Pesquisa de oocistos de Cryptosporidium sp. nas fezes diarreicas de aideticos e de criancas e adultos imunocompetentes, em Sao Paulo / The search for oocysts of Cryptosporidium sp. in loose stools of patients with AIDS and immunocompetent children and adults in Sao Paulo

Com o intuito de cooperar no sentido de ficar melhor conhecida a prevalencia da criptosporidiase no Brasil, foram efetuados em Sao Paulo, exames das fezes diarreicas de pacientes com AIDS e de criancas e adultos imunocompetentes. Atraves de metodologia rigorosa houve encontro de oocisos de Cryptosporium sp. nas avaliacoes referentes a 10,4 por cento dos competentes da casuistica, com deteccao de porcentagens mais elevadas nos grupos de aideticos e de criancas atendidas em creche. Os valoes encontrados podem ser considerados expressivos e indicam a necessidade de incluir, rotineiramente, a pesquisa do referido protozoario na analise parasitologica da materia fecal.

Identification of a 15-kilodalton surface glycoprotein on sporozoites of Cryptosporidium parvum.

An immunoglobulin A monoclonal antibody (MAb5C3) was developed against a 15-kDa surface glycoprotein (GP15) of Cryptosporidium parvum sporozoites. Indirect immunofluorescence and colloidal gold immunoelectron microscopy revealed that the antibody reacted with both the sporozoite and merozoite surface plasma membranes. On Western immunoblots, MAb5C3 binding was found to be strongly inhibited when 200 mM N-acetylglucosamine was used as a competing sugar. N-Acetylgalactosamine inhibited binding of the antibody only slightly, whereas glucose, mannose, and galactose failed to inhibit binding. MAb5C3 was found to recognize a similar 15-kDa epitope associated with a Cryptosporidium sp. isolated from guinea pigs. However, MAb5C3 failed to react with any proteins or glycoproteins associated with C. baileyi from chickens, Cryptosporidium sp. (= bovine C. muris) from cattle, C. serpentis from a rat snake, bradyzoites of Besnoitia darlingi from an opossum, sporozoite/oocyst extracts of Caryospora bigenetica from an eastern diamondback rattlesnake, sporozoites of Eimeria nieschulzi and E. papillata from rats and mice, or tachyzoites of Toxoplasma gondii (RH strain). When hybridoma supernatants containing MAb5C3 were administered orally to suckling mice experimentally infected with C. parvum, a 75% reduction in developmental stages was seen histologically at 72 h postinfection and a 67.5% reduction in mean oocyst output was found at 6 days postinfection.

Identification of isolate-specific sporozoite proteins of Cryptosporidium parvum by two-dimensional gel electrophoresis.

Five isolates of Cryptosporidium parvum collected from human, horse, and calf sources were compared for differences in sporozoite protein patterns by using two-dimensional gel electrophoresis. Silver-stained two-dimensional gels contained over 300 protein spots from detergent-solubilized sporozoites. A distinguishing 106-kilodalton peptide that shifted in isoelectric point was detected in four of the five isolates. Computerized two-dimensional gel analysis was performed to obtain objective quantitation of the pI shift. Three of these four isolates could be differentiated from one other by the pI shift in this peptide. The fifth isolate was distinguished by the absence of the 106-kilodalton peptide and the presence of a 40-kilodalton peptide that was not observed in any other isolate.

Identification of outer oocyst wall proteins of three Cryptosporidium (Apicomplexa: Cryptosporidiidae) species by 125I surface labeling.

Autoradiography of oocyst wall surface proteins of three Cryptosporidium spp. revealed common bands at 285 to 290, 145 to 148, 120, 57, and 32 kilodaltons (kDa). Cryptosporidium baileyi and C. muris share proteins at 180, 100, 80 to 81, 29, and 18 to 19 kDa; C. baileyi and C. parvum share one protein at 46 to 47 kDa; and C. muris and C. parvum share a protein at 67 to 69 kDa. Additional protein bands, each unique to one species, were also observed.

Electrophoretic and immunoblot analysis of Cryptosporidium oocysts.

Cryptosporidium oocysts were recovered by density gradient centrifugation from diarrhoeal faeces of four human patients and one goat kid. Goat-derived oocysts were further treated with excystation medium and the excysted oocyst walls purified by isopycnic ultracentrifugation. Soluble extracts from intact oocysts and the oocyst wall preparation were analysed by SDS-PAGE. Fifty-one polypeptide bands were detected in intact oocyst preparations: 48 were in the range 14,000-200,000 molecular weight (MW), two bands were less than 14,000 MW and one band was above 200,000 MW. Twenty-one bands were detected in the oocyst wall preparation, all within the range 14,000-200,000 MW. Immunoblot analysis of Cryptosporidium polypeptides using acute or convalescent human and goat sera revealed a large number of reactive bands. Varying degrees of heterogeneity were observed within and between the two serum groups. Nine of the 10 human sera and all of the goat kid sera reacted with a 23,000 MW and 32,000 MW antigen. A 15,500 MW antigen was also detected by all the goat and four of the 10 human sera. Both serum groups reacted with various antigens above 40,000 MW. Surface labelling of three human isolates of Cryptosporidium oocysts with 125I was performed using the Bolton and Hunter reagent. The solubilized preparations were separated by SDS-PAGE on 12% and 18% slab gels and autoradiographed. Common bands were seen at 15,500, 32,000, 47,500, 79,000 and 96,000 MW. Some variation in the molecular weight of polypeptides labelled with 125I was observed among the three isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Diagnóstico laboratorial de Cryptos poridium / Laboratory diagnosis of Cryptosporidium

Säo apresentados seis casos de pacientes nos quais foram encontrados oocistos de Cryptosporidium nas fezes. As alternativas para o diagnóstico laboratorial säo apresentadas e discutidas, sendo a modificaçäo da técnica de Ziehl - Neelsen sugerida como a mais conveniente. Os autores sugerem amplo estudo de prevalência para determinar a importância de Cryptosporidium em nosso meio

Cryptosporidium sp: detección de ooquistes en deposiciones preservadas en fenol-alcohol-formalina (PAF) y acetato de sodio, ácido acético y formalina (SAF) / Cryptosperidium sp: oocysts detection in faeces preserved with phenol-alcohol-formaline (PAF) and sodium acetale-acetic acid-formaline (SAF)

Muestras fecales positivas a ooquistes de Cryptosporidium sp fueron preservadas en los fijadores PAF, SAF, Formol-sal y NaCl 0,85%. Después de centrifugar se practicaron frotis con estas muestras y se tiñeron con Ziehl-Neelsen, Aureamina y Giemsa. Con Ziehl-Neelsen y Aureamina se observaron ooquistes en todos los frotis, en cambio con Giemsa sólo en los preservados en SAF. Los solventes de grasas acetato de etilo y éter etílico no afectaron la propiedad ácido-alcohol resistente de los ooquistes. La recuperación de ooquistes preservados en los diferentes fijadores y sometidos a centrifugación fue similar cuando se practicó recuento de su número en frotis teñidos con Ziehl-Neelsen y Aureamina. Se concluye que el diagnóstico de laboratorio de la Cryptosporidiosis intestinal se puede efectuar en muestras de deposiciones preservadas en fijador PAF (método de Burrows) y fijador SAF (método de Yang y Scholten)