Antiviral Rotenoids and Isoflavones Isolated from Millettia oblata ssp. teitensis.

Three new (1-3) and six known rotenoids (5-10), along with three known isoflavones (11-13), were isolated from the leaves of Millettia oblata ssp. teitensis. A new glycosylated isoflavone (4), four known isoflavones (14-18), and one known chalcone (19) were isolated from the root wood extract of the same plant. The structures were elucidated by NMR and mass spectrometric analyses. The absolute configuration of the chiral compounds was established by a comparison of experimental ECD and VCD data with those calculated for the possible stereoisomers. This is the first report on the use of VCD to assign the absolute configuration of rotenoids. The crude leaves and root wood extracts displayed anti-RSV (human respiratory syncytial virus) activity with IC50 values of 0.7 and 3.4 µg/mL, respectively. Compounds 6, 8, 10, 11, and 14 showed anti-RSV activity with IC50 values of 0.4-10 µM, while compound 3 exhibited anti-HRV-2 (human rhinovirus 2) activity with an IC50 of 4.2 µM. Most of the compounds showed low cytotoxicity for laryngeal carcinoma (HEp-2) cells; however compounds 3, 11, and 14 exhibited low cytotoxicity also in primary lung fibroblasts. This is the first report on rotenoids showing antiviral activity against RSV and HRV viruses.

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.

Distinctive Kaposi Sarcoma-Associated Herpesvirus Serological Profile during Acute Plasmodium falciparum Malaria Episodes.

The seroprevalence of Kaposi sarcoma-associated herpesvirus (KSHV) and the incidence of endemic Kaposi sarcoma (KS) overlap with regions of malaria endemicity in sub-Saharan Africa. Multiple studies have shown an increased risk of KSHV seroconversion in children from high malaria compared to low malaria regions; however, the impact of acute episodes of Plasmodium falciparum (P. falciparum) malaria on KSHV's biphasic life cycle and lytic reactivation has not been determined. Here, we examined KSHV serological profiles and viral loads in 134 children with acute malaria and 221 healthy children from high malaria regions in Kisumu, as well as 77 healthy children from low malaria regions in Nandi. We assayed KSHV, Epstein-Barr virus (EBV), and P. falciparum malaria antibody responses in these three by multiplexed Luminex assay. We confirmed that KSHV seroprevalence was significantly associated with malaria endemicity (OR = 1.95, 1.18-3.24 95% CI, p = 0.01) with 71-77% seropositivity in high-malaria (Kisumu) compared to 28% in low-malaria (Nandi) regions. Furthermore, KSHV serological profiles during acute malaria episodes were distinct from age-matched non-malaria-infected children from the same region. Paired IgG levels also varied after malaria treatment, with significantly higher anti-ORF59 at day 0 but elevated ORF38, ORF73, and K8.1 at day 3. Acute malaria episodes is characterized by perturbation of KSHV latency in seropositive children, providing further evidence that malaria endemicity contributes to the observed increase in endemic KS incidence in sub-Saharan Africa.

The emerging paradigm of calcium homeostasis as a new therapeutic target for protozoan parasites.

Calcium channels (CCs), a group of ubiquitously expressed membrane proteins, are involved in many pathophysiological processes of protozoan parasites. Our understanding of CCs in cell signaling, organelle function, cellular homeostasis, and cell cycle control has led to improved insights into their structure and functions. In this article, we discuss CCs characteristics of five major protozoan parasites Plasmodium, Leishmania, Toxoplasma, Trypanosoma, and Cryptosporidium. We provide a comprehensive review of current antiparasitic drugs and the potential of using CCs as new therapeutic targets. Interestingly, previous studies have demonstrated that human CC modulators can kill or sensitize parasites to antiparasitic drugs. Still, none of the parasite CCs, pumps, or transporters has been validated as drug targets. Information for this review draws from extensive data mining of genome sequences, chemical library screenings, and drug design studies. Parasitic resistance to currently approved therapeutics is a serious and emerging threat to both disease control and management efforts. In this article, we suggest that the disruption of calcium homeostasis may be an effective approach to develop new anti-parasite drug candidates and reduce parasite resistance.

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.

The role of complement immune response on artemisinin-based combination therapy in a population from malaria endemic region of Western Kenya.

BACKGROUND: Naturally acquired immunity (NAI), which is characterized by protection against overt clinical disease and high parasitaemia, is acquired with age and transmission intensity. The role of NAI on the efficacy of anti-malarial drugs, including artemisinin-based combinations used as the first-line treatment for uncomplicated Plasmodium falciparum, has not been fully demonstrated. This study investigated the role of NAI in response to artemisinin-based combination therapy (ACT), in symptomatic patients living in western Kenya, a high malaria transmission area. METHODS: Sera samples from malaria immune participants (n = 105) in a therapeutic efficacy study were assessed for in vitro growth inhibitory activity against the 3D7 strain of P. falciparum using a fluorescent-based growth inhibition assay (GIA). Participants' age and parasite clearance parameters were used in the analysis. Pooled sera from malaria naïve participants (n = 6) with no Plasmodium infection from malaria non-endemic regions of Kenya was used as negative control. RESULTS: The key observations of the study were as follows: (1) Sera with intact complement displayed higher GIA activity at lower (1%) serum dilutions (p < 0.0001); (2) there was significant relationship between GIA activity, parasite clearance rate (p = 0.05) and slope half-life (p = 0.025); and (3) age was a confounding factor when comparing the GIA activity with parasite clearance kinetics. CONCLUSION: This study demonstrates for the first time there is synergy of complement, pre-existing immunity, and drug treatment in younger patients with symptomatic malaria in a high-transmission area.

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

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

Four Prenylflavone Derivatives with Antiplasmodial Activities from the Stem of Tephrosia purpurea subsp. leptostachya.

Four new flavones with modified prenyl groups, namely (E)-5-hydroxytephrostachin (1), purleptone (2), (E)-5-hydroxyanhydrotephrostachin (3), and terpurlepflavone (4), along with seven known compounds (5-11), were isolated from the CH2Cl2/MeOH (1:1) extract of the stem of Tephrosia purpurea subsp. leptostachya, a widely used medicinal plant. Their structures were elucidated on the basis of NMR spectroscopic and mass spectrometric evidence. Some of the isolated compounds showed antiplasmodial activity against the chloroquine-sensitive D6 strains of Plasmodium falciparum, with (E)-5-hydroxytephrostachin (1) being the most active, IC50 1.7 ± 0.1 µM, with relatively low cytotoxicity, IC50 > 21 µM, against four cell-lines.

Assessment of the Worldwide Antimalarial Resistance Network Standardized Procedure for In Vitro Malaria Drug Sensitivity Testing Using SYBR Green Assay for Field Samples with Various Initial Parasitemia Levels.

The malaria SYBR green assay, which is used to profilein vitrodrug susceptibility ofPlasmodium falciparum, is a reliable drug screening and surveillance tool. Malaria field surveillance efforts provide isolates with various low levels of parasitemia. To be advantageous, malaria drug sensitivity assays should perform reproducibly among various starting parasitemia levels rather than at one fixed initial value. We examined the SYBR green assay standardized procedure developed by the Worldwide Antimalarial Resistance Network (WWARN) for its sensitivity and ability to accurately determine the drug concentration that inhibits parasite growth by 50% (IC50) in samples with a range of initial parasitemia levels. The initial sensitivity determination of the WWARN procedure yielded a detection limit of 0.019% parasitemia.P. falciparumlaboratory strains and field isolates with various levels of initial parasitemia were then subjected to a range of doses of common antimalarials. The IC50s were comparable for laboratory strains with between 0.0375% and 0.6% parasitemia and for field isolates with between 0.075% and 0.6% parasitemia for all drugs tested. Furthermore, assay quality (Z') analysis indicated that the WWARN procedure displays high robustness, allowing for drug testing of malaria field samples within the derived range of initial parasitemia. The use of the WWARN procedure should allow for the inclusion of more malaria field samples in malaria drug sensitivity screens that would have otherwise been excluded due to low initial parasitemia levels.

Molecular characterization of the cytochrome b gene and in vitro atovaquone susceptibility of Plasmodium falciparum isolates from Kenya.

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

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.

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.

The Walter Reed Project, Kisumu Field Station: Impact of Research on Malaria Policy, Management, and Prevention.

The Walter Reed Project is a collaboration between the Walter Reed Army Institute of Research of the United States Department of Defense and the Kenya Medical Research Institute. The Kisumu field station, comprising four campuses, has until recently been devoted primarily to research on malaria countermeasures. The Kombewa Clinical Research Center is dedicated to conducting regulated clinical trials of therapeutic and vaccine candidates in development. The center's robust population-based surveillance platform, along with an active community engagement strategy, guarantees consistent recruitment and retention of participants in clinical trials. The Malaria Diagnostic Center, backed by WHO-certified microscopists and a large malaria blood film collection, champions high-quality malaria diagnosis and strict quality assurance through standardized microscopy trainings. The Malaria Drug Resistance Laboratory leverages cutting-edge technology such as real-time Polymerase Chain Reaction (qPCR) to conduct comprehensive research on resistance markers and obtain information on drug efficacy. The laboratory has been working on validating artemisinin resistance markers and improving tracking methods for current and future antimalarial compounds. Finally, the Basic Science Laboratory employs advanced genomic technology to examine endpoints such as immunogenicity and genomic fingerprinting for candidate drugs and vaccine efficacy. Herein, we examine the site's significant contributions to malaria policy, management, and prevention practices in Kenya and around the world.

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.