The epidemiology of lymphatic filariasis in Ghana, explained by the possible existence of two strains of Wuchereria bancrofti.

INTRODUCTION: Lymphatic filariasis is a debilitating disease caused by the filarial worm Wuchereria bancrofti. It is earmarked for elimination by the year 2020 through the Global Program for the Elimination of LF (GPELF). In Ghana, mass treatment has been on-going since the year 2000. Earlier studies have revealed differing epidemiology of LF in the North and South of Ghana. This study was therefore aimed at understanding the possible impacts of W. bancrofti diversity on the epidemiology and control of LF in Ghana. METHODS: The Mitochondrial, Cytochrome C Oxidase I gene of W. bancrofti samples was sequenced and analyzed. The test sequences were grouped into infrapopulations, and pairwise differences (π) and mutation rates (θ) were computed. The amount of variance within and among populations was also computed using the AMOVA. The evolutionary history was inferred using the Maximum Parsimony method. RESULTS: Seven samples from the South and 15 samples from the North were sequenced, and submitted to GenBank with accession numbers GQ479497- GQ479518. The results revealed higher mutation frequencies in the southern population, compared to the northern population. Haplotype analyses revealed a total of 11 haplotypes (Hap) in all the 22 DNA sequences, with high genetic variation and polymorphisms within the southern samples. CONCLUSION: This study showed that there is considerable genetic variability within W. bancrofti populations in Ghana, differences that might explain the observed epidemiology of LF. Further studies are however required for an in-depth understanding of LF epidemiology and control.

Monitoring lymphatic filariasis interventions: Adult mosquito sampling, and improved PCR - based pool screening method for Wuchereria bancrofti infection in Anopheles mosquitoes.

BACKGROUND: Monitoring and evaluation are essential to the successful implementation of mass drug administration programmes for LF elimination. Monitoring transmission when it is low requires both large numbers of mosquito vectors and sensitive methods for detecting Wuchereria bancrofti infections in them. PCR-based methods are preferred over classical dissections but the best protocol so far achieved detection of one L3 Wuchereria bancrofti larva in a pool of 35-50 Anopheles mosquitoes. It also lacks consistency and remains still a costly tool. Hence we decided to improve upon this to achieve detection in a pool of 100 or more by enhancing the quality of the template DNA. Prior to this we also evaluated three vector sampling methods in the context of numbers for monitoring. METHODS: Human landing, pyrethrium spray and light traps catches were conducted concurrently at sites in an LF endemic district in Ghana and the numbers obtained compared. Two DNA extraction methods; Bender buffer and phenol/chloroform purification, and DNAeasy Tissue kit (Quaigen Inc) were used on pools of 25, 50, 75 100 and 150 mosquitoes each seeded with one L3 or its quivalent amount of DNA. Then another set of extracted DNA by the two methods was subjected to Dynal bead purification method (using capture oligonucleotide primers). These were used as template DNA in PCR to amplify W. bancrofti sequences. The best PCR result was then evaluated in the field at five sites by comparing its results (infections per 1000 mosquitoes) with that of dissection of roughly equal samples sizes. RESULTS: The largest numbers of mosquitoes were obtained with the human landing catches at all the sites sampled. Although PCR detection of one L3 in pools of 25, 50 and 75 mosquitoes was consistent irrespective of the extraction method, that of one L3 in 100 was only achieved with the kit-extracted DNA/Dynal bead purification method. Infections were found at only two sites by both dissection and pool-screening being 14.3 and 19 versus 13.4 and 20.1 per 1000 Anopheles mosquitoes respectively, which were not statistically significant DISCUSSION AND CONCLUSION: HLC still remains the best option for sampling for the large numbers of mosquitoes required for monitoring transmission during MDA programmes, when vector population densities are high and classical indices of transmission are required. One - in - 100 detection is an improvement on previous PCR pool-screening methods, which in our opinion was a result of the introduction of the extra step of parasite DNA capture using Dynal/beads. As pool sizes increase the insects DNA will swamp parasite DNA making the latter less available for an efficient PCR, therefore we propose either additional steps of parasite DNA capture or real-time PCR to improve further the pool screening method. The study also attests also to the applicability of Katholi et al's algorithm developed for determining onchocerciasis prevalence in LF studies.