Distribution of acetylcholinesterase (Ace-1R) target-site G119S mutation and resistance to carbamates and organophosphates in Anopheles gambiae sensu lato populations from Cameroon

Background Cameroon is considering the implementation of indoor residual spraying (IRS) as a complementary measure to control malaria in the context of high pyrethroid resistance in major malaria vectors. Non-pyrethroid insecticide classes such as organophosphates and carbamates may be utilized in IRS due to widespread pyrethroid resistance. However, the success of this strategy depends on good knowledge of the resistance status of malaria vectors to carbamates and organophosphates. Here, we assessed the susceptibility profile of Anopheles gambiae sensu lato with respect to carbamates and organophosphate and the distribution of the molecular mechanism underlying resistance to these insecticides. Methods Anopheles gambiae s.l. mosquitoes were collected from nine settings across the country and bio-assayed with bendiocarb, propoxur and pirimiphos-methyl. The Ace-1 target-site G119S mutation was genotyped using a TaqMan assay. To investigate the polymorphism in the Ace-1 gene, a region of 924 base pairs in a sequence of the gene was amplified from both live and dead females of An. gambiae exposed to bendiocarb. Results Pirimiphos-methyl induced full mortality in An. gambiae s.l. from all study sites, whereas for carbamates, resistance was observed in four localities, with the lowest mortality rate recorded in Mangoum (17.78 ± 5.02% for bendiocarb and 18.61 ± 3.86% for propoxur) in the southern part of Cameroon. Anopheles coluzzii was found to be the predominant species in the northern tropical part of the country where it is sympatric with Anopheles arabiensis. In the localities situated in southern equatorial regions, this species was predominant in urban settings, while An. gambiae was the most abundant species in rural areas. The G119S Ace-1 target-site mutation was detected only in An. gambiae and only in the sites located in southern Cameroon. Phylogenetic analyses showed a clustering according to the phenotype. Conclusion The occurrence of the Ace-1 target-site substitution G119S in An. gambiae s.l. populations highlights the challenge associated with the impending deployment of IRS in Cameroon using carbamates or organophosphates. It is therefore important to think about a resistance management plan including the use of other insecticide classes such as neonicotinoids or pyrrole to guarantee the implementation of IRS in Cameroon

Diversity and behavioral activity of Anopheles mosquitoes on the slopes of Mount Cameroon.

BACKGROUND: Malaria remains endemic in Cameroon, with heterogeneous transmission related to eco-climatic variations, vector diversity and spatial distribution. The intensification of malaria prevention and control through the free distribution of insecticide-treated nets in recent years may have altered the composition, geographic distribution and natural infection rate of Anopheles species, with implications for malaria transmission dynamics. The present study seeks to assess the vectorial diversity, dynamics and infectivity across different seasons and altitudes in relationship to parasite prevalence around the slopes of Mount Cameroon, southwestern region. METHOD: Mosquitoes were sampled (indoors and outdoors) in 11 eco-epidemiological settings at low (18-197 m), intermediate (371-584 m) and high (740-1067 m) altitude by nightly human landing catches. The mosquitoes were identified morphologically and Anopheles gambiae sibling species identified by PCR. Parity status was ascertained by examining the ovaries and the entomological inoculation rates (EIR) determined by Plasmodium falciparum circumsporozoite antigen ELISA of the head-thorax. The prevalence of Plasmodium infection across target communities was assessed using rapid diagnostic tests. RESULTS: A total of 7327 (18.0 mosquitoes/trap/night) mosquitoes were trapped, mainly during the rainy season (5678, 77.5%) and at low altitude (3669, 50.1%). Anopheles spp. (5079, 69.3%) was the most abundant genera and An. gambiae complex (2691, 36.7%) the major vector, varying with altitude (χ2 = 183.87, df = 8, P < 0.001) and season (χ2 = 28.14, df = 4, P < 0.001). Only An. gambiae (s.s.) was identified following molecular analysis of An. gambiae complex siblings. The overall biting peak for An. gambiae complex was 2-3 a.m. Anopheles cinctus was the most abundant secondary vector in the area. The average EIR in the area was 2.08 infective bites per person per night (ib/p/n), higher at low (2.45 ib/p/n) than at intermediate altitude (1.39 ib/p/n) and during the rainy (1.76 ib/p/n) compared to the dry season (0.34 ib/p/n). Anopheles funestus was most infectious overall (28.1%, 16/57) while An. gambiae had the highest inoculation rates averaging 1.33 ib/p/n. Most Anopheles species across all altitudes and seasons were parous, highest in communities with the highest proportion of malaria parasite infections. CONCLUSION: Anopheles gambiae (s.s.) remains the major malaria vector in the area and An. cinctus possibly a secondary vector of the disease in the slopes of Mt. Cameroon. The seasonal and altitudinal effects on the distribution of these mosquitoes may have implications for the transmission of malaria and its control strategies in the area. Regular monitoring of the bionomics of local Anopheles vector species and targeted control interventions in the 'hotspots' is necessary to curb the prevalence of the infection and incidence of disease

The Effects of HIV Infection on the Immune Response to Malaria Among Pregnant Women in Kumba, Southwest Cameroon: Protocol for a Cross-sectional Study

BackgroundMalaria and HIV, 2 of the world's deadliest diseases, share a lot of territory in sub-Saharan Africa. ObjectiveThis study seeks to investigate the effect of HIV on the immune response to malaria infection among pregnant women in Kumba in the southwest region (SWR) of Cameroon. The study aims to determine the prevalence of malaria infection, assess the occurrence of Plasmodium falciparum genetic diversity, and evaluate the antibody (immunoglobulin [Ig]G and IgM: apical membrane antigen-1 [AMA1], merozoite surface protein [MSP]1, MSP2, MSP3, and erythrocyte-binding antigen [EBA]175) and cytokine (interleukin [IL]-10, tumor necrosis factor alpha [TNF-α], and interferon gamma [IFNγ]) response to malaria infection among pregnant women with and without HIV in Kumba. MethodsThe study will be a hospital-based cross-sectional design that will run from March 2022 to February 2023. It will recruit pregnant women with and without HIV who are in their third trimester of pregnancy. The study will be carried out in 5 health institutions in Kumba: General Hospital Kumba, Presbyterian Hospital Kumba, District Hospital Kumba-town, Kossala Integrated Health Center Kumba, and Catholic Hospital Kumba. About 3 mL of the mother’s venous blood, placental blood, and baby cord blood will be collected from each pregnant women at the point of delivery. Microscopy, rapid diagnostic tests (RDTs), and nested polymerase chain reaction (PCR) will be performed to identify the malaria parasite in all the samples, and nested PCR targeting the different genetic diversity markers for P. falciparum will also be performed. Furthermore, sequencing will be performed to study the nucleotide sequence of different alleles, and the genetic diversity of the alleles responsible for malaria infection among pregnant women will be assessed. A multiplex assay will be conducted to analyze the peripheral blood plasma and cord blood plasma for the cytokine and total antibody response to malaria infection among pregnant women with and without HIV. The questionnaire for data collection will be pretested at the Kumba District Hospital, and ethical clearance will be obtained from the University of Buea and the Regional Delegation of Public Health for the SWR. Data will be analyzed using SPSS Statistics and STATA. All P values <.05 will be considered statistically significant. BioEdit 7.0.0 software will be used to align the nucleotide sequences of different genes after sequencing. Phylogenetic tree searching will be conducted using the maximum-likelihood (ML) method in MEGA V6.0. ResultsThe project started in March 2022 and will end in February 2023. Presently, three-fourth of the project funding has been disbursed to date. A total of 218 participants have been enrolled: 193 (88.5%) women without HIV and 25 (11.5%) women with HIV. Between February 2023 and March 2024, the following results will be ready for publication: maternal-neonatal malaria prevalence among pregnant women and babies in Kumba, the effect of HIV on (1) P. falciparum genetic diversity among pregnant women in Kumba, (2) the maternal and neonatal immune response to MSP1, MSP2, and EBA175 IgG antibody response to P. falciparum–caused malaria infection among pregnant women, and (3) the maternal and neonatal pro-inflammatory and anti-inflammatory cytokine response to malaria infection. ConclusionsHIV infection increases the prevalence of malaria infection among pregnant women and also influences the genetic diversity of P. falciparum, with MSP1 alleles being the most prevalent. HIV infection also reduces the antibody response to malaria infection, as well as altering the level of pro-inflammatory and anti-inflammatory responses to malaria infection. International Registered Report Identifier (IRRID)DERR1-10.2196/3821

Experimental huts trial of the efficacy of pyrethroids/piperonyl butoxide (PBO) net treatments for controlling multi-resistant populations of Anopheles funestus s.s. in Kpomè, Southern Benin

Background: Insecticides resistance in Anopheles mosquitoes limits Long-Lasting Insecticidal Nets (LLIN) used for malaria control in Africa, especially Benin. This study aimed to evaluate the bio-efficacy of current LLINs in an area where An. funestus s.l. and An. gambiae have developed multi-resistance to insecticides, and to assess in experimental huts the performance of a mixed combination of pyrethroids and piperonyl butoxide (PBO) treated nets on these resistant mosquitoes. Methods: The study was conducted at Kpomè, Southern Benin. The bio-efficacy of LLINs against An. funestus and An. gambiae was assessed using the World Health Organization (WHO) cone and tunnel tests. A released/recapture experiment following WHO procedures was conducted to compare the efficacy of conventional LLINs treated with pyrethroids only and LLINs with combinations of pyrethroids and PBO. Prior to huts trials, we confirmed the level of insecticide and PBO residues in tested nets using high performance liquid chromatography (HPLC). Results: Conventional LLINs (Type 2 and Type 4) have the lowest effect against local multi-resistant An. funestus s.s. and An. coluzzii populations from Kpomè. Conversely, when LLINs containing mixtures of pyrethroids and PBO (Type 1 and Type 3) were introduced in trial huts, we recorded a greater effect against the two mosquito populations (P < 0.0001). Tunnel test with An. funestus s.s. revealed mortalities of over 80% with this new generation of LLINs (Type 1 and Type 3),while conventional LLINs produced 65.53 ± 8.33% mortalities for Type 2 and 71.25 ±7.92% mortalities for Type 4. Similarly, mortalities ranging from 77 to 87% were recorded with the local populations of An. coluzzii. Conclusion: This study suggests the reduced efficacy of conventional LLINs (Pyrethroids alone) currently distributed in Benin communities where Anopheles populations have developed multi-insecticide resistance. The new generation nets (pyrethroids+PBO) proved to be more effective on multi-resistant populations of mosquitoes

Water source most suitable for rearing a sensitive malaria vector, Anopheles funestus in the laboratory [version 1; referees: 2 approved]

Background:  The insecticide susceptibility status of Anopheles funestus, one of the main malaria vectors in the Afrotropical regions, remains under-studied due to the difficulty of working with this mosquito species. Collecting their larvae in natural breeding sites, rearing and maintaining them in normal laboratory conditions have been a difficult task. Forced-egg laying technique has been a very good tool to generate eggs from adult mosquitoes collected from the wild but rearing these eggs to obtain satisfying portion as adults has always been the problem. In this study, we optimized the development of mosquito species larvae under standard laboratory conditions for desired production of adult mosquitoes that can be useful for insecticide susceptibility tests. Methods:  A forced-egg laying technique was used to obtain eggs from gravid female Anopheles funestus collected from Kpome locality in Benin. Eggs were reared in three different water samples (water from the borehole,and two mineral water namely FIFA and Possotômè) and larvae were fed with TetraMin baby fish food. The physico-chemical parameters of the waters were investigated prior to use for egg incubation. Results:In contrast to mineral water that had no contamination, the borehole water source was contaminated with lead (2.5mg/L) and nitrate (118.8mg/L). Egg hatching rates ranged as 91.9 ± 4.4%, 89.1 ± 2.5% and 87.9 ± 2.6% in FIFA, Possotômè and borehole water respectively. High emergence of larvae to adult mosquitoes was recorded as in FIFA (74.3%) and Possotômè(79.5%) water. No adult mosquito was obtained from larvae reared in borehole water. Conclusions: This study gave insight on the water sources that could be good for rearing to mass produce An. funestus in the laboratory. More analysis with other local mineral water sources in our environments could be considered in the future, hopefully giving better outputs

Water source most suitable for rearing a sensitive malaria vector, Anopheles funestus in the laboratory [version 2; referees: 2 approved]

Background:  The insecticide susceptibility status of Anopheles funestus, one of the main malaria vectors in the Afrotropical regions, remains under-studied due to the difficulty of working with this mosquito species. Collecting their larvae in natural breeding sites, rearing and maintaining them in normal laboratory conditions have been a difficult task. Forced-egg laying technique has been a very good tool to generate eggs from adult mosquitoes collected from the wild but rearing these eggs to obtain satisfying portion as adults has always been the problem. In this study, we optimized the development of mosquito species larvae under standard laboratory conditions for desired production of adult mosquitoes that can be useful for insecticide susceptibility tests. Methods:  A forced-egg laying technique was used to obtain eggs from gravid female Anopheles funestus collected from Kpome locality in Benin. Eggs were reared in three different water samples (water from the borehole, and two mineral water namely FIFA and Possotômè) and larvae were fed with TetraMin baby fish food. The physico-chemical parameters of the waters were investigated prior to use for egg incubation (introduction of eggs’ batches into water). Results: In contrast to mineral water that had no contamination, the borehole water source was contaminated with lead (2.5mg/L) and nitrate (118.8mg/L). Egg hatching rates ranged as 91.9 ± 4.4%, 89.1 ± 2.5% and 87.9 ± 2.6% in FIFA, Possotômè and borehole water respectively. High emergence of larvae to adult mosquitoes was recorded as in FIFA (74.3%) and Possotômè (79.5%) water. No adult mosquito was obtained from larvae reared in borehole water. Conclusions: This study gave insight on the water sources that could be good for rearing to mass produce An. funestus in the laboratory. More analysis with other local mineral water sources in our environments could be considered in the future, hopefully giving better outputs

First report of the presence of L1014S Knockdown-resistance mutation in Anopheles gambiae s.s and Anopheles coluzzii from Togo, West Africa [version 1; referees: 2 approved, 1 approved with reservations]

Background: To optimize the success of insecticide-based malaria control intervention, knowledge of the distribution of Anopheles gambiae species and insecticide resistance mechanisms is necessary. This paper reported an updated data on pyrethroids/DDT resistance in the An. gambiae s.l population from Togo.  Methods: From December 2013 to April 2015, females of indoor-resting An. gambiae s.l were captured in three locations belonging to three different ecological zones. Resistance to DDT, permethrin and deltamethrin was screened in F1 progeny of collected mosquitoes using WHO susceptibility tests. The identification of species of An. gambiae complex and the detection of kdr and ace.1R allele were carried out using DNA-based molecular techniques. Results: An. gambiae from Kovié and Nangbéto were highly resistant to DDT and permethrin with mortalities rate ranging from 0.83% to 1.58% for DDT and zero to 8.54% for permethrin. Mosquitoes collected in Nangbéto displayed 81.53% mortality with deltamethrin. An. coluzzii and An. gambiae s.s were found in sympatry in Nangbéto and Mango. The allelic frequency of L1014F was high, ranging from 66 to 100% in both An. coluzzii and An. gambiae s.s. For the first time we detected the L1014S allele in both An. coluzzii and An. gambiae s.s. from Togo at the frequency ranging from 5% to 13% in all the sites. The kdr N1575Y was present at various frequencies in both species ranging from 10% to 45%. Both An. gambiae s.s. and An. coluzzii shared the ace1R mutation in all investigated sites with allelic frequency ranging from 4% to 16%. Conclusion: These results showed that multiple mutations are involved in insecticides resistance in An. gambiae populations from Togo including the kdr L1014F, L1014S, and N1575Y and ace.1R G119S mutations

Community-based geographical distribution of Mycobacterium ulcerans VNTR-genotypes from the environment and humans in the Nyong valley, Cameroon

Background: Genotyping is a powerful tool for investigating outbreaks of infectious diseases and it can provide useful information such as identifying the source and route of transmission, and circulating strains involved in the outbreak. Genotyping techniques based on variable number of tandem repeats (VNTR) are instrumental in detecting heterogeneity in Mycobacterium ulcerans (MU) and also for discriminating MU from other mycobacteria species. Here, we describe and map the distribution of MU genotypes in Buruli ulcer (BU) endemic communities of the Nyong valley in Cameroon. We also tested the hypothesis of whether the suspected animal reservoirs of BU that share the human microhabitat are shedding contaminated fecal matters and saliva into their surrounding environments. Methods: Environmental samples from suspected MU-risk factors and lesion swabs from human patients were sampled in BU-endemic communities and tested for the presence of MU by qPCR targeting three independent sequences (IS 2404 , IS 2606 , KR-B). Positive samples to MU were further genotyped by VNTR with confirmation by sequencing of four loci (MIRU1, Locus 6, ST1, Locus 19). Results: MU was detected in environmental samples including water bodies (23%), biofilms (14%), detritus (10%), and in human patients (73%). MU genotypes D, W, and C were found both in environmental and human samples. The micro geo-distribution of MU genotypes from communities showed that genotype D is found both in environmental and human samples, while genotypes W and C are specific to environmental samples and human lesions, respectively. No obvious focal grouping of MU genotypes was observed at the community scale. An additional survey in the human microhabitat suggests that domestic and wild animals do not shed MU in their saliva and feces in sampled communities. Conclusions: VNTR typing uncovered different MU genotypes circulating in the endemic communities of the Akonolinga district. A MU environmental genotype was found in patients, yet the mechanism of contamination remains to be investigated; and recovering MU in culture from the environment remains key priority to enable a better understanding of the mode of transmission of BU. We also conclude that excretions from suspected animals are unlikely to be major sources of MU in the Nyong Valley in Cameroon.</p

Using artificial odors to optimize attractiveness of host decoy traps to malaria vectors

Malaria vector surveillance tools often incorporate features of hosts that are attractive to blood-seeking females. The recently developed host decoy trap (HDT) combines visual, thermal, and olfactory stimuli associated with human hosts and has shown great efficacy in terms of collecting malaria vectors. Synthetic odors and yeast-produced carbon dioxide (CO2) could prove useful by mimicking the human odors currently used in HDTs and provide standardized and easy-to-use olfactory attractants. The objective of this study was to test the attractiveness of various olfactory attractant cues in HDTs to capture malaria vectors. We compared 4 different odor treatments in outdoor field settings in southern Benin and western Burkina Faso: the standard HDT using a human, HDT with yeast-produced CO2, HDT with an artificial odor blend, and HDT with yeast-produced CO2 plus artificial odor blend. In both experimental sites, the standard HDT that incorporated a real human produced the greatest catch of Anopheles gambiae s.l (Diptera: Culicidae). The alternatives tested were still effective at collecting target vector species, although the most effective included CO2, either alone (Benin) or in combination with synthetic odor (Burkina Faso). The trap using synthetic human odor alone caught the fewest An. gambiae s.l. compared to the other baited traps. Both Anopheles coluzzii and Anopheles gambiae were caught by each trap, with a predominance of An. coluzzii. Synthetic baits could, therefore, represent a more standardized and easier-to-deploy approach than using real human odor baits for a robust vector monitoring strategy

Co-infections of Malaria and Geohelminthiasis in Two Rural Communities of Nkassomo and Vian in the Mfou Health District, Cameroon

<div><p>Background</p><p>Human co-infection with malaria and helmimths is ubiquitous throughout Africa. Nevertheless, its public health significance on malaria severity remains poorly understood.</p><p>Methodology/Principal Findings</p><p>To contribute to a better understanding of epidemiology and control of this co-infection in Cameroon, a cross-sectional study was carried out to assess the prevalence of concomitant intestinal geohelminthiasis and malaria, and to evaluate its association with malaria and anaemia in Nkassomo and Vian. Finger prick blood specimens from a total of 263 participants aged 1–95 years were collected for malaria microscopy, assessment of haemoglobin levels, and molecular identification of <i>Plasmodium</i> species by PCR. Fresh stool specimens were also collected for the identification and quantification of geohelminths by the Kato-Katz method. The prevalence of malaria, geohelminths, and co-infections were 77.2%, 28.6%, and 22.1%, respectively. <i>Plasmodium falciparum</i> was the only malaria parasite species identified with mean parasite density of 111 (40; 18,800) parasites/µl of blood. The geohelminths found were <i>Ascaris lumbricoides</i> (21.6%) and <i>Trichuris trichiura</i> (10.8%), with mean parasite densities of 243 (24; 3,552) and 36 (24; 96) eggs/gram of faeces, respectively. Co-infections of <i>A. lumbricoides</i> and <i>P. falciparum</i> were the most frequent and correlated positively. While no significant difference was observed on the prevalences of single and co-infections between the two localities, there was a significant difference in the density of <i>A. lumbricoides</i> infection between the two localities. The overall prevalence of anaemia was 42%, with individuals co-infected with <i>T. trichiura</i> and <i>P. falciparum</i> (60%) being the most at risk. While the prevalence of malaria and anaemia were inversely related to age, children aged 5–14 years were more susceptible to geohelminthiasis and their co-infections with malaria.</p><p>Conclusion/Significance</p><p>Co-existence of geohelminths and malaria parasites in Nkassomo and Vian enhances the occurrence of co-infections, and consequently, increases the risk for anaemia.</p></div