Use of immunoblotting assay improves the sensitivity of paracoccidioidomycosis diagnosis

Abstract

ABSTRACT:The purpose of this work was to evaluate two serological assays: double immunodiffusion (DI) and immunoblotting (IB) in immunodiagnosis of paracoccidioidomycosis (PCM). We evaluated by IB assay 23 sera samples from patients with clinical confirmation of PCM, all of them with negative DI results against culture filtrate from Paracoccidioides brasiliensis isolate 113. For IB, as well as for comparative DI assay, we employed soluble components of the cell wall outer surface (SCCWOS) from P. brasiliensis isolate 113 cultivated at 36°C in Fava-Neto's agar medium for 5 and 10 days. Among the 20 sera samples analyzed by DI, 13 (65%) were negative and 7 (35%) were positive against SCCWOS obtained on the 5 th and 10 th days. By IB assay, 95.4% and 100% of sera reacted against gp43 and gp70 present in SCCWOS from the 5 th day and 95.6% recognized these fractions when evaluated against SCCWOS from the 10 th day. Our results demonstrated that the use of an immunoenzymatic assay significantly improves the sensitivity of PCM immunodiagnosis and also suggests that at least two serological tests for antibody detection should be adopted in cases of questionable diagnosis.

Western Blotting Is an Efficient Tool for Differential Diagnosis of Paracoccidioidomycosis and Pulmonary Tuberculosis

“…On the other hand, WB allowed demonstration of antibodies to the specific antigen (gp43) in sera of 100% of patients positive for PCM. These antibodies have been considered important markers for the diagnosis of PCM (3, 6,12,16).The major contribution of this preliminary study was, therefore, to show that the WB technique can be used as a tool to confirm the differential diagnosis between TB and PCM and to reinforce our previous publication (12), and WB proved to be a particularly useful tool for PCM patients with a negative DID test. The use of this technique resulted in referrals for appropriate treatment of at least six patients who were triaged and referred for AFB investigation.…”

“…On the other hand, WB allowed demonstration of antibodies to the specific antigen (gp43) in sera of 100% of patients positive for PCM. These antibodies have been considered important markers for the diagnosis of PCM (3,6,12,16).…”

Serological Diagnosis of Paracoccidioidomycosis through a Western Blot Technique

“…WB analysis has been used in studies on PCM by several authors (2,10,20,22), but the focus of most studies has been clinical forms and treatment monitoring of PCM (9, 13). There was a previous study similar to ours (9) in which the authors correlated the findings of the same two serological tests, DID and WB analysis, with the clinical diagnosis and follow-up of PCM.…”

“…However, that study analyzed only confirmed patients, whereas in our study, patients were not screened with respect to PCM diagnosis, and yet their results were in accord with ours. Recently, Silva et al (20) compared DID and WB analysis using 23 sera and found that 95.4% of sera negative by DID gave a positive reaction to gp43 from an antigen obtained from a P. brasiliensis culture filtrate by WB analysis. Previously, we found similar results (22).…”

“…Thus, so far none of the procedures used was sufficient to eliminate cross-reactivity of ELISA for PCM. On the other hand, Western blot (WB) analysis offers an advantage over ELISA, since prior protein electrophoresis ensures the presence of markers such as gp43 and, according to some studies, the sensitivity is high (10,20,22), but results have been obtained with a small number of patients, and the technique needs to be tested using a larger sample population. This study aimed at evaluating WB analysis using sera of patients suspected of PCM to determine whether it represents a specific and sensitive serological technique for a rapid and effective diagnosis of this disease.…”