Improving onchocerciasis elimination surveillance: trials of odour baited Esperanza Window Traps to collect black fly vectors and real-time qPCR detection of Onchocerca volvulus in black fly pools

Improving onchocerciasis elimination surveillance: trials of odour baited Esperanza Window Traps to collect black fly vectors and real-time qPCR detection of Onchocerca volvulus in black fly pools | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improving onchocerciasis elimination surveillance: trials of odour baited Esperanza Window Traps to collect black fly vectors and real-time qPCR detection of Onchocerca volvulus in black fly pools M. A Adeleke, K.N Opara, H.B Mafuyai, B.E.B Nwoke, O. A Surakat, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4226239/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Entomological data for onchocerciasis surveillance relies on sampling blackflies through human landing collectors in the field and laboratory testing of the flies for infection using pool screening O-150 PCR-ELISA assay. Both techniques require improvements. This study sought to take promising Esperanza Window Traps (EWT) for blackfly collections and test and optimize them to find alternative carbon dioxide (CO 2 ) mimics, and to test new qPCR methods using mitochondrial DNA targets that have been suggested to improve sensitivity and specificity for Onchocerca volvulus infection in flies. Methods Traps baited with low, medium and high release rates of either 2-butanone or cyclopentanone as CO 2 mimics were field tested against traps baited with organically generated CO 2 in Guinea savannah, derived savannah, rainforest and montane forest ecological zones in Nigeria. The performance of EWTs baited with CO 2 or in combination with 2-butanone (low release) were subsequently evaluated against the human landing collection (HLC). Trap scaling was also pilot tested by comparing double traps to single HLCs. Collected blackflies were used to test detection of O. volvulus in blackflies using Ov ND5 real time PCR (qPCR) in comparison to the conventional pool screening O-150 PCR. Results EWTs baited with 2-butanone caught similar numbers of blackflies to those baited with CO 2 , while cyclopentanone collected significantly fewer flies in all locations. The low release of 2-butanone was the most effective overall, although HLCs collected higher numbers of blackflies than EWT baited with CO 2 either singly or in combination with low release 2-butanone. The combination of two EWTs baited with CO 2 and deployed 100 m apart to each other collected similar numbers of flies as one HLC. More blackfly pools were positive for O. volvulus by Ov ND5 qPCR as compared with O-150 PCR in derived savannah (31.15% vs 15.57%), montane forest (11.54% vs 0%) and rainforest (23.08% vs 2.56%), with only one positive pool in Guinea savannah detected by both methods. Conclusion 2-butanone has potential to be used in xenomonitoring as a standardized replacement to organically generated CO 2 . The positive pools found in foci hitherto considered to have interrupted/eliminated onchocerciasis highlights the need for more sensitive and specific methods that support programmatic assessments that can identify and combat recrudescence. Simulium 2-butanone cyclopentanone carbon dioxide Esperanza Window trap real time PCR onchocerciasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Human onchocerciasis (river blindness) is a debilitating disease of significant public health importance, primarily in Africa, which bears the brunt of the infection [1]. The disease is known for its characteristic skin pigmentation, itching and blindness depending on the strain of the parasite, Onchocerca volvulus , circulating in a transmission zone. The parasite is transmitted by blackflies of the genus Simulium , thus placing the insect as an indispensable indicator for real-time assessment of the transmission of the disease, and for monitoring the impact of control interventions in endemic foci [2]. The control, and now elimination of onchocerciasis has relied mostly on annual mass drug administration (MDA) of ivermectin with some complementary vector control strategies in a few countries [3]. It is expected that the long-term administration of ivermectin of between 12-15 years, with good coverage, has potential to interrupt the transmission of the disease to below the threshold at which recrudescence would be possible [1]. Thus, WHO guidelines place utmost emphasis on epidemiological and entomological impact assessment to determine if the drug could be safely stopped after long-term administration in the transmission zones. Entomological assessments rely on the field collection of blackflies through human landing collection (HLC) technique and subsequent laboratory detection of O. volvulus using O-150 PCR-ELISA technique on pools of the heads of the flies [4]. However, several issues have been raised about these two techniques: the ethical appropriateness and cost of using humans as bait for blackfly collection [5], and the sensitivity of the O-150 PCR-ELISA in detecting the true positive samples in pools of blackfly heads [6]. One of several efforts at developing alternative trapping technique to replace HLC is the Esperanza Window Trap (EWT) [5]. These demonstrated great potential and reasonable logistical advantage for the collection of host-seeking African species of blackflies [7]. It was suggested that one person can monitor multiple traps at a catching site and EWTs can be widely deployed for fly collection in the endemic communities [2]. However, the EWTs need further optimization for consistent performance, which can vary significantly across geographic zones as earlier reported [8,9]. Previous studies have elaborated on the role of CO 2 as an activating attractant to blackflies [10, 11]. Simplifying and standardizing the source of CO 2 to bait the EWT is one of the key improvements recommended for further optimization [8]. The EWT is usually baited with CO 2 generated by yeast fermentation [5], however the type of sugar, yeast strain and environmental conditions affect the production rate of organically generated CO 2 under field conditions [8]. A recent study on another haematophagous Dipteran revealed that traps baited singly with granules of cyclopentanone or in combination with other attractant blends collected more Anopheles arabiensis than those baited with yeast fermentation-produced CO 2 [12].Similar studies by Mburu et al. (2017) [13] reported significant attraction of An. gambiae sensu lato to 2-butanone compared to industrially supplied CO 2 . The potential application to collection of anthropophilic blackflies has not yet been explored. Once collected, blackfly samples must be screened in the laboratory to determine whether they harbour infective (L3-stage) larvae. The current WHO-endorsed molecular technique targets the O-150 repeat for amplification [14], but the reagents require a reliable cold chain which cannot always be guaranteed during shipment or where electrical power supplies are inconsistent. A recently developed real-time qPCR method using Ov ND5, a mitochondrial DNA target, uses heat-stable reagents and is cheaper and faster to complete than the O-150, while also demonstrating increased sensitivity and specificity in initial comparisons [15]. This warrants further field evaluation to determine its suitability for use in programmatic settings. It is against this background of operational roadblocks to verifying onchocerciasis elimination that the present study sought to: (i) evaluate and compare the efficiency of different release rates of cyclopentanone and 2-butanone as attractants for blackfly vectors of onchocerciasis, (ii) evaluate the synergistic potential of EWTs baited with a combination of CO 2 mimic and yeast-fermentation generated CO 2 compared to HLCs, and (iii) evaluate the detection of O. volvulus in head pools of the collected blackflies using O-150 PCR-ELISA versus Ov ND5 qPCR assays. Research took place in four ecological zones in Nigeria, all endemic for onchocerciasis, where there is a diversity of entomological and epidemiological conditions against which to test these new approaches. Methodology a) Study area Four onchocerciasis endemic locations representing four ecological zones with varying Simulium cytospecies present were selected based on previous studies in Nigeria [16-20]. At each of the selected locations, two trap sites (at least 6-10 km apart) were established along the major river at the location for trap trials. The selected locations are described in Figure 1 and Table 1. Field experiments were conducted between September and October 2023. Table 1: Study location characteristics Study Locations/ States Ecological region Latitude/Longitude of trap sites Previously identified blackfly species Onchocerciasis Elimination status Ungwan Habu, Nasarawa State Guinea savannah Trap site 1: 8.944399N, 8.242172E Trap site 2: 8.996879N, 8.256494E S. damnosum s.s. S. sirbanum Interrupted [31,32] Kwa Falls and Ekong Anaku, Cross River State Montane Forest Trap site 1: 5.105748N, 8.630302E Trap site 2: 5.141645N, 8.508362E S. yahense S. squamosum On track to elimination [32] Ike-Isu, Abia State Rain forest Trap site 1: 5.41310N, 7.95398E Trap site 2: 5.416057N, 7.954063E S. squamosum Interrupted [32] Ileogbo, Osun State Transition zone (Forest/derived savannah) Trap site 1: 7.580873N, 4.301190E Trap site 2: 7.587072N, 4.289398E S. soubrense beffa S. damnosum s.s. On track to elimination [32] b) Field trials to improve blackfly catches in EWTs The field trials were divided into three experiments for optimization of EWTs. Experiment 1 and 2 concerned enhancing the olfactory attractivity of EWTs through testing CO 2 mimics and augmenting organically produced CO 2 , and Experiment 2 tested the potential to increase overall blackfly yield through increasing EWT sampling effort. Carbon dioxide and its mimics used in Experiment 1 and 2 were produced as follows. The 2-butanone and cyclopentanone were procured from Chemitica Inc, Costa Rica. Each of the two CO 2 mimics was prepared in semipermeable membrane sachets in three slow-release modes, all of which were formulated for release at temperatures of 20-30 o C as follows: low release (~1-2 mg/day), medium release (~5-10 mg/day), and high release (~45-55 mg/day). The slow release is to enable the blend to mimic natural concentrations [21], last longer and not to be repellent. Most of the semiochemicals used for attracting insect vectors are slow release and can attract vectors over 30 -100 metres away [21]. The organically generated CO 2 was prepared on alternate days (every two days) by mixing 50 g of dry baker yeast, 500 g of sugar and 2.5 litres of water in a 5-litre plastic bottle at least 2 hours before the commencement of trapping as earlier described by [8]. Experiment 1: Identification of optimum CO 2 mimic release rates Two trapping sites, at least 5 km apart and known for the breeding of blackflies, were selected along the river in each of the four study locations (Table 1). Of these, 2-butanone was tested at the first trapping site (Trap site 1), while cyclopentanone was tested at the second (Trap site 2). At each trap site, four points (25 m apart) were selected for the placement of traps: the EWTs comprised a tarpaulin (1 m x 1 m) with blue-black-blue stripes of equal size hung on an upright stand of poles tensioned with string and pegs and suspended about 10 cm above the ground. The two sides of the tarpaulin were coated with TAD All weather (adhesive) (Ladd Research Industries, USA) and baited with a pair of worn socks [7, 8]. The EWTs at the four points were also baited with either low, medium or high release rate CO 2 mimic or organically generated CO 2 (Figures 2 and 3). The traps were set up perpendicular to the breeding site in fairly shaded conditions. The CO 2 or mimic allocated to each trap was changed daily in a randomized Latin square experimental design over a period of 12 days. Flies were recovered from the traps twice daily (12 noon and 5 pm), preserved with 80% ethanol in 30 ml bottles and kept at 4°C until later use. Experiment 2: Optimization of EWTs through augmented odours relative to HLC This experiment was designed to determine whether the number of flies attracted to organically generated CO 2 could be increased through the addition of a CO 2 mimic. For 8 consecutive days at each study location, catches from an EWT baited with the standard organically generated CO 2 were compared with an HLC positioned 25 m away from the trap. The HLC involved two adults collecting blackflies on hourly rotation between 7 am and 5 pm daily in accordance with the standard procedure [22, 8, 9]. The positions of the HLC and EWT were rotated daily in a cross-over design. On the nineth day, the organically generated carbon dioxide at the EWT was augmented with the best CO 2 mimic from Phase 1, identified as 2-butanone at a low release rate, and continued to be compared with the HLC in a cross-over design until day 16. The daily catches were recovered from the traps as described in Phase 1. Experiment 3: Trap scaling pilot As trapping approaches offer the advantage of allowing multiple traps to be operated by a single field entomologist, but the disadvantage of collecting fewer flies, the aim of this experiment was to establish in principle whether doubling the number of EWTs baited with organically generated CO 2 would increase total trap catch relative to an HLC. This phase was conducted at Ileogbo, the derived savannah zone in Osun State only. Two EWTs were placed 100 m apart from each other and from the HLC position. The trial was conducted over 9 days and positions were rotated daily, following the collection and sampling methods described above. The number of flies collected daily by the two traps were added together and compared with the HLC catches. d) Comparison of fly infectivity in pools by O-150 PCR ELISA versus Ov ND5 real-time qPCR All the flies collected in the methods described above were identified morphologically for confirmation of female S. damnosum complex using the standard keys (WHO, 2002). The heads of the flies were separated from the body using entomological pins under the dissecting microscope (Biobase). The batches of the pins used were washed with sodium hypochlorite and sterilized before being re-used to minimize contamination. The heads were arranged in 100 heads per pool and each pool was transferred to a sterilized Eppendorf tube for DNA extraction. The extraction of the DNA was done using Qiagen DNeasy Blood & Tissue Kits (Qiagen, Germany) following the manufacturer’s instruction. The O-150 PCR and Ov ND5 gene real-time qPCR assays for the detection of O. volvulus in the pools were performed as previously described by [2, 23]. e) Statistical analysis The data on blackfly count from the various collection methods were subjected to a generalised linear mixed model (GLMM) with a negative binomial distribution and counts were compared across release rates. Means were then separated using a post-hoc Tukey multiple comparison. A generalised linear model (GLM) was used to compare the means of daily catches between EWT and HLC. The prevalence of infection in the pool screening of fly heads was determined using Poolscreen v2.0 software [24]. All other analysis was performed using statistical package R-4.3.0 [25]. Results Field trials to improve blackfly catches in EWTs Experiment 1: Identification of optimum CO 2 mimic release rates The results obtained from the optimization of the 2-butanone and cyclopentanone against organically generated CO 2 in the four study locations are presented in Fig. 4 . In three of the four study areas, all release rates of 2-butanone performed well in terms of mean daily fly catch relative to the organically produced CO 2 . In the derived savannah (Fig. 4 b), the mean catches of low release (35.58 ± 19.660), medium release (35.41 ± 10.542) and high release (22.16 ± 7.797) rates of 2-butanone were not significantly different to the mean catch by EWT baited with organically generated CO 2 (35.75 ± 9.788) (p = 0.50). Likewise, in montane forest, mean catches from the low (6.08 ± 2.09), medium (4.75 ± 2.09)and high (6.33 ± 2.38) release rates did not differ significantly compared to that from organic CO 2 (8.33 ± 2.87) (p = 0.833) and this was also the case in the rain forest ecoregion, where low (25.58 ± 9.44), medium (24.16 ± 10.63) and high (16.50 ± 5.23) release rates did not yield significantly more blackflies per day than organic CO 2 (32.91 ± 8.40); p = 0.409). However, in Guinea savannah, organically produced CO 2 caught significantly more flies per day (7.16 ± 1.05) than any of the 2-butanone-baited traps (low 3.25 ± 0.67, p = 0.046; medium 3.75 ± 1.08, p = 0.049; high 3.41 ± 0.54, p = 0.048). In contrast, cyclopentanone performed poorly across all ecoregions. The EWT baited with organic CO 2 in derived savannah zone collected a significantly higher mean number of blackflies per day (81.40 ± 36.987,) than EWT baited with cyclopentanone for low release (34.00 ± 12.677; p = 0.047), medium release (28.40 ± 8.704; p = 0.049) and high release (19.20 ± 5.774; p = 0.048). Similar results were obtained in Rainforest (organic CO 2 : 34.25 ± 9.99; Low release: 8.25 ± 2.98, p = 0.048; Medium release: 8.83 ± 3.26, p = 0.048; High release: 12.16 ± 8.22, p = 0.046); Derived Savannah (organic CO 2 : 11.80 ± 4.71; Low release: 2.66 ± 1.40, p = 0.046; Medium release: 5.00 ± 1.09, p = 0.049; High release: 4.00 ± 1.00, p = 0.048); Montane forest (organic CO 2 : 9.08 ± 1.47; Low release: 0.50 ± 0.23, p = 0.000; Medium release: 0.83 ± 0.32, p = 0.002; High release: 0.33 ± 0.28 p = 0.000). Experiment 2: Optimization of EWTs through augmented odours relative to HLC HLC collected a significantly higher number of blackflies per day than EWTs baited with organically generated CO 2 across the locations (p < 0.05). In location I in the four ecozones for example, the mean catches by HLC versus EWT were given as derived savannah = 244.11 ± 22.063 vs 81.000 ± 9.402; rainforest = 58.889 ± 10.671 vs 2.556 ± 1.107; montane forest = 85.667 ± 15.304 vs 9.667 vs ± 0.782; Guinea savannah = 40.778 ± 5.746 vs 8.444 ± 1.591 (Fig. 5 ). The EWTs baited with the combination of 2-butanone (low release) and organically generated CO 2 also collected significantly lower number of blackflies than HLC across the study locations (p < 0.05) (Figs. 5 and 6 ). Experiment 3: Trap scaling pilot The combination of two EWTs (baited with organic CO 2 and deployed 100 m apart to each other and to HLC) collected slightly higher number of flies than the HLC. The pooled mean catch of both traps was 94.011 ± 5.97, which was very similar to the HLC catch of 91.80 ± 9.86, a difference that was not statistically significant (p = 0.788). (Fig. 7 ). Comparison of fly infectivity in pools by O-150 PCR ELISA versus Ov ND5 real-time qPCR The results of the molecular analyses of blackfly infectivity showed that all the study locations had at least one positive pool by both O-150 PCR and Ov ND5 qPCR, with the exception of montane forest, which was positive only by Ov ND5 qPCR (Table 3). More positive pools were detected by Ov ND5 qPCR compared to O-150 PCR in three of the four ecoregions; in Guinea savannah (31.15% vs 15.57%), montane forest (11.54% vs 0%) and rainforest (23.08% vs 2.56%). Only one pool was positive by both Ov ND5 qPCR and O-150 PCR in Guinea savannah. All pools that were positive by O-150 PCR were also positive by Ov ND5 qPCR. The upper limit prevalence was exceedingly higher than 0.05% in all the four locations using both diagnostic methods except at montane forest which has a lower value for O-150 PCR (Table 3). Table 2 Prevalence of O. volvulus in blackflies at four study locations in Nigeria Ecoregions (States) No of pools No of pools positive by O-150 PCR (%) No of pools positive by Ov ND5 qPCR (%) Prevalence by O-150 PCR (95% confidence interval) Prevalence by Ov ND5 qPCR (95% confidence interval) Derived Savannah (Osun) 122 19 (15.57) 38 (31.15) 0.16 (0.1–0.26) 0.37 (0.29–0.51) Montane Forest (Cross River) 78 0 (0) 9 (11.54) 0.00 (0.00) 0.12 (0.06–0.22) Rainforest (Abia) 39 1 (2.56) 9 (23.08) 0.03 (0.00-0.14) 0.26 (0.14–0.48) Guinea savannah (Nasarawa) 06 1 (16.67) 1 (16.67) 0.18 (0.03–0.82) 0.18 (0.03–0.82) Discussion Our study provides important findings for both vector collection and parasite detection within the context of the WHO’s onchocerciasis elimination framework. Our key results indicate that alternative odour baits and using multiple traps could play a role in securing the large number of blackfly vector samples required for infection screening without relying on human collectors, and that the Ov ND5 qPCR technique is a more sensitive method for detecting parasite DNA in these samples than the currently used O-150 method. Carbon dioxide serves as powerful attractant to haematophagous insects, and it has been widely used for traps to lure host-seeking insect vectors [ 10, 11, 13, 26]. In a search to improve the efficiency and easy deployment of EWTs, our field evaluation of various release rates of 2-butanone and cyclopentanone showed that the catches of EWTs baited with 2-butanone were not statistically different to those baited with organic CO 2 across the study locations except at Guinea savannah, where significantly lower catches were recorded for 2-butanone; the reason for this is not clear, however it may be species-specific as unlike other study areas, this Guinea savannah location was dominated by S. damnosum s.s. and S. sirbanum . The lowest release rate of 1–2 mg/day was sufficient to catch similar numbers of blackflies as the medium and high release rates tested. Turner et al. (2011) [ 27 ] reported dose-dependent activation of receptor neurons in the maxillary palps of mosquitoes to 2-butanone; this has not been explored in blackflies and our field results did not indicate a dose effect, so far as the release rate did not significantly affect trap capture rates. 2-butanone is a known natural product emanated by various vertebrates with potential to serve as a long-range attractant to host-seeking insects mimicking the CO 2 produced by vertebrate hosts, including humans [ 13 ]. These properties could have accounted for its effectiveness in collecting flies almost at par with EWTs baited with organically generated CO 2 but not exceeding these. Fermentation of sugar by yeast has been reported to produce not only CO 2 , but other volatile organic compounds, including alcohols, esters, aldehydes and fatty acids, that also attract arthropods [ 5 , 28 ] and may explain its slightly greater success as a lure. However, EWTs baited with cyclopentanone collected significantly lower number of blackflies than those baited with organically generated CO 2 across the four locations. Cyclopentanone is a monoketone which has been reported to activate the cpA CO 2 receptor neuron on the maxillary palp of mosquitoes, and thus serve as a suitable attractant to mosquitoes in semi-field trials [ 29 ]. Subsequent field trials reported poor attraction of the chemical to mosquitoes [ 30 ]. Two reasons were advanced to explain the poor performance of cyclopentanone in the field: i) the diffusion of odour plumes of the chemical due to environmental factors such as relative humidity, temperature and wind and ii) competing chemicals emanating from the surrounding vegetation, detritus and air pollution in the field. The reason may be explained by the fact that cyclopentanone is a monoketone obtainable in plants, and has been used widely in pharmaceuticals and industrial processes including in rubber chemicals [ 26 , 30 ]. Considering the typical ecological habitat of blackflies, which is usually populated with shrubs and vegetation, the competing odour of this chemical from the surrounding vegetation is inevitable. The EWTs baited with organic CO 2 collected significantly lower number of blackflies to HLC across the four ecological zones. Similar observations have also been reported by Hendy et al. (2017) [ 8 ] in Tanzania and Talom et al. (2021) [ 9 ] in Cameroon. In contrast, Toe et al. (2014) [ 7 ] in Burkina Faso and Hendy et al. (2017) [ 8 ] in Uganda reported statistically similar number of flies collection between EWTs and HLC. The reasons for differential trap performance are not clear. In the present study, augmentation of organic CO 2 with low release rates of 2-butanone did not show any additional benefit in terms of catch, whereas in a field study conducted against Anopheles mosquitoes [ 13 ], augmentation of CO 2 sources with 2-butanone significantly improved the catches of the mosquitoes. Interestingly, when two EWTs baited with organic CO 2 were used at a site in the derived savannah, collectively the two traps caught slightly higher numbers of blackflies than two human landing collectors working rotationally on hourly basis. This result may be due to the fact that all the collection methods were well spread out, with approximately 100 m between EWTs and between EWTs and the HLC. The strong selection pressure on blackflies to respond to human-specific cues, and thus the highly attractive profile of human collections, may therefore be outcompeting alternative collection methods that only present partial and artificial attractive cues. This result needs further investigation as it may be crucial for realizing the potential of trapping paradigms in operational contexts. Rodriguez-Perez et al. (2014) [ 2 ] demonstrated the field feasibility of deployment of multiple EWTs for the collection of the required numbers of flies to meet WHO guidelines requirement and our study reinforces this observation, and rigorous investigation in areas with different biting rates and cytospecies should be included as part of future studies. The higher number of blackfly head pools positive for Ov ND5 qPCR as compared with O-150 PCR ELISA showed that the former is more sensitive than the latter. Notably, all the samples positive for O-150 PCR were also positive for Ov ND5 qPCR but the qPCR detected 36 more positive pools that had been scored negative by O-150 PCR. This result is in tandem with the recent studies commissioned by WHO [ 6 ] and published laboratory reports [ 15 ] on the high sensitivity of Ov ND5 qPCR over O-150 qPCR and O-150 PCR ELISA. Importantly, the Ov ND5 qPCR protocol is also faster, more cost effective and easier to use, requiring fewer reagents than the O-150 PCR ELISA. Alongside the growing number of similar reports, our observation has serious implications for the on-going global efforts in onchocerciasis elimination, as accurate detection of infected flies is crucial to informing safe stop-MDA decisions and robust post-elimination surveillance. Flies serve as a link between parasite and host, and they give a real-time indication on whether or not transmission has been interrupted/eliminated in a community [ 5 ]. Erroneous stoppage of MDA in areas with low but existing residual transmission could bring early recrudescence and jeopardize the gains achieved over many years of MDA. Further optimization of qPCR for wide implementation for impact assessment and post-elimination surveillance must be vigorously pursued if the global effort to accelerate towards eliminating onchocerciasis in endemic communities is to be achieved by the year 2030. Notwithstanding the different diagnostic methods used, the results of the pool screening analysis in the four study zones are of significant interest to onchocerciasis elimination efforts in Nigeria. The results obtained in derived savannah (Osun State) and the montane forest (Cross River) showed that there is ongoing transmission in these foci; both zones remain under annual ivermectin coverage, which our results confirm is still required. There is need by the National Onchocerciasis Elimination Programme (NOEP) to aggressively improve the therapeutic coverage in the communities, as this entomological evidence suggests the presence of people harbouring active adult parasite worms at the study locations. However, the observation of positive pools in rainforest (Abia State) and Guinea savannah (Nasarawa State) is a surprise overlaid with serious implications for the ongoing efforts at eliminating onchocerciasis in Nigeria. According to the NOEP [ 31 ] and the available literature [ 32 , 33 ], Nasarawa State is one of the two states where onchocerciasis has been eliminated in Nigeria while Abia State has successfully interrupted the transmission of the disease. The occurrence of the positive pools at these two zones could be attributed to two possibilities: i) that O-150 was not sensitive enough to detect the residual flies carrying infection after the MDA has suppressed active transmission since our study sites were among the communities where the Stop-MDA and Post Stop-MDA evaluations were conducted, or ii) the human migration caused by insecurity in Nasarawa and Abia States in the last six years has introduced sufficient numbers of infected people to the area to cause reinfection in the vector population. Irrespective of driving factors, the pool screening results have shown the possibility of a gradual build-up of recrudescence which requires urgent programmatic evaluation across Nasarawa and Abia States to determine the extent of the spread and instigate implementation strategies to combat it before it erodes the success already recorded in these transmission zones in Nigeria. To the best of our knowledge and available reports, this is the first report of direct comparison of O-150 PCR and Ov ND5 qPCR for the detection of blackfly infectivity in areas with ongoing transmission and in foci where onchocerciasis has been deemed interrupted/eliminated in West Africa. Conclusion In conclusion, our results suggested that 2-butanone was effective as a CO 2 mimic and collected comparable number of S. damnosum s.l. as those attracted by organically generated CO 2 in three out of four selected ecological zones in Nigeria. However, the EWTs baited with cyclopentanone collected significantly fewer flies as compared with those baited with organic CO 2 plausibly suggesting its poor attraction to blackflies in the field. Despite the fact that EWTs baited with organic CO 2 alone and those augmented with 2-butanone at the optimum low release rate collected significantly fewer blackflies as compared with HLC catches, it was found that two EWTs strategically deployed at 100 m apart and to the HLC collected more blackflies than the two human collectors working on hourly rotation. This observation is an indication that EWTs (baited with appropriate odours/lures), if further optimized and strategically deployed with adequate space between competing traps at breeding sites, have potential for replacing HLC for monitoring and surveillance of blackfly vectors in programmatic contexts of onchocerciasis elimination assessment. We observed that Ov ND5 qPCR was more sensitive than O-150 PCR ELISA in detecting infected flies in blackfly head pools and would be a useful tool for accurate entomological assessment for stop-MDA in the context of elimination and circumvention of recrudescence due to poor sensitivity of diagnosis tools. Whether the positive pools found in the foci hitherto thought to have interrupted/eliminated onchocerciasis are caused by poor sensitivity of O-150 PCR ELISA or due to changing epidemiological factors, such as human migration due to insecurity, require further programmatic evaluation by the NOEP in Nigeria. Declarations Acknowledgements The authors thank the NTDs state coordinators, field Entomologists, community members and study participants in the four ecological zones selected for this study. We thank Mr Samson Olumakinde, Oluokun Timileyin Emmanuel and Olaniyan Oluwaseyi for their help in the laboratory. We appreciate Chemitica Inc, Costa Rica for the supply of slow release sachets of 2-butanone and cyclopentanone. Authors contribution : MAA, KNO, BEBN, HBM, OAS conceived and wrote the protocol for the study, FMH reviewed the protocol, MAA, KNO, BEBN, HBM, OAS, CF, YC, MUG, IM, ME, OF, AC, TJD performed the field work, MAA, SBA, IZO performed the molecular analysis, MAA, KNO, BEBN, HBM, OAS drafted the manuscript, all authors reviewed and contributed to the final version of the manuscript. Funding: This project was funded by Bill and Melinda Gates Foundation (INV-047737) but the funder had no influence on the outcomes of the study. Availability of data and materials : The data set for this research is available upon reasonable request from the corresponding author. Ethics approval and consent to participate The study protocol was approved by the National Health Research Ethical Committee of the Federal Ministry of Health, Nigeria (NHREC/01/01/2007-13/03/2023). The Neglected Tropical Diseases Coordinators and the community leaders in the four selected locations were appropriately mobilized for the study. Written informed consent was also obtained from the community leaders and the locally appointed vector collectors after the objectives, benefits and risks had been duly explained. Consent for publication Not Applicable Competing Interest The authors declare no competing interest. Author details 1 Department of Zoology, Osun State University, Osogbo, Nigeria, 2 Department of Animal and Environmental Biology, University of Uyo, Nigeria, 3 Department of Zoology, University of Jos, Nigeria, 4 Department of Animal and Environmental Biology, Imo State University, Owerri Nigeria, 5 Department of Microbiology, Osun State University, Osogbo, Nigeria, 6 Federal Ministry of Health, Nigeria, 7 Department of Biological Science, Nasarawa State University, Lafia, 8 Agriculture, Health & Environment Department, Natural Resources Institute, University of Greenwich, United Kingdom References WHO. Guidelines for stopping Mass Drug Administration and verifying Elimination of Human Onchocerciasis: Criteria and Procedures. WHO/HTM/NTD/PCT/2016. Geneva: WHO; 2016. Rodríguez-Pérez MA, Adeleke MA, Rodrıguez-Luna IC, Cupp EW, Unnasch TR. Evaluation of a Community-Based Trapping Program to Collect Simulium ochraceum sensu lato for Verification of Onchocerciasis Elimination. 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Evaluation of 1-octen-3-ol and carbon dioxide as black fly (Diptera: Simuliidae) attractants in Arkansas. J Am Mosq Control Assoc. 1993;9(2):143-6. PMID: 8350068. Sutcliffe JF. Black fly interactions with their hosts. In: Takken W, Knols B, editors. Ecology and control of vector-borne diseases. Vol 2, Olfaction in vector-host interactions. Netherlands: Wageningen Academic Publishers; 2010. p. 438. Kessy ST, Nyundo BA, Mnyone LL, Lyimo IN. The Use of Granular Cyclopentanone as Alternative to Artificial Source of Carbon Dioxide in Improved Passive Outdoor Host Seeking Device (POHD). The Scientific World Journal. 2020;2020:7620389. DOI: 10.1155/2020/7620389. Mburu MM, Mweresa CK, Omusula P, Hiscox A, Takken W, Mukabana WR. 2-Butanone as a carbon dioxide mimic in attractant blends for the Afrotropical malaria mosquitoes Anopheles gambiae and Anopheles funestus . Malaria Journal. 2017;16(1):351. DOI: 10.1186/s12936-017-1998-2. Meredith SEO, Lando G, Gbakima AA, Zimmerman PA, Unnasch TR. Onchocerca volvulus : Application of the polymerase chain reaction to identification and strain differentiation of the parasite. Exp Parasitol. 1991 Oct 1;73(3):335–44. PMID: 1915748. Doherty M, Grant JR, Pilotte N, Bennuru S, Fischer K, Fischer PU, Lustigman S, Nutman TB, Pfarr K, Hoerauf A, Unnasch TR, Hassan HK, Wanji S, Lammie PJ, Ottesen E, Mackenzie C, Williams SA. Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors. PLoS Negl Trop Dis. 2023 Dec. Mafuyai HB, Post RJ, Vajime CG, Molyneux DH. Cytotaxonomic identifications of the Simulium damnosum complex (Diptera: Simuliidae) from Nigeria. Tropical Medicine & International Health. 1996;1:779-785. DOI: 10.1111/j.1365-3156.1996.tb00110.x Adeleke, M.A ., Mafiana, C.F., Sam-Wobo, S.O., Akinwale, O.P., Olatunde, G.O Sanfo, M., Adjami, A., Toe, L.. Molecular characterization of Simuliumdamnosum Theobald complex (Diptera: Simuliidae) found along Osun River system, Southwestern Nigeria. Annals of Tropical Medicine and Parasitology 2010, 104 (8): 679-683 Tongjura JDC, Amuga GA, Mafuyai HB, Matur MB, Olatunwa JO. Influence of Some Water Physicochemical Parameters on the Distribution of Black Fly (Diptera, Simuliidae) in Some Rivers in Nasarawa State, Nigeria. Advanced Entomology. 2015;3(3):101-111. Oforka LC, Adeleke MA, Anikwe JC, Hardy N, Mathias D, Makanjuola W, Fadamiro H. Poor genetic differentiation but clear cytoform divergence among cryptic species in Simulium damnosum complex (Diptera: Simuliidae). Systematic Entomology. 2017;43(1):123-135. Chkieze FM, Opara KN, Ubulumen PME, Osei-Atweneboana MY. Simulium vectors of the lower cross River Basin: A morphometric and cytotaxonomic identification. International Journal of Entomology Research. 2022;7(2):72-80. Mweresa CK, Mukabana WR, van Loon JJA, Dikke M, Takken W. Use of semiochemicals for surveillance and control of hematophagous insects. Chemoecology. 2020;30:277–286. DOI: 10.1007/s00049-020-00317-1. Adeleke MA, Mafiana CF, Sam-Wobo, Olatunde GO, Ekpo UF, Akinwale OP, Toe L. Biting behaviour of Simulium damnosum Theobald complex and Onchocerca volvulus infection along the Osun River System, Southwest Nigeria. Parasite and Vector. 2010;3(93):1-5. DOI: 10.1186/1756-3305-3-93 Hendy A, Krüger A, Pfarr K, De Witte J, Kibweja A, Mwingira U, Dujardin JC, Post R, Colebunders R, O'Neill S, Kalinga A. The blackfly vectors and transmission of Onchocerca volvulus in Mahenge, southeastern Tanzania. Acta Trop. 2018, 181:50-59. doi: 10.1016/j.actatropica.2018.01.009. Epub 2018 Feb 2. PMID: 29410302 Katholi C. Poolscreen v2.0. Available from: http://www.soph.uab.edu/bst/poolscreen. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2021. Available from: https://www.R-project.org/. Thompson BH. Studies on the attraction of Simulium damnosum s.l. (Diptera: Simuliidae) to its hosts. I. The relative importance of sight, exhaled breath, and smell. Tropenmed Parasitol. 1976;27(4):455-473. Turner SL, Li N, Guda T, Githure J, Cardé RT, Ray A. Ultra-prolonged activation of CO2-sensing neurons disorients mosquitoes. Nature. 2011;4:87–91. Scognamiglio J, Jones L, Letizia CS, Api AM. Fragrance material review on cyclopentanone. Food Chem Toxicol. 2012;50:S608–S612. Tauxe GM, MacWilliam D, Boyle SM, Guda T, Ray A. Targeting a dual detector of skin and CO 2 to modify mosquito host seeking. Cell. 2013;155:1365–1379. Philippe-Janon JC, van den Hurk AF, Francis DP, Shivas MA, Jansen CC. Field Comparison of Cyclopentanone Versus Carbon Dioxide as an Attractant for Adult Mosquitoes in Southeast Queensland, Australia. J Med Entomol. 2015;52(3):483-490. DOI: 10.1093/jme/tjv011. National Onchocerciasis Elimination Committee (NOEC). Minutes of the 15th National Onchocerciasis Elimination Committee Meeting, May 2022. Federal Ministry of Health, Abuja, Nigeria. Available from: https://www.cartercenter.org/resources/pdfs/news/health_publications/river_blindness/nigeria-onchocerciasis-elimination-committee-communique-13.pdf. Miri ES, Eigege A, Kahansim B, Nwodu K, Sambo Y, Mancha B, Adelamo S, Umaru J, Kadimbo J, Danboyi J, Mafuyai H, Makata E, Akpan N, Akilah J, Igbe M, Coalson J, Rakers L, Griswold E, Unnasch TR, Nwoke BEB, Noland GS, Richards FO. Two Nigerian States (Plateau and Nasarawa) Have Eliminated Transmission of Human Onchocerciasis-A Report of Post-Ivermectin Mass Drug Administration Surveillance. Am J Trop Med Hyg. 2022;108(1):37-40. DOI: 10.4269/ajtmh.22-0491. Nwoke BEB. Nigerian Onchocerciasis: from Control to Elimination. African Health. 2023;19-21. Available from: https://africa-health.com/wp-content/uploads/2023/05/19-Onchocerciasis.pdf. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4226239","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288994715,"identity":"5ad90d3a-9c61-43da-872b-1de04c82145d","order_by":0,"name":"M. 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Different letters denote significant differences at p\u0026lt;0.05 (GLM).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4226239/v1/a79bc4294a527654a208b6a6.png"},{"id":54343239,"identity":"3139ee6a-b1a3-43de-b821-07ae75d9c485","added_by":"auto","created_at":"2024-04-09 06:09:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49067,"visible":true,"origin":"","legend":"\u003cp\u003eMean daily blackfly catches (± standard error) by EWTs baited with organic CO\u003csub\u003e2\u003c/sub\u003e versus human landing collection in four different ecoregions of Nigeria. Different letters denote significant differences at p\u0026lt;0.05 (GLM).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4226239/v1/67c31368e2b40fba343bf93c.png"},{"id":54343231,"identity":"d0599797-9e54-46ff-860a-0878b7481c42","added_by":"auto","created_at":"2024-04-09 06:09:42","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39409,"visible":true,"origin":"","legend":"\u003cp\u003eMean daily blackfly catches (± standard error) by EWTs (baited with combination of 2-butanone low release rate plus organic CO\u003csub\u003e2\u003c/sub\u003e) versus human landing collection in four different ecoregions of Nigeria. Different letters denote significant differences at p\u0026lt;0.05 (GLM).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4226239/v1/9c15070e72070f7a98f7342a.png"},{"id":54343235,"identity":"20a68c71-7054-4ba7-8582-5efdc3e8b65c","added_by":"auto","created_at":"2024-04-09 06:09:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":11354,"visible":true,"origin":"","legend":"\u003cp\u003eMean daily blackfly catches (± standard error) by two EWTs (baited with organic CO\u003csub\u003e2\u003c/sub\u003e 100 m apart) versus human landing collection (100 m apart to the trap) at Ileogbo in Osun State, Nigeria, a location in derived savannah. Different letters denote significant differences at p\u0026lt;0.05 (GLM).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4226239/v1/257973ff3747938bef00a7db.png"},{"id":54343586,"identity":"31e4b487-b01c-40f5-9006-3739fab744f2","added_by":"auto","created_at":"2024-04-09 06:17:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1291366,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4226239/v1/d43291af-cf44-4f49-8211-5277469a026a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Improving onchocerciasis elimination surveillance: trials of odour baited Esperanza Window Traps to collect black fly vectors and real-time qPCR detection of Onchocerca volvulus in black fly pools","fulltext":[{"header":"Background","content":"\u003cp\u003eHuman onchocerciasis (river blindness) is a debilitating disease of significant public health importance, primarily in Africa, which bears the brunt of the infection [1]. The disease is known for its characteristic skin pigmentation, itching and blindness depending on the strain of the parasite, \u003cem\u003eOnchocerca volvulus\u003c/em\u003e, circulating in a transmission zone. The parasite is transmitted by blackflies of the genus \u003cem\u003eSimulium\u003c/em\u003e, thus placing the insect as an indispensable indicator for real-time assessment of the transmission of the disease, and for monitoring the impact of control interventions in endemic foci [2].\u003c/p\u003e\n\u003cp\u003eThe control, and now elimination of onchocerciasis has relied mostly on annual mass drug administration (MDA) of ivermectin with some complementary vector control strategies in a few countries [3]. It is expected that the long-term administration of ivermectin of between 12-15 years, with good coverage, has potential to interrupt the transmission of the disease to below the threshold at which recrudescence would be possible [1]. Thus, WHO guidelines place utmost emphasis on epidemiological and entomological impact assessment to determine if the drug could be safely stopped after long-term administration in the transmission zones. Entomological assessments rely on the field collection of blackflies through human landing collection (HLC) technique and subsequent laboratory detection of \u003cem\u003eO. volvulus\u003c/em\u003e using O-150 PCR-ELISA technique on pools of the heads of the flies [4]. However, several issues have been raised about these two techniques: the ethical appropriateness and cost of using humans as bait for blackfly collection [5], and the sensitivity of the O-150 PCR-ELISA in detecting the true positive samples in pools of blackfly heads [6]. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOne of several efforts at developing alternative trapping technique to replace HLC is the Esperanza Window Trap (EWT) [5]. These demonstrated great potential and reasonable logistical advantage for the collection of host-seeking African species of blackflies [7]. It was suggested that one person can monitor multiple traps at a catching site and EWTs can be widely deployed for fly collection in the endemic communities [2]. However, the EWTs need further optimization for consistent performance, which can vary significantly across geographic zones as earlier reported [8,9]. Previous studies have elaborated on the role of\u0026nbsp;CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eas an activating attractant to blackflies [10, 11].\u0026nbsp;Simplifying and standardizing the source of CO\u003csub\u003e2\u003c/sub\u003e to bait the EWT is one of the key improvements recommended for further optimization [8].\u0026nbsp;The EWT is usually baited with CO\u003csub\u003e2\u003c/sub\u003e generated by yeast fermentation [5], however\u0026nbsp;the type of sugar, yeast strain and environmental conditions affect the production rate of organically generated\u0026nbsp;CO\u003csub\u003e2\u003c/sub\u003e under field conditions [8].\u0026nbsp;A recent study on another haematophagous Dipteran revealed that traps baited singly with granules of cyclopentanone or in combination with other attractant blends collected more \u003cem\u003eAnopheles arabiensis\u003c/em\u003e than those baited with yeast fermentation-produced CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e[12].Similar studies by Mburu \u003cem\u003eet al.\u003c/em\u003e (2017) [13] reported significant attraction of \u003cem\u003eAn. gambiae\u003c/em\u003e sensu lato to 2-butanone compared to industrially supplied CO\u003csub\u003e2\u003c/sub\u003e. The potential application to collection of anthropophilic blackflies has not yet been explored.\u003c/p\u003e\n\u003cp\u003eOnce collected, blackfly samples must be screened in the laboratory to determine whether they harbour infective (L3-stage) larvae. The current WHO-endorsed molecular technique targets the O-150 repeat for amplification [14], but the reagents require a reliable cold chain which cannot always be guaranteed during shipment or where electrical power supplies are inconsistent. A recently developed real-time qPCR method using Ov ND5, a mitochondrial DNA target, uses heat-stable reagents and is cheaper and faster to complete than the O-150, while also demonstrating increased sensitivity and specificity in initial comparisons [15]. This warrants further field evaluation to determine its suitability for use in programmatic settings.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt is against this background of operational roadblocks to verifying onchocerciasis elimination that the present study sought to: (i) evaluate and compare the efficiency of different release rates of cyclopentanone and 2-butanone as attractants for blackfly vectors of onchocerciasis, (ii) evaluate the synergistic potential of EWTs baited with a combination of CO\u003csub\u003e2\u003c/sub\u003e mimic and yeast-fermentation generated CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ecompared to HLCs, and (iii) evaluate the detection of \u003cem\u003eO. volvulus\u003c/em\u003e in head pools of the collected blackflies using O-150 PCR-ELISA versus Ov ND5 qPCR assays. Research took place in four ecological zones in Nigeria, all endemic for onchocerciasis, where there is a diversity of entomological and epidemiological conditions against which to test these new approaches.\u003c/p\u003e"},{"header":" Methodology ","content":"\u003cp\u003e\u003cstrong\u003ea)\u0026nbsp; \u0026nbsp;Study area\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour onchocerciasis endemic locations representing four ecological zones with varying \u003cem\u003eSimulium\u0026nbsp;\u003c/em\u003ecytospecies present were selected based on previous studies in Nigeria [16-20]. At each of the selected locations, two trap sites (at least 6-10 km apart) were established along the major river at the location for trap trials. The selected locations are described in Figure 1 and Table 1. Field experiments were conducted between September and October 2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Study location characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"673\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.573551263001487%\"\u003e\n \u003cp\u003eStudy Locations/ States\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.453194650817236%\"\u003e\n \u003cp\u003eEcological region\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.26002971768202%\"\u003e\n \u003cp\u003eLatitude/Longitude\u003c/p\u003e\n \u003cp\u003eof trap sites\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.099554234769688%\"\u003e\n \u003cp\u003ePreviously identified blackfly species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.61367013372957%\"\u003e\n \u003cp\u003eOnchocerciasis Elimination status\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.573551263001487%\"\u003e\n \u003cp\u003eUngwan Habu, Nasarawa State\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.453194650817236%\"\u003e\n \u003cp\u003eGuinea savannah\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.26002971768202%\"\u003e\n \u003cp\u003eTrap site 1:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8.944399N, 8.242172E\u003c/p\u003e\n \u003cp\u003eTrap site 2:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e8.996879N, 8.256494E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.099554234769688%\"\u003e\n \u003cp\u003e\u003cem\u003eS. damnosum\u003c/em\u003e s.s.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. sirbanum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.61367013372957%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eInterrupted [31,32]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.573551263001487%\"\u003e\n \u003cp\u003eKwa Falls and Ekong Anaku, Cross River State\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.453194650817236%\"\u003e\n \u003cp\u003eMontane Forest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.26002971768202%\"\u003e\n \u003cp\u003eTrap site\u0026nbsp;1:\u003c/p\u003e\n \u003cp\u003e5.105748N, 8.630302E\u003c/p\u003e\n \u003cp\u003eTrap site\u0026nbsp;2:\u003c/p\u003e\n \u003cp\u003e5.141645N, 8.508362E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.099554234769688%\"\u003e\n \u003cp\u003e\u003cem\u003eS. yahense\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. squamosum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.61367013372957%\"\u003e\n \u003cp\u003eOn track to elimination [32]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.573551263001487%\"\u003e\n \u003cp\u003eIke-Isu, Abia State\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.453194650817236%\"\u003e\n \u003cp\u003eRain forest\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.26002971768202%\"\u003e\n \u003cp\u003eTrap site 1:\u003c/p\u003e\n \u003cp\u003e5.41310N, 7.95398E\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eTrap site 2:\u003c/p\u003e\n \u003cp\u003e5.416057N, 7.954063E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.099554234769688%\"\u003e\n \u003cp\u003e\u003cem\u003eS. squamosum\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.61367013372957%\"\u003e\n \u003cp\u003eInterrupted [32]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.573551263001487%\"\u003e\n \u003cp\u003eIleogbo, Osun State\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.453194650817236%\"\u003e\n \u003cp\u003eTransition zone (Forest/derived savannah)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.26002971768202%\"\u003e\n \u003cp\u003eTrap site 1:\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e7.580873N, 4.301190E\u003c/p\u003e\n \u003cp\u003eTrap site 2:\u003c/p\u003e\n \u003cp\u003e7.587072N, 4.289398E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.099554234769688%\"\u003e\n \u003cp\u003eS. \u003cem\u003esoubrense beffa\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eS. damnosum\u003c/em\u003e s.s.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.61367013372957%\"\u003e\n \u003cp\u003eOn track to elimination [32]\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eb)\u003c/strong\u003e \u003cstrong\u003eField trials to improve blackfly catches in EWTs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe field trials were divided into three experiments for optimization of EWTs. Experiment 1 and 2 concerned enhancing the olfactory attractivity of EWTs through testing CO\u003csub\u003e2\u003c/sub\u003e mimics and augmenting organically produced CO\u003csub\u003e2\u003c/sub\u003e, and Experiment 2 tested the potential to increase overall blackfly yield through increasing EWT sampling effort.\u003c/p\u003e\n\u003cp\u003eCarbon dioxide and its mimics used in Experiment 1 and 2 were produced as follows. The 2-butanone and cyclopentanone were procured from Chemitica Inc, Costa Rica. Each of the two CO\u003csub\u003e2\u003c/sub\u003e mimics was prepared in semipermeable membrane sachets in three slow-release modes, all of which were formulated for release at temperatures of 20-30\u003csup\u003eo\u003c/sup\u003eC as follows: low release (~1-2 mg/day), medium release (~5-10 mg/day), and high release (~45-55 mg/day). The slow release is to enable the blend to mimic natural concentrations [21], last longer and not to be repellent. Most of the semiochemicals used for attracting insect vectors are slow release and can attract vectors over 30 -100 metres away [21]. The organically generated CO\u003csub\u003e2\u003c/sub\u003e was prepared on alternate days (every two days) by mixing 50 g of dry baker yeast, 500 g of sugar and 2.5 litres of water in a 5-litre plastic bottle at least 2 hours before the commencement of trapping as earlier described by [8].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExperiment 1: Identification of optimum CO\u003csub\u003e2\u003c/sub\u003e\u003c/em\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003e\u003cem\u003emimic release rates\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTwo trapping sites, at least 5 km apart and known for the breeding of blackflies, were selected along the river in each of the four study locations (Table 1). Of these, 2-butanone was tested at the first trapping site (Trap site 1), while cyclopentanone was tested at the second (Trap site 2). At each trap site, four points (25 m apart) were selected for the placement of traps: the EWTs comprised a tarpaulin (1 m x 1 m) with blue-black-blue stripes of equal size hung on an upright stand of poles tensioned with string and pegs and suspended about 10 cm above the ground. The two sides of the tarpaulin were coated with TAD All weather (adhesive) (Ladd Research Industries, USA) and baited with a pair of worn socks [7, 8]. The EWTs at the four points were also baited with either low, medium or high release rate CO\u003csub\u003e2\u003c/sub\u003e mimic or organically generated CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e(Figures 2 and 3). The traps were set up perpendicular to the breeding site in fairly shaded conditions. The CO\u003csub\u003e2\u003c/sub\u003e or mimic allocated to each trap was changed daily in a randomized Latin square experimental design over a period of 12 days. Flies were recovered from the traps twice daily (12 noon and 5 pm), preserved with 80% ethanol in 30 ml bottles and kept at 4\u0026deg;C until later use.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExperiment 2: \u0026nbsp;Optimization of EWTs through augmented odours relative to HLC\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment was designed to determine whether the number of flies attracted to organically generated CO\u003csub\u003e2\u003c/sub\u003e could be increased through the addition of a CO\u003csub\u003e2\u003c/sub\u003e mimic. For 8 consecutive days at each study location, catches from an EWT baited with the standard organically generated CO\u003csub\u003e2\u003c/sub\u003e were compared with an HLC positioned 25 m away from the trap. The HLC involved two adults collecting blackflies on hourly rotation between 7 am and 5 pm daily in accordance with the standard procedure [22, 8, 9]. The positions of the HLC and EWT were rotated daily in a cross-over design. On the nineth day, the organically generated carbon dioxide at the EWT was augmented with the best CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003emimic from Phase 1, identified as 2-butanone at a low release rate, and continued to be compared with the HLC in a cross-over design until day 16. The daily catches were recovered from the traps as described in Phase 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eExperiment 3: Trap scaling pilot\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs trapping approaches offer the advantage of allowing multiple traps to be operated by a single field entomologist, but the disadvantage of collecting fewer flies, the aim of this experiment was to establish in principle whether doubling the number of EWTs baited with organically generated CO\u003csub\u003e2\u003c/sub\u003e would increase total trap catch relative to an HLC. This phase was conducted at Ileogbo, the derived savannah zone in Osun State only. Two EWTs were placed 100 m apart from each other and from the HLC position. The trial was conducted over 9 days and positions were rotated daily, following the collection and sampling methods described above. The number of flies collected daily by the two traps were added together and compared with the HLC catches.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed) Comparison of fly infectivity in pools by O-150 PCR ELISA versus Ov ND5 real-time qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the flies collected in the methods described above were identified morphologically for confirmation of female \u003cem\u003eS. damnosum\u003c/em\u003e complex using the standard keys (WHO, 2002). The heads of the flies were separated from the body using entomological pins under the dissecting microscope (Biobase). The batches of the pins used were washed with sodium hypochlorite and sterilized before being re-used to minimize contamination. The heads were arranged in 100 heads per pool and each pool was transferred to a sterilized Eppendorf tube for DNA extraction. The extraction of the DNA was done using Qiagen DNeasy Blood \u0026amp; Tissue Kits (Qiagen, Germany) following the manufacturer\u0026rsquo;s instruction.\u003c/p\u003e\n\u003cp\u003eThe O-150 PCR and Ov ND5 gene real-time qPCR assays for the detection of \u003cem\u003eO. volvulus\u003c/em\u003e in the pools were performed as previously described by [2, 23].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee) Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data on blackfly count from the various collection methods were subjected to a generalised linear mixed model (GLMM) with a negative binomial distribution and counts were compared across release rates. Means were then separated using a \u003cem\u003epost-hoc\u0026nbsp;\u003c/em\u003eTukey multiple comparison. A generalised linear model (GLM) was used to compare the means of daily catches between EWT and HLC. The prevalence of infection in the pool screening of fly heads was determined using Poolscreen v2.0 software [24]. All other analysis was performed using statistical package R-4.3.0 [25].\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eField trials to improve blackfly catches in EWTs\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e\u003cem\u003eExperiment 1: Identification of optimum CO\u003c/em\u003e\u003csub\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sub\u003e \u003cem\u003emimic release rates\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThe results obtained from the optimization of the 2-butanone and cyclopentanone against organically generated CO\u003csub\u003e2\u003c/sub\u003e in the four study locations are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e. In three of the four study areas, all release rates of 2-butanone performed well in terms of mean daily fly catch relative to the organically produced CO\u003csub\u003e2\u003c/sub\u003e. In the derived savannah (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), the mean catches of low release (35.58\u0026thinsp;\u0026plusmn;\u0026thinsp;19.660), medium release (35.41\u0026thinsp;\u0026plusmn;\u0026thinsp;10.542) and high release (22.16\u0026thinsp;\u0026plusmn;\u0026thinsp;7.797) rates of 2-butanone were not significantly different to the mean catch by EWT baited with organically generated CO\u003csub\u003e2\u003c/sub\u003e (35.75\u0026thinsp;\u0026plusmn;\u0026thinsp;9.788) (p\u0026thinsp;=\u0026thinsp;0.50). Likewise, in montane forest, mean catches from the low (6.08\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09), medium (4.75\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09)and high (6.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38) release rates did not differ significantly compared to that from organic CO\u003csub\u003e2\u003c/sub\u003e (8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;2.87) (p\u0026thinsp;=\u0026thinsp;0.833) and this was also the case in the rain forest ecoregion, where low (25.58\u0026thinsp;\u0026plusmn;\u0026thinsp;9.44), medium (24.16\u0026thinsp;\u0026plusmn;\u0026thinsp;10.63) and high (16.50\u0026thinsp;\u0026plusmn;\u0026thinsp;5.23) release rates did not yield significantly more blackflies per day than organic CO\u003csub\u003e2\u003c/sub\u003e (32.91\u0026thinsp;\u0026plusmn;\u0026thinsp;8.40); p\u0026thinsp;=\u0026thinsp;0.409). However, in Guinea savannah, organically produced CO\u003csub\u003e2\u003c/sub\u003e caught significantly more flies per day (7.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05) than any of the 2-butanone-baited traps (low 3.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67, p\u0026thinsp;=\u0026thinsp;0.046; medium 3.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08, p\u0026thinsp;=\u0026thinsp;0.049; high 3.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54, p\u0026thinsp;=\u0026thinsp;0.048).\u003c/p\u003e \u003cp\u003eIn contrast, cyclopentanone performed poorly across all ecoregions. The EWT baited with organic CO\u003csub\u003e2\u003c/sub\u003e in derived savannah zone collected a significantly higher mean number of blackflies per day (81.40\u0026thinsp;\u0026plusmn;\u0026thinsp;36.987,) than EWT baited with cyclopentanone for low release (34.00\u0026thinsp;\u0026plusmn;\u0026thinsp;12.677; p\u0026thinsp;=\u0026thinsp;0.047), medium release (28.40\u0026thinsp;\u0026plusmn;\u0026thinsp;8.704; p\u0026thinsp;=\u0026thinsp;0.049) and high release (19.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.774; p\u0026thinsp;=\u0026thinsp;0.048). Similar results were obtained in Rainforest (organic CO\u003csub\u003e2\u003c/sub\u003e : 34.25\u0026thinsp;\u0026plusmn;\u0026thinsp;9.99; Low release: 8.25\u0026thinsp;\u0026plusmn;\u0026thinsp;2.98, p\u0026thinsp;=\u0026thinsp;0.048; Medium release: 8.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26, p\u0026thinsp;=\u0026thinsp;0.048; High release: 12.16\u0026thinsp;\u0026plusmn;\u0026thinsp;8.22, p\u0026thinsp;=\u0026thinsp;0.046); Derived Savannah (organic CO\u003csub\u003e2\u003c/sub\u003e : 11.80\u0026thinsp;\u0026plusmn;\u0026thinsp;4.71; Low release: 2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;1.40, p\u0026thinsp;=\u0026thinsp;0.046; Medium release: 5.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09, p\u0026thinsp;=\u0026thinsp;0.049; High release: 4.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.00, p\u0026thinsp;=\u0026thinsp;0.048); Montane forest (organic CO\u003csub\u003e2\u003c/sub\u003e : 9.08\u0026thinsp;\u0026plusmn;\u0026thinsp;1.47; Low release: 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23, p\u0026thinsp;=\u0026thinsp;0.000; Medium release: 0.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32, p\u0026thinsp;=\u0026thinsp;0.002; High release: 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 p\u0026thinsp;=\u0026thinsp;0.000).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 2: Optimization of EWTs through augmented odours relative to HLC\u003c/h2\u003e \u003cp\u003eHLC collected a significantly higher number of blackflies per day than EWTs baited with organically generated CO\u003csub\u003e2\u003c/sub\u003e across the locations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In location I in the four ecozones for example, the mean catches by HLC versus EWT were given as derived savannah\u0026thinsp;=\u0026thinsp;244.11\u0026thinsp;\u0026plusmn;\u0026thinsp;22.063 vs 81.000\u0026thinsp;\u0026plusmn;\u0026thinsp;9.402; rainforest\u0026thinsp;=\u0026thinsp;58.889\u0026thinsp;\u0026plusmn;\u0026thinsp;10.671 vs 2.556\u0026thinsp;\u0026plusmn;\u0026thinsp;1.107; montane forest\u0026thinsp;=\u0026thinsp;85.667\u0026thinsp;\u0026plusmn;\u0026thinsp;15.304 vs 9.667 vs\u0026thinsp;\u0026plusmn;\u0026thinsp;0.782; Guinea savannah\u0026thinsp;=\u0026thinsp;40.778\u0026thinsp;\u0026plusmn;\u0026thinsp;5.746 vs 8.444\u0026thinsp;\u0026plusmn;\u0026thinsp;1.591 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The EWTs baited with the combination of 2-butanone (low release) and organically generated CO\u003csub\u003e2\u003c/sub\u003e also collected significantly lower number of blackflies than HLC across the study locations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExperiment 3: Trap scaling pilot\u003c/h2\u003e \u003cp\u003eThe combination of two EWTs (baited with organic CO\u003csub\u003e2\u003c/sub\u003e and deployed 100 m apart to each other and to HLC) collected slightly higher number of flies than the HLC. The pooled mean catch of both traps was 94.011\u0026thinsp;\u0026plusmn;\u0026thinsp;5.97, which was very similar to the HLC catch of 91.80\u0026thinsp;\u0026plusmn;\u0026thinsp;9.86, a difference that was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.788). (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComparison of fly infectivity in pools by O-150 PCR ELISA versus Ov ND5 real-time qPCR\u003c/h2\u003e \u003cp\u003eThe results of the molecular analyses of blackfly infectivity showed that all the study locations had at least one positive pool by both O-150 PCR and Ov ND5 qPCR, with the exception of montane forest, which was positive only by Ov ND5 qPCR (Table\u0026nbsp;3). More positive pools were detected by Ov ND5 qPCR compared to O-150 PCR in three of the four ecoregions; in Guinea savannah (31.15% vs 15.57%), montane forest (11.54% vs 0%) and rainforest (23.08% vs 2.56%). Only one pool was positive by both Ov ND5 qPCR and O-150 PCR in Guinea savannah. All pools that were positive by O-150 PCR were also positive by Ov ND5 qPCR. The upper limit prevalence was exceedingly higher than 0.05% in all the four locations using both diagnostic methods except at montane forest which has a lower value for O-150 PCR (Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrevalence of \u003cem\u003eO. volvulus\u003c/em\u003e in blackflies at four study locations in Nigeria\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEcoregions (States)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo of pools\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo of pools positive by O-150 PCR\u003c/p\u003e \u003cp\u003e(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo of pools positive by Ov ND5 qPCR (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePrevalence by O-150 PCR (95% confidence interval)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePrevalence by Ov ND5 qPCR\u003c/p\u003e \u003cp\u003e(95% confidence interval)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDerived Savannah (Osun)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19 (15.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38 (31.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003cp\u003e(0.1\u0026ndash;0.26)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.37\u003c/p\u003e \u003cp\u003e(0.29\u0026ndash;0.51)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMontane Forest (Cross River)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9 (11.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.00\u003c/p\u003e \u003cp\u003e(0.00)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003cp\u003e(0.06\u0026ndash;0.22)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRainforest (Abia)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2.56)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9 (23.08)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003cp\u003e(0.00-0.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003cp\u003e(0.14\u0026ndash;0.48)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGuinea savannah (Nasarawa)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (16.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1 (16.67)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003cp\u003e(0.03\u0026ndash;0.82)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.18\u003c/p\u003e \u003cp\u003e(0.03\u0026ndash;0.82)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study provides important findings for both vector collection and parasite detection within the context of the WHO\u0026rsquo;s onchocerciasis elimination framework. Our key results indicate that alternative odour baits and using multiple traps could play a role in securing the large number of blackfly vector samples required for infection screening without relying on human collectors, and that the Ov ND5 qPCR technique is a more sensitive method for detecting parasite DNA in these samples than the currently used O-150 method.\u003c/p\u003e \u003cp\u003eCarbon dioxide serves as powerful attractant to haematophagous insects, and it has been widely used for traps to lure host-seeking insect vectors [ 10, 11, 13, 26]. In a search to improve the efficiency and easy deployment of EWTs, our field evaluation of various release rates of 2-butanone and cyclopentanone showed that the catches of EWTs baited with 2-butanone were not statistically different to those baited with organic CO\u003csub\u003e2\u003c/sub\u003e across the study locations except at Guinea savannah, where significantly lower catches were recorded for 2-butanone; the reason for this is not clear, however it may be species-specific as unlike other study areas, this Guinea savannah location was dominated by \u003cem\u003eS. damnosum s.s.\u003c/em\u003e and \u003cem\u003eS. sirbanum\u003c/em\u003e. The lowest release rate of 1\u0026ndash;2 mg/day was sufficient to catch similar numbers of blackflies as the medium and high release rates tested. Turner \u003cem\u003eet al.\u003c/em\u003e (2011) [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] reported dose-dependent activation of receptor neurons in the maxillary palps of mosquitoes to 2-butanone; this has not been explored in blackflies and our field results did not indicate a dose effect, so far as the release rate did not significantly affect trap capture rates. 2-butanone is a known natural product emanated by various vertebrates with potential to serve as a long-range attractant to host-seeking insects mimicking the CO\u003csub\u003e2\u003c/sub\u003e produced by vertebrate hosts, including humans [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These properties could have accounted for its effectiveness in collecting flies almost at par with EWTs baited with organically generated CO\u003csub\u003e2\u003c/sub\u003e but not exceeding these. Fermentation of sugar by yeast has been reported to produce not only CO\u003csub\u003e2\u003c/sub\u003e, but other volatile organic compounds, including alcohols, esters, aldehydes and fatty acids, that also attract arthropods [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and may explain its slightly greater success as a lure.\u003c/p\u003e \u003cp\u003eHowever, EWTs baited with cyclopentanone collected significantly lower number of blackflies than those baited with organically generated CO\u003csub\u003e2\u003c/sub\u003e across the four locations. Cyclopentanone is a monoketone which has been reported to activate the cpA CO\u003csub\u003e2\u003c/sub\u003e receptor neuron on the maxillary palp of mosquitoes, and thus serve as a suitable attractant to mosquitoes in semi-field trials [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Subsequent field trials reported poor attraction of the chemical to mosquitoes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Two reasons were advanced to explain the poor performance of cyclopentanone in the field: i) the diffusion of odour plumes of the chemical due to environmental factors such as relative humidity, temperature and wind and ii) competing chemicals emanating from the surrounding vegetation, detritus and air pollution in the field. The reason may be explained by the fact that cyclopentanone is a monoketone obtainable in plants, and has been used widely in pharmaceuticals and industrial processes including in rubber chemicals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Considering the typical ecological habitat of blackflies, which is usually populated with shrubs and vegetation, the competing odour of this chemical from the surrounding vegetation is inevitable.\u003c/p\u003e \u003cp\u003eThe EWTs baited with organic CO\u003csub\u003e2\u003c/sub\u003e collected significantly lower number of blackflies to HLC across the four ecological zones. Similar observations have also been reported by Hendy \u003cem\u003eet al.\u003c/em\u003e (2017) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] in Tanzania and Talom \u003cem\u003eet al.\u003c/em\u003e (2021) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] in Cameroon. In contrast, Toe \u003cem\u003eet al.\u003c/em\u003e (2014) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] in Burkina Faso and Hendy \u003cem\u003eet al.\u003c/em\u003e (2017) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] in Uganda reported statistically similar number of flies collection between EWTs and HLC. The reasons for differential trap performance are not clear. In the present study, augmentation of organic CO\u003csub\u003e2\u003c/sub\u003e with low release rates of 2-butanone did not show any additional benefit in terms of catch, whereas in a field study conducted against \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], augmentation of CO\u003csub\u003e2\u003c/sub\u003e sources with 2-butanone significantly improved the catches of the mosquitoes. Interestingly, when two EWTs baited with organic CO\u003csub\u003e2\u003c/sub\u003e were used at a site in the derived savannah, collectively the two traps caught slightly higher numbers of blackflies than two human landing collectors working rotationally on hourly basis. This result may be due to the fact that all the collection methods were well spread out, with approximately 100 m between EWTs and between EWTs and the HLC. The strong selection pressure on blackflies to respond to human-specific cues, and thus the highly attractive profile of human collections, may therefore be outcompeting alternative collection methods that only present partial and artificial attractive cues. This result needs further investigation as it may be crucial for realizing the potential of trapping paradigms in operational contexts. Rodriguez-Perez \u003cem\u003eet al.\u003c/em\u003e (2014) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] demonstrated the field feasibility of deployment of multiple EWTs for the collection of the required numbers of flies to meet WHO guidelines requirement and our study reinforces this observation, and rigorous investigation in areas with different biting rates and cytospecies should be included as part of future studies.\u003c/p\u003e \u003cp\u003eThe higher number of blackfly head pools positive for Ov ND5 qPCR as compared with O-150 PCR ELISA showed that the former is more sensitive than the latter. Notably, all the samples positive for O-150 PCR were also positive for Ov ND5 qPCR but the qPCR detected 36 more positive pools that had been scored negative by O-150 PCR. This result is in tandem with the recent studies commissioned by WHO [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and published laboratory reports [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] on the high sensitivity of Ov ND5 qPCR over O-150 qPCR and O-150 PCR ELISA. Importantly, the Ov ND5 qPCR protocol is also faster, more cost effective and easier to use, requiring fewer reagents than the O-150 PCR ELISA. Alongside the growing number of similar reports, our observation has serious implications for the on-going global efforts in onchocerciasis elimination, as accurate detection of infected flies is crucial to informing safe stop-MDA decisions and robust post-elimination surveillance. Flies serve as a link between parasite and host, and they give a real-time indication on whether or not transmission has been interrupted/eliminated in a community [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Erroneous stoppage of MDA in areas with low but existing residual transmission could bring early recrudescence and jeopardize the gains achieved over many years of MDA. Further optimization of qPCR for wide implementation for impact assessment and post-elimination surveillance must be vigorously pursued if the global effort to accelerate towards eliminating onchocerciasis in endemic communities is to be achieved by the year 2030.\u003c/p\u003e \u003cp\u003eNotwithstanding the different diagnostic methods used, the results of the pool screening analysis in the four study zones are of significant interest to onchocerciasis elimination efforts in Nigeria. The results obtained in derived savannah (Osun State) and the montane forest (Cross River) showed that there is ongoing transmission in these foci; both zones remain under annual ivermectin coverage, which our results confirm is still required. There is need by the National Onchocerciasis Elimination Programme (NOEP) to aggressively improve the therapeutic coverage in the communities, as this entomological evidence suggests the presence of people harbouring active adult parasite worms at the study locations. However, the observation of positive pools in rainforest (Abia State) and Guinea savannah (Nasarawa State) is a surprise overlaid with serious implications for the ongoing efforts at eliminating onchocerciasis in Nigeria. According to the NOEP [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and the available literature [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], Nasarawa State is one of the two states where onchocerciasis has been eliminated in Nigeria while Abia State has successfully interrupted the transmission of the disease. The occurrence of the positive pools at these two zones could be attributed to two possibilities: i) that O-150 was not sensitive enough to detect the residual flies carrying infection after the MDA has suppressed active transmission since our study sites were among the communities where the Stop-MDA and Post Stop-MDA evaluations were conducted, or ii) the human migration caused by insecurity in Nasarawa and Abia States in the last six years has introduced sufficient numbers of infected people to the area to cause reinfection in the vector population. Irrespective of driving factors, the pool screening results have shown the possibility of a gradual build-up of recrudescence which requires urgent programmatic evaluation across Nasarawa and Abia States to determine the extent of the spread and instigate implementation strategies to combat it before it erodes the success already recorded in these transmission zones in Nigeria. To the best of our knowledge and available reports, this is the first report of direct comparison of O-150 PCR and Ov ND5 qPCR for the detection of blackfly infectivity in areas with ongoing transmission and in foci where onchocerciasis has been deemed interrupted/eliminated in West Africa.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our results suggested that 2-butanone was effective as a CO\u003csub\u003e2\u003c/sub\u003e mimic and collected comparable number of \u003cem\u003eS. damnosum\u003c/em\u003e s.l. as those attracted by organically generated CO\u003csub\u003e2\u003c/sub\u003e in three out of four selected ecological zones in Nigeria. However, the EWTs baited with cyclopentanone collected significantly fewer flies as compared with those baited with organic CO\u003csub\u003e2\u003c/sub\u003e plausibly suggesting its poor attraction to blackflies in the field. Despite the fact that EWTs baited with organic CO\u003csub\u003e2\u003c/sub\u003e alone and those augmented with 2-butanone at the optimum low release rate collected significantly fewer blackflies as compared with HLC catches, it was found that two EWTs strategically deployed at 100 m apart and to the HLC collected more blackflies than the two human collectors working on hourly rotation. This observation is an indication that EWTs (baited with appropriate odours/lures), if further optimized and strategically deployed with adequate space between competing traps at breeding sites, have potential for replacing HLC for monitoring and surveillance of blackfly vectors in programmatic contexts of onchocerciasis elimination assessment. We observed that Ov ND5 qPCR was more sensitive than O-150 PCR ELISA in detecting infected flies in blackfly head pools and would be a useful tool for accurate entomological assessment for stop-MDA in the context of elimination and circumvention of recrudescence due to poor sensitivity of diagnosis tools. Whether the positive pools found in the foci hitherto thought to have interrupted/eliminated onchocerciasis are caused by poor sensitivity of O-150 PCR ELISA or due to changing epidemiological factors, such as human migration due to insecurity, require further programmatic evaluation by the NOEP in Nigeria.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the NTDs state coordinators, field Entomologists, community members and study participants in the four ecological zones selected for this study. We thank Mr Samson Olumakinde, Oluokun Timileyin Emmanuel and Olaniyan Oluwaseyi for their help in the laboratory. We appreciate Chemitica Inc, Costa Rica for the supply of slow release sachets of 2-butanone and cyclopentanone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contribution\u003c/strong\u003e: MAA, KNO, BEBN, HBM, OAS conceived and wrote the protocol for the study, FMH reviewed the protocol, \u0026nbsp;MAA, KNO, BEBN, HBM, OAS, CF, YC, MUG, IM, ME, OF, AC, TJD performed the field work, MAA, SBA, IZO performed the molecular analysis, MAA, KNO, BEBN, HBM, OAS drafted the manuscript, all authors reviewed and contributed to the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis project was funded by Bill and Melinda Gates Foundation (INV-047737) but the funder had no influence on the outcomes of the study.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e: The data set for this research is available upon reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the National Health Research Ethical Committee of the Federal Ministry of Health, Nigeria (NHREC/01/01/2007-13/03/2023). The Neglected Tropical Diseases Coordinators and the community leaders in the four selected locations were appropriately mobilized for the study. Written informed consent was also obtained from the community leaders and the locally appointed vector collectors after the objectives, benefits and risks had been duly explained. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Zoology, Osun State University, Osogbo, Nigeria, \u003csup\u003e2\u003c/sup\u003eDepartment of Animal and Environmental Biology, University of Uyo, Nigeria, \u003csup\u003e3\u003c/sup\u003eDepartment of Zoology, University of \u0026nbsp;Jos, \u0026nbsp;Nigeria, \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003csup\u003e4\u003c/sup\u003eDepartment of Animal and Environmental Biology, Imo State University, Owerri Nigeria, \u003csup\u003e5\u003c/sup\u003eDepartment of Microbiology, Osun State University, Osogbo, Nigeria, \u003csup\u003e6\u003c/sup\u003eFederal Ministry of Health, Nigeria, \u003csup\u003e7\u003c/sup\u003eDepartment of Biological Science, Nasarawa State University, Lafia, \u003csup\u003e8\u003c/sup\u003eAgriculture, Health \u0026amp; Environment Department, Natural Resources Institute, University of Greenwich, United Kingdom\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO. 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Cell. 2013;155:1365\u0026ndash;1379.\u003c/li\u003e\n\u003cli\u003ePhilippe-Janon JC, van den Hurk AF, Francis DP, Shivas MA, Jansen CC. Field Comparison of Cyclopentanone Versus Carbon Dioxide as an Attractant for Adult Mosquitoes in Southeast Queensland, Australia. J Med Entomol. 2015;52(3):483-490. DOI: 10.1093/jme/tjv011.\u003c/li\u003e\n\u003cli\u003eNational Onchocerciasis Elimination Committee (NOEC). Minutes of the 15th National Onchocerciasis Elimination Committee Meeting, May 2022. Federal Ministry of Health, Abuja, Nigeria. Available from: https://www.cartercenter.org/resources/pdfs/news/health_publications/river_blindness/nigeria-onchocerciasis-elimination-committee-communique-13.pdf.\u003c/li\u003e\n\u003cli\u003eMiri ES, Eigege A, Kahansim B, Nwodu K, Sambo Y, Mancha B, Adelamo S, Umaru J, Kadimbo J, Danboyi J, Mafuyai H, Makata E, Akpan N, Akilah J, Igbe M, Coalson J, Rakers L, Griswold E, Unnasch TR, Nwoke BEB, Noland GS, Richards FO. 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Available from: https://africa-health.com/wp-content/uploads/2023/05/19-Onchocerciasis.pdf.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Simulium, 2-butanone, cyclopentanone, carbon dioxide, Esperanza Window trap, real time PCR, onchocerciasis ","lastPublishedDoi":"10.21203/rs.3.rs-4226239/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4226239/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e Entomological data for onchocerciasis surveillance relies on sampling blackflies through human landing collectors in the field and laboratory testing of the flies for infection using pool screening O-150 PCR-ELISA assay. Both techniques require improvements. This study sought to take promising Esperanza Window Traps (EWT) for blackfly collections and test and optimize them to find alternative carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e \u003c/sub\u003emimics, and to test new qPCR methods using mitochondrial DNA targets that have been suggested to improve sensitivity and specificity for \u003cem\u003eOnchocerca volvulus \u003c/em\u003einfection in flies. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Traps baited with low, medium and high release rates of either 2-butanone or cyclopentanone as CO\u003csub\u003e2\u003c/sub\u003e mimics were field tested against traps baited with organically generated CO\u003csub\u003e2\u003c/sub\u003e in Guinea savannah, derived savannah, rainforest and montane forest ecological zones in Nigeria. The performance of EWTs baited with CO\u003csub\u003e2 \u003c/sub\u003eor in combination with 2-butanone (low release) were subsequently evaluated against the human landing collection (HLC). Trap scaling was also pilot tested by comparing double traps to single HLCs. Collected blackflies were used to test detection of \u003cem\u003eO. volvulus\u003c/em\u003e in blackflies using Ov ND5 real time PCR (qPCR) in comparison to the conventional pool screening O-150 PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e EWTs baited with 2-butanone caught similar numbers of blackflies to those baited with CO\u003csub\u003e2\u003c/sub\u003e, while cyclopentanone collected significantly fewer flies in all locations. The low release of 2-butanone was the most effective overall, although HLCs collected higher numbers of blackflies than EWT baited with CO\u003csub\u003e2\u003c/sub\u003e either singly or in combination with low release 2-butanone. The combination of two EWTs baited with CO\u003csub\u003e2\u003c/sub\u003e and deployed 100 m apart to each other collected similar numbers of flies as one HLC. More blackfly pools were positive for \u003cem\u003eO. volvulus\u003c/em\u003e by Ov ND5 qPCR as compared with O-150 PCR in derived savannah (31.15% vs 15.57%), montane forest (11.54% vs 0%) and rainforest (23.08% vs 2.56%), with only one positive pool in Guinea savannah detected by both methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e 2-butanone has potential to be used in xenomonitoring as a standardized replacement to organically generated CO\u003csub\u003e2\u003c/sub\u003e. The positive pools found in foci hitherto considered to have interrupted/eliminated onchocerciasis highlights the need for more sensitive and specific methods that support programmatic assessments that can identify and combat recrudescence.\u003c/p\u003e","manuscriptTitle":"Improving onchocerciasis elimination surveillance: trials of odour baited Esperanza Window Traps to collect black fly vectors and real-time qPCR detection of Onchocerca volvulus in black fly pools","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-09 06:09:36","doi":"10.21203/rs.3.rs-4226239/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2241f83-c487-4418-8829-d9709154c281","owner":[],"postedDate":"April 9th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-20T12:48:04+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-09 06:09:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4226239","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4226239","identity":"rs-4226239","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}