Evaluation of the hepatobiliary system in patients with paracoccidioidomycosis treated with cotrimoxazole or itraconazole.

A prospective study was performed in 200 paracoccidioidomycosis (PCM) patients, 51 presenting the acute/subacute form (AF) and 149 the chronic form (CF), submitted to the evaluation of the hepatobiliary system at admission and during the follow-up treatment with cotrimoxazole (CMX) or itraconazole (ITC). This study aimed to better evaluate the involvement of the hepatobiliary system in PCM and the effect of these antifungal compounds on this system. Serum levels of direct bilirubin (DB), total bilirubin (TB), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) were evaluated. At admission, all the variables showed changes with elevated values ranging from 6.2% for TB to 32.6% for GGT. After treatment, the incidence of elevated serum levels ranged from 3.6% for DB to 27.5% for ALT. The course of the alterations during the treatment showed regression to normal values in CMX-treated patients and persistence in ITC-treated patients but without the need to discontinue the therapy. Our findings contribute to the knowledge of the hepatobiliary involvement by Paracoccidioides sp. and to a safe follow-up of PCM patients under treatment.

Increased peripheral blood TCD4+ counts and serum SP-D levels in patients with chronic paracoccidioidomycosis, during and after antifungal therapy.

BACKGROUND: The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE: To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS: Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS: In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION: Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.

Reproducibility of double agar gel immunodiffusion test using stored serum and plasma from paracoccidioidomycosis patients.

Background: Serological evaluation performed by double agar gel immunodiffusion test (DID) is used for diagnosis, evaluation of severity, management of paracoccidioidomycosis patients, and development of new clinical studies. For these reasons, the Botucatu Medical School of UNESP maintains a serum bank at the Experimental Research Unit with patient clinical data. This study aimed to evaluate the influence of the freeze-thaw cycle and different blood matrices on the titration of circulating antibodies. Methods: The study included 207 patients with confirmed (etiology-demonstrated) or probable (serology-demonstrated) paracoccidioidomycosis, and DID was performed with culture filtrate from Paracoccidioides brasiliensis B339 as antigen. First experiment: the antibody levels were determined in serum samples from 160 patients with the chronic form and 20 with the acute/subacute form, stored at -80oC for more than six months. Second experiment: titers of 81 samples of serum and plasma with ethylenediaminetetraacetic acid (EDTA) or heparin, from 27 patients, were compared according to matrix and effect of storage at -20oC for up to six months. Differences of titers higher than one dilution were considered discordant. Results: First experiment: test and retest presented concordant results in serum stored for up to three years, and discordant titers in low incidence in storage for four to six years but high incidence when stored for more than six years, including conversion from reagent test to non-reagent retest. Second experiment: serum, plasma-EDTA and plasma-heparin samples showed concordant titers, presenting direct correlation, with no interference of storage for up to six months. Conclusions: Storage at -80oC for up to six years has no or little influence on the serum titers determined by DID, permitting its safe use in studies depending on this parameter. The concordant titrations in different blood matrices demonstrated that the plasma can be used for immunodiffusion test in paracoccidioidomycosis, with stability for at least six months after storage at -20oC.

Accuracy of serological tests for diagnosis of chronic pulmonary aspergillosis: A systematic review and meta-analysis.

Chronic pulmonary aspergillosis (CPA) is a slow and progressive disease that develops in preexisting lung cavities of patients with tuberculosis sequelae, and it is associated with a high mortality rate. Serological tests such as double agar gel immunodiffusion test (DID) or counterimmunoelectrophoresis (CIE) test have been routinely used for CPA diagnosis in the absence of positive cultures. However, these tests have been replaced with enzyme-linked immunoassay (ELISA) and, a variety of methods. This systematic review compares ELISA accuracy to reference test (DID and/or CIE) accuracy in CPA diagnosis. It was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The study was registered in PROSPERO under the registration number CRD42016046057. We searched the electronic databases MEDLINE (PubMed), EMBASE (Elsevier), LILACS (VHL), Cochrane library, and ISI Web of Science. Gray literature was researched using Google Scholar and conference abstracts. We included articles with patients or serum samples from patients with CPA who underwent two serological tests: ELISA (index test) and IDD and/or CIE (reference test). We used the test accuracy as a result. Original articles were considered without a restriction of date or language. The pooled sensitivity, specificity, and summary receiver operating characteristic curves were estimated. We included 14 studies in the review, but only four were included in the meta-analysis. The pooled sensitivities and specificities were 0.93 and 0.97 for the ELISA test. These values were 0.64 and 0.99 for the reference test (DID and/or CIE). Analyses of summary receiver operating characteristic curves yielded 0.99 for ELISA and 0.99 for the reference test (DID and/or CIE). Our meta-analysis suggests that the diagnostic accuracy of ELISA is greater than the reference tests (DID and/or CIE) for early CPA detection.

Ceruloplasmin, transferrin and apolipoprotein A-II play important role in treatment's follow-up of paracoccidioidomycosis patients.

Paracoccidioidomycosis (PCM) is a systemic disease caused by thermodymorphic fungi of the Paracoccidioides brasiliensis complex, (Paracoccidioides spp.). Patients with PCM reveal specific cellular immune impairment. Despite the effective treatment, quiescent fungi can lead to relapse, usually late, the serological diagnosis of which has been deficient. The present study was carried out with the objective of investigating a biomarker for the identification of PCM relapse and another molecule behaving as an immunological recovery biomarker; therefore, it may be used as a cure criterion. In the evolutionary analysis of the proteins identified in PCM patients, comparing those that presented with those that did not reveal relapse, 29 proteins were identified. The interactions observed between the proteins, using transferrin and haptoglobin, as the main binding protein, were strong with all the others. Patient follow-up suggests that cerulosplamin may be a marker of relapse and that transferrin and apolipoprotein A-II may contribute to the evaluation of the treatment efficacy and avoiding a premature decision.

Serological proteomic biomarkers to identify Paracoccidioides species and risk of relapse.

The sensitivity of the double agar gel immunodiffusion test is about 90% in patients with untreated paracoccidioidomycosis (PCM), but it is much lower in cases of relapse. In addition, serum from patients with PCM caused by Paracoccidioides lutzii, frequent in the Midwest region of Brazil, do not react with the classical antigen obtained from Pb B-339. These findings showed the need for alternative diagnostic methods, such as biological markers through proteomics. The aim of this study was to identify biomarkers for the safe identification of PCM relapse and specific proteins that could distinguish infections caused by Paracoccidioides brasiliensis from those produced by Paracoccidioides lutzii. Proteomic analysis was performed in serum from 9 patients with PCM caused by P. brasiliensis, with and without relapse, from 4 patients with PCM produced by P. lutzii, and from 3 healthy controls. The comparative evaluation of the 29 identified plasma proteins suggested that the presence of the immunoglobulin (Ig) alpha-2 chain C region and the absence of Ig heavy chain V-III TIL indicate infection by P. lutzii. In addition, the absence of complement factor B protein might be a predictor of relapse. The evaluation of these proteins in a higher number of patients should be carried out in order to validate these findings.

Paracoccidioidomycosis: Current Perspectives from Brazil.

BACKGROUND: This review article summarizes and updates the knowledge on paracoccidioidomycosis. P lutzii and the cryptic species of P. brasiliensis and their geographical distribution in Latin America, explaining the difficulties observed in the serological diagnosis. OBJECTIVES: Emphasis has been placed on some genetic factors as predisposing condition for paracoccidioidomycosis. Veterinary aspects were focused, showing the wide distribution of infection among animals. The cell-mediated immunity was better characterized, incorporating the recent findings. METHODS: Serological methods for diagnosis were also compared for their parameters of accuracy, including the analysis of relapse. RESULTS: Clinical forms have been better classified in order to include the pictures less frequently observesiod. CONCLUSION: Itraconazole and the trimethoprim-sulfamethoxazole combination was compared regarding efficacy, effectiveness and safety, demonstrating that azole should be the first choice in the treatment of paracoccidioidomycosis.

Evaluation of the Double Agar Gel Immunodiffusion Test and of the Enzyme-Linked Immunosorbent Assay in the Diagnosis and Follow-Up of Patients with Chronic Pulmonary Aspergillosis.

The diagnosis of chronic pulmonary aspergillosis (CPA) depends on the radiologic image and the identification of specific antibodies. The present study aimed to evaluate accuracy parameters of enzyme-linked immunosorbent assay (ELISA) and of the determination of serum galactomannan level in the diagnosis of patients with CPA, comparing these results with the double agar gel immunodiffusion (DID) test. In addition, the prevalence of cross-reactivity and the serological progression after treatment were evaluated by comparing DID and ELISA. Six study groups were formed: G1: 22 patients with CPA, 17 of whom had Aspergillus fungus ball, one chronic cavitary pulmonary aspergillosis (CCPA) and four chronic fibrosing pulmonary aspergillosis (CFPA); G2: 28 patients with pulmonary tuberculosis (TB); G3: 23 patients with histoplasmosis (HST); G4: 50 patients with paracoccidioidomycosis (PCM); G5: 20 patients with cryptococcosis (CRC); and G6: 200 healthy controls. Serum antibodies were measured by DID and ELISA, with two antigen preparations--Aspergillus fumigatus (DID1, ELISA1) and a pool of A. fumigatus, A. flavus and A. niger antigens (DID2, ELISA2). The Platélia Aspergillus Enzyme Immunoassay (EIA) kit was used to measure galactomannan. The cut-off points of ELISA were determined for each antigen preparation and for the 95% and 99% confidence intervals. Despite the low sensitivity, DID was the technique of choice due to its specificity, positive and negative predictive values and positive likelihood ratio-especially with the antigen pool and due to the low frequency of cross-reactivity. ELISA1 and a 0.090 cut-off showed high sensitivity, specificity and negative predictive value, but a high frequency of cross-reactivity with CRC. The best degree of agreement was observed between ELISA1 and ELISA2. The detection of serum galactomannan showed high sensitivity, comparable to ELISA2. The immunodiffusion test showed an excellent relationship with the progression after treatment, which made it the reaction of choice for patient follow-up.

Comparison between itraconazole and cotrimoxazole in the treatment of paracoccidiodomycosis.

BACKGROUND: There are no published reports on studies comparing itraconazole (ITC), sulfamethoxazole-trimethoprim (cotrimoxazole, CMX), and ITC followed by CMX (ITC/CMX) in the treatment of paracoccidiodomycosis. This study aimed to compare the efficacy, effectiveness, safety and time to clinical and serologic cure in paracoccidioidomycosis patients treated with ITC or CMX, the antifungal agents most widely used. METHODOLOGY: A quasi-experimental study was performed in 177 patients with a confirmed or probable diagnosis of paracoccidioidomycosis. Treatment was divided into two stages: 1) initial, which was continued until clinical cure was achieved and the erythrocyte sedimentation rate decreased to normal values; 2) complementary, which was continued until serologic cure was achieved. Medians were compared via the Mann-Whitney test, and frequencies were compared via the chi-squared test. The assessment of variables as a function of time was performed using Kaplan-Meier curves and Cox regression. The significance level was established as p≤0.05. PRINCIPAL FINDINGS: No difference was found in the efficacy and effectiveness of the initial treatment of 47 individuals given ITC and 130 individuals given CMX; however, the time to clinical cure was shorter in the former compared with the latter group (105 vs. 159 days; p = 0.001), specifically in patients with the chronic form. Efficacy and effectiveness of the three regimens were similar in the complementary treatment; however, the time to serologic cure was shorter when ITC (161 days) or CMX (495 days) was used compared with ITC/CMX (881 days) [p = 0.02]. The independent predictors of a shorter time to serologic cure were treatment with ITC [risk ratio = 6.61 (2.01-21.75)] or with CMX [risk ratio = 5.11 (1.91-13.67)]). The prevalence of side effects was lower with ITC (6.4%) than with CMX (20.0%; p = 0.03). CONCLUSIONS: Since ITC induced earlier clinical cure and was better tolerated than CMX, such triazole should be considered the first-choice for PCM treatment.

Prevalence and serological diagnosis of relapse in paracoccidioidomycosis patients.

A review of 400 clinical records of paracoccidioidomycosis (PCM) patients, 93 with the acute/subacute (AF) and 307 with the chronic form (CF), attended from 1977 to 2011, selected as to the schedule of release for study by the Office of Medical Records at the University Hospital of the Faculdade de Medicina de Botucatu-São Paulo State University--UNESP, was performed to detect cases in relapse. The control of cure was performed by clinical and serological evaluation using the double agar gel immunodiffusion test (DID). In the diagnosis of relapse, DID, enzyme-linked immunosorbent assay (ELISA) and immunoblotting assay (IBgp70 and IBgp43) were evaluated. Out of 400 patients, 21 (5.2%) went through relapse, 18 of them were male and 3 were female, 6∶1 male/female ratio. Out of the 21 patients in relapse, 15 (4.8%) showed the CF, and 6 (6.4%) the AF (p>0.05). The sensitivity of DID and ELISA before treatment was the same (76.1%). DID presented higher sensitivity in pre-treatment (80%) than at relapse (45%; p = 0.017), while ELISA showed the same sensitivity (80% vs 65%; p = 0.125). The serological methods for identifying PCM patients in relapse showed low rates of sensitivity, from 12.5% in IBgp70 to 65.0% in IBgp43 identification and 68.8% in ELISA. The sensitivity of ELISA in diagnosing PCM relapse showed a strong tendency to be higher than DID (p = 0.06) and is equal to IBgp43 (p = 0.11). In sum, prevalence of relapse was not high in PCM patients whose treatment duration was based on immunological parameters. However, the used methods for serological diagnosis present low sensitivity. While more accurate serological methods are not available, we pay special attention to the mycological and histopathological diagnosis of PCM relapse. Hence, direct mycological, cytopathological, and histopathological examinations and isolation in culture for P. brasiliensis must be appropriately and routinely performed when the hypothesis of relapse is considered.

Increased peripheral blood TCD4+ counts and serum SP-D levels in patients with chronic paracoccidioidomycosis, during and after antifungal therapy

BACKGROUND The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.