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Abstract Background Cluster-randomized controlled trials (cluster-RCTs) have demonstrated variation in the epidemiological efficacy of different next-generation insecticide-treated net (ITN) types, with some providing shorter-lived impact than others. Further studies are needed to assess changes in the insecticidal durability of these ITNs over time to complement cluster-RCT results. Methods A series of experimental hut trials were performed to evaluate the bioefficacy of new and field-aged next-generation ITNs (PermaNet® 3.0, Royal Guard®, Interceptor® G2) compared to a pyrethroid-only net (Interceptor®) against pyrethroid-resistant malaria vectors in Covè, southern Benin. Field-aged nets were withdrawn from households at 12, 24 and 36 months. Net pieces cut from whole ITNs were analysed for chemical content, and susceptibility bioassays were performed during each trial to assess changes in insecticide resistance in the Covè vector population. Results Interceptor® G2 induced superior mosquito mortality than the other ITNs across all time points. The improved mortality with Interceptor® G2 compared to Interceptor® was evident across all time points but was greater with new nets (odds ratio (OR) = 8.6, 95% CI [7.4, 10.1]) than field-aged nets (OR = 2.5, 95% CI [1.8, 3.5] at 12 months, OR = 2.4, 95% CI [1.6, 3.7] at 24 months and OR = 2.9, 95% CI [1.6, 5.1] at 36 months). New Royal Guard® reduced mosquito fertility compared to the other ITNs, but this improvement fell after field-ageing, particularly at 24 months when it was similar to Interceptor® (11% vs 3%, p = 0.08). When new, mortality was significantly higher with PermaNet® 3.0 compared to Interceptor® (OR = 3.6, 95% CI [3.0, 4.2]); however, this benefit was lost with field-aged nets at 12 months (OR = 1.1, 95% CI [0.8, 1.5]) and 24 months (OR = 0.6, 95% CI [0.4, 0.9]). Retention of the non-pyrethroid compound in next-generation nets was low after 36 months (27% for PermaNet® 3.0, 26% for Royal Guard® and 15% for Interceptor® G2). Conclusions Interceptor® G2 outperformed the other ITNs, confirming the superiority of pyrethroid-chlorfenapyr nets over other net types. When new, all next-generation ITNs showed superior bioefficacy compared to Interceptor®; however, the size of this improvement fell after field-ageing due to poor durability of the non-pyrethroid compound. These findings emphasize the need to enhance the insecticidal durability of next-generation ITNs.

Abstract Background Studies evaluating the attrition, physical and insecticidal durability of dual active ingredient (AI) insecticide-treated nets (ITNs) are essential for making programmatic decisions regarding their deployment. We performed a prospective study embedded in a cluster randomised controlled trial (cRCT) to evaluate the attrition, fabric integrity and insecticidal durability of Interceptor® G2 (alpha-cypermethrin-chlorfenapyr) and Royal Guard® (alpha-cypermethrin–pyriproxyfen), compared to Interceptor® (alpha-cypermethrin) in Benin. Methods A total of 2428 study nets in 1093 randomly selected households in five clusters per arm of the cRCT were monitored for ITN attrition and fabric integrity every 6–12 months post-distribution. Householders were further surveyed to investigate non-study net use and their preference for ITN fabric types used in the study nets. A second cohort of 120 nets per ITN type were withdrawn every 12 months and assessed for chemical content and insecticidal activity in laboratory bioassays. Alpha-cypermethrin bioefficacy was investigated using the susceptible Anopheles gambiae Kisumu strain, and chlorfenapyr and pyriproxyfen bioefficacy were investigated using the pyrethroid-resistant Anopheles coluzzii Akron strain. Net pieces were tested in WHO cone bioassays and tunnel tests for alpha-cypermethrin and in tunnel tests for chlorfenapyr; pyriproxyfen activity was assessed in cone bioassays as the reduction in fertility of blood-fed survivors using ovary dissection. Bioefficacy was expressed as the proportion of ITNs passing predetermined WHO criteria, namely knock-down ≥ 95% or 24/72 h mortality ≥ 80% or reduction in fertility ≥ 50%. Results Overall ITN survivorship was 52% at 24 months and fell to 15% at 36 months. Median ITN survival time was lower with Royal Guard® relative to Interceptor® [1.6 vs 2.3 years; hazard ratio (HR) 1.49, 95% confidence interval (CI) 1.36–1.66; p

Abstract 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).

Abstract The present study aimed to assess mosquito species diversity, distribution, and ecological preferences in the Covè, Ouinhi, and Zangnanado communes, Southern Benin. Such information is critical to understand mosquito bio-ecology and to focus control efforts in high-risk areas for vector-borne diseases. Mosquito collections occurred quarterly in 60 clusters between June 2020 and April 2021, using human landing catches. In addition to the seasonal mosquito abundance, Shannon's diversity, Simpson, and Pielou's equitability indices were also evaluated to assess mosquito diversity. Ecological niche models were developed with MaxEnt using environmental variables to assess species distribution. Overall, mosquito density was higher in the wet season than in the dry season in all communes. A significantly higher Shannon's diversity index was also observed in the wet season than in the dry seasons in all communes (p

Abstract The present cluster-randomised control trial aims to assess the entomological efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs compared to the standard pyrethroid-only LLINs, in their third year of community usage. Adult mosquito collections were performed every 3 months, in 4 randomly selected houses in each of the 60 trial clusters, using human landing catches. Adult mosquitoes were morphologically identified and Anopheles vectors were molecularly speciated and screened for the presence of the L1014F kdr mutation using PCR. Plasmodium falciparum sporozoite infection was assessed using ELISA. A subset of An. gambiae s.l. was also dissected to examine parity and fertility rates across study arms. There was no evidence of a significant reduction in indoor vector density and entomological inoculation rate by the pyrethroid-pyriproxyfen [DR 0.94 (95% CI 0.46–1.88), p = 0.8527; and RR 1.10 (95% CI 0.44–2.72), p = 0.8380], and pyrethroid-chlorfenapyr [DR 0.74 (95% CI 0.37–1.48), p = 0.3946; and RR 1.00 (95% CI 0.40–2.50), p = 0.9957] LLINs, respectively. The same trend was observed outdoors. Frequencies of the L1014F kdr mutation, as well as parous and fertility rates, were similar between study arms. In the third year after net distribution, entomological indicators show that the two dual active-ingredients nets performed similarly to the standard pyrethroid-only LLIN. To maintain malaria gains, it is crucial that net distribution cycles fit with their operational lifespan.

Regulatory T cells (Treg) play a prominent role in utero tolerating non-inherited maternal antigens and in regulating immune responses against pathogens at birth. This study investigates Treg immunity in newborns in West Africa, where sepsis remains a major public health problem. Treg phenotypes on neonates subgroups with early-onset sepsis (EOS), presumed sepsis, and healthy newborn with and without prenatal risk factors were evaluated. Treg phenotypes varied according to prenatal conditions, with increase in Treg frequency and Foxp3 expression in healthy newborns with prenatal risk factors compared to those with none risk. Compared to healthy newborns with prenatal risk factors, EOS neonates had a significantly reduced frequency of Treg and Foxp3 expression. In the Treg pool, higher frequency of activated Treg was observed in EOS neonates, suggesting an in-utero activation upstream of the sepsis onset. Their migration to the infection site may explain the reduced frequency of circulating Integrin α4β1+ Treg suggestive of homing to the endothelial tissue. EOS neonates show increases expression of CTLA-4, PD-1 and CD39 on Treg, which negatively regulate the activation of effector T cells (Teff) corroborating by the lower frequency of Teff in EOS neonates. The higher frequency of CD39+ Treg and the lower frequency of integrinα4β1+ Treg in EOS non-survivor suggests that Treg exhaustement and endothelial homing are associated with outcome severity. Neonates developing EOS are born with an altered Treg phenotypic profile. Treg expression of CTLA-4, PD-1, CD39, and integrinα4β1 cell markers can be considered as early warning or diagnostic markers of EOS.

Abstract Background Long-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin. Methods Indoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR). Results Overall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii, while the rest were mostly composed of An. gambiae s.s. with a small proportion (

Abstract Background Following the World Health Organization (WHO) endorsement of dual active ingredient (AI) nets, an increased uptake of pyrethroid-chlorfenapyr and pyrethroid-pyriproxyfen nets is expected. Studies evaluating their physical and insecticidal durability are essential for making programmatic and procurement decisions. This paper describes the methodology for a prospective study to evaluate the attrition, fabric integrity, insecticidal durability of Interceptor® G2 (alpha-cypermethrin-chlorfenapyr) and Royal Guard® (alpha-cypermethrin-pyriproxyfen), compared to Interceptor® (alpha-cypermethrin), embedded in a 3-arm cluster randomized controlled trial (cRCT) in the Zou Department of Benin. Methods Ten clusters randomly selected from each arm of the cRCT will be used for the study. A total of 750 ITNs per type will be followed in 5 study clusters per arm to assess ITN attrition and fabric integrity at 6-, 12-, 24- and 36-months post distribution, using standard WHO procedures. A second cohort of 1800 nets per type will be withdrawn every 6 months from all 10 clusters per arm and assessed for chemical content and biological activity in laboratory bioassays at each time point. Alpha-cypermethrin bioefficacy in Interceptor® and Royal Guard® will be monitored in WHO cone bioassays and tunnel tests using the susceptible Anopheles gambiae Kisumu strain. The bioefficacy of the non-pyrethroid insecticides (chlorfenapyr in Interceptor® G2 and pyriproxyfen in Royal Guard®) will be monitored using the pyrethroid-resistant Anopheles coluzzii Akron strain. Chlorfenapyr activity will be assessed in tunnel tests while pyriproxyfen activity will be assessed in cone bioassays in terms of the reduction in fertility of blood-fed survivors observed by dissecting mosquito ovaries. Nets withdrawn at 12, 24 and 36 months will be tested in experimental hut trials within the cRCT study area against wild free-flying pyrethroid resistant An. gambiae sensu lato to investigate their superiority to Interceptor® and to compare them to ITNs washed 20 times for experimental hut evaluation studies. Mechanistic models will also be used to investigate whether entomological outcomes with each dual ITN type in experimental hut trials can predict their epidemiological performance in the cRCT. Conclusion This study will provide information on the durability of two dual AI nets (Interceptor® G2 and Royal Guard®) in Benin and will help identify suitable methods for monitoring the durability of their insecticidal activity under operational conditions. The modelling component will determine the capacity of experimental hut trials to predict the epidemiological performance of dual AI nets across their lifespan.

Abstract Selection of mosquito collection methods is of crucial importance to evaluate the impact of vector control tools on entomological outcomes. During a cluster randomised control trial evaluating the relative efficacy of two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) compared to pyrethroid-only LLINs, we assessed the performance of different mosquito collection methods: Human landing catches (HLC), Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSC). Anopheles mosquitoes were collected using three collection methods in 4 houses, in each of the 60 trial clusters at baseline and every quarter for 24 months using PSCs and HLCs, while CDC light traps were performed during two quarters only. Mean density of vectors collected per method per night was the highest with HLCs (15.9), followed by CDC light traps (6.8); with PSCs (1.1) collecting 10 times less mosquitoes than HLCs. All three collection methods collected fewer mosquitoes in the Interceptor G2® dual a.i. arm, compared to the other trial arms, although only HLCs and PSCs demonstrated strong evidence of this due to a greater number of collection rounds undertaken, than CDC light traps. The broadly similar results regarding the differential impact of the two dual a.i. LLINs showed by the three collection methods suggest that the more ethically acceptable, cheaper, and logistically simpler methods such as CDC light traps could be prioritised for use in large community trials for measuring the efficacy of vector control tools.

The COVID-19 pandemic has sent ripple effects across health systems and impacted the burden of many other diseases, such as malaria in sub-Saharan Africa. This study takes a mixed method approach to assess the impact of COVID-19 on malaria control programs in three rural communes in Benin. We conducted individual semi-structured interviews with key informants who play important roles in malaria control in Benin at three levels of the health system-national, health zone, and commune. Using a purposive sampling technique, informants were interviewed regarding their roles in malaria control, the impact of the pandemic on their activities, and the mitigation strategies adopted. Relevant themes were identified by content analysis. We then formulated an agent-based model of malaria epidemiology to assess the impacts of treatment disruption on malaria burden. The key informant interviews revealed that essential aspects of malaria control were upheld in Benin due to the close collaboration of public health practitioners and health care providers at all levels of the health system. There were some disruptions to case management services for malaria at the start of the pandemic due to the public avoiding health centers and a brief shortage of malaria treatment that may not be entirely attributable to the pandemic. Results from the agent-based model suggest that duration, severity, and timing of treatment disruption can impact malaria burden in a synergistic manner, though the effects are small given the relatively mild disruptions observed. This study highlights the importance of top-down leadership in health emergencies, as well as the critical role of community health workers in preventing negative health outcomes for their communities. We also showcased the integration of qualitative research and mathematical models-an underappreciated form of mixed methods research that offer immense value in the continued evaluation of rapidly evolving health emergencies.

Accurate determination of pre-pregnancy weight is essential for optimal pregnancy monitoring and antenatal care. Determining pre-pregnancy weight in limited-resources settings is challenging for both clinical practice and public health research. From a 2014-2017 pre-pregnancy cohort in Benin, we evaluated the agreement between the measured pre-pregnancy weight (MPPW) and two proxies: (i) the first trimester pregnancy weight (FTPW) and (ii) the estimated pre-pregnancy weight (EPPW) using Thomas & al. formula. We analysed data from 302 pregnant women with both pre-pregnancy weight measured within 3 months before conception and weight measured during the first trimester. Using segmented linear regression, we first assessed up to which gestational age the weight measured during the first trimester could reasonably estimate the MPPW. Then the Bland & Altman method was used to assess agreement between MPPW and the two proxies. Additional analyses were performed to assess the sensitivity of results to the timing of measurement of either MPPW or the two proxies. On average, FTPW did not feature significant difference with MPPW up to 13.03 (11.99-14.06) weeks of gestational age. FTPW, measured on average at 7 ± 2.4 weeks of gestation, and the EPPW showed similar Bland & Altman limits of agreement with the MPPW. However, while the FTPW slightly underestimated the MPPW by a mean of-0.16 (-0.08; +0.39) kg, the EPPW overestimated it by a mean of + 0.43 (+0.20; +0.66) kg. Minor differences in these results were observed when the MPPW was assessed earlier or within three months before pregnancy, or according to the gestational age at the time of the proxy's measurement. In conclusion, in Southern Benin and up to 12-14 weeks of pregnancy, the FTPW appeared to be a good proxy of the MPPW while using Thomas' formula did not enhance pre-pregnancy weight estimation.

Abstract Background Cerebral malaria (CM) is a neuropathology which remains one of the deadliest forms of malaria among African children. The kinetics of the pathophysiological mechanisms leading to neuroinflammation and the death or survival of patients during CM are still poorly understood. The increasing production of cytokines, chemokines and other actors of the inflammatory and oxidative response by various local actors in response to neuroinflammation plays a major role during CM, participating in both the amplification of the neuroinflammation phenomenon and its resolution. In this study, we aimed to identify risk factors for CM death among specific variables of inflammatory and oxidative responses to improve our understanding of CM pathogenesis. Methods Children presenting with CM (n = 70) due to P. falciparum infection were included in southern Benin and divided according to the clinical outcome into 50 children who survived and 20 who died. Clinical examination was complemented by fundoscopic examination and extensive blood biochemical analysis associated with molecular diagnosis by multiplex PCR targeting 14 pathogens in the patients’ cerebrospinal fluid to rule out coinfections. Luminex technology and enzyme immunoassay kits were used to measure 17 plasma and 7 urinary biomarker levels, respectively. Data were analysed by univariate analysis using the nonparametric Mann‒Whitney U test and Pearson’s Chi2 test. Adjusted and multivariate analyses were conducted separately for plasma and urinary biomarkers to identify CM mortality risk factors. Results Univariate analysis revealed higher plasma levels of tumour necrosis factor (TNF), interleukin-1beta (IL-1β), IL-10, IL-8, C-X-C motif chemokine ligand 9 (CXCL9), granzyme B, and angiopoietin-2 and lower urinary levels of prostanglandine E2 metabolite (PGEM) in children who died compared to those who survived CM (Mann–Whitney U-test, P-values between 0.03 and

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.

Globally, negative impacts of the COVID-19 pandemic on malaria prevention and control efforts have been caused by delayed distributions of long-lasting insecticidal nets (LLIN), decreased outpatient attendance, and disruptions to malaria testing and treatment. Using a mixed methods approach, we aimed to evaluate the impact of COVID-19 on community-level malaria prevention and health-seeking practices in Benin more than one year after the start of the COVID-19 pandemic. We collected data through community-based cross-sectional surveys with 4200 households and ten focus group discussions (FGDs). Mixed effect logistic regression models accounting for a clustered sampling design were used to identify variables associated with main outcomes (good COVID-19 knowledge, LLIN usage and access, and avoidance of health centres). Consistent with the experiences of FGD participants, receiving information from radios or televisions was significantly associated with good COVID-19 knowledge and avoiding health centres because of the pandemic (p

Background: Having a maximum number of people vaccinated was the objective to control the COVID-19 pandemic. We report in this manuscript the factors associated with the willingness to be vaccinated against COVID-19 during the pandemic period. Methods: From April to May 2022, a community-based cross-sectional survey was performed. Participants were randomly selected from four districts in Benin (taking into account the COVID-19 prevalence). Mixed-effect logistic regression models were used to identify the variables associated with COVID-19 vaccine acceptance. Results: A total of 2069 participants were included. The proportion of vaccine acceptance was 43.3%. A total of 24.2% were vaccinated and showed proof of vaccination. The population’s request for vaccination was higher after the third epidemic wave. The district of residence, the education level, a fear of being infected, the channel of information, poor medical conditions, a good knowledge of the transmission mode and symptoms, and good behaviors were significantly associated with vaccine acceptance. Conclusion: The overall acceptance of the COVID-19 vaccine in the Beninese population was relatively high. However, vaccine campaigns in areas with a low acceptance as well as the disclosure of information, particularly on our knowledge of the disease and the safety, side effects, and effectiveness of the COVID-19 vaccines, should be strengthened with adapted and consistent messages.

Abstract Background Long-lasting insecticidal nets (LLINs) are currently the primary method of malaria control in sub-Saharan Africa and have contributed to a significant reduction in malaria burden over the past 15 years. However, this progress is threatened by the wide-scale selection of insecticide-resistant malaria vectors. It is, therefore, important to accelerate the generation of evidence for new classes of LLINs. Methods This protocol presents a three-arm superiority, single-blinded, cluster randomized controlled trial to evaluate the impact of 2 novel dual-active ingredient LLINs on epidemiological and entomological outcomes in Benin, a malaria-endemic area with highly pyrethroid-resistant vector populations. The study arms consist of (i) Royal Guard® LLIN, a net combining a pyrethroid (alpha-cypermethrin) plus an insect growth regulator (pyriproxyfen), which in the adult female is known to disrupt reproduction and egg fertility; (ii) Interceptor G2® LLIN, a net incorporating two adulticides (alpha-cypermethrin and chlorfenapyr) with different modes of action; and (iii) the control arm, Interceptor® LLIN, a pyrethroid (alpha-cypermethrin) only LLIN. In all arms, one net for every 2 people will be distributed to each household. Sixty clusters were identified and randomised 1:1:1 to each study arm. The primary outcome is malaria case incidence measured over 24 months through active case detection in a cohort of 25 children aged 6 months to 10 years, randomly selected from each cluster. Secondary outcomes include 1) malaria infection prevalence (all ages) and prevalence of moderate to severe anaemia in children under 5 years old, measured at 6 and 18 months post-intervention; 2) entomological indices measured every 3 months using human landing catches over 24 months. Insecticide resistance intensity will also be monitored over the study period. Discussion This study is the second cluster randomised controlled trial to evaluate the efficacy of these next-generation LLINs to control malaria transmitted by insecticide-resistant mosquitoes. The results of this study will form part of the WHO evidence-based review to support potential public health recommendations of these nets and shape malaria control strategies of sub-Saharan Africa for the next decade. Trial registration ClinicalTrials.gov, NCT03931473 , registered on 30 April 2019.

Abstract Background While sub-microscopic malarial infections are frequent and potentially deleterious during pregnancy, routine molecular detection is still not feasible. This study aimed to assess the performance of a Histidine Rich Protein 2 (HRP2)-based ultrasensitive rapid diagnostic test (uRDT, Alere Malaria Ag Pf) for the detection of infections of low parasite density in pregnant women. Methods This was a retrospective study based on samples collected in Benin from 2014 to 2017. A total of 942 whole blood samples collected in 327 women in the 1st and 3rd trimesters and at delivery were tested by uRDT, conventional RDT (cRDT, SD BIOLINE Malaria Ag Pf), microscopy, quantitative polymerase chain-reaction (qPCR) and Luminex-based suspension array technology targeting P. falciparum HRP2. The performance of each RDT was evaluated using qPCR as reference standard. The association between infections detected by uRDT, but not by cRDT, with poor maternal and birth outcomes was assessed using multivariate regression models. Results The overall positivity rate detected by cRDT, uRDT, and qPCR was 11.6% (109/942), 16.2% (153/942) and 18.3% (172/942), respectively. Out of 172 qPCR-positive samples, 68 were uRDT-negative. uRDT had a significantly better sensitivity (60.5% [52.7–67.8]) than cRDT (44.2% [36.6–51.9]) and a marginally decreased specificity (93.6% [91.7–95.3] versus 95.7% [94.0–97.0]). The gain in sensitivity was particularly high (33%) and statistically significant in the 1st trimester. Only 28 (41%) out of the 68 samples which were qPCR-positive, but uRDT-negative had detectable but very low levels of HRP2 (191 ng/mL). Infections that were detected by uRDT but not by cRDT were associated with a 3.4-times (95%CI 1.29–9.19) increased risk of anaemia during pregnancy. Conclusions This study demonstrates the higher performance of uRDT, as compared to cRDTs, to detect low parasite density P. falciparum infections during pregnancy, particularly in the 1st trimester. uRDT allowed the detection of infections associated with maternal anaemia.

The efficacy of a vector control tool in reducing mosquito biting is crucial for its acceptability. The present study compared the vector density of Culex spp. And Mansonia spp. across clusters, which received two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) and a standard pyrethroid-only LLIN, and assessed the seasonality of these mosquito genera. A total of 85,723 Culex spp. and 144,025 Mansonia spp. were caught over the study period. The density of Culex and Mansonia was reduced in all three arms over the study period. There was no evidence of a significant reduction in the indoor or outdoor density of Culex spp. in either dual-a.i. LLIN arm as compared to the standard pyrethroid-only net arm. A similar trend was observed with Mansonia spp. A high density of Culex spp. was found both in rainy and dry seasons, while for Mansonia spp., this was mainly observed during the rainy season. These results suggest that the novel insecticides in the dual-a.i. LLINs did not have an additional impact on these species and that pyrethroids might still be effective on them. Further work is required to determine whether these species of mosquitoes have resistance to the insecticides tested in this trial.

BackgroundFetal growth restriction is a major complication of pregnancy and is associated with stillbirth, infant death and child morbidity. Ultrasound monitoring of pregnancy is becoming more common in Africa for fetal growth monitoring in clinical care and research, but many countries have no national growth charts. We evaluated the new international fetal growth standards from INTERGROWTH-21st and WHO in a cohort from southern Benin.MethodsRepeated ultrasound and clinical data were collected in women from the preconceptional RECIPAL cohort (241 women with singleton pregnancies, 964 ultrasounds). We modelled fetal biometric parameters including abdominal circumference (AC) and estimated fetal weight (EFW) and compared centiles to INTERGROWTH-21st and WHO standards, using the Bland and Altman method to assess agreement. For EFW, we used INTERGROWTH-21st standards based on their EFW formula (IG21st) as well as a recent update using Hadlock's EFW formula (IG21hl). Proportions of fetuses with measurements under the 10th percentile were compared.ResultsMaternal malaria and anaemia prevalence was 43% and 69% respectively and 11% of women were primigravid. Overall, the centiles in the RECIPAL cohort were higher than that of INTERGROWTH-21st and closer to that of WHO. Consequently, the proportion of fetuses under 10th percentile thresholds was systematically lower when applying IG21st compared to WHO standards. At 27-31 weeks and 33-38 weeks, respectively, 7.4% and 5.6% of fetuses had EFW ConclusionDespite high anemia and malaria prevalence in the cohort, IG21st and WHO standards did not identify higher than expected proportions of fetuses under the 10th percentiles of ultrasound parameters or EFW. The proportions of fetuses under the 10th percentile threshold for IG21st charts were particularly low, raising questions about its use to identify growth-restricted fetuses in Africa.

BackgroundDespite several years of school-based MDA implementation, STH infections remain an important public health problem in Benin, with a country-wide prevalence of 20% in 2015. The DeWorm3 study is designed to assess the feasibility of using community-based MDA with albendazole to interrupt the transmission of STH, through a series of cluster-randomized trials in Benin, India and Malawi. We used the pre-treatment baseline survey data to describe and analyze the factors associated with STH infection in Comé, the study site of the DeWorm3 project in Benin. These data will improve understanding of the challenges that need to be addressed in order to eliminate STH as a public health problem in Benin.MethodsBetween March and April 2018, the prevalence of STH (hookworm spp., Ascaris and Trichuris trichiura) was assessed by Kato-Katz in stool samples collected from 6,153 residents in the community of Comé, Benin using a stratified random sampling procedure. A standardized survey questionnaire was used to collect information from individual households concerning factors potentially associated with the presence and intensity of STH infections in pre-school (PSAC, aged 1-4), school-aged children (SAC, aged 5-14) and adults (aged 15 and above). Multilevel mixed-effects models were used to assess associations between these factors and STH infection.ResultsThe overall prevalence of STH infection was 5.3%; 3.2% hookworm spp., 2.1% Ascaris lumbricoides and 0.1% Trichuris. Hookworm spp. were more prevalent in adults than in SAC (4.4% versus 2.0%, respectively; p = 0.0001) and PSAC (4.4% versus 1.0%, respectively; pConclusionHookworm spp. are the most prevalent STH in Comé, with a persistent reservoir in adults that is not addressed by current control measures based on school MDA. Expanding MDA to target adults and PSAC is necessary to substantially impact population prevalence, particularly for hookworm.Trial registrationClinicalTrials.gov NCT03014167.

BackgroundThis study provides detailed characteristics of vector populations in preparation for a three-arm cluster randomized controlled trial (RCT) aiming to compare the community impact of dual active-ingredient (AI) long-lasting insecticidal nets (LLINs) that combine two novel insecticide classes-chlorfenapyr or pyriproxifen-with alpha-cypermethrin to improve the prevention of malaria transmitted by insecticide-resistant vectors compared to standard pyrethroid LLINs.MethodsThe study was carried out in 60 villages across Cove, Zangnanando and Ouinhi districts, southern Benin. Mosquito collections were performed using human landing catches (HLCs). After morphological identification, a sub-sample of Anopheles gambiae s.l. were dissected for parity, analyzed by PCR for species and presence of L1014F kdr mutation and by ELISA-CSP to identify Plasmodium falciparum sporozoite infection. WHO susceptibility tube tests were performed by exposing adult An. gambiae s.l., collected as larvae from each district, to 0.05% alphacypermethrin, 0.75% permethrin, 0.1% bendiocarb and 0.25% pirimiphos-methyl. Synergist assays were also conducted with exposure first to 4% PBO followed by alpha-cypermethrin.ResultsAn. gambiae s.l. (n = 10807) was the main malaria vector complex found followed by Anopheles funestus s.l. (n = 397) and Anopheles nili (n = 82). An. gambiae s.l. was comprised of An. coluzzii (53.9%) and An. gambiae s.s. (46.1%), both displaying a frequency of the L1014F kdr mutation >80%. Although more than 80% of people slept under standard LLIN, human biting rate (HBR) in An. gambiae s.l. was higher indoors [26.5 bite/person/night (95% CI: 25.2-27.9)] than outdoors [18.5 b/p/n (95% CI: 17.4-19.6)], as were the trends for sporozoite rate (SR) [2.9% (95% CI: 1.7-4.8) vs 1.8% (95% CI: 0.6-3.8)] and entomological inoculation rate (EIR) [21.6 infected bites/person/month (95% CI: 20.4-22.8) vs 5.4 (95% CI: 4.8-6.0)]. Parous rate was 81.6% (95%CI: 75.4-88.4). An. gambiae s.l. was resistant to alpha-cypermethrin and permethrin but, fully susceptible to bendiocarb and pirimiphos-methyl. PBO pre-exposure followed by alpha-cypermethrin treatment induced a higher 24 hours mortality compared to alphacypermethrin alone but not exceeding 40%.ConclusionsDespite a high usage of standard pyrethroid LLINs, the study area is characterized by intense malaria transmission. The main vectors An. coluzzii and An. gambiae s.s. were both highly resistant to pyrethroids and displayed multiple resistance mechanisms, L1014F kdr mutation and mixed function oxidases. These conditions of the study area make it an appropriate site to conduct the trial that aims to assess the effect of novel dual-AI LLINs on malaria transmitted by insecticide-resistant vectors.

Introduction Neonatal sepsis outreaches all causes of neonatal mortality worldwide and remains a major societal burden in low and middle income countries. In addition to limited resources, endemic morbidities, such as malaria and prematurity, predispose neonates and infants to invasive infection by altering neonatal immune response to pathogens. Nevertheless, thoughtful epidemiological, diagnostic and immunological evaluation of neonatal sepsis and the impact of gestational malaria have never been performed.Methods and analysis A prospective longitudinal multicentre follow-up of 580 infants from birth to 3 months of age in urban and suburban Benin will be performed. At delivery, and every other week, all children will be examined and clinically evaluated for occurrence of sepsis. At delivery, cord blood systematic analysis of selected plasma and transcriptomic biomarkers (procalcitonin, interleukin (IL)-6, IL-10, IP10, CD74 and CX3CR1) associated with sepsis pathophysiology will be evaluated in all live births as well as during the follow-up, and when sepsis will be suspected. In addition, whole blood response to selected innate stimuli and extensive peripheral blood mononuclear cells phenotypic characterisation will be performed. Reference intervals specific to sub-Saharan neonates will be determined from this cohort and biomarkers performances for neonatal sepsis diagnosis and prognosis tested.Ethics and dissemination Ethical approval has been obtained from the Comité d’Ethique de la Recherche – Institut des Sciences Biomédicales Appliquées (CER-ISBA 85 - 5 April 2016, extended on 3 February 2017). Results will be disseminated through international presentations at scientific meetings and publications in peer-reviewed journals.Trial registration number ClinicalTrials.gov registration number: NCT03780712.

Abstract Background Malaria in pregnancy is prevalent in Sub-Saharan Africa. The first trimester of pregnancy is a critical period and the best preventive measure is Long Lasting Insecticidal Nets (LLIN). Unfortunately, few studies have been conducted which focuses on the usage and efficacy of LLIN on malaria prevention during the first trimester. Methods We assessed the use and effectiveness of LLIN in early pregnancy in Benin and its impact on malaria infection risk. We followed-up a cohort of 240 pregnant women from pre-conception to the end of the first trimester of pregnancy in Southern Benin. Parasitological, maternal and LLIN data were actively collected before, at the beginning and end of the first trimester of pregnancy. A Cox regression model was used to determine the relationship between the time to onset of the first malaria infection and the use, physical integrity, and bio-efficacy of the LLIN, adjusted for relevant covariables. Results The good use, good physical integrity and biological efficacy of LLIN were associated with a decreased risk of occurrence of the first malaria infection in early pregnancy (HRa = 0.38; (0.18–0.80); p

An amendment to this paper has been published and can be accessed via the original article.

INTRODUCTION: Placental malaria (PM) is an important predictor of infant morbidity and mortality in sub-Saharan Africa. Although placental histology is the gold standard test to diagnose PM, the placenta impression smears remains widely used in epidemiological studies. This study is set to evaluate the performance of placental impression smears to detect PM in pregnant women in southern Benin.

BackgroundMalaria and schistosomiasis represent two of the most prevalent and disabling parasitic infections in developing countries. Few studies have evaluated the effect of maternal schistosomiasis and malaria in the peri-conceptional period on infant's risk of infection.MethodsIn Benin, women were followed from the preconception period until delivery. Subsequently, their children were followed from birth to 3 months of age. Pre-pregnancy malaria, malaria in pregnancy (MiP)-determined monthly using a thick blood smear-and urinary schistosomiasis-determined once before pregnancy and once at delivery using urine filtration-were the main maternal exposures. Infant's febrile infection (fever with respiratory, gastrointestinal and/or cutaneous clinical signs anytime during follow-up) was the main outcome. In a secondary analysis, we checked the relation of malaria and schistosomiasis with infant's hemoglobin (Hb) concentration. Both effects were separately assessed using logistic/mixed linear regression models.ResultsThe prevalence of MiP was 35.7% with 10.8% occurring during the 1st trimester, and the prevalence of schistosomiasis was 21.8%. From birth to 3 months, 25.3% of infants had at least one episode of febrile infection. In multivariate analysis, MiP, particularly malaria in the 1st trimester, was significantly associated with a higher risk of infant's febrile infection (aOR = 4.99 [1.1; 22.6], p = 0.03). In secondary results, pre-pregnancy malaria and schistosomiasis were significantly associated with a lower infant's Hb concentration during the first 3 months.ConclusionWe evidenced the deleterious effect of maternal parasitic infections on infant's health. Our results argue in favor of the implementation of preventive strategies as early as in the peri-conception.

BACKGROUND:At times, ultrasound is not readily available in low resource countries in Africa for accurate determination of gestational age, so using alternative methods is pivotal during pregnancy. These assessments are used to aid the risk analysis for an infant and management strategies for premature delivery, if necessary. Currently, date of last menstrual period, fundal height measurements, and the New Ballard Score are commonly used in resource-limited settings. However, concordance of these measures is unknown for sub-Saharan Africa. We obtained data from an open-label randomized controlled trial, to assess the concordance of these alternative assessment methods. The purpose of our study was to determine the agreement between these alternative methods when used in sub-Saharan African populations. METHODS:A total of 4,390 pregnant women from Benin, Gabon, Mozambique and Tanzania were included in our analysis. The assessment methods compared were: 1) reported last menstrual period, 2) symphysis-fundal height measurement, and 3) the New Ballard Score. The Bland-Altman method and intraclass correlation coefficient (ICC) were used to test the degree of agreement. Survival range gestational age, used as an inclusion criterion for further analysis, was from 22 to 44 weeks. FINDINGS:Plots showed a lack of agreement between methods and the 95% limits of agreement too wide to be clinically useful. ICC = 0.25 indicated poor agreement. A post-hoc analysis, restricted from 32 to 42 weeks, was done to check for better agreement in this near-term population. The plots and ICC = 0.16 still confirmed poor agreement. CONCLUSION:The alternative assessments do not result in comparable outcomes and discrepancies are far beyond the clinically acceptable range. Last menstrual period should not be used as the only estimator of gestational age. In the absence of reliable early ultrasound, symphysis-fundal height measurements may be most useful during pregnancy for fetal risk assessment and the New Ballard Score after delivery as a confirmation of these estimations and for further neonatal management. However, promotion of portable ultrasound devices is required for accurate assessment of gestational age in sub-Sahara Africa.

BackgroundLittle is known about the effects of intermittent preventive treatment of malaria in pregnancy (IPTp) on the health of sub-Saharan African infants. We have evaluated the safety of IPTp with mefloquine (MQ) compared to sulfadoxine-pyrimethamine (SP) for important infant health and developmental outcomes.Methods and findingsIn the context of a multicenter randomized controlled trial evaluating the safety and efficacy of IPTp with MQ compared to SP in pregnancy carried out in four sub-Saharan countries (Mozambique, Benin, Gabon, and Tanzania), 4,247 newborns, 2,815 born to women who received MQ and 1,432 born to women who received SP for IPTp, were followed up until 12 mo of age. Anthropometric parameters and psychomotor development were assessed at 1, 9, and 12 mo of age, and the incidence of malaria, anemia, hospital admissions, outpatient visits, and mortality were determined until 12 mo of age. No significant differences were found in the proportion of infants with stunting, underweight, wasting, and severe acute malnutrition at 1, 9, and 12 mo of age between infants born to women who were on IPTp with MQ versus SP. Except for three items evaluated at 9 mo of age, no significant differences were observed in the psychomotor development milestones assessed. Incidence of malaria, anemia, hospital admissions, outpatient visits, and mortality were similar between the two groups. Information on the outcomes at 12 mo of age was unavailable in 26% of the infants, 761 (27%) from the MQ group and 377 (26%) from the SP group. Reasons for not completing the study were death (4% of total study population), study withdrawal (6%), migration (8%), and loss to follow-up (9%).ConclusionsNo significant differences were found between IPTp with MQ and SP administered in pregnancy on infant mortality, morbidity, and nutritional outcomes. The poorer performance on certain psychomotor development milestones at 9 mo of age in children born to women in the MQ group compared to those in the SP group may deserve further studies.Trial registrationClinicalTrials.gov NCT00811421.

Anaemia is an increasingly recognized health problem in Africa, particularly in infants and pregnant women. Although malaria is known to be the main risk factor of anaemia in both groups, the consequences of maternal factors, particularly malaria in pregnancy (MiP), on infant haemoglobin (Hb) concentrations during the first months of life are still unclear.We followed-up a cohort of 1005 Beninese pregnant women from the beginning of pregnancy until delivery. A subsample composed of the first 400 offspring of these women were selected at birth and followed until the first year of life. Placental histology and blood smear at 1st clinical antenatal visit (ANC), 2nd ANC and delivery were used to assess malaria during pregnancy. Infant Hb concentrations were measured at birth, 6, 9 and 12 months of age. A mixed multi-level model was used to assess the association between MiP and infant Hb variations during the first 12 months of life.Placental malaria (difference mean [dm] = - 2.8 g/L, 95% CI [-5.3, -0.3], P = 0.03) and maternal peripheral parasitaemia at delivery (dm = - 4.6 g/L, 95% CI [-7.9, -1.3], P = 0.007) were the main maternal factors significantly associated with infant Hb concentrations during the first year of life. Poor maternal nutritional status and malaria infection during infancy were also significantly associated with a decrease in infant Hb.Antimalarial control and nutritional interventions before and during pregnancy should be reinforced to reduce specifically the incidence of infant anaemia, particularly in Sub-Saharan countries.

BackgroundIntermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended by WHO to prevent malaria in African pregnant women. The spread of SP parasite resistance has raised concerns regarding long-term use for IPT. Mefloquine (MQ) is the most promising of available alternatives to SP based on safety profile, long half-life, and high efficacy in Africa. We evaluated the safety and efficacy of MQ for IPTp compared to those of SP in HIV-negative women.Methods and findingsA total of 4,749 pregnant women were enrolled in an open-label randomized clinical trial conducted in Benin, Gabon, Mozambique, and Tanzania comparing two-dose MQ or SP for IPTp and MQ tolerability of two different regimens. The study arms were: (1) SP, (2) single dose MQ (15 mg/kg), and (3) split-dose MQ in the context of long lasting insecticide treated nets. There was no difference on low birth weight prevalence (primary study outcome) between groups (360/2,778 [13.0%]) for MQ group and 177/1,398 (12.7%) for SP group; risk ratio [RR], 1.02 (95% CI 0.86-1.22; p=0.80 in the ITT analysis). Women receiving MQ had reduced risks of parasitemia (63/1,372 [4.6%] in the SP group and 88/2,737 [3.2%] in the MQ group; RR, 0.70 [95% CI 0.51-0.96]; p=0.03) and anemia at delivery (609/1,380 [44.1%] in the SP group and 1,110/2743 [40.5%] in the MQ group; RR, 0.92 [95% CI 0.85-0.99]; p=0.03), and reduced incidence of clinical malaria (96/551.8 malaria episodes person/year [PYAR] in the SP group and 130/1,103.2 episodes PYAR in the MQ group; RR, 0.67 [95% CI 0.52-0.88]; p=0.004) and all-cause outpatient attendances during pregnancy (850/557.8 outpatients visits PYAR in the SP group and 1,480/1,110.1 visits PYAR in the MQ group; RR, 0.86 [0.78-0.95]; p=0.003). There were no differences in the prevalence of placental infection and adverse pregnancy outcomes between groups. Tolerability was poorer in the two MQ groups compared to SP. The most frequently reported related adverse events were dizziness (ranging from 33.9% to 35.5% after dose 1; and 16.0% to 20.8% after dose 2) and vomiting (30.2% to 31.7%, after dose 1 and 15.3% to 17.4% after dose 2) with similar proportions in the full and split MQ arms. The open-label design is a limitation of the study that affects mainly the safety assessment.ConclusionsWomen taking MQ IPTp (15 mg/kg) in the context of long lasting insecticide treated nets had similar prevalence rates of low birth weight as those taking SP IPTp. MQ recipients had less clinical malaria than SP recipients, and the pregnancy outcomes and safety profile were similar. MQ had poorer tolerability even when splitting the dose over two days. These results do not support a change in the current IPTp policy.Trial registrationClinicalTrials.gov NCT 00811421; Pan African Clinical Trials Registry PACTR 2010020001429343 Please see later in the article for the Editors' Summary.