SARS-CoV-2 seroprevalence among Beninese pregnant women in the third year of the pandemic.

BACKGROUND: Pregnant women are a vulnerable population to COVID-19 given an increased susceptibility to severe SARS-CoV-2 infection and pregnancy complications. However, few SARS-CoV-2 serological surveys have been performed among this population to assess the extent of the infection in sub-Saharan countries. The objectives of this study were to determine SARS-CoV-2 seroprevalence among Beninese pregnant women, to identify spatial seropositivity clusters and to analyse factors associated with the infection. METHODS: A cross-sectional study including women in their third trimester of pregnancy attending the antenatal care (ANC) clinics at Allada (south Benin) and Natitingou (north Benin) was conducted. Rapid diagnostic tests (RDT) for detection of IgG/IgM against the SARS-CoV-2 spike protein were performed using capillary blood. Seroprevalence of SARS-CoV-2 antibodies and associations between SARS-CoV-2 serostatus and maternal characteristics were analyzed by multivariate logistic regression. Spatial analyses were performed using the spatial scan statistics to identify spatial clusters of SARS-CoV-2 infection. RESULTS: A total of 861 pregnant women were enrolled between May 4 and June 29, 2022. 58/861 (6.7%) participants reported having received COVID-19 vaccine. None of the participants had been diagnosed with COVID-19 during their pregnancy. SARS-CoV-2 antibodies were detected in 607/802 (75.7%; 95% CI 72.56%-78.62%) of unvaccinated participants. Several urban and rural spatial clusters of SARS-CoV-2 cases were identified in Allada and one urban spatial cluster was identified in Natitingou. Unvaccinated participants from Allada with at least one previous morbidity were at a three-times higher risk of presenting SARS-CoV-2 antibodies (OR = 2.89; 95%CI 1.19%-7.00%). CONCLUSION: Three out of four pregnant women had SARS-CoV-2 antibodies, suggesting a high virus circulation among pregnant women in Benin, while COVID-19 vaccination coverage was low. Pregnant women with comorbidities may be at increased risk of SARS-CoV-2 infection. This population should be prioritized for COVID-19 diagnosis and vaccination in order to prevent its deleterious effects. TRIAL REGISTRATION: NCT06170320 (retrospectively registered on December 21, 2023).

Interest of seroprevalence surveys for the epidemiological surveillance of the SARS-CoV-2 pandemic in African populations: Insights from the ARIACOV project in Benin.

BACKGROUND: Many SARS-CoV-2 seroprevalence surveys since the end of 2020 have disqualified the first misconception that Africa had been spared by the pandemic. Through the analysis of three SARS-CoV-2 seroprevalence surveys carried out in Benin as part of the ARIACOV project, we argue that the integration of epidemiological serosurveillance of the SARS-CoV-2 infection in the national surveillance packages would be of great use to refine the understanding of the COVID-19 pandemic in Africa. METHODS: We carried out three repeated cross-sectional surveys in Benin: two in Cotonou, the economic capital in March and May 2021, and one in Natitingou, a semi-rural city in the north of the country in August 2021. Total and weighted-by-age-group seroprevalences were estimated and the risk factors for SARS-CoV-2 infection were assessed by multivariate logistic regression. RESULTS: In Cotonou, a slight increase in overall age-standardised SARS-CoV-2 seroprevalence from 29.77% (95% CI: 23.12%-37.41%) at the first survey to 34.86% (95% CI: 31.57%-38.30%) at the second survey was observed. In Natitingou, the globally adjusted seroprevalence was 33.34% (95% CI: 27.75%-39.44%). A trend of high risk for SARS-CoV 2 seropositivity was observed in adults over 40 versus the young (less than 18 years old) during the first survey in Cotonou but no longer in the second survey. CONCLUSIONS: Our results show that, however, rapid organisation of preventive measures aimed at breaking the chains of transmission, they were ultimately unable to prevent a wide spread of the virus in the population. Routine serological surveillance on strategic sentinel sites and/or populations could constitute a cost-effective compromise to better anticipate the onset of new waves and define public health strategies.

Emergence of SARS-CoV-2 Delta Variant, Benin, May-July 2021.

Severe acute respiratory syndrome coronavirus 2 Delta variant epidemiology in Africa is unknown. We found Delta variant was introduced in Benin during April-May 2021 and became predominant within 2 months, after which a steep increase in reported coronavirus disease incidence occurred. Benin might require increased nonpharmaceutical interventions and vaccination coverage.

Limited Specificity of Serologic Tests for SARS-CoV-2 Antibody Detection, Benin.

We used commercially available ELISAs to test 68 samples from coronavirus disease cases and prepandemic controls from Benin. We noted <25% false-positive results among controls, likely due to unspecific immune responses elicited by acute malaria. Serologic tests must be carefully evaluated to assess coronavirus disease spread and immunity in tropical regions.

Mutations Associated with SARS-CoV-2 Variants of Concern, Benin, Early 2021.

Intense transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Africa might promote emergence of variants. We describe 10 SARS-CoV-2 lineages in Benin during early 2021 that harbored mutations associated with variants of concern. Benin-derived SARS-CoV-2 strains were more efficiently neutralized by antibodies derived from vaccinees than patients, warranting accelerated vaccination in Africa.

Attrition, physical integrity and insecticidal activity of long-lasting insecticidal nets in sub-Saharan Africa and modelling of their impact on vectorial capacity.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear. METHODS: Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission-in terms of vectorial capacity (VC)-provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels. RESULTS: There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss. CONCLUSIONS: There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

Lassa Virus in Pygmy Mice, Benin, 2016-2017.

Lassa virus has been identified in 3 pygmy mice, Mus baoulei, in central Benin. The glycoprotein and nucleoprotein sequences cluster with the Togo strain. These mice may be a new reservoir for Lassa virus in Ghana, Togo, and Benin.

Empowering public health: building advanced molecular surveillance in resource-limited settings through collaboration and capacity-building.

The rapid detection and continuous surveillance of infectious diseases are important components of an effective public health response. However, establishing advanced molecular surveillance systems, crucial for monitoring and mitigating pandemics, poses significant challenges in resource-limited developing countries. In a collaborative effort, research institutions from Benin joined forces with Mali's National Institute of Public Health to implement a state-of-the-art molecular surveillance system in Mali. This approach was characterized by collaboration, multidisciplinarity, and tutoring. Key activities included a comprehensive assessment of infrastructure and human resources through document reviews, interviews, and laboratory visits; the development and validation of Standard Operating Procedures (SOPs) for advanced molecular surveillance following an inclusive approach; capacity-building initiatives for 25 biologists in Mali on sequencing techniques; and international tutoring sessions for eight Malian professionals held in Benin. These collective efforts enabled Mali to establish an advanced molecular surveillance system aligned with the WHO's global strategy for genomic surveillance. This manuscript aims to share experiences, insights, and outcomes from this initiative, with the hope of contributing to the broader discussion on strengthening global health security through collaborative approaches and capacity-building efforts, particularly in developing countries.

Development of rice farming: a cause of the emergence of multiple insecticide resistance in populations of Anopheles gambiae s.l and its impact on human health in Malanville, Bénin.

Aim: The rise in rice production in the district of Malanville, Northen Benin, is a present concern, as it has resulted in the widespread usage of pesticides for crop protection. This could impact human health but also life cycle of Anopheles gambiae, the main vector of malaria. Methods: Therefore, insecticide susceptibility bioassays were carried out on populations of An. gambiae s.l aged to 3-5 days old (two from areas where insecticide is highly used and other two areas of low insecticide use) and subjected to insecticide-impregnated papers (Permethrin 0.75%; deltamethrin 0.05%; DDT 4% and bendiocarb 0.1%) following WHO protocol. Polymerase Chain Reactions (PCRs) were used for the detection of Acethlylcholinestrase (Ace-1) and the knock down resistance (kdr) L1014F mutations in An. gambiae populations. Finally, indirect bioassays were conducted for the investigating on the factors affecting the life cycle of An. gambiae due to the use of pesticides. Results: An. gambiae from the four sites were resistant to DDT (6 to 8% and 10 to 14% respectively from areas of high and low dose), pyrethroids (22 to 26% and 30 to 36% for permethrin, from areas of high and low dose respectively and 66 to 70% and 72 to 80% for deltamethrin, from high and low dose) but susceptible to carbamate. The kdr L1014F mutation was detected in An. gambiae populations (0.88 to 0.90 and 0.84 to 0.88 from high and low dose, respectively). The ace-1 was detected at low frequencies (<0.002). Bioassays on the impacts of the use of pesticides in the life cycle of An. gambiae showed that soil substrates with pesticides residues have a negative impact on the life cycle eggs of An. gambiae. Conclusion: These findings confirmed the negative impacts of pesticides use in rice farming and its impacts on the life cycle of An. gambiae.

Genetic diversity and wing geometric morphometrics among four populations of Aedes aegypti (Diptera: Culicidae) from Benin.

BACKGROUND: The impact of the arbovirus vector Aedes aegypti is of major concern for global public health as the viruses that it transmits affect millions of people each year worldwide. Originating in Africa, Ae. aegypti has now spread throughout much of the world. While the genetic makeup of Ae. aegypti in the New World has been extensively studied, there is limited knowledge on its genetic diversity in Africa, particularly at a microgeographical level. METHODS: We investigated mitochondrial cytochrome oxidase I of four Ae. aegypti populations from Benin and employed wing morphometric analyses as a cost-effective and reliable tool to explore population structure. Our sampling encompassed various areas of Benin, from the southern to the northern borders of the country, and included urban, semi-urban, and sylvatic sites. RESULTS: We observed a notable level of genetic diversity (haplotype diversity of 0.8333) and nucleotide diversity (0.00421986), and identified seven distinct haplotypes. Sylvatic and semi-urban sites exhibited a greater number of haplotypes compared to urban sites. Utilizing 18 wing landmarks, we calculated the centroid size, which revealed significant variation among the three landscape types. However, principal component analysis, employed to assess wing shape variation, did not demonstrate significant differences between populations based on landscape type. CONCLUSIONS: Our findings indicate substantial genetic and morphological diversity among Ae. aegypti populations in Benin, and provide insight into important biological characteristics of these populations with respect to their potential to transmit viruses. To the best of our knowledge, this is the first study undertaken in Africa to integrate genetics with morphology to analyse the population structure of the major arbovirus vector Ae. aegypti.

A prospective, multi-site, cohort study to estimate incidence of infection and disease due to Lassa fever virus in West African countries (the Enable Lassa research programme)-Study protocol.

BACKGROUND: Lassa fever (LF), a haemorrhagic illness caused by the Lassa fever virus (LASV), is endemic in West Africa and causes 5000 fatalities every year. The true prevalence and incidence rates of LF are unknown as infections are often asymptomatic, clinical presentations are varied, and surveillance systems are not robust. The aim of the Enable Lassa research programme is to estimate the incidences of LASV infection and LF disease in five West African countries. The core protocol described here harmonises key study components, such as eligibility criteria, case definitions, outcome measures, and laboratory tests, which will maximise the comparability of data for between-country analyses. METHOD: We are conducting a prospective cohort study in Benin, Guinea, Liberia, Nigeria (three sites), and Sierra Leone from 2020 to 2023, with 24 months of follow-up. Each site will assess the incidence of LASV infection, LF disease, or both. When both incidences are assessed the LASV cohort (nmin = 1000 per site) will be drawn from the LF cohort (nmin = 5000 per site). During recruitment participants will complete questionnaires on household composition, socioeconomic status, demographic characteristics, and LF history, and blood samples will be collected to determine IgG LASV serostatus. LF disease cohort participants will be contacted biweekly to identify acute febrile cases, from whom blood samples will be drawn to test for active LASV infection using RT-PCR. Symptom and treatment data will be abstracted from medical records of LF cases. LF survivors will be followed up after four months to assess sequelae, specifically sensorineural hearing loss. LASV infection cohort participants will be asked for a blood sample every six months to assess LASV serostatus (IgG and IgM). DISCUSSION: Data on LASV infection and LF disease incidence in West Africa from this research programme will determine the feasibility of future Phase IIb or III clinical trials for LF vaccine candidates.

First Detection of the Invasive Mosquito Vector Aedes albopictus (Diptera: Culicidae) in Benin, West Africa, 2021.

Aedes albopictus (Skuse) is native to Southeast Asia and has colonized tropical and temperate regions worldwide in the last three to four decades. In Africa, data on its distribution is incomplete. Most studies having focused on the abundance, competition to other species, and phylogenetics of this vector are from the central African region. Here, we report the first detection of Ae. albopictus in Benin, West Africa. A total of 13 specimens were collected during the study period in 2021. The phylogenetic analysis of a cytochrome oxidase c subunit I gene fragment revealed a close relationship to populations from tropical India. Because of its close geographical proximity to areas where it has been found, it is assumed that the species was introduced several years before and is currently widely distributed in Benin. Additional studies are needed to explore its distribution, expansion range, and competitive effects on native species.

Factors enhancing the transmission of mosquito-borne arboviruses in Africa.

Arthropod-borne viruses (Arboviruses) replicate in vertebrates and invertebrates and are mainly transmitted by mosquitoes. Between 2000 and 2021, several arbovirus outbreaks were recorded in African countries, including dengue, yellow fever, Chikungunya, Zika, and O'nyong nyong. Most often, the causes and factors involved in these outbreaks are unknown. We aimed to understand current knowledge regarding factors responsible for the persistent transmission and emergence of mosquito-borne arboviruses in Africa and to identify critical research gaps important for preventing future outbreaks. We used a systematic literature review between 2020 and 2021, to show that the main identified factors favoring the arbovirus outbreak in Africa are low vaccination coverage, high density and diversity of competent mosquitoes, insecticide resistance of mosquito vectors, and a scarcity of data on arboviruses. Further studies on arboviruses may include studies of competence to viral strains and the susceptibility of mosquito vectors to insecticides. Because of the detrimental effects of insecticides on human health and the environment, viral paratransgenesis and other biological control methods should be explored as alternatives or as supplements to insecticides. Graphical abstract: Illustration of factors identified for promoting the transmission of arbovirus in Africa. The main factors are the lack of drugs and vaccines, low coverage of vaccination when a vaccine exists, competence of mosquitoes to viruses, diversity and high density of vectors. Climate change, urbanization, deforestation and agricultural practices, lead to a richness and high density of vectors.

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century.

RETRACTED: Gradual emergence followed by exponential spread of the SARS-CoV-2 Omicron variant in Africa.

The geographic and evolutionary origins of the SARS-CoV-2 Omicron variant (BA.1), which was first detected mid-November 2021 in Southern Africa, remain unknown. We tested 13,097 COVID-19 patients sampled between mid-2021 to early 2022 from 22 African countries for BA.1 by real-time RT-PCR. By November-December 2021, BA.1 had replaced the Delta variant in all African sub-regions following a South-North gradient, with a peak Rt of 4.1. Polymerase chain reaction and near-full genome sequencing data revealed genetically diverse Omicron ancestors already existed across Africa by August 2021. Mutations, altering viral tropism, replication and immune escape, gradually accumulated in the spike gene. Omicron ancestors were therefore present in several African countries months before Omicron dominated transmission. These data also indicate that travel bans are ineffective in the face of undetected and widespread infection.

Is biological larviciding against malaria a starting point for integrated multi-disease control? Observations from a cluster randomized trial in rural Burkina Faso.

OBJECTIVES: To evaluate the impact of anti-malaria biological larviciding with Bacillus thuringiensis israelensis on non-primary target mosquito species in a rural African setting. METHODS: A total of 127 villages were distributed in three study arms, each with different larviciding options in public spaces: i) no treatment, ii) full or iii) guided intervention. Geographically close villages were grouped in clusters to avoid contamination between treated and untreated villages. Adult mosquitoes were captured in light traps inside and outside houses during the rainy seasons of a baseline and an intervention year. After enumeration, a negative binomial regression was used to determine the reductions achieved in the different mosquito species through larviciding. RESULTS: Malaria larviciding interventions showed only limited or no impact against Culex mosquitoes; by contrast, reductions of up to 34% were achieved against Aedes when all detected breeding sites were treated. Culex mosquitoes were captured in high abundance in semi-urban settings while more Aedes were found in rural villages. CONCLUSIONS: Future malaria larviciding programs should consider expanding onto the breeding habitats of other disease vectors, such as Aedes and Culex and evaluate their potential impact. Since the major cost components of such interventions are labor and transport, other disease vectors could be targeted at little additional cost.

An Observational Laboratory-Based Assessment of SARS-CoV-2 Molecular Diagnostics in Benin, Western Africa.

Information on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spread in Africa is limited by insufficient diagnostic capacity. Here, we assessed the coronavirus disease (COVID-19)-related diagnostic workload during the onset of the pandemic in the central laboratory of Benin, Western Africa; characterized 12 SARS-CoV-2 genomes from returning travelers; and validated the Da An RT-PCR-based diagnostic kit that is widely used across Africa. We found a 15-fold increase in the monthly laboratory workload due to COVID-19, dealt with at the cost of routine activities. Genomic surveillance showed near-simultaneous introduction of distinct SARS-CoV-2 lineages termed A.4 and B.1, including the D614G spike protein variant potentially associated with higher transmissibility from travelers from six different European and African countries during March-April 2020. We decoded the target regions within the ORF1ab and N genes of the Da An dual-target kit by MinION-based amplicon sequencing. Despite relatively high similarity between SARS-CoV-2 and endemic human coronaviruses (HCoVs) within the ORF1ab target domain, no cross-detection of high-titered cell culture supernatants of HCoVs was observed, suggesting high analytical specificity. The Da An kit was highly sensitive, detecting 3.2 to 9.0 copies of target-specific in vitro transcripts/reaction. Although discrepant test results were observed in low-titered clinical samples, clinical sensitivity of the Da An kit was at least comparable to that of commercial kits from affluent settings. In sum, virologic diagnostics are achievable in a resource-limited setting, but unprecedented pressure resulting from COVID-19-related diagnostics requires rapid and sustainable support of national and supranational stakeholders addressing limited laboratory capacity.IMPORTANCE Months after the start of the COVID-19 pandemic, case numbers from Africa are surprisingly low, potentially because the number of SARS-CoV-2 tests performed in Africa is lower than in other regions. Here, we show an overload of COVID-19-related diagnostics in the central laboratory of Benin, Western Africa, with a stagnating average number of positive samples irrespective of daily sample counts. SARS-CoV-2 genomic surveillance confirmed a high genomic diversity in Benin introduced by travelers returning from Europe and other African countries, including early circulation of the D614G spike mutation associated with potentially higher transmissibility. We validated a widely used RT-PCR kit donated by the Chinese Jack Ma Foundation and confirmed high analytical specificity and clinical sensitivity equivalent to tests used in affluent settings. Our assessment shows that although achievable in an African setting, the burden from COVID-19-related diagnostics on national reference laboratories is very high.

Bendiocarb, a potential alternative against pyrethroid resistant Anopheles gambiae in Benin, West Africa.

BACKGROUND: Anopheles gambiae, the main malaria vector in Benin has developed high level of resistance to pyrethroid insecticides, which is a serious concern to the future use of long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS). In this context, one of the pathways available for malaria vector control would be to investigate alternative classes of insecticides with different mode of action than that of pyrethroids. The goal of this study was to evaluate under field conditions the efficacy of a carbamate (bendiocarb) and an organophosphate (fenitrothion) against pyrethroid-resistant An. gambiae s.s. METHODS: Wild populations and females from laboratory colonies of five days old An. gambiae were bio-assayed during this study. Two pyrethroids (deltamethrin and alphacypermethrin), an organophosphate (fenitrothion), a carbamate (bendiocarb) and a mixture of an organophosphate (chlorpyriphos + a pyrethroid deltamethrin) were compared in experimental huts as IRS treatments. Insecticides were applied in the huts using a hand-operated compression sprayer. The deterrency, exophily, blood feeding rate and mortality induced by these insecticides against An. gambiae were compared to the untreated control huts. RESULTS: Deltamethrin, alphacypermethrin and bendiocarb treatment significantly reduced mosquito entry into the huts (p < 0.05) compared to untreated huts. Blood feeding rates in huts treated with fenitrothion and the mixture chlorpyriphos/deltamethrin were reduced from 10.95% respectively to 3.7% and 4.47% three months after treatment and from 10.20% to 4.4% and 2.04% four months after treatment. Exophily rates in huts with deltamethrin, alphacypermethrin and the mixture chlorpyriphos/deltamethrin were significantly higher than in the huts with fenitrothion. Deltamethrin and alphacypermethrin had the lowest mortality rate while fenitrothion killed 100% of An. gambiae (in the first month) and 77.8% (in the fourth month). Bendiocarb and the mixture chlorpyriphos/deltamethrin mortality rates ranged from 97.9 to 100% the first month and 77.7-88% the third month respectively. CONCLUSION: After four months, fenitrothion, bendiocarb and the mixture chlorpyriphos/deltamethrin performed effectively against pyrethroid-resistant Anopheles. These results showed that bendiocarb could be recommended as an effective insecticide for use in IRS operations in Benin, particularly as the mixture chlorpyriphos/deltamethrin does not have WHOPES authorization and complaints were mentioned by the sleepers about the safety and smell of fenitrothion.

Insecticide resistance status in Anopheles gambiae in southern Benin.

BACKGROUND: The emergence of pyrethroid resistance in Anopheles gambiae has become a serious concern to the future success of malaria control. In Benin, the National Malaria Control Programme has recently planned to scaling up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) for malaria prevention. It is, therefore, crucial to monitor the level and type of insecticide resistance in An. gambiae, particularly in southern Benin where reduced efficacy of insecticide-treated nets (ITNs) and IRS has previously been reported. METHODS: The protocol was based on mosquito collection during both dry and rainy seasons across forty districts selected in southern Benin. Bioassay were performed on adults collected from the field to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. The species within An. gambiae complex, molecular form and presence of kdr and ace-1 mutations were determined by PCR. RESULTS: Strong resistance to permethrin and DDT was found in An. gambiae populations from southern Benin, except in Aglangandan where mosquitoes were fully susceptible (mortality 100%) to all insecticides tested. PCR showed the presence of two sub-species of An. gambiae, namely An. gambiae s.s, and Anopheles melas, with a predominance for An. gambiae s.s (98%). The molecular M form of An. gambiae was predominant in southern Benin (97%). The kdr mutation was detected in all districts at various frequency (1% to 95%) whereas the Ace-1 mutation was found at a very low frequency (