Genetic variation in interleukin-7 is associated with a reduced erythropoietic response in Kenyan children infected with Plasmodium falciparum.

BACKGROUND: Severe malarial anemia (SMA) is a leading cause of malaria-related morbidity and mortality in children. The genetic factors that influence development of SMA and inefficient erythropoiesis, a central pathogenic feature of SMA, are only partially understood. METHODS: We performed a pilot Genome-wide Association Study (GWAS) on children with Plasmodium falciparum. The GWAS was performed using the Illumina® Infinium® HD Super Assay in conjunction with Illumina's® Human Omni2.5-8v1 BeadChip (with > 2.45 M markers). Data were analyzed using single SNP logistic regression analysis with an additive model of inheritance controlling for covariates. Results from our pilot global genomics study identified that variation in interleukin (IL)-7 was associated with enhanced risk of SMA. To validate this finding, we investigated the relationship between genotypes and/or haplotypes of two single nucleotide polymorphisms (SNPs) in IL7 [72194 T/C and - 2440 A/G] and susceptibility to both SMA and inefficient erythropoiesis [i.e., reticulocyte production index (RPI) < 2.0 in anemic children (Hb < 11.0 g/dL). Children presenting with P. falciparum malaria (< 3 years, n = 883) were stratified into two groups: Uncomplicated malaria (UM, n = 718) and SMA (n = 165). RESULTS: Regression modeling, controlling for anemia-related confounders, revealed that carriage of the TC genotype at position 72194 T/C was associated with enhanced susceptibility to inefficient erythropoiesis (OR = 1.90; 95% CI 1.09-3.30; P = 0.02) as was homozygous CC (OR 5.14; 95% CI = 1.20-21.99; P = 0.03). Consistent with this finding, individuals with the CA (72194C/-2440A) haplotype had an increased risk of inefficient erythropoiesis (OR = 1.90; 95% CI = 1.10-3.30; P = 0.02), whereas TA haplotype carriers had marginal protection against inefficient erythropoiesis (OR = 0.24; 95% CI = 0.06-1.21; P = 0.05). These observations were supported by Cochran-Armitage trend test for inefficient erythropoiesis (CA > TA > CG; P < 0.01). Although none of the genotype and/or haplotypic variants were significantly associated with SMA, the direction of the risk profiles were consistent with the erythropoiesis results. CONCLUSION: Taken together, variation in IL7 is associated with erythropoietic responses in children with falciparum malaria, a central physiological feature contributing to development of SMA.

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.

Cigarette Smoking Behaviors and Beliefs in Persons Living With HIV in Nairobi, Kenya.

INTRODUCTION: Persons living with HIV (PLWH) use tobacco at higher rates than the general population in both high-income countries and low- and middle-income countries. Tobacco use rates are increasing in sub-Saharan Africa, the home to most of the world's PLWH. As the reach of antiretroviral therapy (ART) expands and HIV-related morbidity and mortality wanes, tobacco use is emerging as a leading cause of disease and death in PLWH. A better understanding of tobacco use behaviors in various settings will be crucial to designing optimal tobacco control strategies. METHODS: In late 2019, we enrolled 50 PLWH cigarette smokers from 6 clinical sites in Nairobi, Kenya (4 HIV care clinics and 2 methadone maintenance programs) for one-on-one interviews focusing on their behaviors and beliefs related to tobacco use. RESULTS: Fifty PLWH smokers completed the interviews. The mean age was 38.5 ± 9.7 years (range 20-57 years) and 68% were male. All were currently receiving ART. They smoked a mean of 14.9 ± 12.4 cigarettes per day, and 82% reported smoking every day. Only 6% reported dual use of smokeless tobacco products. Nicotine dependence was moderate or high in 74%. More than a third (36%) reported a prior history of tuberculosis. In our sample, use of other substances was common, especially alcohol, marijuana, and methadone. On the motivation to quit scale, 90% were at least in the contemplation stage, but only 2% had ever received behavioral cessation counseling, and only 8% had ever used pharmacotherapy (exclusively nicotine replacement therapy). Participants reported significant concern about developing smoking-related illness, exposing others to secondary smoke, and the financial burden associated with their tobacco use. Measures of intrinsic and extrinsic motivation to quit, smoker and abstainer self-concept, and social support yielded encouraging results regarding the possibility of successful quitting. CONCLUSIONS: Tobacco use is an important health concern in PLWH in Kenya. A more thorough understanding of their tobacco use behaviors and beliefs will provide critical information for providers, public health officials, and policy makers as they redouble their efforts to confront this urgent health challenge.

Author Correction: Synergistic blending of high-valued heterocycles inhibits growth of Plasmodium falciparum in culture and P. berghei infection in mouse model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Molecular basis of reduced LAIR1 expression in childhood severe malarial anaemia: Implications for leukocyte inhibitory signalling.

BACKGROUND: Leukocyte-associated immunoglobulin like receptor-1 (LAIR1) is a transmembrane inhibitory receptor that influences susceptibility to a myriad of inflammatory diseases. Our recent investigations of severe malarial anaemia (SMA) pathogenesis in Kenyan children discovered that novel LAIR1 genetic variants which were associated with decreased LAIR1 transcripts enhanced the longitudinal risk of SMA and all-cause mortality. METHODS: To characterize the molecular mechanism(s) responsible for altered LAIR1 signalling in severe malaria, we determined LAIR1 transcripts and protein, sLAIR1, sLAIR2, and complement component 1q (C1q) in children with malarial anaemia, followed by a series of in vitro experiments investigating the LAIR1 signalling cascade. FINDINGS: Kenyan children with SMA had elevated circulating levels of soluble LAIR1 (sLAIR1) relative to non-SMA (1.69-fold P < .0001). The LAIR1 antagonist, sLAIR2, was also elevated in the circulation of children with SMA (1.59 fold-change, P < .0001). There was a positive correlation between sLAIR1 and sLAIR2 (ρ = 0.741, P < .0001). Conversely, circulating levels of complement component 1q (C1q), a LAIR1 natural ligand, were lower in SMA (-1.21-fold P = .048). These in vivo findings suggest that reduced membrane-bound LAIR1 expression in SMA is associated with elevated production of sLAIR1, sLAIR2 (antagonist), and limited C1q (agonist) availability. Since reduced LAIR1 transcripts in SMA were associated with increased acquisition of haemozoin (PfHz) by monocytes (P = .028), we explored the relationship between acquisition of intraleukocytic PfHz, LAIR1 expression, and subsequent impacts on leukocyte signalling in cultured PBMCs from malaria-naïve donors stimulated with physiological concentrations of PfHz (10 µg/mL). Phagocytosis of PfHz reduced LAIR1 transcript and protein expression in a time-dependent manner (P < .050), and inhibited LAIR1 signalling through decreased phosphorylation of LAIR1 (P < .0001) and SH2-domain containing phosphatase-1 (SHP-1) (P < .001). This process was associated with NF-κB activation (P < .0001) and enhanced production of IL-6, IL-1ß, and TNF-α (all P < .0001). INTERPRETATION: Collectively, these findings demonstrate that SMA is characterized by reduced LAIR1 transmembrane expression, reduced C1q, and enhanced production of sLAIR1 and sLAIR2, molecular events which can promote enhanced production of cytokines that contribute to the pathogenesis of SMA. These investigations are important for discovering immune checkpoints that could be future targets of immunotherapy to improve disease outcomes.

Antimalarials: Review of Plasmepsins as Drug Targets and HIV Protease Inhibitors Interactions.

Malaria is a major global health concern with the majority of cases reported in regions of South-East Asia, Eastern Mediterranean, Western Pacific, the Americas, and Sub-Saharan Africa. The World Health Organization (WHO) estimated 216 million worldwide reported cases of malaria in 2016. It is an infection of the red blood cells by parasites of the genus Plasmodium with most severe and common forms caused by Plasmodium falciparum (P. falciparum or Pf) and Plasmodium vivax (P. vivax or Pv). Emerging parasite resistance to available antimalarial drugs poses great challenges to treatment. Currently, the first line of defense includes artemisinin combination therapies (ACTs), increasingly becoming less effective and challenging to combat new occurrences of drug-resistant parasites. This necessitates the urgent need for novel antimalarials that target new molecular pathways with a different mechanism of action from the traditional antimalarials. Several new inhibitors and potential drug targets of the parasites have been reported over the years. This review focuses on the malarial aspartic proteases known as plasmepsins (Plms) as novel drug targets and antimalarials targeting Plms. It further discusses inhibitors of hemoglobin-degrading plasmepsins Plm I, Plm II, Plm IV and Histo-aspartic proteases (HAP), as well as HIV protease inhibitors of plasmepsins.

Integrated OMICS platforms identify LAIR1 genetic variants as novel predictors of cross-sectional and longitudinal susceptibility to severe malaria and all-cause mortality in Kenyan children.

BACKGROUND: Severe malarial anaemia (SMA) is a leading cause of childhood mortality in holoendemic Plasmodium falciparum regions. METHODS: To gain an improved understanding of SMA pathogenesis, whole genome and transcriptome profiling was performed in Kenyan children (n=144, 3-36months) with discrete non-SMA and SMA phenotypes. Leukocyte associated immunoglobulin like receptor 1 (LAIR1) emerged as a predictor of susceptibility to SMA (P<1×10-2, OR: 0.44-1.37), and was suppressed in severe disease (-1.69-fold, P=0.004). To extend these findings, the relationship between LAIR1 polymorphisms [rs6509867 (16231C>A); rs2287827 (18835G>A)] and clinical outcomes were investigated in individuals (n=1512, <5years) at enrolment and during a 36-month longitudinal follow-up. FINDINGS: Inheritance of the 16,231 recessive genotype (AA) increased susceptibility to SMA at enrolment (OR=1.903, 95%CI: 1.252-2.891, P=0.003), and longitudinally (RR=1.527, 95%CI: 1.119-2.083, P=0.008). Carriage of the 18,835 GA genotype protected against SMA cross-sectionally (OR=0.672, 95%CI: 0.480-0.9439, P=0.020). Haplotype carriage (C16231A/G18835A) also altered cross-sectional susceptibility to SMA: CG (OR=0.717, 95%CI: 0.527-0.9675, P=0.034), CA (OR=0.745, 95%CI: 0.536-1.036, P=0.080), and AG (OR=1.641, 95%CI: 1.160-2.321, P=0.005). Longitudinally, CA carriage was protective against SMA (RR=0.715, 95%CI: 0.554-0.923, P=0.010), while AG carriage had an additive effect on enhanced SMA risk (RR=1.283, 95%CI: 1.057-1.557, P=0.011). Variants that protected against SMA had elevated LAIR1 transcripts, while those with enhanced risk had lower expression (P<0.05). Inheritance of 18,835 GA reduced all-cause mortality by 44.8% (HR=0.552, 95%CI: 0.329-0.925, P=0.024), while AG haplotype carriage increased susceptibility by 68% (HR=1.680, 95%CI: 1.020-2.770, P=0.040). INTERPRETATION: These findings suggest LAIR1 is important for modulating susceptibility to SMA and all-cause childhood mortality.

Fast-Acting Small Molecules Targeting Malarial Aspartyl Proteases, Plasmepsins, Inhibit Malaria Infection at Multiple Life Stages.

The eradication of malaria remains challenging due to the complex life cycle of Plasmodium and the rapid emergence of drug-resistant forms of Plasmodium falciparum and Plasmodium vivax. New, effective, and inexpensive antimalarials against multiple life stages of the parasite are urgently needed to combat the spread of malaria. Here, we synthesized a set of novel hydroxyethylamines and investigated their activities in vitro and in vivo. All of the compounds tested had an inhibitory effect on the blood stage of P. falciparum at submicromolar concentrations, with the best showing 50% inhibitory concentrations (IC50) of around 500 nM against drug-resistant P. falciparum parasites. These compounds showed inhibitory actions against plasmepsins, a family of malarial aspartyl proteases, and exhibited a marked killing effect on blood stage Plasmodium. In chloroquine-resistant Plasmodium berghei and P. berghei ANKA infected mouse models, treating mice with both compounds led to a significant decrease in blood parasite load. Importantly, two of the compounds displayed an inhibitory effect on the gametocyte stages (III-V) of P. falciparum in culture and the liver-stage infection of P. berghei both in in vitro and in vivo. Altogether, our findings suggest that fast-acting hydroxyethylamine-phthalimide analogs targeting multiple life stages of the parasite could be a valuable chemical lead for the development of novel antimalarial drugs.

Antimalarials: Molecular Drug Targets and Mechanism of Action.

BACKGROUND: Despite a reduction in the global burden of malaria, the disease remains responsible for 214 million cases and 438,000 deaths annually with 88% of the mortality occurring in sub-Saharan Africa. Malaria control largely depends on effective chemotherapy. However, the historic and current emergence and spread of multi-drug resistant parasite strains provides significant challenges to malaria control and consequently, reduction of malaria-associated morbidity and mortality. Combating parasite drug resistance requires pharmacological compounds that target both known and novel metabolic pathways that are crucial for parasite survival. In addition, the identification of novel therapeutic agents that target distinct molecular pathways, apart from those of the conventional antimalarials, offers an approach for minimizing drug resistance. CONCLUSION: This review summarizes current anti-malarial approaches and strategies, therapeutic efficacy for conventional and non-conventional antimalarials, parasitic targets, and the mechanisms responsible for the development of drug resistance.

Synergistic blending of high-valued heterocycles inhibits growth of Plasmodium falciparum in culture and P. berghei infection in mouse model.

A series of phthalimide analogues, novelized with high-valued bioactive scaffolds was synthesized by means of click-chemistry under non-conventional microwave heating and evaluated as noteworthy growth inhibitors of Plasmodium falciparum (3D7 and W2) in culture. Analogues 6a, 6h and 6 u showed highest activity to inhibit the growth of the parasite with IC50 values in submicromolar range. Structure-activity correlation indicated the necessity of unsubstituted triazoles and leucine linker to obtain maximal growth inhibition of the parasite. Notably, phthalimide 6a and 6u selectively inhibited the ring-stage growth and parasite maturation. On other hand, phthalimide 6h displayed selective schizonticidal activity. Besides, they displayed synergistic interactions with chloroquine and dihydroartemisinin against parasite. Additional in vivo experiments using P. berghei infected mice showed that administration of 6h and 6u alone, as well as in combination with dihydroartemisinin, substantially reduced the parasite load. The high antimalarial activity of 6h and 6u, coupled with low toxicity advocate their potential role as novel antimalarial agents, either as standalone or combination therapies.

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

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

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

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

Five-year tracking of Plasmodium falciparum allele frequencies in a holoendemic area with indistinct seasonal transitions.

BACKGROUND: The renewed malaria eradication efforts require an understanding of the seasonal patterns of frequency of polymorphic variants in order to focus limited funds productively. Although cross-sectional studies in holoendemic areas spanning a single year could be useful in describing parasite genotype status at a given point, such information is inadequate in describing temporal trends in genotype polymorphisms. For Plasmodium falciparum isolates from Kisumu District Hospital, Plasmodium falciparum chloroquine-resistance transporter gene (Pfcrt-K76T) and P. falciparum multidrug resistance gene 1 (PfMDR1-N86Y), were analyzed for polymorphisms and parasitemia changes in the 53 months from March 2008 to August 2012. Observations were compared with prevailing climatic factors, including humidity, rainfall, and temperature. METHODS: Parasitemia (the percentage of infected red blood cells per total red blood cells) was established by microscopy for P. falciparum malaria-positive samples. P. falciparum DNA was extracted from whole blood using a Qiagen DNA Blood Mini Kit. Single nucleotide polymorphism identification at positions Pfcrt-K76T and PfMDR1-N86Y was performed using real-time polymerase chain reaction and/or sequencing. Data on climatic variables were obtained from http://www.tutiempo.net/en/. RESULTS: A total of 895 field isolates from 2008 (n=169), 2009 (n=161), 2010 (n=216), 2011 (n=223), and 2012 (n=126) showed large variations in monthly frequency of PfMDR1-N86Y and Pfcrt-K76T as the mutant genotypes decreased from 68.4%±15% and 38.1%±13% to 29.8%±18% and 13.3%±9%, respectively. The mean percentage of parasitemia was 2.61%±1.01% (coefficient of variation 115.86%; n=895). There was no correlation between genotype or parasitemia and climatic factors. CONCLUSION: This study shows variability in the frequency of Pfcrt-K76T and PfMDR1-N86Y polymorphisms during the study period, bringing into focus the role of cross-sectional studies in describing temporal genotype trends. The lack of correlation between genotypes and climatic changes, especially precipitation, emphasizes the cost of investment in genotype change.