Abstract
Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.
Highlights
The species of dimorphic fungi of the Paracoccidioides brasiliensis complex and P. lutzii are the etiological agents of paracoccidioidomycosis (PCM) [1], which is a human systemic mycosis highly prevalent in South American countries [2]
To detect anti-Hsp60 and anti-gp43 antibodies in sera from patients with PCM, we produced His-tagged the receiver-operating characteristics (ROC) curve, based on the absorbance at 405 nm obtained with sera from PCM patients and negative control (NC) subjects that were submitted to reaction with gp43
The titers of the sera from PCM patients with rgp43 were significantly higher than those from NC subjects (Figure 3A) and about 87% of the sera from PCM patients were positive for rgp43
Summary
The species of dimorphic fungi of the Paracoccidioides brasiliensis complex and P. lutzii are the etiological agents of paracoccidioidomycosis (PCM) [1], which is a human systemic mycosis highly prevalent in South American countries [2]. Brazil has been considered one of the most endemic areas of the world, with about 80% of all active cases of PCM [3]. The polyclonal activation of B cells is a characteristic of PCM that frequently results in hypergammaglobulinemia [4]. Serological assays have proven helpful in clinical monitoring and followup of therapy in PCM patients [5]. Many serological techniques have been widely used to evaluate the concentration of antibodies in sera from PCM patients, such as immunodiffusion, counterimmunoelectrophoresis, double immunodiffusion and immunoenzymatic assays (reviewed in Ref. 5). The 43-kDa glycoprotein (gp43) is unambiguously the most studied P. brasiliensis antigen. Besides being one of the most predominant glycoproteins in P. brasiliensis, gp is recognized by most sera from PCM patients [7]
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