Oral exfoliative cytology in the diagnosis of paracoccidioidomycosis

OBJECTIVE: To assess the efficacy of conventional oral exfoliative cytology as a diagnostic tool in paracoccidioidomycosis. STUDY DESIGN: Cytologic smears and incisional biopsies were obtained from 10 patients with a clinical suspicion and oral manifestations of paracoccidioidomycosis. Cytologic smears and sections of the incisional biopsy underwent methenamine silver staining for fungi according to the Gomori-Grocott method. The dry glass slides were examined at 400 or 1,000 x magnification, and the presence and shape of yeasts of Paracoccidioides brasiliensis were investigated. RESULTS: Yeasts of the fungus P brasiliensis were clearly identified in cytologic smears and sections from incisional biopsies in all cases analyzed (100.0%). CONCLUSION: Cytology of oral samples proved an effective diagnostic method for the detection of paracoccidioidomycosis in humans.     

Binding of laminin to Paracoccidioides brasiliensis induces a less severe pulmonary paracoccidioidomycosis caused by virulent and low-virulence isolates

The pathogenic fungus Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis (PCM). This pulmonary mycosis, acquired by inhalation of airborne propagules, may disseminate to several internal organs and tissues, leading to severe disease. Adhesion to host cell components is the first step involved in dissemination of pathogens. Previous studies showed that laminin, the most abundant glycoprotein of the basement membrane, binds to P. brasiliensis yeast cells, enhancing their pathogenicity in the hamster testicle model. As PCM is primarily a pulmonary infection, we studied the influence of previous treatment of yeast cells with laminin on the course of the intratracheal infection of resistant and susceptible mice using high-virulence (Pb18) and low-virulence (Pb265) P. brasiliensis isolates. Laminin treatment did not alter fungal loads, delayed-type hypersensitivity reactions, levels of pulmonary cytokines and production of specific antibodies in any group of Pb18-infected mice. However, early in the infection, a less intense inflammatory reaction was detected in the lungs of the laminin-treated groups. In addition, laminin treatment of Pb265 resulted in a less severe infection as revealed by the lower fungal loads recovered from lungs. Antibody and cytokine levels, however, did not change after laminin treatment. Altogether, our results demonstrate that laminin binding to yeast cells diminishes P. brasiliensis pathogenicity. The lower inflammatory response observed with the virulent isolate and the decreased pulmonary fungal burden with the low-virulence isolate indicate an inhibitory effect of laminin treatment on P. brasiliensis infectivity and interaction with pulmonary host cells or extracellular matrix proteins.     

A novel method for estimating killing ability and digestion of Paracoccidioides brasiliensis by phagocytic cells in vitro

We describe a novel method by which phagocytosis, digestion and killing of Paracoccidioides brasiliensis yeast cells by polymorphonuclear leukocytes or other phagocytic cells may be estimated simultaneously. Suspensions of P. brasiliensis (yeast-like phase) were sonicated, counted and incubated at 37 degrees C with known numbers of phagocytes. Control preparations contained no phagocytic cells. At given intervals samples were incorporated into gelatin nutrient medium and droplets of the mixtures were incubated at room temperature. Live yeast-like P. brasiliensis germinate in vitro and produce filaments. After incubation, droplets may be melted and examined under phase contrast optics, or the cells may be washed and stained by a variation of Papanicolaou's method. Digested P. brasiliensis, intact but non-germinating yeasts and filamented (viable) yeasts may be identified and counted. Killing and digestive abilities of phagocytes may be estimated by the difference between values obtained from phagocyte-containing and control preparations.     

Paracoccidioidomycosis in a Woman with Idiopathic Hirsutism

Paracoccidioidomycosis, especially the chronic pulmonary form of the disease, is not commonly described in females. Data from in vitro and vivo studies support the hypothesis that estrogens might influence the pathogenesis of paracoccidioidomycosis in humans by inhibition of transition of conidia or mycelia to yeast form of Paracoccidioides brasiliensis. The authors describe a chronic progressive pulmonary form of paracoccidioidomycosis in a woman with idiopathic hirsutism. In addition to estrogens, the present report suggests that other hormonal factors might play an important role in the pathogenesis of paracoccidioidomycosis, including the increased production of 5alpha-dehydrotestosterone frequently described in individuals with idiopathic hirsutism.     

Neoplasia and paracoccidioidomycosis

Published studies on the association between cancer and paracoccidioidomycosis consist either isolated cases or clinical data based on hospital cohorts of paracoccidioidomycosis. The frequency of neoplasia in series of ≥80 patients with paracoccidioidomycosis ranges from 0.16 to 14.1%, mean of 3.96%. There are only two retrospective controlled studies, one of them showing greater incidence of carcinoma in biopsy and necropsy samples of paracoccidioidomycosis (12 cases in 147 patients with the mycosis: 8.2%) than in the necropsies of the control group (320 cases in 7,302 necropsies: 4.9%). In the other, 22,409 autopsies were reviewed and 4,372 cases of cancer were found; of the 85 patients with paracoccidioidomycosis, 12 were diagnosed with cancer. No differences were observed in the frequency of malignancies between the group of patients with paracoccidioidomycosis (14.1%) and the control group (19.5%). Considering all the reported cases, carcinoma was more frequent than hematological malignancies, and was more often found at the same site or in a neighboring site affected by the mycosis, usually occurring after the diagnosis of the mycosis. Commonly, the basic cause of death was related to secondary infections or neoplasia. Lymphoma was associated with poorly organized rich in fungi granuloma. The clinical course and mortality were related to the cancer evolution or secondary infections and was worse in lymphoid series, metastatic carcinoma or in patients under cytotoxic chemotherapy. Additionally, as in several cases the clinical and histopathological data may mimick neoplasia, the correct diagnosis of both diseases is essential to guarantee an early and safe intervention.     

Detection of gp43 of Paracoccidioides brasiliensis by the loop-mediated isothermal amplification (LAMP) method

Paracoccidioidomycosis is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. We detected the species specific gp43 gene of P. brasiliensis by loop-mediated isothermal amplification (LAMP) in 22 clinical and seven armadillo-derived isolates. The amplified DNA appeared as a ladder with a specific banding pattern. The advantage of the LAMP method is speed; only 3 h were necessary for identification of the organism and diagnosis of the disease. We were also able to obtain positive results from DNA extracted from a paraffin-embedded tissue sample of paracoccidioidomycosis, suggesting that this method may achieve clinical application in the near future.     

Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells

Paracoccidioides brasiliensis is a dimorphic fungus known to produce invasive systemic disease in humans. The 43-kDa glycoprotein of P. brasiliensis is the major diagnostic antigen of paracoccidioidomycosis and may act as a virulence factor, since it is a receptor for laminin.Very little is known about early interactions between this fungus and the host cells, so we developed in vitro a model system employing cultured mammalian cells (Vero cells), in order to investigate the factors and virulence mechanisms of P.brasiliensis related to the adhesion and invasion process. We found that there is a permanent interaction after 30 min of contact between the fungus and the cells. The yeasts multiply in the cells for between 5 and 24 h. Different strains of P. brasiliensis were compared, and strain 18 (high virulence) was the most strongly adherent, followed by strain 113 (virulent), 265 (considered of low virulence) and 113M (mutant obtained by ultraviolet radiation, deficient in gp43). P. brasiliensis adhered to the epithelial cells by a narrow tube, while depressions were noticed in the cell surface, suggesting an active cavitation process. An inhibition assay was performed and it was verified that anti-gp43 serum and a pool of sera from individuals with paracoccidioidomycosis were able to inhibit the adhesion of P. brasiliensis to the Vero cells. Glycoprotein 43 (gp43) antiserum abolished 85% of the binding activity of P. brasiliensis. This fungus can also invade the Vero cells, and intraepithelial parasitism could be an escape mechanism in paracoccidioidomycosis.     

Report of an imported cutaneous disseminated case of paracoccidioidomycosis

Paracoccidioidomycosis is a chronic progressive infection. It affects mainly the elderly and it is geographically limited to certain areas of Latin America. In Europe it is considered a rare imported infection. Here we report a case of paracoccidioidomycosis that occurred in a 27-year-old Ecuadorian patient living in Spain initially misdiagnosed as blastomycosis. The typical multi-budding yeast cells of Paracoccidioides brasiliensis were observed in Grocott stained samples. This case should alert Spanish mycologists, clinicians and pathologists about the possibility of patients who have travelled or lived outside Spain may suffer paracoccidioidomycosis or other imported mycoses.     

Sputum cytology in the diagnosis of pulmonary paracoccidioidomycosis

The presence of Paracoccidioides brasiliensis was determined in sputum samples from 50 patients with paracoccidioidomycosis using four different techniques: (a) cell-block preparations stained with silver methenamine, (b) direct microbiologie examination, (c) smears stained with Shorr, and (d) smears stained with silver methenamine. Overall, cell-block preparations and smears stained with silver methenamine proved to be the most sensitive techniques, followed by smears stained with Shorr and direct microbiologic examination in decreasing order of sensitivity. Sputum cytology tended to be less positive in patients with interstitial pulmonary lesions as determined by chest X-ray than in patients with alveolar lesions. In addition to its high sensitivity, cell-block preparation technique allows storage of blocks and slides for further studies.     

Chronic murine paracoccidioidomycosis: effect of ketoconazole on clearance of Paracoccidioides brasiliensis and immune response

In a murine model of chronic pulmonary and disseminated paracoccidioidomycosis, ketoconazole (100 mg kg-1 in 0.3% agar) given by gavage twice daily for 1 or 2 months enabled all mice to clear disseminated Paracoccidioides brasiliensis from the spleen. Clearance of P. brasiliensis from the lungs was more difficult, and was achieved in 60% of the mice treated for 2 months. Sera from agar-treated control mice at days 77 and 103 post-infection demonstrated precipitating antibodies to P. brasiliensis antigens, but sera from ketoconazole-treated mice were precipitin-negative, indicating a favorable prognosis. Delayed hypersensitivity reactions to P. brasiliensis antigens in ketoconazole-treated mice were not significantly greater than in controls; consequently this test correlated less well with response than levels of serum antibody. This is the first use of this animal model of paracoccidioidomycosis to study the effect of antifungal drug protocols on the resolution of the disease. It also demonstrates the utility of this model in addressing clinically relevant questions about this disease and its treatment.     

Virulence attenuation and phenotypic variation of Paracoccidioides brasiliensis isolates obtained from armadillos and patients

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, the most important systemic mycosis in Latin America. The virulence profiles of five isolates of P. brasiliensis were studied in two different moments and correlated with some colonial phenotypic aspects. We observed a significant decrease in the virulence and an intense phenotypic variation in the mycelial colony. The recognition of all ranges of phenotypic and virulence variation of P. brasiliensis, as well as its physiological and genetic basis, will be important for a better comprehension of its pathogenic and epidemiological features.     

Synchronous Oral Paracoccidioidomycosis and Esophageal Carcinoma

Paracoccidioidomycosis is the most common deep mycosis in South America and is caused by Paracoccidioides brasiliensis (P. brasiliensis), a thermally dimorphic fungus. Infections usually occur by inhalation of conidia, which more often cause respiratory, mucocutaneous, and lymph nodal changes. Chronic features of this mycosis can mimic diverse infections and malignancies and constitute diagnosis challenges. Squamous cell carcinoma deserves special attention in this setting. We describe the case of a patient with synchronous diagnosis of oral paracoccidioidomycosis and esophageal squamous cell carcinoma. Concomitance of these conditions may be a casual event, but a not fully understood causal relationship can be involved.     

Evaluation of DNA polymorphisms amplified by arbitrary primers (RAPD) as genetically associated elements to differentiate virulent and non-virulent Paracoccidioides brasiliensis isolates

Randomly amplified polymorphic DNA (RAPD) analysis of 35 Paracoccidioides brasiliensis isolates was carried out to evaluate the correlation of RAPD profiles with the virulence degree or the type of the clinical manifestations of human paracoccidioidomycosis. The dendrogram presented two main groups sharing 64% genetic similarity. Group A included two isolates from patients with chronic paracoccidioidomycosis; group B comprised the following isolates showing 65% similarity: two non-virulent, six attenuated, five virulent, eight from patients with chronic paracoccidioidomycosis and two from patients with acute paracoccidioidomycosis. The virulent Pb18 isolate and six attenuated or non-virulent samples derived from it were genetically indistinguishable (100% of similarity). Thus, in our study, RAPD patterns could not discriminate among 35 P. brasiliensis isolates according to their differences either in the degree of virulence or in the type of the clinical manifestation of this fungal infection.     

Immunocytochemical characterization of large-cell carcinomas of the lung. Role, limitations and technical considerations.

To clarify the use of cytologic preparations, particularly those previously stained by the Papanicolaou method, for the immunocytochemical evaluation of large-cell carcinomas (LCCs), 37 cytologic preparations from cases diagnosed as LCC were examined using a battery of immunocytochemical stains for keratin, chromogranin, common leukocyte antigen (CLA) and B72.3. Thirty-two specimens were from the thoracopulmonary region (12 fine needle aspirates of the lung, 7 bronchial brushings, 5 bronchial washings, 2 sputa and 6 pleural fluids); the remaining specimens were fine needle aspirates of 3 lymph nodes, 1 vertebral body and 1 liver. Of the specimens analyzed, 30 of 37 were positive for keratin and 7 of 35 were positive for B72.3 (6 were positive for both). Only 1 of 37 was positive for CLA while none of 37 was positive for chromogranin. Six specimens showed no reaction with either keratin, B72.3 or chromogranin. These results confirm that the majority of LCCs consist of epithelial cells of either a squamous or an adenocarcinomatous type. They also show that immunocytochemistry is a useful diagnostic adjunct that can be applied to cytologic preparations previously stained by the Papanicolaou method; this is important since immunostaining is often considered after undifferentiated malignant cells are encountered in a previously stained preparation. However, a thorough understanding of some technical limitations is critical in the evaluation of the results of this technique when it is applied to cytologic specimens.     

Amyloid in cytologic specimens. Differential diagnosis and diagnostic pitfalls.

OBJECTIVE: To describe and illustrate the characteristic features of amyloid in cytologic preparations and point out its diagnostic pitfalls. STUDY DESIGN: Five fine needle aspirates and one bronchial washing that contained amyloid were retrospectively reviewed. The aspirates were obtained from each of the five following sites: lung, occipital lymph node, thyroid gland, proximal humerus and subcutaneous soft tissue. Smears of all of the aspirates were stained with Papanicolaou stain, and in two cases they were also stained with Diff-Quik. Cell block sections were stained with hematoxylin and eosin. Congo red, CD45 and CD20 were used on selected cases. RESULTS: Amyloid appears as either flocculent material or irregularly shaped fragments with scalloped and pointed edges. The amorphous fragments are acellular and frequently associated with connective tissue cells. They stain eosinophilic to cyanophilic with Papanicolaou stain and deep blue with Diff-Quik. In two cases an exuberant giant cell reaction almost obscured the amyloid. In the thyroid aspirate, the amyloid was misinterpreted as colloid. In bronchial washings and lung aspirates, amyloid has to be distinguished from mucus, alveolar proteinosis, chondroid material and corpora amylacea. When circumferentially surrounded by lymphocytes or plasma cells, flocculent amyloid deposits may simulate adenoid cystic carcinoma. CONCLUSION: Amyloid can be easily overlooked or mistaken for other entities with similar staining qualities. Congo red staining can help to confirm the diagnosis.     

Paracoccidioides brasiliensis induces secretion of IL-6 and IL-8 by lung epithelial cells. Modulation of host cytokine levels by fungal proteases

Paracoccidioides brasiliensis is a pathogenic, dimorphic fungus that causes paracoccidioidomycosis, a systemic human mycosis that is highly prevalent in Latin America. In this study, we demonstrated that P. brasiliensis yeasts induced interleukin (IL)-8 and IL-6 secretion by human lung epithelial A549 cells. However, tumor necrosis factor-α and interferon-γ were undetectable in these cultures. Moreover, P. brasiliensis yeasts induced activation of p38 mitogen-activated protein kinase (MAPK), c-Jun NH(2)-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 in A549 cells, and IL-8 and IL-6 secretion promoted by this fungus was dependent on activation of p38 MAPK and ERK 1/2. In addition, IL-8 and IL-6 levels were significantly higher in culture supernatants of A549 cells that were incubated with formaldehyde-fixed P. brasiliensis compared to cultures of cells that were infected with live yeasts. Our results indicate that the observed cytokine level differences were due to protease expression, in live yeasts, that degraded these cytokines. Degradation of human recombinant IL-8 and IL-6 by live P. brasiliensis was inhibited by AEBSF and aprotinin, suggesting that these proteases belong to a family of serine proteases. This is the first report showing that P. brasiliensis may modulate host inflammation by expressing proteases that degrade proinflammatory cytokines.     

A surface 75-kDa protein with acid phosphatase activity recognized by monoclonal antibodies that inhibit Paracoccidioides brasiliensis growth

Paracoccidioides brasiliensis is a thermo-dimorphic fungus responsible for paracoccidioidomycosis (PCM), a systemic granulomatous mycosis prevalent in Latin America. The fungus releases many antigens which may be transiently bound to its cell surface. Some of them may show enzymatic functions essential for maintaining many cell processes and survival of the microorganism at different conditions. In this study, we report the characterization of a secreted 75kDa protein from P. brasiliensis with phosphatase activity. Biologic function of the molecule was demonstrated using two specific mAbs produced and characterized as IgM and IgG isotypes. Confocal microscopy and flow cytometry analysis demonstrated that both mAbs recognized the protein on the fungus surface, mainly in its budding sites. In vitro experiments showed that fungal growth was inhibited by blocking the protein with mAbs. In addition, opsonized yeast cells with both mAbs facilitated phagocytosis by murine peritoneal macrophages. Passive immunization using mAbs before P. brasiliensis mice infection reduced colony-forming units (CFU) in the lungs as compared with controls. Histopathology showed smaller inflammation, absence of yeast cells and no granuloma formation.     

Serological diagnosis of paracoccidioidomycosis in HIV-coinfected patients

Paracoccidioidomycosis should be differentiated from other opportunistic diseases in human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients who live in Latin America. Laboratory investigation can begin with serological tests, which are rapid and efficient. In the present study, double immunodiffusion (DID), counterimmunoelectrophoresis (CIEP) and an enzyme linked immunosorbent assay (ELISA) tests were assessed for the detection of anti-Paracoccidioides brasiliensis antibodies in 40 patients coinfected with HIV. The results were compared to those obtained for 75 non-HIV-infected patients with endemic paracoccidioidomycosis. Anti-P. brasiliensis antibodies were detected in 65% (DID), 79% (CIEP) and 95% (ELISA) of the patients with HIV/AIDS, significantly lower rates than those detected in cases of endemic paracoccidioidomycosis, which were 89%, 99% and 100%, respectively. The reactive sera of HIV-infected patients also showed lower anti-P. brasiliensis antibody titres than those of non-HIV-infected patients. Despite the lower intensity of the specific humoral response, serological tests are useful for the diagnosis of opportunistic paracoccidioidomycosis in the HIV/AIDS population. We suggest optimization of the laboratory diagnosis by combining the ELISA test with CIEP or DID.     

Evaluation of polymerase chain reaction for the detection of Paracoccidioides brasiliensis DNA on serum samples from patients with paracoccidioidomycosis

The aim of this study was to demonstrate the DNA of Paracoccidioides brasiliensis in human serum samples of patients with paracoccidioidomycosis using the polymerase chain reaction (PCR). The diagnosis of paracoccidioidomycosis (PCM) was defined by microscopic observation of the fungus on direct exam or histopathology, culture, and serological positivity. DNA from serum of 33 patients with PCM was extracted and submitted to nested-PCR using primers from the gp 43 gene. Only one sample was positive on nested-PCR. We conclude that the prevalence of fungemia in patients with different clinical forms of PCM is low, limiting the use of serum DNA detection as an alternative diagnostic tool.