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.

High immunological response against a Trypanosoma equiperdum protein that exhibits homology with the regulatory subunits of mammalian cAMP-dependent protein kinases.

Previously, we have identified a protein in Trypanosoma equiperdum that possesses homology with the regulatory (R) subunits of the mammalian cAMP-dependent protein kinase (PKA). The recombinant T. equiperdum PKA R-like protein was expressed in bacteria and purified to homogeneity. Mice polyclonal antibodies were raised against the recombinant R-like protein to serologically evaluate its humoral immune response. High titers of specific sera antibodies were obtained against the parasite R-like protein by indirect enzyme-linked immunosorbent assay (ELISA), and immunoblots revealed that this protein was specifically recognized by the hyperimmune mice sera. Cellular proliferation assays using splenic B cells from the immunized mice showed higher values when the recombinant T. equiperdum R-like protein was employed than when concanavalin A was utilized as an unspecific mitogen. Two healthy horses that were experimentally infected using either T. equiperdum or Trypanosoma evansi showed a curve response characterized by the appearance of anti-T. equiperdum PKA R-like protein antibody production in sera using indirect ELISA. The recombinant parasite PKA R-like protein was also recognized by sera from naturally trypanosome-infected horses using western blotting. These findings demonstrated that the T. equiperdum PKA R-like protein is an antigen that exhibits cross-reaction with T. equiperdum and T. evansi.

Serodiagnosis of bovine trypanosomosis caused by non-tsetse transmitted Trypanosoma (Duttonella) vivax parasites using the soluble form of a Trypanozoon variant surface glycoprotein antigen.

Previous studies have shown that a 64-kDa antigen (p64) that was purified from the Venezuelan TeAp-N/D1 isolate of Trypanosoma (Trypanozoon) equiperdum corresponds to the soluble form of its predominant variant surface glycoprotein (VSG), and exhibited cross-reactivity with Trypanosoma (Duttonella) vivax. The course of experimental acute infections of bovines with T. vivax were followed by measuring whole anti-p64 antibodies and specific anti-p64 IgG and IgM antibodies in animal sera by indirect enzyme-linked immunosorbent assay (ELISA). The value of p64 to diagnose bovine trypanosomosis was also examined using 350 sera from healthy and T. vivax-infected cows living in a trypanosomosis-endemic and enzootic stable area, and 48 sera obtained during a trypanosomosis outbreak. Serological assays showed that ∼ 70-80% of the infected sera contained anti-p64 antibodies, based on the comparative immunodetection of the T. equiperdum clarified antigenic fraction used as a reference test. In the absence of a gold standard, Bayesian analysis for multiple testing estimated a sensitivity and specificity of 71.6% and 98.8%, respectively, for the indirect ELISA using p64 as antigen. An apparent prevalence of 37.7% for bovine trypanosomosis infection was also estimated with a Bayesian approach when the p64 ELISA test was used. Employing blood from acute infected cows, the indirect ELISA response against p64 was contrasted with the microhematocrit centrifuge method and analyses by polymerase chain reaction (PCR) using specific primers targeting the inter-specific length variation of the internal transcribed spacer 1 region of the 18S ribosomal gene. The efficiency of p64 for the detection of anti-trypanosome antibodies in acute infected bovines was also corroborated serologically by comparing its response to that of the Indonesian Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2 VSG, which possesses high specificity and sensitivity. As expected, PCR was the best method to detect parasites and diagnose bovine trypanosomosis; however, a substantial level of concordance (Cohen's κ=0.667) was obtained when serological tests using p64 and RoTat 1.2 VSG were compared. Additionally, an agglutination assay was designed using p64 covalently coupled to carboxylate-modified latex microparticles, which was proven here to be suitable for a fast qualitative diagnosis of bovine trypanosomosis.