Peninah Muiruri

Bio: Peninah Muiruri is an academic researcher from Jomo Kenyatta University of Agriculture and Technology. The author has contributed to research in topics: Plasmodium falciparum & Drug resistance. The author has an hindex of 5, co-authored 6 publications receiving 93 citations. Previous affiliations of Peninah Muiruri include Kenya Medical Research Institute & United States Army Medical Research Unit-Kenya.

Temporal trends in prevalence of Plasmodium falciparum molecular markers selected for by artemether–lumefantrine treatment in pre-ACT and post-ACT parasites in western Kenya

Abstract: Artemether–lumefantrine (AL) became the first-line treatment for uncomplicated malaria in Kenya in 2006. Studies have shown AL selects for SNPs in pfcrt and pfmdr1 genes in recurring parasites compared to the baseline infections. The genotypes associated with AL selection are K76 in pfcrt and N86, 184F and D1246 in pfmdr1. To assess the temporal change of these genotypes in western Kenya, 47 parasite isolates collected before (pre-ACT; 1995–2003) and 745 after (post-ACT; 2008–2014) introduction of AL were analyzed. In addition, the associations of parasite haplotype against the IC50 of artemether and lumefantrine, and clearance rates were determined. Parasite genomic DNA collected between 1995 and 2014 was analyzed by sequencing or PCR-based single-base extension on Sequenom MassARRAY. IC50s were determined for a subset of the samples. One hundred eighteen samples from 2013 to 2014 were from an efficacy trial of which 68 had clearance half-lives. Data revealed there were significant differences between pre-ACT and post-ACT genotypes at the four codons (chi-square analysis; p < 0.0001). The prevalence of pfcrt K76 and N86 increased from 6.4% in 1995–1996 to 93.2% in 2014 and 0.0% in 2002–2003 to 92.4% in 2014 respectively. Analysis of parasites carrying pure alleles of K + NFD or T + YYY haplotypes revealed that 100.0% of the pre-ACT parasites carried T + YYY and 99.3% of post-ACT parasites carried K + NFD. There was significant correlation (p = 0.04) between lumefantrine IC50 and polymorphism at pfmdr1 codon 184. There was no difference in parasite clearance half-lives based on genetic haplotype profiles. This study shows there is a significant change in parasite genotype, with key molecular determinants of AL selection almost reaching saturation. The implications of these findings are not clear since AL remains highly efficacious. However, there is need to closely monitor parasite genotypic, phenotypic and clinical dynamics in response to continued use of AL in western Kenya.

Analysis of major genome loci underlying artemisinin resistance and pfmdr1 copy number in pre- and post-ACTs in western Kenya.

Abstract: Genetic analysis of molecular markers is critical in tracking the emergence and/or spread of artemisinin resistant parasites. Clinical isolates collected in western Kenya pre- and post- introduction of artemisinin combination therapies (ACTs) were genotyped at SNP positions in regions of strong selection signatures on chromosome 13 and 14, as described in Southeast Asia (SEA). Twenty five SNPs were genotyped using Sequenom MassArray and pfmdr1 gene copy number by real-time PCR. Parasite clearance half-life and in vitro drug sensitivity testing were performed using standard methods. One hundred twenty nine isolates were successfully analyzed. Fifteen SNPs were present in pre-ACTs isolates and six in post-ACTs. None of the SNPs showed association with parasite clearance half-life. Post-ACTs parasites had significantly higher pfmdr1 copy number compared to pre-ACTs. Seven of eight parasites with multiple pfmdr1 were post-ACTs. When in vitro IC50s were compared for parasites with single vs. multiple gene copies, only amodiaquine and piperaquine reached statistical significance. Data showed SNPs on chromosome 13 and 14 had different frequency and trend in western Kenya parasites compared SEA. Increase in pfmdr1 gene copy is consistent with recent studies in African parasites. Data suggests genetic signature of artemisinin resistance in Africa might be different from SEA.

Polymorphisms in Pfmdr1, Pfcrt, and Pfnhe1 Genes Are Associated with Reduced In Vitro Activities of Quinine in Plasmodium falciparum Isolates from Western Kenya

Abstract: In combination with antibiotics, quinine is recommended as the second-line treatment for uncomplicated malaria, an alternative first-line treatment for severe malaria, and for treatment of malaria in the first trimester of pregnancy. Quinine has been shown to have frequent clinical failures, and yet the mechanisms of action and resistance have not been fully elucidated. However, resistance is linked to polymorphisms in multiple genes, including multidrug resistance 1 (Pfmdr1), the chloroquine resistance transporter (Pfcrt), and the sodium/hydrogen exchanger gene (Pfnhe1). Here, we investigated the association between in vitro quinine susceptibility and genetic polymorphisms in Pfmdr1codons 86 and 184, Pfcrt codon 76, and Pfnhe1 ms4760 in 88 field isolates from western Kenya. In vitro activity was assessed based on the drug concentration that inhibited 50% of parasite growth (the IC50), and parasite genetic polymorphisms were determined from DNA sequencing. Data revealed there were significant associations between polymorphism in Pfmdr1-86Y, Pfmdr1-184F, or Pfcrt-76T and quinine susceptibility (P < 0.0001 for all three associations). Eighty-two percent of parasites resistant to quinine carried mutant alleles at these codons (Pfmdr1-86Y, Pfmdr1-184F, and Pfcrt-76T), whereas 74% of parasites susceptible to quinine carried the wild-type allele (Pfmdr1-N86, Pfmdr1-Y184, and Pfcrt-K76, respectively). In addition, quinine IC50 values for parasites with Pfnhe1 ms4760 3 DNNND repeats were significantly higher than for those with 1 or 2 repeats (P = 0.033 and P = 0.0043, respectively). Clinical efficacy studies are now required to confirm the validity of these markers and the importance of parasite genetic background.

The prevalence and antifolate drug resistance profiles of Plasmodium falciparum in study participants randomized to discontinue or continue cotrimoxazole prophylaxis.

Abstract: 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

Selective sweeps and genetic lineages of Plasmodium falciparum multi-drug resistance ( pfmdr1 ) gene in Kenya

Abstract: There are concerns that resistance to artemisinin-based combination therapy might emerge in Kenya and sub-Saharan Africa (SSA) in the same pattern as was with chloroquine and sulfadoxine–pyrimethamine. Single nucleotide polymorphisms (SNPs) in critical alleles of pfmdr1 gene have been associated with resistance to artemisinin and its partner drugs. Microsatellite analysis of loci flanking genes associated with anti-malarial drug resistance has been used in defining the geographic origins, dissemination of resistant parasites and identifying regions in the genome that have been under selection. This study set out to investigate evidence of selective sweep and genetic lineages in pfmdr1 genotypes associated with the use of artemether–lumefantrine (AL), as the first-line treatment in Kenya. Parasites (n = 252) from different regions in Kenya were assayed for SNPs at codons 86, 184 and 1246 and typed for 7 neutral microsatellites and 13 microsatellites loci flanking (± 99 kb) pfmdr1 in Plasmodium falciparum infections. The data showed differential site and region specific prevalence of SNPs associated with drug resistance in the pfmdr1 gene. The prevalence of pfmdr1 N86, 184F, and D1246 in western Kenya (Kisumu, Kericho and Kisii) compared to the coast of Kenya (Malindi) was 92.9% vs. 66.7%, 53.5% vs. to 24.2% and 96% vs. to 87.9%, respectively. The NFD haplotype which is consistent with AL selection was at 51% in western Kenya compared to 25% in coastal Kenya. Selection pressures were observed to be different in different regions of Kenya, especially the western region compared to the coastal region. The data showed independent genetic lineages for all the pfmdr1 alleles. The evidence of soft sweeps in pfmdr1 observed varied in direction from one region to another. This is challenging for malaria control programs in SSA which clearly indicate effective malaria control policies should be based on the region and not at a country wide level.

Antimalarial drug resistance in Africa: the calm before the storm?

Abstract: Antimalarial drug resistance, in particular resistance to Plasmodium falciparum, challenges the treatment and control of malaria. In this Review, we summarise evolving patterns of antimalarial drug resistance in Africa. Resistance to aminoquinolines and antifolates is long-standing, yet with greatly decreased use of chloroquine to treat malaria, the prevalence of resistance to chloroquine has decreased. Resistance to antifolates, which are used to prevent malaria in some settings, remains widespread. Resistance to artemisinin-based combination therapies, the standard treatments for malaria in Africa, has emerged in southeast Asia. At present, resistance to artemisinins or key partner drugs included in combination therapies does not appear to be a substantial problem in Africa. However, emergence of resistance to artemisinin-based combination therapies in Africa would probably have devastating consequences, and continued surveillance for the emergence of resistance on this continent is a high priority.

Review of malaria situation in Cameroon: technical viewpoint on challenges and prospects for disease elimination.

Abstract: Malaria still has a devastating impact on public health and welfare in Cameroon. Despite the increasing number of studies conducted on disease prevalence, transmission patterns or treatment, there are to date, not enough studies summarising findings from previous works in order to identify gaps in knowledge and areas of interest where further evidence is needed to drive malaria elimination efforts. The present study seeks to address these gaps by providing a review of studies conducted so far on malaria in Cameroon since the 1940s to date. Over 250 scientific publications were consulted for this purpose. Although there has been increased scale-up of vector control interventions which significantly reduced the morbidity and mortality to malaria across the country from a prevalence of 41% of the population reporting at least one malaria case episode in 2000 to a prevalence of 24% in 2017, the situation is not yet under control. There is a high variability in disease endemicity between epidemiological settings with prevalence of Plasmodium parasitaemia varying from 7 to 85% in children aged 6 months to 15 years after long-lasting insecticidal nets (LLINs) scale-up. Four species of Plasmodium have been recorded across the country: Plasmodium falciparum, P. malariae, P. ovale and P. vivax. Several primate-infecting Plasmodium spp. are also circulating in Cameroon. A decline of artemisinin-based combinations therapeutic efficacy from 97% in 2006 to 90% in 2016 have been reported. Several mutations in the P. falciparum chloroquine resistance (Pfcrt) and P. falciparum multidrug resistance 1 (Pfmdr1) genes conferring resistance to either 4-amino-quinoleine, mefloquine, halofanthrine and quinine have been documented. Mutations in the Pfdhfr and Pfdhps genes involved in sulfadoxine-pyrimethamine are also on the rise. No mutation associated with artemisinin resistance has been recorded. Sixteen anopheline species contribute to malaria parasite transmission with six recognized as major vectors: An. gambiae, An. coluzzii, An. arabiensis, An. funestus, An. nili and An. moucheti. Studies conducted so far, indicated rapid expansion of DDT, pyrethroid and carbamate resistance in An. gambiae, An. coluzzii, An. arabiensis and An. funestus threatening the performance of LLINs. This review highlights the complex situation of malaria in Cameroon and the need to urgently implement and reinforce integrated control strategies in different epidemiological settings, as part of the substantial efforts to consolidate gains and advance towards malaria elimination in the country.

A Variant PfCRT Isoform Can Contribute to Plasmodium falciparum Resistance to the First-Line Partner Drug Piperaquine.

Abstract: Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC90) or 50% parasite killing (50% lethal dose [LD50]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite's acidic digestive vacuole as the primary mode of both the bis-aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates.IMPORTANCE The global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite Plasmodium Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparum resistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparum chloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

Abstract: BACKGROUND In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Multi-omics Based Identification of Specific Biochemical Changes Associated With PfKelch13-Mutant Artemisinin-Resistant Plasmodium falciparum.

Abstract: Background The emergence of artemisinin resistance in the malaria parasite Plasmodium falciparum poses a major threat to the control and elimination of malaria. Certain point mutations in the propeller domain of PfKelch13 are associated with resistance, but PfKelch13 mutations do not always result in clinical resistance. The underlying mechanisms associated with artemisinin resistance are poorly understood, and the impact of PfKelch13 mutations on cellular biochemistry is not defined. Methods This study aimed to identify global biochemical differences between PfKelch13-mutant artemisinin-resistant and -sensitive strains of P. falciparum by combining liquid chromatography-mass spectrometry (LC-MS)-based proteomics, peptidomics, and metabolomics. Results Proteomics analysis found both PfKelch13 mutations examined to be specifically associated with decreased abundance of PfKelch13 protein. Metabolomics analysis demonstrated accumulation of glutathione and its precursor, gamma-glutamylcysteine, and significant depletion of 1 other putative metabolite in resistant strains. Peptidomics analysis revealed lower abundance of several endogenous peptides derived from hemoglobin (HBα and HBβ) in the artemisinin-resistant strains. Conclusion PfKelch13 mutations associated with artemisinin resistance lead to decreased abundance of PfKelch13 protein, decreased hemoglobin digestion, and enhanced glutathione production.