Impact of glutathione-s transferase and cytochrome P450 metabolic resistance on the effectiveness of various bed nets against Anopheles funestus, a major malaria vector in Africa

The scale-up of insecticide-based interventions, including Indoor Residual Spraying (IRS) and Long Lasting Insecticidal Nets (LLINs), has contributed to the significant reduction of malaria burden in the past decade. Unfortunately, growing insecticide resistance in malaria vectors is threatening these successes. However, the impact of resistance, especially metabolic resistance, on the effectiveness of vector control tools against pyrethroid-resistant mosquito populations, remains a topic of debate. One of the key challenges of assessing the impact of metabolic resistance on the effectiveness of these insecticide-based interventions has been the lack of molecular markers for insecticide resistance, notably metabolic resistance. The recent detection of key genetic markers conferring metabolic-mediated resistance (GSTe2 marker and CYP6P9a/b markers) is providing the opportunity to now address this question. In this work, we used an experimental hut trial, and the markers recently made available to investigate the direct impact of metabolic resistance on the effectiveness of insecticide-based interventions. The characterization of the area selected for experimental hut trial revealed that Anopheles funestus sensu stricto (s.s.) (80%) and Anopheles gambiae s.s. (20%) are the two main species. High levels of resistance to pyrethroids and organochlorines were noticed. Moderate resistance was observed against bendiocarb (carbamate) in both species, but relatively higher in Anopheles gambiae s.s. In contrast, full susceptibility was observed for the organophosphate malathion. The PBO synergist assays with pyrethroid type I and II revealed a significant recovery of the susceptibility in Anopheles funestus s.s. population (48.8 to 98.1% mortality and 38.3 to 96.5% mortality, respectively). The DDT/pyrethroid 119F-GSTe2 resistant allele (28.1%) and the dieldrin 296S-RDL resistance (9.7%) were detected in Anopheles funestus s.s. The high pyrethroid/DDT resistance in Anopheles gambiae correlated with the high frequency of the 1014F knockdown resistance allele (63.9%). The 1014S-kdr allele was detected at low frequency (1.97%). The Plasmodium infection rate was 20% in Anopheles gambiae, whereas Anopheles funestus exhibited an oocyst infection rate of 15 and 5% for the sporozoite infection rate. Using experimental hut trials and genotyping of a glutathione S-transferase resistance marker (L119F-GSTe2), we noticed that PBO-based nets induced a significantly higher mortality rate than pyrethroid-only nets. Blood feeding rate and deterrence were significantly higher in all LLINs than control, although greater for PermaNet 3.0 than PermaNet 2.0 but not between Olyset nets. Genotyping the the L119F-GSTe2 mutation revealed that, for permethrin-based nets, 119F-GSTe2 resistant mosquitoes have a greater ability to blood feed than susceptible [Olyset Plus (OR= 3.1; P=0.00007) and Olyset (OR=3.0; P=0.00007)] while the opposite effect is observed for deltamethrin-based nets. For Olyset Plus, a significant association with exophily was observed in resistant mosquitoes (OR=11.7; P=0.003). Furthermore, GSTe2-resistant mosquitoes (cone assays) significantly survived with PermaNet 2.0 (OR=2.1; P=0.002) and even more with PermaNet 3.0 (OR=30.1; P<0.0001) whereas no association was observed for Olyset Plus. L119F-GSTe2 was confirmed to confer resistance to permethrin, Deltamethrin, and DDT. Also, genotyping of the CYP6P9a/b markers revealed that homozygous resistant (RR) were better able to survive exposure to Olyset nets than homozygous susceptible (SS) : (OR=7.03; P< 0.01) for CYP6P9a and (OR=5.1; P<0.01) for CYP6P9b. In addition, homozygous resistant (RR) show a greater ability to blood feed when compared to homozygous susceptible (SS) (OR= 3.1; p<0.001) for CYP6P9a and (OR= 8.01; p<0.001) for CYP6P9b. Further analysis demonstrates that mosquitoes double homozygous resistant (RR/RR) at both genes had by far a significant ability to reduce LLINs efficacy (Blood feeding: RR/RR v SS/SS; OR= 9.3; p<0.001) compared to single homozygous (Blood feeding RR v SS; OR= 4.5; p<0.001). This study shows the greater efficacy of PBO-based nets against pyrethroid-resistant malaria vectors. Reduced efficiency of LLINs and the increased ability to survive and blood feed of CYP6P9a/b and GSTe2 resistant mosquitoes highlight the impact of metabolic resistance

Awareness, attitudes and prevention of malaria in the cities of Douala and Yaoundé (Cameroon)

<p>Abstract</p> <p>Background</p> <p>There is little information on the social perception of malaria and the use of prevention methods in Cameroon. This study was designed to assess knowledge, attitude and management of malaria in households living in the cities of Douala and Yaoundé.</p> <p>Results</p> <p>Over 82% of people interviewed associated malaria transmission to mosquito bites. Methods used for malaria prevention were: environmental sanitation 1645 (76.1%), use of bed nets 1491 (69%), insecticide spray/coils 265 (12.3%) and netting of doors or windows 42 (1.9%). Bed net ownership was significantly high in Yaoundé (73.8%) (P < 0.0001), whereas the use of insecticide spray or coils was significantly important in Douala (16.3%) (P < 0.0001). Some of the problems experienced by families using ITN were the difficulty in finding chemicals for the retreatment of nets 702 (47%), insufficient financial means to buy new bed nets to replace old ones 366 (24.5%) or, to provide bed nets to everybody in the household 289 (19.4%) and the sensation of feeling excessive heat when sleeping under a bed net 74 (5%). The amount spent monthly by a household for vector control and malaria treatment was estimated at 2377 fcfa (3.6 euros) and 4562 fcfa (6.95 euros) respectively. These amounts were not significantly different between households of Douala and Yaoundé. Concerning management of malaria cases, 18.6% of people declare going to the hospital when suffering from malaria. The majority of people (81.4%) do self medication - they either buy drugs from the pharmacists, street sellers or they use plants to cure malaria.</p> <p>Conclusion</p> <p>The study revealed a high awareness of populations on malaria and ITNs. However some attitudes hindering the use of ITN or related to the management of clinical cases need further attention.</p

Physiology-based simulation of the retinal vasculature enables annotation-free segmentation of OCT angiographs

Optical coherence tomography angiography (OCTA) can non-invasively image the eye's circulatory system. In order to reliably characterize the retinal vasculature, there is a need to automatically extract quantitative metrics from these images. The calculation of such biomarkers requires a precise semantic segmentation of the blood vessels. However, deep-learning-based methods for segmentation mostly rely on supervised training with voxel-level annotations, which are costly to obtain. In this work, we present a pipeline to synthesize large amounts of realistic OCTA images with intrinsically matching ground truth labels; thereby obviating the need for manual annotation of training data. Our proposed method is based on two novel components: 1) a physiology-based simulation that models the various retinal vascular plexuses and 2) a suite of physics-based image augmentations that emulate the OCTA image acquisition process including typical artifacts. In extensive benchmarking experiments, we demonstrate the utility of our synthetic data by successfully training retinal vessel segmentation algorithms. Encouraged by our method's competitive quantitative and superior qualitative performance, we believe that it constitutes a versatile tool to advance the quantitative analysis of OCTA images.Comment: Accepted at MICCAI 202

Insecticide Resistance in Malaria Vectors: An Update at a Global Scale

Malaria remains the deadliest vector-borne disease in the world. With nearly half of the world’s population at risk, 216 million people suffered from malaria in 2016, with over 400,000 deaths, mainly in sub-Saharan Africa. Important global efforts have been made to eliminate malaria leading to significant reduction in malaria cases and mortality in Africa by 42% and 66%, respectively. Early diagnosis, improved drug therapies and better health infrastructure are key components, but this extraordinary success is mainly due the use of long-lasting insecticidal nets (LLINs) and indoor residual sprayings (IRS) of insecticide. Unfortunately, the emergence and spread of resistance in mosquito populations against insecticides is jeopardising the effectiveness of the most efficient malaria control interventions. To help establish suitable resistance management strategies, it is vital to better understand the distribution of resistance, its mechanisms and impact on effectiveness of control interventions and malaria transmission. In this chapter, we present the current status of insecticide resistance worldwide in main malaria vectors as well as its impact on malaria transmission, and discuss the molecular mechanisms and future perspectives

Multiple Insecticide Resistance in the Malaria Vector Anopheles funestus from Northern Cameroon Is Mediated by Metabolic Resistance Alongside Potential Target Site Insensitivity Mutations

Background Despite the recent progress in establishing the patterns of insecticide resistance in the major malaria vector Anopheles funestus, Central African populations of this species remain largely uncharacterised. To bridge this important gap and facilitate the implementation of suitable control strategies against this vector, we characterised the resistance patterns of An. funestus population from northern Cameroon. Methods and Findings Collection of indoor-resting female mosquitoes in Gounougou (northern Cameroon) in 2012 and 2015 revealed a predominance of An. funestus during dry season. WHO bioassays performed using F1 An. funestus revealed that the population was multiple resistant to several insecticide classes including pyrethroids (permethrin, deltamethrin, lambda-cyhalothrin and etofenprox), carbamates (bendiocarb) and organochlorines (DDT and dieldrin). However, a full susceptibility was observed against the organophosphate malathion. Bioassays performed with 2015 collection revealed that resistance against pyrethroids and DDT is increasing. PBO synergist assays revealed a significant recovery of susceptibility for all pyrethroids but less for DDT. Analysis of the polymorphism of a portion of the voltage-gated sodium channel gene (VGSC) revealed the absence of the L1014F/S kdr mutation but identified 3 novel amino acid changes I877L, V881L and A1007S. However, no association was established between VGSC polymorphism and pyrethroid/DDT resistance. The DDT resistant 119F-GSTe2 allele (52%) and the dieldrin resistant 296S-RDL allele (45%) were detected in Gounougou. Temporal analysis between 2006, 2012 and 2015 collections revealed that the 119F-GSTe2 allele was relatively stable whereas a significant decrease is observed for 296S-RDL allele. Conclusion This multiple resistance coupled with the temporal increased in resistance intensity highlights the need to take urgent measures to prolong the efficacy of current insecticide-based interventions against An. funestus in this African region

Rapid evolution of pyrethroid resistance prevalence in Anopheles gambiae populations from the cities of Douala and Yaoundé (Cameroon)

Background The adaptation of malaria vectors to urban areas is becoming a serious challenge for malaria control. The study presents the evolution of pyrethroid resistance in mosquito populations from the cities of Douala and Yaoundé between 2010 and 2013. Methods Susceptibility tests to permethrin and deltamethrin were carried out with two- to four-day old unfed Anopheles gambiae sensu lato adults raised from larvae collected from the field. Mosquitoes resistant to permethrin and deltamethrin and control were screened to detect the presence of the kdr alleles using the TaqMan assays. Mosquitoes belonging to the An. gambiae complex were subjected to PCR assays designed for species and molecular forms identifications. The genomic region containing the upstream of intron-1 of the voltage-gated sodium channel was sequenced and compared between mosquitoes originating from different breeding habitats. Results Anopheles gambiae s.l. specimens collected from the city of Douala were all Anopheles coluzzii. In Yaoundé, both An. gambiae and An. coluzzii were recorded. A rapid decrease of mosquito mortality to permethrin and deltamethrin was recorded between 2010 and 2013 in the two cities. The mortality rate varied from 80.3 to 22.3% and 94.4 to 59.7% for permethrin and deltamethrin, respectively. Both kdr alleles L1014F and L1014S were recorded. The frequency of kdr alleles increased rapidly over the study period, varying from 44 to 88.9% in Yaoundé and from 68 to 81% in Douala. The sequencing of a 1,228 bp region of intro-1 of the voltage-gated sodium channel revealed the presence of five different haplotypes. A high number of these haplotypes were recorded in An. coluzzii samples. No evidence for a recent selective sweep on intron-1 sequence within samples originating from different breeding habitat was detected using Fu’s and Tajima Fs statistics. Conclusion The present study supports rapid evolution of pyrethroid resistance in vector populations from the cities of Douala and Yaoundé and calls for immediate action to fight against the increasing prevalence of pyrethroid-resistant mosquitoes

Uncertainty-Aware and Lesion-Specific Image Synthesis in Multiple Sclerosis Magnetic Resonance Imaging: A Multicentric Validation Study

Generative adversarial networks (GANs) can synthesize high-contrast MRI from lower-contrast input. Targeted translation of parenchymal lesions in multiple sclerosis (MS), as well as visualization of model confidence further augment their utility, provided that the GAN generalizes reliably across different scanners. We here investigate the generalizability of a refined GAN for synthesizing high-contrast double inversion recovery (DIR) images and propose the use of uncertainty maps to further enhance its clinical utility and trustworthiness. A GAN was trained to synthesize DIR from input fluid-attenuated inversion recovery (FLAIR) and T1w of 50 MS patients (training data). In another 50 patients (test data), two blinded readers (R1 and R2) independently quantified lesions in synthetic DIR (synthDIR), acquired DIR (trueDIR) and FLAIR. Of the 50 test patients, 20 were acquired on the same scanner as training data (internal data), while 30 were scanned at different scanners with heterogeneous field strengths and protocols (external data). Lesion-to-Background ratios (LBR) for MS-lesions vs. normal appearing white matter, as well as image quality parameters were calculated. Uncertainty maps were generated to visualize model confidence. Significantly more MS-specific lesions were found in synthDIR compared to FLAIR (R1: 26.7 ± 2.6 vs. 22.5 ± 2.2 p &lt; 0.0001; R2: 22.8 ± 2.2 vs. 19.9 ± 2.0, p = 0.0005). While trueDIR remained superior to synthDIR in R1 [28.6 ± 2.9 vs. 26.7 ± 2.6 (p = 0.0021)], both sequences showed comparable lesion conspicuity in R2 [23.3 ± 2.4 vs. 22.8 ± 2.2 (p = 0.98)]. Importantly, improvements in lesion counts were similar in internal and external data. Measurements of LBR confirmed that lesion-focused GAN training significantly improved lesion conspicuity. The use of uncertainty maps furthermore helped discriminate between MS lesions and artifacts. In conclusion, this multicentric study confirms the external validity of a lesion-focused Deep-Learning tool aimed at MS imaging. When implemented, uncertainty maps are promising to increase the trustworthiness of synthetic MRI

Reduced performance of community bednets against pyrethroid-resistant Anopheles funestus and Anopheles gambiae, major malaria vectors in Cameroon

Background: Long-lasting insecticidal nets (LLINs) are a vital tool in the fight against malaria vectors. However, their efficacy in the field can be impacted by several factors, including patterns of usage, net age, mosquito resistance and the delayed mortality effect, all of which could influence malaria transmission. We have investigated the effectiveness of the various brands of LLINs available in markets and households in Cameroon on pyrethroid-resistant mosquitoes and assessed their post-exposure effect. Methods: Following quality control assessment on a susceptible laboratory mosquito strain, we evaluated the immediate and delayed mortality effects of exposure to LLINs (both newly bough LLINst and used ones collected from households in Elende village, Cameroon, in 2019) using standard WHO cone tests on Anopheles gambiae and Anopheles funestus populations collected from the Centre region of Cameroon. Alive female mosquitoes were genotyped for various resistance markers at different time points post-exposure to evaluate the impact of insecticide resistance on the efficacy of bednets. Results: The laboratory-susceptible strain experienced high mortality rates when exposed to all pyrethroid-only brands of purchased nets (Olyset® Net, Super Net, PermaNet® 2.0, Yorkool®, Royal Sentry®) (Mean±SEM: 68.66 ± 8.35% to 93.33 ± 2.90%). However, low mortality was observed among wild An. funestus mosquitoes exposed to the bednets (0 ± 0 to 28 ± 6.7%), indicating a reduced performance of these nets against field mosquitoes. Bednets collected from households also showed reduced efficacy on the laboratory strain (mortality: 19–66%), as well as displaying a significant loss of efficacy against the local wild strains (mortality: 0 ± 0% to 4 ± 2.6% for An. gambiae sensu lato and 0 ± 0% to 8 ± 3.2% for An. funestus). However, compared to the unexposed group, mosquitoes exposed to bednets showed a significantly reduced longevity, indicating that the efficacy of these nets was not completely lost. Mosquitoes with the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance showed greater longevity after exposure to the Olyset net than their susceptible counterparts, indicating the impact of resistance on bednet efficacy and delayed mortality. Conclusion: These findings show that although standard bednets drastically lose their efficacy against pyrethroid-resistant field mosquitoes, they still are able to induce delayed mortality in exposed populations. The results of this study also provide evidence of the actual impact of resistance on the quality and efficacy of LLINs in use in the community, with mosquitoes carrying the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance living longer than their susceptible counterparts. These results highlight the need to use new-generation nets that do not rely solely on pyrethroids

Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in Anopheles funestus across Africa

New insecticides have recently been produced to help control pyrethroid-resistant malaria vectors including the pyrrole, chlorfenapyr. Monitoring the susceptibility of mosquito populations against this new product and potential cross-resistance with current insecticides is vital for better resistance management. In this study, we assessed the resistance status of the major malaria vectors Anopheles gambiae and Anopheles funestus to chlorfenapyr across Africa and explored potential cross-resistance with known pyrethroid resistance markers. Efficacy of chlorfenapyr 100 µg/ml against An. gambiae and An. funestus from five Cameroonian locations, the Democratic Republic of Congo, Ghana, Uganda, and Malawi was assessed using CDC bottle assays. Synergist assays were performed with PBO (4%), DEM (8%) and DEF (0.25%) and several pyrethroid-resistant markers were genotyped in both species to assess potential cross-resistance between pyrethroids and chlorfenapyr. Resistance to chlorfenapyr was detected in An. gambiae populations from DRC (Kinshasa) (mortality rate: 64.3 ± 7.1%) Ghana (Obuasi) (65.9 ± 7.4%), Cameroon (Mangoum; 75.2 ± 7.7% and Nkolondom; 86.1 ± 7.4). In contrast, all An. funestus populations were fully susceptible. A negative association was observed between the L1014F-kdr mutation and chlorfenapyr resistance with a greater frequency of homozygote resistant mosquitoes among the dead mosquitoes after exposure compared to alive (OR 0.5; P = 0.02) whereas no association was found between GSTe2 (I114T in An. gambiae; L119F in An. funestus) and resistance to chlorfenapyr. A significant increase of mortality to chlorfenapyr 10 µg/ml was observed in An. funestus after to PBO, DEM and DEF whereas a trend for a decreased mortality was observed in An. gambiae after PBO pre-exposure. This study reveals a greater risk of chlorfenapyr resistance in An. gambiae populations than in An. funestus. However, the higher susceptibility in kdr-resistant mosquitoes points to higher efficacy of chlorfenapyr against the widespread kdr-based pyrethroid resistance

Increased prevalence of insecticide resistance in Anopheles coluzzii populations in the city of Yaoundé, Cameroon and influence on pyrethroid-only treated bed net efficacy

In Cameroon, pyrethroid-only long-lasting insecticidal nets (LLINs) are still largely used for malaria control. The present study assessed the efficacy of such LLINs against a multiple-resistant population of the major malaria vector, Anopheles coluzzii, in the city of Yaoundé via a cone bioassay and release-recapture experimental hut trial. Susceptibility of field mosquitoes in Yaoundé to pyrethroids, DDT, carbamates and organophosphate insecticides was investigated using World Health Organization (WHO) bioassay tube tests. Mechanisms of insecticide resistance were characterised molecularly. Efficacy of unwashed PermaNet 2.0 was evaluated against untreated control nets using a resistant colonised strain of An. coluzzii. Mortality, exophily and blood feeding inhibition were estimated. Field collected An. coluzzii displayed high resistance with mortality rates of 3.5% for propoxur (0.1%), 4.16% for DDT (4%), 26.9% for permethrin (0.75%), 50.8% for deltamethrin (0.05%), and 80% for bendiocarb (0.1%). High frequency of the 1014F west-Africa kdr allele was recorded in addition to the overexpression of several detoxification genes, such as Cyp6P3, Cyp6M2, Cyp9K1, Cyp6P4 Cyp6Z1 and GSTe2. A low mortality rate (23.2%) and high blood feeding inhibition rate (65%) were observed when resistant An. coluzzii were exposed to unwashed PermaNet 2.0 net compared to control untreated net (p < 0.001). Furthermore, low personal protection (52.4%) was observed with the resistant strain, indicating reduction of efficacy. The study highlights the loss of efficacy of pyrethroid-only nets against mosquitoes exhibiting high insecticide resistance and suggests a switch to new generation bed nets to improve control of malaria vector populations in Yaoundé