Pyrethroid Resistance Situation across Different Eco-Epidemiological Settings in Cameroon.

Rapid emergence and spread of pyrethroid resistance in Anopheles gambiae populations is among the main factors affecting malaria vector control in Cameroon, but there is still not enough data on the exact pyrethroid resistance status across Cameroon. The present study assessed pyrethroid resistance profile in different eco-epidemiological settings in Cameroon. Susceptibility bioassay tests were performed with F0 An. gambiae females aged three to five days. Mosquito susceptibility to both permethrin and deltamethrin was assessed. Species of the An. gambiae s.l. complex were identified using molecular diagnostic tools. Target site mutations conferring resistance were detected using Taqman assays. Quantitative reverse transcription-real-time PCR (qRT-PCR) 3-plex TaqMan® assays were used for the quantification of detoxification genes implicated in pyrethroid resistance. An. gambiae, An. coluzzii and An. arabiensis were identified in the different settings. An. gambiae was dominant in Santchou, Kékem, Bélabo, Bertoua and Njombé, while An. coluzzii was abundant in Tibati and Kaélé. High frequencies of the kdr L1014F allele ranging from 43% to 100% were recorded in almost all sites. The L1014S kdr allele was detected at low frequency (4.10-10%) only in mosquito populations from Njombé and Tibati. The N1575Y mutation was recorded in Kaélé, Santchou, Tibati and Bertoua with a frequency varying from 2.10% to 11.70%. Six Cytochrome P450 genes (Cyp6p3, Cyp6m2, Cyp9k1, Cyp6p4, Cyp6z1, and Cyp4g16) were found to be overexpressed in at least one population. Analysis of cuticular hydrocarbon lipids indicated a significant increase in CHC content in mosquito populations from Kaélé and Njombé compared to Kékem, Bélabo and Bertoua populations. The study indicated high pyrethroid resistance across different ecological settings in Cameroon with different profile of resistance across the country. The present situation calls for further actions in order to mitigate the impact of insecticide resistance on vector control measures.

Diversity of trypanosomes in tsetse fly caught in two "silent" sleeping sickness foci of Bafia and the Manoka Island in Cameroon.

BACKGROUND: Surveillance of "silent" human African Trypanosomiasis (HAT) foci is important for the achievement of the World Health Organization (WHO) goal of interrupting the transmission of this disease by 2030. It is in this context that this study was carried out to determine the trypanosome species circulating in the "silent" HAT foci of Bafia and the Manoka island in Cameroon. METHODS: In the Bafia and Manoka HAT foci, georeferenced pyramidal traps were used to trap tsetse flies. After DNA extraction from each whole fly, molecular tools were used to detect different trypanosome species as well as the origin of tsetse fly blood meals. Geographical information system was used to map the trypanosome infections and entomological data and to localize areas at high risk for trypanosome transmission. RESULTS: For this study, 1683 tsetse flies were caught and the relative apparent densities was 2.96: 0.03 in the Bafia HAT focus and 5.23 in the Manoka island. For the molecular identification of trypanosomes, 708 non-teneral tsetse flies (8 from Bafia and 700 from Manoka) were randomly selected. The overall trypanosome infection rate was 7.34 % with no infection in the Bafia HAT focus. Among the analysed flies, 4.57 % had trypanosomes of the subgenus Trypanozoon while 4.1 % and 1.13 % had respectively T. congolense and T. vivax. The most common mixed infections were the combination of trypanosomes of the subgenus Trypanozoon and T. congolense. Of the 708 tsetse flies analysed, 134 (18.93 %) tsetse flies were found with residual blood meals, 94 % and 6 % were respectively from humans and dogs. The trapping sites of Plateau, Sandje and Hospital appeared as the areas where contact with tsetse flies is most common. CONCLUSION: This study revealed a discrepancy in the abundance tsetse flies as well as the trypanosome infection rates in tsetse of the two "silent" HAT foci of Cameroon. The detection of different trypanosome species in tsetse from the Manoka Island highlights their transmission. The high percentage of human blood meals in tsetse flies indicates an important contact between tsetse flies and human; emphasizing the risk of trypanosome transmission to human in this island.

[Anopheles gambiae s.l. larval habitats and resistance mechanisms in Kribi, Cameroon]. / Habitats larvaires d'Anopheles gambiae s.l. et mécanismes de résistance à Kribi (Cameroun).

Background: The effectiveness of Long-Lasting Insecticidal Nets (LLINs) and indoor residual spraying (IRS) in controlling malaria vectors is hampered by the resistance of anopheles to insecticides. A good knowledge of the breeding sites and of the resistance profile of the vectors could facilitate the development of an appropriate control strategy. This study looks at the larval ecology and the susceptibility profile of An. gambiae s.l. to insecticides in urban and rural areas in Kribi, South Region of Cameroon. Methods: Mosquito breeding sites were categorized and geo-referenced. For each site, larvae were collected and reared and physicochemical parameters were measured in situ. The susceptibility of anopheles to dichlorodiphenyltrichloroethane (DDT), deltamethrin and permethrin, after pre-exposure to piperonyl butoxide (PBO) or not, was evaluated on the reared larvae. The Kdr mutation was detected using the Hot Oligonucleotide Ligation Assay (HOLA). Results: Natural breeding sites of An. gambiae s.l. were tyre tracks (12%, n=10), unbuilt wells (5%, n=4), pools of residual water (57%, n=48), foot and hoof prints, gullies, streams and the banks of the Kienké River (15%, n=13). Artificial breeding sites were abandoned dugouts (11%, n=9). Breeding sites in urban areas were characterized by higher mean values of temperature, conductivity, salinity and turbidity compared to the breeding sites in the rural area. The breeding sites of An. gambiae s.l. in urban Kribi were found to be sunnier than those in rural Kribi. A total of 4320 adults were used for testing, 1 440 mosquitoes from rural Kribi, 1 440 from urban Kribi and 1 440 specimens from the laboratory Kisumu strain. For DDT and deltamethrin, susceptibility tests showed that mortality was lower in a situation of no pre-exposure to PBO than in a situation of pre-exposure to PBO in the two study areas. The frequency of the resistant allele (R) was high for the Kdr West mutation in both urban (0.94) and rural areas in Kribi (0.93). Conclusion: An. gambiae s.l. colonizes a wide range of breeding sites and develops metabolic and mutation resistance to recommended insecticides. The search of alternative molecules for vector control is a necessity.

Bendiocarb and Malathion Resistance in Two Major Malaria Vector Populations in Cameroon Is Associated with High Frequency of the G119S Mutation (Ace-1) and Overexpression of Detoxification Genes.

The spread of pyrethroid resistance in malaria vectors is a major threat affecting the performance of current control measures. However, there is still not enough information on the resistance profile of mosquitoes to carbamates and organophosphates which could be used as alternatives. The present study assessed the resistance profile of Anopheles gambiae s.l. to bendiocarb and malathion, at the phenotypic and molecular levels, in different eco-epidemiological settings in Cameroon. Anopheles gambiae s.l. mosquitoes were collected from four eco-epidemiological settings across the country and their susceptibility level to bendiocarb and malathion was determined using WHO tubes bioassays. The ace-1 target site G119S mutation was screened by PCR. Reverse Transcription quantitative PCR 3-plex TaqMan assays were used to quantify the level of expression of eight genes associated with metabolic resistance. Resistance to malathion and/or bendiocarb was recorded in all study sites except in mosquitoes collected in Kaélé and Njombé. The Ace-1 (G119S) mutation was detected in high frequencies (>40%) in Kékem and Santchou. Both An. gambiae and An. coluzzii were detected carrying this mutation. The cytochrome P450s gene Cyp6p3 associated with carbamate resistance and the glutathione S-transferase gene Gste2 associated with organophosphate resistance were found to be overexpressed. Genes associated with pyrethroid (Cyp6m2, Cyp9k1, Cyp6p3) and organochlorine (Gste2, Cyp6z1, Cyp6m2) and cuticle resistance (Cyp4g16) were also overexpressed. The rapid spread of resistance to organophosphates and carbamates could seriously compromise future control strategies based on IRS. It is therefore becoming important to assess the magnitude of bendiocarb and malathion resistance countrywide.

Variations of insecticide residual bio-efficacy on different types of walls: results from a community-based trial in south Cameroon.

BACKGROUND: Determination of residual activity of insecticides is essential information for the selection of appropriate indoor spraying operation. The present study was undertaken to evaluate the residual effect of three candidate insecticide formulations on different indoor surfaces in order to guide future interventions, in the context of Cameroon and other African countries. METHODS: The study was conducted in the Ntougou neighbourhood in Yaoundé (capital city of Cameroon). Bendiocarb WP, lambda-cyhalothrin CS and deltamethrin WG were sprayed on the indoor wall surfaces of local cement, wood and mud houses. Their effects on the knockdown and mortality of the Kisumu susceptible strain of Anopheles gambiae s.s were assessed each month from March to September 2009, using the WHO plastic cones test. Knockdown and mortality rates were compared between different surfaces using Chi-square test. A Kaplan-Meir model was used to estimate the time of treatment failure. RESULTS: With bendiocarb WP, the knockdown rates were frequently above 98% during 13 weeks after spraying, except on mud walls where it significantly decreased at the 13th week (P < 0.05). With lambda cyhalothrin CS, the knockdown rates remained 100% on wood surfaces during the 26 weeks trial. However, it significantly decreased on concrete and mud surfaces from the 11th (83%) and the 20th (88%) weeks respectively (P < 0.05). With deltamethrin WG, it remained high on concrete surfaces during 26 weeks (> 98%); while it varied between 60 and 100% on wood or mud surfaces. The survival estimates of bendiocarb WP treatments remaining effective in killing An. gambiae s.s. (mortality rate ≥ 80%) was > 13 weeks on cement and wood surfaces and 13 weeks on mud surfaces. Those of lambda-cyhalothrin CS were > 26 weeks on wood surfaces, and 20 weeks on concrete and mud surfaces. By contrast, those of deltamethrin WG were 26 weeks on concrete, 20 weeks on mud surfaces and 15 weeks on wood surfaces. CONCLUSION: Current data suggest variable durations of spray cycles for each product, according to the type of wall surfaces, highlighting the importance of testing candidate products in local context before using them in large scale.

Impact of vegetable crop agriculture on anopheline agressivity and malaria transmission in urban and less urbanized settings of the South region of Cameroon.

BACKGROUND: The use of inland valley swamps for vegetable crop agriculture contributes to food security in urban and less urbanized settings in Africa. The impact of this agriculture on aggressive mosquitoes' diversity and malaria transmission in central Africa is poorly documented. This study is aimed at assessing the impact of vegetable crop agriculture on these entomological parameters in urban and less urbanized settings of the forest area, south of Cameroon. METHODS: The human bait technique was used for the capture of aggressive mosquitoes from January to December 2012. For three consecutive days each month, captures were performed on volunteers in hydro-agricultural and river bank sites of Akonolinga and Yaoundé. Physico-chemical characteristics of mosquito breeding sites were recorded. Molecular alongside morpho-taxonomic techniques were used for the identification of mosquito species; ELISA test was used to reveal Plasmodium falciparum infected mosquitoes through the detection of CSP. Mosquito diversity, aggressivity and malaria transmission in sites and settings were determined and compared. RESULTS: Biting rates were higher in hydro-agricultural sites of less urbanized and urban settings (31.8 b/p/n and 28.6 b/p/n respectively) than in river banks sites (6.83 b/p/n and 3.64 b/p/n respectively; p < 0.0001). Physico-chemical parameters of breeding sites were not fundamentally different. Five anopheline species were identified; An. gambiae, An. funestus s.s., An. moucheti s.s., An. hancocki and An. nili s.s. In hydro-agricultural sites 2 species were captured in the urban setting versus 4 in the less urbanized setting, meanwhile in river bank sites, 3 species were captured in the urban setting versus 4 species in the less urbanized setting. An. nili s.s. was found in river banks only. An. hancocki was not found to insure Plasmodium falciparum Welch transmission. EIR in hydro-agricultural sites varied from 1.86 ib/p/n (urban area) to 2.13 ib/p/n (less urbanized area) with higher rates in April/May and August. Overall, EIR was higher in less urbanized areas (p < 0.0001) but the difference was nullified with the practice of vegetable crop agriculture (p = 0.2). CONCLUSION: These results highlight the need for specific preventive measures that take into account the ecological peculiarities related to vegetable crop agriculture on hydro-agricultural lands, in order to protect inhabitants from malaria.

Challenges towards the elimination of Human African Trypanosomiasis in the sleeping sickness focus of Campo in southern Cameroon.

The sleeping sickness focus of Campo lies along the Atlantic coast and extends along the Ntem River, which constitutes the Cameroonian and Equatorial Guinean border. It is a hypo-endemic focus with the disease prevalence varying from 0.3 to 0.86% during the last few decades. Investigations on animal reservoirs revealed a prevalence of Trypanosoma brucei gambiense of 0.6% in wild animals and 4.83% in domestic animals of this focus. From 2001 to 2012, about 19 931 tsetse were collected in this focus and five tsetse species including Glossina palpalis palpalis, G. pallicera, G. nigrofusca, G. tabaniformis and G. caliginea were identified. The analysis of blood meals of these flies showed that they feed on human, pig, goat, sheep, and wild animals such as antelope, duiker, wild pig, turtle and snake. The percentage of blood meals taken on these hosts varies according to sampling periods. For instance, 6.8% of blood meals from pig were reported in 2004 and 22% in 2008. This variation is subjected to considerable evolutions because the Campo HAT focus is submitted to socio-economic mutations including the reopening of a new wood company, the construction of autonomous port at "Kribi" as well as the dam at "Memve ele". These activities will bring more that 3000 inhabitants around Campo and induce the deforestation for the implementation of farmlands as well as breeding of domestic animals. Such mutations have impacts on the transmission and the epidemiology of sleeping sickness due to the modification of the fauna composition, the nutritional behavior of tsetse, the zoophilic/anthropophilic index. To achieve the elimination goal in the sleeping sickness focus of Campo, we report in this paper the current epidemiological situation of the disease, the research findings of the last decades notably on the population genetics of trypanosomes, the modifications of nutritional behavior of tsetse, the prevalence of T. b. gambiense in humans, domestic and wild animals. An overview on the types of mutations occurring in the region has been raised and a discussion on the strategies that can be implemented to achieve the elimination of the disease has been made.