Intense malarial transmission during the dry season in irrigated rice-growing areas: a case study in Sakassou, Côte d'Ivoire.

The health district of Sakassou is one of the 83 health districts in Côte d'Ivoire, located in a zone with very high malarial transmission rates, with an incidence rate of ≥40% Therefore, to guide vector control methods more effectively, it was crucial to have a good understanding of the vectors in the area. This study aimed to determine the level of malarial transmission during the dry season in Sakassou, Côte d'Ivoire. Female Anopheles mosquitoes were sampled using human landing catches (HLCs) and pyrethrum spraying catches (PSCs). The larvae were collected using the 'dipping' method. A total of 10,875 adult female mosquitoes of Anopheles gambiae were collected. The PCR analysis revealed that all individuals were Anopheles coluzzii. The geographical distribution of potential breeding sites of Anopheles showed the presence of An. coluzzii in all the wetlands of the city of Sakassou. During the dry season, the human-biting rate of An. coluzzii was 139.1 bites/person/night. An exophagic trend was displayed by an adult female of An. coluzzii. The entomological inoculation rate during the dry season was 1.49 infectious bites/person/night. This study demonstrated that An. coluzzii was the main vector of malarial transmission in Sakassou, and the intensity of transmission remains high throughout the dry season.

High Levels of Admixture in Anopheles gambiae Populations from Côte d'Ivoire Revealed by Multilocus Genotyping.

Anopheles coluzzii and An. gambiae-the two most recently radiated species of the An. gambiae complex and the major Afrotropical malaria vector species-are identified by markers in the X-centromeric IGS rDNA region. Putative IGS-hybrids are rarely found in the field, except in restricted areas where genomic studies have led to the hypothesis that the observed IGS-patterns are due to cryptic taxa rather than to hybridization between the two species. We investigated the genome-wide levels of admixture in two villages in Côte d'Ivoire where high levels of IGS-hybrids have been detected, confirming unparalleled high frequencies in the coastal village. Genotyping of 24 Ancestry Informative Markers (AIMs) along the three chromosomes produced discordant results between the IGS-marker and the multilocus genotype obtained for AIMs across the whole genome (29%) as well as AIMs on chromosome-X (considered to be fundamental for species reproductive isolation) only (21%). Results highlight a complicated pattern of admixture that deserves deeper genomic analyses to understand better possible underlying causes (from extensive processes of hybridization to the existence of different cryptic taxa), and stress the need of developing advanced diagnostics for An. coluzzii, An. gambiae and putative new taxa, instrumental for assessing taxon-specific epidemiological characters.

Seasonal prevalence of Plasmodium falciparum infection and use of insecticide-treated nets among children in three agroecosystems in Aboisso, Côte d'Ivoire.

Agroecosystems have been associated with risk of malaria. The aim of this study was to determine the relationship between three agroecosystems: (i) rubber plantation (RP); (ii) oil palm plantation (OPP); (iii) no cash crop plantation (NCCP) and the prevalence of Plasmodium falciparum infection among children living in the Aboisso region. In the three villages within (Ehania-V5) or close (N'zikro) or far from (Ayébo) to each agroecosystem (RP, OPP, and NCCP), two cross-sectional parasitological surveys were carried out during the dry and the peak of the long wet seasons. A total of 586 children aged 1-14 years were recruited in the three villages to determine the prevalence of malaria using conventional microscopy. Plasmodium falciparum was the dominant species with an overall infection prevalence of 40.8%. There was a significant difference in prevalence between agroecosystems, during both the dry (p = 0.002) and wet seasons (p < 0.001), which was higher in agricultural settings compared with the NCCP environment, whatever the season. The prevalence of P. falciparum infection increased from the dry to the wet season in agricultural settings (RP and OPP), whereas no difference was noted for NCCP. Less than 18% of children use insecticide-treated nets (ITNs) in the three villages, ranging from 6 (in RP) to 30% (in OPP). Multivariate analysis indicated that age (1-4; 5-9; and 10-14 years) was not associated with malaria risk, but the season and living in agricultural villages were associated with a greater risk of malaria infection. Risk of malaria exposure was fourfold higher in children from agricultural villages than their counterpart from the non-agricultural area. Our findings highlight significant variations in the prevalence of P. falciparum according to agroecosystem and season. The findings will be useful in designing and implementing malaria control interventions by the National Malaria Control Program.

Role of Anopheles gambiae s.s. and Anopheles coluzzii (Diptera: Culicidae) in Human Malaria Transmission in Rural Areas of Bouaké, in Côte d'Ivoire.

Studies done in Bouaké (Côte d'Ivoire) about 20-yr ago reported that Anopheles gambiae s.l. Giles was the major malaria vector. The present study aimed to update these data and to identify the main vectors. Mosquitoes were collected in Allokokro and Petessou villages between June 2014 and December 2015 using the human landing catching method. Potential breeding sites of An. gambiae s.l. were identified in August and October 2014 and mapped using GPS. Anopheles species were morphologically and molecularly [polymerase chain reaction (PCR)] identified. Ovaries of female were dissected to determine the parity and infection with Plasmodium was detected in head and thorax by quantitative PCR. In Allokokro, the biting rate of An. gambiae s.s was significantly greater than Anopheles coluzzii, whereas, in Petessou, biting rates of both species were comparable. Plasmodium falciparum (Haemosporida: Plasmodiidae), Plasmodium malariae (Haemosporida: Plasmodiidae), and Plasmodium ovale (Haemosporida: Plasmodiidae) identified in both villages. The infection rates of An. gambiae s.s. and An. coluzzii were not significantly different. The entomological inoculation rate (EIR) of An. gambiae s.s. for P. falciparum was 9-fold greater than that of An. coluzzii in Allokokro; however, in Petessou, the EIRs of both species were comparable. In both village, An. gambiae s.s was responsible for P. falciparum and P. ovale transmission whereas An. coluzzii transmitted all three Plasmodium species.

Presence of susceptible wild strains of Anopheles gambiae in a large industrial palm farm located in Aboisso, South-Eastern of Côte d'Ivoire.

BACKGROUND: The effectiveness of malaria control programmes through implementation of vector control activities is challenged by the emergence of insecticide resistance. In the South-Eastern region of Côte d'Ivoire, where palm oil plantations remain the predominant agricultural crop, the susceptibility of wild Anopheles gambiae sensu lato species is still unknown and thus requires a particular attention. The current study was carried out to address the gap by in-depth characterization of susceptibility level of An. gambiae mosquitoes from Ehania-V1 to WHO-recommended doses of six insecticides belonging to available classes and also to screen a subset for target site mutations and possible inhibition of P450 enzymes. RESULTS: Overall results showed variable resistance profile across WHO-recommended insecticides tested. Mortalities ranged from 8.3% (the lowest mortality was recorded with DDT) to 98% (the highest mortality was recorded with fenitrothion). Importantly, mortality to deltamethrin, an important pyrethroid used in public health for impregnation of mosquito nets was close to 98%, precluding a possible susceptibility to this insecticide, albeit further investigations are required. Pre-exposure of An. gambiae s.l. to PBO did not show any significant variation across insecticides (p = 0.002), although a partial increase was detected for alphacypermethrin and bendiocarb, suggesting a low of activity of cytochrome P450 enzymes (p = 0.277). High frequency of kdr L1014F was recorded in both Anopheles coluzzii (91%) and in An. gambiae (96%), associated with ace-1 R G119S mutation at low frequency (<20%). CONCLUSION: The high mortality rate to deltamethrin, organophosphate and the non-detection of P450 activity in resistance observed in Ehania-V1 appears as a positive outcome for further control strategies as metabolic-based P450 resistance remains major challenge to manage. These results should help the National Malaria Control Programme when designing strategies for vector control in palm oil areas of Côte d'Ivoire.

Oviposition ecology and species composition of Aedes spp. and Aedes aegypti dynamics in variously urbanized settings in arbovirus foci in southeastern Côte d'Ivoire.

BACKGROUND: Aedes mosquito-transmitted outbreaks of dengue and yellow fever have been reported from rural and urban parts of Côte d'Ivoire. The present study aimed at assessing Aedes spp. oviposition ecology in variously urbanized settings within arbovirus foci in southeastern Côte d'Ivoire. METHODS: Aedes spp. eggs were sampled using a standard ovitrap method from January 2013 to April 2014 in different ecosystems of rural, suburban and urban areas. Emerged larvae were reared until the adult stage for species identification. RESULTS: Aedes spp. oviposition ecology significantly varied from rural-to-urban areas and according to the ecozones and the seasons. Species richness of Aedes spp. gradually decreased from rural (eight species) to suburban (three species) and urban (one species) areas. Conversely, emerged adult Aedes spp. mean numbers were higher in the urban (1.97 Aedes/ovitrap/week), followed by the suburban (1.44 Aedes/ovitrap/week) and rural (0.89 Aedes/ovitrap/week) areas. Aedes aegypti was the only species in the urban setting (100 %), and was also the predominant species in suburban (85.5 %) and rural (63.3 %) areas. The highest Ae. aegypti mean number was observed in the urban (1.97 Ae. aegypti/ovitrap/week), followed by the suburban (1.20 Ae. aegypti/ovitrap/week) and rural (0.57 Ae. aegypti/ovitrap/week) areas. Aedes africanus (9.4 %), Ae. dendrophilus (8.0 %), Ae. metallicus (1.3 %) in the rural, and Ae. vittatus (6.5 %) and Ae. metallicus (1.2 %) in the suburban areas each represented more than 1 % of the total Aedes fauna. In all areas, Aedes species richness and abundance were higher in the peridomestic zones and during the rainy season, with stronger variations in species richness in the rural and in abundance in the urban areas. Besides, the highest Culex quinquefasciatus abundance was found in the urban areas, while Eretmapodites chrysogaster was restricted to the rural areas. CONCLUSIONS: Urbanization correlates with a substantially higher abundance in Aedes mosquitoes and a regression of the Aedes wild species towards a unique presence of Ae. aegypti in urban areas. Aedes wild species serve as bridge vectors of arboviruses in rural areas, while Ae. aegypti amplifies arbovirus transmission in urban areas. Our results have important ramifications for dengue and yellow fever vector control and surveillance strategies in arbovirus foci in southeastern Côte d'Ivoire.

Insecticide resistance status of Anopheles gambiae s.s population from M'Bé: a WHOPES-labelled experimental hut station, 10 years after the political crisis in Côte d'Ivoire.

BACKGROUND: An experimental hut station built at M'Bé in 1998 was used for many years for the evaluation of insecticidal product for public health until the civil war broke out in 2002. Breeding sites of mosquitoes and selection pressure in the area were maintained by local farming practices and the West African Rice Development Association (WARDA, actually AfricaRice) in a large rice growing area. Ten years after the crisis, bioassays, molecular and biochemical analyses were conducted to update the resistance status and study the evolution of resistance mechanisms of Anopheles gambiae s.s population. METHODS: Anopheles gambiae s.s larvae from M'Bé were collected in breeding sites and reared until emergence. Resistance status of this population to conventional insecticides was assessed using WHO bioassay test kits for adult mosquitoes, with 10 insecticides belonging to pyrethroids, pseudo-pyrethroid, organochlorides, carbamates and organophosphates with and without the inhibitor piperonyl butoxyde (PBO). Molecular and biochemical assays were carried out to identify the L1014F kdr, L1014S kdr and ace-1(R) alleles in individual mosquitoes and to detect potential increase in mixed function oxidases (MFO) level, non-specific esterases (NSE) and glutathione S-transferases (GST) activities. RESULTS AND DISCUSSION: Anopheles gambiae s.s from M'Bé exerted high resistance levels to organochlorides, pyrethroids, and carbamates. Mortalities ranged from 3% to 21% for organochlorides, from 50% to 75% for pyrethroids, 34% for etofenprox, the pseudo-pyrethroid, and from 7% to 80% for carbamates. Tolerance to organophosphates was observed with mortalities ranging from 95% to 98%. Bioassays run with a pre-exposition of mosquitoes to PBO induced very high levels of mortalities compared to the bioassays without PBO, suggesting that the resistance to pyrethroid and carbamate relied largely on detoxifying enzymes' activities. The L1014F kdr allelic frequency was 0.33 in 2012 compared to 0.05 before the crisis in 2002. Neither the L1014S kdr nor ace-1(R) mutations were detected. An increased activity of NSE and level of MFO was found relative to the reference strain Kisumu. This was the first evidence of metabolic resistance based resistance in An. gambiae s.s from M'Bé. CONCLUSION: The An. gambiae s.s population showed very high resistance to organochlorides, pyrethroids and carbamates. This resistance level relied largely on two major types of resistance: metabolic and target-site mutation. This multifactorial resistance offers a unique opportunity to evaluate the impact of both mechanisms and their interaction with the vector control tools currently used or in development.

Status of pyrethroid resistance in Anopheles gambiae s. s. M form prior to the scaling up of Long Lasting Insecticidal Nets (LLINs) in Adzopé, Eastern Côte d'Ivoire.

BACKGROUND: The growing development of pyrethroid resistance constitutes a serious threat to malaria control programmes and if measures are not taken in time, resistance may compromise control efforts in the foreseeable future. Prior to Long Lasting Insecticidal Nets (LLINs) distribution in Eastern Cote d'Ivoire, we conducted bioassays to inform the National Malaria Control Programme of the resistance status of the main malaria vector, Anopheles gambiae s. s. and the need for close surveillance of resistance. METHODS: Larvae of An. gambiae s. s. were collected in two areas of Adzopé (Port-Bouët and Tsassodji) and reared to adults. WHO susceptibility tests with impregnated filter papers were carried out to detect resistance to three pyrethroids commonly used to develop LLINs: permethrin 1%, deltamethrin 0.05% and lambda-cyhalothrin 0.05%. Molecular assays were conducted to detect M and S forms and the L1014F kdr allele in individual mosquitoes. RESULTS: Resistance, at various degrees was detected in both areas of Adzopé. Overall, populations of An. gambiae at both sites surveyed showed equivalent frequency of the L1014F kdr allele (0.67) but for all tested pyrethroids, there were significantly higher survival rates for mosquitoes from Tsassodji (32-58%) than those from Port-Bouët (3-32%) (p < 0.001), indicating the implication of resistance mechanisms other than kdr alone. During the survey period (May-June) in this forested area of Côte d'Ivoire, An. gambiae s. s. found were exclusively of the M form and were apparently selected for pyrethroid resistance through agricultural and household usage of insecticides. CONCLUSION: Prior to LLINs scaling up in Eastern Côte d'Ivoire, resistance was largely present at various levels in An. gambiae. Underlying mechanisms included the high frequency of the L1014F kdr mutation and other unidentified components, probably metabolic detoxifiers. Their impact on the efficacy of the planned strategy (LLINs) in the area should be investigated alongside careful monitoring of the trend in that resistance over time. The need for alternative insecticides to supplement or replace pyrethroids on nets must be stressed.

Update on resistance status of Anopheles gambiae s.s. to conventional insecticides at a previous WHOPES field site, "Yaokoffikro", 6 years after the political crisis in Côte d'Ivoire.

BACKGROUND: At Yaokoffikro field site near Bouaké, in central Côte d'Ivoire, a group of experimental huts built in 1996 served over many years for the evaluation of insecticides against highly resistant mosquitoes. Breeding sites of mosquitoes and selection pressure in the area were maintained by local farming practices until a war broke out in September 2002. Six years after the crisis, we conducted bioassays and biochemical analysis to update the resistance status of Anopheles gambiae s.s. populations and detect other potential mechanisms of resistance that might have evolved. METHODS: An. gambiae s.s. larvae from Yaokoffikro were collected in breeding sites and reared to adults. Resistance status of this population to insecticides was assessed using WHO bioassay test kits for adult mosquitoes with seven insecticides: two pyrethroids, a pseudo-pyrethroid, an organochloride, two carbamates and an organophosphate.Molecular and biochemical assays were carried out to identify the L1014F kdr and ace-1R alleles in individual mosquitoes and to detect potential increase in mixed function oxidases (MFO), non-specific esterases (NSE) and glutathione S-transferases (GST) activity. RESULTS: High pyrethroids, DDT and carbamate resistance was confirmed in An. gambiae s.s. populations from Yaokoffikro. Mortality rates were less than 70% with pyrethroids and etofenprox, 12% with DDT, and less than 22% with the carbamates. Tolerance to fenitrothion was observed, with 95% mortality after 24 h.PCR analysis of samples from the site showed high allelic frequency of the L1014F kdr (0.94) and the ace-1R (0.50) as before the crisis. In addition, increased activity of NSE, GST and to a lesser extent MFO was found relative to the reference strain Kisumu. This was the first report detecting enhanced activity of these enzymes in An. gambiae s.s from Yaokoffikro, which could have serious implications in detoxification of insecticides. Their specific roles in resistance should be investigated using additional tools. CONCLUSION: The insecticide resistance profile at Yaokoffikro appears multifactorial. The site presents a unique opportunity to evaluate its impact on the protective efficacy of insecticidal products as well as new tools to manage these complex mechanisms. It calls for innovative research on the behaviour of the local vector, its biology and genetics that drive resistance.

Distribution of ace-1R and resistance to carbamates and organophosphates in Anopheles gambiae s.s. populations from Côte d'Ivoire.

BACKGROUND: The spread of pyrethroid resistance in Anopheles gambiae s.s. is a critical issue for malaria vector control based on the use of insecticide-treated nets. Carbamates and organophosphates insecticides are regarded as alternatives or supplements to pyrethroids used in nets treatment. It is, therefore, essential to investigate on the susceptibility of pyrethroid resistant populations of An. gambiae s.s. to these alternative products. METHODS: In September 2004, a cross sectional survey was conducted in six localities in Côte d'Ivoire: Toumbokro, Yamoussoukro, Toumodi in the Southern Guinea savannah, Tiassalé in semi-deciduous forest, then Nieky and Abidjan in evergreen forest area. An. gambiae populations from these localities were previously reported to be highly resistant to pyrethroids insecticides. Anopheline larvae were collected from the field and reared to adults. Resistance/susceptibility to carbamates (0.4% carbosulfan, 0.1% propoxur) and organophosphates (0.4% chlorpyrifos-methyl, 1% fenitrothion) was assessed using WHO bioassay test kits for adult mosquitoes. Then, PCR assays were run to determine the molecular forms (M) and (S), as well as phenotypes for insensitive acetylcholinesterase (AChE1) due to G119S mutation. RESULTS: Bioassays showed carbamates (carbosulfan and propoxur) resistance in all tested populations of An. gambiae s.s. In addition, two out of the six tested populations (Toumodi and Tiassalé) were also resistant to organophosphates (mortality rates ranged from 29.5% to 93.3%). The M-form was predominant in tested samples (91.8%). M and S molecular forms were sympatric at two localities but no M/S hybrids were detected. The highest proportion of S-form (7.9% of An. gambiae identified) was in sample from Toumbokro, in the southern Guinea savannah. The G119S mutation was found in both M and S molecular forms with frequency from 30.9 to 35.2%. CONCLUSION: This study revealed a wide distribution of insensitive acetylcholinesterase due to the G119S mutation in both M and S molecular forms of the populations of An. gambiae s.s. tested. The low cross-resistance between carbamates and organophosphates highly suggests involvement of other resistance mechanisms such as metabolic detoxification or F290V mutation.