Vectorial competence, insecticide resistance in Anopheles funestus and operational implications for malaria vector control strategies in Benin Republic.

The primary reason for the failure of malaria vector control across endemic regions is the widespread insecticide resistance observed in Anopheles vectors. The most dominant African vectors of malaria parasites are Anopheles gambiae and Anopheles funestus mosquitoes. These species often exhibit divergent behaviours and adaptive changes underscoring the importance of deploying active and effective measures in their control. Unlike An. gambiae, An. funestus mosquitoes are poorly studied in Benin Republic. However, recent reports indicated that An. funestus can adapt and colonize various ecological niches owing to its resistance against insecticides and adaptation to changing breeding habitats. Unfortunately, scientific investigations on the contribution of An. funestus to malaria transmission, their susceptibility to insecticide and resistance mechanism developed are currently insufficient for the design of better control strategies. In an attempt to gather valuable information on An. funestus, the present review examines the progress made on this malaria vector species in Benin Republic and highlights future research perspectives on insecticide resistance profiles and related mechanisms, as well as new potential control strategies against An. funestus. Literature analysis revealed that An. funestus is distributed all over the country, although present in low density compared to other dominant malaria vectors. Interestingly, An. funestus is being found in abundance during the dry seasons, suggesting an adaptation to desiccation. Among the An. funestus group, only An. funestus sensu stricto (s.s.) and Anopheles leesoni were found in the country with An. funestus s.s. being the most abundant species. Furthermore, An. funestus s.s. is the only one species in the group contributing to malaria transmission and have adapted biting times that allow them to bite at dawn. In addition, across the country, An. funestus were found resistant to pyrethroid insecticides used for bed nets impregnation and also resistant to bendiocarb which is currently being introduced in indoor residual spraying formulation in malaria endemic regions. All these findings highlight the challenges faced in controlling this malaria vector. Therefore, advancing the knowledge of vectorial competence of An. funestus, understanding the dynamics of insecticide resistance in this malaria vector, and exploring alternative vector control measures, are critical for sustainable malaria control efforts in Benin Republic.

Multiple Resistance Mechanisms to Pyrethroids Insecticides in Anopheles gambiae sensu lato Population From Mali, West Africa.

BACKGROUND: Insecticide-based vector control is responsible for reducing malaria mortality and morbidity. Its success depends on a better knowledge of the vector, its distribution, and resistance status to the insecticides used. In this paper, we assessed Anopheles gambiae sensu lato (A gambiae s.l.) population resistance to pyrethroids in different ecological settings. METHODS: The World Health Organization standard bioassay test was used to assess F0A gambiae s.l. susceptibility to pyrethroids. Biochemical Synergist assays were conducted with piperonyl butoxide (PBO), S,S,S-tributyl phosphotritioate, and diethyl maleate. L1014F, L1014S, and N1575Y knockdown resistance (kdr) mutations were investigated using TaqMan genotyping. RESULTS: Anopheles gambiae sensu lato was composed of Anopheles arabienisis, Anopheles coluzzii, and A gambiae in all study sites. Anopheles gambiae sensu lato showed a strong phenotypic resistance to deltamethrin and permethrin in all sites (13% to 41% mortality). In many sites, pre-exposure to synergists partially improved the mortality rate suggesting the presence of detoxifying enzymes. The 3 kdr (L1014F, L1014S, and N1575Y) mutations were found, with a predominance of L1014F, in all species. CONCLUSIONS: Multiple resistance mechanisms to pyrethroids were observed in A gambiae s.l. in Mali. The PBO provided a better partial restoration of susceptibility to pyrethroids, suggesting that the efficacy of long-lasting insecticidal nets may be improved with PBO.

Evidences of the Low Implication of Mosquitoes in the Transmission of Mycobacterium ulcerans, the Causative Agent of Buruli Ulcer.

BACKGROUND: Buruli ulcer (BU) continues to be a serious public health threat in wet tropical regions and the mode of transmission of its etiological agent, Mycobacterium ulcerans (MU), remains poorly understood. In this study, mosquito species collected in endemic villages in Benin were screened for the presence of MU. In addition, the ability of mosquitoes larvae to pick up MU from their environment and remain colonized through the larval developmental stages to the adult stage was investigated. METHODS: 7,218 adults and larvae mosquitoes were sampled from endemic and nonendemic villages and screened for MU DNA targets (IS2404, IS2606, and KR-B) using qPCR. Results. MU was not detected in any of the field collected samples. Additional studies of artificially infected larvae of Anopheles kisumu with MU strains revealed that mosquitoes larvae are able to ingest and host MU during L1, L2, L3, and L4 developmental stages. However, we noticed an absence of these bacteria at both pupae and adult stages, certainly revealing the low ability of infected or colonized mosquitoes to vertically transmit MU to their offspring. CONCLUSION: The overall findings highlight the low implication of mosquitoes as biological vectors in the transmission cycle of MU from the risk environments to humans.

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.

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.

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 (IS2404, IS2606, 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.

Acetylcholinesterase (ace-1R) target site mutation G119S and resistance to carbamates in Anopheles gambiae (sensu lato) populations from Mali.

BACKGROUND: The long-lasting insecticidal nets (LLINs) and indoor residual spraying of insecticide (IRS) are major malaria vector control strategies in Mali. The success of control strategies depends on a better understanding of the status of malaria vectors with respect to the insecticides used. In this study we evaluate the level of resistance of Anopheles gambiae (sensu lato) to bendiocarb and the molecular mechanism that underlies it. METHODS: Larvae of An. gambiae (s.l.) were collected from breeding habitats encountered in the three study sites and bioassayed with bendiocarb. The ace-1 target site substitution G119S was genotyped using a TaqMan assay. RESULTS: The three species of the An. gambiae complex in Mali, i.e. An. arabiensis, An. coluzzii and An. gambiae (s.s.) were found in sympatry in the three surveyed localities with different frequencies. We observed a resistance and suspicious resistance of the three species to bendiocarb with a mortality rate ranging from 37% to 86%. The allelic frequency of the G119S mutation was higher in An. gambiae (s.s.) compared to the other two species; 42.86%, 25.61% and 16.67% respectively in Dangassa, Koula, and Karadié. The allelic frequency of G119S in An. coluzzii ranged from 4.5% to 8.33% and from 1.43% to 21.15% for An. arabiensis. After exposure to bendiocarb, the G119S mutation was found only in survivors. The survival of Anopheles gambiae (s.l) populations from the three surveyed localities was associated with the presence of the mutation. CONCLUSIONS: The study highlights the implication of G119S mutation in bendiocarb resistance in An. gambiae (s.s.), An. arabiensis and An. coluzzii populations from the three surveyed localities.

Case Definitions of Clinical Malaria in Children from Three Health Districts in the North Region of Cameroon.

Malaria endemicity in Cameroon greatly varies according to ecological environment. In such conditions, parasitaemia, which is associated with fever, may not always suffice to define an episode of clinical malaria. The evaluation of malaria control intervention strategies mostly consists of identifying cases of clinical malaria and is crucial to promote better diagnosis for accurate measurement of the impact of the intervention. We sought out to define and quantify clinical malaria cases in children from three health districts in the Northern region of Cameroon. A cohort study of 6,195 children aged between 6 and 120 months was carried out during the raining season (July to October) between 2013 and 2014. Differential diagnosis of clinical malaria was performed using the parasite density and axillary temperature. At recruitment, patients with malaria-related symptoms (fever [axillary temperature ≥ 37.5°C], chills, severe malaise, headache, or vomiting) and a malaria positive blood smear were classified under clinical malaria group. The malaria attributable fraction was calculated using logistic regression models. Plasmodium falciparum was responsible for over 91% of infections. Children from Pitoa health district had the highest number of asymptomatic infections (45.60%) compared to those from Garoua and Mayo Oulo. The most suitable cut-off for the association between parasite densities and fever was found among children less than 24 months. Overall, parasite densities that ranged above 3,200 parasites per µl of blood could be used to define the malaria attributable fever cases. In groups of children aged between 24 and 59 months and 60 and 94 months, the optimum cut-off parasite density was 6,400 parasites per µl of blood, while children aged between 95 and 120 months had a cut-off of 800 parasites per µl of blood. In the same ecoepidemiological zone, clinical malaria case definitions are influenced by age and location (health district) and this could be considered when evaluating malaria intervention strategies in endemic areas.

Susceptibility Profiles of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) to Deltamethrin Reveal a Contrast between the Northern and the Southern Benin.

Helicoverpa armigera is an indigenous species in Africa and has been reported in the destruction of several crops in Benin. Management of H. armigera pest is mainly focused on the use of synthetic pyrethroids, which may contribute to resistance selection. This study aimed to screen the susceptibility pattern of field populations of H. armigera to deltamethrin in Benin. Relevant information on the type of pesticides used by farmers were gathered through surveys. Collected samples of Helicoverpa (F0) were reared to F1. F0 were subjected to morphological speciation followed by a confirmation using restriction fragment length polymorphism coupled with a polymerase chain reaction (RFLP-PCR). F1 (larvae) were used for insecticide susceptibility with deltamethrin alone and in the presence of the P450 inhibitor Piperonyl Butoxide (PBO). Deltamethrin and lambda-cyhalothrin were the most used pyrethroids in tomato and cotton farms respectively. All field-sampled Helicoverpa were found to be H. armigera. Susceptibility assays of H. armigera to deltamethrin revealed a high resistance pattern in cowpea (resistance factor (RF) = 2340), cotton (RF varying from 12 to 516) and tomato (RF=85) farms which is a concern for the control of this major polyphagous agricultural pest. There was a significant increase of mortality when deltamethrin insecticide was combined with piperonyl butoxide (PBO), suggesting the possible involvement of detoxification enzymes such as oxidase. This study highlights the presence of P450 induced metabolic resistance in H. armigera populations from diverse cropping systems in Benin. The recorded high levels of deltamethrin resistance in H. armigera is a concern for the control of this major agricultural pest in Benin as the country is currently embarking into economical expansion of cotton, vegetables and grain-legumes cropping systems.

First report of the presence of L1014S Knockdown-resistance mutation in Anopheles gambiae s.s and Anopheles coluzzii from Togo, West Africa.

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.1 R 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. gambiaes.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 ace1 R 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.1 R G119S mutations.

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. funestuss.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.

The Rapid Degradation of Lambda-Cyhalothrin Makes Treated Vegetables Relatively Safe for Consumption.

Lambda-cyhalothrin (λ-cyhalothrin) is the most commonly used pyrethroid insecticide for vegetable farming in Benin. This insecticide is misused and overused by farmers, and hence may pose health hazards to consumers. We monitored λ-cyhalothrin residues in lettuce and cabbage from farms at the market gates in Cotonou and Parakou using high performance liquid chromatography (HPLC) analysis techniques. These residues were also monitored on samples directly from farms (on-farm sampling) for 14 days post-treatment. Potential factors such as photolysis and hydrolysis involved in λ-cyhalothrin degradation were also screened. Results revealed that the level of λ-cyhalothrin residue concentrations in lettuce from Houeyiho decreased from 4.2 mg/kg on Day 1 to about 0.2 mg/kg on Day 7. On Day 9, analyzed lettuces were all λ-cyhalothrin free. In contrast, even 14 days after treatment of cabbage from Bawera (Parakou), we still recorded the presence of λ-cyhalothrin residues in analyzed samples. For samples from market gates, λ-cyhalothrin residues were found in lettuce from two markets out of the nine surveyed in Cotonou. Interestingly, none of these contaminated samples had residues above the maximum residue limit for lettuce (MRL = 0.5 mg/kg). Similarly, in Parakou, samples from all five surveyed vegetable markets were contaminated with λ-cyhalothrin residues at concentrations below the MRL for cabbage (MRL = 0.2 mg/kg). We conclude that λ-cyhalothrin residues in lettuce and cabbage from farms and markets in Parakou and Cotonou are within the MRL, and hence are relatively safe for consumption.

Domestic animals infected with Mycobacterium ulcerans-Implications for transmission to humans.

BACKGROUND: The environmental pathogen, Mycobacterium ulcerans (MU) can infect both humans and animals and cause Buruli ulcer (BU) disease. However, its mode(s) of transmission from the colonized environment to human/animal hosts remain unclear. In Australia, MU can infect both wildlife and domestic mammals. Till date, BU-like lesions have only been reported in wildlife in Africa. This warrants a thorough assessment of possible MU in domestic animals in Africa. Here, we screened roaming domesticated animals that share the human microhabitat in two different BU endemic sites, Sedje-Denou in Benin and Akonolinga in Cameroon, for MU lesions. METHODOLOGY/PRINCIPAL FINDINGS: We screened roaming mammals and birds across 3 endemic villages of Sedje-Denou in Southern Benin and 6 endemic villages of Akonolinga in Cameroon. After approval from relevant authorities, specimens (wound swabs and tissue fragments) were collected from animals with open or active lesion and systematically screened to detect the presence of MU though the diagnostic DNA targets IS2404, IS2606 and KR-B. Out of 397 animals surveyed in Akonolinga, 44 (11.08%) carried skin lesions and all were negative for MU DNA. For Sedje-Denou, only 25 (6.93%) out of 361 animals surveyed carried external skin lesions of which 2 (8%) were positive for MU DNA targets. These MU infected lesions were found in two different villages on a goat (abdominal part) and on a dog (nape area of the neck). Source-tracking of MU isolates within infected animal lesions was performed using VNTR genotyping and further confirmed with sequencing. One MU VNTR genotype (Z) was successfully typed from the goat lesion. The evolutionary history inferred from sequenced data revealed a clustering of animal MU isolates within isolates from human lesions. CONCLUSION/SIGNIFICANCE: This study describes the first report of two MU infected lesions in domestic animals in Africa. Their DNA sequence analyses show close relationship to isolates from human cases. It suggests that MU infection should be suspected in domestic hosts and these could play a role in transmission. The findings further support the hypothesis that MU is a ubiquitous environmental pathogen found in endemic areas, and probably involved in a multiple transmission pathway.

Water source most suitable for rearing a sensitive malaria vector, Anopheles funestus in the laboratory.

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.

Co-infections of malaria and geohelminthiasis in two rural communities of Nkassomo and Vian in the Mfou health district, Cameroon.

BACKGROUND: Human co-infection with malaria and helmimths is ubiquitous throughout Africa. Nevertheless, its public health significance on malaria severity remains poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: 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 Plasmodium 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. Plasmodium falciparum was the only malaria parasite species identified with mean parasite density of 111 (40; 18,800) parasites/µl of blood. The geohelminths found were Ascaris lumbricoides (21.6%) and Trichuris trichiura (10.8%), with mean parasite densities of 243 (24; 3,552) and 36 (24; 96) eggs/gram of faeces, respectively. Co-infections of A. lumbricoides and P. falciparum 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 A. lumbricoides infection between the two localities. The overall prevalence of anaemia was 42%, with individuals co-infected with T. trichiura and P. falciparum (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. CONCLUSION/SIGNIFICANCE: Co-existence of geohelminths and malaria parasites in Nkassomo and Vian enhances the occurrence of co-infections, and consequently, increases the risk for anaemia.

Factors associated to bed net use in Cameroon: a retrospective study in Mfou health district in the Centre Region.

INTRODUCTION: Insecticide treated net remains a tool of choice for malaria prevention in Cameroon. However, data suggests that its use by the population, especially vulnerable groups remains low. Moreover, there is a paucity of information about factors influencing its use. We sought out to identify factors associated with net use in Mfou health district, prior to distribution of long lasting insecticides treated nets (LLINs) in households. METHODS: A two-stage cluster random sampling was conducted in 4 health areas with an average of 13 villages each. A total of 541 households were selected and heads interviewed using a structured household questionnaire. Data collected were entered into a database and multivariate logistic regression analyses of the association between net use and explanatory factors were performed using SPSS. RESULTS: Net possession and use were respectively, 59.7 and 42.6%; thus, 2 out of 5 people who spent the previous night in households, slept under a net. Factors associated with net use included: net density≥0.5 (OR=8.88, 95% CI: 6.24-12.64), age≥5 years (OR=0.37, 95%CI: 0.28-0.47), secondary education (OR=1.41, 95% CI: 1.11-1.80) compared to primary/no education, parent status (OR=3.32, 95% CI: 2.31-4.76), house construction (OR=1.37, 95% CI: 1.10-1.71) and environment characteristics (OR=1.46, 95% CI: 1.18-1.80). CONCLUSION: These data suggest that a universal coverage with one LLIN for two people should be achieved in households. Then, malaria health education should be conducted to re-enforce net use among school-aged children and adolescents, as well as older household members. Moreover, management of environment and improvement in houses construction are necessary.