Genome-wide association study identifies novel candidate malaria resistance genes in Cameroon.

Recent data suggest that only a small fraction of severe malaria heritability is explained by the totality of genetic markers discovered so far. The extensive genetic diversity within African populations means that significant associations are likely to be found in Africa. In their series of multi-site genome-wide association studies (GWAS) across sub-Saharan Africa, the Malaria Genomic Epidemiology Network (MalariaGEN) observed specific limitations and encouraged country-specific analyses. Here, we present findings of a GWAS of Cameroonian participants that contributed to MalariaGEN projects (n = 1103). We identified protective associations at polymorphisms within the enhancer region of CHST15 [Benjamin-Hochberg false discovery rate (FDR) < 0.02] that are specific to populations of African ancestry, and that tag strong eQTLs of CHST15 in hepatic cells. In-silico functional analysis revealed a signature of epigenetic regulation of CHST15 that is preserved in populations in historically malaria endemic regions, with haplotype analysis revealing a haplotype that is specific to these populations. Association analysis by ethnolinguistic group identified protective associations within SOD2 (FDR < 0.04), a gene previously shown to be significantly induced in pre-asymptomatic malaria patients from Cameroon. Haplotype analysis revealed substantial heterogeneity within the beta-like globin (HBB) gene cluster amongst the major ethnic groups in Cameroon confirming differential malaria pressure and underscoring age-old fine-scale genetic structure within the country. Our findings revealed novel insights in the evolutionary genetics of populations living in Cameroon under malaria pressure with new significant protective loci (CHST15 and SOD2) and emphasized the significant attenuation of genetic association signals by fine-scale genetic structure.

Effect of predators on Anopheles arabiensis and Anopheles funestus larval survivorship in Homa Bay County Western Kenya.

BACKGROUND: The rise of insecticide resistance against malaria vectors in sub-Saharan Africa has resulted in the need to consider other methods of vector control. The potential use of biological methods, including larvivorous fish, Bacillus thuringiensis israelensis (Bti) and plant shading, is sustainable and environmentally friendly options. This study examined the survivorship of Anopheles arabiensis and Anopheles funestus larvae and habitat productivity in four permanent habitat types in Homa Bay county, western Kenya. METHODS: Predator densities were studied in a laboratory setup while habitat productivity and larval survivorship was studied in field setup. RESULTS: Fish were observed as the most efficient predator (75.8% larval reduction rate) followed by water boatman (69%), and dragonfly nymph (69.5%) in predation rates. Lower predation rates were observed in backswimmers (31%), water beetles (14.9%), water spiders (12.2%), mayflies (7.3%), and tadpoles (6.9%). Increase in predator density in the field setup resulted in decreased Culex larval density. Larval and pupa age-specific distribution was determined and their survivorship curves constructed. Combined larvae (Stage I-IV) to pupa mortality was over 97% for An. arabiensis and 100% for An. funestus. The highest larval stage survival rate was from larval stages I to II and the lowest from larval stage IV to pupa. Stage-specific life tables indicated high mortality rates at every developmental stage, especially at the larval stage II and III. CONCLUSION: Determination of the efficiency of various larval predators and habitat productivity will help with the correct identification of productive habitats and selection of complementary vector control methods through environmental management and/or predator introduction (for instance fish) in the habitats.

Community-based surveys for Plasmodium falciparum pfhrp2 and pfhrp3 gene deletions in selected regions of mainland Tanzania.

BACKGROUND: Histidine-rich protein 2 (HRP2)-based malaria rapid diagnostic tests (RDTs) are effective and widely used for the detection of wild-type Plasmodium falciparum infections. Although recent studies have reported false negative HRP2 RDT results due to pfhrp2 and pfhrp3 gene deletions in different countries, there is a paucity of data on the deletions of these genes in Tanzania. METHODS: A community-based cross-sectional survey was conducted between July and November 2017 in four regions: Geita, Kigoma, Mtwara and Ruvuma. All participants had microscopy and RDT performed in the field and provided a blood sample for laboratory multiplex antigen detection (for Plasmodium lactate dehydrogenase, aldolase, and P. falciparum HRP2). Samples showing RDT false negativity or aberrant relationship of HRP2 to pan-Plasmodium antigens were genotyped to detect the presence/absence of pfhrp2/3 genes. RESULTS: Of all samples screened by the multiplex antigen assay (n = 7543), 2417 (32.0%) were positive for any Plasmodium antigens while 5126 (68.0%) were negative for all antigens. The vast majority of the antigen positive samples contained HRP2 (2411, 99.8%), but 6 (0.2%) had only pLDH and/or aldolase without HRP2. Overall, 13 samples had an atypical relationship between a pan-Plasmodium antigen and HRP2, but were positive by PCR. An additional 16 samples with negative HRP2 RDT results but P. falciparum positive by microscopy were also chosen for pfhrp2/3 genotyping. The summation of false negative RDT results and laboratory antigen results provided 35 total samples with confirmed P. falciparum DNA for pfhrp2/3 genotyping. Of the 35 samples, 4 (11.4%) failed to consistently amplify positive control genes; pfmsp1 and pfmsp2 and were excluded from the analysis. The pfhrp2 and pfhrp3 genes were successfully amplified in the remaining 31 (88.6%) samples, confirming an absence of deletions in these genes. CONCLUSIONS: This study provides evidence that P. falciparum parasites in the study area have no deletions of both pfhrp2 and pfhrp3 genes. Although single gene deletions could have been missed by the multiplex antigen assay, the findings support the continued use of HRP2-based RDTs in Tanzania for routine malaria diagnosis. There is a need for the surveillance to monitor the status of pfhrp2 and/or pfhrp3 deletions in the future.

Comparative Evaluation of LAMP, qPCR, Conventional PCR, and ELISA to Detect Ralstonia solanacearum in Kenyan Potato Fields.

Bacterial wilt caused by Ralstonia solanacearum is considered among the most damaging diseases of potato in Sub-Saharan Africa and the most significant biotic constraint of potato production alongside late blight. Unlike late blight, which can be managed by chemical means, R. solanacearum can only be managed through cultural methods and clean seed. Laboratory testing to certify seed before planting is required to confirm the absence of the pathogen in Kenya. A loop-mediated isothermal amplification (LAMP) assay was developed using the UDP-(3-O-acyl)-N-acetylglucosamine deacetylase gene (IpxC) to screen seed potato for R. solanacearum strains. The assay was assessed using DNA extracted from R. solanacearum and other soil and potato pathogens to demonstrate specificity and sensitivity. The LAMP assay was validated using field samples from different potato growing regions of Kenya collected over two growing seasons and compared with established nucleic acid and protein-based assays. The IpxC LAMP assay was found to be specific and sensitive to R. solanacearum, detecting as low as 2.5 pg/µl of R. solanacearum DNA. Of the 47 potentially infected field samples collected, both IpxC LAMP and quantitative polymerase chain reaction (PCR) detected R. solanacearum DNA in 90% of the samples, followed by conventional PCR (86%) and ELISA (75%). This IpxC LAMP assay is a promising diagnostic tool to rapidly screen for R. solanacearum in seed potato with high sensitivity in Kenya. Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Effects of bacterial composition and aquatic habitat metabolites on malaria vector larval availability in irrigated and non-irrigated sites of Homa Bay county, western Kenya.

Gravid Anopheles malaria vectors depend on both chemical and physical (including microbial) cues for selection of preferred habitats for oviposition. This study focused on assessing the effects of bacterial composition and habitat metabolites on malaria vector larval availability in irrigated and non-irrigated potential larval sources. Water samples were collected from larval positive and negative habitats in the irrigated and non- irrigated areas of Homa Bay county. Bacteria cultured from the water samples were subjected to Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) for species identification. DNA was extracted from the colonies and polymerase chain reaction (PCR) and sequencing done. Finally, the metabolite composition of larval positive and negative habitats was determined. MALDI-TOF MS results revealed that Bacillus was the only genera identified from larval sources in the non-irrigated zone. In the irrigated area, Shigella was the dominant genera (47%) while Escherichia coli was the abundant species (13/51). Of the sequenced isolates, 65% were Bacillus. Larvicidal isolates Brevibacillus brevis, Bacillus subtilis, and Exiguobacterium profundum were isolated and grouped with Bacillus mojavensis, Bacillus tequilensis, Bacillus stercoris, and Brevibacillus agri. Irrigated areas with larvae had reduced crude fat (0.01%) and protein content (0.13%) in comparison to those without larvae. In irrigated and non- irrigated areas, larval presence was evident in habitats with high total chlorophyll content (1.12 µg/g vs 0.81µg/g and 3.37 µg/g vs 0.82). Aquatic habitats with larvae in both irrigated and non-irrigated areas exhibited higher sugar concentration than habitats without larvae; however, when compared, non-irrigated areas with larvae had higher sugar concentration than similar habitats in irrigated areas. In addition, substantial concentrations of Manganese, Calcium, and Copper were found in aquatic habitats containing larvae in both irrigated and non-irrigated areas. These results allow for prospective examination as potential larvicidal or adulticidal agents and could be considered when designing potential vector control interventions.

Habitat Diversity, Stability, and Productivity of Malaria Vectors in Irrigated and Nonirrigated Ecosystems in Western Kenya.

Several sub-Saharan African countries rely on irrigation for food production. This study examined the impact of environmental modifications resulting from irrigation on the ecology of aquatic stages of malaria vectors in a semi-arid region of western Kenya. Mosquito larvae were collected from irrigated and non-irrigated ecosystems during seasonal cross-sectional and monthly longitudinal studies to assess habitat availability, stability, and productivity of anophelines in temporary, semipermanent, and permanent habitats during the dry and wet seasons. The duration of habitat stability was also compared between selected habitats. Emergence traps were used to determine the daily production of female adult mosquitoes from different habitat types. Malaria vectors were morphologically identified and sibling species subjected to molecular analysis. Data was statistically compared between the two ecosystems. After aggregating the data, the overall malaria vector productivity for habitats in the two ecosystems was estimated. Immatures of the malaria vector (Anopheles arabiensis) Patton (Diptera: Culicidae) comprised 98.3% of the Anopheles in both the irrigated and non-irrigated habitats. The irrigated ecosystem had the most habitats, higher larval densities, and produced 85.8% of emerged adult females. These results showed that irrigation provided conditions that increased habitat availability, stability, and diversity, consequently increasing the An. arabiensis production and potential risk of malaria transmission throughout the year. The irrigated ecosystems increased the number of habitats suitable for Anopheles breeding by about 3-fold compared to non-irrigated ecosystems. These results suggest that water management in the irrigation systems of western Kenya would serve as an effective method for malaria vector control.

Insecticide resistance status of Anopheles arabiensis in irrigated and non-irrigated areas in western Kenya.

BACKGROUND: Malaria control in Kenya is based on case management and vector control using long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the development of insecticide resistance compromises the effectiveness of insecticide-based vector control programs. The use of pesticides for agricultural purposes has been implicated as one of the sources driving the selection of resistance. The current study was undertaken to assess the status and mechanism of insecticide resistance in malaria vectors in irrigated and non-irrigated areas with varying agrochemical use in western Kenya. METHODS: The study was carried out in 2018-2019 in Homa Bay County, western Kenya. The bioassay was performed on adults reared from larvae collected from irrigated and non-irrigated fields in order to assess the susceptibility of malaria vectors to different classes of insecticides following the standard WHO guidelines. Characterization of knockdown resistance (kdr) and acetylcholinesterase-inhibiting enzyme/angiotensin-converting enzyme (Ace-1) mutations within Anopheles gambiae s.l. species was performed using the polymerase chain reaction (PCR) method. To determine the agricultural and public health insecticide usage pattern, a questionnaire was administered to farmers, households, and veterinary officers in the study area. RESULTS: Anopheles arabiensis was the predominant species in the irrigated (100%, n = 154) area and the dominant species in the non-irrigated areas (97.5%, n = 162), the rest being An. gambiae sensu stricto. In 2018, Anopheles arabiensis in the irrigated region were susceptible to all insecticides tested, while in the non-irrigated region reduced mortality was observed (84%) against deltamethrin. In 2019, phenotypic mortality was decreased (97.8-84% to 83.3-78.2%). In contrast, high mortality from malathion (100%), DDT (98.98%), and piperonyl butoxide (PBO)-deltamethrin (100%) was observed. Molecular analysis of the vectors from the irrigated and non-irrigated areas revealed low levels of leucine-serine/phenylalanine substitution at position 1014 (L1014S/L1014F), with mutation frequencies of 1-16%, and low-frequency mutation in the Ace-1R gene (0.7%). In addition to very high coverage of LLINs impregnated with pyrethroids and IRS with organophosphate insecticides, pyrethroids were the predominant chemical class of pesticides used for crop and animal protection. CONCLUSION: Anopheles arabiensis from irrigated areas showed increased phenotypic resistance, and the intensive use of pesticides for crop protection in this region may have contributed to the selection of resistance genes observed. The susceptibility of these malaria vectors to organophosphates and PBO synergists in pyrethroids offers a promising future for IRS and insecticide-treated net-based vector control interventions. These findings emphasize the need for integrated vector control strategies, with particular attention to agricultural practices to mitigate mosquito resistance to insecticides.

Fine scale human genetic structure in three regions of Cameroon reveals episodic diversifying selection.

Inferences from genetic association studies rely largely on the definition and description of the underlying populations that highlight their genetic similarities and differences. The clustering of human populations into subgroups (population structure) can significantly confound disease associations. This study investigated the fine-scale genetic structure within Cameroon that may underlie disparities observed with Cameroonian ethnicities in malaria genome-wide association studies in sub-Saharan Africa. Genotype data of 1073 individuals from three regions and three ethnic groups in Cameroon were analyzed using measures of genetic proximity to ascertain fine-scale genetic structure. Model-based clustering revealed distinct ancestral proportions among the Bantu, Semi-Bantu and Foulbe ethnic groups, while haplotype-based coancestry estimation revealed possible longstanding and ongoing sympatric differentiation among individuals of the Foulbe ethnic group, and their Bantu and Semi-Bantu counterparts. A genome scan found strong selection signatures in the HLA gene region, confirming longstanding knowledge of natural selection on this genomic region in African populations following immense disease pressure. Signatures of selection were also observed in the HBB gene cluster, a genomic region known to be under strong balancing selection in sub-Saharan Africa due to its co-evolution with malaria. This study further supports the role of evolution in shaping genomes of Cameroonian populations and reveals fine-scale hierarchical structure among and within Cameroonian ethnicities that may impact genetic association studies in the country.

Single-nucleotide polymorphism characterization of gametocyte development 1 gene in Plasmodium falciparum isolates from Baringo, Uasin Gishu, and Nandi Counties, Kenya.

INTRODUCTION: Plasmodium falciparum relies on gametocytogenesis to transmit from humans to mosquitoes. Gametocyte development 1 (Pfgdv1) is an upstream activator and epigenetic controller of gametocytogenesis. The emergence of drug resistance is a major public health concern and this requires the development of new strategies that target the transmission of malaria. As a putative drug target, Pfgdv1 has not been characterized to identify its polymorphisms and alleles under selection and how such polymorphisms influence protein structure. METHODS: This study characterized single-nucleotide polymorphisms (SNPs) in primary sequences (n = 30) of Pfgdv1 gene generated from thirty blood samples collected from patients infected with P. falciparum and secondary sequences (n = 216) retrieved from PlasmoDB. ChromasPro, MUSCLE, Tajima's D statistic, SLAC, and STRUM were used in editing raw sequences, performing multiple sequence alignment (MSA), identifying signatures of selection, detecting codon sites under selection pressure, and determining the effect of SNPs, respectively. RESULTS: MSA of primary and secondary sequences established the existence of five SNPs, consisting of four non-synonymous substitutions (nsSNPs) (p.P217H, p.R398Q, p.H417N, and p.D497E), and a synonymous substitution (p.S514S). The analysis of amino acid changes reveals that p.P217H, p.R398Q, and p.H417N comprise non-conservative changes. Tajima's D statistic showed that these SNPs were under balancing selection, while SLAC analysis identified p.P217H to be under the strongest positive selection. . Further analysis based on thermodynamics indicated that p.P217H has a destabilizing effect, while p.R398Q and p.D497E have stabilizing effects on the protein structure. CONCLUSIONS: The existence of four nsSNPs implies that Pfgdv1 has a minimal diversity in the encoded protein. Selection analysis demonstrates that these nsSNPs are under balancing selection in both local and global populations. However, p.P217H exhibits positive directional selection consistent with previous reports where it showed differentiatial selection of P. falciparum in low and high transmission regions. Therefore, in-silico prediction and experimental determination of protein structure are necessary to evaluate Pfgdv1 as a target candidate for drug design and development.

Transcriptomics reveal potential vaccine antigens and a drastic increase of upregulated genes during Theileria parva development from arthropod to bovine infective stages.

Theileria parva is a protozoan parasite transmitted by the brown ear tick Rhipicephalus appendiculatus that causes East Coast fever (ECF) in cattle, resulting in substantial economic losses in the regions of southern, eastern and central Africa. The schizont form of the parasite transforms the bovine host lymphocytes into actively proliferating cancer-like cells. However, how T. parva causes bovine host cells to proliferate and maintain a cancerous phenotype following infection is still poorly understood. On the other hand, current efforts to develop improved vaccines have identified only a few candidate antigens. In the present paper, we report the first comparative transcriptomic analysis throughout the course of T. parva infection. We observed that the development of sporoblast into sporozoite and then the establishment in the host cells as schizont is accompanied by a drastic increase of upregulated genes in the schizont stage of the parasite. In contrast, the ten highest gene expression values occurred in the arthropod vector stages. A comparative analysis showed that 2845 genes were upregulated in both sporozoite and schizont stages compared to the sporoblast. In addition, 647 were upregulated only in the sporozoite whereas 310 were only upregulated in the schizont. We detected low p67 expression in the schizont stage, an unexpected finding considering that p67 has been reported as a sporozoite stage-specific gene. In contrast, we found that transcription of p67 was 20 times higher in the sporoblast than in the sporozoite. Using the expression profiles of recently identified candidate vaccine antigens as a benchmark for selection for novel potential vaccine candidates, we identified three genes with expression similar to p67 and several other genes similar to Tp1-Tp10 schizont vaccine antigens. We propose that the antigenicity or chemotherapeutic potential of this panel of new candidate antigens be further investigated. Structural comparisons of the transcripts generated here with the existing gene models for the respective loci revealed indels. Our findings can be used to improve the structural annotation of the T. parva genome, and the identification of alternatively spliced transcripts.

Green tea proanthocyanidins cause impairment of hormone-regulated larval development and reproductive fitness via repression of juvenile hormone acid methyltransferase, insulin-like peptide and cytochrome P450 genes in Anopheles gambiae sensu stricto.

Successful optimization of plant-derived compounds into control of nuisance insects would benefit from scientifically validated targets. However, the close association between the genotypic responses and physiological toxicity effects mediated by these compounds remains underexplored. In this study, we evaluated the sublethal dose effects of proanthocyanidins (PAs) sourced from green tea (Camellia sinensis) on life history traits of Anopheles gambiae (sensu stricto) mosquitoes with an aim to unravel the probable molecular targets. Based on the induced phenotypic effects, genes selected for study targeted juvenile hormone (JH) biosynthesis, signal transduction, oxidative stress response and xenobiotic detoxification in addition to vitellogenesis in females. Our findings suggest that chronic exposure of larval stages (L3/L4) to sublethal dose of 5 ppm dramatically extended larval developmental period for up to 12 days, slowed down pupation rates, induced abnormal larval-pupal intermediates and caused 100% inhibition of adult emergence. Further, females exhibited significant interference of fecundity and egg hatchability relative to controls (p < 0.001). Using reverse transcription quantitative polymerase chain reaction (RT-qPCR), our findings show that PA-treated larvae exhibited significant repression of AgamJHAMT (p < 0.001), AgamILP1 (p < 0.001) and AgamCYP6M2 (p < 0.001) with up-regulation of Hsp70 (p < 0.001). Females exposed as larvae demonstrated down-regulation of AgamVg (p = 0.03), AgamILP1 (p = 0.009), AgamCYP6M2 (p = 0.05) and AgamJHAMT (p = 0.02). Our findings support that C. sinensis proanthocyanidins affect important vectorial capacity components such as mosquito survival rates and reproductive fitness thus could be potentially used for controlling populations of malaria vectors.

Target-similarity search using Plasmodium falciparum proteome identifies approved drugs with anti-malarial activity and their possible targets.

Malaria causes about half a million deaths annually, with Plasmodium falciparum being responsible for 90% of all the cases. Recent reports on artemisinin resistance in Southeast Asia warrant urgent discovery of novel drugs for the treatment of malaria. However, most bioactive compounds fail to progress to treatments due to safety concerns. Drug repositioning offers an alternative strategy where drugs that have already been approved as safe for other diseases could be used to treat malaria. This study screened approved drugs for antimalarial activity using an in silico chemogenomics approach prior to in vitro verification. All the P. falciparum proteins sequences available in NCBI RefSeq were mined and used to perform a similarity search against DrugBank, TTD and STITCH databases to identify similar putative drug targets. Druggability indices of the potential P. falciparum drug targets were obtained from TDR targets database. Functional amino acid residues of the drug targets were determined using ConSurf server which was used to fine tune the similarity search. This study predicted 133 approved drugs that could target 34 P. falciparum proteins. A literature search done at PubMed and Google Scholar showed 105 out of the 133 drugs to have been previously tested against malaria, with most showing activity. For further validation, drug susceptibility assays using SYBR Green I method were done on a representative group of 10 predicted drugs, eight of which did show activity against P. falciparum 3D7 clone. Seven had IC50 values ranging from 1 µM to 50 µM. This study also suggests drug-target association and hence possible mechanisms of action of drugs that did show antiplasmodial activity. The study results validate the use of proteome-wide target similarity approach in identifying approved drugs with activity against P. falciparum and could be adapted for other pathogens.

Potential of Camellia sinensis proanthocyanidins-rich fraction for controlling malaria mosquito populations through disruption of larval development.

BACKGROUND: Anopheles arabiensis and A. gambiae (sensu stricto) are the most prolific Afrotropical malaria vectors. Population control efforts of these two vectors have been hampered by extremely diverse larval breeding sites and widespread resistance to currently available insecticides. Control of mosquito larval stages using bioactive compounds of plant origin has the potential to suppress vector populations leading to concomitant reduction in disease transmission rates. In this study, we evaluated the efficacy of Camellia sinensis crude leaf extract and its fraction against the larvae of A. arabiensis and A. gambiae (s.s.). METHODS: Late third/early fourth instar larvae (L3/L4) of A. arabiensis and A. gambiae (s.s.) were exposed to increasing doses of C. sinensis leaf extract and its active fraction for 72 h, with mortality rates recorded every 24 h in both control and test groups. Ultra performance liquid chromatography electron spray ionization quadruple time of flight coupled with mass spectrometry (UPLC/ESI-Qtof/MS) was used to determine the main active constituents in the fraction. RESULTS: The major bioactive chemical constituents in the C. sinensis leaf extract were identified to be proanthocyanidins. The extract significantly interfered with larval survival and adult emergence in both species (ANOVA, F (5,24) = 1435.92, P < 0.001). Additionally, larval exposure to crude extract at 250 ppm and 500 ppm for 24 h resulted in larval mortality rates of over 90 % in A. gambiae (s.s.) and 75 % in A. arabiensis. A relatively lower concentration of 100 ppm resulted in moderate mortality rates of < 50 % in both species, but induced growth disruption effects evident as abnormal larval-pupal intermediates and disrupted adult emergence. The estimated LC50 concentrations of the crude leaf extract against A. arabiensis and A. gambiae (s.s.) larvae at 24 h were 154.58 ppm (95 % CI: 152.37-158.22) and 117.15 ppm (95 % CI: 112.86-127.04), respectively. The bioactive polar fraction caused 100 % larval mortality in both vector species at 25 ppm. CONCLUSIONS: Our findings demonstrate the potential of green tea extract and its active constituents in disrupting mosquito larval development. This could contribute to the control of mosquito populations and improved management of malaria.