Development and Validation of Rapid Molecular Assays for Diagnosis and Surveillance of Wuchereria Bancrofti in Human Blood and Mosquito Vectors in Kenya

Abstract

Background: Lymphatic filariasis is caused by Wuchereria and Brugia species. Elimination control programme aims at eliminating the disease as a public health problem. Point-of-care, affordable, user friendly and accurate diagnostic tools are needed. Molecular characterization of Wuchereria bancrofti 18S rRNA gene was characterized for diagnostics targets analysis. Loop-mediated and Helicase dependent isothermal amplification assays were developed, evaluated and validated against PCR assay. Materials and methods: Human blood samples and mosquito were collected from Malindi, Tana River and Busia Counties which have a high prevalence of lymphatic filariasis. Whole genomic DNA were extracted and W. bancrofti 18S rRNA fragments were amplified by PCR and sequenced. LAMP and HDA assays were used for screening and detection of W. bancrofti DNA in human blood and mosquitoes. Kappa statistics were calculated to determine congruence between the two tests. The sensitivity and specificity of LAMP and HDA assays were compared using 2X2 contingency table. Chromas version 2.6.5 and BioEdit softwares were used for manual editing and multiple sequence alignment of the 18S rRNA nucleotide sequences. Phylogenetic analysis was done in MEGA 7. Results: Malindi and Tana River 18S rRNA isolates had a mean genetic distance of 0.99. Percent identity of the 18S rRNA nucleotide sequence with GenBank reference sequences ranged from 79-98%. The sensitivity of LAMP assay was 92.3%, while that of HDA was 76.6%. The specificity of the LAMP assay was 97.3% and that of HDA was 93.7%. Kappa statistics was at 0.84 and 0.67 for LAMP and HDA respectively. Conclusion: Wuchereria bancrofti 18S rRNA gene were highly conserved among the 14 isolates from Malindi and Tana River and thus, suitable for diagnostic targets. LAMP assay was found to be more sensitive and comparable to PCR than HDA. Recommendations: More genetic studies to be undertaken to establish the degree of diversity in W. bancrofti strains in Kenyan endemic regions. LAMP assay should be adopted in local health clinics at endemic regions for diagnosis of lymphatic filariasis. Key words; genetic, characterization, variation, LAMP, HDA, Wuchereria, bancrofti, isotherma