Improved serodiagnosis of histoplasmosis by use of deglycosylated extracellular released antigens of Histoplasma capsulatum.

The diagnosis of histoplasmosis depends on various approaches: direct clinical examination, fungus isolation from cultures of clinical samples, histopathological evaluation, and serological testing. In serodiagnostic assays, the Histoplasma capsulatum H and M antigenic glycoproteins have been extensively used. However, both antigens showed limitations attributed mainly to their cross-reactivity with glycoproteins from other pathogenic fungi, which compromises specificity, and generates false positives, misdiagnosis, and therapeutic failure. In this work, we deglycosylated extracellular released antigens from the Venezuelan 7090 H. capsulatum clinical isolate, using chemical and enzymatic methods and evaluated their effectiveness by indirect enzyme-linked immunosorbent assay (ELISA) with sera from patients with either histoplasmosis or PCM. Prior to deglycosylation, the extracellular released antigen showed 62% of sensitivity 66% of specificity and 68% of cross-reactivity with paracoccidioidomicosis sera. The chemically deglycosylated extracellular released antigen, for 8 or 18  h showed 72 and 52% sensitivity with 98% and 92% specificity, respectively. Moreover, cross-reactivity with Paracoccidioides decreased to 4 and 16%, following deglycosylation for 8 or 18 h, respectively. The enzymatically treated antigen showed 52% of sensitivity, 92% of specificity and 8% cross-reactivity against Paracoccidioides. Deglycosylation of the H. capsulatum antigen improves its specificity and decreases its cross-reactivity against Paracoccidioides when using indirect ELISA for serodiagnosis. Therefore, it is recommended to deglycosylate the fungal extracellular released antigen for clinical serodiagnosis, and to monitor humoral immune responses during therapy of patients with the different clinical forms of histoplasmosis.

[Description of an acidic peptidase, insensitive to classical inhibitors, in protein extracts of Trypanosoma cruzi, from a rural area of Venezuela, where Chagas disease is endemic]. / Una peptidasa ácida, insensible a inhibidores clásicos, en extractos proteicos de Trypanosoma cruzi, proveniente de una zona rural de Venezuela, endémica para la enfermedad de Chagas.

Through two peptidase assay methods, one in liquid-phase and another, in gel-phase (gel zymography), an acid peptidase was detected in protein crude extracts of epimastigotes of Trypanosoma cruzi, from a rural area of Venezuela where Chagas disease is endemic. The peptidase shows activity at a pH range between 2.0 and 2.9. Under the experimental conditions described, the acid peptidase was insensitive to usual concentrations of peptidase inhibitors of the types: serine, cysteine, aspartic and metallopeptidases. Nevertheless, like porcine pepsin at pH 2.9, the peptidase was inhibited in the presence of 5mM DTT.

Una peptidasa ácida, insensible a inhibidores clásicos, en extractos proteicos de Trypanosoma cruzi, proveniente de una zona rural de Venezuela, endémica para la enfermedad de Chagas / Description of an acidic peptidase, insensitive to classical inhibitors, in protein extracts of Trypanosoma cruzi, from a rural area of Venezuela, where Chagas disease is endemic

Mediante dos métodos de ensayo de peptidasas, uno en fase líquida y otro en fase gel (zimografía en geles), se detectó una peptidasa, en extractos proteicos crudos de epimastigotes de Trypanosoma cruzi, provenientes de un área rural de Venezuela endémica para el mal de Chagas. La peptidasa mostró actividad en el intervalo de pH comprendido entre 2,0 y 2,9. Bajo las condiciones experimentales descritas, la peptidasa resultó insensible a concentraciones usuales de inhibidores clásicos de peptidasas de tipo: serina, cisteína, metalo-peptidasas y aspártico. No obstante, a semejanza de la pepsina porcina a pH 2,9, la peptidasa es inhibida en presencia de 5mM DTT.

Through two peptidase assay methods, one in liquid-phase and another, in gel-phase (gel zymography), an acid peptidase was detected in protein crude extracts of epimastigotes of Trypanosoma cruzi, from a rural area of Venezuela where Chagas disease is endemic. The peptidase shows activity at a pH range between 2.0 and 2.9. Under the experimental conditions described, the acid peptidase was insensitive to usual concentrations of peptidase inhibitors of the types: serine, cysteine, aspartic and metallo-peptidases. Nevertheless, like porcine pepsin at pH 2.9, the peptidase was inhibited in the presence of 5mM DTT.

Gelatinase activity in Mycobacterium bovis protein extract.

Proteases are well-recognized as virulence factors in different pathologies, resulting in tissue damage potential. Despite efforts over the past few years to identify mycobacterial protein antigens, there is little information regarding the role of mycobacterial proteinase activities. In this study, by zymography techniques, we have detected and partially studied some biochemical properties of Mycobacterium bovis proteases, such as pH dependency of activity and susceptibility to classical proteinase inhibitors. We observed optimal proteolytic activity at pH 8. Some proteinases were inhibited by classic inhibitors of serine proteases, such as PMSF, AEBSF, and 3-4 DCI. In some AEBSF pre-treated preparations we observed residual gelatinase activity in Rf 0.32. This gelatinase was stimulated by Zn2+ and inhibited by OPA (1 mM). This last effect was reversed by exposure to equimolar quantitative OPA/Zn+2 (1 mM/1 mM). These results suggest the existence of serine proteinase and metalloproteinase types in protein extracts of Mycobacterium bovis.