Evaluation of two PCR protocols for the detection of Entamoeba histolytica/E. dispar/E. moshkovskii in stool samples.

The diagnosis of amoebiasis (caused by Entamoeba histolytica) is performed using coprological techniques, but these have low sensitivity in cases of low parasite loads. Furthermore, other species such as Entamoeba dispar and E. moshkovskii cannot be morphologically differentiated. PCR could be an alternative. In this study, four DNA extraction techniques were compared, and two PCR techniques (based on the 18S sequence, PCR-Eh for E. histolytica and multiplex PCR-Ehdm for E. histolytica, E. dispar, and E. moshkovskii) were standardized for molecular diagnosis in stool samples. The specificity of these techniques were determined. The two standardized PCR assays were applied to 180 stool samples from individuals from a rural Venezuelan community endemic for Entamoeba spp. The PCR results were compared with those obtained by coprology. Extraction with the Wizard kit was the most suitable due to the quality of the DNA obtained. Standardization of the PCR assays allowed for obtaining the optimal reagent concentrations for the best amplification, which were lower than those used by the original authors. 18S sequence analysis showed genetic variability. The sensitivity of the two standardized PCR assays was lower than coprology in the evaluated samples, perhaps due to genetic variability of the parasites circulating the area. Therefore, PCRs must be previously evaluated with samples from the regions where they will be used. The high prevalence of this infection detected by coprology highlights the need of molecular techniques for diagnosis.