Insecticide resistance in indoor and outdoor-resting Anopheles gambiae in Northern Ghana.

BACKGROUND: Selection pressure from continued exposure to insecticides drives development of insecticide resistance and changes in resting behaviour of malaria vectors. There is need to understand how resistance drives changes in resting behaviour within vector species. The association between insecticide resistance and resting behaviour of Anopheles gambiae sensu lato (s.l.) in Northern Ghana was examined. METHODS: F1 progenies from adult mosquitoes collected indoors and outdoors were exposed to DDT, deltamethrin, malathion and bendiocarb using WHO insecticide susceptibility tests. Insecticide resistance markers including voltage-gated sodium channel (Vgsc)-1014F, Vgsc-1014S, Vgsc-1575Y, glutathione-S-transferase epsilon 2 (GSTe2)-114T and acetylcholinesterase (Ace1)-119S, as well as blood meal sources were investigated using PCR methods. Activities of metabolic enzymes, acetylcholine esterase (AChE), non-specific β-esterases, glutathione-S-transferase (GST) and monooxygenases were measured from unexposed F1 progenies using microplate assays. RESULTS: Susceptibility of Anopheles coluzzii to deltamethrin 24 h post-exposure was significantly higher in indoor (mortality = 5%) than outdoor (mortality = 2.5%) populations (P = 0.02). Mosquitoes were fully susceptible to malathion (mortality: indoor = 98%, outdoor = 100%). Susceptibility to DDT was significantly higher in outdoor (mortality = 9%) than indoor (mortality = 0%) mosquitoes (P = 0.006). Mosquitoes were also found with suspected resistance to bendiocarb but mortality was not statistically different (mortality: indoor = 90%, outdoor = 95%. P = 0.30). Frequencies of all resistance alleles were higher in F1 outdoor (0.11-0.85) than indoor (0.04-0.65) mosquito populations, while Vgsc-1014F in F0 An. gambiae sensu stricto (s.s) was significantly associated with outdoor-resting behaviour (P = 0.01). Activities of non-specific β-esterase enzymes were significantly higher in outdoor than indoor mosquitoes (Mean enzyme activity: Outdoor = : 1.70/mg protein; Indoor = 1.35/mg protein. P < 0.0001). AChE activity was also more elevated in outdoor (0.62/mg protein) than indoor (0.57/mg protein) mosquitoes but this was not significant (P = 0.08). Human blood index (HBI) was predominantly detected in indoor (18%) than outdoor mosquito populations (3%). CONCLUSIONS: The overall results did not establish that there was a significant preference of resistant malaria vectors to solely rest indoors or outdoors, but varied depending on the resistant alleles present. Phenotypic resistance was higher in indoor than outdoor-resting mosquitoes, but genotypic and metabolic resistance levels were higher in outdoor than the indoor populations. Continued monitoring of changes in resting behaviour within An. gambiae s.l. populations is recommended

Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum.

BACKGROUND: The burden of falciparum malaria is especially high in sub-Saharan Africa. Differences in pressure from host immunity and antimalarial drugs lead to adaptive changes responsible for high level of genetic variations within and between the parasite populations. Population-specific genetic studies to survey for genes under positive or balancing selection resulting from drug pressure or host immunity will allow for refinement of interventions. METHODS: We performed a pooled sequencing (pool-seq) of the genomes of 100 Plasmodium falciparum isolates from Nigeria. We explored allele-frequency based neutrality test (Tajima's D) and integrated haplotype score (iHS) to identify genes under selection. RESULTS: Fourteen shared iHS regions that had at least 2 SNPs with a score > 2.5 were identified. These regions code for genes that were likely to have been under strong directional selection. Two of these genes were the chloroquine resistance transporter (CRT) on chromosome 7 and the multidrug resistance 1 (MDR1) on chromosome 5. There was a weak signature of selection in the dihydrofolate reductase (DHFR) gene on chromosome 4 and MDR5 genes on chromosome 13, with only 2 and 3 SNPs respectively identified within the iHS window. We observed strong selection pressure attributable to continued chloroquine and sulfadoxine-pyrimethamine use despite their official proscription for the treatment of uncomplicated malaria. There was also a major selective sweep on chromosome 6 which had 32 SNPs within the shared iHS region. Tajima's D of circumsporozoite protein (CSP), erythrocyte-binding antigen (EBA-175), merozoite surface proteins - MSP3 and MSP7, merozoite surface protein duffy binding-like (MSPDBL2) and serine repeat antigen (SERA-5) were 1.38, 1.29, 0.73, 0.84 and 0.21, respectively. CONCLUSION: We have demonstrated the use of pool-seq to understand genomic patterns of selection and variability in P. falciparum from Nigeria, which bears the highest burden of infections. This investigation identified known genomic signatures of selection from drug pressure and host immunity. This is evidence that P. falciparum populations explore common adaptive strategies that can be targeted for the development of new interventions

estMOI: estimating multiplicity of infection using parasite deep sequencing data.

Individuals living in endemic areas generally harbour multiple parasite strains. Multiplicity of infection (MOI) can be an indicator of immune status and transmission intensity. It has a potentially confounding effect on a number of population genetic analyses, which often assume isolates are clonal. Polymerase chain reaction-based approaches to estimate MOI can lack sensitivity. For example, in the human malaria parasite Plasmodium falciparum, genotyping of the merozoite surface protein (MSP1/2) genes is a standard method for assessing MOI, despite the apparent problem of underestimation. The availability of deep coverage data from massively parallizable sequencing technologies means that MOI can be detected genome wide by considering the abundance of heterozygous genotypes. Here, we present a method to estimate MOI, which considers unique combinations of polymorphisms from sequence reads. The method is implemented within the estMOI software. When applied to clinical P.falciparum isolates from three continents, we find that multiple infections are common, especially in regions with high transmission

Prospective Identification of Malaria Parasite Genes under Balancing Selection

BACKGROUND: Endemic human pathogens are subject to strong immune selection, and interrogation of pathogen genome variation for signatures of balancing selection can identify important target antigens. Several major antigen genes in the malaria parasite Plasmodium falciparum have shown such signatures in polymorphism-versus-divergence indices (comparing with the chimpanzee parasite P. reichenowi), and in allele frequency based indices. METHODOLOGY/PRINCIPAL FINDINGS: To compare methods for prospective identification of genes under balancing selection, 26 additional genes known or predicted to encode surface-exposed proteins of the invasive blood stage merozoite were first sequenced from a panel of 14 independent P. falciparum cultured lines and P. reichenowi. Six genes at the positive extremes of one or both of the Hudson-Kreitman-Aguade (HKA) and McDonald-Kreitman (MK) indices were identified. Allele frequency based analysis was then performed on a Gambian P. falciparum population sample for these six genes and three others as controls. Tajima's D (TjD) index was most highly positive for the msp3/6-like PF10_0348 (TjD = 1.96) as well as the positive control ama1 antigen gene (TjD = 1.22). Across the genes there was a strong correlation between population TjD values and the relative HKA indices (whether derived from the population or the panel of cultured laboratory isolates), but no correlation with the MK indices. CONCLUSIONS/SIGNIFICANCE: Although few individual parasite genes show significant evidence of balancing selection, analysis of population genomic and comparative sequence data with the HKA and TjD indices should discriminate those that do, and thereby identify likely targets of immunity

High cases of submicroscopic Plasmodium falciparum infections in a suburban population of Lagos, Nigeria.

BACKGROUND: Asymptomatic malaria parasites are significant sources of infections for onward malaria transmission. Conventional tools for malaria diagnosis such as microscopy and rapid diagnostic test kits (RDT) have relatively low sensitivity, hence the need for alternative tools for active screening of such low-density infections. METHODS: This study tested var acidic terminal sequence-based (varATS) quantitative polymerase chain reaction (qPCR) for screening asymptomatic Plasmodium falciparum infections among dwellers of a sub-urban community in Lagos, Nigeria. Clinically healthy participants were screened for malaria using microscopy, RDT and varATS qPCR techniques. Participants were stratified into three age groups: 1-5, 6-14 and > 14 years old. RESULTS: Of the 316 participants screened for asymptomatic malaria infection, 78 (24.68%) were positive by microscopy, 99 (31.33%) were positive by RDT and 112 (35.44%) by varATS qPCR. Participants aged 6-14 years had the highest prevalence of asymptomatic malaria, with geometric means of ~ 116 parasites/µL and ~ 6689 parasites/µL as detected by microscopy and varATS, respectively. CONCLUSION: This study has revealed high prevalence of asymptomatic malaria in the study population, with varATS detecting additional sub-microscopic infections. The highest concentration of asymptomatic malaria was observed among school-age children between 6 and 14 years old. A large-scale screening to identify other potential hotspots of asymptomatic parasites in the country is recommended

Population genetic analysis of the Plasmodium falciparum circumsporozoite protein in two distinct ecological regions in Ghana.

BACKGROUND: Extensive genetic diversity in the Plasmodium falciparum circumsporozoite protein (PfCSP) is a major contributing factor to the moderate efficacy of the RTS,S/AS01 vaccine. The transmission intensity and rates of recombination within and between populations influence the extent of its genetic diversity. Understanding the extent and dynamics of PfCSP genetic diversity in different transmission settings will help to interpret the results of current RTS,S efficacy and Phase IV implementation trials conducted within and between populations in malaria-endemic areas such as Ghana. METHODS: Pfcsp sequences were retrieved from the Illumina-generated paired-end short-read sequences of 101 and 131 malaria samples from children aged 6-59 months presenting with clinical malaria at health facilities in Cape Coast (in the coastal belt) and Navrongo (Guinea savannah region), respectively, in Ghana. The sequences were mapped onto the 3D7 reference strain genome to yield high-quality genome-wide coding sequence data. Following data filtering and quality checks to remove missing data, 220 sequences were retained and analysed for the allele frequency spectrum, genetic diversity both within the host and between populations and signatures of selection. Population genetics tools were used to determine the extent and dynamics of Pfcsp diversity in P. falciparum from the two geographically distinct locations in Ghana. RESULTS: Pfcsp showed extensive diversity at the two sites, with the higher transmission site, Navrongo, exhibiting higher within-host and population-level diversity. The vaccine strain C-terminal epitope of Pfcsp was found in only 5.9% and 45.7% of the Navrongo and Cape Coast sequences, respectively. Between 1 and 6 amino acid variations were observed in the TH2R and TH3R epitope regions of PfCSP. Tajima's D was negatively skewed, especially for the population from Cape Coast, given the expected historical population expansion. In contrast, a positive Tajima's D was observed for the Navrongo P. falciparum population, consistent with balancing selection acting on the immuno-dominant TH2R and TH3R vaccine epitopes. CONCLUSION: The low frequencies of the Pfcsp vaccine haplotype in the analysed populations indicate a need for additional molecular and immuno-epidemiological studies with broader temporal and geographic sampling in endemic populations targeted for RTS,S application. These results have implications for the efficacy of the vaccine in Ghana and will inform the choice of alleles to be included in future multivalent or chimeric vaccines

Seroprevalence and Parasite Rates of Plasmodium malariae in a High Malaria Transmission Setting of Southern Nigeria.

Although Plasmodium falciparum continues to be the main target for malaria elimination, other Plasmodium species persist in Africa. Their clinical diagnosis is uncommon, whereas rapid diagnostic tests (RDTs), the most widely used malaria diagnostic tools, are only able to distinguish between P. falciparum and non-falciparum species, the latter as "pan-species." Blood samples from health facilities were collected in southern Nigeria (Lagos and Calabar) in 2017 (October-December) and Calabar only in 2018 (October-November), and analyzed by several methods, namely, microscopy, quantitative real-time PCR (qPCR), and peptide serology targeting candidate antigens (Plasmodium malariae apical membrane antigen, P. malariae lactose dehydrogenase, and P. malariae circumsporozoite surface protein). Both microscopy and qPCR diagnostic approaches detected comparable proportions (∼80%) of all RDT-positive samples infected with the dominant P. falciparum malaria parasite. However, higher proportions of non-falciparum species were detected by qPCR than microscopy, 10% against 3% infections for P. malariae and 3% against 0% for Plasmodium ovale, respectively. No Plasmodium vivax infection was detected. Infection rates for P. malariae varied between age-groups, with the highest rates in individuals aged > 5 years. Plasmodium malariae-specific seroprevalence rates fluctuated in those aged < 10 years but generally reached the peak around 20 years of age for all peptides. The heterogeneity and rates of these non-falciparum species call for increased specific diagnosis and targeting by elimination strategies

Influence of insecticide resistance on the biting and resting preferences of malaria vectors in the Gambia.

BACKGROUND: The scale-up of indoor residual spraying and long-lasting insecticidal nets, together with other interventions have considerably reduced the malaria burden in The Gambia. This study examined the biting and resting preferences of the local insecticide-resistant vector populations few years following scale-up of anti-vector interventions. METHOD: Indoor and outdoor-resting Anopheles gambiae mosquitoes were collected between July and October 2019 from ten villages in five regions in The Gambia using pyrethrum spray collection (indoor) and prokopack aspirator from pit traps (outdoor). Polymerase chain reaction assays were performed to identify molecular species, insecticide resistance mutations, Plasmodium infection rate and host blood meal. RESULTS: A total of 844 mosquitoes were collected both indoors (421, 49.9%) and outdoors (423, 50.1%). Four main vector species were identified, including An. arabiensis (indoor: 15%, outdoor: 26%); An. coluzzii (indoor: 19%, outdoor: 6%), An. gambiae s.s. (indoor: 11%, outdoor: 16%), An. melas (indoor: 2%, outdoor: 0.1%) and hybrids of An. coluzzii-An. gambiae s.s (indoors: 3%, outdoors: 2%). A significant preference for outdoor resting was observed in An. arabiensis (Pearson X2 = 22.7, df = 4, P<0.001) and for indoor resting in An. coluzzii (Pearson X2 = 55.0, df = 4, P<0.001). Prevalence of the voltage-gated sodium channel (Vgsc)-1014S was significantly higher in the indoor-resting (allele freq. = 0.96, 95%CI: 0.78-1, P = 0.03) than outdoor-resting (allele freq. = 0.82, 95%CI: 0.76-0.87) An. arabiensis population. For An. coluzzii, the prevalence of most mutation markers was higher in the outdoor (allele freq. = 0.92, 95%CI: 0.81-0.98) than indoor-resting (allele freq. = 0.78, 95%CI: 0.56-0.86) mosquitoes. However, in An. gambiae s.s., the prevalence of Vgsc-1014F, Vgsc-1575Y and GSTe2-114T was high (allele freq. = 0.96-1), but did not vary by resting location. The overall sporozoite positivity rate was 1.3% (95% CI: 0.5-2%) in mosquito populations. Indoor-resting An. coluzzii had mainly fed on human blood while indoor-resting An. arabiensis fed on animal blood. CONCLUSION: In this study, high levels of resistance mutations were observed that could be influencing the mosquito populations to rest indoors or outdoors. The prevalent animal-biting behaviour demonstrated in the mosquito populations suggest that larval source management could be an intervention to complement vector control in this setting

Population genetic analysis of Plasmodium falciparum cell-traversal protein for ookinetes and sporozoite among malaria patients from southern Nigeria

Plasmodium falciparum immune escape mechanisms affect antigens being prioritized for vaccine design. As a result of the multiple surface antigens the parasite exhibits at different life cycle stages, designing a vaccine that would efficiently boost the immune system in clearing infections has been challenging. The P. falciparum cell-traversal protein for ookinetes and sporozoite (Pfceltos) is instrumental for ookinete traversal of the mosquito midgut and sporozoites invasion of the human liver cells. Pfceltos elicits both humoral and cellular immune response but has been reported with multiple single nucleotide polymorphisms in global isolates. A cross-sectional survey, conducted in southern Nigeria, between January-March 2021 recruited 283 individuals. Of this, 166 demonstrated P. falciparum infections (86 from Cross River and 80 from Edo), 48 (55.8%) while only 36 (45%) were amplified for Pfceltos gene from both sites respectively. Fifty amplified samples were sequenced and analysed for their diversity, polymorphisms and population structure of the gene. The number of segregating sites in Edo State was higher (34) than that of Cross River State. Though nucleotide diversity was higher for Edo compared to Cross River State (θw = 0.02505; π = 0.03993 versus θw = 0.00930; π = 0.01033 respectively), the reverse was the case for haplotype diversity (0.757 versus 0.890 for Edo and Cross River respectively). Of the twelve haplotypes observed from both states, only two (KASLPVEK and NAFLSFEK) were shared, with haplotype prevalence higher in Edo (16% and 36%) than Cross River (8% and 4%). The Tajima's D test was positive for both states, with Fst value showing a strong genetic differentiation (Fst = 0.25599), indicating the occurrence of balancing selection favoring haplotype circulation at a low frequency. The shared haplotypes, low Hst and Fst values presents a challenge to predict the extent of gene flow. High LD values present a grim public health consequence should a Pfceltos-conjugated vaccine be considered for prophylaxis in Nigeria

Prevalence of asymptomatic malaria parasitaemia following mass testing and treatment in Pakro sub-district of Ghana.

BACKGROUND: Global efforts to scale-up malaria control interventions are gaining steam. These include the use of Long-Lasting Insecticide Nets, Indoor Residual Spraying, Intermittent Preventive Treatment and Test, Treat and Track. Despite these, the drive for malaria elimination is far from being realistic in endemic communities in Africa. This is partly due to the fact that asymptomatic parasite carriage, not specifically targeted by most interventions, remains the bedrock that fuels transmission. This has led to mass testing, treatment and tracking (MTTT) as an alternative strategy to target asymptomatic individuals. We report the impact of MTTT on the prevalence of asymptomatic malaria parasitaemia over a one-year period in Ghana, hypothesizing that implementing MTTT could reduce the rate of asymptomatic parasitaemia. METHODS: A population of about 5000 individuals in seven communities in the Pakro sub-district of Ghana participated in this study. A register was developed for each community following a census. MTTT engaged trained community-based health volunteers who conducted house-to-house testing using RDTs every 4 months and treated positive cases with Artemisinin-based Combination Therapy. Between interventions, community-based management of malaria was implemented for symptomatic cases. RESULTS: MTTT Coverage was 98.8% in July 2017 and 79.3% in July 2018. Of those tested, asymptomatic infection with malaria parasites reduced from 36.3% (1795/4941) in July 2017 to 32.9% (1303/3966) in July 2018 (p = 0.001). Prevalence of asymptomatic parasitaemia among children under 15 years declined from 52.6% (1043/1984) in July 2017 to 47.5% (820/1728) in July 2018 (p = 0.002). Implementing MTTT significantly reduced asymptomatic parasitaemia by 24% from July 2017 to July 2018 after adjusting for age, ITN use and axillary temperature (OR = 0.76, CI = 0.67, 0.85 p ≤ 0.001). CONCLUSION: This study has demonstrated that implementing MTTT is feasible and could reduce the prevalence of asymptomatic malaria parasitaemia in children under 15 years of age. Furthermore, the use of community-based health volunteers could ensure high coverage at lower cost of implementation