REACTIVITY OF ANTI-GP43 ANTIBODIES FROM PARACOCCIDIOIDES BRASILIENSIS ANTISERUM WITH EXTRACTS FROM CUTANEOUS LESIONS OF LOBO'S DISEASE: PRELIMINARY NOTE

We demonstrated through several immunochemical tests the presence of GP-43 from P. brasiliensis in extracts of cutaneous lesions from Jorge Lobo's disease. This glicoprotein is one of the immunodominant antigens in this species, and is used to identify it. The demonstration of GP-43 tissues infected by the agent of Jorge Lobo's disease is an additional evidence for classifying it in the genera Paracoccidioides, species loboi

Paracoccidioides brasiliensis: A MYCOLOGIC AND IMMUNOCHEMICAL STUDY OF TWO STRAINS

The authors conducted a mycologic, immunochemical and molecular biology study on two strains of Paracoccidioides brasiliensis, one of them, called IBIÁ, isolated from soil in the municipality of IBIÁ (Minas Gerais) by Silva-Vergara et al. (l996,1998)20,21, and the other, BAT, cultivated from a human case of paracoccidioidomycosis in Ribeirão Preto (São Paulo/Brazil) by Freitas da Silva (l996)6. Both strains showed cotton-like (M) and yeast-like (Y) forms and were pathogenic for testicularly inoculated guinea pigs, producing granulomatous and/or suppurative orchitis. Immunochemically was demonstrated the presence of gp43 by double immunodiffusion, immunoelectrophoresis and immunoblotting.

PCR with Paracoccidioides brasiliensis specific primers: potential use in ecological studies

The precise microenvironment of Paracoccidioides brasiliensis has not yet been discovered perhaps because the methods used are not sensitive enough. We applied to this purpose the polymerase chain reaction (PCR) using three sets of specific primers corresponding to two P. brasiliensis genes. This fungus as well as several other fungi, were grown and their DNA obtained by mechanical disruption and a phenol chloroform isoamylalcohol-based purification method. The DNA served for a PCR reaction that employed specific primers from two P. brasiliensis genes that codify for antigenic proteins, namely, the 27 kDa and the 43 kDa. The lowest detection range for the 27 kDa gene was 3 pg. The amplification for both genes was positive only with DNA from P. brasiliensis; additionally, the mRNA for the 27 kDa gene was present only in P. brasiliensis, as indicated by the Northern analysis. The standardization of PCR technology permitted the amplification of P. brasiliensis DNA in artificially contaminated soils and in tissues of armadillos naturally infected with the fungus. These results indicate that PCR technology could play an important role in the search for P. brasiliensis’ habitat and could also be used in other ecological studies.

Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected with viable and non-viable Paracoccidioides brasiliensis propagules

Patients with paracoccidioidomycosis often present pulmonary fibrosis and exhibit important respiratory limitations. Based on an already established animal model, the contribution of viable and non-viable P. brasiliensis propagules to the development of fibrosis was investigated. BALB/c male mice, 4-6 weeks old were inoculated intranasally either with 4x10(6 )viable conidia (Group I), or 6.5x10(6) fragmented yeast cells (Group II). Control animals received PBS. Six mice per period were sacrificed at 24, 48, 72h (initial) and 1, 2, 4, 8, 12 and 16 weeks post-challenge (late). Paraffin embedded lungs were sectioned and stained with H&E, trichromic (Masson), reticulin and Grocott´s. During the initial period PMNs influx was important in both groups and acute inflammation involving 34% to 45% of the lungs was noticed. Later on, mononuclear cells predominated. In group I, the inflammation progressed and granulomas were formed and by the 12th week they fussed and became loose. Thick collagen I fibers were observed in 66.6% and 83.3% of the animals at 8 and 12 weeks, respectively. Collagen III, thick fibers became apparent in some animals at 4weeks and by 12 weeks, 83% of them exhibited alterations in the organization and thickness of these elements. In group II mice, this pattern was different with stepwise decrease in the number of inflammatory foci and lack of granulomas. Although initially most animals in this group had minor alterations in thin collagen I fibers, they disappeared by the 4th week. Results indicate that tissue response to fragmented yeast cells was transitory while viable conidia evoked a progressive inflammatory reaction leading to granuloma formation and to excess production and/or disarrangement of collagens I and III; the latter led to fibrosis.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing: Paracoccidioides cerebriformis revisited

Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.

Septicemia caused by Paracoccidioides brasiliensis (Lutz, 1908) as the cause of death of an aids patient from Santos, São Paulo State, Brazil – a nonendemic area

The first case of Paracoccidioides brasiliensis in Santos (Brazil) leading to septicemia and death of an HIV-positive patient is reported here. The patient was a 34-year-old female that presented essential fever and was only diagnosed after death by positive blood culture. The authors underscore the atypical nature of the case, since the patient was a female at fertile age who was born and had always lived in Santos, which is a nonendemic area for this infection.

Virulence profile of ten Paracoccidioides brasiliensis isolates: association with morphologic and genetic patterns

Ten isolates of Paracoccidioides brasiliensis were examined for differences in virulence in outbred mice intravenously inoculated with the fungus, associated with mycelial morphology, and genetic patterns measured by random amplified polymorphic DNA (RAPD). Virulence was evaluated by viable yeast cell recovery from lungs and demonstration of histopathologic lesions in different organs. The results showed that the isolates presented four virulence degrees: high virulence, intermediate, low and non-virulence. RAPD clustered the isolates studied in two main groups with 56% of genetic similarity. Strains with low virulence, Pb265 or the non-virulent, Pb192, showed glabrous/cerebriform morphology and high genetic similarity (98.7%) when compared to the other isolates studied. The same was observed with Bt79 and Bt83 that shared 96% genetic similarity, cottony colonies and high virulence. The RAPD technique could only discriminate P. brasiliensis isolates according to glabrous/cerebriform or cottony colonies, and also high from low virulence strains. Isolates with intermediate virulence such as Pb18, Pb18B6, Bt32 and Bt56 showed variability in their similarity coefficient suggesting that RAPD was able to detect genetic variability in this fungal specie. Virulence profile of P. brasiliensis demonstrated that both mycelial morphologic extreme phenotypes may be associated with fungal virulence and their in vitro subculture time. Thus, RAPD technique analysis employed in association with virulence, morphologic and immunologic aspects might prove adequate to detect differences between P. brasiliensis isolates.

Inhibitory effect of deferoxamine on Paracoccidioides brasiliensis survival in human monocytes: reversal by holotransferrin not by apotransferrin

The mechanisms used by Paracoccidioides brasiliensis to survive into phagocytic cells are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Thus, the objective of this paper was to investigate the role of intracellular iron in regulating the capacity of P. brasiliensis yeast cells to survive within human monocytes. Treatment of monocytes with deferoxamine, an iron chelator, suppressed the survival of yeasts in a concentration-dependent manner. The effect of deferoxamine was reversed by iron-saturated transferrin (holotransferrin) but not by nonsaturated transferrin (apotransferrin). These results strongly suggest that P. brasiliensis survival in human monocytes is iron dependent.

Sensitivity of an immunoenzymatic test for detection of ant-L. brasiliensis antibodies compared to other tests used for the diagnosis of American cutaneous leishmaniasis

The diagnosis of American cutaneous leishmaniasis (ACL) is frequently based on clinical and epidemiological data associated with the results of laboratory tests. Some laboratory methods are currently being applied for the diagnosis of ACL, among them the indirect immunofluorescence reaction (IIFR), the Montenegro skin test (MST), histopathological examination, and the polymerase chain reaction (PCR). The performance of these methods varies in a considerable proportion of patients. After the standardization of an immunoenzymatic test (ELISA) for the detection of IgG in the serum of patients with ACL using a crude Leishmania braziliensis antigen, the results obtained were compared to those of other tests routinely used for the diagnosis. The tests revealed the following sensitivity, when analyzed separately: 85% for ELISA IgG, 81% for PCR, 64.4% for MST, 58.1% for IIFR, and 34% for the presence of parasites in the biopsy. ELISA was positive in 75% of patients with ACL presenting a negative MST, in 84.8% of ACL patients with negative skin or mucous biopsies for the presence of the parasite, and in 100% of cases with a negative PCR. Thus, ELISA presented a higher sensitivity than the other tests and was useful as a complementary method for the diagnosis of ACL.

Role of iron in the nitric oxide-mediated fungicidal mechanism of IFN-gamma-activated murine macrophages against Paracoccidioides brasiliensis conidia

Iron is an essential growth element of virtually all microorganisms and its restriction is one of the mechanisms used by macrophages to control microbial multiplication. Paracoccidioides brasiliensis, the agent of paracoccidioidomycosis, an important systemic mycosis in Latin America, is inhibited in its conidia-to-yeast conversion in the absence of iron. We studied the participation of iron in the nitric oxide (NO)-mediated fungicidal mechanism against conidia. Peritoneal murine macrophages activated with 50U/mL of IFN-gamma or treated with 35 µM Deferoxamine (DEX) and infected with P. brasiliensis conidia, were co-cultured and incubated for 96 h in the presence of different concentrations of holotransferrin (HOLO) and FeS0(4). The supernatants were withdrawn in order to assess NO2 production by the Griess method. The monolayers were fixed, stained and observed microscopically. The percentage of the conidia-to-yeast transition was estimated by counting 200 intracellular propagules. IFN-gamma-activated or DEX-treated Mthetas presented marked inhibition of the conidia-to-yeast conversion (19 and 56%, respectively) in comparison with non-activated or untreated Mthetas (80%). IFN-gamma-activated macrophages produced high NO levels in comparison with the controls. Additionally, when the activated or treated-macrophages were supplemented with iron donors (HOLO or FeSO4), the inhibitory action was reversed, although NO production remained intact. These results suggest that the NO-mediated fungicidal mechanism exerted by IFN-gamma-activated macrophages against P. brasiliensis conidia, is dependent of an iron interaction.

On the unknown ecological niche of Paracoccidioides brasiliensis: our hypothesis of 1989: present status and perspectives

In 1989, CONTI DÍAZ & RILLA published a hypothesis concerning the as yet unknown ecological niche of Paracoccidioides brasiliensis. The hypothesis proposed a highly efficient paracoccidioidal ecological strategy based on an important natural reservoir of the parasite, probably in heterothermic animals from fresh water environments. Further, the hypothesis proposed, a transient and variable residence in the soil with a wide aleuriospore dispersion throughout the environment together with an elevated capability of infecting humans, and domestic and wild animals. This paper analyzes scientific publications from the IX International Meeting on Paracoccidioidomycosis held in Águas de Lindoia, São Paulo, Brazil from 2-5 October 2005, providing a comparative study among this articles and with other recently published papers and the hypothesis' postulates. Since various findings and observations appear to agree with the postulates, the pursuit of novel, specific research projects in the supposed reservoirs is recommended partially or fully to confirm the hypothesis using classical laboratorial methods and modern molecular biology techniques.

Lysozyme plays a dual role against the dimorphic fungus Paracoccidioides brasiliensis

In order to determine the role of lysozyme, an antimicrobial peptide belonging to the innate immune system, against the dimorphic fungus Paracoccidioides brasiliensis, co-cultures of the MH-S murine alveolar macrophages cell line with P. brasiliensis conidia were done; assays to evaluate the effect of physiological and inflammatory concentrations of lysozyme directly on the fungus life cycle were also undertaken. We observed that TNF-α-activated macrophages significantly inhibited the conidia to yeast transition (p = 0.0043) and exerted an important fungicidal effect (p = 0.0044), killing 27% more fungal propagules in comparison with controls. Nonetheless, after adding a selective inhibitor of lysozyme, the fungicidal effect was reverted. When P. brasiliensis propagules were exposed directly to different concentrations of lysozyme, a dual effect was observed. Physiologic concentrations of the enzyme facilitated the conidia-to-yeast transition process (p < 0.05). On the contrary, inflammatory concentrations impaired the normal temperature-dependant fungal transition (p < 0.0001). When yeast cells were exposed to lysozyme, irrespective of concentration, the multiple-budding ability was badly impaired (p < 0.0001). In addition, ultra-structural changes such as subcellular degradation, fusion of lipid vacuoles, lamellar structures and interruption of the fibrilar layer were observed in lysozyme exposed conidia. These results suggest that lysozyme appears to exert a dual role as part of the anti-P. brasiliensis defense mechanisms.