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.

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.

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.

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.

Early sexual debut is associated with drug use and decreased educational attainment among males and females in Kisumu County, Kenya.

Differing global sociocultural contexts of sexual relationships influence age at first sexual intercourse with potentially long-lasting region-specific effects such as increased risk of contracting HIV and other sexually transmitted infections (STIs). In these cross-sectional analyses of data from the screening and enrollment visits for an HIV incidence study in Kisumu County, Kenya, we evaluated factors associated with having experienced an early sexual debut (ESD) among males and females aged 18-35 years. Clinical evaluation was performed and sexual behaviors were assessed via questionnaire. ESD was defined as self-reported age 15 years or younger at first sexual intercourse. Robust Poisson regression was used to estimate prevalence ratios (PRs) and 95% confidence intervals (95% CIs) for factors associated with ESD. Of 1057 participants, 542 (51.3%) were female. Participants' median age at study screening was 25 years (interquartile range [IQR]: 22-29), and at sexual debut was 16 years (IQR: 14-17). Five hundred and four participants (47.7%) reported ESD. ESD was less common among females (PR 0.78, CI 0.67-0.90) and participants with more than primary education (PR 0.56, CI 0.47-0.66). ESD was more common in participants with a history of drug use (PR 1.28, CI 1.10-1.49). Drug use removed the protective effect of education (some secondary education or less, no drug use: PR 0.72, CI 0.61-0.85; some secondary education or less, drug use: PR 0.94, CI 0.74-1.18). ESD was common in our study and associated with lower educational attainment and increased likelihood of drug use. Interventions are needed early in life, well before 15 years of age, to encourage engagement in schooling and prevent drug use. Comprehensive sexual education and interventions to prevent drug use may be beneficial before the age of 15 years.

Early sexual debut can be defined as first sexual intercourse at or before 15 years of age. There are many social and cultural factors that influence the age of sexual debut. People who start having sex early in life may exhibit behaviors that increase risk for HIV and other sexually transmitted infections. We conducted a study of men and women aged 18­35 years in Kisumu County, Kenya, which included documentation of medical history, physical examination, laboratory tests, and a questionnaire to assess sexual behaviors. Among the 1057 people studied, the average age of sexual debut was 16.0 years for females and 15.4 years for males. A total of 504 (47.7%) participants reported early sexual debut. The data showed that early sexual debut was less common in females and in participants with more years of education. Early sexual debut was more common in participants with a history of drug use. The findings suggest that interventions to prevent early sexual debut might be improved if they focus on educational attainment and prevention of drug use.

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.

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.

Pf7: an open dataset of Plasmodium falciparum genome variation in 20,000 worldwide samples.

We describe the MalariaGEN Pf7 data resource, the seventh release of Plasmodium falciparum genome variation data from the MalariaGEN network.  It comprises over 20,000 samples from 82 partner studies in 33 countries, including several malaria endemic regions that were previously underrepresented.  For the first time we include dried blood spot samples that were sequenced after selective whole genome amplification, necessitating new methods to genotype copy number variations.  We identify a large number of newly emerging crt mutations in parts of Southeast Asia, and show examples of heterogeneities in patterns of drug resistance within Africa and within the Indian subcontinent.  We describe the profile of variations in the C-terminal of the csp gene and relate this to the sequence used in the RTS,S and R21 malaria vaccines.  Pf7 provides high-quality data on genotype calls for 6 million SNPs and short indels, analysis of large deletions that cause failure of rapid diagnostic tests, and systematic characterisation of six major drug resistance loci, all of which can be freely downloaded from the MalariaGEN website.

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.

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.

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.

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.

A new benzophenone, and the antiplasmodial activities of the constituents of Securidaca longipedunculata fresen (Polygalaceae).

Extracts from Securidaca longipedunculata showed antiplasmodial activities against reference clones and clinical isolates using SYBR Green I method. A new benzophenone, 2,3,4,5-tetramethoxybenzophenone (1) was isolated and characterized along with seven known compounds: 4-hydroxy-2,3-dimethoxybenzophenone (2); 3-hydroxy-5-methoxybiphenyl (3), methyl-2-hydroxy-6-methoxybenzoate (4), benzyl-2-hydroxy-6-methoxybenzoate (5), 2-hydroxy-6-methoxybenzoic acid (6), 2,4,5-trimethoxybenzophenone (7) and 2-methoxy-3,4-methylenedioxybenzophenone (8). Compounds 1 and 2 showed ex vivo antiplasmodial activities (IC50 28.8 µM and 18.6 µM, respectively); while 5 and 8 showed in vivo activities (IC50 19.7 µM and 14.5 µM, respectively) against D6 strain. In a cytotoxicity assay, all the extracts (with an exception of the MeOH extract of the leaves) and pure compounds were not toxic to the normal LO2 and BEAS cell-lines, while the methanol roots extract (IC50 66.4 µg/mL against A549, and 77.4 µg/mL against HepG2), compounds 6 (IC50 22.2 µM against A549) and 7 (IC50 45.2 µM against HepG2) were weakly active against cancerous cell-lines.

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.

An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples.

MalariaGEN is a data-sharing network that enables groups around the world to work together on the genomic epidemiology of malaria. Here we describe a new release of curated genome variation data on 7,000 Plasmodium falciparum samples from MalariaGEN partner studies in 28 malaria-endemic countries. High-quality genotype calls on 3 million single nucleotide polymorphisms (SNPs) and short indels were produced using a standardised analysis pipeline. Copy number variants associated with drug resistance and structural variants that cause failure of rapid diagnostic tests were also analysed.  Almost all samples showed genetic evidence of resistance to at least one antimalarial drug, and some samples from Southeast Asia carried markers of resistance to six commonly-used drugs. Genes expressed during the mosquito stage of the parasite life-cycle are prominent among loci that show strong geographic differentiation. By continuing to enlarge this open data resource we aim to facilitate research into the evolutionary processes affecting malaria control and to accelerate development of the surveillance toolkit required for malaria elimination.

A review of the frequencies of Plasmodium falciparum Kelch 13 artemisinin resistance mutations in Africa.

Artemisinin resistance (AR) emerged in South East Asia 13 years ago and the identification of the resistance conferring molecular marker, Plasmodium falciparum Kelch 13 (Pfk13), 7 years ago has provided an invaluable tool for monitoring AR in malaria endemic countries. Molecular Pfk13 surveillance revealed the resistance foci in the Greater Mekong Subregion, an independent emergence in Guyana, South America, and a low frequency of mutations in Africa. The recent identification of the R561H Pfk13 AR associated mutation in Tanzania, Uganda and in Rwanda, where it has been associated with delayed parasite clearance, should be a concern for the continent. In this review, we provide a summary of Pfk13 resistance associated propeller domain mutation frequencies across Africa from 2012 to 2020, to examine how many other countries have identified these mutations. Only four African countries reported a recent identification of the M476I, P553L, R561H, P574L, C580Y and A675V Pfk13 mutations at low frequencies and with no reports of clinical treatment failure, except for Rwanda. These mutations present a threat to malaria control across the continent, since the greatest burden of malaria remains in Africa. A rise in the frequency of these mutations and their spread would reverse the gains made in the reduction of malaria over the last 20 years, given the lack of new antimalarial treatments in the event artemisinin-based combination therapies fail. The review highlights the frequency of Pfk13 propeller domain mutations across Africa, providing an up-to-date perspective of Pfk13 mutations, and appeals for an urgent and concerted effort to monitoring antimalarial resistance markers in Africa and the efficacy of antimalarials by re-establishing sentinel surveillance systems.

Clinical laboratory reference values in adults in Kisumu County, Western Kenya; hematology, chemistry and CD4.

BACKGROUND: Clinical laboratory reference intervals (RIs) are essential for diagnosing and managing patients in routine clinical care as well as establishing eligibility criteria and defining adverse events in clinical trials, but may vary by age, gender, genetics, nutrition and geographic location. It is, therefore, critical to establish region-specific reference values in order to inform clinical decision-making. METHODS: We analyzed data from a prospective observational HIV incidence cohort study in Kombewa, Kenya. Study participants were healthy males and females, aged 18-35 years, without HIV. Median and 95% reference values (2.5th percentile to 97.5th percentile) were calculated for laboratory parameters including hematology, chemistry studies, and CD4 T cell count. Standard Deviation Ratios (SDR) and Bias Ratios (BR) are presented as measures of effect magnitude. Findings were compared with those from the United States and other Kenyan studies. RESULTS: A total of 299 participants were analyzed with a median age of 24 years (interquartile range: 21-28). Ratio of males to females was 0.9:1. Hemoglobin range (2.5th-97.5th percentiles) was 12.0-17.9 g/dL and 9.5-15.3 g/dL in men and women respectively. In the cohort, MCV range was 59-95fL, WBC 3.7-9.2×103/µL, and platelet 154-401×103/µL. Chemistry values were higher in males; the creatinine RI was 59-103 µmol/L in males vs. 46-76 µmol/L in females (BRUL>.3); and the alanine transferase range was 8.8-45.3 U/L in males vs. 7.5-36.8 U/L in females (SDR>.3). The overall CD4 T cell count RI was 491-1381 cells/µL. Some parameters including hemoglobin, neutrophil, creatinine and ALT varied with that from prior studies in Kenya and the US. CONCLUSION: This study not only provides clinical reference intervals for a population in Kisumu County but also highlights the variations in comparable settings, accentuating the requirement for region-specific reference values to improve patient care, scientific validity, and quality of clinical trials in Africa.

Clinical laboratory hematology reference values among infants aged 1month to 17 months in Kombewa Sub-County, Kisumu: A cross sectional study of rural population in Western Kenya.

There is an urgent need for reliable region-specific hematological reference values for clinical monitoring. Laboratory reference ranges are important for assessing study participant eligibility, toxicity grading and management of adverse events in clinical trials and clinical diagnosis. Most clinical laboratories in Kenya rely on hematological reference values provided by instrument manufacturers and/or textbooks, which are based on population from Europe or North America. The use of such values in medical practice could result in improper patient management, selection bias in selection of appropriate participants for clinical trials and flawed classification of the clinical adverse events when applied to African populations. The aim of this study was to establish local laboratory hematological reference values in infants aged 1 month to 17 months from Kombewa Sub-county that could be true representative of the existing rural population. The study participants in the current study were those who had previously been recruited from GSK-sponsored study. This study was a phase III, Double Blind, Randomized, GSK-sponsored, Malaria Vaccine Clinical Trial that was conducted in infants aged 1month to 17months. 1,509 participants were included in the study analysis. Data were partitioned into 3 different age groups (1-6 months[m], 6-12 m and 12-17 m) and differences between gender were compared within each group. Data were analyzed using Graphpad prism V5 to generate 95% reference ranges (2.5th-97.5th percentile). There was evidence of gender differences in hemoglobin values (p = 0.0189) and platelet counts (p = 0.0005) in the 1 to 6m group. For the 12-17m group, there were differences in MCV (p<0.0001) and MCH (p = 0.0003). Comparing gender differences for all age groups, differences were noted in percent lymphocytes (p = 0.0396), percent monocytes (p = 0.0479), percent granulocytes (p = 0.0044), hemoglobin (p = 0.0204), hematocrit (p = 0.0448), MCV (p = 0.0092), MCH (p = 0.0089), MCHC (p = 0.0336) and absolute granulocytes (p = 0.0237). In 1 to 6m age group and all age groups assessed, for WBCs, hemoglobin, hematocrit, MCV and lymphocytes absolute counts, both 2.5th and 97.5th percentiles for Kisumu infants were higher than those from Kilifi. Platelet ranges for Kisumu children were narrower compared to Kilifi ranges. Kisumu hematology reference ranges were observed to be higher than the ranges of Tanzanian children for the WBCs, absolute lymphocyte and monocyte counts, hemoglobin, hematocrit and MCV. Higher ranges of WBCs, absolute lymphocyte and monocyte counts were observed compared to the values in US/Europe. Wider ranges were observed in hemoglobin, hematocrit, and MCV. Wider ranges were observed in platelet counts in Kisumu infants compared to the US/Europe ranges. Compared to Harriet Lane Handbook reference values that are used in the area, higher counts were observed in WBC counts, both absolute and percent lymphocyte counts, as well as monocyte counts for current study. Wider ranges were observed in RBC, platelets and RDW, while lower ranges noted in the current study for hemoglobin, hematocrit and granulocyte counts. This study underscores the importance of using locally established hematology reference ranges of different age groups in support of proper patient management and for clinical trials.

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.