Malaria Transmission Dynamics in a High-Transmission Setting of Western Kenya and the Inadequate Treatment Response to Artemether-Lumefantrine in an Asymptomatic Population.

BACKGROUND: Assessing the infectious reservoir is critical in malaria control and elimination strategies. We conducted a longitudinal epidemiological study in a high-malaria-burden region in Kenya to characterize transmission in an asymptomatic population. METHODS: 488 study participants encompassing all ages in 120 households within 30 clusters were followed for 1 year with monthly sampling. Malaria was diagnosed by microscopy and molecular methods. Transmission potential in gametocytemic participants was assessed using direct skin and/or membrane mosquito feeding assays, then treated with artemether-lumefantrine. Study variables were assessed using mixed-effects generalized linear models. RESULTS: Asexual and sexual parasite data were collected from 3792 participant visits, with 903 linked with feeding assays. Univariate analysis revealed that the 6-11-year-old age group was at higher risk of harboring asexual and sexual infections than those <6 years old (odds ratio [OR] 1.68, P < .001; and OR 1.81, P < .001), respectively. Participants with submicroscopic parasitemia were at a lower risk of gametocytemia compared with microscopic parasitemia (OR 0.04, P < .001), but they transmitted at a significantly higher rate (OR 2.00, P = .002). A large proportion of the study population who were infected at least once remained infected (despite treatment) with asexual (71.7%, 291/406) or sexual (37.4%, 152/406) parasites. 88.6% (365/412) of feeding assays conducted in individuals who failed treatment the previous month resulted in transmissions. CONCLUSIONS: Individuals with asymptomatic infection sustain the transmission cycle, with the 6-11-year age group serving as an important reservoir. The high rates of artemether-lumefantrine treatment failures suggest surveillance programs using molecular methods need to be expanded for accurate monitoring and evaluation of treatment outcomes.

Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates.

BACKGROUND: The unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages of Plasmodium necessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, from Cissampelos pariera (Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity. METHODS: Malaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collected P. falciparum isolates. To determine the speed- and stage-of-action of isoliensinine, an IC50 speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico. RESULTS: Isoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexual of 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50IEV = 1.433 µM (95% CI 0.917-2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties. CONCLUSION: These findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.

Impact of parasite genomic dynamics on the sensitivity of Plasmodium falciparum isolates to piperaquine and other antimalarial drugs.

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 IC50 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 IC50s 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 IC50s 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 IC50 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.

Burden of malaria infection among individuals of varied blood groups in Kenya.

BACKGROUND: The ABO blood groups consist of A, B, and H carbohydrate antigens, which regulate protein activities during malaria infection in humans. Understanding the interplay between the malaria parasite and blood group antigens is essential in understanding new interventions to reduce the global burden of malaria. This study assessed the burden of malaria infection among individuals with varying blood groups seeking treatment at selected hospitals in Kenya. METHODS: A total of 366 samples from an ongoing malaria surveillance study were diagnosed for malaria by microscopy and further typed for blood group using ABO blood grouping. Age and sex were recorded in a data sheet, and analysed using R software version 4. Groups' proportions (blood group, malaria infection, age and sex) were compared using Pearson's Chi-square and Fischer exact tests. Wilcoxon and Kruskal-Wallis tests were performed and P-value < 0.05 was considered significant after Bonferroni correction for multiple comparisons. To understand the effect of each blood group on parasitaemia, multivariate logistic regression was used to model ABO blood group in relation to parasitaemia. RESULTS: Of the 366 samples analysed, 312 were malaria positive, mean age was 9.83 years (< 5 years n = 152 (48.41%), 6 to 17 years n = 101 (32.16%) and > 18 years n = 61 (19.43%)). Malaria prevalence was higher among females than males, 54.46% and 45.54%, respectively. Kisumu enrolled the highest number 109 (35%)) of malaria cases, Kombewa 108 (35%), Malindi 32 (10%), Kisii 28 (9%), Marigat 23 (7%), and Kericho 12 (4%). Blood group O+ was the most prevalent among the enrolled individuals (46.50%), A+ (27.71%), B+ (21.02%) and AB+ (4.78%) respectively. Compared to blood group O+, blood group B+ individuals were (14%) were more likely to habour Plasmodium falciparum infection as opposed to A+ and AB+ individuals, that were 7% and 20%, respectively,. Those living in malaria-endemic zones presented with higher parasite densities compared to those living in malaria-epidemic (p = 0.0061). Individuals bearing B + blood group are more likely to habour high parasitaemia compared to O + blood group bearers (OR = 4.47, CI = 1.53-13.05, p = 0.006). CONCLUSION: Individuals of blood group B harbour high parasitaemia compared with the blood group O, Additionally, blood group A and B present with symptoms at lower parasitaemia than blood group O. Regardles of malaria transmission zones, individuals from endemic zones showed up with high parasitaemia and among them were more individuals of blood groups A and B than individuals of blood group O. Implying that these individuals were more at risk and require additional attention and effective case management.

Epidemiological and clinical implications of asymptomatic malaria and schistosomiasis co-infections in a rural community in western Kenya.

BACKGROUND: Malaria and schistosomiasis present considerable disease burden in tropical and sub-tropical areas and severity is worsened by co-infections in areas where both diseases are endemic. Although pathogenesis of these infections separately is well studied, there is limited information on the pathogenic disease mechanisms and clinical disease outcomes in co-infections. In this study, we investigated the prevalence of malaria and schistosomiasis co-infections, and the hematologic and blood chemistry abnormalities in asymptomatic adults in a rural fishing community in western Kenya. METHODS: This sub-study used samples and data collected at enrollment from a prospective observational cohort study (RV393) conducted in Kisumu County, Kenya. The presence of malaria parasites was determined using microscopy and real-time-PCR, and schistosomiasis infection by urine antigen analysis (CCA). Hematological analysis and blood chemistries were performed using standard methods. Statistical analyses were performed to compare demographic and infection data distribution, and hematologic and blood chemistry parameters based on different groups of infection categories. Clinically relevant hematologic conditions were analyzed using general linear and multivariable Poisson regression models. RESULTS: From February 2017 to May 2018, we enrolled 671 participants. The prevalence of asymptomatic Plasmodium falciparum was 28.2% (157/556) and schistosomiasis 41.2% (229/562), with 18.0% (100/556) of participants co-infected. When we analyzed hematological parameters using Wilcoxon rank sum test to evaluate median (IQR) distribution based on malarial parasites and/or schistosomiasis infection status, there were significant differences in platelet counts (p = 0.0002), percent neutrophils, monocytes, eosinophils, and basophils (p < 0.0001 each). Amongst clinically relevant hematological abnormalities, eosinophilia was the most prevalent at 20.6% (116/562), whereas thrombocytopenia was the least prevalent at 4.3% (24/562). In univariate model, Chi-Square test performed for independence between participant distribution in different malaria parasitemia/schistosomiasis infection categories within each clinical hematological condition revealed significant differences for thrombocytopenia and eosinophilia (p = 0.006 and p < 0.0001, respectively), which was confirmed in multivariable models. Analysis of the pairwise mean differences of liver enzyme (ALT) and kidney function (Creatinine Clearance) indicated the presence of significant differences in ALT across the infection groups (parasite + /CCA + vs all other groups p < .003), but no differences in mean Creatinine Clearance across the infection groups. CONCLUSIONS: Our study demonstrates the high burden of asymptomatic malaria parasitemia and schistosomiasis infection in this rural population in Western Kenya. Asymptomatic infection with malaria or schistosomiasis was associated with laboratory abnormalities including neutropenia, leukopenia and thrombocytopenia. These abnormalities could be erroneously attributed to other diseases processes during evaluation of diseases processes. Therefore, evaluating for co-infections is key when assessing individuals with laboratory abnormalities. Additionally, asymptomatic infection needs to be considered in control and elimination programs given high prevalence documented here.

Reduced in vitro doxycycline susceptibility in plasmodium falciparum field isolates from Kenya is associated with PfTetQ KYNNNN sequence polymorphism.

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.

Polymorphisms in Pfmdr1, Pfcrt, and Pfnhe1 genes are associated with reduced in vitro activities of quinine in Plasmodium falciparum isolates from western Kenya.

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.

Sample-ready multiplex qPCR assay for detection of malaria.

BACKGROUND: Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. METHODS: A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, "wet" assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. RESULTS: The limit of detection for the MMSR assay was 0.244 parasites/µL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to "wet" assay which was 0.39 and 3.13 parasites/µL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the "wet" assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the "wet" assay. CONCLUSION: The MMSR assay has the same robust performance characteristics as the "wet" assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.

Trends in drug resistance codons in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Kenyan parasites from 2008 to 2012.

BACKGROUND: Sulphadoxine-pyrimethamine (SP), an antifolate, was replaced by artemether-lumefantrine as the first-line malaria drug treatment in Kenya in 2004 due to the wide spread of resistance. However, SP still remains the recommended drug for intermittent preventive treatment in pregnant women and infants (IPTP/I) owing to its safety profile. This study assessed the prevalence of mutations in dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes associated with SP resistance in samples collected in Kenya between 2008 and 2012. METHODS: Field isolates collected from Kisumu, Kisii, Kericho and Malindi district hospitals were assessed for genetic polymorphism at various loci within Pfdhfr and Pfdhps genes by sequencing. RESULTS: Among the Pfdhfr mutations, codons N51I, C59R, S108N showed highest prevalence in all the field sites at 95.5%, 84.1% and 98.6% respectively. Pfdhfr S108N prevalence was highest in Kisii at 100%. A temporal trend analysis showed steady prevalence of mutations over time except for codon Pfdhps 581 which showed an increase in mixed genotypes. Triple Pfdhfr N51I/C59R/S108N and double Pfdhps A437G/ K540E had high prevalence rates of 86.6% and 87.9% respectively. The Pfdhfr/Pfdhps quintuple, N51I/C59R/S108N/A437G/K540E mutant which has been shown to be the most clinically relevant marker for SP resistance was observed in 75.7% of the samples. CONCLUSION: SP resistance is still persistently high in western Kenya, which is likely due to fixation of key mutations in the Pfdhfr and Pfdhps genes as well as drug pressure from other antifolate drugs being used for the treatment of malaria and other infections. In addition, there is emergence and increasing prevalence of new mutations in Kenyan parasite population. Since SP is used for IPTP/I, molecular surveillance and in vitro susceptibility assays must be sustained to provide information on the emergence and spread of SP resistance.

Comparative analysis of peripheral whole blood transcriptome from asymptomatic carriers reveals upregulation of subsets of surface proteins implicated in Plasmodium falciparum phenotypic plasticity.

The molecular mechanism underlying Plasmodium falciparum's persistence in the asymptomatic phase of infection remains largely unknown. However, large-scale shifts in the parasites' gene expression during asymptomatic infections may enhance phenotypic plasticity, maximizing their fitness and leading to the persistence of the asymptomatic infections. To uncover these mechanisms, we aimed to identify parasite genetic factors implicated in asymptomatic infections through whole transcriptome analysis. We analyzed publicly available transcriptome datasets containing asymptomatic malaria (ASM), uncomplicated malaria (SM), and malaria-naïve (NSM) samples from 35 subjects for differentially expressed genes (DEGs) and long noncoding RNAs. Our analysis identified 755 and 1773 DEGs in ASM vs SM and NSM, respectively. These DEGs revealed sets of genes coding for proteins of unknown functions (PUFs) upregulated in ASM vs SM and ASM, suggesting their role in underlying fundamental molecular mechanisms during asymptomatic infections. Upregulated genes in ASM vs SM revealed a subset of 24 clonal variant genes (CVGs) involved in host-parasite and symbiotic interactions and modulation of the symbiont of host erythrocyte aggregation pathways. Moreover, we identified 237 differentially expressed noncoding RNAs in ASM vs SM, of which 11 were found to interact with CVGs, suggesting their possible role in regulating the expression of CVGs. Our results suggest that P. falciparum utilizes phenotypic plasticity as an adaptive mechanism during asymptomatic infections by upregulating clonal variant genes, with long noncoding RNAs possibly playing a crucial role in their regulation. Thus, our study provides insights into the parasites' genetic factors that confer a fitness advantage during asymptomatic infections.

Protein target similarity is positive predictor of in vitro antipathogenic activity: a drug repurposing strategy for Plasmodium falciparum.

Drug discovery is an intricate and costly process. Repurposing existing drugs and active compounds offers a viable pathway to develop new therapies for various diseases. By leveraging publicly available biomedical information, it is possible to predict compounds' activity and identify their potential targets across diverse organisms. In this study, we aimed to assess the antiplasmodial activity of compounds from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library using in vitro and bioinformatics approaches. We assessed the in vitro antiplasmodial activity of the compounds using blood-stage and liver-stage drug susceptibility assays. We used protein sequences of known targets of the ReFRAME compounds with high antiplasmodial activity (EC50 < 10 uM) to conduct a protein-pairwise search to identify similar Plasmodium falciparum 3D7 proteins (from PlasmoDB) using NCBI protein BLAST. We further assessed the association between the compounds' in vitro antiplasmodial activity and level of similarity between their known and predicted P. falciparum target proteins using simple linear regression analyses. BLAST analyses revealed 735 P. falciparum proteins that were similar to the 226 known protein targets associated with the ReFRAME compounds. Antiplasmodial activity of the compounds was positively associated with the degree of similarity between the compounds' known targets and predicted P. falciparum protein targets (percentage identity, E value, and bit score), the number of the predicted P. falciparum targets, and their respective mutagenesis index and fitness scores (R2 between 0.066 and 0.92, P < 0.05). Compounds predicted to target essential P. falciparum proteins or those with a druggability index of 1 showed the highest antiplasmodial activity.

Plasmodium ovale spp dhfr mutations associated with reduced susceptibility to pyrimethamine in sub-Saharan Africa: a retrospective genetic epidemiology and functional study.

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.

Increased sensitivity of malaria parasites to common antimalaria drugs after the introduction of artemether-lumefantrine: Implication of policy change and implementation of more effective drugs in fight against malaria.

Single nucleotide polymorphisms (SNPs) in the Plasmodium falciparum multi-drug resistance protein 1 (Pfmrp1) gene have previously been reported to confer resistance to Artemisinin-based Combination Therapies (ACTs) in Southeast Asia. A total of 300 samples collected from six sites between 2008 and 2019 under an ongoing malaria drug sensitivity patterns in Kenya study were evaluated for the presence of SNPs at Pfmrp1 gene codons: H191Y, S437A, I876V, and F1390I using the Agena MassARRAY® platform. Each isolate was further tested against artemisinin (ART), lumefantrine (LU), amodiaquine (AQ), mefloquine (MQ), quinine (QN), and chloroquine (CQ) using malaria the SYBR Green I-based method to determine their in vitro drug sensitivity. Of the samples genotyped, polymorphism at Pfmrp1 codon I876V was the most frequent, with 59.3% (163/275) mutants, followed by F1390I, 7.2% (20/278), H191Y, 4.0% (6/151), and S437A, 3.3% (9/274). A significant decrease in median 50% inhibition concentrations (IC50s) and interquartile range (IQR) was noted; AQ from 2.996 ng/ml [IQR = 2.604-4.747, n = 51] in 2008 to 1.495 ng/ml [IQR = 0.7134-3.318, n = 40] (P<0.001) in 2019, QN from 59.64 ng/ml [IQR = 29.88-80.89, n = 51] in 2008 to 18.10 ng/ml [IQR = 11.81-26.92, n = 42] (P<0.001) in 2019, CQ from 35.19 ng/ml [IQR = 16.99-71.20, n = 30] in 2008 to 6.699 ng/ml [IQR = 4.976-9.875, n = 37] (P<0.001) in 2019, and ART from 2.680 ng/ml [IQR = 1.608-4.857, n = 57] in 2008 to 2.105 ng/ml [IQR = 1.266-3.267, n = 47] (P = 0.0012) in 2019, implying increasing parasite sensitivity to the drugs over time. However, no significant variations were observed in LU (P = 0.2692) and MQ (P = 0.0939) respectively, suggesting stable parasite responses over time. There was no statistical significance between the mutation at 876 and parasite sensitivity to selected antimalarials tested, suggesting stable sensitivity for the parasites with 876V mutations. These findings show that Kenyan parasite strains are still sensitive to AQ, QN, CQ, ART, LU, and MQ. Despite the presence of Pfmrp1 mutations in parasites among the population.

Therapeutic response to artemisinin combination therapies among individuals with Plasmodium falciparum single infection vs mixed Plasmodium species infections: a retrospective posthoc analysis in Kisumu County, western Kenya.

OBJECTIVES: This study examined the treatment response of mixed vs single-species Plasmodium falciparum infections to artemisinin-based combination therapies (ACTs). METHODS: A total of 1211 blood samples collected on days 0, 7, 14, 21, 28, 35, and 42 from 173 individuals enrolled in two randomized ACT efficacy studies were tested for malaria using 18s ribosomal RNA-based real-time polymerase chain reaction. All recurrent parasitemia were characterized for Plasmodium species composition and time to reinfection during 42-day follow-up compared across ACTs. RESULTS: Day 0 samples had 71.1% (116/163) single P. falciparum infections and 28.2% (46/163) coinfections. A total of 54.0% (88/163) of individuals tested positive for Plasmodium at least once between days 7-42. A total of 19.3% (17/88) of individuals with recurrent infections were infected with a different Plasmodium species than observed at day 0, with 76.5% (13/17) of these "hidden" infections appearing after clearing P. falciparum present at day 0. Artesunate-mefloquine (16.4 hours) and dihydroartemisinin-piperaquine (17.6 hours) had increased clearance rates over artemether-lumefantrine (21.0 hours). Dihydroartemisinin-piperaquine exhibited the longest duration of reinfection prophylaxis. Cure rates were comparable across each species composition. CONCLUSION: No differences in clearance rates were found depending on whether the infection contained species other than P. falciparum. Significantly longer durations of protection were observed for individuals treated with dihydroartemisinin-piperaquine.

Implications of asymptomatic malaria infections on hematologic parameters in adults living with HIV in malaria-endemic regions with varying transmission intensities.

OBJECTIVES: HIV and malaria coinfection impacts disease management and clinical outcomes. This study investigated hematologic abnormalities in malaria-asymptomatic people living with HIV (PLHIV) in regions with differing malaria transmission. METHODS: Study participants were enrolled in the African Cohort Study: two sites in Kenya, one in Uganda, and one in Nigeria. Data was collected at enrollment and every 6 months. Logistic regression estimated odds ratios for associations between HIV/malaria status and anemia, thrombocytopenia, and leucopenia. RESULTS: Samples from 1587 participants with one or more visits comprising 1471 (92.7%) from PLHIV and 116 (7.3%) without HIV were analyzed. Parasite point prevalence significantly differed across the study sites (P <0.001). PLHIV had higher odds of anemia, with males at lower odds compared to females; the odds of anemia decreased with age, reaching significance in those ≥50 years old. Participants in Kisumu, Kenya had higher odds of anemia compared to other sites. PLHIV had higher odds of leucopenia, but malaria co-infection was not associated with worsened leucopenia. The odds of thrombocytopenia were decreased in HIV/malaria co-infection compared to the uninfected group. CONCLUSION: Hematological parameters are important indicators of health and disease. In PLHIV with asymptomatic malaria co-infection enrolled across four geographic sites in three African countries, abnormalities in hematologic parameters differ in different malaria transmission settings and are region-specific.

Age-dependent antibody profiles to plasmodium antigens are differentially associated with two artemisinin combination therapy outcomes in high transmission setting.

The impact of pre-existing immunity on the efficacy of artemisinin combination therapy is largely unknown. We performed in-depth profiling of serological responses in a therapeutic efficacy study [comparing artesunate-mefloquine (ASMQ) and artemether-lumefantrine (AL)] using a proteomic microarray. Responses to over 200 Plasmodium antigens were significantly associated with ASMQ treatment outcome but not AL. We used machine learning to develop predictive models of treatment outcome based on the immunoprofile data. The models predict treatment outcome for ASMQ with high (72-85%) accuracy, but could not predict treatment outcome for AL. This divergent treatment outcome suggests that humoral immunity may synergize with the longer mefloquine half-life to provide a prophylactic effect at 28-42 days post-treatment, which was further supported by simulated pharmacokinetic profiling. Our computational approach and modeling revealed the synergistic effect of pre-existing immunity in patients with drug combination that has an extended efficacy on providing long term treatment efficacy of ASMQ.

Synergism in Antiplasmodial Activities of Artemether and Lumefantrine in Combination with Securidaca longipedunculata Fresen (Polygalaceae).

Malaria is the most lethal parasitic disease in the world. The frequent emergence of resistance by malaria parasites to any drug is the hallmark of sustained malaria burden. Since the deployment of artemisinin-based combination therapies (ACTs) it is clear that for a sustained fight against malaria, drug combination is one of the strategies toward malaria elimination. In Sub-Saharan Africa where malaria prevalence is the highest, the identification of plants with a novel mechanism of action that is devoid of cross-resistance is a feasible strategy in drug combination therapy. Thus, artemether and lumefantrine were separately combined and tested with extracts of Securidaca longipedunculata, a plant widely used to treat malaria, at fixed extract-drug ratios of 4:1, 3:1, 1:1, 1:2, 1:3, and 1:4. These combinations were tested for antiplasmodial activity against three strains of Plasmodium falciparum (W2, D6, and DD2), and seven field isolates that were characterized for molecular and ex vivo drug resistance profiles. The mean sum of fifty-percent fractional inhibition concentration (FIC50) of each combination and singly was determined. Synergism was observed across all fixed doses when roots extracts were combined with artemether against D6 strain (FIC50 0.403 ± 0.068) and stems extract combined with lumefantrine against DD2 strain (FIC50 0.376 ± 0.096) as well as field isolates (FIC50 0.656 ± 0.067). Similarly, synergism was observed in all ratios when leaves extract were combined with lumefantrine against W2 strain (FIC50 0.456 ± 0.165). Synergism was observed in most combinations indicating the potential use of S. longipedunculata in combination with artemether and lumefantrine in combating resistance.

Plasmodium interspecies interactions during a period of increasing prevalence of Plasmodium ovale in symptomatic individuals seeking treatment: an observational study.

BACKGROUND: The epidemiology and severity of non-falciparum malaria in endemic settings has garnered little attention. We aimed to characterise the prevalence, interaction, clinical risk factors, and temporal trends of non-falciparum Plasmodium species among symptomatic individuals presenting at health-care facilities in endemic settings of Kenya. METHODS: We diagnosed and analysed infecting malaria species (Plasmodium falciparum, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, and Plasmodium malariae) via PCR in clinical samples collected between March 1, 2008, and Dec 31, 2016, from six hospitals located in different regions of Kenya. We recruited patients aged 6 months or older who presented at outpatient departments with symptoms of malaria or tested positive for uncomplicated malaria by malaria rapid diagnostic test. Descriptive statistics were used to describe the prevalence and distribution of Plasmodium species. A statistical model was designed and used for estimating the frequency of Plasmodium species and assessing interspecies interactions. Mixed-effect linear regression models with random slopes for each location were used to test for change in prevalence over time. FINDINGS: Samples from 2027 symptomatic participants presenting at care facilities were successfully analysed for all Plasmodium species. 1469 (72·5%) of the samples were P falciparum single-species infections, 523 (25·8%) were mixed infections, and only 35 (1·7%) were single non-falciparum species infections. 452 (22·3%) were mixed infections containing P ovale spp. A likelihood-based model calculation of the population frequency of each species estimated a significant within-host interference between P falciparum and P ovale curtisi. Mixed-effect logistic regression models identified a significant increase in P ovale wallikeri (2·1% per year; p=0·043) and P ovale curtisi (0·7% per year; p=0·0002) species over time, with a reciprocal decrease in P falciparum single-species infections (2·5% per year; p=0·0065). The frequency of P malariae infections did not significantly change over time. Risk of P falciparum infections presenting with fever was lower if co-infected with P malariae (adjusted odds ratio 0·43, 95% CI 0·25-0·74; p=0·0023). INTERPRETATION: Our results show a prevalence of non-falciparum species infections of 27·5% among symptomatic individuals presenting at care facilities, which is higher than expected from previous cross-sectional surveys. The proportion of infections with P ovale wallikeri and P ovale curtisi was observed to significantly increase over the period of study, which could be due to attenuated responsiveness of these species to malaria drug treatment. The increase in frequency of P ovale spp could threaten the malaria control efforts in Kenya and pose increased risk of malaria to travellers. FUNDING: Armed Forces Health Surveillance Branch and its Global Emerging Infections Surveillance Section.

A seven-year surveillance of epidemiology of malaria reveals travel and gender are the key drivers of dispersion of drug resistant genotypes in Kenya.

Malaria drug resistance is a global public health concern. Though parasite mutations have been associated with resistance, other factors could influence the resistance. A robust surveillance system is required to monitor and help contain the resistance. This study established the role of travel and gender in dispersion of chloroquine resistant genotypes in malaria epidemic zones in Kenya. A total of 1,776 individuals presenting with uncomplicated malaria at hospitals selected from four malaria transmission zones in Kenya between 2008 and 2014 were enrolled in a prospective surveillance study assessing the epidemiology of malaria drug resistance patterns. Demographic and clinical information per individual was obtained using a structured questionnaire. Further, 2 mL of blood was collected for malaria diagnosis, parasitemia quantification and molecular analysis. DNA extracted from dried blood spots collected from each of the individuals was genotyped for polymorphisms in Plasmodium falciparum chloroquine transporter gene (Pfcrt 76), Plasmodium falciparum multidrug resistant gene 1 (Pfmdr1 86 and Pfmdr1 184) regions that are putative drug resistance genes using both conventional polymerase chain reaction (PCR) and real-time PCR. The molecular and demographic data was analyzed using Stata version 13 (College Station, TX: StataCorp LP) while mapping of cases at the selected geographic zones was done in QGIS version 2.18. Chloroquine resistant (CQR) genotypes across gender revealed an association with chloroquine resistance by both univariate model (p = 0.027) and by multivariate model (p = 0.025), female as reference group in both models. Prior treatment with antimalarial drugs within the last 6 weeks before enrollment was associated with carriage of CQR genotype by multivariate model (p = 0.034). Further, a significant relationship was observed between travel and CQR carriage both by univariate model (p = 0.001) and multivariate model (p = 0.002). These findings suggest that gender and travel are significantly associated with chloroquine resistance. From a gender perspective, males are more likely to harbor resistant strains than females hence involved in strain dispersion. On the other hand, travel underscores the role of transport network in introducing spread of resistant genotypes, bringing in to focus the need to monitor gene flow and establish strategies to minimize the introduction of resistance strains by controlling malaria among frequent transporters.