Enhanced mosquito vectorial capacity underlies the Cape Verde Zika epidemic.

The explosive emergence of Zika virus (ZIKV) across the Pacific and Americas since 2007 was associated with hundreds of thousands of human cases and severe outcomes, including congenital microcephaly caused by ZIKV infection during pregnancy. Although ZIKV was first isolated in Uganda, Africa has so far been exempt from large-scale ZIKV epidemics, despite widespread susceptibility among African human populations. A possible explanation for this pattern is natural variation among populations of the primary vector of ZIKV, the mosquito Aedes aegypti. Globally invasive populations of Ae. aegypti outside of Africa are considered effective ZIKV vectors because they are human specialists with high intrinsic ZIKV susceptibility, whereas African populations of Ae. aegypti across the species' native range are predominantly generalists with low intrinsic ZIKV susceptibility, making them less likely to spread viruses in the human population. We test this idea by studying a notable exception to the patterns observed across most of Africa: Cape Verde experienced a large ZIKV outbreak in 2015 to 2016. We find that local Ae. aegypti in Cape Verde have substantial human-specialist ancestry, show a robust behavioral preference for human hosts, and exhibit increased susceptibility to ZIKV infection, consistent with a key role for variation among mosquito populations in ZIKV epidemiology. These findings suggest that similar human-specialist populations of Ae. aegypti in the nearby Sahel region of West Africa, which may be expanding in response to rapid urbanization, could serve as effective vectors for ZIKV in the future.

Reemergence of Sylvatic Dengue Virus Serotype 2 in Kedougou, Senegal, 2020.

In 2020, a sylvatic dengue virus serotype 2 infection outbreak resulted in 59 confirmed dengue cases in Kedougou, Senegal, suggesting those strains might not require adaptation to reemerge into urban transmission cycles. Large-scale genomic surveillance and updated molecular diagnostic tools are needed to effectively prevent dengue virus infections in Senegal.

Ebola Virus Transmission Initiated by Relapse of Systemic Ebola Virus Disease.

During the 2018-2020 Ebola virus disease (EVD) outbreak in North Kivu province in the Democratic Republic of Congo, EVD was diagnosed in a patient who had received the recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) (Merck). His treatment included an Ebola virus (EBOV)-specific monoclonal antibody (mAb114), and he recovered within 14 days. However, 6 months later, he presented again with severe EVD-like illness and EBOV viremia, and he died. We initiated epidemiologic and genomic investigations that showed that the patient had had a relapse of acute EVD that led to a transmission chain resulting in 91 cases across six health zones over 4 months. (Funded by the Bill and Melinda Gates Foundation and others.).

Rapid detection of Ebolavirus using isothermal recombinase-aided amplification.

Ebolavirus disease (EVD) is an often-lethal disease caused by the genus Ebolavirus (EBOV). Although vaccines are being developed and recently used, outbreak control still relies on a combination of various factors, including rapid identification of EVD cases. This allows rapid patient isolation and control measure implementation. Ebolavirus diagnosis is performed in treatment centers or reference laboratories, which usually takes a few hours to days to confirm the outbreak or deliver a clear result. A fast and field-deployable molecular detection method, such as the isothermal amplification recombinase-aided amplification (RAA), could significantly reduce sample-to-result time. In this study, a RT-RAA assay was evaluated for EBOV detection. Various primer and probe combinations were screened; analytical sensitivity and cross-specificity were tested. A total of 40 archived samples from the 2014 to 2016 Ebola outbreak in West Africa were tested with both the reference method real-time RT-PCR and the established RT-RAA assay. The assay could detect down to 22.6 molecular copies per microliter. No other pathogens were detected with the Ebolavirus RT-RAA assay. Testing 40 samples yield clinical sensitivity and specificity of 100% each. This rapid isothermal RT-RAA assay can replace the previous RT-RPA and continue to offer rapid EBOV diagnostics.

An amplicon-based sequencing approach for Usutu virus characterization.

Usutu virus (USUV), an arbovirus from the Flaviviridae family, genus Flavivirus, has recently gained increasing attention because of its potential for emergence. After his discovery in South Africa, USUV spread to other African countries, then emerged in Europe where it was responsible for epizootics. The virus has recently been found in Asia. USUV infection in humans is considered to be most often asymptomatic or to cause mild clinical signs. However, a few cases of neurological complications such as encephalitis or meningo-encephalitis have been reported in both immunocompromised and immunocompetent patients. USUV natural life cycle involves Culex mosquitoes as its main vector, and multiple bird species as natural viral reservoirs or amplifying hosts, humans and horses can be incidental hosts. Phylogenetic studies carried out showed eight lineages, showing an increasing genetic diversity for USUV. This work describes the development and validation of a novel whole-genome amplicon-based sequencing approach to Usutu virus. This study was carried out on different strains from Senegal and Italy. The new approach showed good coverage using samples derived from several vertebrate hosts and may be valuable for Usutu virus genomic surveillance to better understand the dynamics of evolution and transmission of the virus.

Atopic dermatitis: A global health perspective.

The International Society of AD (ISAD) organized a roundtable on global aspects of AD at the WCD 2023 in Singapore. According to the Global Burden of Disease (GBD) consortium, at least 171 million individuals were affected with AD in 2019, corresponding to 2.23% of the world population, with age-standardized prevalence and incidence rates that were relatively stable from 1990 to 2019. Based on the panel experience, most AD cases are mild-to-moderate. Without parallel data on disease prevalence and severity, the GBD data are difficult to interpret in many regions. This gap is particularly important in countries with limited medical infrastructure, but indirect evidence suggests a significant burden of AD in low-and-medium resource settings, especially urban areas. The Singapore roundtable was an opportunity to compare experiences in World Bank category 1 (Madagascar and Mali), 3 (Brazil, China) and 4 (Australia, Germany, Qatar, USA, Singapore, Japan) countries. The panel concluded that current AD guidelines are not adapted for low resource settings and a more pragmatic approach, as developed by WHO for skin NTDs, would be advisable for minimal access to moisturizers and topical corticosteroids. The panel also recommended prioritizing prevention studies, regardless of the level of existing resources. For disease long-term control in World Bank category 3 and most category 4 countries, the main problem is not access to drugs for most mild-to-moderate cases, but rather poor compliance due to insufficient time at visits. Collaboration with WHO, patient advocacy groups and industry may promote global change, improve capacity training and fight current inequalities. Finally, optimizing management of AD and its comorbidities needs more action at the primary care level, because reaching specialist care is merely aspirational in most settings. Primary care empowerment with store and forward telemedicine and algorithms based on augmented intelligence is a future goal.

Molecular characterization of circulating Dengue virus 2 during an outbreak in Northern Senegal's Saint-Louis region in 2018.

Globally, 390 million people are at risk of dengue infection and over the past 50 years, the virus incidence increased thirty-fold. In Senegal, an unprecedented occurrence of outbreaks and sporadic cases have been noticed since 2017. In October 2018, an outbreak of Dengue virus 2 (DENV-2) was reported in the north of Senegal affecting multiple areas including Saint-Louis, Richard Toll, and Rosso which are located at the border with Mauritania. Of these 173 blood specimen samples collected from patients, 27 were positive for dengue by quantitative reverse transcription PCR (qRT-PCR), and eight were serologically confirmed to be positive for DENV immunoglobulin M (IgM). Serotyping using qRT-PCR reveals that isolates were positive for DENV-2. A subset of DENV-2 positive samples was selected and subjected to whole-genome sequencing followed by phylogenetic analysis. Analysis of six nearly complete genome sequences revealed that the isolates belong to the cosmopolitan genotype and are closely related to the Mauritanian strains detected between 2017 and 2018 and those detected in many West African countries such as Burkina Faso or Cote d'Ivoire. Our results suggest a transboundary circulation of the DENV-2 cosmopolitan genotype between Senegal and Mauritania and call for a need for coordinated surveillance of arboviruses between these two countries. Interestingly, a high level of homology between West African isolates highlights endemicity and calls for the set-up of subregional viral genomic surveillance which will lead to a better understanding of viral dynamics, transmission, and spread across Africa.

Performance assessment and validation of a plaque reduction neutralization test (PRNT) in support to yellow fever diagnostic and vaccine clinical trials.

Yellow fever (YF) virus is a mosquito-borne virus belonging to the Flaviviridae family that circulates in tropical and subtropical areas of Africa and South America. Despite the availability of an effective vaccine, YF remains a threat to travelers, residents of endemic areas, and unvaccinated populations. YF vaccination and natural infection both induce the production of neutralizing antibodies. Serological diagnostic methods detecting YF virus-specific antibodies demonstrate high levels of cross-reactivities with other flaviviruses. To date, the plaque reduction neutralization test (PRNT) is the most specific serological test for the differentiation of flavivirus infections and is considered the reference method for detecting YF neutralizing antibodies and assessing the protective immune response following vaccination. In this study, we developed and validated a YF PRNT. We optimized different parameters including cell concentration and virus-serum neutralization time period and then assessed the intra- and inter-assay precisions, dilutability, specificity, and lower limit of quantification (LLOQ) using international standard YF serum, sera from vaccinees and human specimens collected through YF surveillance. The YF PRNT has shown good robustness and 100% of intra-assay precision, 95.6% of inter-assay precision, 100% of specificity, 100% of LLOQ, and 95.3% of dilutability. The test is, therefore, suitable for use in the YF diagnostic as well as evaluation of the YF vaccine neutralizing antibody response and risk assessment studies.

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

Importation and Circulation of Vaccine-Derived Poliovirus Serotype 2, Senegal, 2020-2021.

Environmental surveillance for poliovirus is increasingly used in poliovirus eradication efforts as a supplement to acute flaccid paralysis (AFP) surveillance. Environmental surveillance was officially established in 2017 in Senegal, where no poliovirus had been detected since 2010. We tested sewage samples from 2 sites in Dakar monthly for polioviruses. We identified a vaccine-derived poliovirus serotype 2 on January 19, 2021, from a sample collected on December 24, 2020; by December 31, 2021, we had detected 70 vaccine-derived poliovirus serotype 2 isolates circulating in 7 of 14 regions in Senegal. Sources included 18 AFP cases, 20 direct contacts, 17 contacts in the community, and 15 sewage samples. Phylogenetic analysis revealed the circulation of 2 clusters and provided evidence on the virus introduction from Guinea. Because novel oral polio vaccine serotype 2 was used for response activities throughout Senegal, we recommend expanding environmental surveillance into other regions.

SARS-CoV-2 Circulation, Guinea, March 2020-July 2021.

This overview of severe acute respiratory syndrome coronavirus 2 circulation over 1.5 years in Guinea demonstrates that virus clades and variants of interest and concern were progressively introduced, mostly by travellers through Conakry, before spreading through the country. Sequencing is key to following virus evolution and establishing efficient control strategies.

Human Herpesvirus 8 seroprevalence among blood donors in Mali.

In sub-Saharan Africa, the Human Herpesvirus 8 (HHV-8) is endemic but with disparities between regions and population studied. Although the virus remains mostly latent, there is some evidence that blood transfusion may represents one of the transmission way for this virus. Here, we evaluated HHV-8 seroprevalence among blood donors in Mali. This cross-sectional study recruited blood donors from the Blood Transfusion Center at Gabriel Touré Hospital, Bamako. Serum was used for the detection of latent HHV-8 immunoglobulin G directed against latent associated nuclear antigen 1 by an indirect immunofluorescence assay. Human immunodeficiency virus 1 (HIV-1), Hepatitis B Virus (HBV), HCV, and Treponema pallidum were also screened. HHV-8 seroprevalence was 10.4% in Malian blood donors. None of the sociodemographic characteristics were associated with HHV-8 infection, although there is a tendency of a higher HHV-8 seroprevalence among participants living in Bamako than those not living there. One individual had coinfection HHV-8/HBV, another HHV-8/HCV while another had HCV and T. pallidum. None has been tested positive for HIV infection. This intermediate seroprevalence in Malian blood donors suggests that the risk of HHV-8 transmission by transfusion should be considered. Further investigations are needed to assess impact of HHV-8 in polytransfused patients residing in an endemic area for this virus.

Full genome analysis of circulating DENV-2 in Senegal reveals a regional diversification into separate clades.

To assess the genetic diversity of circulating dengue virus 2 (DENV-2) in Senegal, we analyzed nine newly generated complete genomes of strains isolated during the 2018 outbreaks and 06 sequences obtained in 2018 and 2019 from Thiès and Rosso, respectively. Phylogenetic analyses revealed that Senegalese strains belonged to the cosmopolitan genotype of DENV-2, but we observed intragenotype variability leading to a divergence in two clades associated with specific geographic distribution. We report two DENV-2 variants belonging to two distinct clades. Isolates from the "Northern clade" (n = 8) harbored three nonsynonymous mutations (V1183M, R1405K, P2266T) located respectively on NS2A, NS2B, and NS4A, while isolates from the "Western clade" (n = 7) had two nonsynonymous mutations (V1185E, V3214E) located respectively in the NS2A and NS5 genes. These findings call for phylogeographic analysis to investigate routes of introductions, dispersal patterns, and in-depth in vitro and functional study to elucidate the impact of observed mutations on viral fitness, spread, epidemiology, and pathology.

Evaluation of the residual efficacy and physical durability of five long-lasting insecticidal nets (LLINs) in Senegal.

BACKGROUND: The preventive and curative strategies of malaria are based on promoting the use of long-lasting insecticidal nets (LLINs) and treating confirmed cases with artemisinin-based combination therapy. These strategies have led to a sharp decline in the burden of malaria, which remains a significant public health problem in sub-Saharan countries. The objective of this study was to determine and compare the residual efficacy of LLINs recommended by the World Health Organization. METHODS: The study was conducted in six villages in two sites in Senegal located in the Sahelo-Sudanian area of the Thiès region, 70 km from Dakar and in Mbagame, a semi-urban zone in the Senegal River Valley. A census was conducted of all sleeping places in each household to be covered by LLINs. Five brands of LLIN were distributed, and every six months, retention rates, net use, maintenance, physical integrity, insecticide chemical content, and biological efficacy were examined for each type of LLIN. RESULTS: A total of 3012 LLINs were distributed in 1249 households in both sites, with an average coverage rate of 94% (95% CI 92.68-95.3). After 36 months, the average retention rate was 12.5% and this rate was respectively 20.5%, 15.1%, 10%, 7%, and 3% for Olyset Net®, Dawa Plus® 2.0, PermaNet® 2.0, NetProtect® and Life Net®, respectively. The proportion of LLINs with holes and the average number of holes per mosquito net increased significantly during each follow-up, with a large predominance of size 1 (small) holes for all types of LLINs distributed. During the three-year follow-up, bioassay mortality rates of a susceptible strain of insectary reared Anopheles coluzzii decreased in the following net types: in Dawa Plus® 2.0 (100% to 51.7%), PermaNet® 2.0 (96.6% to 83%), and Olyset Net® (96.6% to 33.3%). Mortality rates remained at 100% in Life Net® over the same time period. After 36 months, the average insecticide content per brand of LLIN decreased by 40.9% for Dawa Plus® 2.0, 31% for PermaNet® 2.0, 39.6% for NetProtect® and 51.9% for Olyset Net® and 40.1% for Life Net. CONCLUSIONS: Although some net types retained sufficient insecticidal activity, based on all durability parameters measured, none of the net types survived longer than 2 years.

Real-time, portable genome sequencing for Ebola surveillance.

The Ebola virus disease epidemic in West Africa is the largest on record, responsible for over 28,599 cases and more than 11,299 deaths. Genome sequencing in viral outbreaks is desirable to characterize the infectious agent and determine its evolutionary rate. Genome sequencing also allows the identification of signatures of host adaptation, identification and monitoring of diagnostic targets, and characterization of responses to vaccines and treatments. The Ebola virus (EBOV) genome substitution rate in the Makona strain has been estimated at between 0.87 × 10(-3) and 1.42 × 10(-3) mutations per site per year. This is equivalent to 16-27 mutations in each genome, meaning that sequences diverge rapidly enough to identify distinct sub-lineages during a prolonged epidemic. Genome sequencing provides a high-resolution view of pathogen evolution and is increasingly sought after for outbreak surveillance. Sequence data may be used to guide control measures, but only if the results are generated quickly enough to inform interventions. Genomic surveillance during the epidemic has been sporadic owing to a lack of local sequencing capacity coupled with practical difficulties transporting samples to remote sequencing facilities. To address this problem, here we devise a genomic surveillance system that utilizes a novel nanopore DNA sequencing instrument. In April 2015 this system was transported in standard airline luggage to Guinea and used for real-time genomic surveillance of the ongoing epidemic. We present sequence data and analysis of 142 EBOV samples collected during the period March to October 2015. We were able to generate results less than 24 h after receiving an Ebola-positive sample, with the sequencing process taking as little as 15-60 min. We show that real-time genomic surveillance is possible in resource-limited settings and can be established rapidly to monitor outbreaks.

Survival of eight LLIN brands 6, 12, 24 and 36 months after a mass distribution campaign in rural and urban settings in Senegal.

BACKGROUND: Long lasting insecticidal nets (LLIN) are one of the core components of global malaria prevention and control. The lifespan of LLIN varies widely depending on the population or environment, and randomized studies are required to compare LLIN inaccording to arbitrary thresholds households under different field conditions. This study investigated survival of different LLIN brands in Senegal. METHODS: Ten thousand six hundred eight LLINs were distributed in five regions, each stratified by rural and urban setting. As part of the longitudinal follow-up, 2222 nets were randomly sampled and monitored from 6 to 36 months. Using random effects for households, Bayesian models were used to estimate independent survival by net type (Interceptor®, Life Net®, MAGNet™, Netprotect®, Olyset® Net, PermaNet® 2.0 R, PermaNet® 2.0 C, Yorkool® LN) and by area (rural/urban). In addition to survival, median survival time and attrition of each LLIN brand was determined. Attrition was defined as nets that were missing because they were reported given away, destroyed and thrown away, or repurposed. RESULTS: Three net types had a proportion of survival above 80% after 24 months: Interceptor®87.8% (95% CI 80-93.4); conical PermaNet® 2.0 86.9% (95% CI 79.3-92.4) and Life Net® 85.6% (95% CI 75-93). At 36 months, conical PermaNet® 2.0 maintained a good survival rate, 79.5% (95% CI 65.9-88.8). The attrition due to redistributed nets showed that the two conical net types (PermaNet® 2.0 and Interceptor®) were more often retained by households and their median retention time was well above 3 years (median survival time = 3.5 years for PermaNet® 2.0 and median survival time = 4 years for Interceptor®). Despite this good retention, Interceptor® had weak physical integrity and its median survival due to wear and tear was below 3 years (median survival time = 2.4 years). The odds ratio of survival was 2.5 times higher in rural settings than in urban settings (OR 2.5; 95% CI 1.7-3.7). CONCLUSIONS: Differences in survival among LLIN may be driven by brand, shape or environmental setting. In this study in Senegal, conical PermaNet® 2.0 were retained in households while rectangular PermaNet® 2.0 had lower retention, suggesting that net shape may play a role in retention and should be further investigated. Distribution of preferred LLIN shape, accompanied by good communication on care and repair, could lead to increased effective lifespan, and allow for longer intervals between universal coverage campaigns.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

New assessment of Anopheles vector species identification using MALDI-TOF MS.

BACKGROUND: Anopheles species identification is essential for an effective malaria vector control programme. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) has been developed to identify adult Anopheles species, using the legs or the cephalothorax. The protein repertoire from arthropods can vary according to compartment, but there is no general consensus regarding the anatomic part to be used. METHODS: To determine the body part of the Anopheles mosquitoes best suited for the identification of field specimens, a mass spectral library was generated with head, thorax with wings and legs of Anopheles gambiae, Anopheles arabiensis and Anopheles funestus obtained from reference centres. The MSL was evaluated using two independent panels of 52 and 40 An. gambiae field-collected in Mali and Guinea, respectively. Geographic variability was also tested using the panel from Mali and several databases containing added specimens from Mali and Senegal. RESULTS: Using the head and a database without specimens from the same field collection, the proportion of interpretable and correct identifications was significantly higher than using the other body parts at a threshold value of 1.7 (p < 0.0001). The thorax of engorged specimens was negatively impacted by the blood meal after frozen storage. The addition of specimens from Mali into the database significantly improved the results of Mali panel (p < 0.0001), which became comparable between head and legs. With higher identification scores, the using of the head will allow to decrease the number of technical replicates of protein extract per specimen, which represents a significant improvement for routine use of MALDI-TOF MS. CONCLUSIONS: The using of the head of Anopheles may improve the performance of MALDI-TOF MS. Region-specific mass spectrum databases will have to be produced. Further research is needed to improve the standardization in order to share online spectral databases.

Determination of the discriminating concentration of chlorfenapyr (pyrrole) and Anopheles gambiae sensu lato susceptibility testing in preparation for distribution of Interceptor® G2 insecticide-treated nets.

BACKGROUND: Following agricultural use and large-scale distribution of insecticide-treated nets (ITNs), malaria vector resistance to pyrethroids is widespread in sub-Saharan Africa. Interceptor® G2 is a new dual active ingredient (AI) ITN treated with alpha-cypermethrin and chlorfenapyr for the control of pyrethroid-resistant malaria vectors. In anticipation of these new nets being more widely distributed, testing was conducted to develop a chlorfenapyr susceptibility bioassay protocol and gather susceptibility information. METHODS: Bottle bioassay tests were conducted using five concentrations of chlorfenapyr at 12.5, 25, 50, 100, and 200 µg AI/bottle in 10 countries in sub-Saharan Africa using 13,639 wild-collected Anopheles gambiae sensu lato (s.l.) (56 vector populations per dose) and 4,494 pyrethroid-susceptible insectary mosquitoes from 8 colonized strains. In parallel, susceptibility tests were conducted using a provisional discriminating concentration of 100 µg AI/bottle in 16 countries using 23,422 wild-collected, pyrethroid-resistant An. gambiae s.l. (259 vector populations). Exposure time was 60 min, with mortality recorded at 24, 48 and 72 h after exposure. RESULTS: Median mortality rates (up to 72 h after exposure) of insectary colony mosquitoes was 100% at all five concentrations tested, but the lowest dose to kill all mosquitoes tested was 50 µg AI/bottle. The median 72-h mortality of wild An. gambiae s.l. in 10 countries was 71.5, 90.5, 96.5, 100, and 100% at concentrations of 12.5, 25, 50, 100, and 200 µg AI/bottle, respectively. Log-probit analysis of the five concentrations tested determined that the LC95 of wild An. gambiae s.l. was 67.9 µg AI/bottle (95% CI: 48.8-119.5). The discriminating concentration of 203.8 µg AI/bottle (95% CI: 146-359) was calculated by multiplying the LC95 by three. However, the difference in mortality between 100 and 200 µg AI/bottle was minimal and large-scale testing using 100 µg AI/bottle with wild An. gambiae s.l. in 16 countries showed that this concentration was generally suitable, with a median mortality rate of 100% at 72 h. CONCLUSIONS: This study determined that 100 or 200 µg AI/bottle chlorfenapyr in bottle bioassays are suitable discriminating concentrations for monitoring susceptibility of wild An. gambiae s.l., using mortality recorded up to 72 h. Testing in 16 countries in sub-Saharan Africa demonstrated vector susceptibility to chlorfenapyr, including mosquitoes with multiple resistance mechanisms to pyrethroids.