Use of the 27-Kilodalton Recombinant Protein fromParacoccidioides brasiliensisin Serodiagnosis of Paracoccidioidomycosis

Abstract

Paracoccidioidomycosis (PCM) is one of the most important endemic mycoses in Latin America; it is usually diagnosed by observation and/or isolation of the etiologic agent, Paracoccidioides brasiliensis, as well as by a variety of immunological methods. Although the latter are effective, two circumstances, cross-reactions with other mycotic agents and antigen preparation that is marked by extreme variability among lots, hinder proper standardization of the procedures. To circumvent this lack of reproducibility, molecular biology tools were used to produce a recombinant 27-kDa-molecular-mass antigen from this fungus; a sizable quantity of this antigen was obtained through fermentation of Escherichia coli DH5α, which is capable of expressing the fungal protein. The latter was purified by the Prep-Cell System (Bio-Rad); the recovery rate of the pure protein was approximately 6%. A battery of 160 human serum samples, consisting of 64 specimens taken at the time of diagnosis from patients with PCM representing the various clinical forms plus 15 serum specimens each from patients with histoplasmosis and aspergillosis, 10 each from patients with cryptococcosis and tuberculosis, 6 from patients with coccidioidomycosis, and 40 from healthy subjects, were all tested by an indirect enzyme-linked immunosorbent assay with the purified 27-kDa recombinant protein. The latter was used at a concentration of 1.0 μg/well; there were three serum dilutions (1:1,000, 1:2,000, and 1:4,000). The experiment was repeated at least twice. The average sensitivity for both experiments was 73.4%; in comparison with the healthy subjects, the specificity for PCM patients was 87.5% while for patients with other mycoses, it was 58.7%. Important cross-reactions with sera from patients with aspergillosis and histoplasmosis were detected. The positive predictive value of the test was 90.4%. These results indicate that it is possible to employ recombinant antigenic proteins for the immunologic diagnosis of PCM and, by so doing, achieve high coverage rates. Furthermore, antigen reproducibility can now be ensured, thus facilitating inter- and intralaboratory standardization.

Diagnosis of paracoccidioidomycosis by a dot blot assay using a recombinant Paracoccidioides brasiliensis p27 protein

“…The p27 recombinant protein was previously cloned in pBluescript IISK and expressed in E. coli DH5a cells, produced by fermentation techniques and purified by preparative electrophoresis. 10,11,13 Although, protein obtained in this system showed to be reactive when evaluated with sera from PCM patients, an important cross-reactivity was detected with sera from patients with aspergillosis and histoplasmosis; in addition, this process was expensive and time consuming.…”

“…Expression and purification of recombinant antigens have appeared as a valuable alternative to the use of crude antigenic extracts. 9,10,11 In our search for more reproducible results, we subcloned the gene coding for the p27 immunodominant antigen of P. brasiliensis and evaluated this recombinant protein in a dot blot assay, to explore its usefulness in the diagnosis of this mycosis. The use of a well-characterised antigen in the form of a recombinant protein and the appropriate test format to be used in PCM diagnosis would allow more reproducible results, making possible the standardisation of those diagnostic techniques in different laboratories.…”

“…It is known that PCM diagnosis has been based mostly on microbiological and immunological methods, 1,5,6,16,17 but both these alternatives present problems and, although, immunological tests have proved useful, one of the difficulties encountered has been the high level of cross reactivity found among the patients with other mycosis, especially histoplasmosis, mostly because of the use of crude antigens and their high associated variability. 2,8,10,11 Being aware of the need for more sensitive and specific methods and also the need of more cost-efficient and less time-consuming tests, we used the newly produced p27 protein on a dot blot format. The availability of an appropriate antigen used in combination with a standardised test, would allow its evaluation with a greater number of sera from patients with PCM in different stages of the disease to validate its use in diagnosis.…”

“…8 An alternative to obtaining suitable antigens is the cloning, expression and characterisation of inmunodominant antigens derived from the fungus. [9][10][11] Cisalpino et al [9] cloned a P. brasiliensis immunodominant antigen, the glycoprotein of 43 kDa (gp43); this recombinant antigen showed high reactivity when evaluated with sera from patients with PCM. 12 McEwen et al [13] cloned the protein of 27 kDa (p27) on the plasmid pBluescript; immunological characterisation of this recombinant protein showed that it was recognised by a high proportion of sera of patients with PCM.…”

“…10 When this p27 recombinant protein was evaluated on an ELISA format, the values of sensitivity and specificity obtained were of 73.4% and 87.5% respectively. 11 One of the main advantages of using recombinant antigens is the reduction of the cross-reactions that occur with other mycoses when crude antigens are used, as previously indicated. Additionally, recombinant proteins facilitate production of antigenic preparations that display a little variability and can be used in different tests and different laboratories.…”