Evaluation of different amplification targets for the detection of Necator americanus by PCR in stool samples

Hookworm infection, caused by Necator americanus and Ancylostoma duodenale, is prevalent in areas with poor sanitation. Coprology is the gold standard for diagnosis, but it has low sensitivity in low parasite loads. The objective of this study was to evaluate four Polymerase Chain Reaction (PCR) targets for molecular diagnosis. Stool samples were collected from 180 residents of the rural community Tierra Blanca, Bolívar State, Venezuela, in which coproparasitological tests were performed. Four DNA extraction methods were standardized, and four PCR techniques were studied: NaITS1F/NaITS1R (ITS1), Na58/Na158R (ITS2), NA/NC2 (ITS2-28S) and NC1/NC2 (ITS2-28S) for N. americanus. Only two of these techniques could be standardized and evaluated, as amplification was not achieved with the other two. The two standardized techniques were applied to all samples, and diagnostic indices were obtained. The results showed that extraction with the Wizard kit was the most suitable due to the quality of the DNA obtained. Primer analysis revealed high identity, but with sequences mainly from Asia and Africa, since there are very few available from America. PCR-NaITS1F/NaITS1R (ITS1) demonstrated the best diagnostic indices, significantly surpassing coprology. PCR-Na58/Na158R (ITS2) showed lower sensitivity than coprology. Therefore, PCR-NaITS1F/NaITS1R (ITS1) is recommended for molecular diagnosis. The prevalence of N. americanus in the Tierra Blanca community was 20.6 % by coprology, 88.3 % by PCR-NaITS1F/NaITS1R (ITS1) and 94.4% by the combination of these techniques. The high prevalence of infections underscores the need for molecular techniques but these must be previously evaluated with samples from the regions where they will be used.