USE OF IMMUNOBLOTTING ASSAY IMPROVES THE SENSITIVITY OF PARACOCCIDIOIDOMYCOSIS DIAGNOSIS

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 and 10 days. By IB assay, 95.4% and 100% of sera reacted against gp43 and gp70 present in SCCWOS from the 5 day and 95.6% recognized these fractions when evaluated against SCCWOS from the 10 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.


INTRODUCTION
Paracoccidioidomycosis (PCM) is a systemic, chronic, granulomatous, progressive disease caused by the dimorphic fungus Paracoccidioides brasiliensis (5).The definitive diagnosis of PCM is usually based on the demonstration of multibudding yeast cells in different biological specimens and/or the isolation of the fungus by culturing; however, the former is insensitive, and the latter is time-consuming (5).
Consequently, serological techniques are an important tool not only for disease diagnosis but also for monitoring the patients' response to treatment (6,8).
Circulating antibodies to P. brasiliensis can be detected in the patients' sera by serological assays such as DI, counterimmunoelectrophoresis and immunoenzymatic assays (6,20).DI test is routinely used by clinical laboratories due to its easy procedure, low cost involved in its execution as well as its high specificity (about 100%) and sensitivity (65%-90%) (6,8).The use of undefined antigens has imposed important limitations on DI test; cross-reactivity has been a problem, as has the absence of antigen standardization (6,8).Variation in antigen production can arise from differences in the used strains, in the chosen fungus growth phase, in the incubation time, and in the employed culture media (6,8).Among the antigens used in serological approaches, gp43 is the most frequently studied P. brasiliensis component (6,8,11).Del Negro et al. (9) described that rarely a patient presents negative DI results.However, when further analyzed by an immunoenzymatic assay such as IB, these sera demonstrate anti-gp43 antibodies, probably due to the relatively low sensitivity of precipitation assays.The purpose of this work was to evaluate two serological tests: DI and IB in immunodiagnosis of PCM.

Paracoccidioides brasiliensis Antigens
Two types of antigens were used in this study.The first was a 20-day culture filtrate (Ag CF) obtained according to Garcia et al. (10) from the yeast phase of P. brasiliensis.Briefly, the fungus was cultured in NGTA liquid medium for 20 days at 36 o C with shaking.After incubation time, the culture was treated with boratethimerosal solution (1:5,000), filtered, divided into small volumes and stored at 4 o C until use.The second antigen employed was P. brasiliensis SCCWOS obtained 15 years ago by Assis (1)(2)(3)(4).Yeast cells were grown in Fava Netto's agar at 36 o C. The cells were collected on days 5 and 10, suspended in 0.85% NaCl solution and pelleted by centrifugation.Supernatants, treated with borate-thimerosal solution (1:5,000) and containing the soluble components, were divided into small volumes and stored at 4 o C until use.

Sera Samples
Twenty-three sera samples from patients with proven cases of active PCM (13 sera obtained from patients with chronic and 10 from patients with acute form of the disease), but non-reactive, with culture filtrate or SCCWOS antigens by DI assay, were analyzed.The diagnosis was established by histological or direct microscopic examination and/or by isolation of P. brasiliensis.

Double Immunodifusion Assay
Reactions were performed according to the modified method of Ouchterlony (18).
Glass slides were covered with 3.0ml of a gel composed of 1% agarose type II medium (Sigma Chemical Co.St. Louis, Mo, USA) in a buffered saline (pH 6.9) containing 0.4% sodium citrate and 7.5% glycine.Antigen (12μl) was placed in the central well, reference and patients' sera (12μl) in the surrounding wells.The slides were incubated in a humid chamber at room temperature for 48h.They were then washed in saline with several changes over a 24h period.The gels were dried and stained with 0.4% Coomassie Brilliant Blue R-250 (Sigma Chemical Co.St. Louis, Mo, USA) in an ethanol-acetic acid-water mixture as the solvent.

SDS-PAGE and Immunoblotting Assay
For sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), P. brasiliensis antigens were diluted in a buffer containing 62mM Tris-HCl (pH 6.8); 2% (wt/vol) SDS, 50mM 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue were boiled for 3min and centrifuged before application to gels.Cell wall antigens were then subjected to electrophoresis (20mA at room temperature) on a 10% discontinuous SDS buffer system (13) in a Mini-protean II, Electrophoresis Cell (Bio Rad Laboratories, Richmond, CA, USA), and the molecular mass was determined by the use of a 6.5-175kDa standard prestained protein marker (New England BioLabs).Immunobloting assays were conducted as previously described (19).

RESULTS
Table 1 summarizes the DI results.Among the 23 sera samples analyzed, 100% showed negative DI results against culture filtrate antigen; 13 (65%) were negative and 7 (35%) were positive against SCCWOS antigens obtained on the 5 th and 10 th days of culture.However, when these negative sera were further analyzed by IB assay, we observed that 95.4% and 100% of sera reacted against gp43 and gp70 present in SCCWOS from 5 days, and 95.6% recognized these fractions when evaluated against SCCWOS from 10 days.(Figure 1

DISCUSSION
For several years, many different antigenic preparations have been used for the immunodiagnosis of PCM by DI assay.However, these various antigenic preparations lacked a standardized preparation from one laboratory to another and included cytoplasmatic extracts from the yeast forms, concentrated filtrates, lyophilized filtrates, and cell wall components to cite a few approaches (7).Moreover, these antigens were prepared from different P. brasiliensis isolates grown in different culture media as well as under different growth conditions (6,12,16).Distinct P.
Paracoccidioides brasiliensis isolate 113, kindly provided by Professor Dr. Carlos da Silva Lacaz-Culture Collection of the São Paulo Institute of Tropical Medicine [Instituto de Medicina Tropical de São Paulo], Brazil, was used.The isolate was maintained in Potato agar medium at 25 o C and subcultured every three months at the Laboratory of Immunodiagnosis in Mycosis, Adolfo Lutz Institute, São Paulo, Brazil.
Proteins from SDS-PAGE were electrotransferred onto 0.20μm nitrocellulose membranes (Sigma Chemical Co.St. Louis, Mo, USA) in a Mini Trans-Blot Cell (Bio Rad Laboratories, Richmond, CA, USA), with 25mM Tris, 192mM glycine, pH 8.3, and methanol 20% (v:v).The nitrocellulose membranes containing electrophoresed antigens were blocked with 5% non-fat dry milk in PBS for 1h at room temperature.Membranes were incubated for 2h at room temperature with human sera diluted 1:40 in PBS containing 0.05% Tween-20 (PBS-T), then washed six times with PBS-T and developed with peroxidase-conjugated goat anti-human IgG antibody (Sigma Chemical Co.St. Louis, Mo, USA) for 2h at room temperature.The reactions were visualized with 4-chloro-1-naphtol substrate (Sigma Chemical Co.St. Louis, Mo, USA).

Figure 1 .
Figure 1.Antibody response to a soluble component of the cell wall outer surface of Paracoccidioides brasiliensis obtained on the 5 th (Panel A) and 10 th (Panel B) days of culture probed with 23 negative double immunodiffusion paracoccidioidomycosis sera samples (Lanes 1 to 23).Goat anti-human IgG serum conjugated with peroxidase was used as the second antibody.

Table 1 .
) Reactivity pattern of paracoccidioidiomycosis sera samples by double immunodiffusion and immunoblotting assays employing culture filtrate antigen (Ag CF) and soluble components of the cell wall outer surface (SCCWOS) of Paracoccidioides brasiliensis antigens obtained from isolate113.