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 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.

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 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.(AU)

Genotyping of clinical isolates of Acanthamoeba genus in Venezuela.

Free-living amoebae of Acanthamoeba genus are opportunistic pathogens distributed worldwide. Strains included in this genus are causative agents of a fatal encephalitis and a sight-threating keratitis in humans and other animals. In this study, 550 clinical samples which were collected between 1984 and 2014 from different patients with suspected infections due to Acanthamoeba were initially screened for the presence of this amoebic genus at the Laboratorio de Amibiasis-Escuela de Bioanálisis at the Universidad Central de Venezuela. Samples were cultured in 2% Non-Nutrient agar plates seeded with a layer of heat killed Escherichia coli. From the 550 clinical samples included in this study, 18 of them were positive for Acanthamoeba genus after culture identification. Moreover, positive samples were confirmed after amplification of the Diagnostic Fragment 3 (DF3) of the Acanthamoeba18S rDNA genus and sequencing was carried out in order to genotype the isolated strains of Acanthamoeba. Furthermore, the pathogenic potential of the strains was checked by performing thermotolerance and osmotolerance assays. Sequencing of the DF3 region resulted in the identification of genotype T4 in all the isolated strains. Moreover, most isolates were thermotolerant or both thermotolerant and osmotolerant and thus were classified as potentially pathogenic strains. To the best of our knowledge, this is the first report on the molecular characterization at the genotype level of Acanthamoeba strains in Venezuela.