Dot-Blot Methodology for Rapid Diagnosis of Paracoccidioidomycosis Caused by Paracoccidioides brasiliensis

Paracoccidioidomycosis (PCM), a mycosis which clinical diagnosis can be confused with other infectious diseases like leishmaniasis and tuberculosis, presents significant rates of mortality, estimated between 2-23% in severe cases, reaching 30% when associated with AIDS [1], and considered to lead mortality among mycosis in Brazil, with an average of 111.5 deaths/year, according to the Mortality Information System of the Ministry of Health [2]. Given the above, an early and accurate diagnosis of this infectious disease is very important because it allows the establishment of appropriate antifungal therapy, reduction of unnecessary use of toxic drugs; decreasing the use of empiric therapy, thereby minimizing the emergence of multidrug-resistant fungal strains. In addition, the technique used to immunodiagnostic of PCM has to combine sensitivity and specificity, so that the predictive value is high and reproducible. In this study, Dot-blot (DB) method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity, as well as a rapid diagnostic test, given the release of true negative results in less time (one day) compared to the double immunodiffusion assay in agarose gel (DI, seven days), considered the gold standard serological method and, therefore, of great diagnostic value, particularly for the disposal of clinical suspicion for the this mycosis. Furthermore, Dot-blot method shows the prospect of being transferred to laboratories of public service including those with fewer resources of facilities such as photometers, plate washers and others, or even be used directly in the field, with an excellent shelf life validity, ie membranes coated with antigen can be used for testing, without changes in the pattern of reactivity among laboratories and present reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological gold standard methodology, therefore to be used in routine diagnosis for Public Health.


Introduction
Paracoccidioidomycosis, a highly endemic mycosis in Brazil, is caused by fungal species of Paracoccidioides (P. brasiliensis and P. lutzii) [3,4]. The gold standard method for definitive diagnosis is based on budding yeast cells observation, by conducting direct examination, isolation and identification of the etiologic agent after culture of biological samples. However, isolation and culture are not easy and rapid to be performed, with often serial collection of samples, that contributes to the delay in the establishment of appropriate antifungal treatment; also adds to these observations, the low sensitivity of mycological methods [5]. Thus, serological techniques have been used as important diagnostic and prognostic tools, optimizing time for obtaining results [6]. The double immunodiffusion assay is widely used for PCM diagnosis due its high specificity and easily to perform with no automation, also allowing qualitative and semi-quantitative detection of species-specific antibodies, for monitoring the effectiveness of therapeutic treatment. Although DI is highly specific, its sensitivity is weak, so substantial number of false negative results have been reported, beside the long time period until the release of result (120 hours). The Dot-Blot technique has been successfully used in the diagnosis of many infectious and parasitic diseases, such as toxoplasmosis and visceral leishmaniasis, aiming to offer a faster response [7]. For the immunodiagnosis of PCM, the DB has shown to be considerable promise on monitoring patients during anti-fungal treatment and seroepidemiological surveys [8].

Objectives
The objective of this study was to optimize, standardize and validate the Dot-Blot test for rapid diagnosis of PCM, and to propose it as serological screening tool for patients with clinical suspicion for Paracoccidiodomycosis, by comparing to DI and evaluating the shelf life of coated membranes storage over the time.

Methodology
We evaluated 443 serum samples from patients, as follow: 143 grouped as control (23 samples from apparently healthy patients, 77 samples from patients with PCM confirmed by mycological and/or histophatological evaluations and 43 samples with other infectious diseases [tuberculosis, aspergillosis and/or histoplasmosis]) and 300 serum samples from patients with clinical suspicion of PCM, forwarded to the Immunodiagnostic of Mycoses Laboratory, Immunology Center, Instituto Adolfo Lutz. Tested exoantigens, were obtained following the methodology proposed by Garcia et al. and modified by Silva, using culture filtrates of sample 113 and B-339 strain of P. brasiliensis. Protein concentration was determined by Bradford method using NanoDrop® Spectrophotomer equipment ND-1000 (Thermo Fisher Scientific, DE, USA) [9][10][11]. Detection of anti-Paracoccidioides brasiliensis circulating antibodies was carried out using double immunodiffusion in agarose gel assay, proposed by Ouchterlony [12]. The Dot-Blot method was optimized based on the protocol described by Hawkes et al. and Pappas [7,13]. For the serological test validation, we calculated intrinsic parameters of sensitivity, specificity, predictive values, prevalence, accuracy/ efficiency, co-positivity (relative sensitivity) and co-negativity (relative specificity) according to Ferreira and Ávila. Analysis of concordance of results was evaluated by Kappa index Cohen obtained using table proposed by Landis and Koch [14,15]. Finally, for proportions comparison we used the chi-square test, performed by the Epi Info 6.1 program (Center for Disease Control and Prevention-http:// www.cdc.gov) with p ≤ 005 significance value.

Results
The Dot-blot method standardization showed better results using nitrocellulose membranes coated with antigen obtained from culture filtrate of B-339 isolated of P. brasiliensis and serum and conjugate diluted at 1:40 and 1:2000, respectively, both incubating in PBS-M 3% solution. Sensitivity, specificity, positive and negative predictive values and accuracy for DI and DB were calculated over results of 143 samples from control group, as follows: 98.2%, 75.5%, 72.7%, 98.4%, 40% and 84.6% for DI, 95.9%, 90%, 91%, 95.4%, 51% and 93% for DB, respectively. Performance of DB compared to DI standard method were evaluated using 300 samples from patients with clinical suspicion of PCM. Anti-P. brasiliensis circulating antibodies were detected in 34% of samples, while DB had detected these antibody in 47.3%. Discriminatory capacity of both methods suggested that DB test has a tendency to increase the reactivity pattern, as it was recognized by 40 (13.3%) more samples than DI. Copositivity (relative sensitivity) and co-negativity (relative specificity) values, using the DI assay as the standard serological test, were 68.3% and 96.8%, respectively, achieving an agreement level between them of κ=0.66, considered good.

Conclusion
A statistical analysis, demonstrated that Dot-blot technique was significantly more sensitive (p=0.000067) than Double Immunodiffusion methodology, proving to be an excellent tool for the serological screening of serum samples from patients with clinical suspicion and/orconfirmed paracoccidioidomycosis. Dot-blot test showed lower specificity (95.4%) than that calculated for the reaction of double immunodiffusion (98.5%), confirming the applicability of the DI assay as diagnostic test. Dot-blot method showed alow percentage of cross-reactivity when serum samples from patients with other lunginfections such as histoplasmosis, tuberculosis and aspergillosis were evaluatedThese results suggest a possible new algorithm for serological diagnosis of PCM, carrying out the serological screening with Dot-blot methodology instead of the indirect ELISA test.