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.

Abstract

Background: Entomological data for onchocerciasis surveillance relies on sampling black flies through human landing collectors in the field and laboratory testing of the flies for infection using pooled screening O-150 PCR-ELISA assay. Both techniques require improvements. This study aimed to optimize the Esperanza Window Trap (EWT) for black fly collection. We tested alternative carbon dioxide (CO₂) mimics to attract black flies to the traps. Additionally, we evaluated new quantitative PCR (qPCR) methods that target mitochondrial DNA markers and have been proposed to enhance the sensitivity and specificity for detecting Onchocerca volvulus infections in blackflies.

Methods: Traps baited with low, medium and high release rates of either 2-butanone or cyclopentanone as CO₂ mimics were field tested against traps baited with organically generated CO₂ in four ecological zones in Nigeria: Guinea savannah, derived savannah, rainforest and montane forest. The performance of EWTs baited with CO₂ 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 two EWTs to a single HLC team. Collected black flies were used to test detection of O. volvulus in black flies using Ov ND5 real-time PCR (qPCR) compared to the conventional pool screening O-150 PCR.

Results: EWTs baited with 2-butanone caught similar numbers of black flies (Simulium damnosum s.l.) to those baited with CO₂, 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 black flies than EWT baited with CO₂ either singly or in combination with low-release 2-butanone. The combination of two EWTs baited with CO₂ and deployed 100 m apart from each other collected similar numbers of flies as one HLC. More black fly pools were positive for O. volvulus by Ov ND5 qPCR 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.

Conclusions: The 2-butanone has potential to be used in xenomonitoring as a standardized replacement for organically generated CO₂. Ov ND5 qPCR detected more positive pools than O-150 PCR. The positive pools found in foci hitherto considered to have interrupted/eliminated onchocerciasis highlight the need for more sensitive and specific methods that support programmatic assessments to identify and combat recrudescence.