Comparing reliability of skin microfilarial load with first internal transcribed spacer primers DNA amplification in monitoring onchocerciasis treatment control

Abstract

Treatment compliance is a serious challenge to community-directed treatment intervention (CDTI) targeting onchocerciasis eradication. Isolated cases of active infections could jeopardise decision to stop mass drug distribution. Onchocerciasis co-endemicity with lymphatic filariasis in the study-area informed the use of primers capable of differentiating sympatric infections in a single assay. The study-population was hitherto found to be negative for presence of skin microfilaria. DNA extracted from skin snips of patients (n=63) comprised of males, n=21 and females, n=42 were analysed using 18S first internal transcribed spacer (ITS1) in polymerase chain reaction (PCR) assay. Amplified DNA sequences were subjected to basic local alignment search tool. Spectrophotometric analysis of DNA concentrations ranged between 1.5- and 24.5 Nano gram (ng) and the two positive controls (adult worm DNA extract) were 10.5 and 105 ng at 260 nanometer (nm) wave length. The 260/ 280 nm ratios were between 1.44 and 1.68. Four samples (6.35%), two males and two females, and the two positive controls showed PCR amplification products. Two of the positive samples indicated double bands in an agarose gel electrophoregram with molecular weight of 350 and 500 base pairs (bp), while two had single band of 350bp, which is within the expected Mw range of 344 bp for Onchocerca volvulus. The double bands may not be due to unspecific amplification. Moreover, the DNA sequence of a sample had 69.1% homology with control sequence but not with O. volvulus sequences available in the GenBank database. This study underscored the need to assess effectiveness of CDTI with highly sensitive test and not depend on microscopy of emerged microfilaria from skin snips. Whether the DNA was from dead or living microfilaria remained a conjecture.

Keywords: Skin microfilaria; DNA sequences.