Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria.

OBJECTIVE: Nigeria bears 25% of global malaria burden despite concerted efforts towards its control and elimination. The emergence of drug resistance to first line drugs, artemisinin combination therapies (ACTs), indicates an urgent need for continuous molecular surveillance of drug resistance especially in high burden countries where drug interventions are heavily relied on. This study describes mutations in Plasmodium falciparum genes associated with drug resistance in malaria; Pfk13, Pfmdr1, PfATPase6 and Pfcrt in isolates obtained from 83 symptomatic malaria patients collected in August 2014, aged 1-61 years old from South-west Nigeria. RESULTS: Two Pfmdr1, N86 and Y184 variants were present at a prevalence of 56% and 13.25% of isolates respectively. There was one synonymous (S679S) and two non-synonymous (M699V, S769M) mutations in the PATPase6 gene, while Pfcrt genotype (CVIET), had a prevalence of 45%. The Pfk13 C580Y mutant allele was suspected by allelic discrimination in two samples with mixed genotypes although this could not be validated with independent isolation or additional methods. Our findings call for robust molecular surveillance of antimalarial drug resistance markers in west Africa especially with increased use of antimalarial drugs as prophylaxis for Covid-19

A barcode of multilocus nuclear DNA identifies genetic relatedness in pre- and post-Artemether/Lumefantrine treated Plasmodium falciparum in Nigeria.

BACKGROUND: The decline in the efficacy of artemisinin-based combination treatment (ACT) in some endemic regions threatens the progress towards global elimination of malaria. Molecular surveillance of drug resistance in malaria-endemic regions is vital to detect the emergence and spread of mutant strains. METHODS: We observed 89 malaria patients for the efficacy of artemether-lumefantrine for the treatment of uncomplicated Plasmodium falciparum infections in Lagos, Nigeria and determined the prevalence of drug resistant strains in the population. Parasite clearance rates were determined by microscopy and the highly sensitive var gene acidic terminal sequence (varATS) polymerase chain reaction for 65 patients with samples on days 0, 1, 3, 7, 14, 21 and 28 after commencement of treatment. The genomic finger print of parasite DNA from pre- and post-treatment samples were determined using 24 nuclear single nucleotide polymorphisms (SNP) barcode for P. falciparum. Drug resistance associated alleles in chloroquine resistance transporter gene (crt-76), multidrug resistance genes (mdr1-86 and mdr1-184), dihydropteroate synthase (dhps-540), dihydrofolate reductase (dhfr-108) and kelch domain (K-13580) were genotyped by high resolution melt analysis of polymerase chain reaction (PCR) fragments. RESULTS: By varATS qPCR, 12 (18.5%) of the participants had detectable parasite DNA in their blood three days after treatment, while eight (12.3%) individuals presented with genotypable day 28 parasitaemia. Complexity of infection (CoI) was 1.30 on day 0 and 1.34 on day 28, the mean expected heterozygosity (HE) values across all barcodes were 0.50 ± 0.05 and 0.56 ± 0.05 on days 0 and 28 respectively. Barcode (π) pairwise comparisons showed high genetic relatedness of day 0 and day 28 parasite isolates in three (37.5%) of the eight individuals who presented with re-appearing infections. Crt-76 mutant allele was present in 38 (58.5%) isolates. The mdr1-86 mutant allele was found in 56 (86.2%) isolates. No mutation in the K-13580 was observed. CONCLUSIONS: Persistence of DNA-detectable parasitaemia in more than 18% of cases after treatment and indications of genetic relatedness between pre- and post-treatment infections warrants further investigation of a larger population for signs of reduced ACT efficacy in Nigeria

Design and methods for a quasi-experimental pilot study to evaluate the impact of dual active ingredient insecticide-treated nets on malaria burden in five regions in sub-Saharan Africa

Background:Vector control tools have contributed significantly to a reduction in malaria burden since 2000, primar‑ily through insecticidal‑treated bed nets (ITNs) and indoor residual spraying. In the face of increasing insecticide resist‑ance in key malaria vector species, global progress in malaria control has stalled. Innovative tools, such as dual active ingredient (dual‑AI) ITNs that are effective at killing insecticide‑resistant mosquitoes have recently been introduced. However, large‑scale uptake has been slow for several reasons, including higher costs and limited evidence on their incremental effectiveness and cost‑effectiveness. The present report describes the design of several observational studies aimed to determine the effectiveness and cost‑effectiveness of dual‑AI ITNs, compared to standard pyre‑throid‑only ITNs, at reducing malaria transmission across a variety of transmission settings.Methods:Observational pilot studies are ongoing in Burkina Faso, Mozambique, Nigeria, and Rwanda, leveraging dual‑AI ITN rollouts nested within the 2019 and 2020 mass distribution campaigns in each country. Enhanced surveil‑lance occurring in select study districts include annual cross‑sectional surveys during peak transmission seasons, monthly entomological surveillance, passive case detection using routine health facility surveillance systems, and studies on human behaviour and ITN use patterns. Data will compare changes in malaria transmission and disease burden in districts receiving dual‑AI ITNs to similar districts receiving standard pyrethroid‑only ITNs over three years. The costs of net distribution will be calculated using the provider perspective including financial and economic costs, and a cost‑effectiveness analysis will assess incremental cost‑effectiveness ratios for Interceptor® G2, Royal Guard®, and piperonyl butoxide ITNs in comparison to standard pyrethroid‑only ITNs, based on incidence rate ratios calcu‑lated from routine data.Conclusions:Evidence of the effectiveness and cost‑effectiveness of the dual‑AI ITNs from these pilot studies will complement evidence from two contemporary cluster randomized control trials, one in Benin and one in Tanzania, to provide key information to malaria control programmes, policymakers, and donors to help guide decision‑making and planning for local malaria control and elimination strategies. Understanding the breadth of contexts where these dual‑AI ITNs are most effective and collecting robust information on factors influencing comparative effectiveness could improve uptake and availability and help maximize their impact

Additional file 2 of Molecular detection of drug resistant polymorphisms in Plasmodium falciparum isolates from Southwest, Nigeria

Dataset containing transcript level determination and allelic discrimination analyses performed with Bio-Rad CFX96 manager software

Optimizing odor-baited trap methods for collecting mosquitoes during the malaria season in The Gambia.

BACKGROUND: Baited traps are potential tools for removal or surveillance of disease vectors. To optimize the use of counter-flow traps baited with human odor (nylon socks that had been worn for a single day) to capture wild mosquitoes in the Gambia, investigations were conducted at a field experimental site. METHODOLOGY/PRINCIPAL FINDINGS: Experiments employing Latin square design were conducted with a set of six huts to investigate the effects of the following on overnight mosquito trap catches: (1) placement of traps indoors or immediately outdoors, CO(2) supply, and presence of a human subject in the hut; (2) trap height for collecting mosquitoes immediately outdoors; (3) height and distance from hut; (4) interaction between multiple traps around a single hut and entry of mosquitoes into huts. A total of 106,600 adult mosquitoes (9.1% Anopheles gambiae s.l., 4.0% other Anopheles species) were collected over 42 nights. The high numbers of An. gambiae s.l. and other mosquitoes collected by odor-baited traps required CO(2) but were largely independent of the presence of a person sleeping in the hut or of trap placement indoors or outdoors. For outdoor collection that is considered less intrusive, traps opening 15 cm above the floor of the hut veranda were more highly effective than traps at other heights or further from the hut. There was no significant evidence of saturation or competition by the traps, with multiple traps around a hut each collecting almost as many mosquitoes as single traps and no effect on the numbers of mosquitoes entering the huts. CONCLUSIONS/SIGNIFICANCE: The outdoor trapping protocol is convenient to compare attractiveness of different odors or synthetic chemicals to malaria vectors and other wild mosquitoes. The finding that such traps are reliably attractive in the presence or absence of a human volunteer encourages their potential development as standardised surveillance tools

Pyrethroids resistance intensity and resistance mechanisms in Anopheles gambiae from malaria vector surveillance sites in Nigeria.

Anopheles gambiae, An. coluzzii and An. arabiensis are the three major vectors of malaria in Nigeria. These mosquitoes have developed resistance to different insecticides. Insecticides resistance intensity assay was recently introduced to provide insight into the potential operational significance of insecticide resistance. Here, we present data on pyrethroids resistance intensity and resistance mechanisms from six vector surveillance sites (Lagos, Ogun, Edo, Anambra, Kwara and Niger) in Nigeria. Adult Anopheles reared from larval collections were tested using WHO insecticides susceptibility protocol with 1x concentration of permethrin and deltamethrin followed with intensity assays with 5x and 10x concentrations of both insecticides. Synergistic and biochemical assays were carried out and underlying resistance mechanisms determined following standard protocols. Anopheles gambiae constituted >50% samples tested in five sites. Permethrin and deltamethrin resistance was observed at all the sites. The Kdt50 varied from 15 minutes (CI = 13.6-17.2) in deltamethrin to 42.1 minutes (CI = 39.4-44.1) in permethrin. For both insecticides, Kdt95 was >30 minutes with 25% to 87% post exposure mortality at the different sites. The West Africa knock down resistance (kdr-w) mechanism was found at each site. Resistant An. gambiae from Lagos, Ogun and Niger synergized prior to permethrin or deltamethrin exposure showed significant mortality (89-100%) compared to unsynergized mosquitoes (Lagos, p = 0.031; Ogun, p = 0.025; Niger, p = 0.018). Biochemical analyses revealed significant increased levels of P450 enzymes in resistant Anopheles gambiae from Lagos (p = 0.038); Ogun (p = 0.042) and Niger (p = 0.028) in addition to GST in Lagos (p = 0.028) and Ogun (p = 0.033). Overall, the results revealed high pyrethroid resistance associated with increased activities of metabolic enzymes (P450 + GST) in An. gambiae and An. coluzzii from Lagos and Ogun. The presence of kdr + P450 conferred moderate resistance whereas low resistance was the case where kdr was the sole resistance mechanism. Findings thus suggests that elevated levels of cytochrome P450 enzymes together with GST were responsible for high or severe pyrethroid resistance

The development of insecticide-treated durable wall lining for malaria control: insights from rural and urban populations in Angola and Nigeria.

BACKGROUND: Durable lining (DL) is a deltamethrin-impregnated polyethylene material, which is designed to cover domestic walls that would normally be sprayed with residual insecticide. The operational success of DL as a long-lasting insecticidal substrate will be dependent on a high level of user acceptability as households must maintain correctly installed linings on their walls for several years. Preliminary trials were undertaken to identify a material to develop into a marketable wall lining and to assess its level of acceptability among rural and urban populations. METHODS: In Angola (n=60), prototype DL and insecticide-treated plastic sheeting (ITPS) were installed on urban house walls and ceilings, respectively, and acceptability was compared to indoor residual spraying (IRS) (n=20) using a knowledge, attitude and practice (KAP) questionnaire. In Nigeria (n=178), three materials (prototype DL, ITPS and insecticide-treated wall netting) were distributed among rural and urban households. User opinions were gathered from focus group discussions, in-depth interviews and KAP questionnaires. RESULTS: In Angola, after two weeks, the majority of participants (98%) expressed satisfaction with the products and identified the killing of insects as the materials' principal benefits (73%). After one year, despite a loss of almost 50% of households to refugee repatriation, all 32 remaining households still asserted that they had liked the DL/ITPS in their homes and given the choice of intervention preferred DL/ITPS to IRS (94%) or insecticide-treated nets (78%). In Nigeria, a dichotomy between rural and urban respondents emerged. Rural participants favoured wall adornments and accepted wall linings because of their perceived decorative value and entomological efficacy. By contrast, urban households preferred minimal wall decoration and rejected the materials based upon objections to their aesthetics and installation feasibility. CONCLUSIONS: The high level of acceptability among rural inhabitants in Nigeria identifies these communities as the ideal target consumer group for durable wall linings. The poorer compliance among urban participants suggests that wall linings would not be readily adopted or sustained in these regions. If DL is as well received by other rural populations it could overcome some of the logistical constraints associated with spray campaigns and has the potential to become a long-lasting alternative to IRS in malaria endemic areas