An allele specific PCR assay for screening for drug resistance among Wuchereria bancrofti populations in India.

BACKGROUND & OBJECTIVE Albendazole, a commonly used anthelminthic drug that targets the polymerization of alpha- and beta-tubulin dimer is currently co-administered with the antifilarial drug, diethylcarbamazine citrate (DEC) in the ongoing Global Programme for Elimination of Lymphatic Filariasis (GPELF). The experience in veterinary field has shown that there can be a rapid development of resistance to this drug, which therefore, needs to be monitored regularly in GPELF. Hence, we investigated the nucleotide polymorphism in the albendazole-binding domain of the isotype 1 beta-tubulin gene from several populations of Wuchereria bancrofti and developed an AS-PCR assay useful in screening for sensitive/resistance alleles among parasite populations and also evaluated its utility. METHODS For studying the polymorphism of isotype 1 beta-tubulin gene, a 475 bp fragment spanning exon 5 and 6 of the gene was amplified and sequenced from the genomic DNA of W. bancrofti collected from six geographic regions of India. An allele specific (AS) PCR for screening albendazole sensitivity/resistance was developed and a total of 55 mf samples from blood smears on slides collected from Thiruvannamalai, Thanjavur and Puducherry were screened. Selective therapy with DEC was in place in three areas, mass drug administration (MDA) with DEC alone was implemented in four areas, while DEC plus albendazole was administered in one district. RESULTS The analysis of the nucleotide sequence of the fragment from 20 W. bancrofti populations showed the domain to be highly conserved. An allele-specific PCR assay developed was used to detect sensitive/resistance alleles among 55 isolates of W. bancrofti and no albendazole resistance alleles were detected among the populations tested. INTERPRETATION & CONCLUSION The drug-binding domain of isotype 1 beta-tubulin gene of W. bancrofti from different geographical locations was highly conserved. The AS-PCR developed showed potential application as a tool for monitoring albendazole sensitivity/resistance alleles among W. bancrofti populations, in areas where combination therapy of DEC-albendazole is being mass administered in the LF elimination programme.