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

Abstract

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 G. duodenalis , 12.5 pg for Entamoeba histolytica or Entamoeba dispar , 50 fg for Cryptosporidium spp., 225 pg for Cyclospora spp. and 800 fg or 8 fg for Blastocystis spp. after performing a 1780 bp PCR or 310 bp nested PCR, respectively. The sensitivity B was 100 cysts for G. duodenalis, 500 cysts for E. histolytica or E. dispar , 1000 oocysts for Cyclospora spp. and 3600 or four vegetatives forms for PCR or nested PCR of Blastocystis 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 Giardia duodenalis , Cryptosporidium spp., and Blastocystis 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.

Intestinal Parasitic Infections Among Pediatric Patients in a Metropolitan City of Bangladesh With Emphasis on Cryptosporidiosis

“…Our findings were similar to earlier studies [44,168]. The finding suggests the transmission mode could be the person-to-person contact-no association of the pathogen with the rural resident, supporting some previous studies [118,124,169,170].…”

“…A prospective study on children under 16 years showed that 7.7% of patients were detected by PCR [ 60 ]. Another study found the prevalence of this parasite is about 1.3% [ 118 ]. Improved sanitation, safe drinking water, and awareness about health and hygiene enable a lower prevalence of these parasitic diseases in the community [ 119 ].…”