP primer sets amplify DNA of T. parva isolates from cattle and buffalo from different countries including Kenya, South Africa, Tanzania, Rwanda, Uganda and Burundi, indicating their ability to detect T. parva from different countries. With the advantages of simplicity, rapidity and cost effectiveness, these LAMP assays are good candidates for molecular epidemiology studies and for monitoring control programs in ECF-endemic, resource poor countries., (Copyright 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Whether the number of concurrent clones in asymptomatic Plasmodium falciparum infections reflects the degree of host protection was investigated in children living in areas with different levels of transmission on the coast of Kenya. The number of concurrent clones was determined on the basis of polymorphism in msp2, which encodes the vaccine candidate antigen merozoite surface protein 2. In a low-transmission area, most children had monoclonal infections, and diversity did not predict a risk of clinical malaria. In an area of moderate transmission, asymptomatic infections with 2 clones were, compared with 1 clone, associated with an increased risk of subsequent malaria. In a comparative assessment in a high-transmission area in Tanzania, multiclonal infections conferred a reduced risk. The different nonlinear associations between the number of clones and malaria morbidity suggest that levels of tolerance to multiclonal infections are transmission dependent as a result of cumulative exposure to antigenically diverse P. falciparum infections.

Human travel to malaria endemic lowlands from epidemic highlands has been shown to increase the risk of malaria infections in the highlands. In order to gain insight on the impact of human travel, we examined prevalence, genetic variability and population genetic structure of Plasmodium falciparum in asymptomatic children from one highland site and three surrounding malaria endemic lowland sites in Western Kenya, using multilocus microsatellite genotyping. We further analyzed the frequencies of mutations at the genes conferring resistance to chloroquine and sulfadoxine-pyrimethamine. We found a significant decrease in malaria prevalence in the highland site from 2006 to 2007, 1 year after the introduction of the artemisinin-based combination therapy as first-line treatment for uncomplicated malaria and the scale-up of insecticide-treated bed nets. Population genetic diversity, measured by the number of observed and effective microsatellite alleles and Nei's unbiased genetic diversity, was high and comparable for both highland and lowland populations. Analysis of molecular variance did not detect a significant genetic structure across highland and lowland regions. Similarly, mutations at key antimalarial-resistance codons of the pfcrt, pfmdr1, pfdhfr and pfdhps genes were found at comparable high frequencies in all four sites. High level of gene flow and lack of significant genetic structure in malaria parasites between highland and lowland areas suggest the importance of human travel in shaping parasite population structure.

Background: The spread of resistance to chloroquine (CQ) led to its withdrawal from use in most countries in sub-Saharan Africa in the 1990s. In Malawi, this withdrawal was followed by a rapid reduction in the frequency of resistance to the point where the drug is now considered to be effective once again, just nine years after its withdrawal. In this report, the polymorphisms of markers associated with CQ-resistance against Plasmodium falciparum isolates from coastal Kenya (Kilifi) were investigated, from 1993, prior to the withdrawal of CQ, to 2006, seven years after its withdrawal. Changes to those that occurred in the dihydrofolate reductase gene (dhfr) that confers resistance to the replacement drug, pyrimethamine/sulphadoxine were also compared., Methods: Mutations associated with CQ resistance, at codons 76 of pfcrt, at 86 of pfmdr1, and at codons 51, 59 and 164 of dhfr were analysed using PCR-restriction enzyme methods. In total, 406, 240 and 323 isolates were genotyped for pfcrt-76, pfmdr1-86 and dhfr, respectively., Results: From 1993 to 2006, the frequency of the pfcrt-76 mutant significantly decreased from around 95% to 60%, while the frequency of pfmdr1-86 did not decline, remaining around 75%. Though the frequency of dhfr mutants was already high (around 80%) at the start of the study, this frequency increased to above 95% during the study period. Mutation at codon 164 of dhfr was analysed in 2006 samples, and none of them had this mutation., Conclusion: In accord with the study in Malawi, a reduction in resistance to CQ following official withdrawal in 1999 was found, but unlike Malawi, the decline of resistance to CQ in Kilifi was much slower. It is estimated that, at current rates of decline, it will take 13 more years for the clinical efficacy of CQ to be restored in Kilifi. In addition, CQ resistance was declining before the drug's official withdrawal, suggesting that, prior to the official ban, the use of CQ had decreased, probably due to its poor clinical effectiveness.

Objectives: Resistance to pyrimethamine in Plasmodium falciparum is conferred by mutations in the gene encoding dihydrofolate reductase (DHFR). It is known that DHFR double mutants have evolved independently in multiple geographic areas, whereas the triple mutant prevalent in Africa appears to have originated in south-east Asia. In this study, we investigated whether other triple mutants may have evolved independently in Africa., Methods: We determined the DHFR genotypes and haplotypes of five microsatellite loci flanking the DHFR locus between 4.49 kb upstream and 1.48 kb downstream of 159 isolates collected from three African countries (Republic of Congo, Ghana and Kenya)., Results: The CIRNI type of DHFR triple mutant (with mutations underlined at amino acid positions 51, 59 and 108) was predominant in the Republic of Congo (82%) and Ghana (81%) and was the second most prevalent in Kenya (27%), where the CICNI type of DHFR double mutant was dominant. Three distinct microsatellite haplotypes were identified in the DHFR triple mutant. One haplotype was identical to that originating in south-east Asia. The other two haplotypes occurred in Ghana and Kenya, which were unique, previously undescribed and identical to those of the two DHFR double mutants found in the same locations., Conclusions: This study presents strong evidence for the unique, multiple independent evolution of pyrimethamine resistance in Africa. Indigenous evolution of the triple mutant from the double mutant appears to have occurred in a step-wise manner in Kenya and Ghana or in nearby countries in east and west Africa.