Performance of novel onchocerciasis rapid diagnostic tests in a field setting in South Sudan

Introduction
Onchocerciasis is a neglected tropical disease caused by Onchocerca volvulus and is primarily controlled through mass drug administration (MDA) of ivermectin. Current diagnostic tests perform sub-optimally for onchocerciasis elimination programs. According to the WHO, a test needs a sensitivity of ≥60% and a specificity of ≥99.8% for mapping, and a sensitivity of ≥89% and a specificity of ≥99.8% for MDA stopping decisions. We assessed the performance of the commercially available Ov16 SD Bioline rapid diagnostic test (RDT) and three novel RDTs: DDTD biplex type A, type C, and the monoplex type GADx.

Methods
The study included 319 pregnant/post-partum women in Maridi, an onchocerciasis-endemic area in South Sudan. Locally trained healthcare workers conducted RDT with whole blood and skin snip testing. Ov16 SD Bioline RDT was also performed using dried blood spots (DBS). Diagnostic performances were evaluated using three individual reference tests (anti-Ov16 ELISA, O-150 qPCR, skin snip microscopy) and two composite reference standards. DBS were tested for Mansonella spp. using qPCR to account for potential false positives.

Results
Anti-Ov16 ELISA detected O. volvulus antibodies in 49.6% (123/248) participants, while O. volvulus infection was documented by skin snip microscopy in 31.9% (46/144) participants and in 41.8% (50/141) by qPCR. Mansonella spp. infection prevalence was 13.4% (34/253). Monoplex RTDs (Ov16 SD Bioline, GADx) detected O. volvulus antibodies in >60%, while the biplex tests (DDTD A and C) detected 50% seropositivity. Estimated sensitivities of RDTs ranged from 69.4% (Ov16 SD Bioline) to 48.9% (DDTD A), while specificity ranged from 73.2% (DDTD C) to 42.5% (Ov16 SD Bioline).

Conclusion
O. volvulus and Mansonella spp. prevalence was high among pregnant and post-partum women in Maridi. The novel RDTs demonstrated sensitivities within the range of the commercially available Ov16 SD Bioline RDT, meeting the WHO threshold for onchocerciasis mapping but not for MDA stopping decisions. No RDT met the specificity threshold of 99.8%, although some exhibited slightly higher specificity than Ov16 SD Bioline RDT. This suboptimal specificity raises concerns, underscoring the need for better diagnostic tools to support onchocerciasis elimination efforts.

Author summary
Onchocerciasis, or river blindness, is a parasitic disease transmitted by blackflies and controlled through mass treatment with ivermectin. As countries move toward eliminating the disease, highly accurate diagnostic tools are urgently needed to map infection areas and decide when to stop mass drug administration. In this study, we evaluated the accuracy of four rapid diagnostic tests (RDTs) in detecting infection in pregnant and post-partum women in South Sudan. We compared the RDT results to multiple standard laboratory tests for the parasite, including antibody detection, skin microscopy, and DNA testing. We also tested for co-infection with Mansonella parasites, which could interfere with diagnosis. While some new tests performed comparably to the currently available Ov16 SD Bioline RDT, none achieved the high specificity required by the World Health Organization for decisions about stopping treatment programs. Our findings highlight the continued need for improved diagnostic tools that are both sensitive and specific, especially in areas where similar parasitic infections may lead to false-positive results.