Comparison of PCR Methods for Onchocerca volvulus Detection in Skin Snip Biopsies from the Tshopo Province, Democratic Republic of the Congo

Abstract

Defining the optimal diagnostic tools for evaluating onchocerciasis elimination efforts in areas co-endemic for other filarial nematodes is imperative. This study compared three published polymerase chain reaction (PCR) methods: the -specific qPCR-O150, the pan-filarial qPCR melt curve analysis (MCA), and the O150-PCR enzyme-linked immunosorbent assay (ELISA) currently used for vector surveillance in skin snip biopsies (skin snips) collected from the Democratic Republic of the Congo. The pan-filarial qPCR-MCA was compared with species-specific qPCRs for and . Among the 471 skin snips, 47.5%, 43.5%, and 27.0% were positive by qPCR-O150, qPCR-MCA, and O150-PCR ELISA, respectively. Using qPCR-O150 as the comparator, the sensitivity and specificity of qPCR-MCA were 89.3% and 98.0%, respectively, whereas for O150-PCR ELISA, they were 56.7% and 100%, respectively. Although qPCR-MCA identified the presence of and spp. in skin snips, species-specific qPCRs had greater sensitivity and were needed to identify . Most of the qPCR-MCA misclassifications occurred in mixed infections. The reduced sensitivity of O150-PCR ELISA was associated with lower microfilaria burden and with lower amounts of DNA. Although qPCR-MCA identified most of the -positive skin snips, it is not sufficiently robust to be used for stop-mass drug administration (MDA) evaluations in areas co-endemic for other filariae. Because O150-PCR ELISA missed 43.3% of qPCR-O150-positive skin snips, the qPCR-O150 assay is more appropriate for evaluating skin snips of OV-16 + children in stop-MDA assessments. Although improving the sensitivity of the O150-PCR ELISA as an alternative to qPCR might be possible, qPCR-O150 offers distinct advantages aside from increased sensitivity.

NTD Diagnostics for Disease Elimination: A Review

“…Many papers sought to explore new testing approaches or ways to incrementally improve current methods [39,[84][85][86][87][88][89][90][91]. In addition, common practice and official guidance were questioned [39,78,92].…”

Onchodermatitis: Where Are We Now?

“…Real-time PCR and isothermal loop amplification (LAMP) assays have also been developed [ 45 , 46 ]. On comparison of three PCR methods for evaluating onchocerciasis elimination efforts in areas co-endemic with other filarial nemaodes, the qPCR-O150 assay was deemed to be more appropriate for evaluating skin snips of OV-16 positive children when deciding when to stop MDA [ 47 ]. A novel O-5S qPCR assay targeting the O. volvulus O-5S rRNA gene, had 100% specificity and proved more sensitive than O-150 qPCR assay (66.5% vs 39% positivity rate) [ 48 ].…”

Differential susceptibility of Onchocerca volvulus microfilaria to ivermectin in two areas of contrasting history of mass drug administration in Cameroon: Relevance of microscopy and molecular techniques for the monitoring of skin microfilarial repopulation within six months of direct observed treatment

“…Besides microscopy [10], immunological [17,18] and nucleic acid-based methods, speci cally PCR [19,20] and LAMP [21,22] have been used to diagnose O. volvulus in skin. Although there have been advances in the development of immunological methods involving detection of either antibody or antigen [17,18], cross-reactivity with other larial species has been reported [23].…”

“…However, PCR-based methods can be time consuming, require highly puri ed DNA and require expensive equipment and material compared to microscopy. Nevertheless, this technology was shown to be more sensitive than microscopy [11,20]. Alternatively, loop-mediated isothermal ampli cation (LAMP) is a simple molecular method which rapidly synthesizes large amounts of DNA within 60 minutes and which is less sensitive to impure DNA preparations and has higher ampli cation e ciency than PCR [21,22,[29][30][31][32].…”