Standardization of molecular techniques for the detection and characterization of intestinal protozoa and other pathogens in humans.

BACKGROUND

The intrinsic sensitivity limitations of basic parasitological methods, along with the particular biological characteristics of parasites, make these methods ineffective to differentiate morphologically indistinguishable species. Molecular detection and characterization techniques could be used to overcome these problems. The purpose of this work was to standardize molecular polymerase chain reaction (PCR) techniques, described in the literature, for the detection and molecular characterization of intestinal protozoa and other pathogens in humans.

METHODS

DNA was extracted from human or animal feces, previously washed or cultured in Boeck Drbohlav's Modified Medium. DNA extraction was performed with Machery-Nagel extraction kits. The standardization of the PCR, nested-PCR or RFLP techniques was carried out according to the literature. For each molecular technique performed, the sensitivity of the test was determined based on the minimun quantity required of DNA (sensitivity A) and the minimum quantity of life forms that the test detected (sensitivity B).

RESULTS

Sensitivity A was 10 fg for , 12.5 pg for or , 50 fg for spp., 225 pg for spp. and 800 fg or 8 fg for spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for 500 cysts for or , 1000 oocysts for spp. and 3600 or four vegetatives forms for PCR or nested PCR of spp., respectively.

CONCLUSIONS

The molecular detection of protozoa and chromist was achieved and the molecular characterization allowed the genotyping of some of the parasites such as , spp., and spp. This study summarizes the molecular techniques for epidemiological studies in humans and animals, and helps in the investigation of their transmission sources in countries where intestinal parasites are a public health problem.