DifferentiatingDracunculusmedinensisfromD.insignis, by the sequence analysis of the 18S rRNA gene

Abstract

This study, undertaken as a component of the global Dracunculiasis Eradication Program (DEP), was designed to provide molecular tools to distinguish Dracunculus medinensis, the nematode causing human dracunculiasis, from other tissue-dwelling nematodes, including other Dracunculus species that infect humans and other animals. DNA was extracted from D. medinensis and from a closely related species that infects North American carnivores, D. insignis, so that the genes coding for the small-subunit ribosomal RNA (18S rRNA) of the parasites could be amplified, sequenced and compared. Sequences were obtained for 20 specimens of D. medinensis (from humans in Pakistan, Yemen and six African countries endemic for dracunculiasis) and three of D. insignis (from raccoons trapped in the state of Georgia in the southern U.S.A.). All of the D. medinensis 18S-rRNA sequences were found to be 1819 bases long and identical. The three D. insignis 18S-rRNA sequences were also found to be identical to each other but were 1821 bases long and differed from the D. medinensis 18S-rRNA sequence at eight positions (representing a difference of 0.44%). The 18S-rRNA coding region of a Guinea worm extracted from a dog in Ghana was indistinguishable from that of the D. medinensis isolates from human cases. These results provide the basis for the molecular differentiation of D. medinensis that will permit the DEP to determine, rapidly and accurately, whether a worm recovered from an area considered dracunculiasis-free is a specimen of D. medinensis or not.

The Peculiar Epidemiology of Dracunculiasis in Chad

“…Morphological distinction (and thus confirmatory diagnosis) is difficult as there are no clearly defined morphologic differences evident on emergent female worms or L1 larvae to distinguish species2,4; the most salient morphological features distinguishing species of Dracunculus are present in the male worms, which are rarely recovered. Thus, it is only recently that North American dracunculid infections of wildlife, classically considered D. medinensis , have been distinguished through molecular biology as distinct species ( D. insignis and D. lutrae ) 6,7,13,14. To date, there have been no descriptions of other Dracunculus species in mammals in Africa except D. medinensis , nor is there evidence to date that suggests animals act as reservoir hosts for human Guinea worms.…”

“…Initially, worms were evaluated through polymerase chain reaction (PCR) amplification and DNA sequence analysis of the 18S small subunit ribosomal RNA locus. This method was previously described to distinguish D. medinensis from Dracunculus insignis ,6 a closely related species that occurs in wildlife in North America. This method amplifies 1.8 Kb of the gene, with a difference in both total length (1,819 bases long in D. medinensis , and 1,821 in D. insignis ) and at eight positions (= difference of 0.44%).…”

“…All worms from Chad were subjected to evaluation of both genes. To confirm the species identity of the specimens recovered from people and dogs in Chad, DNA from female worms or larvae was amplified as previously described and sequenced in both forward and reverse directions6,7; the resulting sequences were assembled and compared with GenBank references using the basic local alignment search tool (BLAST) algorithm.…”

Population Genetics and Molecular Epidemiology of Eukaryotes

“…In several recent cases, the zoonotic contribution to human disease could only be appreciated by molecular techniques. Morphologically similar parasites may infect human and/or animal hosts without belonging to the same species (15). In other cases, there may be new or unappreciated hosts, and these must be verified using molecular techniques.…”

Emergence of Onchocerca volvulus from Skin Mimicking Dracunculiasis medinensis

“…To assist in this identification, a molecular method was developed at CDC to distinguish D. medinensis from other potential zoonotic Dracunculus spp. 4 To date, 58 specimens have been submitted for verification, and most have proven to be D. medinensis . However, not all submitted worms are D. medinensis .…”