Specific amplification of Necator americanus or Ancylostoma duodenale DNA by PCR using markers in ITS-1 rDNA, and its...

Necator americanus and Ancylostoma duodenale are the two most important species of human hookworm, and occur in sympatry over much of their distribution. The specific diagnosis of hookworm infections is central to control. Diagnosis currently relies on the detection of hookworm eggs in human faeces and/or the specific identification of larvae by 'copro-culture' combined with microscopic examination. However, the eggs of the two species are morphologically indistinguishable, and the procedure of copro-culture is tedious and time-consuming to carry out. To work toward overcoming these limitations, a molecular approach utilizing genetic markers in the first internal transcribed spacer (ITS-1) of ribosomal DNA (rDNA) was established. The ITS-1 sequences of both hookworm species were determined, and specific oligonucleotide primers designed to regions of major sequence difference between the species were evaluated in polymerase chain reaction (PCR). Using a range of control samples, the primers allowed the specific identification of as little as 10 pg DNA of A. duodenale or N. americanus. The findings indicate clearly the potential for specific PCR to confirm the identity of eggs from faeces and larvae from the environment or host tissues. This should have important implications for studying fundamental aspects relating to anthelmintic efficacy and the epidemiology of hookworms.