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.

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.

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.

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.

Characterization of sulfated polysaccharide activity against virulent Plasmodium falciparum PHISTb/RLP1 protein.

Background: The emergence of artemisinin resistance in South East Asia calls for urgent discovery of new drug compounds that have antiplasmodial activity. Unlike the classical compound screening drug discovery methods, the rational approach involving targeted drug discovery is less cumbersome and therefore key for innovation of new antiplasmodial compounds.  Plasmodium falciparum (Pf) utilizes the process of host erythrocyte remodeling using Plasmodium-helical interspersed sub-telomeric domain (PHIST) containing proteins, which are amenable drug targets. The aim of this study is to identify inhibitors of PHIST from sulfated polysaccharides as new antimalarials. Methods: 251 samples from an ongoing study of epidemiology of malaria and drug resistance sensitivity patterns in Kenya were sequenced for PHISTb/RLP1 gene using Sanger sequencing. The sequenced reads were mapped to the reference Pf3D7 protein sequence of PHISTb/RLP1 using CLC Main Workbench. Homology modeling of both reference and mutant protein structures was achieved using the LOMETs tool. The models were refined using ModRefiner for energy minimization. Ramachandran plot was generated by ProCheck to assess the conformation of amino acids in the protein model. Protein binding sites predictions were assessed using FT SITE software. We searched for prospective antimalarials from PubChem. Docking experiments were achieved using AutoDock Vina and analysis results visualized in PyMOL. Results: Sanger sequencing generated 86 complete sequences. Upon mapping of the sequences to the reference, 12 non-synonymous single nucleotide polymorphisms were considered for mutant protein structure analysis. Eleven drug compounds with antiplasmodial activity were identified. Both modelled PHISTb/RLP1 reference and mutant structures had a Ramachandran score of >90% of the amino acids in the favored region. Ten of the drug compounds interacted with amino acid residues in PHISTb and RESA domains, showing potential activity against these proteins. Conclusion: These interactions provide lead compounds for new anti-malarial molecules. Further in vivo testing is recommended.