Heterologous expression of three antigenic proteins from Angiostrongylus cantonensis: ES-7, Lec-5, and 14-3-3 in mammalian cells.

Angiostrongylus cantonensis is a parasitic nematode and the main causative agent of human cerebral eosinophilic meningoencephalitis (EoM). A definitive diagnosis of EoM usually requires serologic or molecular analysis of the patient's clinical sample. Currently, a 31 kDa antigen is used in immunological tests for this purpose, however as a crude antigen preparation it may present cross-reactivity with other helminthic infections, especially echinococcosis. Heterologous expression studies using prokaryotic systems failed on producing antigenic proteins. The aim of this study was to express and purify three recombinant glycoproteins representing A. cantonensis antigens: ES-7, Lec-5, and 14-3-3, in Chinese hamster ovary (CHO) cells and ES-7 in human embryonic kidney (HEK) cells to develop a source of specific antigens to be used in the diagnosis of angiostrongyliasis. The potential diagnostic value of these three proteins was subsequently characterized in one- and two-dimensional electrophoresis and Western blot to dot blot analyses, with Angiostrongylus-positive sera, normal human sera (NHS), and a pool of Echinococcus-positive sera (included as a specificity control) used for detection. In addition, recognition of these three proteins following treatment with N-glycosidase F was examined. The ES-7 proteins that were expressed in HEK and CHO cells, and the Lec-5 protein that was expressed in CHO cells, were specifically recognized by A. cantonensis-positive sera in the 2D electrophoresis analysis. This recognition was shown to be dependent on the presence of glycidic portions, making mammalian cells a very promising source of heterologous expression antigenic proteins from Angiostrongylus.

LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools.

BACKGROUND: Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species. METHODS: PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools. RESULTS: Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli. CONCLUSION: These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.

Genotypic identification of Cryptosporidium spp. isolated from HIV-infected patients and immunocompetent children of São Paulo, Brazil.

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the State of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that corresponded to Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil / Identificação genotípica de Cryptosporidium spp. isolados a partir de pacientes com HIV e crianças imunocompetentes de São Paulo, Brasil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Isolados de Cryptosporidium identificados em quatorze amostras de fezes, coletadas de cinco pacientes com infecção por HIV e de nove crianças imunocompetentes, residentes no estado de São Paulo, Brasil, foram submetidos a análise molecular por Nested-PCR, seguido da caracterização genética por polimorfismo do tamanho do fragmento de restrição (RFLP). A análise foi baseada na digestão, com a enzima de restrição RsaI, de um fragmento de DNA amplificado do gene que codifica a proteína de parede do oocisto de Cryptosporidium (COWP). Baseado nesta análise, quando comparadas aos padrões utilizados, quatro amostras foram identificadas como Cryptosporidium parvum, oito como Cryptosporidium hominis e duas apresentaram um perfil correspondente ao de Cryptosporidium meleagridis. O uso de métodos moleculares pode ser útil para identificar a fonte das infecções e os fatores de risco relacionados à infecção por Cryptosporidium em nossas comunidades.