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)

[The immunosuppressant effect of T. lewisi (Kinetoplastidae) infection on the multiplication of Toxoplasma gondii (Sarcocystidae) on alveolar and peritoneal macrophages of the white rat]. / Efecto inmunosupresor de Trypanosoma lewisi (Kinetoplastidae) sobre la multiplicación de Toxoplasma gondii (Sarcocystidae) en macrófagos alveolares y peritoneales de rata blanca.

The immunosuppressant effect of T. lewisi infection on the multiplication of T. gondii was compared in peritoneal (MP) and alveolar macrophages (MA) of white rat. Two animal groups were infected with T. lewisi and sacrificed after four days and seven days post infection. A group without infection was maintained as a control. The number of intracellular parasites (tachyzoites) (IT) was counted by light microscopy, calculating the rate infection rate per 100 total cells (TC) and per infected cells (IC) for each group of phagocyte cells. The relation quotient IT, TC or IC multiplied percent, provided a statistical ratio (RE) of the relative number of parasites in both cellular types for each time interval. MA as well as MP obtained after 4 days showed a significant increase in the multiplication of T. gondii with respect to the control. Unlike the MP (which had an increase in the multiplication of T. gondii the fourth day of infection with T. lewisi diminishing towards the seventh day), the MA had an increase in the multiplication of the parasite from the fourth to the seventh day. This difference can be related to the route of infection used for the experiments, that affect the MP directly with a greater effect in comparison with the MA of the lungs. Lung compartment will be affected later, when the infection becomes systemic between the fourth and sixth day of infection. The immunity against T. gondii is similar between both phagocytes, but the time of infection and the compartment where the cells are located, makes the difference in the response time against T. gondii. Supernatants from macrophage cultures or T. lewisi by rat did not induced any immunosuppression.

[Intestinal parasites in howler monkeys Alouatta palliata (Primates: Cebidae) of Costa Rica]. / Paráisitos intestinales en monos congo Alouatta palliata (Primates: Cebidae) de Costa Rica.

Fecal samples of 102 howler monkeys (Alouatta palliata) from several sites of Costa Rica were studied for intestinal parasites. The zones studied were: Central Valley (San Ramón, Alajuela), Central Pacific (Chomes and Manuel Antonio National Park. Puntarenas), North Pacific (Palo Verde Park and Playa Potrero, Guanacaste). Chira Island in the Nicoya Gulf and Caribean area (Cahuita. Limón). Animals were anesthetized with dards containing Telazol in order to collect the fecal material; some monkeys defecated spontaneously and others by direct stimulation. Samples were studied in saline solution (0.85%) and a Iodine solution, or stained with Haematoxylin. The material was also cultured in Dobell culture medium to determine the presence of amoeba and flagellates. Strongvloides. Controrchis. Trypanoxyuris genera were found in 3.4% of the samples. In addition 16.7% to 80% of the animals showed protozoa infection with Endolimax, Entamoeba, Trichomonas and Giardia. It is discussed the relationships of parasite infection with environmental conditions, animal population and human presence, specially in the monkey conservation programs point of view.