Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis.

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

Schistosoma mansoni: molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages.

Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase (SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composed of 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPI anchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blot experiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalization analysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of this enzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.

Schistosoma mansoni Molecular characterization of Alkaline Phosphatase and expression patterns across life cycle stages

Here we describe the cloning and characterization of the Schistosoma mansoni Alkaline Phosphatase(SmAP), previously identified in the tegument of adult worms. SmAP encodes a complete sequence composedof 536 amino acids containing an N-terminal signal peptide, five N-glycosylation sites, and a GPIanchor signal, similar to that described for mammalian orthologs. Real-time RT-PCR and Western blotexperiments suggest a rapid translation as soon as cercariae are transformed into schistosomula. Immunolocalizationanalysis shows that the protein is widely distributed in the worm tissues, with increased concentration in the vitelline glands of female parasites. Furthermore, the surface localization of thisenzyme was quantitatively supported by its enzymatic activity in live ex vivo or cultured parasites throughout the life cycle stages. The fact that cercariae accumulate large amounts of SmAP mRNA, which rapidly translates into protein upon schistosomula transformation, indicates it may have an important role in host invasion.