Multiple insecticide resistance in the major malaria vector Anopheles funestus in southern Ghana: implications for malaria control

Background Understanding the dynamics of insecticide resistance in African malaria vectors is crucial for successful implementation of resistance management strategies in the continent. This study reports a high and multiple insecticide resistance in Anopheles funestus from southern Ghana which could compromise the Malaria Operational Plan in this country, if not tackled. Adult Anopheles mosquitoes were collected in Obuasi and Adawukwa, in southern Ghana. Plasmodium infection rates, susceptibility to the main insecticides used in public health and the molecular basis of insecticide resistance were established. Results An. funestus (sensu stricto) (s.s.) was the predominant mosquito species found resting inside the houses in Obuasi, while at Adawukwa it was found together with An. coluzzii. Parasite rates were high in An. funestus (s.s.) populations from both localities, with Plasmodium infection rates greater than 12.5 %. Both, An. funestus (s.s.) and An. coluzzii, from the two sites exhibited high resistance to the insecticide from various classes including the pyrethroids, carbamates and DDT, but remained fully susceptible to the organophosphates. A preliminary characterization of the underlying molecular mechanisms of resistance in An. funestus (s.s.) populations from both sites revealed that CYP6P9a, CYP6P9b, CYP6M7 and GSTe2 genes are upregulated, markedly higher in Obuasi (between 3.35 and 1.83 times) than in Adawukwa population. The frequency of L119F-GSTe2 and A296S-RDL resistance markers were also higher in Obuasi (42.5 and 68.95 % higher), compared with An. funestus (s.s.) populations from Adawukwa. These findings suggest that the similar resistance pattern observed in both An. funestus (s.s.) populations are driven by different mechanisms. Conclusions Resistance to multiple insecticides in public health use is present in malaria vectors from Ghana with major resistance genes already operating in the field. This should be taken into consideration in the design of resistance management strategies to avoid operational failure

Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions

Genome-wide association studies reveal novel loci associated with pyrethroid and organophosphate resistance in Anopheles gambiae

Resistance to insecticides in Anopheles mosquitoes threatens the effectiveness of malaria control, but the genetics of resistance are only partially understood. We performed a large scale multi-country genome-wide association study of resistance to two widely used insecticides: deltamethrin and pirimiphos-methyl, using sequencing data from An. gambiae and An. coluzzii from ten locations in West Africa. Resistance was highly multi-genic, multiallelic and variable between populations. While the strongest and most consistent association with deltamethrin resistance came from Cyp6aa1, this was based on several independent copy number variants (CNVs) in An. coluzzii, and on a non-CNV haplotype in An. gambiae. For pirimiphos-methyl, signals included Ace1, cytochrome P450s, glutathione S-transferases and the nAChR target site of neonicotinoid insecticides. The regions around Cyp9k1 and the Tep family of immune genes showed evidence of cross-resistance to both insecticides. These locally-varying, multi-allelic patterns highlight the challenges involved in genomic monitoring of resistance, and form the basis for improved surveillance methods

A cis-regulatory sequence driving metabolic insecticide resistance in mosquitoes: Functional characterisation and signatures of selection

Although cytochrome P450 (CYP450) enzymes are frequently up-regulated in mosquitoes resistant to insecticides, no regulatory motifs driving these expression differences with relevance to wild populations have been identified. Transposable elements (TEs) are often enriched upstream of those CYP450s involved in insecticide resistance, leading to the assumption that they contribute regulatory motifs that directly underlie the resistance phenotype. A partial CuRE1 (Culex Repetitive Element 1) transposable element is found directly upstream of CYP9M10, a cytochrome P450 implicated previously in larval resistance to permethrin in the ISOP450 strain of Cx. quinquefasciatus, but is absent from the equivalent genomic region of a susceptible strain. Via expression of CYP9M10 in E.coli we have now demonstrated time- and NADPH-dependant permethrin metabolism, prerequisites for confirmation of a role in metabolic resistance, and through qPCR shown that CYP9M10 is >20-fold over-expressed in ISOP450 compared to a susceptible strain. In a fluorescent reporter assay the region upstream of CYP9M10 from ISOP450 drove 10x expression compared to the equivalent region (lacking CuRE1) from the susceptible strain. Close correspondence with the gene expression fold-change implicates the upstream region including CuRE1 as a cis-regulatory element involved in resistance. Only a single CuRE1 bearing allele, identical to the CuRE1 bearing allele in the resistant strain, is found throughout Sub-Saharan Africa, in contrast to the diversity encountered in non-CuRE1 alleles. This suggests a single origin and subsequent spread due to selective advantage. CuRE1 is detectable using a simple diagnostic. When applied to Cx. quinquefasciatus larvae from Ghana we have demonstrated a significant association with permethrin resistance in multiple field sites (mean Odds Ratio = 3.86) suggesting this marker has relevance to natural populations of vector mosquitoes. However, when CuRE1 was excised from the allele used in the reporter assay through fusion PCR, expression was unaffected, indicating that the TE has no direct role in resistance and hence that CuRE1 is acting only as a marker of an as yet unidentified regulatory motif in the association analysis. This suggests that a re-evaluation of the assumption that TEs contribute regulatory motifs involved in gene expression may be necessary

Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus

Publisher Copyright: © 2021 Grau-Bové et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions.publishersversionpublishe

Resistance to pirimiphos-methyl in West African Anopheles is spreading via duplication and introgression of the Ace1 locus

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Coˆte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based interventions

Elevated IL-12, TNF-α, and TNF-α/IL-10 Ratios in Stored Plasmodium falciparum-Infected Whole Blood: Implications for Safe Haemotransfusion

Although Plasmodium falciparum infections in blood donors have been reported, the impact of parasitaemia on cytokine levels in stored whole blood has not been explored. This study evaluated the effect of P. falciparum parasitaemia on circulating cytokines and their relationship with haematological parameters in banked blood. In this case-control study, two groups of donor whole blood were recruited: P. falciparum-infected donors (parasitaemia: 515–1877 parasites/μL) and noninfected blood donors (control). At day 0 (baseline), 7, 14, 21, and 35 of banking circulating cytokine levels of tumor necrosis factor alpha (TNF-α), interleukin- (IL-) 12, IL-10, and IL-6 levels and haematological parameters were determined. Kruskal-Wallis test determined differences in weekly cytokine levels while Dunn’s post hoc test determined exact significant points. At baseline, the mean TNF-α (33.81 pg/mL vs. 22.70 pg/mL), IL-12 (28.39 pg/mL vs. 16.15 pg/mL), IL-10 (51.04 pg/mL vs. 18.95 pg/mL), and IL-6 (71.03 pg/mL vs. 30.89 pg/mL) levels were significantly higher in infected donor whole blood. Significant rate of increase was observed in TNF-α, IL-12 levels, and TNF-α/IL-10 ratios in infected blood, while decreased levels were observed in IL-10. IL-6 peaked at day 21 and fell below baseline level at day 35. Significant changes in TNF-α, IL-12, IL-10, IL-6 levels, and TNF-α/IL-10 ratios in infected donor blood were observed 7 days after storage. Unlike in noninfected stored whole blood, TNF-α, IL-6, IL-12, and TNF-α/IL-10 ratio levels in infected stored whole blood related inversely to haematological parameters (white cells, red cells, platelets, and haemoglobin levels) during storage. However, in both groups, significant direct relationship was observed in IL-10 and haematological parameters. In conclusion, banking of P. falciparum-infected donor whole blood may lead to infusion of large quantities of inflammatory cytokines with potential adverse immunological response in recipients

Identification of Sand Flies (Diptera: Psychodidae) Collected from Cutaneous Leishmaniasis Endemic Focus in the Ho Municipality, Ghana

Leishmaniasis, is a vector-borne disease transmitted to humans through the bite of infected female sand flies. Active and continuous monitoring of the sand fly is an important aspect of disease control. Thus, the correct identification of its vectors is paramount in this regard. Objective: The study was conducted to morphologically and molecularly identify female sand fly species in a cutaneous leishmaniasis endemic focus collected in three villages in the Ho Municipality of the Volta region. CDC light traps and sticky paper traps was used for the collection of the sand flies. The morphologically identified sand flies was molecularly confirmed by running PCR with the mitochondrial cytochrome c oxidase gene subunit I (COI) primers and DNA sequenced. A total of 537 sand flies was collected, made up of 363 females and 174 males.  Eleven different species of sand flies was morphologically identified – one Phlebotomus species and ten Sergentomyia species. The PCR amplified products showed bands of molecular weights 658 base pairs for the primers. The molecular identification using the 658-bp fragment of the (COI) gene was congruent with the morphological identification

Effectiveness of Intermittent Preventive Treatment in Pregnancy with Sulphadoxine-Pyrimethamine against Submicroscopic falciparum

Malaria infections undetectable by microscopy but detectable by Polymerase Chain Reaction (PCR) (submicroscopic malaria) are common in endemic areas like Ghana. Submicroscopic malaria has been linked with severe pregnancy outcomes as well as contributing to malaria transmission. In this cross-sectional study 872 consenting pregnant women (gestation ≥ 20 weeks) were recruited from 8 hospitals in Central Region, Ghana, between July and December 2009. Malaria infection was detected by microscopy and PCR. Haemoglobin was measured and anaemia was defined as haemoglobin lower than 11 g/dL. Majority of the women, 555 (63.6%), were Intermittent Preventive Treatment in Pregnancy with Sulphadoxine-Pyrimethamine (IPTp-SP) users while 234 (36.4%) were nonusers. The prevalence of malaria by microscopy was 20.9% (182/872) and 9.7% (67/688) of microscopy negative women had submicroscopic malaria. IPTp-SP usage significantly (odds ratio = 0.13, 95% confidence interval = 0.07–0.23, p=0.005) reduced the prevalence of submicroscopic malaria as more nonusers (51/234) than users (16/454) were PCR positive. After controlling for other variables the effect of IPTp-SP remained statistically significant (odds ratio = 0.11, 95% confidence interval = 0.02–0.22, p=0.006). These results suggest that Intermittent Preventive Treatment with Sulphadoxine-Pyrimethamine is useful in the reduction of submicroscopic malaria in pregnancy