Paracocci

Acta Microbiol Pol, 2000, 49(3-4), 265 - 70
Plasmid occurrence and diversity in the genus Paracoccus; Baj J et al.; The results of screening for the occurrence of plasmids in several strains representing 11 out of 13 species of the genus Paracoccus are presented . We show that plasmids (ranging in size from 2.7 to above 450 kb) are widely distributed in this genus . Only one tested strain (P . alkenifer) appears to be plasmid-free . The majority of the strains harbour at least two plasmids, one of which usually fits into the class of megaplasmids.

J Inorg Biochem, 2001 Feb, 83(4), 281 - 6
Perturbations at the high spin heme b center in the membrane-bound nitric oxide reductase; Kurose S et al.; The effects of lowering pH from 7 to 5 on the absorption, circular dichroism (MCD) and EPR spectra were studied for Paracoccus halodenitrificans nitric oxide reductase (NOR) . Intensities of the characteristic bands for the high spin heme b, that at 592 nm in the absorption spectrum and those at 591 (+) and 606 (-) in the MCD spectrum decreased considerably . Concomitant cryogenic EPR spectrum indicated a drastic increase in the signal intensity due to the high spin heme b at g approximately 6, of which less than 5% had been EPR detectable at pH 7 . Cyanide (x40) bound to the high spin heme b center in the reduced NOR irrespective of pH, while a much larger amount of azide (x1000) was necessary to bind to the reduced NOR at an acidic pH, ca . 5 . Based on these results the structure and function of the high spin heme b center as the active site of NOR was discussed.

Arch Microbiol, 2001 Feb, 175(2), 102 - 11
Evidence for two pathways of thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus); Kappler U et al.; The pathway of thiosulfate oxidation in the facultatively chemolithotrophic, sulfur-oxidizing bacterium Starkeya novella (formerly Thiobacillus novellus) has not been established beyond doubt . Recently, isolation of the sorAB genes, which encode a soluble sulfite:cytochrome c oxidoreductase, has been reported, indicating that a thiosulfate-oxidizing pathway not involving a multienzyme complex may exist in this organism . Here we report the cloning and sequencing of the soxBCD genes from S . novella, which are closely related to the corresponding genes encoding the thiosulfate-oxidizing multienzyme complex from Paracoccus pantotrophus . These findings suggest two distinct pathways for thiosulfate oxidation in S . novella . The expression of sorAB and soxC in cells grown on thiosulfate- and/or glucose-containing media was studied by Western blot analysis . The results showed that the SorAB protein is synthesized in the presence of thiosulfate irrespective of the presence of glucose . In contrast, the SoxC protein is subject to repression by glucose; the repression, however, appears to be dependent on the relative amounts of glucose and thiosulfate present . The regulatory effects observed for the expression of sorAB are likely to be mediated by an extracytoplasmic function sigma factor encoded by the sigE gene identified upstream of sorAB.

Braz J Med Biol Res, 2001 Apr, 34(4), 529 - 37
The low efficiency of dendritic cells and macrophages from mice susceptible to Paracoccidioides brasiliensis in inducing a Th1 response; Almeida SR et al.; In the present study we evaluated T cell proliferation and Th lymphokine patterns in response to gp43 from Paracoccidioides brasiliensis presented by isolated dendritic cells from susceptible and resistant mice . T cell proliferation assays showed that dendritic cells from susceptible mice were less efficient than those from resistant mice . The pattern of T cell lymphokines stimulated by dendritic cells was always Th1, although the levels of IL-2 and IFN-gamma were lower in T cell cultures from susceptible mice . To determine whether different antigen-presenting cells such as macrophages and dendritic cells stimulated different concentrations of Th1 lymphokines, the production of IFN-gamma and IL-2 was measured . It was observed that dendritic cells were more efficient than macrophages in stimulating lymphoproliferation in resistant mice . However, no significant difference was observed for IFN-gamma or IL-2 production . When cells from susceptible mice were used, macrophages were more efficient in stimulating lymphoproliferation than dendritic cells, but no difference was observed in the production of Th1 cytokine . Taken together, these results suggest the lower efficiency of dendritic cells and macrophages from B10.A mice in stimulating T cells that secrete Th1 lymphokines in vitro, an effect that may be involved in the progression of the disease in vivo.

J Am Acad Dermatol, 2001 Apr, 44(4), 585 - 92
Chromoblastomycosis: a review of 100 cases in the state of Rio Grande do Sul, Brazil; Minotto R et al.; BACKGROUND: If not diagnosed earlier, chromoblastomycosis has a chronic evolutional course that may cause several problems, such as difficulty in managing therapy because of the recrudescent character of the disease, potential association with the growth of epidermoid carcinoma in affected regions, and poor quality of life and work incapacity to the patient . Although infrequent, new cases are reported in the state of Rio Grande do Sul every year, ratifying the necessity for further studies on this disease . OBJECTIVE: The purpose of this study was to review clinical features and response to therapy in patients with chromoblastomycosis and present data on the demography and history of this disease in the state of Rio Grande do Sul, Brazil . METHODS: We reviewed case records of 100 patients with skin lesions caused by chromoblastomycosis, who were treated between 1963 and 1998 . The cases were confirmed by the histopathologic and mycologic analyses made by the Dermatology Service of the Universidade Federal do Rio Grande do Sul at the Santa Casa de Misericordia Hospital . RESULTS: There was a predominance of male patients (4:1) and of white farmers whose ages ranged from 50 to 59 years, with lesions on their lower limbs . Most of them were from the northern regions of the state . The average time between the appearance of the disease and medical diagnosis was 14 years . The verrucous type proved to be the most frequently reported lesion (53%) . Thorn wounds were associated with the disease in 16% of the cases . Lesions uncommon to some parts of the body were also reported . In two of the cases, cutaneous lesions caused by paracoccidioidomycosis and chromoblastomycosis were found in the same patient . Epidermoid carcinoma was found in the same parts of the body affected by chromoblastomycosis . Eumycotic mycetoma and chromoblastomycosis were associated . Fonsecaea pedrosoi was found in 96% of the cases, and Phialophora verrucosa in 4% of the cases . CONCLUSION: In our study, we observed a predominance of cases in the regions of Missoes and Alto Uruguay, followed by the upper and lower northeastern slopes and the lowlands . Severe cases of chromoblastomycosis with intense skin involvement (eg, lesions with carcinoma) were observed . Statistical analysis showed recrudescence of the disease in 43% of cases despite the treatment used.

Med Trop (Mars), 2000, 60(3), 281 - 90
{Systemic tropical mycoses}; Marty P et al.; Systemic tropical mycoses have been emerging since the beginning of the AIDS epidemic . The incidence of these infections is probably even underestimated since most cases occur in populations with poor access to medical care in regions where modern diagnostic methods are unavailable . In Europe, this pathology is sometimes observed in returning travelers, aid workers, and immigrants . Differential diagnosis of imported systemic tropical mycosis may be difficult for uniformed physicians . The purpose of this review of the literature is to provide up-to-date epidemiological, clinical, diagnostic, and therapeutic data on the six main systemic tropical mycoses with disseminated forms . The six mycoses described in this study are histoplasmosis, coccidioidomycosis, paracoccidioidomycosis, blastomycosis, sporotrichosis, and penicilliosis due to Penicillium marneffei . Strictly superficial and subcutaneous mycoses are not covered.

Expert Opin Pharmacother, 2000 Jan, 1(2), 287 - 304
Itraconazole; Pierard GE et al.; Itraconazole is a broad spectrum triazole antifungal agent . It has favourable pharmacodynamic and pharmacokinetic profiles and is available as both oral and i.v . formulations . Over the last two decades, clinical and animal infection studies have demonstrated the efficacy of itraconazole in a wide range of superficial fungal infections including difficult-to-treat dermatophytoses and onychomycoses . Furthermore, shortened treatment regimens have proven to be effective, ranging from 1-day treatment for vaginal candidosis to 1-week pulse therapy per month, for 2-4 months, in onychomycosis and follicular dermatophytosis . Clinical experience with itraconazole in the treatment of deep mycoses is less comprehensive . However, results in systemic candidosis, sporotrichosis, blastomycosis, paracoccidioiodomycosis, certain types of histoplasmosis and aspergillosis are extremely encouraging . Itraconazole is less effective in the treatment of chromomycosis and coccidioidomycosis . Nevertheless, considering the refractory nature of these diseases, itraconazole has proven to be a valuable addition to the antifungal drugs currently available for treatment . Itraconazole has been well-tolerated with doses of up to 400 mg/day being generally free of serious adverse effects . However, a potential for drug interactions exists, mediated through the cytochrome P450 enzyme 3A4 system, which should be considered when itraconazole is used as part of a multi-drug regimen.

Biochem Biophys Res Commun, 2001 Mar 2, 281(3), 788 - 94
The cytochrome c domain of dimeric cytochrome cd(1) of Paracoccus pantotrophus can be produced at high levels as a monomeric holoprotein using an improved c-type cytochrome expression system in Escherichia coli; Gordon EH et al.; Cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus is a dimer; within each monomer there is a largely alpha-helical domain that contains the c-type cytochrome centre . The structure of this domain changes significantly upon reduction of the heme iron, for which the ligands change from His17/His69 to Met106/His69 . Overproduction, using an improved Escherichia coli expression system, of this c-type cytochrome domain as an independent monomer is reported here . The properties of the independent domain are compared with those when it is part of dimeric holo or semi-apo cytochrome cd(1) .

Cytokine, 2001 Feb 21, 13(4), 248 - 52
Imbalance of IL-2, IFN-gamma and IL-10 secretion in the immunosuppression associated with human paracoccidioidomycosis; Benard G et al.; Patients with paracoccidioidomycosis (PCM) display a certain degree of immunecompromise characterized by lymphocyte hyporesponsiveness to the main Paracoccidioides brasiliensis antigen (gp43) . To determine whether cytokines are involved in this state, we evaluated the secretion of IL-2, IL-10 and IFN-gamma by peripheral blood mononuclear cells (PBMC) from patients with the acute (AF) and chronic (CF) forms of PCM and from healthy, P . brasiliensis-sensitized subjects . gp43-stimulated PBMC from healthy subjects produced substantial amounts of IL-2, IFN-gamma and IL-10, whereas PBMC from AF and CF patients produced low levels of IL-2 and IFN-gamma but substantial amounts of IL-10 . Phytohaemagglutinin-induced cytokine secretion was comparable among AF and CF patients and healthy subjects, suggesting integrity of non-specific cellular immune mechanisms in PCM . gp43-pulsed adherent cells, but not non-adherent cells, were the main source of IL-10 . Moreover, IL-2 and IFN-gamma secretion correlated inversely with the amount of specific antibodies produced by patients and healthy subjects . Our results suggest that the imbalance in cytokine production of patients with PCM plays a role in the gp43-hyporesponsiveness and the marked (non-protective) antibody production of these patients .

J Med Microbiol, 2001 Feb, 50(2), 127 - 34
Antibody isotypes to a Paracoccidioides brasiliensis somatic antigen in sub-acute and chronic form paracoccidioidomycosis; Juvenale M et al.; This report describes the differences in isotype antibody reactivity against a crude Paracoccidioides brasiliensis antigenic preparation in the sub-acute (SAF) and chronic (CF) forms of paracoccidioidomycosis before treatment . IgG antibodies were detected in all patients, with a slightly but not significantly higher reactivity in the SAF . IgG1 antibodies were present, frequently at high levels, in both forms, whereas IgG3 was always low or absent . IgG2 antibodies were detectable in most patients, but at high levels in only a few CF patients . IgG4 was found mainly in SAF patients, whereas IgA was detected almost only in CF patients, probably due to a Th2 pattern of immune response in the more severe SAF, and the characteristic mucosal involvement of the CF, respectively . Immunoblot analysis showed that, in addition to the 43-kDa immunodominant fraction, other less well-characterised fractions were also recognised differentially by the isotypes and deserve further investigation.

Med Mycol, 2000 Dec, 38(6), 437 - 41
RFLP analysis reveals marked geographical isolation between strains of Paracoccidioides brasiliensis; Nino-Vega GA et al.; Restriction fragment length polymorphism (RFLP) was performed on 32 isolates of the pathogenic fungus Paracoccidioides brasiliensis from geographically separated regions of South America . The use of HinfI and HincII gave clear RFLP patterns, for which high discriminatory indices could be calculated . Computational analysis of the RFLP patterns for the 32 isolates suggested that at least five groups of strains existed, each of which was geographically distinct and corresponded closely with present country borders . These results underline the belief that P . brasiliensis infections are acquired from exogenous sources and that this fungus occupies specialist endemic niches within the natural environment.

Mycoses, 2000, 43(11-12), 403 - 7
In vitro susceptibilities of Paracoccidioides brasiliensis yeast form to antifungal drugs; Hahn RC et al.; A total of 12 Paracoccidioides brasiliensis yeast form strains, including clinical and environmental isolates, were tested for their susceptibilities to amphotericin B, ketoconazole, fluconazole, and itraconazole . The tests were determined using a broth macrodilution procedure . In general, there was a remarkable homogeneity of results for all strains, and comparable MICs were found for environmental and clinical isolates.

Med Mycol, 2000, 38 Suppl 1, 189 - 97
Molecular typing of pathogenic fungi; McEwen JG et al.; In this Round Table, the application of several methods of molecular typing were discussed in reference to four important pathogenic fungi: Coccidioides immitis, Histoplasma capsulatum, Candida albicans and Paracoccidioides brasiliensis . Among the different methods the following were discussed: restriction fragment length polymorphisms (RFLP), single nucleotide polymorphisms, random amplified polymorphic DNA (RAPD), polymerase chain reaction (PCR)-RFLP and microsatellites . By means of these methods, several important biological questions related to speciation, mode of reproduction and population genetics could be approached . The basic information obtained from this approach has implications in the understanding of these pathogenic fungi in relation to their behavior and the development of pathogenic features, such as resistance to antimicrobials and virulence factors used for colonization of mammalian hosts . The knowledge obtained from these studies could also be used for the development of innovative diagnostic methods, as well as for novel therapeutic approaches and production of vaccines.

Med Mycol, 2000, 38 Suppl 1, 139 - 45
Conventional versus molecular diagnostic tests; Elias Costa MR et al.; In this session, emphasis was placed on the diagnosis of various mycoses through the identification of antibodies and antigens in sera, as well as on new techniques to properly identify medically important fungi through molecular biological procedures . The use of restriction fragment length polymorphism (RFLP) on fungal mitochondrial DNA (mtDNA) has enabled the identification of different strains of Sporothrix schenkii, several dermatophytes, Candida spp . and black fungi according to their species-specific mtDNA-RFLP patterns . In some species, distinct specific types where found in relation to the geographic origin of the patients . These particular molecular diagnostic tests are useful in the identification of strains and in epidemiological studies . An account of the applications of serological methods in the diagnosis of paracoccidioidomycosis was presented . Serology has been used in the identification of paracoccidioidomycosis using a specific, sensitive and rapid antibody-based immunodiagnosis method . Using the gp43 antigen, the diagnostic coverage of inmunodifussion has been improved from the 93-95% achieved with crude antigens, to 100% in an enzyme-linked immunodiffusion assay capture test . Cross-reactions were eliminated by treatment of the antigen with sodium metaperiodate . Antibody detection is useful, especially in paracoccidioidomycosis and histoplasmosis.

Med Mycol, 2000, 38 Suppl 1, 113 - 23
Pathogenesis II: fungal responses to host responses: interaction of host cells with fungi; Mendes-Giannini MJ et al.; Most of our knowledge concerning the virulence determinants of pathogenic fungi comes from the infected host, mainly from animal models and more recently from in vitro studies with cell cultures . The fungi usually present intra- and/or extracellular host-parasite interfaces, with the parasitism phenomenon dependent on complementary surface molecules . Among living organisms, this has been characterized as a cohabitation event, where the fungus is able to recognize specific host tissues acting as an attractant, creating stable conditions for its survival . Several fungi pathogenic for humans and animals have evolved special strategies to deliver elements to their cellular targets that may be relevant to their pathogenicity . Most of these pathogens express surface factors that mediate binding to host cells either directly or indirectly, in the latter case binding to host adhesion components such as extracellular matrix (ECM) proteins, which act as 'interlinking' molecules . The entry of the pathogen into the host cell is initiated by fungal adherence to the cell surface, which generates an uptake signal that may induce its cytoplasmic internalization . Once this is accomplished, some fungi are able to alter the host cytoskeletal architecture, as manifested by a rearrangement of microtubule and microfilament proteins, and this can also induce epithelial host cells to become apoptotic . It is possible that fungal pathogens induce modulation of different host cell pathways in order to evade host defences and to foster their own proliferation . For a number of pathogens, the ability to bind ECM glycoproteins, the capability of internalization and the induction of apoptosis are considered important factors in virulence . Furthermore, specific recognition between fungal parasites and their host cell targets may be mediated by the interaction of carbohydrate-binding proteins, e.g., lectins on the surface of one type of cell, probably a parasite, that combine with complementary sugars on the surface of host-cell . These interactions supply precise models to study putative adhesins and receptor-containing molecules in the context of the fungus-host interface . The recognition of the host molecules by fungi such as Aspergillus fumigatus, Paracoccidioides brasiliensis and Histoplasma capsulatum, and their molecular mechanisms of adhesion and invasion, are reviewed in this paper.

Med Mycol, 2000, 38 Suppl 1, 1 - 8
From magic to science: a journey throughout Latin American medical mycology; San-Blas G; The start of Latin America's love story with fungi may be placed in pre-Hispanic times when the use of fungi in both ritual ceremonies and daily life were common to the native civilizations . But the medical mycology discipline in Latin America started at the end of the 19th Century . At that time, scholars such as A . Posadas, R . Seeber, A . Lutz and P . Almeida, discovered agents of fungal diseases, the study of which has influenced the regional research ever since . Heirs to them are the researchers that today thrive in regional Universities and Research Institutes . Two current initiatives improve cooperation among Latin American medical mycologists . First, the periodical organization of International Paracoccidioidomycosis Meetings (seven so far, from 1979 to 1999); second, the creation of the Latin American Association for Mycology in 1991 (three Congresses, from 1993 to 1999) . Latin American publications have increased in international specialized journals such as that from our Society (ISHAM) (from 8% in 1967 to 19% in 1999), and the Iberoamerican Journal of Mycology (Revista Iberoamericana de Micologia; > 40% from 1997 to 1999) . In addition, Latin American participation at ISHAM International Congresses has risen from 6.9% in 1975 to 21.3% in 1997, and 43.2% at the 14th ISHAM Congress, held for the first time in a Latin American country, Argentina . A significant contribution of women to the scientific establishment of Latin American medical mycology (e.g., 45% of Latin American papers vs . 18% of other regions published in Journal of Medical and Veterinary Mycology in 1987, had women as authors or coauthors) suggests a better academic consideration of Latin American women against their counterparts in the developed world . Taken together, all these figures reflect the enthusiasm of our Latin American colleagues in the field, despite the difficulties that afflict our region, and affect our work.

Analyst, 2000 Nov, 125(11), 1993 - 9
Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in a sol-gel matrix; Ferretti S et al.; Nitrite is an important human health and environmental analyte . As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM) . In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus . The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined . The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9 . Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection . The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity . The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM . In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1 . This structure enabled the determination of nitrite concentrations within ca . 5 min . The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C.

J Biol Inorg Chem, 2001 Jan, 6(1), 23 - 6
An amicyanin C-terminal loop mutant where the active-site histidine donor cannot be protonated; Remenyi R et al.; A novel blue copper protein was constructed by replacing the C-terminal loop of amicyanin (Paracoccus versutus) by the homologous loop of rusticyanin . The C-terminal loop of both amicyanin and rusticyanin contains three (His, Cys, Met) of the four copper ligands . The amicyanin mutant exhibits all spectroscopic properties normally encountered for blue copper sites . The midpoint potential (369 mV) is the highest reported value for an amicyanin mutant . Cyclic voltammetry and NMR studies of the reduced form indicate that, in contrast to wild-type amicyanin and all amicyanin mutants described so far, the C-terminal histidine ligand does not protonate in the accessible pH range (pKa<4.5).

Mycopathologia, 1999 Dec, 148(3), 123 - 30
Paracoccidioides brasiliensis isolated from armadillos is virulent to Syrian hamsters; Peracoli MT et al.; Isolates of Paracoccidioides brasiliensis may vary in virulence according to time of in vitro subcultivation . The present study compared the morphology and pathogenicity to hamsters of two P . brasiliensis isolates: one obtained from human lesions and maintained in the laboratory for several years (Pb-18) and the other isolate recovered from hamsters inoculated with organ homogenates from armadillos (Pb-T) . The microscopic morphology of Pb-18 and Pb-T showed yeast cells with similar diameter . However, Pb-T produced a significantly higher number of buds per mother cell than Pb-18 . Besides, the mycelial form of Pb-T developed abundant sporulation during 8 weeks of culture which was absent in the Pb-18 isolate . Virulence studies demonstrated that mortality rates, antibody levels, fungal load and extent of lesions in the organs were significantly higher in animals infected with Pb-T . The results demonstrated that Pb-T recently isolated from an animal was more virulent than Pb-18 . These differences between the two P . brasiliensis isolates may be indicators of virulence attenuation in this fungal species.

Nippon Ishinkin Gakkai Zasshi, 2001, 42(1), 23 - 7
Detection of gp43 and ITS1-5.8S-ITS2 ribosomal RNA genes of Paracoccidioides brasiliensis in paraffin-embedded tissue; Sano A et al.; Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries . An increase in PCM has been reported in recent years and the disease is now recognized as one of the imported fungal infections in Japan . To date, more than 15 cases of PCM have been reported in our country, and five of them were diagnosed by clinical and histopathological findings without mycological study . We applied 2 nested polymerase chain reaction (PCR) amplification methods for detecting P . brasiliensis genes from paraffin-embedded tissue specimens . Successfully amplified were: a 473 base pairs fragment of gp43 gene of P . brasiliensis (located from 741st to 1,213rd base), and a 418 base pairs fragment of 5.8S ribosomal RNA gene of P . brasilienisis which included internal transcribed spacers (ITS) 1 and 2 (located from 131st at ITS1 to 195th at ITS2) in paraffin-embedded murine tissues infected with P . brasiliensis yeast cells . The authenticity of the PCR products was confirmed by nucleotide sequence analysis . These results indicate that the two nested PCR methods may be useful for diagnosis of PCM.

Biochem Biophys Res Commun, 2001 Jan 12, 280(1), 19 - 24
Structure elucidation of sphingolipids from the mycopathogen Sporothrix schenckii: identification of novel glycosylinositol phosphorylceramides with core manalpha1-->6Ins linkage; Toledo MS et al.; Acidic glycosphingolipid components were extracted from the mycelium form of the thermally dimorphic mycopathogen Sporothrix schenckii . Two fractions from the mycelium form (Ss-M1 and Ss-M2), having the highest Rf values on HPTLC analysis, were isolated and their structures elucidated by 1- and 2-D 13C- and 1H-nuclear magnetic resonance spectroscopy, and electrospray ionization mass spectrometry with lithium adduction of molecular ions . The structures of Ss-M1 and Ss-M2 were determined to be Manalpha1-->Ins1-P-1Cer and Manalpha1--> 3Manalpha1-->Ins1-P-1Cer, respectively (where Ins = myo-inositol, P = phosphodiester) . The Manalpha1-->6Ins motif is found normally in diacylglycerol-based glycophosphatidylinositols of Mycobacteria, but this is the first unambiguous identification of the same linkage making up the core structure of fungal glycosylinositol phosphorylceramides (GIPCs) . These results are discussed in relation to the structures of GIPCs of other mycopathogens, including Histoplasma capsulatum and Paracoccidioides brasiliensis .

J Clin Microbiol, 2001 Jan, 39(1), 309 - 14
Phylogenetic analysis of Lacazia loboi places this previously uncharacterized pathogen within the dimorphic Onygenales; Herr RA et al.; Lacazia loboi is the last of the classical fungal pathogens to remain a taxonomic enigma, primarily because it has resisted cultivation and only causes cutaneous and subcutaneous infections in humans and dolphins in the New World tropics . To place it in the evolutionary tree of life, as has been done for the other enigmatic human pathogens Pneumocystis carinii and Rhinosporidium seeberi, we amplified its 18S small-subunit ribosomal DNA (SSU rDNA) and 600 bp of its chitin synthase-2 gene . Our phylogenetic analysis indicated that L . loboi is the sister taxon of the human dimorphic fungal pathogen Paracoccidioides brasiliensis and that both species belong with the other dimorphic fungal pathogens in the order Onygenales . The low nucleotide variation among three P . brasiliensis 18S SSU rDNA sequences contrasts with the surprising amount of nucleotide differences between the two sequences of L . loboi used in this study, suggesting that the nucleic acid epidemiology of this hydrophilic pathogen will be rewarding.

Biochem Biophys Res Commun, 2000 Dec 20, 279(2), 674 - 7
A novel conformer of oxidized Paracoccus pantotrophus cytochrome cd(1) observed by freeze-quench NIR-MCD spectroscopy; Allen JW et al.; Paracoccus pantotrophus cytochrome cd(1) is a physiological nitrite reductase and an in vitro hydroxylamine reductase . The oxidised "as isolated" form of the enzyme has bis-histidinyl coordinated c-heme and upon reduction its coordination changes to histidine/methionine . Following treatment of reduced enzyme with hydroxylamine, a novel, oxidised, conformer of the enzyme is obtained . We have devised protocols for freeze-quench near-ir-MCD spectroscopy that have allowed us to establish unequivocally the c-heme coordination of this species as His/Met . Thus it is shown that the catalytically competent, hydroxylamine reoxidised, form of P . pantotrophus cytochrome cd(1) has different axial ligands to the c-heme than "as isolated" enzyme .

J Bacteriol, 2001 Jan, 183(1), 257 - 63
Identification of ccdA in Paracoccus pantotrophus GB17: disruption of ccdA causes complete deficiency in c-type cytochromes; Bardischewsky F et al.; A transposon Tn5-mob insertional mutant of Paracoccus pantotrophus GB17, strain TP43, was unable to oxidize thiosulfate aerobically or to reduce nitrite anaerobically, and the cellular yields were generally decreased by 11 to 20% . Strain TP43 was unable to form functional c-type cytochromes, as determined by difference spectroscopy and heme staining . However, formation of apocytochromes and their transport to the periplasm were not affected, as seen with SoxD, a c-type cytochrome associated with the periplasmic sulfite dehydrogenase homologue . The Tn5-mob-containing DNA region of strain TP43 was cloned into pSUP205 to produce pE18TP43 . With the aid of pE18TP43 the corresponding wild-type gene region of 15 kb was isolated from a heterogenote recombinant to produce pEF15 . Sequence analysis of 2.8 kb of the relevant region uncovered three open reading frames, designated ORFA, ccdA, and ORFB, with the latter being oriented divergently . ORFA and ccdA were constitutively cotranscribed as determined by primer extension analysis . In strain TP43 Tn5-mob was inserted into ccdA . The deduced ORFA product showed no similarity to any protein in databases . However, the ccdA gene product exhibited similarities to proteins assigned to different functions in bacteria, such as cytochrome c biogenesis . For these proteins at least six transmembrane helices are predicted with the potential to form a channel with two conserved cysteines . This structural identity suggests that these proteins transfer reducing equivalents from the cytoplasm to the periplasm and that the cysteines bring about this transfer to enable the various specific functions via specific redox mediators such as thioredoxins . CcdA of P . pantotrophus is 42% identical to a protein predicted by ORF2, and its location within the sox gene cluster coding for lithotrophic sulfur oxidation suggested a different function.

Biochem J, 2000 Dec 15, 352 Pt 3, 859 - 64
Thiocyanate binding to the molybdenum centre of the periplasmic nitrate reductase from Paracoccus pantotrophus; Butler CS et al.; The periplasmic nitrate reductase (NAP) from Paracoccus pantotrophus is a soluble two-subunit enzyme (NapAB) that binds two haem groups, a {4Fe-4S} cluster and a bis(molybdopterin guanine dinucleotide) (MGD) cofactor that catalyses the reduction of nitrate to nitrite . In the present study the effect of KSCN (potassium thiocyanate) as an inhibitor and Mo ligand has been investigated . Results are presented that show NAP is sensitive to SCN(-) (thiocyanate) inhibition, with SCN(-) acting as a competitive inhibitor of nitrate (K(i) approximately 4.0 mM) . The formation of a novel EPR Mo(V) species with an elevated g(av) value (g(av) approximately 1.994) compared to the Mo(V) High-g (resting) species was observed upon redox cycling in the presence of SCN(-) . Mo K-edge EXAFS analysis of the dithionite-reduced NAP was best fitted as a mono-oxo Mo(IV) species with three Mo-S ligands at 2.35 A (1 A=0.1 nm) and a Mo-O ligand at 2.14 A . The addition of SCN(-) to the reduced Mo(IV) NAP generated a sample that was best fitted as a mono-oxo (1.70 A) Mo(IV) species with four Mo-S ligands at 2.34 A . Taken together, the competitive nature of SCN(-) inhibition of periplasmic nitrate reductase activity, the elevated Mo(V) EPR g(av) value following redox cycling in the presence of SCN(-) and the increase in sulphur co-ordination of Mo(IV) upon SCN(-) binding, provide strong evidence for the direct binding of SCN(-) via a sulphur atom to Mo.

Braz J Infect Dis, 1998 Jun, 2(3), 118 - 127
Management of Opportunistic Infections in HIV(+) Patients: Contrasts Between Europe and South America; Levi GC; The author discusses the management of some opportunistic diseases more commonly observed in South American AIDS patients than in European ones . Characteristics of coinfection with HIV and leprosy, paracoccidioidomycosis, Chagas' disease, mucocutaneous leishmaniasis, malaria, disseminated BCG and strongyloidiasis are reviewed, with special emphasis on preferred therapeutic schedules for these conditions.

Med Mycol, 2000 Oct, 38(5), 371 - 7
Correlation among immune response, morphogenesis of the granulomatous reaction and spleen lymphoid structure in murine experimental paracoccidioidomycosis; Soares AM et al.; We studied the correlation among cellular immune response, the pattern of lung granulomatous lesions and alterations in spleen lymphoid structure in Swiss mice inoculated intravenously with Paracoccidioides brasiliensis strain 18 . The animals were evaluated at 24, 48 and 96 h after infection and further studied weekly for 18 weeks by: (i) the macrophage migration inhibition test with phytohemagglutinin (PHA) and P . brasiliensis antigen (PbAg); and (ii) histopathology of the lung and spleen lesions . One group of animals was gamma-irradiated (8 Gy), infected under the same conditions and evaluated for the pattern of lung granulomatous lesions and spleen lymphoid structure at 24, 48 and 96 h after infection . During the first week of infection, the non-irradiated animals presented a positive response to PHA and PbAg, compact granulomas in the lungs and a typical hyperplasia of the spleen white pulp . However, from weeks 2 to 5, a depression of the cell-mediated immunity (CMI) response to PHA and PbAg was observed in association with granulomas presenting only large mononuclear cells and lacking both giant cells and a peripheral halo of small mononuclear cells . This pattern of granuloma formation was similar to that seen in gamma-irradiated animals, whose cells involved in CMI were absent . After week 7, the non-irradiated animals showed granulomas characterized by the presence of giant cells and a peripheral halo of small mononuclear cells . This type of granuloma was formed concomitantly with recovery of the CMI and of the lymphoid structure of the spleen . The results showed a correlation among granulomas composed of large mononuclear cells, hypoplasia of the splenic tissue and impaired CMI . This correlation indicated that although granuloma morphogenesis per se does not depend on the activation of CMI, this response is important at later stages during modulation of the cellular composition of the granulomas.

Plasmid, 2000 Nov, 44(3), 209 - 19
Sequence diversity of the plasmid replication gene repC in the Rhizobiaceae; Palmer KM et al.; The repABC operon is essential for stable maintenance of some Rhizobiaceae plasmids and of pTAV320 from Paracoccus versutus . These plasmids are the largest described family of homologous, yet compatible replicons . The repC gene is essential for plasmid replication, and previous work identified four distinct sequence groups (repC1, repC2, repC3, and repC4) that appear to define different compatibility classes . Probes for these different groups were used to characterize plasmids in Rhizobium leguminosarum population studies and three new repC sequence groups, repC5, repC6, and repC7 were identified . The general repC primers were modified to amplify a wider range of repC sequences and repC sequences were identified in Sinorhizobium and Mesorhizobium type strains . We also showed that the repC3 group-specific primers described previously do not amplify all repC3 sequences and developed a new repC3 amplification strategy .

J Clin Microbiol, 2000 Nov, 38(11), 3960 - 6
Polymorphism in the gene coding for the immunodominant antigen gp43 from the pathogenic fungus Paracoccidioides brasiliensis; Morais FV et al.; The gp43 glycoprotein is an immune-dominant antigen in patients with paracoccidioidomycosis (PCM) . It is protective against murine PCM and is a putative virulence factor . The gp43 gene of Paracoccidioides brasiliensis B-339 is located in a 1,329-bp DNA fragment that includes two exons, a 78-bp intron, and a leader peptide-coding region of 105 bp . Polymorphism in gp43 has been suggested by the occurrence, in the same isolate or among different fungal samples, of isoforms with distinct isoelectric points . In the present study we aligned and compared with a consensus sequence the gp43 precursor genes of 17 P . brasiliensis isolates after sequencing two PCR products from each fungal sample . The genotypic types detected showed 1 to 4 or 14 to 15 informative substitution sites, preferentially localized between 578 and 1166 bp . Some nucleotide differences within individual isolates (noninformative sites) resulted in a second isoelectric point for the deduced protein . The most polymorphic sequences were also phylogenetically distant from the others and encoded basic gp43 isoforms . The three isolates in this group were from patients with chronic PCM, and their DNA restriction patterns were distinct in Southern blots . The nucleotides encoding the inner core of the murine T-cell-protective epitope of gp43 were conserved, offering hope for the development of a universal vaccine.

Rev Inst Med Trop Sao Paulo, 2000 Sep-Oct, 42(5), 239 - 43
Inoculation of BALB/c mice with Lacazia loboi; Madeira S et al.; In a previous study, the authors inoculated Swiss mice with Lacazia loboi (L . loboi) and succeeded in maintaining a granulomatous infiltrate and viable fungal cells up to one year and six months after inoculation . Considering the experimental work on paracoccidioidomycosis, 0.03 ml of a fungal suspension obtained from a biopsy of a Jorge Lobo's Disease patient were inoculated into both hind foot pads of 32 six week-old BALB/c mice of both sexes . The animals were sacrificed 1, 4, 7 and 10 months post inoculation . The suspension contained 1.3 x 10(6) fungi/ml and presented 38% viability . Seven months after inoculation, most of the animals presented profuse infiltrates consisting of isolated histiocytes, foreign body and Langhans' giant cells and a large number of fungi, most of them viable . Emergence of macroscopic lesions was observed during the 8th month . Based on fungal count, viability index before and after inoculation, presence of macroscopic lesions and histopathological findings similar to the findings in humans, the authors believe that BALB/c mice may be a good experimental model to study Jorge Lobo's Disease, mainly regarding therapeutic evaluation.

Clin Infect Dis, 2000 Oct, 31(4), 1032 - 9 Epub 2000 Oct 20.
Paracoccidioidomycosis: a model for evaluation of the effects of human immunodeficiency virus infection on the natural history of endemic tropical diseases; Benard G et al.; The interaction of human immunodeficiency virus (HIV) infection with endemic tropical diseases has become a major concern, but its mechanisms are still poorly understood . Paracoccidioidomycosis (PCM), a South America endemic deep mycosis, may provide an interesting model to investigate this interaction, as clinical-epidemiological features of most HIV-PCM-coinfected patients are difficult to classify into the standard acute and chronic forms of PCM . Such patients have presented clinical features indicative of an uncontrolled infection with lymphohematogenous dissemination, similar to the more severe, acute form . However, this infection probably resulted from reactivated latent foci that, in nonimmunocompromised hosts, leads to the less severe chronic form, characterized by mucosal lesions . We propose that a new outcome of the Paracoccidioides brasiliensis-host interaction is induced by concomitant HIV infection . This outcome probably reflects an impaired anti-P . brasiliensis immune response during coinfection that is similar to that seen in the acute form, although the patients have a chronic P . brasiliensis infection.

Int J Syst Evol Microbiol, 2000 Sep, 50 Pt 5, 1797 - 802
Proposal for the reclassification of Thiobacillus novellus as Starkeya novella gen . nov., comb . nov., in the alpha-subclass of the Proteobacteria; Kelly DP et al.; Thiobacillus novellus is a facultatively chemolithoautotrophic and methylotrophic, Gram-negative, rod-shaped sulfur bacterium, shown by 16S rRNA gene sequence analysis to be a member of the alpha-2 subclass of the Proteobacteria . As such, it must be excluded from the genus Thiobacillus, whose species are members of the beta-Proteobacteria . It closest phylogenetic neighbour appears to be Ancylobacter, from which it is distinct morphologically and in some physiological characteristics . It is distinct physiologically and biochemically in a number of diagnostic features from Paracoccus versutus, in the alpha-3 subclass of the Proteobacteria and does not appear to be sufficiently closely related to any other genus of the alpha-Proteobacteria to be reassigned to a known genus . The new genus and species name Starkeya novella is proposed for T . novellus . The type strain is ATCC 8093T (= NCIMB 10456T = NCIMB 9113T = DSM 506T = IAM 12100T = IFO 12443T = CCM 1077T).

J Biol Chem, 2001 Feb 23, 276(8), 5846 - 55 Epub 2000 Oct 16.
Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase; Steensma E et al.; The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy . In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase . In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17 . An equilibrium mixture of heme ligands is present in isolated oxidized c domain . Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains . This form is in equilibrium with a high-spin form in which Met106 has left the heme iron . Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form . The thermally denatured forms are high-spin in both oxidation states . The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme . This process is not solely a function of the properties of the c domain . The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.

J Eur Acad Dermatol Venereol, 2000 May, 14(3), 166 - 71
The role of HLA antigens in the development of paracoccidioidomycosis; Dias MF et al.; BACKGROUND: Paracoccidioidomycosis is a systemic granulomatous disease that involves primarily the lungs and may disseminate to other organs and systems . It is caused by Paracoccidioides brasiliensis, a fungus that exhibits reversible thermal dimorphism and whose natural habitat is presently unknown . There are two main clinical forms: the acute (subacute) juvenile form and the chronic adult form . The former runs a more rapid course and is more severe than the latter . This mycosis is found throughout Latin America . Brazil accounts for 80% of reported cases . Presumably P . brasiliensis thrives in humid and hot places, especially near forests or farms . The infection is endemic in certain areas, especially in Brazil, Colombia and Venezuela, where nearly 100% of the population show cutaneous paracoccidioidina positive skin tests, indicating previous contact with the fungus, although a small percentage show clinical manifestations of the disease . METHODS: We compared the expression of HLA class I antigens in a healthy group (control) and in a group of patients with paracoccidioidomycosis (chronic adult form) using the Terasaki lymphocytotoxicity test modified by Amos for HLA antigen analysis . AIMS: To discover indications of whether or not individual susceptibility to P . brasiliensis might depend on some specific immunological defect . RESULTS: There is no evidence of association between a specific HLA antigen and paracoccidioidomycosis in the subjects studied . Further investigations are recommended.

Nat Struct Biol, 2000 Oct, 7(10), 885 - 8
A switch in heme axial ligation prepares Paracoccus pantotrophus cytochrome cd1 for catalysis; Allen JW et al.; Cytochrome cd1 nitrite reductase (cd1) from Paracoccus pantotrophus is a respiratory enzyme capable of using nitrite, hydroxylamine and oxygen as electron accepting substrates . Structural studies have shown that when the enzyme is reduced there is a change in the axial ligation of both hemes, which has been proposed to form part of the catalytic cycle . Here we report the use of a physiological electron donor, pseudoazurin, to investigate the relationship between heme ligation and catalysis . A combination of visible absorption and electron paramagnetic resonance spectroscopies reveals the formation of a catalytically competent state of oxidized cd1 with 'switched' axial ligands immediately after complete reoxidation of reduced cd1 with hydroxylamine . This activated conformer returns over 20 min at 25 degrees C to the state previously observed for oxidized 'as isolated' cd1, which is catalytically inactive towards the same substrates.

Rev Argent Microbiol, 2000 Jul-Sep, 32(3), 116 - 22
{In vitro infection by different strains of Paracoccidioides brasiliensis}; Canteros CE et al.; We analyzed the in vitro infection process by P . brasiliensis and the effect of extracellular factor(s) produced on monolayers of mammalian Vero cell lines . The yeast phase of four strains was studied: B339 (avirulent or slightly virulent), U, (intermediate virulence), 93745 and 63265 (both highly virulent) . Strains of intermediate and high virulence had higher adherence at first contact (about 16%) . Strain B339 had a slower adherence at first contact (8%) than the others during the same period . The production of extracellular proteases, soluble extracellular factor(s) and extracellular antigen gP43 showed no correlation with the in vitro physiopathogenicity of the analyzed strains . We demonstrate that the Vero model presented in this paper is a suitable system to study infection and virulence in vitro . We are currently assessing its usefulness as a tool for the analysis of the interaction between pathogen, host and antifungal agents.

Oral Dis, 2000 Sep, 6(5), 327 - 9
Paracoccidioidomycosis in an HIV-positive patient: a case report with gingival aspects; Giovani EM et al.; Paracoccidioidomycosis is an important deep mycosis, endemic in some areas of the South American countryside, with great incidence in males bearing rural activities, being unusual in urban developed centres . Cell-mediated immunity is the main host defence against the P . brasiliensis, and HIV-positive patients have been increasingly affected by the disease, although only a few reports are available in the literature . We present a case of paracoccidiodomicosis in an HIV-positive female, with unusual clinical history and histopathological aspects.

Biochemistry, 2000 Sep 12, 39(36), 10967 - 74
Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study; Sjogren T et al.; We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha) . Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed . This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0 . Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm . The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5) . After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release . The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A . The structure shows that CO binds to the iron of the d(1) heme in the active site . The ligation of the c heme is unchanged in the complex . A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme . Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site . Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).

Scand J Gastroenterol, 2000 Aug, 35(8), 824 - 31
Presence of bacterial 16S ribosomal RNA gene segments in human intestinal lymph follicles; Chiba M et al.; BACKGROUND: There is currently no information regarding microbial agents inside the intestinal lymph follicles . METHODS: Biopsy or resected specimens, mostly from macroscopically normal areas, were sectioned with a cryostat . DNA was extracted from microdissected samples, exclusively from the lymph follicle . Amplification of DNA was performed using universal primers designed from conserved regions of bacterial 16S ribosomal RNA (rRNA) . Several clones with inserts of around 400 base pairs were subjected to DNA sequence analysis followed by a database homology search . RESULTS: Bacterial 16S rRNA gene segments were detected in the lymph follicle in 2 of 14 (14%) non-inflammatory bowel disease (IBD) cases, 4 of 14 (28%) Crohn disease cases, and in 2 of 5 (40%) ulcerative colitis cases . Nineteen 16S rRNA gene segments were recognized in the eight positive cases . Five segments showed 100% identity to known bacterial 16S rRNAs, namely staphylococcus species, Streptococcus sanguis, and Paracoccus marcusii . However, the other 14 segments showed below 100% identity, indicating either the presence of unknown bacteria or of bacteria without known DNA data . No single identified or unidentified bacterium, characteristic of IBD, including Mycobacterium paratuberculosis and Listeria monocytogenes, was detected . CONCLUSIONS: The present study confirms the presence of bacterial 16S rRNA gene segments in human intestinal lymph follicles and paves the way for new investigations into the microbiology of the lymph follicle . Whether or not bacteria inside the lymph follicle is a primary stimulus in IBD has yet to be clarified.

Med Mycol, 2000 Aug, 38(4), 317 - 22
Morphological aspects of Paracoccidioides brasiliensis in lymph nodes: implications for the prolonged latency of paracoccidioidomycosis?
Restrepo A.
In order to determine if fungal morphology in tissues would furnish indications on the viability of Paracoccidioides brasiliensis yeast cells, lymph node biopsies from five patients with paracoccidioidomycosis, including one with residual circumscribed lesions, were examined . A program that allows transferring of microscopic images to the computer for further processing was used . In the four active cases, the infected lymph nodes had over 49% of healthy-looking yeast cells while in the case of the residual lesion, this figure was smaller (21%) . The residual had a larger proportion of aberrant yeast cells, with predominance of shell-like, empty cells (33%) and crescent bodies (30%); balloon-like yeasts were also seen (16%) . The last two types of cells were also seen in the active lesions, but in smaller proportions, < or = 8% and < or = 9%, respectively . The number of multiple budding yeast cells, which clearly demonstrate fungal viability, ranged 24-33% in active cases but was only 5% in the residual lesion . Although the number of biopsies examined is small, the results tend to indicate that the morphology of P . brasiliensis yeast cells in walled-off tissues is abnormal and that the number of viable elements is small . There might be a connection between these findings and the long latency period illustrated by those patients with paracoccidioidomycosis that have been diagnosed in non-endemic areas . Additionally, if P . brasiliensis yeast cells were to be subjected to the microaerophilic environment present in walled-off lesions, they would probably require a long time to multiply . Under these circumstances, the mycosis would also need many years to manifest.

Med Mycol, 2000 Aug, 38(4), 309 - 15
Experimental paracoccidioidomycosis in high and low antibody responder mice of Selection IV-A; Soares AM et al.; High (H) and low (L) responder mice were selected for their ability to produce antibodies against sheep and human erythrocytes (Selection IV-A) . In this selection, the difference in antibody responsiveness between H and L lines (HIV-A and LIV-A mice, respectively) was shown to depend mainly on macrophage function . The more rapid catabolism of antigens by macrophages in L mice has been suggested as the main cause of the low antibody production . Due to this high macrophage activity, L animals have been described as more resistant than H animals to intracellular pathogens . These animals were utilized as an experimental model of paracoccidioidomycosis . HIV-A and LIV-A mice were infected with Paracoccidioides brasiliensis by the intravenous route . As expected, H mice were more susceptible to P . brasiliensis with a shorter survival time and higher levels of specific antibodies when compared to L mice . Contrasting with the survival time, the lungs, spleen and liver from H mice showed typical nodular granulomas containing epithelioid and giant cells and few fungi . On the other hand, in LIV-A mice, the lesions of these organs were characterized by looser granulomas with irregular borders and the presence of a large number of fungi . However, the adrenal gland showed different lesion patterns . In H mice these lesions were extensive and characterized by loose granulomas with numerous fungi, while in LIV-A mice the lesions were small and limited to the cortex . Moreover the HIV-A mice presented higher levels of serum corticosterone when compared to LIV-A ones . The higher susceptibility of H mice could be attributed to the extensive lesions of the adrenal glands . These results suggest the use of the H line from the IV-A Selection as an experimental model for further studies of adrenal involvement in paracoccidioidomycosis.

Arq Neuropsiquiatr, 2000 Sep, 58(3A), 741 - 7
{Paracoccidioidomycosis of the central nervous system: case report}; da Silva CE et al.; The involvement of the central nervous system in paracoccidioidomycosis is more frequent than previously thought . The first reference to the possibility that Paracoccidioides brasiliensis could affect the central nervous system was by Pereira & Jacobs in 1919 . Since then, a great number of other studies has showed this form of clinical behavior and, in some of them, the frequency has ranged 27.27% . We report a clinical case of a 34-year-old white Brazilian woman admitted because of bacterial pneumonia . In the sixth day of admission, the patient developed cerebellar symptomatology with nausea, vomiting, dysmetria and gait disturbance . Central nervous system computer tomographic scanning disclosed a hypodense lesion in the right cerebellar hemisphere . The patient was submitted to surgery with total excision of the lesion . Histopathological examination confirmed the diagnosis of neuroparacoccidioidomycosis . Coadjuvant treatment with sulfamethoxazole-trimetoprim was introduced . The patient had a good outcome and was discharge 30 days after surgery.

J Eur Acad Dermatol Venereol, 2000 Mar, 14(2), 113 - 7
Cutaneous primary paracoccidioidomycosis; Garcia Bustinduy M et al.; Paracoccidioidomycosis is a common fungal infection in Latin America . Few cases have been described in non-endemic countries and their diagnosis without the correct suspicion, bearing in mind that the latency period might be up to 60 years, may not be easy . We report the case of a 59-year-old man who worked as a taxi driver for 25 years in Venezuela . Cutaneous paracoccidioidomycosis was diagnosed one year after he returned to Spain . No internal signs or symptoms of the disease were found and cell-mediated immunity showed no depression . Our case suggested us to consider direct cutaneous infection, although respiratory tract is believed to be the most frequent portal of entry for this infection.

J Clin Microbiol, 2000 Sep, 38(9), 3478 - 80
PCR for diagnosis of paracoccidioidomycosis; Gomes GM et al.; A PCR assay based on oligonucleotide primers derived from the sequence of the gene coding for the 43,000-Da (gp43) antigen was developed to detect Paracoccidioides brasiliensis DNA in sputa . In the standardized conditions, it could detect 10 cells/ml of sputum, providing sufficient accuracy to be useful for diagnosis of paracoccidioidomycosis.

J Clin Microbiol, 2000 Sep, 38(9), 3190 - 3
Small subunit ribosomal DNA sequence shows Paracoccidioides brasiliensis closely related to Blastomyces dermatitidis; Bialek R et al.; The similarities of paracoccidioidomycosis and blastomycosis are highly suggestive of a close relation of the two etiological agents . Whereas the agent of the first disease is exclusively endemic in Latin America, the agent of the latter one is endemic in North America and Africa . In symptomatic travelers visiting both areas of endemicity, differentiation of the diseases might be impossible, even though therapy and prognosis for these two diseases differ significantly . In order to identify differences in the 18S rRNA gene (rDNA) for use as molecular diagnostic tools, we sequenced this gene from five isolates of Paracoccidioides brasiliensis and compared them to known sequences of other fungi . Neighbor-joining, maximum parsimony, and maximum likelihood analyses and, finally, the Kishino-Hasegawa test revealed that P . brasiliensis, Blastomyces dermatitidis, and Emmonsia parva are more closely related than Histoplasma capsulatum and B . dermatitidis, whose teleomorphic forms belong to one genus, Ajellomyces . In accordance with the work of other investigators who have used internal transcribed spacer and large subunit rDNA sequences, our small subunit rDNA data show that the dimorphic fungus P . brasiliensis must be grouped within the order Onygenales and is closely related to members of the family Onygenaceae . There are hints in the molecular phylogenetic analysis that the family Onygenaceae might be further divided into two families . The subgroup that includes P . brasiliensis comprises all zoopathogenic species . The differences in the 18S rDNAs appear to be too small to allow species identification of the members of the family Onygenaceae pathogenic for humans by use of target sequences within this gene.

Rev Inst Med Trop Sao Paulo, 2000 Jul-Aug, 42(4), 231 - 4
Central nervous system paracoccidioidomycosis . Report of a case successfully treated with itraconazol; Villa LA et al.; Paracoccidioidomycosis (PCM) is a primary pulmonary infection that often disseminates to other organs and systems . Involvement of the central nervous system (CNS) is rare and due to the fact that both clinical alertness and establishment of the diagnosis are delayed, the disease progresses causing serious problems . We report here a case of neuroparacoccidioidomycosis (NPCM), observed in a 55 year-old male, who consulted due to neurological symptoms (left hemiparesis, paresthesias, right palpebral ptosis, headache, vomiting and tonic clonic seizures) of a month duration . Upon physical examination, an ulcerated granulomatous lesion was observed in the abdomen . To confirm the diagnosis a stereotactic biopsy was taken; additionally, mycological tests from the ulcerated lesion and a bronchoalveolar lavage were performed . In the latter specimens, P . brasiliensis yeast cells were visualized and later on, the brain biopsy revealed the presence of the fungus . Treatment with itraconazole (ITZ) was initiated but clinical improvement was unremarkable; due to the fact that the patient was taking sodium valproate for seizure control, drug interactions were suspected and confirmed by absence of ITZ plasma levels . The latter medication was changed to clonazepam and after several weeks, clinical improvement began to be noticed and was accompanied by diminishing P . brasiliensis antigen and antibody titers . In the PCM endemic areas, CNS involvement should be considered more often and the efficacy of itraconazole therapy should also be taken into consideration.

Rev Soc Bras Med Trop, 2000 May-Jun, 33(3), 309 - 12
Intestinal paracoccidioidomycosis simulating colon cancer; Chojniak R et al.; We report a case of intestinal involvement of Paracoccidioidomycosis, in a patient considered to have colonic cancer . The diagnosis of this mycosis should be considered when an abdominal mass associated with intra-lesional calcifications on X-ray is observed . CT scans increase the findings.

Microbes Infect, 2000 Jul, 2(8), 877 - 84
Adherence and intracellular parasitism of Paracoccidioides brasiliensis in Vero cells; Hanna SA et al.; 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.

J Bacteriol, 2000 Sep, 182(17), 4677 - 87
Novel genes coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17; Friedrich CG et al.; The gene region coding for lithotrophic sulfur oxidation of Paracoccus pantotrophus GB17 is located on a 13-kb insert of plasmid pEG12 . Upstream of the previously described six open reading frames (ORFs) soxABCDEF with a partial sequence of soxA and soxF (C . Wodara, F . Bardischewsky, and C . G . Friedrich, J . Bacteriol . 179:5014-5023, 1997), 4,350 bp were sequenced . The sequence completed soxA, and uncovered six new ORFs upstream of soxA, designated ORF1, ORF2, and ORF3, and soxXYZ . ORF1 could encode a 275-amino-acid polypeptide of 29,332 Da with a 61 to 63% similarity to LysR transcriptional regulators . ORF2 could encode a 245-amino-acid polypeptide of 26,022 Da with the potential to form six transmembrane helices and with a 48 to 51% similarity to proteins involved in redox transport in cytochrome c biogenesis . ORF3 could encode a periplasmic polypeptide of 186 amino acids of 20,638 Da with a similarity to thioredoxin-like proteins and with a putative signal peptide of 21 amino acids . Purified SoxXA, SoxYZ, and SoxB are essential for thiosulfate or sulfite-dependent cytochrome c reduction in vitro . N-terminal and internal amino acid sequences identified SoxX, SoxY, SoxZ, and SoxA to be coded by the respective genes . The molecular masses of the mature proteins determined by electrospray ionization spectroscopy (SoxX, 14,834 Da; SoxY, 11,094 Da; SoxZ, 11,717 Da; and SoxA, 30,452 Da) were identical or close to those deduced from the nucleotide sequence with differences for the covalent heme moieties . SoxXA represents a novel type of periplasmic c-type cytochromes, with SoxX as a monoheme and SoxA as a hybrid diheme cytochrome c . SoxYZ is an as-yet-unprecedented soluble protein . SoxY has a putative signal peptide with a twin arginine motif and possibly cotransports SoxZ to the periplasm . SoxYZ neither contains a metal nor a complex redox center, as proposed for proteins likely to be transported via the Tat system.

Rev Latinoam Microbiol, 1999 Jul-Sep, 41(3), 139 - 43
Random amplified polymorphic DNA (RAPD) analysis of Paracoccidioides brasiliensis isolates; Totti DO et al.; Five Paracoccidioides brasiliensis isolates of humans origin were analyzed using three arbitrary primers (3301, 3304 and 3307 of 10, 9 and 10 oligonucleotides respectively) in random amplified polymorphic DNA (RAPD) analysis . The analysis of the complex RAPD profiles obtained were carried out using the Dice similarity coefficient that distinguished the isolated Pb 02 from the others (Pb 18, Pb 192, Pb 265 and Pb SN) . The results revealed limited intraspecific genomic variations in these P . brasiliensis isolates and indicate that RAPD can be useful for analysis of P . brasiliensis genome for characterization or differentiation within this genus.

Biochemistry, 2000 Aug 8, 39(31), 9551 - 60
The Met99Gln mutant of amicyanin from Paracoccus versutus; Diederix RE et al.; The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus . Dynamic and structural characteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR, cyclic voltammetry, and isomorphous metal replacement . M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin . In addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been assessed and compared to values for M121Q azurin . For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M . These values are very similar to the midpoint potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin . A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand.

J Biol Chem, 2000 Oct 27, 275(43), 33231 - 7
Time-resolved infrared spectroscopy reveals a stable ferric heme-NO intermediate in the reaction of Paracoccus pantotrophus cytochrome cd1 nitrite reductase with nitrite; George SJ et al.; Cytochrome cd(1) is a respiratory enzyme that catalyzes the physiological one-electron reduction of nitrite to nitric oxide . The enzyme is a dimer, each monomer containing one c-type cytochrome center and one active site d(1) heme . We present stopped-flow Fourier transform infrared data showing the formation of a stable ferric heme d(1)-NO complex (formally d(1)Fe(II)-NO(+)) as a product of the reaction between fully reduced Paracoccus pantotrophus cytochrome cd(1) and nitrite, in the absence of excess reductant . The Fe-(14)NO nu(NO) stretching mode is observed at 1913 cm(-1) with the corresponding Fe-(15)NO band at 1876 cm(-1) . This d(1) heme-NO complex is still readily observed after 15 min . EPR and visible absorption spectroscopic data show that within 4 ms of the initiation of the reaction, nitrite is reduced at the d(1) heme, and a cFe(III) d(1)Fe(II)-NO complex is formed . Over the next 100 ms there is an electron redistribution within the enzyme to give a mixed species, 55% cFe(III) d(1)Fe(II)-NO and 45% cFe(II) d(1)Fe(II)-NO(+) . No kinetically competent release of NO could be detected, indicating that at least one additional factor is required for product release by the enzyme . Implications for the mechanism of P . pantotrophus cytochrome cd(1) are discussed.

J Clin Microbiol, 2000 Aug, 38(8), 3106 - 9
Molecular identification of Paracoccidioides brasiliensis by PCR amplification of ribosomal DNA; Motoyama AB et al.; We have amplified and sequenced the 5.8S and 28S ribosomal DNA genes and intergenic regions of Paracoccidioides brasiliensis, strain Pb01 . Using primers specifically designed for both ribosomal DNA regions, we were able to discriminate between P . brasiliensis and other human pathogenic fungi by PCR . The use of this molecular marker could be important for paracoccidiodomycosis diagnosis and ecological and molecular epidemiological studies of P . brasiliensis in Latin America.

J Clin Microbiol, 2000 Aug, 38(8), 2940 - 2
Detection of Paracoccidioides brasiliensis in tissue samples by a nested PCR assay; Bialek R et al.; A nested PCR assay for the detection of Paracoccidioides brasiliensis DNA was evaluated, using a sequence of the immunogenic gp43 gene as a target . This gene encodes an outer membrane protein unique to this dimorphic fungus . DNA from six clinical isolates and the ATCC strain 60885 of P . brasiliensis, as well as DNA from closely related fungi, was examined to determine detection limits and cross-reactions . PCR was done on DNA extracts of lung homogenates from 23 experimentally P . brasiliensis-infected and two uninfected BALB/c mice and from 20 Histoplasma capsulatum-infected ICR mice . The results were compared to quantitative cultures . A detection limit of 0.5 fg of specific DNA was determined using cloned plasmid DNA . In all seven P . brasiliensis isolates, the expected 196-bp nested PCR product was found . Their sequences were 100% identical to the gp43 gene sequence in GenBank . DNA extracts of all other, related fungi were negative . The PCR assay was positive in 21 out of 23 culture-positive lung homogenates with concentrations of 1 x 10(3) to 1.3 x 10(7) CFU of P . brasiliensis per g of lung . Uninfected BALB/c mice and H . capsulatum-infected mice samples gave negative results . The high sensitivity and specificity of this new nested PCR assay for the detection of P . brasiliensis in tissue samples were demonstrated . The assay may be useful for diagnosis in human tissue samples.

J Clin Microbiol, 2000 Aug, 38(8), 2909 - 13
Comparison of an established antibody sandwich method with an inhibition method of Histoplasma capsulatum antigen detection; Garringer TO et al.; The Histoplasma antigen immunoassay utilizes an antibody sandwich method that provides a rapid and reliable means of diagnosing the more severe forms of histoplasmosis . Inhibition assays have been developed for antigen detection and offer at least one potential advantage, namely, reduced antibody requirements . We have developed an inhibition assay using the polyclonal antibody employed in our standard sandwich assay . Urine and serum specimens from patients with culture-proven histoplasmosis and controls were tested using both methods . The two methods had similar sensitivities for detection of antigen in urine (antibody sandwich = 92.5% versus inhibition = 87.5%, P = 0.500) and serum (82.5% versus 80.0%, P = 1 . 000) . With serum, the specificities of both methods were similar (antibody sandwich assay = 95.0% versus inhibition assay = 92.5%, P = 1.000), and with urine, the specificity of the antibody sandwich method was superior (97.5% versus 80.0%, P = 0.039) . While the overall reproducibility of both methods was excellent (with urine, antibody sandwich assay intraclass correlation coefficient = 0.9975 and with serum = 0.9949; correlation coefficient of the inhibition assay with urine = 0.9736 and with serum = 0.9850), that of the inhibition method was only fair to poor for the controls: urine = -0 . 0152, serum = 0.5595 . Reproducibility was good for the controls using the sandwich method: urine = 0.7717, serum = 0.9470 . Cross-reactivity was observed in specimens from patients infected with Blastomyces dermatitidis, Paracoccidioides brasiliensis, and Penicillium marneffei . In conclusion, the decreased specificity and inferior reproducibility with control specimens suggest that the inhibition assay has poorer precision toward the lower end of the detection range.

Br J Dermatol, 2000 Jul, 143(1), 188 - 91
Paracoccidioidomycosis (South American blastomycosis) successfully treated with terbinafine: first case report; Ollague JM et al.; We describe a 63-year-old man who presented with painful malodorous lesions in the perianal, perineal and scrotal regions . Following definitive diagnosis of paracoccidioidomycosis, he was treated initially with trimethoprim/sulphamethoxazole, but there was no clinical improvement . He then received terbinafine (Lamisil) 250 mg twice daily for 6 months . There was rapid resolution of all lesions and complete relief of symptoms, without any associated side-effects . The patient remains clinically well and without any evidence of infection 2 years after discontinuation of terbinafine treatment.

Med Mycol, 2000 Jun, 38(3), 225 - 9
Production of IFN-gamma is impaired in patients with paracoccidioidomycosis during active disease and is restored after clinical remission; Karhawi AS et al.; Cellular immunity is usually suppressed during paracoccidioidomycosis (PCM) and is restored after treatment . In this study we evaluated the induction of a type 1 (interferon gamma (IFN-gamma)), a type 2 (interleukin (IL)-10) and a primarily macrophage derived cytokine (tumor necrosis factor (TNF)-alpha) in peripheral blood mononuclear cells (PBMC) from patients with PCM . Eight male patients with active PCM, nine male patients with clinical remission of the disease and 10 healthy control subjects were enrolled in the study . Cytokines were induced with non-specific stimuli --phytohaemagglutin (PHA) (induces IL-10 and IFN-gamma), Lipopolysaccharide (induces TNF-alpha)--and Paracoccidioides brasiliensis antigen (PbAg) (induces IL-10, IFN-gamma and TNF-alpha) . Induction of IFN-gamma with PHA differed among the three groups (P < 0.01; Kruskal-Wallis test) and with PbAg was lower in patients with active disease compared to those in clinical remission (P = 0.05; Mann-Whitney) . Induction of IL-10 and of TNF-alpha was similar in the three groups . The suppressed production of IFN-gamma in patients with active disease may underscore the cellular immune deficiency seen in these patients.

Med Mycol, 2000 Jun, 38(3), 193 - 9
Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus novemcinctus) in an area where the fungus was recently isolated from soil; Silva-Vergara ML et al.; Natural infection of armadillos (Dasypus novemcinctus) with Paracoccidioides brasiliensis in Northern Brazil was reported in 1986, raising great interest in the understanding of the role of this mammal in the epidemiological cycle of the fungus . Recently, P . brasiliensis was isolated from the soil of Ibia, State of Minas Gerais, southeastern Brazil . Armadillos captured in this area were evaluated for the presence of P . brasiliensis in the viscera and infection was detected in 4/16 animals (25%) . Fungal yeast phase cells were observed in three of the four infected armadillos by direct microscopic examination and by the indirect immunofluorescence test carried out on homogenized tissues . P . brasiliensis was isolated from three armadillos whose homogenized viscera had been injected into Swiss mice . The new strains (Ibia-T1, Ibia-T2 and Ibia-T3) were identified as P . brasiliensis on the basis of macro- and micromorphology, thermodimorphism, production and serologic activity of exoantigens, and by polymerase chain reaction (PCR)-detection of the gp43 gene . The lethality and lesions caused to the mice from which the strains were recovered confirmed the virulence of the isolates . We conclude that P . brasiliensis infects armadillos in locations with different geoclimatic characteristics and vegetation cover . The direct observation of yeast cells in tissues and the multiple visceral involvement, including the lungs, suggests the occurrence of paracoccidioidomycosis disease in these mammals and supports their role as wild hosts in the epidemiological cycle of the fungus.

Med Mycol, 2000 Jun, 38(3), 185 - 91
A critical analysis of isolation of Paracoccidioides brasiliensis from soil; Franco M et al.; The present review is a critical analysis of positive and negative reports of the isolation of Paracoccidioides brasiliensis from soil . The strains isolated from soil or soil-contaminated material (dogfood, penguin feces) by Batista et al . in Recife, Brazil, Negroni in the Argentinian Chaco, Albornoz in rural Venezuela, Silva-Vergara et al . in Ibia, Brazil, Ferreira et al . in Uberlandia, Brazil, and Gezuele et al . at the Uruguayan base in the Antarctic region, presented mycological characteristics consistent with P . brasiliensis . In most of these studies, morphological characterization was complemented with an evaluation of virulence and antigenicity, and biochemical or molecular analysis . These isolations, therefore, can be considered true, supporting the concept of soil as an important element in the ecology of the pathogen . The large number of negative reports in attempts involving soil samples and the low repeatability of isolation of the fungus from the same area indicate that the specific conditions supporting growth of the pathogen in soil have not been fully clarified.

Rev Inst Med Trop Sao Paulo, 2000 May-Jun, 42(3), 167 - 70
Gallium-67 imaging in a patient with paracoccidioidomycosis: a case report; Teixeira AB et al.; A 26 year-old female was admitted with abdominal pain, fever and weight loss . The clinical and laboratory investigations led to the diagnosis of paracoccidioidomycosis . Gallium-67 whole body images correlated well with the clinical course of the disease and with the patient's prognosis.

Rev Soc Bras Med Trop, 1999 Sep-Oct, 32(5), 571 - 5
{Paracoccidioidomycosis in Brazilian Indians of the Suruí tribe: clinical-laboratory study of 2 cases}; Forjaz MH et al.; Paracoccidioidomycosis has been considered the most frequent endemic systemic mycosis in Latin America . Although most cases of paracoccidioidomycosis involve rural workers, this systemic fungal disease has been scarcely reported among Amerindian populations from Brazil . We report two cases of paracoccidioidomycosis in Tupi-Monde Amerindians from Cacoal, state of Rondonia, Brazil . Both cases exhibited positive serological results by a specific immunodiffusion test only when the assay was performed with antigens obtained from the mycelial form of P . brasiliensis . The authors present a literature review of paracoccidioidomycosis in Brazilian Amerindians and discuss the need for further investigations about the impact of the antigenic diversity of P . brasiliensis from different geographic areas on the serological diagnosis of PCM.

Mycoses, 1999, 42 Suppl 2, 11 - 8
Yeast tissue phase of Emmonsia pasteuriana inoculated in golden hamster by intratesticular way; Drouhet E et al.; The scope of our study was to present an experimental model reproducing the dimorphic yeast-like population (as for Histoplasma capsulatum, Blastomyces dermatitidis) similar to that observed in the cutaneous biopsy of an Italian woman who had never traveled abroad, being intravenous drug user and HIV positive for 10 years, finally infected with the new dimorphic fungus Emmonsia pasteuriana . Experimental inoculation was unsuccessful by intraperitoneal (i.p.) and intravenous (i.v.) ways in a mouse and in a guinea-pig model inoculated by cutaneous or subcutaneous routes, reason for that we chose the golden hamster, highly sensitive to dimorphic fungi as agents of systemic mycoses as histoplasmosis, blastomycosis, sporotrichosis, penicilliosis marneffei, paracoccidioidomycosis when the inoculation was done by intraperitoneal route . We inoculated young golden hamsters by i.p . and intratesticular ways . Only by this last route we reproduced an orchiepididymitis with necrosis, haemorrhages and a polymorphic yeast-like population similar to the polymorphism observed in the cutaneous biopsy of the patient . The intratesticular affinity of E . pasteuriana provided an interesting model for this infection.

Antimicrob Agents Chemother, 2000 Jul, 44(7), 1997 - 2000
Effects of amphotericin B and three azole derivatives on the lipids of yeast cells of Paracoccidioides brasiliensis; Hahn RC et al.; Yeast cells of five different strains of Paracoccidioides brasiliensis were obtained for partial analysis of lipid composition, and sterol content was determined quantitatively and qualitatively . The determinations were conducted with cells cultured in the presence and absence of amphotericin B and azole derivatives at levels below the MIC.

Protein Sci, 2000 May, 9(5), 846 - 58
The structure and dynamics in solution of Cu(I) pseudoazurin from Paracoccus pantotrophus; Thompson GS et al.; The solution structure and backbone dynamics of Cu(I) pseudoazurin, a 123 amino acid electron transfer protein from Paracoccus pantotrophus, have been determined using NMR methods . The structure was calculated to high precision, with a backbone RMS deviation for secondary structure elements of 0.35+/-0.06 A, using 1,498 distance and 55 torsion angle constraints . The protein has a double-wound Greek-key fold with two alpha-helices toward its C-terminus, similar to that of its oxidized counterpart determined by X-ray crystallography . Comparison of the Cu(I) solution structure with the X-ray structure of the Cu(II) protein shows only small differences in the positions of some of the secondary structure elements . Order parameters S2, measured for amide nitrogens, indicate that the backbone of the protein is rigid on the picosecond to nanosecond timescale.

J Biol Chem, 2000 Aug 18, 275(33), 25089 - 94
X-ray crystallographic study of cyanide binding provides insights into the structure-function relationship for cytochrome cd1 nitrite reductase from Paracoccus pantotrophus; Jafferji A et al.; We present a 1.59-A resolution crystal structure of reduced Paracoccus pantotrophus cytochrome cd(1) with cyanide bound to the d(1) heme and His/Met coordination of the c heme . Fe-C-N bond angles are 146 degrees for the A subunit and 164 degrees for the B subunit of the dimer . The nitrogen atom of bound cyanide is within hydrogen bonding distance of His(345) and His(388) and either a water molecule in subunit A or Tyr(25) in subunit B . The ferrous heme-cyanide complex is unusually stable (K(d) approximately 10(-6) m); we propose that this reflects both the design of the specialized d(1) heme ring and a general feature of anion reductases with active site heme . Oxidation of crystals of reduced, cyanide-bound, cytochrome cd(1) results in loss of cyanide and return to the native structure with Tyr(25) as a ligand to the d(1) heme iron and switching to His/His coordination at the c-type heme . No reason for unusually weak binding of cyanide to the ferric state can be identified; rather it is argued that the protein is designed such that a chelate-based effect drives displacement by tyrosine of cyanide or a weaker ligand, like reaction product nitric oxide, from the ferric d(1) heme.

Vaccine, 2000 Jul 1, 18(26), 3050 - 8
DNA-based vaccination against murine paracoccidioidomycosis using the gp43 gene from paracoccidioides brasiliensis; Pinto AR et al.; Gp43, the major 43-kDa antigenic glycoprotein of Paracoccidioides brasiliensis, or its 15-amino acid inner peptide (P10), induces a T-CD4(+), Th1 cellular immune response which protects BALB/c mice from intratracheal infection by virulent yeast forms . We investigated whether DNA vaccination using the gp43 gene could elicit protective immunity against P . brasiliensis . Animals immunised intramuscularly (i.m.) or intradermally (i.d.) with plasmid DNA containing the gp43 gene induced a specific, long lasting humoral and cellular immune response . A mixed Th1/Th2 cellular immune response in DNA-immunized mice was modulated in vivo by IFN-gamma and was protective in BALB/c mice . A significant decrease in the lung colony forming units (CFUs) and reduced, or no dissemination to the spleen and liver of immunised mice were observed.

Mycopathologia, 1999, 146(3), 131 - 4
Comparative efficacy of fluconazole and amphotericin B in the parenteral treatment of experimental paracoccidioidomycosis in the rat; Martinez R et al.; Patients with severe and complicated paracoccidioidomycosis are treated with amphotericin B by the intravenous route . Fluconazole is active in vitro against Paracoccidioides brasiliensis and can also be administered intravenously, but few clinical or experimental data are available about its action against the infection caused by this fungus . In the present study, the efficacy of fluconazole and amphotericin B was assessed comparatively in rats inoculated parenterally with P . brasiliensis . The treatment was performed 3 times a week for 4 weeks starting one week after infection . Fluconazole administered intraperitoneally (14 mg/kg body weight/dose) was more effective (P < 0.001) than amphotericin B (2 mg/kg body weight/dose) in reducing the number of colony forming units in the lungs and spleen . When administered intravenously at the dose of 3 mg/kg body weight, fluconazole was as effective as amphotericin B (0.8 mg/kg body weight) in reducing the pulmonary fungal burden . Under these conditions, the rats treated with fluconazole had a smaller number of colony forming units than untreated animals (P < 0.001), but amphotericin B was more effective than fluconazole in reducing spleen infection (P < 0.005) . Except for this result obtained with a low dose, fluconazole showed an antifungal action equal to or higher than that of amphotericin B . The activity of fluconazole at doses equivalent to those used for human treatment suggests that this antifungal agent may be an alternative to amphotericin B for the early intravenous treatment of patients with paracoccidioidomycosis.

Med Mycol, 2000 Apr, 38(2), 177 - 82
Effect of cytokines on antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis; Kurita N et al.; In our previous study, it was observed that human peripheral blood polymorphonuclear leucocytes (PMNs) exhibited a fungistatic effect on yeast cells of Paracoccidioides brasiliensis, and that interferon-gamma (IFN-gamma), but not tumor necrosis factor-alpha or interleukin-8 (IL-8), enhanced the antifungal activity of PMNs . In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) also enhanced the PMN activity . GM-CSF-activated PMNs exhibited a killing effect on P . brasiliensis isolate Bt-4 and an enhanced fungistatic effect on isolate Aoki . IL-1beta activated PMNs to kill isolate Bt-4 . Granulocyte colony-stimulating factor had no effect . Combinations of IFN-gamma with GM-CSF or IL-1beta, but not a combination of GM-CSF and IL-1beta, exhibited a synergistic effect in enhancing the antifungal activity of PMNs . These results strongly suggest that PMNs activated with IFN-gamma, GM-CSF and/or IL-1beta might play an important role in host defense in early infection with P . brasiliensis due to their enhanced antifungal activity.

Rev Inst Med Trop Sao Paulo, 2000 Mar-Apr, 42(2), 59 - 66
Fibrotic sequelae in pulmonary paracoccidioidomycosis: histopathological aspects in BALB/c mice infected with viable and non-viable paracoccidioides brasiliensis propagules; Cock AM et al.; 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 Grocot&tacute;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 4 weeks 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.

Am J Pathol, 2000 May, 156(5), 1811 - 20
Interferon-gamma and tumor necrosis factor-alpha determine resistance to Paracoccidioides brasiliensis infection in mice; Souto JT et al.; To investigate the role of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the resistance to Paracoccidioides brasiliensis (Pb) infection, mice with homologous disruption of the IFN-gamma (GKO) or TNF-alpha receptor p55 (p55KO) were infected with the parasite . GKO and p55KO, but not wild-type (WT) mice, were unable to control the growth of yeast cells and the mice succumbed to infection by days 16 and 90 after infection, respectively . Typical inflammatory granulomas were found only in WT mice . In contrast, knockout mice presented an inflammatory infiltrate composed of a few neutrophils, mononuclear, epithelioid, and multinuclear giant cells forming incipient granulomas in GKO mice and without granuloma formation in p55KO mice . Besides, both groups of knockout mice exhibited elevated numbers of yeast forms in agreement with colony-forming unit counts in organs . Compared with WT, splenocytes from infected GKO mice cultured with the Pb F1 fraction produced lower TNF-alpha levels, whereas leukocytes from infected p55KO mice produced similar amounts of TNF-alpha but higher levels of IFN-gamma . Moreover, splenocytes from infected WT mice produced higher levels of nitric oxide (NO) resulting in a lower T-cell proliferative response to Con A than uninfected WT, or infected p55KO and GKO mice . On the contrary, the addition of IFN-gamma to splenocytes from infected GKO mice resulted in higher NO production and lower T cell proliferation . Taken together, these findings suggests that endogenous TNF-alpha, acting through the p55 receptor, and IFN-gamma mediate resistance to Pb infection and induce NO production that determines marked T cell unresponsiveness.

Infection, 2000 Mar-Apr, 28(2), 119 - 20
A case of paracoccidioidomycosis: experience with long-term therapy; Borgia G et al.; We describe long-term therapy for paracoccidioidomycosis occurring in a 61-year-old house-painter from Venezuela . The diagnostic examinations made in South America had shown pulmonary granulomatous lesions and an osteolytic pattern of the left knee that had been considered suspect of malignant disease with an indication for limb amputation . With the aid of fine needle aspiration biopsy (FNAB) and culture examination we diagnosed an osteomyelitis by Paracoccidioides brasiliensis and initiated therapy with itraconazole, 400 mg per day, reduced to 200 mg per day after 2 months . At the end of 2 years of drug therapy, we observed complete regression of the pulmonary lesions and of the osteolytic area of the left knee . Moreover, we have periodically observed our patient to verify his clinical development and he is still in good health . We suggest that this pathology be considered in differential diagnosis of leprosy, tuberculosis, leishmaniasis, and systemic mycoses, even in non-endemic areas.

Infect Immun, 2000 May, 68(5), 2546 - 52
Nitric oxide participation in the fungicidal mechanism of gamma interferon-activated murine macrophages against Paracoccidioides brasiliensis conidia; Gonzalez A et al.; Paracoccidioidomycosis, a systemic mycosis restricted to Latin America and produced by the dimorphic fungus Paracoccidioides brasiliensis, is probably acquired by inhalation of conidia produced by the mycelial form . The macrophage (Mphi) represents the major cell defense against this pathogen; when activated with gamma interferon (IFN-gamma), murine Mphis kill the fungus by an oxygen-independent mechanism . Our goal was to determine the role of nitric oxide in the fungicidal effect of Mphis on P . brasiliensis conidia . The results revealed that IFN-gamma-activated murine Mphis inhibited the conidium-to-yeast transformation process in a dose-dependent manner; maximal inhibition was observed in Mphis activated with 50 U/ml and incubated for 96 h at 37 degrees C . When Mphis were activated with 150 to 200 U of cytokine per ml, the number of CFU was 70% lower than in nonactivated controls, indicating that there was a fungicidal effect . The inhibitory effect was reversed by the addition of anti-IFN-gamma monoclonal antibodies . Activation by IFN-gamma also enhanced Mphi nitric oxide production, as revealed by increasing NO(2) values (8 +/- 3 microM in nonactivated Mphis versus 43 +/- 13 microM in activated Mphis) . The neutralization of IFN-gamma also reversed nitric oxide production at basal levels (8 +/- 5 microM) . Additionally, we found that there was a significant inverse correlation (r = -0.8975) between NO(2)(-) concentration and transformation of P . brasiliensis conidia . Additionally, treatment with any of the three different nitric oxide inhibitors used (arginase, N(G)-monomethyl-L-arginine, and aminoguanidine), reverted the inhibition of the transformation process with 40 to 70% of intracellular yeast and significantly reduced nitric oxide production . These results show that IFN-gamma-activated murine Mphis kill P . brasiliensis conidia through the L-arginine-nitric oxide pathway.

Biochemistry, 2000 Apr 18, 39(15), 4243 - 9
Cytochrome cd(1) from Paracoccus pantotrophus exhibits kinetically gated, conformationally dependent, highly cooperative two-electron redox behavior; Koppenhofer A et al.; Each monomer of the dimeric cytochrome cd(1) nitrite reductase from Paracoccus pantotrophus contains two hemes: one c-type center and one noncovalently bound d(1) center . Potentiometric analysis at 20 degrees C shows substantial cooperativity between the two redox centers in terms of their joint co-reduction (or co-oxidation) at a single apparent potential with an n value of 1.4 +/- 0.1 . Reproducible hysteresis is demonstrated in the redox titrations . In a reductive titration both centers titrate with an apparent midpoint potential of +60 +/- 5 mV while in the oxidative titration the apparent potential is +210 +/- 5 mV . However, at 40 degrees C the reductive and oxidative titrations are shifted such that they almost superimpose; each has n = 2 . A kinetically gated process that can be correlated with oxidation/reduction-dependent ligand changes at the two heme centers, previously seen by crystallography, is implicated . In contrast, a semi-apoenzyme, lacking the d(1) heme, exhibits a reversible redox titration with a midpoint potential of +242 +/- 5 mV (n = 1) . The data with the holoenzyme show how redox changes can themselves generate a gating of the type that is minimally required to account for redox-linked proton pumping by membrane-bound cytochromes.

Biochemistry, 2000 Apr 11, 39(14), 4028 - 36
Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus; Koppenhofer A et al.; Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction) . Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration . Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron . Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen . On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed . Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically . These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme . At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme . Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen . The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species . This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical . Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle.

Med Mycol, 2000 Feb, 38(1), 51 - 60
Experimental paracoccidioidomycosis of the Syrian hamster: fungicidal activity and production of inflammatory cytokines by macrophages; Parise-Fortes MR et al.; Phagocytic cells play an important role in nonspecific resistance to fungal infection by mediating an inflammatory response and by a direct fungicidal action . In this study, the functional activity of peritoneal macrophages obtained from hamsters experimentally infected with strain Pb18 of Paracoccidioides brasiliensis was evaluated during 16 weeks of infection . The results showed that macrophages had a higher spreading ability associated with increased production of tumor necrosis factor alpha (TNF-alpha) and enhanced fungicidal activity during the early periods of infection . TNF-alpha levels remained elevated during all periods studied, while low levels of interleukin-1 beta (IL-1 beta) were produced during the infection . A necrotic area with dead fungi was observed at the inoculation site and the infection disseminated only to liver and lymph nodes in a few animals . These results suggest that during the early stages of infection with P . brasiliensis, macrophage activation by the high levels of TNF-alpha limited fungal dissemination . In contrast, in the later stages of infection, high levels of TNF-alpha were observed while the fungicidal activity of macrophages was lower and the animals presented loss of vitality resulting in their death . These observations suggest a complex role of TNF-alpha in experimental paracoccidioidomycosis of Syrian hamsters, involving not only resistance but also pathogenesis.

Med Mycol, 2000 Feb, 38(1), 31 - 9
Differential expression of chitin synthase genes during temperature-induced dimorphic transitions in Paracoccidioides brasiliensis; Nino-Vega GA et al.; Fragments of five genes encoding chitin synthase enzymes were identified in the dimorphic fungus Paracoccidioides brasiliensis by polymerase chain reaction (PCR) amplification of conserved CHS gene domains . These represent several classes of enzyme: PbrCHS1, class I; PbrCHS2, class II; PbrCHS3, class IV; and PbrCHS4 and PbrCHS5, class V . Expression of these genes during the temperature regulated dimorphic transition from yeast to mycelium and from mycelium to yeast was determined by Northern analysis . One gene (PbrCHS3) was not expressed at detectable levels . The others were regulated by morphology and/or by the growth phase of the organism . Despite the fact that yeast cells contain more chitin than hyphal cells, the levels of mRNA for PbrCHS1, PbrCHS2, PbrCHS4, and PbrCHS5 were higher in hyphal cells than in yeast cells . This supports observations in other fungi that transcript levels often do not correlate with chitin content and that post-transcriptional regulation of CHS gene expression is important for morphogenesis.

Rev Inst Med Trop Sao Paulo, 2000 Jan-Feb, 42(1), 38 - 40
Paracoccidioidomycosis of the male genital tract . Report of eleven cases and a review of Brazilian literature; Severo LC et al.; Eleven cases of involvement of the genital tract in paracoccidioidomycosis were collected in a retrospective study of the clinical records of 683 patients seen in Porto Alegre, Rio Grande do Sul, Brazil . These cases are herein summarily reported . Eighteen similar cases were gathered in review of the Brazilian literature . Obtained data are discussed.

Mycopathologia, 1999, 146(1), 13 - 7
Production of proteinase and phospholipase by Paracoccidioides brasiliensis; de Assis CM et al.; We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis . Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 degrees C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 degrees C . After 30 days net enzyme activity was visualized and quantitatively evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it . Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P . brasiliensis.

Clin Diagn Lab Immunol, 2000 Mar, 7(2), 175 - 81
Anti-idiotypic antibodies in patients with different clinical forms of paracoccidioidomycosis; Souza AR et al.; Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America . Patients with PCM show a wide spectrum of clinical and pathological manifestations depending on both host and pathogen factors . Two clinical forms of the disease are recognized: the acute or juvenile form and the chronic or adult form . The major antigenic component of the parasite is a glycoprotein of 43 kDa (gp43) . All patient sera present antibodies against gp43 (anti-gp43) and, as demonstrated before by our group, spontaneous anti-idiotypic (anti-Id) antibodies (Ab2) can be detected in patient sera with high titers of anti-gp43 . Since it has been postulated that anti-Id antibodies may have a modulating function, we decided to purify and characterize anti-Id antibodies in this system . The possible correlation of Ab2 titers with different clinical forms of disease was also verified . Results showed that purified human anti-Id antibodies (human Ab2) recognized specifically the idiotype of some murine monoclonal anti-gp43 (17c and 3e) but not others (40.d7, 27a, and 8a) . Spontaneous anti-Id antibodies were found in all clinical forms of disease . The majority of patients (88%, n = 8) with the acute form of PCM had high titers of Ab2 . However, among patients with the multifocal chronic form of the disease, only 29% (n = 14) had high titers of Ab2; 70% (n = 10) of patients with the unifocal chronic form had low titers of Ab2 . A correlation between Ab2 titers and anti-gp43 titers was observed before and during antimycotic treatment . Our results suggest that titers of anti-Id antibodies correlate with the severity of PCM in humans.

Med Microbiol Immunol (Berl), 1999 Aug, 188(1), 41 - 9
Influence of the genetic background on the pattern of lesions developed by resistant and susceptible mice infected with Paracoccidioides brasiliensis; Xidieh CF et al.; To compare the sequential evolution of lesions developed by resistant (A/Sn) and susceptible (B10.A) mice to Paracoccidioides brasiliensis infection we inoculated a virulent isolate of the fungus and collected the pancreas/peripancreatic omentum monthly (from 1 to 6 months) post infection . After fixation, tissue sections were stained by conventional methods for light microscopy to investigate the cellular composition, the extracellular matrix (ECM) patterns and the morphology of the yeasts in the lesions . In both strains, the fungal lesions were localized mostly in the omentum; a few lesions in the pancreatic parenchyma were observed, mostly in B10.A mice . In both strains, macrophages and plasmocytes were the predominant cells in all lesions, followed by neutrophils (PMN) and macrophages transformed into giant and epithelioid cells . Remarkable differences were observed between resistant and susceptible mice, specially related to the ECM structure of the granulomatous lesions . In A/Sn mice, from the 1st month on, the coexistence of two types of lesions was observed: one type showed a well-defined encapsulated nodule, constituted mainly of type I collagen . Neutrophils were abundant in areas of massive fungal destruction and few viable yeasts were observed . The other type showed residual characteristics, with sparse collagen deposits and presence of xantomatous-like macrophages, containing degenerated fungi . Such residual lesions predominated after the 2nd month and were the only type observed from the 4th month on, indicating the control of the infection . In B10.A mice, on the contrary, only one type of lesion was observed, showing less tendency to encapsulation and the formation of multiple small granulomatous foci, individualized by reticular type III collagen fibers . There were many plasmocytes in the periphery and large numbers of budding yeasts, with no evidence of fungal destruction . In the course of the infection the lesions progressively increased in number and size . Altogether, the comparative histopathological analysis demonstrates the influence of the genetic pattern of the host on the lesions developed by resistant and susceptible mice to P . brasiliensis infection.

Rev Inst Med Trop Sao Paulo, 1999 Nov-Dec, 41(6), 359 - 64
Jorge Lobo's disease: experimental inoculation in Swiss mice; Opromolla DV et al.; Sixty-four isogenic Swiss mice were intradermically inoculated in both hind foot pads . The inocula, consisting of fungal suspensions from biopsies obtained from Jorge Lobo's Disease patients, had the total number of fungi and the viability index determined using a Neubauer chamber and the fluorescein diacetate-ethidium bromide technique (FD-EB), respectively . The animals were sacrificed at times ranging from ten days to eighteen months after inoculation . The cellular infiltrate, mainly consisting of macrophages containing fungi, increased progressively up to end of the study; however, no macroscopic alterations were observed in the inoculated feet . After nine months, small numbers of Langhans' giant cells started to appear in the infiltrate . A considerable number of fungi was observed at the end of the experimental period, but only a few were viable when stained by the FD-EB technique . This fact suggests that there is a multiplication of fungal cells, which are destroyed by the macrophages but remain in the tissue for a long time due perhaps to the difficulties in their elimination . These findings led us to conclude that in spite of the maintenance of the infection in these animals, Swiss mice cannot be considered an ideal model to study Jorge Lobo's Disease . However, the authors call attention to the possibility of other mouse strains being more susceptible to Paracoccidioides loboi.

Rev Inst Med Trop Sao Paulo, 1999 Nov-Dec, 41(6), 351 - 8
PCR with Paracoccidioides brasiliensis specific primers: potential use in ecological studies; Diez S et al.; 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.

J Interferon Cytokine Res, 2000 Jan, 20(1), 89 - 97
Resistance to Paracoccidioides brasiliensis infection is linked to a preferential Th1 immune response, whereas susceptibility is associated with absence of IFN-gamma production; Kashino SS et al.; The secretion of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-4, IL-5, and IL-10 by antigen-stimulated lymph node cells, eosinophil maturation, and the antibody isotypes produced were examined during intraperitoneal infection of susceptible (B10.A) and resistant (A/Sn) mice with Paracoccidioides brasiliensis . Lymph node cells from resistant mice produced early and sustained levels of IFN-gamma and IL-2, whereas susceptible animals secreted low to undetectable amounts of these type 1 cytokines . Both mouse strains presented late and transient production of IL-4, whereas IL-10 was produced constantly throughout the course of disease . Resistant animals produced increasing levels of IL-5 in the chronic phase of the infection (from the eighth week on), whereas susceptible mice showed two peaks of IL-5 production, at the first and twelfth weeks after infection . Only the susceptible strain presented medullary and splenic eosinophilia concomitant with the raised IL-5 production . In resistant mice, the levels of IgG2a antibodies were significantly higher than those observed in susceptible mice, which preferentially secreted IgG2b and IgA isotypes . Taken together, these results demonstrate that a sustained production of IFN-gamma and IL-2 and a predominant secretion of IgG2a antibodies are associated with resistance to P . brasiliensis . In contrast, the production of low levels of IFN-gamma, early secretion of high levels of IL-5 and IL-10, eosinophilia, and a preferential secretion of IgG2b and IgA isotypes characterize the progressive disease in susceptible animals.

J Med Microbiol, 2000 Jan, 49(1), 37 - 46
Evaluation of tests for antibody response in the follow-up of patients with acute and chronic forms of paracoccidioidomycosis; Del Negro GM et al.; Several serological tests have been used successfully in the diagnosis of paracoccidioidomycosis (PCM) . In contrast, data about the use of these tests in the follow-up of PCM patients have been heterogeneous . In this study, serum samples from 43 PCM patients with different clinical forms were analysed by counter-immuno-electrophoresis (CIE), complement fixation (CF) and ELISA before treatment . With CIE and ELISA, the chronic unifocal form showed significantly lower antibody levels compared with chronic multifocal and acute forms . Acute form patients had significantly higher titres than patients with multifocal disease by CIE but not by ELISA . No significant differences were observed with CF . Twenty-seven of these patients were followed-up for 2 years and showed a decline in antibody levels by all three tests, paralleling clinical improvement . However, only patients with unifocal disease cleared their antibodies after 1 year of treatment as analysed by CF and ELISA and after 2 years by CIE, suggesting that these patients may need shorter courses of therapy . Patients with the other clinical form of the disease needed > or =2 years of therapy to clear their antibodies . Sera from a further five patients who presented with a relapse were analysed . At the time of relapse all showed increases in antibody levels by CIE and ELISA, but only three showed increases by CF tests . Therefore, CIE and ELISA demonstrated a better clinical correlation than CF, probably reflecting the fungal burden of PCM patients more accurately.

Br J Radiol, 1999 Jul, 72(859), 717 - 22
MRI of head and neck paracoccidioidomycosis; de Castro CC et al.; Paracoccidioidomycosis, caused by the dimorphic fungus Paracoccidioides brasiliensis, is the most important systemic mycosis in Latin America . Imported cases have been reported in North America, Asia and Europe, in individuals who lived in endemic areas, sometimes many years before the development of clinical manifestations . The disease causes cutaneous and/or respiratory tract mucosal lesions as well as lymph node enlargement . Involvement of the oropharynx and/or the larynx, either alone or in association with pulmonary involvement, is one of the commonest clinical presentations . On MRI, the major features are mucosal lesions, usually hypointense on T1 weighted images and hyperintense on T2 weighted or fat suppressed images, affecting the oral cavity, oropharynx and larynx, with head and neck lymph node enlargement . Differential diagnosis includes other granulomatous infectious diseases, especially tuberculosis, and cancers such as squamous cell carcinoma and lymphomas.

Arch Otolaryngol Head Neck Surg, 1999 Dec, 125(12), 1375 - 8
Laryngeal manifestations of paracoccidioidomycosis (South American blastomycosis); Sant'Anna GD et al.; OBJECTIVE: To report clinical manifestations, diagnosis, and epidemiologic characteristics of laryngeal paracoccidioidomycosis . DESIGN: Case series . SETTINGS: Tertiary care institutional hospital . PATIENTS: We reviewed the hospital records of 7 patients with laryngeal paracoccidioidomycosis diagnosed by histopathological examination . MAIN OUTCOME MEASURE: Clinical manifestations of laryngeal paracoccidioidomycosis . RESULTS: All patients were men and were middle-aged (range, 43-65 years), and most (86% {6/7}) were farm workers . All 7 patients regularly used tobacco, but only (43% {3/7}) were alcohol users . Clinical manifestations were dysphonia (86% {6/7}), dyspnea (71% {5/7}), dysphagia (43% {3/7}), and cough (29% {2/7}) . Laryngeal examination revealed ulcerative lesions with a mulberrylike appearance in 3 patients and vegetative lesions in 4 patients . Many had multiple laryngeal lesions with involvement of the true and false vocal cords, the epiglottis, and the arytenoid and interarytenoid areas . The first diagnostic impression was carcinoma in all patients . CONCLUSIONS: Laryngeal paracoccidioidomycosis may be a difficult diagnosis for the unsuspecting clinician to make . Examination of the larynx can reveal lesions similar to laryngeal cancer; therefore, diagnosis of carcinoma must be ruled out by histopathological examination or culture of a specimen.

Microbes Infect, 1999 Apr, 1(5), 353 - 60
In vitro human immune reactivity of fast protein liquid chromatography fractionated Paracoccidioides brasiliensis soluble antigens; Diniz SN et al.; Soluble antigens of Paracoccidioides brasiliensis yeast cells (PbAg) were fractionated in a fast protein liquid chromatography (FPLC) system, using Q-Sepharose anion-exchange resin, in order to characterize antigenic fractions that could elicit cell reactivity and antibody recognition in human paracoccidioidomycosis (PCM) . PbAg fractions were eluted by 20 mM Tris-HCl solution (pH 9.6) with an increasing gradient up to 1 M NaCl . The FPLC system was able to resolve 7 fractions, enumerated from 0 to VI, according to the elution on the NaCl gradient . The analysis of each fraction on SDS-PAGE showed that fractions 0 to V were constituted by multiple protein bands with molecular mass ranging from 18 to 114 kDa . Large amounts of nucleic acids were evidenced in fraction VI, as revealed by agarose gel stained with ethidium bromide . Sera from PCM patients presenting different clinical forms contained antibodies that recognized antigens in all fractions with the exception of fraction VI as detected by ELISA . Further studies were designed to investigate the capacity of these fractions to induce cell proliferation . It was demonstrated that fractions III and V (200 and 450 mM NaCl, respectively) stimulated a significant proliferative response of peripheral blood mononuclear cells, while fraction 0 induced the lowest proliferative response among patients with PCM, in either acute, acute treated, or chronic forms.

Microbes Infect, 1999 Apr, 1(4), 273 - 8
Differential antibody isotype expression to the major Paracoccidioides brasiliensis antigen in juvenile and adult form paracoccidioidomycosis; Baida H et al.; We investigated the relationship between antibody response to the major Paracoccidioides brasiliensis antigen, a 43-kDa glycoprotein, and the two paracoccidioidomycosis (PCM) clinical presentations, the juvenile and the adult forms . Total immunoglobulin G (IgG), IgG isotypes, and IgA anti-gp43 antibodies were determined by enzyme-linked immunosorbent assay in patients'sera . Juvenile PCM patients had higher (P =.003) IgG anti-gp43 levels than adult form patients . IgG1 subclass levels, however, were comparable between the two clinical forms . Patients with the juvenile form had higher (P < . 001) IgG4, but lower (P =.03) IgG2 levels than patients with the adult form . The IgG4 isotype, regulated by interleukin 4, was found in all juvenile form patients but in only 12% of the adult form patients . In contrast, high levels of the IgG2 isotype, regulated by interferon-gamma, were found in 41% of the adult PCM patients, mainly those with a more benign disease, but in only 12% of the juvenile patients . IgG3 was either absent or detected at low levels . These results demonstrate, for the first time, specific IgG4 antibodies in the humoral immune response of patients with an endemic deep mycosis and suggest that the switch to the IgG subclasses in PCM is regulated by the patients' T-helper subset (Th-1 or Th-2) dominant cytokine profile . A possible role for IgG4 in the immunopathogenesis of the juvenile, more severe form of the disease is discussed . Finally, IgA was found mainly in adult form patients, probably as a result of the chronic mucosal antigenic stimulation characteristic of this form.

Rev Inst Med Trop Sao Paulo, 1999 Sep-Oct, 41(5), 279 - 83
Paracoccidioides brasilienses isolates obtained from patients with acute and chronic disease exhibit morphological differences after animal passage; Svidzinski TI et al.; The basis for virulence in Paracoccidioides brasiliensis is not completely understood . There is a consensus that the sequential in vitro subcultivation of P . brasiliensis leads to loss of its pathogenicity, which can be reverted by reisolation from animal passage . Attention to morphological and biochemical properties that are regained or demonstrated after animal passage may provide new insights into factors related to the pathogenicity and virulence of P . brasiliensis . We evaluated morphological characters: the percentage of budding cells, number of buds by cell and the diameter of 100 mother cells of yeast-like cells of 30 P . brasiliensis isolates, before and after animal passage . The isolates were obtained from patients with different clinical forms of paracoccidioidomycosis (PCM): acute form (group A, n=15) and chronic form (group C, n=15) . The measurement of the yeast cell sizes was carried out with the aid of an Olympus CBB microscope coupled with a micrometer disc . We measured the major transverse and longitudinal axes of 100 viable cells of each preparation . The percentage of budding cells as also the number of buds by cell was not influenced by animal passage, regardless of the source of the strain (acute or chronic groups) . The size values of P . brasiliensis isolates from groups A and C, measured before the animal passage exhibited the same behavior . After animal passage, there was a statistically significant difference between the cell sizes of P . brasiliensis isolates recovered from testicles inoculated with strains from groups A and C . The maximum diameter of mother cells from group A isolates exhibited a size of 42.1 microm in contrast with 32.9 microm exhibited by mother cells from group C (p<0.05) . The diameter of 1500 mother cells from group A isolates exhibited a medium size of 16.0 microm (SD +/- 4.0), a value significantly higher than the 14.1 microm (SD = +/- 3.3) exhibited by 1500 mother cells from group C isolates (p<0.05) . Our results reinforce the polymorphism exhibited by P . brasiliensis in biological material and the need for further investigations to elucidate the role of morphological parameters of the fungus in the natural history of the disease.

Infect Immun, 2000 Jan, 68(1), 352 - 9
Depletion of CD8(+) T cells in vivo impairs host defense of mice resistant and susceptible to pulmonary paracoccidioidomycosis; Cano LE et al.; Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection . Employing the same experimental model, we examined herein the role of CD8(+) T cells in the course of paracoccidioidomycosis . Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8(+) T cells in both mouse strains . The number of pulmonary CD4(+) T cells and immunoglobulin-positive cells was independent of the number of CD8(+) T cells . In susceptible mice, the loss of CD8(+) T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen . The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load . Furthermore, CD8(+) T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice . The production of P . brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8(+) T cells was similar to that of mice given control antibody . Histopathologically, depletion of CD8(+) T cells did not disorganize the focal granulomatous lesions developed by both mouse strains . These results indicate that CD8(+) T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P . brasiliensis and demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.

Mycopathologia, 1999, 145(2), 81 - 7
Experimental paracoccidioidomycosis in hamsters (Mesocricetus auratus): gestational interactions; Freire de Carvalho MG et al.; Paracoccidioides brasiliensis is a dimorphic fungus presenting specific steroid hormone receptors, both in the yeast and mycelial forms and estrogen inhibits the transition from mycelium to yeast . In the acute phase, the disease occurs with equal frequency in both sexes but in adults, females are spared . Placental fungal infection has been reported, but references to fetal infection have not been confirmed . We used 78 Syrian female hamsters divided into 3 groups: GI consisted of 30 infected mated females, GII of 20 infected unmated females and GIII of 28 uninfected mated females . Animals of group I were mated 4 weeks after infection and half of them were submitted to cesarean section on day 15 after successful mating; the other half was maintained and submitted to cesarean section and sacrificed 14 weeks after infection . Half of the animals of group II were sacrificed seven weeks and the other half 14 weeks after infection . Uninfected animals of group III were treated the same as the animals of group I . The animals were infected with strain 18 of P . brasiliensis by the intracardiac route . We evaluated the disease by the volume of granulomas in different organs, number of fungi in liver and spleen and the immunologic responses {ELISA, Double Immunodifusion (DID), Delayed Hypersensitivity Skin Test (DHT) and Macrophage Migration Inhibition (MMI)} . We studied the infection through the gestation by evaluation of the abortions, morphologic and clinic examinations of the fetuses . Our results showed that the infection did not transfer to the fetus through the placenta, but the number of abortions was larger among infected females . The newborns of GI females were smaller, weighed less and showed little vitality . The disease was more severe and disseminated in infected mated females, especially in the second sacrifice 14 weeks after inoculation, when the total volume of granulomas in them (56.3 mm) was much greater than in the infected unmated females (12 mm).

Mycopathologia, 1999, 145(2), 63 - 7
Chronic pulmonary paracoccidioidomycosis in the state of Rio Grande do Sul, Brazil; dos Santos JW et al.; Since 1942, when paracoccidioidomycosis was first identified in the state of Rio Grande do Sul, paracoccidioidal pulmonary lesions became a great concern to physicians . The present study focuses on 53 patients diagnosed over a seven-year period who presented paracoccidioidal lesions circumscribed to the lungs . These patients presented clinical and radiological features that simulated several pulmonary infectious and non-infectious conditions . Four unusual cases are briefly discussed . A sequence of laboratorial tests should be established for the diagnosis of pulmonary paracoccidioidomycosis.

Mycopathologia, 1999, 145(1), 7 - 14
Subcutaneous infection of mice with Paracoccidiodes brasiliensis induces a peculiar pattern of inflammatory and immune responses; Gesztesi JL et al.; It is well established that resistance or susceptibility to Paracoccidioidis brasiliensis infection in mice is under strict host's genetic control . Mice from A/Sn strain inoculated by the ip route are resistant to fungal infection while infection induced in mice from B10.A strain results in a fatal disease . The early cellular events of infection in both strains are characterized by a marked neutrophilic infiltration that is more prominent in B10.A mice . A peculiar characteristic of the Paracoccidioides brasiliensis-mouse model is that the subcutaneous (sc) inoculations of the fungus either in resistant (A/Sn) or susceptible (B10.A) mice is self-curing and tums mice from the B10.A strain able to express typical DTH reaction to fungal antigens, as observed in A/Sn mice . Here we report the investigation on the early events of the inflammatory response induced by the inoculation of live fungus into the hind footpad of A/Sn (resistant) and B10.A (susceptible) mice . The influence of neutrophils on the inflammatory response and antibody titers or DTH response to gp43, the major fungal antigen, was also evaluated . Results showed a different course of the inflammatory response induced by fungal inoculation in A/Sn and B10.A mice . Neutrophil depletion before infection differently influenced the kinetics of the inflammatory process in both mice strains but did not modify the fungal load in the lesions . In neutrophil depleted mice from both strains, a decrease in DTH response and an increase in total antibody titers to gp43 were observed . The significant increase in the fungal load in lesions seen in nude mice indicates that the self-limited infection evoked by fungal inoculation into the subcutaneous tissue is a T-cell dependent phenomenon . The implications of these observations in the pathogenesis of paracoccidioidomycosis are discussed.

Mycopathologia, 1998-99, 144(3), 131 - 3
Role of the armadillo Dasypus novemcinctus in the epidemiology of paracoccidioidomycosis; Vergara ML et al.; A study conducted in a rural area of Ibia, State of Minas Gerais, Brazil, where Paracoccidioides brasiliensis was recently isolated from soil, sought to determine if the armadillo Dasypus novemcinctus developed paracoccidioidomycosis . Out of 21 armadillos captured in the area, one had a lung granuloma containing fungal cells attributable to those of P . brasiliensis . The present report presents the first histopathological evidence for the presence of this pathogen in the lungs of an armadillo . This finding permitted us to establish a linkage between the two species and to suggest that fungal infections of humans and of armadillos occur in the same agricultural and forest microenvironment that they share.

Am J Trop Med Hyg, 1999 Sep, 61(3), 390 - 4
Endemic regions of paracoccidioidomycosis in Brazil: a clinical and epidemiologic study of 584 cases in the southeast region; Blotta MH et al.; This paper describes the clinical-seroepidemiologic characteristics of patients with paracoccidioidomycosis (PCM) who visited the University Hospital at the State University of Campinas (Campinas, Sao Paulo, Brazil) . The study group consisted of 584 individuals (492 males and 92 females) with ages ranging from 5 to 87 years . The highest incidence of the disease occurred between the ages of 41 and 50 years for men and between 11 and 40 years for women . Rural activities were the principal occupation of 46% of the patients . The diagnosis was confirmed by histopathologic examination and demonstration of fungus in scrapings, secretions, or in the sputum . Serologic test results for PCM were positive in 80% of the 584 patients studied . The significant number of patients, including 33 children less than 14 years old, indicates the presence of the fungus in the area and that this region is an important endemic area for PCM.

Rev Soc Bras Med Trop, 1999 Jul-Aug, 32(4), 439 - 42
{Systemic paracoccidioidomycosis with central nervous system involvement}; Duarte AL et al.; A clinical case of a patient bearing systemic paracoccidioidomycosis with regional ganglionic and oral exposure and later pulmonary involvement is presented . The patient was treated with specific drugs (amphotericin B, itraconazole, sulfamethoxazole-trimethoprim) and followed throughout a 6-year period and eventually died showing an extensive involvement of the central nervous system.

Mycopathologia, 1998-99, 144(2), 61 - 5
Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo (Dasypus novemcinctus); Sano A et al.; We studied three different isolates of Paracoccidioides brasiliensis obtained from the mesenteric lymph node (D3LY1), the spleen (D3S1) and the liver (D3LIV1) of the same armadillo (Dasypus novemcinctus) . Pulmonal inflammatory area was evaluated by intravenous inoculation of 10(6) yeast cells of each isolates in young, male, ddY mice . Moreover, the partial sequence of GP43kDa gene of P . brasiliensis was analyzed . The lung inflammatory area was greater in animals inoculated with isolate D3S1 . The partial sequence of GP43kDa gene indicated that isolate D3S1 is different from isolates D3LY1 and D3LIV1 . This study suggested that the same armadillo might be susceptible to multiple P . brasiliensis isolates simultaneously.

Bull Soc Pathol Exot, 1999 Jul, 92(3), 173 - 6
{Paracoccidioides brasiliensis infection in a subtropical region with important environmental changes}; Mangiaterra ML et al.; Paracoccidioidomycosis is a systemic mycoses caused by Paracoccidioides brasiliensis, a dimorphic fungus that infects man through respiratory ducts . It has been accepted that its ecological niche is located in the soil and plants of subtropical forests of Latin America . The Province of Corrientes is located at the northeastern border of Argentina, in a subtropical area where important environmental modifications have been introduced in the last decade as consequence of damming the Parana river at Yacyreta, one of the biggest hydroelectric dams in the world . Since there are no data on human infection provoqued by this fungal agent in Corrientes, the purpose of this study was to obtain information at present time about infection indexes and to assess if environmental changes introduced in the area could impact on the epidemiology of the disease . Skin tests with paracoccidioidin and histoplasmin were performed on 455 persons of both sexes, from 1 to 73 years of age, who live permanently in the area and voluntarily accepted to be included in the study . Both antigens were employed at the same time in order to evaluate crossed type reactions . Of the 455 persons, 52 (39 males-13 females) were reactive to paracoccidioidin (11.4%), with an increasing prevalence with age . According to previous data, these results would indicate an increase in the index of human infection by P . brasiliensis, and this may be related to the important changes in climatic and environmental conditions introduced in the area in the last years.

Am J Trop Med Hyg, 1999 Aug, 61(2), 280 - 7
Macrophage expression of class II major histocompatibility complex gene products in Paracoccidioides brasiliensis-infected mice; Bocca AL et al.; C57B1/6 isogenic mice infected with Paracoccidioides brasiliensis strains showed a disruption in the expression of Ia antigen . Expression slowly decreased during the course of the infection with a slight variation dependent on the route of inoculation and the fungal strain used, but production of interferon-gamma and tumor necrosis factor-alpha were observed . Suppression of Ia antigen expression and depression of the immunoproliferative responses of spleen cells were strongly correlated with nitric oxide levels . These parameters were inhibited when the animals were treated with nitro-L-arginine, which resulted in inhibition the activation of nitric oxide (NO) production . Analysis of the data showed that changes in the expression of the Ia antigen occur in P . brasiliensis infection and are strongly correlated with NO levels . These phenomena may be interrelated and reflect macrophage activation that contributes to the control of the disease and to the immunosuppression observed during the course of the infection.

Curr Opin Pulm Med, 1999 Sep, 5(5), 319 - 25
Paracoccidioidomycosis; Bethlem EP et al.; Paracoccidioidomycosis is a systemic infection caused by the dimorphic fungus Paracoccidioides brasiliensis . It is commonly an endemic disease in Latin America, but several cases have been reported outside this area, particularly now in this time of world globalization . Primary pulmonary infection occurs commonly in the first and second decades of life and usually has a benign, self-limited respiratory infection course . The adult chronic manifestation of the disease is usually the result of reactivation of quiescent lesions with diffuse lung infiltrates, predominately of the interstitial pattern, with or without involvement of various other organs . The finding of this disease in a patient is an important step for the large differential diagnosis of the interstitial lung diseases group.

Rev Inst Med Trop Sao Paulo, 1999 Jan-Feb, 41(1), 9 - 12
Constitutive melanin in the cell wall of the etiologic agent of Lobo's disease; Taborda VB et al.; Lobo's disease is a chronic granulomatous disease caused by the obligate pathogenic fungus, whose cell walls contain constitutive melanin . In contrast, melanin does not occur in the cell walls of Paracoccidioides brasiliensis when stained by the Fontana-Masson stain.

Rev Inst Med Trop Sao Paulo, 1998 Nov-Dec, 40(6), 377 - 81
Paracoccidioidomycosis and AIDS: report of the first two Colombian cases; Tobon AM et al.; The records of the first two Colombian patients with AIDS and paracoccidioidomycosis are presented . Both patients were males and had no known risk factors for HIV although in the past they had worked in the field where they could have been infected with the fungus . They exhibited the juvenile type of disease with multiple organ system involvement and symptoms of short duration . They were deeply immunodepressed as indicated by less than 100 CD4 T lymphocytes per mL; however, serologic tests revealed circulating anti-Paracoccidioides brasiliensis antibodies and in one patient the first diagnostic clue came from such tests . In one case, the mycosis preceded the AIDS diagnosis while in the other, both pathologies were discovered simultaneously . Antimycotic therapy with itraconazole was administered for over 10 months, with an initial dose of 200 mg/day followed by 100 mg/day; marked improvement of the mycotic signs and symptoms was soon noticed an there have been no signs of relapse . The patients improvement was also due to the combined retroviral treatment that was instituted . In spite of the rarity of the AIDS-paracoccidioidomycosis association, physicians practicing in endemic areas should consider the presence of the mycosis in immunosuppressed patients, since a prompt diagnosis and institution of combined antimycotic-anti-retroviral treatments would result in patient improvement and survival . It appears possible that the longer survival time of today's AIDS patients would give the quiescent fungus the opportunity to revive, multiply and cause overt disease.

Rev Argent Microbiol, 1999 Apr-Jun, 31(2), 78 - 81
{In vitro sensitivity of Paracoccidioides brasiliensis to systemically used antifungal agents}; Rodero L et al.; In 1992, the NCCLS proposed a broth macrodilution method for determining the in vitro susceptibility of yeast . However, for dimorphic fungi no standardised method is available . The aim of our study was to evaluate the reference broth macrodilution method with isolates of Paracoccidioides brasiliensis . The in vitro susceptibility of two ATCC strains (32069 and 36324) and nine clinical isolates were determined against serial dilutions of amphotericin B (AMB), ketoconazole (KTZ), and itraconazole (ITZ) from 0.02 to 20 mg/l and fluconazole (FCZ) from 0.1 to 100 mg/l . The inoculum used was 0.5 x 10(4)-2.5 x 10(5) CFU/ml, employing the yeast phase in order to obtain homogeneous growth . The test was incubated with agitation in a rotating shaker at 35 degrees C, and 7 days was the optimal time for MIC final reading . Average MIC50/MIC90 (mg/l) for the eleven isolates tested were the following: AMB: < 0.02/0.16, FCZ: 1.6/6.2, KTZ: 0.08/0.31, ITZ: 0.31/1.3 . One clinical isolate showed high MIC for azole drugs (KTZ: 1.3 mg/ml, FCZ: 100 mg/l and ITZ 5 mg/l) . Although further studies are necessary to evaluate the usefulness of this methodology for P . brasiliensis, the latter may be clinically useful to predict the development of resistance.

Mycoses, 1999, 42(4), 281 - 3
Ultrastructural differences observed in Paracoccidioides brasiliensis yeast phase cells grown on solid and in liquid medium; Borba Cde M et al.; An ultrastructural study was conducted on, yeast-like Paracoccidioides brasiliensis cells grown on liquid and solid peptone--yeast extract--glucose medium . A large proportion of cells grown in liquid medium presented cytoplasmic damage compared with the cells grown on solid medium, which remained intact, suggesting that agar plays an important role in the development of this fungus.

Med Mycol, 1999 Aug, 37(4), 277 - 84
Correlation between antigenemia of Paracoccidioides brasiliensis and inhibiting effects of plasma in patients with paracoccidioidomycosis; Sugizaki MF et al.; Metabolites produced by pathogenic fungi may be involved in the pathogenesis of fungal infections consequently altering the defence mechanisms of the host . In this study the levels of Paracoccidioides brasiliensis antigens detected in the plasma of patients with paracoccidioidomycosis correlated with the suppression index detected by the low mitogenic response of peripheral blood mononuclear cells (PBMC) to phytohaemaglutinin (PHA) . This inhibitory effect on lymphoproliferation was observed in the plasma of 58% of the patients, suggesting the presence of inhibitory factors . Plasma samples from paracoccidioidomycosis patients having or not having inhibitory factors showed no significant effect on chromosomes of lymphocytes from healthy individuals . However, these plasmas had a suppressive activity on the blastogenic response of these lymphocytes stimulated with PHA, that was independent of a cytotoxic effect . P . brasiliensis antigens added to the proliferative response of PBMC from healthy individuals stimulated or not stimulated with PHA showed a dose-dependent suppressor effect, reproducing the inhibitory effect of patients' plasma . We suggest that the antigens of P . brasiliensis present in the plasma of patients, even at low concentrations, can play an important role in the reduction of the cellular immune response and in the genesis of the immunoregulatory disturbances observed in paracoccidioidomycosis.

Med Mycol, 1999 Aug, 37(4), 269 - 76
Virulence of Paracoccidioides brasiliensis isolates can be correlated to groups defined by random amplified polymorphic DNA analysis; Molinari-Madlum EE et al.; Fifteen Paracoccidioides brasiliensis isolates were discriminated by the RAPD analysis into two groups with only 17% of genomic identity . The ability of P . brasiliensis isolates to invade tissues was studied in an experimental model using susceptible B10.A mice . The analysis was performed according to the severity of the lesions including the number and size of the granuloma, the number and dissemination of fungi to different organs . The isolates from two RAPD groups demonstrated a marked difference in their virulence patterns for B10.A mice . The isolates Pb S, 662, Bt and 166 (group I) elicited localized infection restricted to the liver showing compact epithelioid granuloma with few fungi in the early post-infection period (slightly virulent) . On the other hand the isolates Pb 01 and 7455 (group II) elicited a disseminated infection with a mixed suppurative and looser granulomatous inflammation, showing extensive areas of necrosis and large numbers of viable fungal cells (highly virulent) . These results are strong evidence for correlation between RAPD patterns and the virulence degree of P . brasiliensis.

Med Mycol, 1999 Aug, 37(4), 261 - 7
Antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis; Kurita N et al.; Human peripheral blood polymorphonuclear leucocytes (PMN) were examined in vitro for antifungal activity against yeast cells of Paracoccidioides brasiliensis . The yeast cell of this fungus was resistant to killing by PMN . However, PMN exhibited a fungistatic effect on the fungal isolates employed in a long-term ( approximately 72 h) assay . Lysates of PMN did not show a fungistatic or fungicidal effect, indicating that live PMN are necessary for the antifungal effect . Interferon-Gamma (IFN-Gamma) enhanced the antifungal activity of PMN . IFN-Gamma-treated PMN killed isolate Bt-4 within 2 h of coculture, and after 24 h a still greater killing effect was observed . By contrast, IFN-Gamma-treated PMN did not show a significant killing effect on isolate Tatu, but did exhibit an enhanced fungistatic effect on this isolate . In contrast, tumor necrosis factor-alpha and interleukin 8 had no effect on the antifungal activity of PMN . Scanning electron microscopy revealed that the surface structure of the fungal cell was apparently damaged by PMN within 24 h of cocultures . Based on these novel findings, we speculate that human PMN might play a role in host resistance in early infection with this fungus due to their antifungal activity.

Med Mycol, 1999 Jun, 37(3), 219 - 22
Electrophoretic karyotype of environmental isolates of Paracoccidioides brasiliensis; Montoya AE et al.; Five of the 12 environmental isolates of the dimorphic fungus Paracoccidioides brasiliensis known to date, were analysed by contour-clamped homogeneous electric field gel electrophoresis (CHEF) . The electrophoretic pattern was shown to consist of five bands, with molecular size ranging from 3.2 to 10 Mb, a model quite similar to the one found in the clinical isolates previously tested and used here as controls . However, one of the bands in the environmental isolates had a lesser weight (7.2 Mb), than the one corresponding to the clinical counterparts (8.8 Mb) . This resulted in a smaller genome, approximately 29.7 Mb . The small differences that were found indicate the presence of chromosome polymorphism in this fungus.

Med Mycol, 1999 Jun, 37(3), 207 - 11
In situ hybridization in paracoccidioidomycosis; De Brito T et al.; In situ hybridization (ISH) was performed using oral biopsies from patients with paracoccidioidomycosis and guinea pig testes inoculated with a culture of Paracoccidioides brasiliensis isolated from soil, employing both a 14 base-pair specific oligoprobe (ACT CCC CCG TGG TC) and its complementary sequence . When combining ISH with the Gridley stain which detects fungal cell walls, about 2-3% of the fungal cells present in the tissues were labelled . When the complementary probe was used, labelling was higher, reaching the 3% level.

Eur J Epidemiol, 1999 Apr, 15(4), 383 - 8
Histoplasmosis and Paracoccidioidomycosis in northwestern Argentina III . Epidemiological survey in Vipos, La Toma, and Choromoro - Trancas, Tucumán, Argentina; van Gelderen de Komaid A et al.; The present work was undertaken to obtain epidemiological data on the extent and distribution of Histoplasma capsulatum var . capsulatum and Paracoccidiodes brasiliensis diseases in the Vipos, La Toma and Choromoro areas . Skin test surveys with histoplasmin and paracoccidioidin were carried out in the permanent human population of those localities . Mycological sputum studies and serological tests were performed to skin test reactors to determine if there were signs or symptoms of active mycotic disease . La Toma and Choromoro are highly prevalent areas of histoplasmosis (>30% the histoplasmin positive individuals) whereas Vipos can be relatively considered a highly prevalent area (between 20-30% the histoplasmin reactors) according to the normally accepted range used to define an endemic disease {2} . Early Histoplasma capsulatum infection (<10 years old) is reported for Vipos and Choromoro . La Toma has the highest rate of previous exposure to P . brasiliensis detected in the studied area (10.2%) . Vipos residents are not infected with P . brasiliensis.

Rev Inst Med Trop Sao Paulo, 1999 Mar-Apr, 41(2), 79 - 86
Paracoccidioides brasiliensis . A mycologic and immunochemical study of two strains; Lacaz Cda S et al.; The authors conducted a mycologic, immunochemical and molecular biology study on two strains of Paracoccidioides brasiliensis, one of them, called IBIA, isolated from soil in the municipality of IBIA (Minas Gerais) by Silva-Vergara et al . (1996, 1998), and the other, BAT, cultivated from a human case of paracoccidioidomycosis in Ribeirao Preto (Sao Paulo/Brazil) by Freitas Da Silva (1996) . 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.

Braz J Med Biol Res, 1999 May, 32(5), 645 - 9
Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp; Puccia R et al.; We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro . In the present study, we separated the components of P . brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa . The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37 degrees C, and detection of fluorescent bands with a UV transilluminator . Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature . A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component . Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

AJR Am J Roentgenol, 1999 Jul, 173(1), 59 - 64
Chronic pulmonary paracoccidioidomycosis (South American blastomycosis): high-resolution CT findings in 41 patients; Funari M et al.; OBJECTIVE: To assess the pulmonary parenchymal findings on high-resolution CT in 41 patients with the chronic form of paracoccidioidomycosis (South American blastomycosis) . SUBJECTS AND METHODS: The study included 41 consecutive patients in whom chronic paracoccidioidomycosis had been proven . All patients underwent high-resolution CT (1-mm collimation, high-spatial-frequency reconstruction algorithm) at 12 equally spaced intervals through the chest . The images were analyzed by two radiologists, and each final decision was reached by consensus . RESULTS: Thirty-eight (93%) of the 41 patients had CT scans with abnormal findings . The findings included interlobular septal thickening in 36 patients (88%), 1-25 mm diameter nodules in 34 (83%), peribronchovascular interstitial thickening in 32 (78%), centrilobular opacities in 26 (63%), intralobular lines in 24 (59%), ground-glass opacities in 14 (34%), cavitation in seven (17%), air-space consolidation in five (12%), traction bronchiectasis in 34 (83%), and paracicatricial emphysema in 28 (68%) . In approximately 90% of patients, the abnormalities were bilateral and symmetrical and involved all lung zones . CONCLUSION: High-resolution CT findings of paracoccidioidomycosis consist predominantly of interstitial abnormalities and nodules associated with traction bronchiectasis and paracicatricial emphysema in a bilaterally symmetrical distribution.

Microbiology, 1999 May, 145 ( Pt 5), 1153 - 60
Anaerobic oxidations of cysteate: degradation via L-cysteate:2-oxoglutarate aminotransferase in Paracoccus pantotrophus; Mikosch CA et al.; Anoxic, fresh-water enrichment cultures to oxidize different organosulfonates were set up with nitrate, ferric iron or sulfate as electron acceptors . Pure cultures were easily obtained with two naturally occurring sulfonates, cysteate (2-amino-3-sulfopropionate) and taurine (2-aminoethanesulfonate), under nitrate-reducing conditions . These two sulfonates were also oxidized during reduction of iron(III), though isolation of pure cultures was not successful . One nitrate-reducing cysteate-oxidizing bacterium, strain NKNCYSA, was studied in detail . It was identified as Paracoccus pantotrophus . Eighteen sulfonates were tested, and the organism degraded cysteate, taurine, isethionate (2-hydroxyethanesulfonate), sulfoacetate or 3-aminopropanesulfonate with concomitant reduction of nitrate, presumably to molecular nitrogen . The carbon skeleton of these substrates was converted to cell material and, presumably, CO2 . The amino group was released as ammonia and the sulfono moiety was recovered as sulfate . Cell-free extracts of P . pantotrophus NKNCYSA contained constitutive L-cysteate:2-oxoglutarate aminotransferase (EC 2.6.1.-) and glutamate dehydrogenase (EC 1.4.1.4) . Taurine:pyruvate aminotransferase, in contrast, was inducible.

Med Mycol, 1999 Apr, 37(2), 115 - 21
Identification, N-terminal region sequencing and similarity analysis of differentially expressed proteins in Paracoccidioides brasiliensis; Cunha AF et al.; Paracoccidioides brasiliensis is the causal agent of paracoccidioidomycosis, which is a systemic mycosis in Latin America . This human pathogen is a dimorphic fungus existing as mycelium (26 degrees C) and in infected tissues as a yeast form (36 degrees C) . The in vitro differentiation process is reversible and dependent on temperature shift . In the present study, the total proteins from both forms of P . brasiliensis (isolate Pb01) were analysed by two-dimensional electrophoresis . Differentially expressed proteins were identified . Two of these proteins, PbM46 (mycelium) and PbY20 (yeast), were submitted to automated protein sequencing of their N-terminal regions . The 15 amino acid residue sequence of PbM46, AITKIFALKVYDSSG, is similar to enolases from several sources, and specially those from Saccharomyces cerevisiae (80%) and Candida albicans (67%), when compared to the NR database at NCBI using the BLASTP program . The 34 amino acid residue sequence of PbY20, APKIAIVFYSLYGHIQKLAEAQKKGIEAAGGTAD, could probably represent an allergen protein since it is very similar (90%) to the minor allergen protein of Alternaria alternata and 82% similar to the allergen protein of Cladosporium herbarum . This comparative analysis of proteins from mycelium and yeast forms has allowed the identification and characterization of differentially expressed proteins, probably related to differential gene expression in P . brasiliensis.

Zentralbl Bakteriol, 1999 Apr, 289(2), 211 - 6
Histopathological evolution of experimental paracoccidioidomycosis in Wistar rats; Iovannitti CA et al.; Twenty Wistar rats were inoculated, by the intracardiac route, with 0.5 ml each of a yeast phase suspension of Paracoccidioides brasiliensis Utero strain . The rats were sacrificed at regular intervals post-infection, at which time their lungs, heart, liver, spleen and kidneys were removed, fixed and stained for study . The parameters of interest for the lung specimens were: (a) extent of the lesions; (b) number of fungi; (c) presence of a lymphomononuclear halo . Extrapulmonary lesions were also sought . Until the fourth month post-infection, the lesions were progressive in nature, contained great numbers of viable fungi, and were surrounded by an important lymphomononuclear halo which tended to be confluent . At four and a half months p.i., the extent of the pulmonary lesions was reduced, the granulomas were less compact with fewer viable fungi, macrophages showed microvacuolation, and the lymphomononuclear halo was less pronounced . Extrapulmonary lesions, which were frequently identified in the first months post-infection, diminished from the seventh month onwards . The histological characteristics of extrapulmonary lesions were always the same as those found in the lungs . Infection tended to be controlled by the animals from the fourth month, but without complete resolution of the lesions.

Biochemistry, 1999 Jun 1, 38(22), 7294 - 306
Characterization of sphingolipids from mycopathogens: factors correlating with expression of 2-hydroxy fatty acyl (E)-Delta 3-unsaturation in cerebrosides of Paracoccidioides brasiliensis and Aspergillus fumigatus; Toledo MS et al.; Significant differences exist between mammals and fungi with respect to glycosphingolipid (GSL) structure and biosynthesis . Thus, these compounds, as well as the cellular machinery regulating their expression, have considerable potential as targets for the diagnosis and treatment of fungal diseases . In this study, the major neutral GSL components extracted from both yeast and mycelium forms of the thermally dimorphic mycopathogen Paracoccidioides brasiliensis were purified and characterized by 1H and 13C NMR spectroscopy, ESI-MS and ESI-MS/CID-MS, and GC-MS . The major GSLs of both forms were identified as beta-glucopyranosylceramides (GlcCer) having (4E, 8E)-9-methyl-4,8-sphingadienine as long chain base in combination with either N-2'-hydroxyoctadecanoate or N-2'-hydroxy-(E)-3'-octadecenoate . The mycelium form GlcCer had both fatty acids in a approximately 1:1 ratio, while that of the yeast form had on average only approximately 15% of the (E)-Delta 3-unsaturated fatty acid . Cerebrosides from two strains of Aspergillus fumigatus (237 and ATCC 9197) expressing both GalCer and GlcCer were also purified and characterized by similar methods . The GalCer fractions were found to have approximately 70% and approximately 90% N-2'-hydroxy-(E)-3'-octadecenoate, respectively, in the two strains . In contrast, the GlcCer fractions had N-2'-hydroxy-(E)-3'-octadecenoate at only approximately 20 and approximately 50%, respectively . The remainder in all cases was the saturated 2-OH fatty acid, which has not been previously reported in cerebrosides from A . fumigatus . The availability of detailed structures of both glycosylinositol phosphorylceramides {Levery, S . B., Toledo, M . S., Straus, A . H., and Takahashi, H . K . (1998) Biochemistry 37, 8764-8775} and cerebrosides from P . brasiliensis revealed parallel quantitative differences in expression between yeast and mycelium forms, as well as a striking general partitioning of ceramide structure between the two classes of GSLs . These results are discussed with respect to possible functional roles for fungal sphingolipids, particularly as they relate to the morphological transitions exhibited by P . brasiliensis.

Mycopathologia, 1998-99, 143(3), 165 - 9
Comparison between human and armadillo Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis; Sano A et al.; Sixty-three Paracoccidioides brasiliensis isolates obtained from three nine-banded armadillos (Dasypus novemcinctus), one Amazonian armadillo's and 19 clinical isolates were compared by random amplified polymorphic DNA analysis with the primer OPG-19 . The isolates were divided into three major clusters, I, II and III . Coincidences between human and armadillo isolates were observed in clusters I and II . Cluster III consisted only of armadillos' isolates . The results suggested that (I) humans may acquire P . brasiliensis infection by contact with armadillo's environment, (II) there may be P . brasiliensis genotypes peculiar to the animal, and (III) individual armadillos may be infected with P . brasiliensis cells with different genotypes.

Am J Trop Med Hyg, 1999 May, 60(5), 837 - 9
Failure of amphotericin B colloidal dispersion in the treatment of paracoccidioidomycosis; Dietze R et al.; Although amphotericin B desoxycholate is considered the most effective treatment for disseminated Paracoccidioides brasiliensis infections, little is known about the efficacy of lipid-based formulations of amphotericin B in this infection . In this study, we treated four adults with the juvenile form of paracoccidioidomycosis with 3 mg/kg/day of amphotericin B colloidal dispersion for at least 28 days . Although all of the patients initially responded by clinical observation, all four patients relapsed within six months . The use of amphotericin B colloidal dispersion for the initial induction of paracoccidioidomycosis failed to cure this infection . Possible reasons for failure include dose, duration, or reduced efficacy of this lipid preparation . For many fungal infections, lipid-based preparations have been shown to have a therapeutic-toxic advantage, but our experience with Paracoccidioides infections suggests that more careful studies will need to be performed before they can be recommended for use in this mycosis.

Diagn Microbiol Infect Dis, 1999 May, 34(1), 27 - 32
"Giant" blastoconidia of Candida albicans: morphologic presentation and concepts regarding their production; Bottone EJ et al.; Candida albicans normally produces blastoconidia measuring 2 to 8 microns in diameter . Markedly enlarged "giant" (approximately 30 microns) blastoconidia of a C . albicans isolate (designated BH) were observed after growth on commercially prepared chocolate agar already supplemented with IsoVitalex (BBL-Microbiology Systems, Cockeysville, MD, USA) . Morphologically, "giant" blastoconidia presented a spectrum of forms such as blastoconidia with linear creases, with a single broad-based bud resembling Blastomyces dermatididis, with multiple buds resembling Paracoccidioides brasiliensis, or elliptical in shape . "Giant" blastoconidia contained a large oval clear vacuole occupying greater than 50% of the blastoconidium . Pseudohyphae emanating from these blastoconidia were also enlarged and contained a similar oval inclusion . Rarely observed were "giant" blastoconidia with either adherent or internalized blastoconidia uniformly arranged within the blastoconidium . "Giant" or enlarged blastoconidia production was constant, usually approaching 10 to 20% of the blastoconidial units comprising a single colony, irrespective of the number of subcultures . IsoVitalex supplementation of Remel (Lexana, KS, USA) chocolate agar but not a variety of other media also resulted in "giant" blastoconidia production . It is, therefore, theorized that a component(s) of IsoVitalex activates/blocks a gene present in select clones of C . albicans blastoconidia resulting in "giant" or enlarged blastoconidiogenesis.

J Clin Microbiol, 1999 Jun, 37(6), 2031 - 3
Lacazia loboi gen . nov., comb . nov., the etiologic agent of lobomycosis; Taborda PR et al.; The new genus Lacazia P . Taborda, V . Taborda, et McGinnis is proposed to accommodate Lacazia loboi (O . M . Fonseca et Lacaz) P . Taborda, V . Taborda, et McGinnis, the obligate pathogen that causes lobomycosis in mammals . The continued placement of that fungus in the genus Paracoccidioides Almeida as Paracoccidioides loboi is taxonomically inappropriate . Loboa loboi Ciferri et al . is a synonym of Paracoccidioides brasiliensis.

Nippon Ishinkin Gakkai Zasshi, 1999, 40(2), 103 - 9
Epidemiological survey of the imported mycoses in Japan; Fujio J et al.; The authors surveyed the present situation for imported (introduced) mycoses from papers published in Japan as of the end of October, 1998 . There were 16 cases of coccidioidomycosis, 22 of histoplasmosis, 13 of paracoccidioidomycosis and one of penicilliosis marneffei . In coccidioidomycosis the ages ranged from 17 to 55, for 14 males and two females . There were eight cases of primary pulmonary coccidioidomycosis and the remaining eight were of the disseminated type, of which two were fatal . For histoplasmosis, the ages ranged from 17 to 74 of which five cases were female and 17 male . They consisted of 13 pulmonary histoplasmosis, seven disseminated type and two cutaneous type . All cases of the disseminated type died . All of the patients with paracoccidioidomycosis were infected in Latin American countries . They consisted of 12 males and one female, and the ages ranged from 34 to 68 . The clinical types were dominated by mucocutaneous-lymphangitic paracoccidioidomycoses (10 cases) . A single case of penicilliosis marneffei (38 years old, male) was reported very recently . The case was complicated by AIDS and became critical . Blastomycosis has not yet been reported . This survey indicates that the number of the imported mycoses in Japan is increasing . It is necessary for the responsible authorities to take counter measures to cope with this situation.

J Antimicrob Chemother, 1999 Mar, 43(3), 321 - 31
Endemic mycoses: a treatment update; Lortholary O et al.; Endemic mycoses remain a major public health problem in several countries and they are becoming increasingly frequent with the spread of HIV infection . Amphotericin B remains the drug of choice during the acute stage of life-threatening endemic mycoses occurring in both immunocompetent and immunocompromised hosts . Ketoconazole is effective in non-AIDS patients with non-life-threatening histoplasmosis, blastomycosis, or paracoccidioidomycosis . Itraconazole is the treatment of choice for non-life-threatening Histoplasma capsulatum or Blastomyces dermatitidis infections occurring in immunocompetent individuals and is the most efficient secondary prophylaxis of histoplasmosis in AIDS patients . Itraconazole is also effective in lymphocutaneous and visceral sporotrichosis, in paracoccidioidomycosis, for Penicillum marneffei infection, and is an alternative to amphotericin B for Histoplasma duboisii infection . Coccidioidomycosis may be effectively treated with prolonged and sometimes life-long itraconazole or fluconazole therapy . Fluconazole has relatively poor efficacy against histoplasmosis, blastomycosis and sporotrichosis . New antifungal agents have been tested in vitro or in animal models and may soon be evaluated in clinical trials.

Med Mycol, 1998 Dec, 36(6), 413 - 7
Antigenic relationship between Loboa loboi and Paracoccidioides brasiliensis as shown by serological methods; Camargo ZP et al.; Lobomycosis and paracoccidioidomycosis are two different mycoses caused by Loboa loboi and Paracoccidioides brasiliensis, respectively . To verify cross-antigenicity between them, lobomycosis sera were tested by immunoblotting, ELISA and capture-EIA against crude exo-antigen, 'cell-free antigen' and gp43 from P . brasiliensis . The majority of lobomycosis serum samples recognized crude exo-antigens and gp43 from P . brasiliensis . Gp43 was eluted from an affinity column prepared with IgG from a patient with active lobomycosis . In lower frequencies and intensities, lobomycosis sera also recognized proteins of 29 kDa, 36 kDa, 39 kDa, 52 kDa, 63 kDa, 70 kDa, 83 kDa, and 108 kDa from P . brasiliensis.

Med Mycol, 1998 Dec, 36(6), 405 - 11
Involvement of the major glycoprotein (gp43) of Paracoccidioides brasiliensis in attachment to macrophages; Almeida SR et al.; The yeast form of Paracoccidioides brasiliensis, the causative agent of a deep mycosis in humans, is known to be phagocytized by, and to multiply inside, macrophages . In this work we describe the involvement of gp43, a major antigenic protein of P . brasiliensis, in the initial steps of attachment of the fungus to macrophages . Anti-gp43 F(ab) polyclonal fragments were capable of inhibiting phagocytosis in a concentration-dependent manner . Sheep red blood cells sensitized with purified gp43 were more endocytized than SRBC alone, and this process was also inhibited by anti-gp43 F(ab) fragments . Inhibition tests indicated the involvement of fucose and mannose residues in the phagocytosis of the fungus and of SRBC-gp43 by macrophages . Taken together, these results suggest that gp43 may be involved in the adherence and uptake of the fungus by murine peritoneal macrophages, and that this binding may be dependent on monosaccharide residues that are part of the gp43 glycoprotein.

Med Mycol, 1998 Dec, 36(6), 351 - 64
Serodiagnosis of histoplasmosis, paracoccidioidomycosis and penicilliosis marneffei; current status and future trends; Hamilton AJ; Effective serodiagnosis of systemic fungal infections is of increasing importance, particularly with regard to the identification of infection with Histoplasma capsulatum, Paracoccidioides brasiliensis and Penicillium marneffei . Methodology has been based either around antibody or antigen detection, although there is clear overlap between the two . Antibody-based detection systems for the diagnosis of histoplasmosis, paracoccidioidomycosis and penicilliosis marneffei have now begun to incorporate a range of highly purified and well-characterized antigens, in contrast to the situation of a few years ago when relatively crude preparations derived from either whole cells or culture filtrate were used . The application of such antigens offers improvements in reproducibility and specificity, although the detection of meaningful antibody responses in immunosuppressed individuals remains a problem . Partly as a consequence of this a great deal of attention has focused on the development of antigen detection assays, and such methods have proved particularly successful, as for instance in the serodiagnosis of histoplasmosis in AIDS patients . The recent utilization of monoclonal antibodies in the development of antigen detection methods for the diagnosis of histoplasmosis and paracoccidioidomycosis offers further scope for improvement in this area.

Mycopathologia, 1998, 143(2), 65 - 9
Fine needle aspiration in the diagnosis of pulmonary paracoccidioidomycosis; dos Santos JW et al.; Six cases of pulmonary paracoccidioidomycosis diagnosed only by transthoracic fine needle aspiration are presented . The clinical and radiological presentation is varied . The most frequent use of this technique will permit the diagnosis of early lesions of mycosis.

Med Mycol, 1999 Feb, 37(1), 19 - 24
Fungistatic and fungicidal activities of murine polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis; Kurita N et al.; Recently, a novel culture medium for detecting live yeast cells of Paracoccidioides brasiliensis was developed by Kurita et al . Using this culture medium, murine peritoneal polymorphonuclear leucocytes (PMN) were examined for fungistatic and fungicidal activities against P . brasiliensis yeast cells . The magnitude of the antifungal effect of PMN varied depending upon the fungal isolates used . PMN exhibited a killing effect on P . brasiliensis isolate Bt-4 in 2 h of coculture . In contrast, the other three fungal isolates employed were resistant to killing by PMN . However, PMN considerably suppressed the growth of isolates Tatu and Recife in a long-term assay (approximately 72 h) . The growth of isolate Bt-9 was also suppressed by PMN during the first 24 h, but was found to be considerably promoted at 72 h of coculture . Interferon-gamma (IFN-gamma), but not tumour-necrosis factor-alpha, significantly augmented the antifungal activity of PMN . IFN-gamma-treated PMN exhibited a killing effect on isolates Tatu, Recife and Bt-9 after 24 h of coculture, and showed an enhanced killing effect on isolate Bt-4 . Contact between PMN and fungal cells was required for PMN to exert the antifungal effect . Our results suggest that PMN, whether activated with cytokines or not, might play a critical role in host resistance in early infection with this fungus by buying time for development of more effective immunologic responses.

J Neuroimaging, 1993 Oct, 3(4), 216 - 9
Magnetic resonance imaging in intracranial paracoccidioidomycosis; Magalhaes AC et al.; Paracoccidioidomycosis is a systemic mycosis, endemic in South and Central America, that affects the central nervous system (CNS) in almost 10% of patients . Neurological involvement includes two different clinical forms: meningeal and granulomatous, also known as the pseudotumor form . Five patients with biopsy-proved systemic paracoccidioidomycosis and neurological complaints were studied by magnetic resonance imaging . CNS involvement was detected in all patients in the form of multiple round or lobulated lesions, predominantly hypointense on T2-weighted images and ring or nodular enhancement on post-gadolinium T1-weighted images . The lesions were distributed diffusely, with a slight predominance in the supratentorial compartment, although infratentorial lesions were also observed, mainly in the cerebellum . Hypointense lesions on T2-weighted images persisted in all 3 patients reexamined after treatment, whereas enhancing lesions on post-gadolinium T1-weighted images turned isointense in 2 patients . Magnetic resonance imaging is a sensitive method in documenting CNS paracoccidioidomycosis, most frequently as supratentorial and infratentorial multiple, round or lobulated hypointense lesions on T2-weighted images.

Mol Microbiol, 1999 Feb, 31(4), 1039 - 50
Differential expression of an hsp70 gene during transition from the mycelial to the infective yeast form of the human pathogenic fungus Paracoccidioides brasiliensis; da Silva SP et al.; We have isolated and characterized cDNA and genomic clones that encode a 70 kDa heat shock protein (Hsp70) from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis . The gene encodes a 649-amino-acid protein showing high identity with other members of the hsp70 gene family . The hsp70 gene is induced during both heat shock of yeast cells at 42 degrees C and the mycelial to yeast transition . A differential expression of this gene can be observed between mycelial and yeast forms, with a much higher level of expression in the yeast . We found two introns of 178 and 72 nucleotides in the P . brasiliensis hsp70 gene . Splicing of these introns is regulated during the heat shock process and possibly during infection . In order to analyse the differential accumulation of unspliced mRNA following cellular differentiation and/or heat shock, reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were carried out . The temperature-induced mycelial to yeast transition results in the transient accumulation of unspliced hsp70 mRNA transcripts . Yeast cells, after adaptation at 36 degrees C, seem to be more proficient at splicing, at least with respect to hsp70 mRNA because, during a severe heat shock (42 degrees C), the unspliced form of this mRNA does not accumulate . The mycelial to yeast differentiation will have the adaptational effect of increasing the resistance of the organism to environmental stress, which may be necessary for parasite survival in the mammalian host.

Med Mycol, 1998 Oct, 36(5), 345 - 8
Exocellular proteolytic activity of Paracoccidioides brasiliensis: cleavage of components associated with the basement membrane; Puccia R et al.; We have previously characterized an exocellular serine-thiol proteinase activity in Paracoccidioides brasiliensis, using as substrates peptides analogous of the internally quenched fluorogenic peptide Abz-MKRLTL-EDDnp . In this communication, detection of maximal proteinase activity in the culture supernatant fluids followed the abrupt increase in the medium pH, owing to the accumulation of ammonia generated by urease activity . Culture supernatant fluids collected at the peak of proteinase activity against Abz-MRKLTL-EDDnp were able to cleave components of the basal membrane of the extracellular matrix (EM), including laminin, fibronectin, collagen type IV and proteoglycans, and the proteolytic activity was selectively inhibited both by PMSF and p-HMB (sodium 7-hydroxymercuribenzoate), which are also specific inhibitors of the serine-thiol proteinase . Human collagen I, bovine fibrinogen, human immunoglobulin G, BSA or P . brasiliensis gp43 were resistant to proteolysis . The kinetics of appearance of the proteinase activity against EM substrates coincided with that of proteolysis of Abz-MKRLTL-EDDnp . Moreover, chromatographic fractions of culture supernatants containing the serine-thiol proteinase at high specific activity were also active against EM substrates . These data suggest the involvement of this enzyme activity in the degradation of the basement membrane, which is the first step for fungal tissue invasion.

Mycopathologia, 1998, 142(3), 139 - 42
An unusual presentation of paracoccidioidomycosis in an AIDS patient: a case report; dos Santos JW et al.; A case of paracoccidioidomycosis presenting as a solitary pulmonary nodular lesion in a patient with acquired immunodeficiency syndrome (AIDS) is presented . This case illustrates that restricted lung lesions can also be found and diagnosed in immunodeficient patients.

Int J Syst Bacteriol, 1999 Jan, 49 Pt 1, 277 - 82
Paracoccus carotinifaciens sp . nov., a new aerobic gram-negative astaxanthin-producing bacterium; Tsubokura A et al.; The strain E-396T, isolated from soil, was Gram-negative, aerobic, orange-pigmented, rod-shaped, motile by peritrichous flagella and astaxanthin-producing . This organism produced carotenoids, mainly astaxanthin, and did not produce bacteriochlorophyll . The ubiquinone system was Q-10 . Analysis of the 16S rRNA sequence of strain E-396T showed it to be a member of the alpha-3 subclass of the Proteobacteria, forming a cluster with the species of the genus Paracoccus . On the basis of the production of orange pigments and motility by peritrichous flagella, together with DNA-DNA reassociation data, it is concluded that the new isolate should be classified into a new species of the genus Paracoccus, Paracoccus carotinifaciens sp . nov . The type strain is E-396T (= IFO 16121T).

Med Mycol, 1998, 36 Suppl 1, 45 - 51
Stress proteins in fungal diseases; Matthews RC et al.; Heat shock proteins (hsps) are ubiquitous families of proteins, found in all organisms studied so far . They are highly conserved across the species barrier and serve fundamental functions in cell physiology . The term 'heat shock' was adopted because of the early observation of the heat-inducible nature of these proteins, although, as it is now realized that they can be induced by a variety of stressful stimuli, it is probably more appropriate to call them 'stress proteins' . The nomenclature of many hsps, for example hsp90, hsp70 and hsp60, reflects the approximate molecular mass of hsps within each of these families . For many bacterial and parasitic infections, hsps were first recognized as immunodominant antigens on immunoblots of extracts from the organism probed with immune sera, or in T-cell proliferation assays . They have now been identified in a range of fungal pathogens, again often linked to an immune response . In this symposium, we review the association of hsps with humoral immunity to candidosis and aspergillosis, cellular immunity to histoplasmosis, and the identification of hsp70 in another dimorphic fungus, Paracoccidioides brasiliensis . Finally, the crucial role of the membrane in setting the temperature of the heat shock response in yeasts is discussed.

J Clin Microbiol, 1999 Mar, 37(3), 653 - 63
Phylogenetic relationships of varieties and geographical groups of the human pathogenic fungus Histoplasma capsulatum Darling; Kasuga T et al.; The phylogeny of 46 geographically diverse Histoplasma capsulatum isolates representing the three varieties capsulatum, duboisii, and farciminosum was evaluated using partial DNA sequences of four protein coding genes . Parsimony and distance analysis of the separate genes were generally congruent and analysis of the combined data identified six clades: (i) class 1 North American H . capsulatum var . capsulatum, (ii) class 2 North American H . capsulatum var . capsulatum, (iii) Central American H . capsulatum var . capsulatum, (iv) South American H . capsulatum var . capsulatum group A, (v) South American H . capsulatum var . capsulatum group B, and (vi) H . capsulatum var . duboisii . Although the clades were generally well supported, the relationships among them were not resolved and the nearest outgroups (Blastomyces and Paracoccidioides) were too distant to unequivocally root the H . capsulatum tree . H . capsulatum var . farciminosum was found within the South American H . capsulatum var . capsulatum group A clade . With the exception of the South American H . capsulatum var . capsulatum group A clade, genetic distances within clades were an order of magnitude lower than those between clades, and each clade was supported by a number of shared derived nucleotide substitutions, leading to the conclusion that each clade was genetically isolated from the others . Under a phylogenetic species concept based on possession of multiple shared derived characters, as well as concordance of four gene genealogies, H . capsulatum could be considered to harbor six species instead of three varieties.

Nippon Ishinkin Gakkai Zasshi, 1999, 40(1), 1 - 8
{The Research Encouragement Award . Effects of sex hormones on sexual difference of experimental paracoccidioidomycosis}; Sano A et al.; Paracoccidioidomycosis is a deep mycosis in Latin America . The causative agent is Paracoccidioides brasiliensis, a thermal dimorphic fungus . The incidence of the disease is much higher in men than in women . Although one explanation is the inhibitive effect of estradiol on fungal growth, its effect on the yeast form growth of P . brasiliensis is still unclear . There is limited information on progesterone and testosterone, which shows weak or no effects . On the other hand, numerous studies on sexual differences between male and female animals have been made with experimental paracoccidioidomycosis . The conclusions however, remain unclear . The present study shows the effects of estradiol (17 beta-estradiol), progesterone and testosterone on the yeast form growth of P . brasiliensis, and the sexual difference of the susceptibility in adult BALB/c mice at the initial stages of infection with view of their estrous cycles and body weight . The inhibitive effects of estradiol, progesterone and testosterone on the yeast form growth of P . brasiliensis were examined using a brain heart infusion broth with different doses of these hormones . The inhibition by estradiol was dose-dependent within the range of physiological concentrations . While the inhibitive effect of progesterone and testosterone was very weak . The phenomenon that estradiol inhibited the yeast form growth of P . brasiliensis may be one important reason for the sexual differences of paracoccidioidomycosis . The initial stages of the yeast form cells of P . brasiliensis infection were evaluated by three inoculation routes: intravenous, intraperitoneal and intratracheal . The mice were divided into 6 groups, including male and female at proestrus, estrus, metestrus- I, metestrus- II and diestrus . Four hours after inoculation, blood samples, peritoneal lavage and pulmonary homogenates were collected and their colony forming units were examined on brain heart infusion agar plates with 1 % of dextrose and 50 mg per liter of chloramphenicol . In all inoculation routes, the clearance of the yeast cells was influenced by the estrous cycles . In particular, female mice at estrus, thought to have high blood estradiol levels, showed a marked clearance of the yeast cells from the blood, peritoneal cavity and lung . All female groups inoculated by any of the three different routes, showed much higher clearance than the male groups . These results suggest that non-specific host resistance to the yeast form cells of P . brasiliensis in females was much higher than in males . There are two explanations for the tendency of higher occurrence of paracoccidioidomycosis in men . One is the inhibitive effect of estradiol on the growth of P . brasiliensis and the other is the superior tendency of non-specific host resistance in females than in males . These two factors seem to have synergistic actions in the sexual difference of paracoccidioidomycosis.

Rev Soc Bras Med Trop, 1999 Jan-Feb, 32(1), 31 - 3
{Paracoccidioidomycosis in children in Belem, Para}; Fonseca ER et al.; During the period from January/85 to July/96, 102 cases of paracoccidioidomycosis were observed in a hospital in Belem, PA . Thirteen of these cases were children between 3 and 13 years of age, with a predominance of females (8:5) . All patients presented the disseminated subacute form of the mycosis . The high prevalence of the mycosis in children in endemic areas of the Amazon region is discussed.

Am J Trop Med Hyg, 1998 Dec, 59(6), 971 - 7
Comparative studies on the antibody repertoire produced by susceptible and resistant mice to virulent and nonvirulent Paracoccidioides brasiliensis isolates; Vaz CA et al.; The specific recognition pattern of antibodies produced by susceptible and resistant mice infected with the low virulence Paracoccidioides brasiliensis isolate (Pb265) was examined by an immunoblotting procedure and compared with that of antibodies produced by highly virulent isolate (Pb18) in infected mice . Both mouse strains produced IgG antibodies to 13 of the 16 major antigen bands, and showed a recognition pattern similar to sera from mice infected with the virulent isolate . Nevertheless, the reaction to components interposed among major bands (intermediate antigen bands of 75, 73, 68, 64, 33, 23, 22, and 12.5 kD) were detected exclusively with antibodies raised in response to the virulent P . brasiliensis isolate independent of the resistance pattern of the host . It was also demonstrated here that the most diversified repertoire of specific IgA was produced when the susceptible host and virulent fungus were associated.

Rev Soc Bras Med Trop, 1998 Nov-Dec, 31(6), 563 - 7
{Acute/subacute disseminated paracoccidioidomycoses . First case in Rio Grande do Sul}; Colares SM et al.; The first autochthonous case of acute/subacute disseminated paracoccidioidomycosis observed in a child in Rio Grande do Sul (Brazil) is reported . The disease started with widespread superficial lymphadenopathy six months before the patient was admitted to the hospital . The diagnosis was made through a cervical lymph node biopsy . The spectrum of the clinical forms of the mycosis observed in this State is commented upon.

J Cardiovasc Surg (Torino), 1998 Oct, 39(5), 573 - 6
Paracoccidioidomycotic aortitis with embolization to the lower limbs . Report of a case and review of the literature; Cherri J et al.; The authors report a clinical case of a 60-year-old Caucasian man, with two episodes of arterial embolization in the lower limbs . A microscope investigation of the emboli revealed that they originated from fungal aortitis caused by Paracoccidioides brasiliensis . A review of aortic infections showed only one similar report of this rare clinical expression of blastomycosis . The authors suggest a routine postoperative search for emboli followed by culture and histopathology.

Rev Inst Med Trop Sao Paulo, 1998 May-Jun, 40(3), 203 - 7
A case report of intraspinal paracoccidioidomycosis; do Valle AC et al.; The authors report a case of paraplegia caused by a lumbar intraspinal paracoccidioidomycosis (PCM) granuloma . Clinical neurological diagnosis of a compressive spinal cord lesion was confirmed by spinal magnetic resonance imaging (MRI) . Patient was submitted to surgery with total excision of the lesion . Histopathological analysis confirmed the diagnosis of PCM . Patient is on sulfamethoxazole/trimethoprim combined with fluconazole and is experiencing positive neurological recovery.

Rev Inst Med Trop Sao Paulo, 1998 May-Jun, 40(3), 155 - 64
Epidemiologic surveys of histoplasmin and paracoccidioidin sensitivity in Brazil; Fava S di C et al.; We report here the results obtained in epidemiologic surveys of histoplasmosis and paracoccidioidomycosis carried out in Brazil using the histoplasmin and paracoccidioidin delayed hypersensitivity skin tests . Most of these data have not been previously published in scientific journals and are now reported here in two tables respectively concerning histoplasmosis (88 surveys) and paracoccidioidomycosis (58 surveys) . The guidelines to be followed in surveys of this nature are also commented upon.

Cell Immunol, 1998 Nov 25, 190(1), 68 - 76
Pattern of immune response to GP43 from Paracoccidioides brasiliensis in susceptible and resistant mice is influenced by antigen-presenting cells; de Almeida SR et al.; Paracoccidioidomycosis (PCM), endemic in Latin America, is a progressive systemic mycosis caused by Paracoccidioides brasiliensis . The infection can evolve to different clinical forms that are associated with various degrees of suppressed cell-mediated immunity . In the murine model, A/Sn and B10.A isogenic strains of mice are known to be resistant and susceptible, respectively, to this fungal infection . Assuming that the effector immune response is a consequence of the preferential activation of either Th1 or Th2 subsets, in the present work we evaluated the importance of two antigen-presenting cells (APCs), macrophages and B cells, in the development of the immune response to P . brasiliensis . In resistant mice, purified gp43, the main antigenic component of P . brasiliensis, seems to have been preferentially presented by macrophages and stimulated Th1 lymphokine production . On the other hand, in susceptible animals gp43 was distinguishably presented by B cells, which led to stronger activation of Th2 subsets . Moreover, T cells from resistant mice responded as those from susceptible animals when stimulated by gp43 presented by APCs from susceptible mice and vice versa, indicating that there are no significant differences in the T cell repertoires from A/Sn and B10.A mice . When T cells from F1 (A/Sn x B10.A) mice were stimulated by gp43 presented by APCs from A/Sn or B10.A, impaired behavior of B10.A macrophages in activating Th1 cells and a B10.A B cell tendency to stimulate T cells that secrete higher levels of IL-10 were observed . Taken together, our results suggest that APCs may be implicated in the outcome of P . brasiliensis infection .

Rev Argent Microbiol, 1998 Jul-Sep, 30(3), 115 - 21
{Specific fungal antisera production in rabbits}; Perrotta D et al.; A method for fungic antisera production against Aspergillus fumigatus, Histoplasma capsulatum, Paracoccidioides brasiliensis and Coccidioides immitis in rabbits was evaluated . Intradermic via and antigen in the dilution used for routine tests (UD) were employed to produce positive control serum for immunodiffusion test in agar gel . A . fumigatus, H . capsulatum and C . immitis antigens were prepared as described in CDC's Procedure Manual, P . brasiliensis antigen was prepared as previously described by Pires de Camargo . All rabbits produced antibodies against the different specific antigens in the primary response peak and after each booster . The titer obtained in secondary response was similar or smaller than the primary response in all cases . However, bands of similar quality and intensity were obtained by immunoprecipitation in agar gel tests . Although the antibody titers proved to be similar, higher or lower concentration of antigen used in the primary immunization produced fewer and smeared bands, respectively . This effect was evaluated in A . fumigatus only . Specific antisera production with this method proved to be easy and yielded high quality antisera . The major advantages of this method are: a) reduced number of inoculations, b) fast and simple standardisation of the antigen needed, c) equally useful for all the fungal species used so far . Therefore we strongly recommend this method.

Cell Immunol . 1998 Nov 1;189(2):169.
Papers to appear in forthcoming issues
Roles of four iron centers in Paracoccus halodenitrificans nitric oxide reductase.
Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, 920-1192, Japan . ts0513@kenroku.kanazawa-u.ac.jp

Reactions of Paracoccus halodenitrificans nitric oxide reductase (NOR) containing four iron centers, a low spin hemec, a low spin heme b, a high spin heme b and a non-heme iron, have been studied to show the roles of each iron center . Soon after reacting the resting (oxidized) NOR with L-ascorbate, the low spin heme c and low spin heme b were reduced to a considerable extent but the high spin heme b was still in the oxidized form and was reduced slowly . When CO acted on the reduced NOR, the high spin heme b center changed to a low spin state . On the other hand, when NO acted on the resting NOR, no apparent spectral change was observed . However, when NO acted on the reduced NOR (a steady state condition, excess dithionite is present), both of the low spin centers changed to be partly in the oxidized form . A small but clear new EPR signal with g = 4.1 appeared together with some new signals at the g = 2 region soon after the action of NO on the reduced NOR . During incubation at room temperature the nitrosyl-heme signal typical of 5-coordination developed . These results suggested that both the high spin-heme b center and the non-heme iron are the reaction centers and their reductions are indispensable for the enzyme process in contrast to the reaction mechanism proposed for the P-450 type NOR(P-450nor) .

Infect Immun, 1998 Nov, 66(11), 5587 - 91
Morphological transition of Paracoccidioides brasiliensis conidia to yeast cells: in vivo inhibition in females; Aristizabal BH et al.; Clinical paracoccidioidomycosis is 13 times more common in men than in women . Estrogen inhibits the transition of mycelia or conidia (the saprophytic form of Paracoccidioides brasiliensis) to yeasts (the parasitic form) in vitro . Here, we show that, in male mice that were infected intranasally (mimicking natural infection) the transition of conidia in bronchoalveolar lavage fluids to intermediate forms and yeasts occurred over 24 to 96 h; CFU and yeasts (shown by histopathology) increased subsequently . In females, transition did not occur and infection cleared . These events in vivo are consistent with epidemiological and in vitro observations, suggesting that female hormones block transition and are responsible for resistance.

Enantiomer, 1998, 3(2), 95 - 102
Pressure and temperature dependence of enantioselective excited-state quenching of chiral Tb(III) and Eu(III) tris(pyridine-2,6-dicarboxylate) chelates by various C-type ferricytochromes; Meskers SC et al.; For mitochondrial ferricytochrome c from horse, cow and tuna and for bacterial cytochrome c-550 from Paracoccus versutus, the pressure and temperature dependence of their quenching of racemic Tb(DPA)3(3-) and Eu(DPA)3(3-) (DPA = pyridine-2,6-dicarboxylate) luminescence in aqueous solution is investigated . Of these energy transfer reactions the activation volumes (delta V#) and energies (Ea) are determined for the ranges P = 0-3 kbar and T = 15-40 degrees C . For the lambda enantiomers of Tb(DPA)3(3-) and Eu(DPA)3(3-), delta V# and Ea are almost the same for all proteins: 0.4 < or = delta V# < or = 1.1 cm3/mol and 0 < or = Ea < or = 2 kJ/mol . For the delta enantiomer, whose luminescence is quenched faster, the activation parameters vary significantly with the origin of the protein and they are different for Tb(DPA)3(3-) and Eu(DPA)3(3-) . Values for delta V#, delta range from +4.9 +/- 0.5 (Eu/horse) to +0.5 +/- 0.5 cm3/mol (Tb/tuna); Ea ranges from +8 +/- 1 (Tb/cow) to -1 +/- 1 kj/mol (Tb/tuna) . The degree of enantioselectivity in the quenching (Eq) by cytc from horse, cow, chicken and pigeon is almost the same and markedly higher than from tuna . The differing quenching characteristics of the tuna protein are ascribed to the amino acid variations near the exposed heme edge: Ile9-->Thr, Thr28-->Val and/or Phe46-->Tyr . Other variations in this region, Thr47-->Ser and Ala15-->Ser, do not affect Eq.

Med Mycol, 1998 Apr, 36(2), 75 - 9
Lysis of Paracoccidioides brasiliensis by Zygosporium geminatum; San-Blas G et al.; Zygosporium geminatum, isolated as a contaminant in a culture of the mycelial phase of Paracoccidioides brasiliensis, was lethal to the latter organism . Its lytic action was due to exocellular alpha-1,3- and beta-1,3-glucanases which degraded the P . brasiliensis cell wall . The alpha-1,3-glucanase was more active at 30 degrees C and the beta-1,3-glucanase at 23 degrees C, each having pH 6.0 as its optimum.

Med Mycol, 1998 Feb, 36(1), 37 - 42
Isolation of a Paracoccidioides brasiliensis strain from the soil of a coffee plantation in Ibiá, State of Minas Gerais, Brazil; Silva-Vergara ML et al.; Paracoccidioides brasiliensis has rarely been isolated from its habitat in rural areas . In order to investigate the hypothesis that human infection with this fungus is linked to coffee plantations (Coffea arabica), material was collected monthly over a period of 1 year from farms in the town of Ibia, State of Minas Gerais, Brazil . A total of 760 samples of soil, coffee leaves and fruits was cultured and inoculated into mice . A fungus isolated from the liver of a mouse inoculated with soil showed temperature-dependent dimorphism and in vitro mycelium and yeast phases characteristic of P . brasiliensis . Yeast cells of this fungus caused disseminated infection after intraperitoneal inoculation in Wistar rats from which the fungus was re-isolated . An antigen reacting with sera from patients with paracoccidioidomycosis was obtained from this P . brasiliensis strain; antigenic identity with strain 339 and with four other P . brasiliensis strains was detected by gel immunodiffusion . However, when the exo-antigen was submitted to SDS-PAGE, we observed low gp43 expression in this new strain, which we called Ibia . The isolation of P . brasiliensis from the soil at a coffee plantation suggests that this is one of its habitats and supports the hypothesis of acquisition of paracoccidioidomycosis during agricultural activity in these areas.

J Clin Microbiol, 1998 Nov, 36(11), 3309 - 16
Antigenemia in patients with paracoccidioidomycosis: detection of the 87-kilodalton determinant during and after antifungal therapy; Gomez BL et al.; Serological diagnosis and follow-up of paracoccidioidomycosis (PCM) patients have relied mainly on the detection of antibody responses by using techniques such as complement fixation (CF) and immunodiffusion . We recently described a novel inhibition enzyme-linked immunosorbent assay (inh-ELISA) which proved to be useful in the diagnosis of PCM via the detection of an 87-kDa determinant in patient sera (B . L . Gomez, J . I . Figueroa, A . J . Hamilton, B . Ortiz, M . A . Robledo, R . J . Hay, and A . Restrepo, J . Clin . Microbiol . 35:3278-3283, 1997) . This test has now been assessed as a means of following up PCM patients . A total of 24 PCM patients, classified according to their clinical presentation (6 with the acute form of the disease, of whom two had AIDS, 12 with the multifocal form of the disease, and 6 with the unifocal form of the disease), were studied . The four human immunodeficiency virus-negative patients with acute PCM showed a statistically significant decrease in circulating antigen levels after the start of antifungal therapy . Antigen levels in this group became negative by our criteria (</=2.3 microgram/ml) before week 20 and remained so in three of four of these patients . In contrast, the two AIDS patients who also presented with the acute form of PCM showed no statistically significant decrease in circulating antigen levels even after 68 weeks of therapy . Taken together as a group, the patients with the multifocal form showed a statistically significant decrease in antigenemia after 28 weeks of therapy . In addition, five of six patients with the unifocal form became antigen negative by week 40 . Antigen level decrease mirrored clinical cure in the majority of patients in all clinical groups; in contrast, measurement of anti-PCM antibodies via the CF test showed wide fluctuations in titers during the follow-up period . The inh-ELISA for the detection of the 87-kDa Paracoccidioides brasiliensis determinant would appear to be a valuable additional tool in the follow-up of PCM patients.

Clin Exp Immunol, 1998 Oct, 114(1), 40 - 8
Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans; Souza AR et al.; Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela . The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis . A glycoprotein of 43,000 D (gp43) is the major antigen of P . brasiliensis . Antibodies directed to this antigen are detected in the sera of all patients with PCM . Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus . As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH) . Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization . Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c . AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a . It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies . These data suggest that the immune response to P . brasiliensis can be spontaneously modulated by the idiotypic network.

Mycopathologia, 1998, 141(2), 79 - 91
Study of pulmonary experimental paracoccidioidomycosis by analysis of bronchoalveolar lavage cells: resistant vs . susceptible mice; Vilani-Moreno F et al.; Adult Swiss (susceptible) and BALB/c (non-susceptible) mice were inoculated by the intravenous route with 1 x 10(6) yeast cells of Paracoccidioides brasiliensis, strain 18 . Immunologic parameters, histopathology and features of the bronchoalveolar lavage (BAL) were evaluated at week 2, 4, 8 and 16 post-infection . The pulmonary infection was progressive in Swiss mice and regressive in Balb/c mice . The numbers of total cells, lymphocytes and polymorphonuclear neutrophils increased in BAL, as well as the percentages of giant cells, and CD4 and CD8 positive cells . The ultrastructural study of BAL cells revealed a predominance of macrophages and a frequency of 13.2% of type II pneumocytes . As the infection progressed, the number of fungal cells and spreading macrophages, as well as the stimulated release of H2O2 by macrophages, increased . The animals exhibited an exacerbation of the humoral immune response and a depression of cellular immunity during the infection . There was a good correlation between the intensity and the pattern of the pulmonary histopathology and the cellular findings in the BAL . The present model reproduces some anatomoclinical patterns of the human disease and shows that BAL may be a useful tool in monitoring the pulmonary infection caused by P . brasiliensis.

J Immunol, 1998 Sep 15, 161(6), 3056 - 63
Treatment of Paracoccidioides brasiliensis-infected mice with a nitric oxide inhibitor prevents the failure of cell-mediated immune response; Bocca AL et al.; The activation of the nitric oxide (NO) production system and its involvement in the control of the lung fungal burden and in immunosuppression mechanisms were studied during the course of Paracoccidioides brasiliensis-infected mice . Mice that had been infected with the fungus were treated daily with a specific inhibitor of NO synthesis, N omega-nitro-L-arginine, or with buffered saline (control); NO production was assessed on the basis of spontaneous NO2- production by bronchoalveolar and peritoneal macrophages (Mphi) and of serum NO3- levels . The infection coursed with an elevation of NO3- levels . The Mphi produced NO2- and released TNF-alpha only after stimulation with LPS . In addition, the immunoproliferative responses of spleen cells that had been stimulated with the fungus Ag or with Con A were depressed . An examination of the lungs of infected animals showed a progressive increase in the size of the lesions . Treatment of the animals, which resulted in an inhibition of NO2- production by Mphi and a reduction of serum NO3- levels, caused the spontaneous release of TNF-alpha from infected animals and prevented the failure of the lymphoproliferative capacity of spleen cells . Furthermore, the treatment resulted in less pulmonary damage despite the fact that the lung fungal burden increased . It was also demonstrated that the NO donors S-nitroso-acetyl penicillamine and 3-morpholino-sydnonimine-hydrochloride were able to inhibit the growth of P . brasiliensis in vitro . These results suggest that although NO is important for the killing of the fungi, the activation of NO production in P . brasiliensis infection contributes to the occurrence of the immunosuppression observed during the course of the infection.

J Clin Microbiol, 1998 Oct, 36(10), 2918 - 25
Molecular genetic variation in Emmonsia crescens and Emmonsia parva, etiologic agents of adiaspiromycosis, and their phylogenetic relationship to Blastomyces dermatitidis (Ajellomyces dermatitidis) and other systemic fungal pathogens; Peterson SW et al.; Emmonsia crescens, an agent of adiaspiromycosis, Blastomyces dermatitidis, the agent of blastomycosis, and Histoplasma capsulatum, the agent of histoplasmosis, are known to form meiotic (sexual) stages in the ascomycete genus Ajellomyces (Onygenaceae, Onygenales), but no sexual stage is known for E . parva, the type species of the genus Emmonsia . To evaluate relationships among members of the putative Ajellomyces clade, large-subunit ribosomal and internal transcribed spacer region DNA sequences were determined from PCR-amplified DNA fragments . Sequences were analyzed phylogenetically to evaluate the genetic variation within the genus Emmonsia and evolutionary relationships to other taxa . E . crescens and E . parva are distinct species . E . crescens isolates are placed into two groups that correlate with their continents of origin . Considerable variation occurred among isolates previously classified as E . parva . Most isolates are placed into two closely related groups, but the remaining isolates, including some from human sources, are phylogenetically distinct and represent undescribed species . Strains of B . dermatitidis are a sister species of E . parva . Paracoccidioides brasiliensis and Histoplasma capsulatum are ancestral to most Emmonsia isolates, and P . brasiliensis, which has no known teleomorph, falls within the Ajellomyces clade.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 543 - 8
Paracoccus marcusii sp . nov., an orange gram-negative coccus; Harker M et al.; Phenotypic, chemotaxonomic and 16S rDNA sequence analysis of an orange Gram-negative coccus that appeared as a contaminant on a nutrient agar plate delineated a new species of the genus Paracoccus . Phenotypic features of the strain that differ from all or most of the previously described Paracoccus species include its bright orange colour, caused by the synthesis of large amounts of carotenoids (mainly astaxanthin), and its inability to use nitrate as an electron acceptor in respiration . The name Paracoccus marcusii is proposed for this organism . The type strain is DSM 11574T.

Rev Inst Med Trop Sao Paulo, 1998 Jan-Feb, 40(1), 15 - 8
Periodontal aspects of the juvenile form of paracoccidioidomycosis; Migliari DA et al.; Three cases of the juvenile form of paracoccidioidomycosis are reported . Emphasis has been given to the oral manifestations, particularly the periodontal involvement . The main periodontal findings were: generalized and progressive alveolar bone destruction leading to gingival recession with exposure of the tooth roots, and spontaneous tooth losses . The gingival mucosa was predominantly smooth, erythematous and slightly swollen . These aspects, although rare, may be the earliest signs of the disease and sometimes its only manifestations.

Rev Inst Med Trop Sao Paulo, 1998 Jan-Feb, 40(1), 11 - 3
Paracoccidioidomycosis in children in the state of Rio de Janeiro (Brazil) . Geographic distribution and the study of a "reservarea"; Goncalves AJ et al.; Thirty six cases of acute disseminated paracoccidioidomycosis in 3 to 12 year-old children, natives of the state of Rio de Janeiro, were seen in the period 1981-1996 . All patients were residents in the rural region of 15 counties, scattered on the Southwestern part of this state . The rural region of two neighboring counties, where 16 cases (44.4%) occurred, was visited . It exhibited the environmental conditions that are considered favorable to the survival of P . brasiliensis . The most important of these conditions, abundant watercourses and autochthonous forest, are distributed on well defined and limited areas, in which the dwellings are also localized . Probably, a careful epidemiological study of forthcoming cases of the disease in children may facilitate the search for the micro-niche of the fungus.

Braz J Med Biol Res, 1998 May, 31(5), 615 - 23
Cytokines produced by susceptible and resistant mice in the course of Paracoccidioides brasiliensis infection; Calich VL et al.; Paracoccidioidomycosis (PCM) is the most prevalent deep mycosis in Latin America and presents a wide spectrum of clinical manifestations . We established a genetically controlled murine model of PCM, where A/Sn mice develop an infection which mimics the benign disease (immune responses which favor cellular immunity) and B10.A animals present the progressive disseminated form of PCM (preferential activation of B cells and impairment of cellular immune responses) . To understand the immunoregulatory phenomena associated with resistance and susceptibility in experimental PCM, A/Sn and B10.A mice were studied regarding antigen-elicited secretion of monokines (TNF-alpha and TGF-beta) and type-1 (IL-2 and IFN-gamma) and type-2 (IL-4,5,10) cytokines . Total lymph node cells from resistant mice infected i.p . with P . brasiliensis produced early and sustained levels of IFN-gamma and IL-2; type-2 cytokines (IL-4 and IL-5) started to appear 8 weeks after infection . In contrast, susceptible mice produced low levels of IFN-gamma concomitant with significant levels of IL-5 and IL-10 early in the infection . In the chronic phase of the disease, susceptible animals presented a transitory secretion of IL-2, and IL-4 . In the pulmonary infection IL-4, IL-5 and IL-10 were preferentially detected in the lung cells washings of susceptible animals . After in vitro challenge with fungal antigens, normal peritoneal macrophages from B10.A mice secreted high levels of TGF-beta and low levels of TNF-alpha . In contrast, macrophages from A/Sn animals released high levels of TNF-alpha associated with a small production of TGF-beta . The in vivo depletion of IFN-gamma not only abrogated the resistance of A/Sn mice but also diminished the relative resistance of B10.A animals . The in vivo depletion of IL-4 did not alter the disease outcome, whereas administration of rIL-12 significantly enhanced resistance in susceptible animals . Taken together, these results suggest that an early secretion of high levels of TNF-alpha and IFN-gamma followed by a sustained secretion of IL-2 and IFN-gamma plays a dominant role in the resistance mechanisms to P . brasiliensis infection . In contrast, an early and ephemeral secretion of low levels of TNF-alpha and IFN-gamma associated with production of IL-5, IL-10 and TGF-beta characterizes the progressive disease of susceptible animals.

Am J Trop Med Hyg, 1998 Jul, 59(1), 66 - 72
Localization of eosinophil granule major basic protein in paracoccidioidomycosis lesions; Wagner JM et al.; Paracoccidioidomycosis is a chronic granulomatous disease caused by the fungus Paracoccidioides brasiliensis . Although eosinophils have long been associated with the immune defense against helminths, the role of eosinophils in the immune response to fungal diseases is not as well studied . The eosinophil granule major basic protein is toxic to helminths and mammalian cells in vitro, and its release has been used as a marker of eosinophil localization and degranulation . To determine whether eosinophil infiltration and degranulation, as evidenced by the deposition of major basic protein, occur in lesions of P . brasiliensis, we used an immunofluorescence technique to localize the P . brasiliensis organisms and eosinophils and major basic protein . Initially, all tissues were stained with polyclonal antibody to major basic protein; subsequently, colocalization of major basic protein and P . brasiliensis by double staining with mouse and rabbit antibodies, respectively, was performed . Nine biopsy tissues from seven patients were analyzed . All nine biopsies showed infiltration of intact eosinophils using both the monoclonal and the polyclonal anti-major basic protein antibodies, along with the presence of P . brasiliensis . Furthermore, using the polyclonal anti-major basic protein antibody, nine of nine tissues showed extracellular major basic protein deposition (granular or diffuse fluorescence staining outside of intact eosinophils) . The double staining procedure using the anti-major basic protein monoclonal antibody showed extracellular deposition in five of eight biopsies; in these five biopsies, approximately 60% of the areas containing P . brasiliensis had extracellular major basic protein deposited on the organisms . These observations support the hypothesis that the eosinophil, through toxic granule proteins such as major basic protein, participates in the pathophysiology of paracoccidioidomycosis.

Curr Microbiol, 1998 Aug, 37(2), 141 - 3
Cytosolic neutral proteinases of Paracoccidioides brasiliensis; San-Blas G et al.; Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis . Preparations from the mycelial phase were more active in vitro than those from the yeast cells . Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45 degrees C . Gelatin-SDS-PAGE electrophoresis separated several bands (58-112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations . Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases . Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases . The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process.

Rev Inst Med Trop Sao Paulo, 1997 Sep-Oct, 39(5), 261 - 9
Serological follow-up of patients with paracoccidioidomycosis treated with itraconazole using Dot-blot, ELISA and western-blot; Martins R et al.; Twenty-seven mycologically proven cases of paracoccidioidomycosis (PCM) were treated with itraconazole (100-200 mg/day in month 1 and 100 mg/day until month 6-8) and evaluated clinically and serologically, up to 3.5 years post-therapy, using Dot-blot and ELISA for measuring the titers of IgG, IgA and IgM anti-P.brasiliensis antibodies and Western-blot for determining IgG, IgA and IgM antibodies against the antigen components of the fungus . Before treatment, 81.5% (Dot-blot) and 84% (ELISA) of the patients presented elevated IgG anti-P.brasiliensis antibody titers which dropped slightly with treatment . On the other hand, the percentages of pre-treatment high-titered sera for IgA and IgM anti-P.brasiliensis were lower (51.9% and 51.8%: Dot-blot; 16.5 and 36%: ELISA, respectively) but the titers tended to become negative more frequently with treatment . Prior to treatment, the percentages of positivity for IgG, IgA and IgM anti-P.brasiliensis antibodies in Western-blot were 96%, 20.8% and 41.6%, respectively . Antigens with molecular weights varying from 16-78 kDa, from 21-76 kDa and from 27-78 kDa were reactive for IgG, IgA and IgM antibodies, respectively . The most frequently reactive antigenic components had molecular weights of 27, 33 and 43 kDa for IgG, and 70 for IgA and IgM antibodies . During the period of study, the patients responded well to treatment . The present data confirm the diversity and complexity of the humoral response in PCM, and the importance of utilizing different serological tests to detect IgG, IgA and IgM anti-P . brasiliensis antibodies.

Rev Inst Med Trop Sao Paulo, 1997 Sep-Oct, 39(5), 257 - 60
Histoplasmin reaction . Comparison of a polysaccharide antigen to the filtrate antigen; Fava S di C et al.; This work was planned by taking into account all the knowledge accumulated from the immunological study of paracoccidioidomycosis . It aimed at comparing a polysaccharide antigen from Histoplasma capsulatum to a classic histoplasmin with the help of intradermal tests of delayed type of hypersensitivity . Tests were applied to 115 individuals in Santo Amaro, a town in the State of Sao Paulo . Positive results using classic histoplasmin were obtained in 46.0% cases whereas positive results using the polysaccharide antigen at its highest concentration were obtained in 51.30% cases . The major conclusion in this investigation is that it is possible to use the polysaccharide antigen as histoplasmin instead of the filtrate antigen.

Biochemistry, 1998 Jun 16, 37(24), 8764 - 75
Structure elucidation of sphingolipids from the mycopathogen Paracoccidioides brasiliensis: an immunodominant beta-galactofuranose residue is carried by a novel glycosylinositol phosphorylceramide antigen; Levery SB et al.; Two major acidic glycolipid components (Pb-1 and Pb-2) have been extracted from the mycopathogen Paracoccidioides brasiliensis, a thermally dimorphic fungus endemic to rural areas of South and Central America . Sera of all patients exhibiting paracoccidioidomycosis were found to be reactive with Pb-1, but not with Pb-2; no reactivity was observed with sera of healthy patients or those with histoplasmosis {Toledo, M . S., Suzuki, S., Straus, A . H., and Takahashi, H . K . (1995) J . Med . Vet . Mycol . 33, 247-251} . We report here the complete structure elucidation of both P . brasiliensis glycolipids using monosaccharide, fatty acid, sphingosine, and inositol component analysis by GC-MS; 1H- and 31P NMR spectroscopy; ESI-MS and -MS/CID-MS, linkage analysis, and exoglycosidase digestion . The compounds were found to be glycosylinositol phosphorylceramides (GIPCs) with the following structures: Pb-2, Manpalpha1-->3Manpalpha1-->2Ins1-P-1Cer; and Pb-1, Manpalpha1-->3{Galfbeta1-->6}Manpalpha1-->2Ins1- P-1Cer . The serologically nonreactive Pb-2 appears to be a biosynthetic intermediate between mannosylinositol phosphorylceramide (MIPC), which is widely distributed among fungi, and the antigenic Pb-1 . Pb-1 is a novel glycosphingolipid, similar to a triglycosyl IPC (Hc-VI) reported from Histoplasma capsulatum {Barr, K., Laine, R.A, and Lester, R . L . (1984) Biochemistry 23, 5589-5596}, but differing in the anomeric configuration of the terminal Galf1-->6 residue, which is immunodominant . The significance of these structures as serological and taxonomic markers, as well as their potential utility as targets for immunodiagnostic agents, is discussed.

J Clin Microbiol, 1998 Jun, 36(6), 1733 - 6
Geographic discrimination of Paracoccidioides brasiliensis strains by randomly amplified polymorphic DNA analysis; Calcagno AM et al.; Randomly amplified polymorphic DNA (RAPD) analysis of 33 Paracoccidioides brasiliensis strains from Argentina, Brazil, Colombia, Peru, and Venezuela produced reproducible amplification products which were sufficiently polymorphic to allow differentiation of the strains . Types generated with five primers (OPG 03, OPG 05, OPG 14, OPG 16, and OPG 18) resulted in a high discriminatory index (0.956) . The discriminatory index was slightly reduced (0.940) when only two primers (OPG 3 and OPG 14) were used . A dendrogram based on these results showed a high degree of similarity among the strains, and genetic differences were expressed in clusters related to geographical regions but not to pathological features of the disease . With a few exceptions, strains were sorted into five groups by geographical origin as follows: group I, Venezuelan strains; group II, Brazilian strains; group III, Peruvian strains; group IV, Colombian strains; and group V, Argentinian strains . The group containing the most disparate strains was group V (discriminatory index, 0.633); the discriminatory index for the other four groups was 0.824 . The use of primer OPG 18 by itself was sufficient to discriminate species specificity, and the use of primer OPG 14 by itself was sufficient to discriminate among the geographical locations of the strains in the sample . This method may be helpful for epidemiological studies of P . brasiliensis.

J Clin Microbiol, 1998 Jun, 36(6), 1723 - 8
Detection of circulating Paracoccidioides brasiliensis antigen in urine of paracoccidioidomycosis patients before and during treatment; Salina MA et al.; For the diagnosis and follow-up of paracoccidioidomycosis patients undergoing therapy, we evaluated two methods (immunoblotting and competition enzyme immunoassay) for the detection of circulating antigen in urine samples . A complex pattern of reactivity was observed in the immunoblot test . Bands of 70 and 43 kDa were detected more often in urine samples from patients before treatment . The immunoblot method detected gp43 and gp70 separately or concurrently in 11 (91.7%) of 12 patients, whereas the competition enzyme immunoassay detected antigenuria in 9 (75%) of 12 patients . Both tests appeared to be highly specific (100%), considering that neither fraction detectable by immunoblotting was present in urine samples from the control group . gp43 remained present in the urine samples collected during the treatment period, with a significant decrease in reactivity in samples collected during clinical recovery and increased reactivity in samples collected during relapses . Reactivity of some bands was also detected in urine specimens from patients with "apparent cure." The detection of Paracoccidioides brasiliensis antigens in urine appears to be a promising method for diagnosing infection, for evaluating the efficacy of treatment, and for detecting relapse.

Biochemistry, 1998 May 19, 37(20), 7378 - 89
Understanding the electronic properties of the CuA site from the soluble domain of cytochrome c oxidase through paramagnetic 1H NMR; Salgado J et al.; The soluble domain of the subunit II of cytochrome c oxidase from Paracoccus versutus was cloned, expressed, and studied by 1H NMR at 600 MHz . The properties of the redox-active dinuclear CuA site in the paramagnetic mixed-valence Cu(I)-Cu(II) state were investigated in detail . A group of relatively sharp signals found between 30 and 15 ppm in the 1H NMR spectrum correspond to the imidazole protons of the coordinated histidines (H181 and H224) . A second group of broader and farther shifted signals between 50 and 300 ppm are assigned to Hbeta protons of the bridging cysteines (C216 and C220); the protons from the weak M227 and E218 ligands do not shift outside of the diamagnetic envelope . About 40% of the total spin density appears delocalized over the cysteine-bridging ligands while a much smaller amount is delocalized on the two ligand histidines . The latter have similar spin density distributions . Analysis of the pattern of the hyperfine shifts of the Cys H beta protons shows that the ground state bears 2B3u character, in which the sulfur lobes in the singly occupied molecular orbital are aligned with the Cu-Cu axis . Analysis of the temperature dependence of the shifts of the Cys H beta signals leads to the conclusion that the 2B2u excited state is thermally accessible at room temperature (Delta E approximately kT).

Mycopathologia, 1997, 140(1), 29 - 33
Palpebral paracoccidioidomycosis; Burnier SV et al.; This paper describes two cases of eyelid paracoccidioidomycosis (South American blastomycosis) in which it was the first signal of the disease . In both cases the first clinical diagnosis made was not a fungal infection, but a neoplastic disease that was not confirmed by the pathology study . In the first patient we suspected a primary infection on the eyelid, because there was no other systemic signs of the disease, and in the second patient we noted a very advanced pulmonary lesions caused by the Paracoccidioides brasiliensis . We believe that, in endemic areas, the histopathological study should be made before every excisional procedures to avoid unnecessary palpebral mutilation.

Mycopathologia, 1997, 140(1), 19 - 27
Interactions of F1 fractions from different strains of Paracoccidioides brasiliensis with human complement and with human neutrophils; Crott LS et al.; The aim of our study was to investigate differences that might exist in the activation of the human complement system by F1 fractions from four different isolates of P . brasiliensis . Isolates HC and 18 (virulent), 265 (low virulence), and 9 (intermediate virulence, attenuated) were used; before the experiments, the virulence of isolates HC and 18 was recovered by in vivo passage in guinea pigs . The four isolates of the fungus were processed for purification of F1 fractions and the activation of the human complement system was studied by a kinetic method of hemolytic activity measurement . The incubation of F1 fractions in normal human serum resulted in different degrees of inhibition of the classical and alternative pathways . The F1 fraction from the low virulence isolate was more efficient than the F1 fraction from the virulent isolates (HC and 18) . Previous absorption of sera with F1 fractions completely abolished classical pathway activation . Using zymosan, instead of F1, in the absorption process caused the same phenomenon, suggesting that natural or nonspecific antibodies are responsible for the classical pathway activation . The alternative pathway activation did not depend on these antibodies, but was enhanced by their presence . On the other hand, F1 fractions from virulent isolates were more active in the stimulation of neutrophil chemiluminescence compared with the F1 fraction from the low virulence isolate . Whole P . brasiliensis yeast cells (WYC) from two distinct strains, 18 and 265, showed the same patterns of response of those observed with the F1 fractions in the functions tested . These differences in the behavior of the F1 fractions as well as WYC in relation to human complement activation and consequently to neutrophil stimulation may correlate with the virulence of individual isolates and may contribute to the understanding of the inflammatory response generation and maintenance processes in paracoccidioidomycosis.

Am J Trop Med Hyg, 1998 Apr, 58(4), 505 - 12
Isolation of Paracoccidioides brasiliensis from armadillos (Dasypus noveminctus) captured in an endemic area of paracoccidioidomycosis; Bagagli E et al.; Paracoccidioides brasiliensis, the causative agent of paracoccidioidomycosis (PCM), was first isolated from armadillos from the Amazonian region where the mycosis is uncommon . In the present study, we report on the high incidence of PCM infection in armadillos from a hyperendemic region of the disease . Four nine-banded armadillos (Dasypus novemcinctus) were captured in the endemic area of Botucatu, Sao Paulo, Brazil, killed by manual cervical dislocation and autopsied under sterile conditions . Fragments of lung, spleen, liver, and mesenteric lymph nodes were processed for histology, cultured on Mycosel agar at 37 degrees C, and homogenized for inoculation into the testis and peritoneum of hamsters . The animals were killed from week 6 to week 20 postinoculation and fragments of liver, lung, spleen, testis, and lymph nodes were cultured on brain heart infusion agar at 37 degrees C . Paracoccidioides brasiliensis was isolated from three armadillos both by direct organ culture and from the liver, spleen, lung, and mesenteric lymph nodes of hamsters . In addition, one positive armadillo presented histologically proven PCM disease in a mesenteric lymph node . The three armadillos isolates (Pb-A1, Pb-A2, and Pb-A4) presented thermodependent dimorphism, urease activity, and casein assimilation, showed amplification of the gp43 gene, and were highly virulent in intratesticularly inoculated hamsters . The isolates expressed the gp43 glycoprotein, the immunodominant antigen of the fungus, and reacted with a pool of sera from PCM patients . Taken together, the present data confirm that armadillos are a natural reservoir of P . brasiliensis and demonstrate that the animal is a sylvan host to the fungus.

Am J Trop Med Hyg, 1998 Apr, 58(4), 424 - 30
Experimental pulmonary fibrosis induced by Paracoccidioides brasiliensis conidia: measurement of local host responses; Franco L et al.; Pulmonary fibrosis was induced following inoculation of Paracoccidioides brasiliensis conidia intranasally in BALB/c mice . Fibrosis was associated with formation of granulomas, increase in lung hydroxyproline, and sustained increases in tissue tumor necrosis factor-alpha and transforming growth factor-beta . This study suggests a role for these cytokines in generation of pulmonary fibrosis associated with chronic granulomatous infectious diseases.

Rev Invest Clin, 1997 Nov-Dec, 49(6), 501 - 5
{Production of fungal antigens from local strains for the immunodiagnosis of mycoses in Mexico}; Toriello C et al.; Fungal antigens with good reactivity and specificity are essential for the immunodiagnosis of systemic mycoses . We give here data on crude and purified antigens of Histoplasma capsulatum, Coccidioidis immitis and Paracoccidioides brasiliensis from local strains and which are the more prevalent in Mexico . The crude antigens had good reactivity in precipitating and skin testing whereas the purified antigens (DPPC: deproteinized polysaccharide protein complex) had a higher specificity in more sensitive tests such as ELISA and complement fixation . Our efficiency analysis showed that the crude antigens are best for epidemiologic studies due to their low cost, easy handling and fast detection . The purified ones are suited to establish with more precision the diagnosis of systemic mycoses.

Acta Microbiol Pol, 1997, 46(4), 387 - 92
Construction of mobilizable cloning vectors derived from pBGS18 and their application for analysis of replicator region of a pTAV202 mini-derivative of Paracoccus versutus pTAV1 plasmid; Bartosik D et al.; Two mobilizable cloning vectors, designated pABW1 and pAWB2, were constructed basing on the E . coli vector pBGS18 and oriT originating from RK2 . In pABW2 the kanamycin resistance gene was replaced by a novel tetracycline resistance cassette derived from Tn1721 . Both vectors, specific for E . coli, allow to perform the cloning steps in E . coli and then to efficiently transfer the constructs by conjugation to the host of choice . A vector which cannot propagate in the given host can be applied for identification of the host specific plasmid replicator regions . With the use of pABW2 we defined the minimal replicator region of pTAV202-a mini-derivative of the large pTAV1 plasmid of P . versutus . We also proved that RepC' encoded on this fragment is the principal initiator replication protein and that oriV is located along its coding sequence.

J Clin Microbiol, 1998 Mar, 36(3), 742 - 7
Electrophoretic karyotypes and genome sizing of the pathogenic fungus Paracoccidioides brasiliensis; Cano MI et al.; Here we present the karyotype analysis and genome sizing of Paracoccidioides brasiliensis, a pathogen refractory to conventional genetic analysis . We have established pulsed-field gel electrophoresis (PFGE) conditions to resolve the high-molecular-weight chromosomal bands of two clinical isolates of P . brasiliensis . Both isolates showed four megabase-sized bands, ranging from 2.0 to 10.0 Mbp . Significant differences in chromosome sizes and in the chromosomal location of genes for the gp43 antigen and chitin synthase were found . Different technical approaches were employed to estimate the DNA content and to define the ploidy of P . brasiliensis . An estimated genome size in the range of 45.7 to 60.9 Mbp was provided by the analysis of data generated by measuring the amplitude of fluorescence intensity of DAPI (4',6-diamidino-2-phenylindole)-stained nuclei (by confocal microscopy) . The nuclear genome size estimated by confocal microscopy is twice that estimated by the average sum of the molecular weight of chromosome-sized DNA molecules by PFGE, suggesting that each separated P . brasiliensis chromosomal band is diploid.

Am J Trop Med Hyg, 1998 Feb, 58(2), 152 - 3
Short report: use of the polymerase chain reaction to detect Paracoccidioides brasiliensis in murine paracoccidioidomycosis; Goldani LZ et al.; The polymerase chain reaction (PCR) was used to detect the presence of Paracoccidioides brasiliensis in a murine model of disseminated paracoccidioidomycosis . Using a previously identified P . brasiliensis-specific DNA sequence, P . brasiliensis DNA was detected in serum of five experimentally infected mice . The PCR method was able to detect as little as 10 pg of P . brasiliensis DNA in serum, and it was more sensitive than blood culture isolation (five of five were PCR positive versus two of five blood culture positive) . There were no amplified fragments in serum from three noninfected control mice . Lung colony counts were similar in all infected mice and reflected a similar degree of P . brasiliensis infection at the time the samples were drawn . The relatively short processing time for the PCR, when compared with culture, its sensitivity, and the possibility of using serum samples for analysis, are important factors favoring this method for the diagnosis of paracoccidioidomycosis . Future studies should include the detection of P . brasiliensis in patients with different clinical forms of paracoccidioidomycosis.

Braz J Med Biol Res, 1997 Oct, 30(10), 1175 - 9
Lung tissue mechanics in the early stages of induced paracoccidioidomycosis in rats; Shikanai-Yasuda MA et al.; Pulmonary dysfunction represents the most important cause of death in patients with paracoccidioidomycosis (PBM) . In order to investigate the functional changes of the lungs in the early stages of PBM, a model of benign disease was developed by intratracheal challenge of 12-week old isogenic Wistar rats with 1 x 10(6) yeast forms of Paracoccidioides brasiliensis . Animals were studied 30 and 60 days after infection, when fully developed granulomas were demonstrable in the lungs . Measurements of airway resistance, lung elastance and tissue hysteresis were made during sinusoidal deformations (100 breaths/min, tidal volume = 2 ml) with direct measurement of alveolar pressure using the alveolar capsule technique . Infection caused a significant increase in hysteresis (infected: 1.69, N = 13; control: 1.13, N = 12, P = 0.024, ANOVA), with no alterations in airway resistance or lung elastance . Histopathological analysis revealed the presence of fully developed granulomas located in the axial compartment of the lung interstitial space . These results suggest that alterations of tissue mechanics represent an early event in experimental PBM.

Microbiology, 1998 Feb, 144 ( Pt 2), 309 - 14
Anionogenic groups and surface sialoglycoconjugate structures of yeast forms of the human pathogen Paracoccidioides brasiliensis; Soares RM et al.; The surface anionogenic groups and sialoglycoconjugate structures of Paracoccidioides brasiliensis yeast forms were analysed by cell microelectrophoresis, binding assays with lectins and viral particles, ultrastructural cytochemistry, enzymic digestion and flow cytofluorimetry . P . brasiliensis yeast forms, particularly the budding primordia, reacted strongly with cationized ferritin . Binding assays showed that the reaction with sialic-acid-specific Limax flavus lectin (LFA) was distributed over the entire P . brasiliensis cell wall . Treatment of yeast forms with neuraminidase significantly reduced their negative surface charge and LFA labelling, which suggests that sialic acid residues are major anionogenic groups exposed on the P . brasiliensis surface . Furthermore, after neuraminidase treatment, labelling with Arachis hypogaea (peanut) agglutinin increased due to unmasking of subterminal beta-D-galactopyranosyl residues . The sialic acid linkages to galactose are alpha 2,6 and alpha 2,3 as assessed, respectively, by fungal attachment to M1/5 and M1/5 HS8 strains of influenza A virus and binding of Sambucus niger and Maackia amurensis agglutinins . The alpha 2,6 linkage clearly predominated in both experiments . Flow cytofluorimetry analysis revealed the heterogenicity of P . brasiliensis yeast cell populations, which comprised young and mature budding yeasts . Both express binding sites to LFA and Limulus polyphemus agglutinin.

Yeast, 1998 Jan 30, 14(2), 181 - 7
Molecular cloning and sequencing of a chitin synthase gene (CHS2) of Paracoccidioides brasiliensis; Nino-Vega GA et al.; The nucleotide sequence of a chitin synthase gene (CHS2) of the dimorphic fungal human pathogen Paracoccidioides brasiliensis has been determined . The deduced amino acid sequence of Chs2p consists of 1043 residues and is highly homologous to other class II fungal chitin synthases . Computational structural analyses suggest very high similarity to other fungal chitin synthases with a highly variable region at the cytosolic amino-terminal region which may be related to its possible zymogenic nature, and the putative catalytic region close to seven membrane-spanning regions at the carboxyl terminus.

Mycoses, 1997 Dec, 40(9-10), 343 - 4
Paracoccidioidomycosis in a boy infected with HIV; Cimerman S et al.; Paracoccidioidomycosis has been rarely reported among HIV patients, despite being an endemic mycosis in Latin America . The present report illustrates a case of PM in a teenager infected with HIV . Clinicians must be aware that this mycosis may occur in young HIV infected patients who live in endemic areas.

Mycoses, 1997 Dec, 40(11-12), 415 - 8
Paracoccidioidomycosis: an uncommon localization in the scrotum; Tomimori-Yamashita J et al.; Paracoccidioidomycosis is a systemic fungal infection caused by the thermally dimorphic fungus Paracoccidioides brasiliensis . The authors present a case of a 49-year-old man who developed verrucous lesions on nasal mucosa and on genital localization (inguinal and scrotal), without signs of systemic disease . Direct mycological examination, culture on Sabouraud glucose medium, and biopsy were positive for P . brasiliensis . The interest of this case is the genital localization which is uncommon in this infection . Moreover, there are few cases described in the literature.

Rev Inst Med Trop Sao Paulo, 1997 May-Jun, 39(3), 141 - 4
Paracoccidioides cerebriformis Moore, 1935 . Mycologic and immunochemical study; Lacaz Cda S et al.; The present study concern on mycologic and immunochemical data obtained from two samples of a fungus considered as belonging to the species Paracoccidioides cerebriformis described by Moore in 1935, and maintained since then on Sabouraud's agar in the mycology collection of the Instituto de Medicina Tropical de Sao Paulo . After 60 years, the samples exhibited the same characteristics described by MOORE (1935) . However, experimental lesions did not resulted in guinea-pigs inoculated intratesticularly . The dominant antigen in Paracoccidioides brasiliensis, 43 kDa glicoprotein (gp43), could not be demonstrated by SDS PAGE and Western blotting . Immunoelectrophoresis did not demonstrated the E arch of cathodic migration using a policlonal anti gp43 serum . According to these findings, it is concluded that the fungus described by MOORE (1935) as P . cerebriformis does not belong to the genus Paracoccidioides . Paracoccidioidomycosis should therefore be considered as resulting from infection by a single species, Paracoccidioides brasiliensis (Splendore, 1912) as asserted by ALMEIDA (1930) . Further studies, through molecular biology methods, could identify the mentioned fungus.

Rev Soc Bras Med Trop, 1997 Sep-Oct, 30(5), 393 - 5
{Monoarthritis of the knee as an isolated manifestation of paracoccidioidomycosis}; Silvestre MT et al.; Osteoarthritis in paracoccidioidomycosis has been rarely reported . The present case describes a 36-"year"-old woman, with chronic monoarthritis in the knee lasting 4 years . The diagnosis was achieved only after synovial biopsy, by anatomopathological examination showing granulomatous reaction with a large number of the characteristic "pilot wheel"Paracoccidioides brasiliensis yeast cells . Specific therapy, initially with ketoconazole and followed by cotrimoxazole led to complete functional recovery of the compromised joint . No other affected site was detected by various propaedeutic methods, including computed axial tomography of the thorax and abdomen . The authors emphasize the rarity of the case and discuss its possible pathophysiology.

Infect Immun, 1998 Feb, 66(2), 800 - 6
Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis; Cano LE et al.; We have developed a murine model of pulmonary infection by Paracoccidioides brasiliensis in which resistance was associated with immunological activities governed by gamma interferon (IFN-gamma) . To better characterize this model, we measured type 1 and type 2 cytokines in the lungs and investigated the effect of endogenous IFN-gamma depletion by monoclonal antibodies in the course of infection of susceptible (B10.A) and resistant (A/Sn) mice . At weeks 4 and 8 after infection, lungs from susceptible animals presented levels of IFN-gamma, interleukin-4 (IL-4), IL-5, and IL-10 higher than those in resistant mice . In both mouse strains, neutralization of endogenous IFN-gamma induced exacerbation of the pulmonary infection, earlier fungal dissemination to the liver and spleen, impairment of the specific cellular immune response resulting in significantly lower delayed-type hypersensitivity reactions, and increased levels of immunoglobulin G1 (IgG1)- and IgG2b-specific antibodies . Histopathological analysis demonstrated that depletion of IFN-gamma changes the focal granulomatous lesions found in the lungs of B10.A and A/Sn mice into coalescent granulomata which destroy the pulmonary architecture . These results suggest that irrespective of the mouse strain, IFN-gamma plays a protective role and that this cytokine is one major mediator of resistance against P . brasiliensis infection in mice.

Infect Immun, 1998 Feb, 66(2), 786 - 93
Mapping of the T-cell epitope in the major 43-kilodalton glycoprotein of Paracoccidioides brasiliensis which induces a Th-1 response protective against fungal infection in BALB/c mice; Taborda CP et al.; The 43-kDa glycoprotein of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis, the prevalent systemic mycosis of Latin America . Apart from eliciting high antibody titers, gp43 is also immunodominant in delayed-type hypersensitivity reactions in infected animals and humans . The cellular immune response in mice to gp43 administered in complete Freund's adjuvant involves CD4+ Th-1 lymphocytes, secreting gamma interferon (IFN-gamma) and interleukin 2 (IL-2) but not IL-4 and IL-10 . The T-cell epitope of this antigen was mapped to a 15-amino-acid peptide (P10) based on lymphoproliferations with primed cells from three different haplotypes and on a computer-assisted protein analysis . The structural requirements of the T-cell epitope were determined by assaying a series of P10 analogous and truncated peptides . Only 12-mer or longer sequences were active, confirming presentation by major histocompatibility complex II . The HTLAIR inner core of P10 is the essential domain of the epitope, with various flanking regions possible . Immunization of mice with both gp43 and P10 led to vigorous protection against intratracheal challenge by virulent P . brasiliensis, with a >200-fold decrease in lung CFU and halting of dissemination to the spleen and liver . The protective effect of P10 is mainly attributed to an IFN-gamma-mediated cellular immune response . Unlike gp43, which induces an antibody response compatible with both Th-1 and Th-2 activation in infected BALB/c mice, P10 does not induce a humoral response . Protection by gp43 and P10 was characterized by a few well-demarcated lung granulomas with numerous nonviable yeast forms or resolved lesions with no detectable fungal cells.

Mycopathologia, 1997, 138(2), 65 - 9
Monocyte adherence to Paracoccidioides brasiliensis, zymosan-C3b and erythrocyte-hemolysin in patients with paracoccidioidomycosis; Shikanai-Yasuda MA et al.; Independent and dependent (C3b/Fc receptors) opsonic adherence ability of monocytes from thirty-three patients with acute or chronic paracoccidioidomycosis and from 13 healthy individuals were studied in the presence of Paracoccidioides brasiliensis (Pb), Paracoccidioides brasiliensis opsonized by patient's serum (PbPS) or normal serum (PbNS), zymosan opsonized by fresh sera from healthy donors (ZyNS) and erythrocytes opsonized by hemolysin (EA) . Statistically significant differences concerning the percentage of adhered monocytes to PbPS (number of adhered monocytes/total number of monocytes) were detected between control and chronic (active and inactive) groups . Significant differences in relationship to the mean number of PbPS (number of fungi in monocytes/total number of monocytes) were also observed between control and chronic active mycosis . Present data suggest that patients with chronic disease have more ability in the first step of phagocytic activity, considered as the main effector mechanism to control the dissemination and severity of paracoccidiodomycosis.

Mycoses, 1997 Sep, 40(3-4), 91 - 4
Localization of glycoconjugates and glycogen in yeast-like cells and protoplasts of Paracoccidioides brasiliensis; Borba Cde M et al.; The presence and localization of storage polysaccharides and of polysaccharides as cell structure constituents of Paracoccidioides brasiliensis yeast-like cells and protoplasts were studied using the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) technique . Yeast-like cells presented glycogen particles in the form of rosettes, which were mostly concentrated in regions of the cytoplasm . Cells in the budding process presented small amounts of glycogen in their matrix . The intracellular membranes and the attachment of the mother cell to the bud were clearly labelled by silver, demonstrating a large amount of glyco-conjugates . The protoplasts presented a small amount of glycogen in their cytoplasm, a reduction probably due to the enzymatic treatment to which the cells were submitted.

Mycoses, 1997 Sep, 40(3-4), 69 - 81
Molecular taxonomy and epidemiology of Blastomyces and Histoplasma species; Gueho E et al.; Cladistic analysis of partial 26S rRNA sequences was used to estimate evolutionary distances among species and varieties of the dimorphic onygenalean genera Blastomyces, Coccidioides, Emmonsia, Histoplasma and Paracoccidioides . With the exception of Coccidioides, all genera were closely related, with about 5% base differences and even less (1-2%) between Blastomyces and Emmonsia . These data were supported by a teleomorph in the same genus Ajellomyces . In a phylogenic study of a wide range of ascomycete orders and families, Coccidioides immitis was found to be closest to Aphanoascus fulvescens and Chrysosporium keratinophilum, and to have relative distances to the remaining dimorphic genera (family Onygenaceae) similar to those of the dermatophytes (family Arthrodermataceae) . The sequencing data were confirmed by genomic comparisons . All dimorphic genera had a nuclear DNA base composition in the same range of 46.6-47.3% G + C . The DNA melting curves of Blastomyces and Histoplasma strains showed irregularities that were ascribed to the presence of AT-rich stretches in satellite DNA rather than in mitochondrial DNA . Derivative profiles proved to be highly reproducible within regional populations and coincided with differences in clinical behaviour of each species . Blastomyces dermatitidis generated two kinds of curves, corresponding to the geographically distinct serotypes 1 and 2 . The African type (serotype 2) was characterized by a classical sigmoidal melting curve similar to that for all strains of Coccidioides, Emmonsia and Paracoccidioides . In contrast, the American type (serotype 1) contained satellite DNA (27% G + C) . A rRNA base difference of 1.5% was observed between geographical types, a value slightly higher than that noted between Histoplasma capsulatum and its variety farciminosum (0.9%) . All three H . capsulatum varieties presented irregularities in their DNA melting curves . The molecular data support the recognition of two of them as agents of blastomycosis and the assignment of more than one species and two varieties to the genus Emmonsia.

Mycopathologia, 1997, 138(1), 37 - 41
Antigenic similarities to Paracoccidioides brasiliensis in thermo-dependent dimorphic fungi isolated from soil in Botucatu, SP, Brazil; Sano A et al.; We compared the antigenic characteristics of two thermo-dependent dimorphic fungi isolated from soil in Botucatu, an endemic area of paracoccidioidomycosis (PCM) and Paracoccidioides brasiliensis . The soil isolates grew as cerebriform colonies at 37 degrees C (yeast form) and as cottonous colonies at 25 degrees C (mycelial form) . No pathogenicity for ddY mice or hamsters were observed . In immunodiffusion test, there were precipitation bands between the 2 soil isolates and pooled PCM patient sera . There were also common precipitation bands at 21, 50 and 58 kDa between the soil isolates antigens and PCM patient sera by Western-blotting, but no gp43 kDa band . No gene for gp 43 kDa protein was detected in the soil isolates by PCR . The fact that these isolates were obtained from an endemic area of PCM and there were some antigenic similarities between the soil isolates and P . brasiliensis in immunodiffusion test and Western-blotting may have some importance in epidemiological surveys done with paracoccidioidin as well interfering with the immune response of the exposed population.

J Med Vet Mycol, 1997 Sep-Oct, 35(5), 341 - 5
Expression of glycoprotein gp43 in stage-specific forms and during dimorphic differentiation of Paracoccidioides brasiliensis; Mattar-Filho R et al.; Expression of the 43 kDa glycoprotein (gp43) was analysed in several Paracoccidioides brasiliensis isolates . Using one- and two-dimensional analysis of crude cellular extracts, it was shown that protein expression in yeast and mycelium was dependent on the isolate analysed . In two strains, in both yeast and mycelium cells . gp43 was present, whereas expression was restricted to the yeast phase of two other strains . The clinical implications of this phase-specific gp43 expression are uncertain.

J Clin Microbiol, 1997 Dec, 35(12), 3278 - 83
Use of monoclonal antibodies in diagnosis of paracoccidioidomycosis: new strategies for detection of circulating antigens; Gomez BL et al.; The precise diagnosis of paracoccidioidomycosis, in most cases, is established by direct methods and indirect immunological tests . The latter method is reliant on the identification of the host's humoral responses, which are usually impaired or absent in patients with severe juvenile forms of the disease and in immunocompromised patients . Determining disease activity or assessing treatment responses by measuring antibody levels is difficult, since antibody titer may remain elevated or persist at stationary levels, even in the presence of clinical improvement . Consequently, there is a need for alternative tests aimed at the identification of circulating antigens . A modification of the standard hybridoma production method was used to raise a panel of murine monoclonal antibodies (MAbs) against the yeast form of Paracoccidioides brasiliensis . Of these, MAb PIB, directed against an 87-kDa determinant, was used to develop an inhibition ELISA (inh-ELISA) capable of detecting as little as 5.8 ng of circulating antigen per ml of serum . Sera from 46 patients with paracoccidioidomycosis or other mycoses and sera from healthy individuals were evaluated by the inh-ELISA; overall sensitivity was 80.4% (37 of 46 paracoccidioidomycosis patients tested positive), and specificity compared with that of normal controls from areas of endemicity was 81.4% . The inh-ELISA detected circulating antigen in 100% of patients with the acute form of paracoccidioidomycosis and in 83.3 and 60% of patients with the chronic multifocal and unifocal forms of paracoccidioidomycosis according to the patients' clinical presentation . These results indicate that the inh-ELISA with MAb PIB is effective in the detection of circulating antigen and that this test may be useful for monitoring responses to treatment and establishing disease prognoses.

Rev Inst Med Trop Sao Paulo, 1997 Jan-Feb, 39(1), 35 - 7
Reactivity of anti-gp43 antibodies from Paracoccidioides brasiliensis antiserum with extracts from cutaneous lesions of Lobo's disease . Preliminary note; Vidal MS et al.; We demonstrated through several immunochemical tests the presence of gp43 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 gp43 tissues infected by the agent of Jorge Lobo's disease is an additional evidence for classifying it in the genera Paracoccidioides, species loboi.

Eur Respir J, 1997 Oct, 10(10), 2419 - 22
c-ANCA positivity in a Belgian patient with pulmonary paracoccidioidomycosis; Stappaerts I et al.; We present the case of a 69 yr old, white male, suffering from diffuse interstitial lung disease, finally diagnosed as paracoccidioidomycosis or South American blastomycosis . During the course of his disease, antineutrophil cytoplasmic antibodies (c-ANCAs) became positive, suggesting the possibility of a Wegener's granulomatosis . Transbronchial biopsies and a video-assisted thoracoscopic lung biopsy revealed only the pulmonary yeast infection, without other co-existing pathology . During treatment with itraconazole, the patient improved clinically and functionally, and c-ANCAs became negative . Serological monitoring confirmed the diagnosis . To our knowledge, this is the first report describing positive c-ANCAs in a patient with paracoccidioidomycosis . It re-emphasizes the fact that cautious interpretation of c-ANCAs in patients without convincing clinical signs or pathological evidence of a granulomatous vasculitis is absolutely necessary . In this era of increased mobility, a thorough medical history, including documentation of travel, remains an inexpensive tool in making a diagnosis and is still the cornerstone of good medical practice.

Mycopathologia, 1997, 137(3), 129 - 36
Model of in vitro granulomatous hypersensitivity in human paracoccidioidomycosis; Franco MV et al.; With the purpose of studying the immunological components of granulomatous hypersensitivity in patients infected with Paracoccidioides brasiliensis, we used the model of in vitro granuloma formation developed for schistosomiasis studies, that correlates with in vivo granulomatous reactivity occurring around eggs trapped in organs of infected donors . In this case, granuloma formation can be determined examining cellular reactivity manifested as multiple cell layers surrounding antigen-conjugated polyacrilamide beads . Our results showed that peripheral blood mononuclear cells (PBMC) obtained from acute treated and chronic paracoccidioidomycosis patients proliferate and generate in vitro granulomas in response to P . brasiliensis antigens (PbAg) . In contrast, no proliferation or granuloma formation were observed when PBMC from acute non-treated patients were used . These studies demonstrate the feasibility of investigating granulomatous hypersensitivity in P . brasiliensis-infected patients by using an in vitro granuloma model.

Chirurg, 1997 Jun, 68(6), 633 - 7
{Endosonography control of percutaneous paracoccygeal drainage of deep pelvic abscesses after rectum resection}; Truong S et al.; Pelvic abscesses are severe complications after rectal surgery . In recent years, surgical drainage has been edged out by percutaneous drainage techniques . We report our experience with the drainage of postoperative pelvic abscesses via the paracoccygeal route controlled by endosonography . In eight patients the diagnosis of a retrorectal pelvic abscess was established by endosonography and confirmed by endosonographically controlled exploratory puncture . Drainage of the abscesses was performed via the paracoccygeal access route by trocar or Seldinger technique under permanent visual control by endoluminal sonography . Irrigation of the abscess cavity was than performed daily . In all cases drainage was successful without complications . Duration of drainage was 9-14 days with a mean of 10.8 days . In one patient there was a recurrence of the abscess because of early removal of the drainage catheter . Because of its overall availability and its good results, paracoccygeal percutaneous endosonographically controlled drainage seems to be a suitable uncomplicated method for drainage of postoperative pelvic abscesses.

Mycopathologia, 1997, 137(2), 83 - 5
Paracoccidioidoma: case record and review; dos Santos JW et al.; A case of solitary pulmonary paracoccidioidal lesion-paracoccidioidoma-is related . It is the first reported case in Brazil . The literature on spontaneously regressive lesion of paracoccidioidomycosis is commented upon.

Rev Inst Med Trop Sao Paulo, 1996 Sep-Oct, 38(5), 349 - 53
Histoplasmin and paracoccidioidin skin reactivity in infantile population of northern Argentina (1); Mangiaterra M et al.; In order to estimate ages at which etiological agents of systemic mycoses initiate infection, histoplasmin and paracoccidioidin skin tests were performed in 344 children of both sexes, between 2 and 15 years old . They were selected from a statistically significant population sample Gral . San Martin city (Northeast Argentina) . Tests were read 48 h after injection and considered positive if a 5 mm on larger induration was present . Circulating antibodies were also evaluated by agar gel immunodiffusion . The overall infection rate for H . capsulatum was 9.2%, belonging to children from 4 to 14 years old, without significant differences among sexes . Five children from 2 to 14 years old were positive to paracoccidioidin (1.6%) . None of the children had specific antibodies neither signs of active mycosis . Results show H . capsulatum infection can be found from age 4, while for P . brasiliensis the lower limit was two years old . These findings may contribute to better knowledge on infantile fungal infection in a geographical region where no previous references can be found.

J Med Vet Mycol, 1997 Jul-Aug, 35(4), 237 - 41
Changes in plasmatic biochemical parameters during experimental murine paracoccidioidomycosis; Meloni-Bruneri LH et al.; Determinations of plasma cholesterol, HDL-cholesterol, triglycerides, total proteins, bilirubin, aspartate transaminases (AST), glucose, iron and total iron binding capacity were evaluated after 15 and 90 days of intraperitoneal infection with 5 x 10(6) virulent yeasts of Paracoccidioides brasiliensis Pb 18 isolate in resistant (A/J) and susceptible (B10.A) mice . Significant differences between control and infected groups were seen: an increase of triglycerides and total proteins for both strains, a decrease of glucose and HDL-cholesterol in B10.A mice and of iron in both strains, while AST, bilirubin and TIBC increased only in B10.A mice . While glucose, AST, bilirubin and TIBC were strongly modified in susceptible mice, no differences were seen in the resistant infected group, thus confirming the usefulness of biochemical parameters as an index of infection severity, as well as of recovery.

J Clin Microbiol, 1997 Sep, 35(9), 2252 - 5
Comparison of radioimmunoassay and enzyme-linked immunoassay methods for detection of Histoplasma capsulatum var . capsulatum antigen; Durkin MM et al.; The purpose of this study was to compare the conventional radioimmunoassay (RIA) to an enzyme-linked immunoassay (EIA) for the measurement of Histoplasma antigen in banked urine specimens . A correlation between the two methods would allow the EIA to be used as a nonradioactive alternative to the established 125I RIA . The study used stored urine from patients diagnosed with histoplasmosis during an outbreak in Indianapolis which began in 1988 . Control specimens from healthy adults, patients with other fungal infections, urinary tract infections, or nonfungal pneumonia were also tested . Both the RIA and EIA were run concurrently . The RIA system measured antigen levels of 0.4 to 27.0 RIA units, while the EIA measured antigen levels of 0.6 to 20.1 units . Both the EIA and RIA detected measurable antigen levels in urine from 50 of 56 patients (89%) with disseminated disease and 11 of 30 patients (37%) with self-limiting disease . One of 96 control specimens, from a patient with paracoccidioidomycosis, was positive with both systems . Antigen levels measured by EIA correlated well with those measured by the established RIA method (correlation coefficient, 0.974) . The EIA is an acceptable alternative to the RIA for measuring Histoplasma antigen levels in urine specimens.

Clin Exp Immunol, 1997 Aug, 109(2), 261 - 71
Cellular requirements for immunomodulatory effects caused by cell wall components of Paracoccidioides brasiliensis on antibody production; Silva MF et al.; In a previous study, we reported an increase in the number of immunoglobulin-secreting cells and the augmentation of antibody production (IgM and IgG3) against unrelated antigens (sheep erythrocytes or bovine serum albumin (BSA)) in mice infected with the fungus Paracoccidioides brasiliensis as well as in mice inoculated with its cell wall preparation (CW) . The immunomodulatory effect of the live fungus and CW preparation was dose-dependent and mainly restricted to the i.p . inoculation simultaneously to the BSA challenge by the i.v . route . In the present study, we investigated the active component of CW preparation upon the phenotype and also the degree of activation of possible target peritoneal cells involved in those phenomena . An insoluble polysaccharide fraction (F1 fraction) mainly composed of beta-glucan and chitin, and the purified beta-glucan (BGPb) behaved as CW in the augmentation of early antibody production . The peritoneal mononuclear inflammatory cells induced by CW, F1 fraction and BGPb were highly positive to alpha-naphthyl esterase staining; released low H2O2; expressed high levels of MHC-Ia(d) molecules and produced inflammatory cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-6 . Phenotypic analysis by flow cytometry and immunohistochemical techniques of the inflammatory cells responding to F1 fraction showed a prevalence of (CD11b/CD18, Mac-1)+ peritoneal macrophages . In addition, s.c . inoculation of F1 fraction resulted in the formation of nodular, localized and not progressive granulomatous lesions with an accumulation of (CD11b/C18)+ macrophages . Adoptive transferred Mac-1 macrophages to immunized syngeneic recipient mice were able to cause an increase in anti-BSA antibody production . These results suggest that inflammatory (CD11b/CD18)+ macrophages may be related to immunological disturbances, caused by cell wall components of P . brasiliensis.

J Clin Microbiol, 1997 Jul, 35(7), 1894 - 6
Molecular detection and identification of Paracoccidioides brasiliensis; Sandhu GS et al.; Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified . Hybridization results showed that the probe identified P . brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.

Clin Infect Dis, 1997 Jun, 24(6), 1169 - 71
Cross-reactivity in Histoplasma capsulatum variety capsulatum antigen assays of urine samples from patients with endemic mycoses; Wheat J et al.; We evaluated cross-reactivity in the antigen assay used for the diagnosis of histoplasmosis by testing urine samples from patients with disseminated fungal infections . The mycoses chosen for this study were selected on the basis of the observation that during clinical testing, cross-reactions may occur between Histoplasma capsulatum var . capsulatum, Paracoccidioides brasiliensis, Blastomyces dermatitidis, Coccidioides immitis, and Penicillium marneffei . We detected antigen in 12 of 19 patients with blastomycosis, 8 of 9 with paracoccidioidomycois, in 17 of 18 with P . marneffei infection, and in one with disseminated H . capsulatum var . duboisii infection . Cross-reactions were not observed in the assays for six patients with disseminated coccidioidomycosis . Cross-reactivity between the agents of other endemic mycoses should be considered in interpreting a positive H . capsulatum var . capsulatum antigen assay . Antigen detection may provide a rapid, provisional diagnosis for patients with serious infections caused by one of these organisms.

Glycobiology, 1997 Jun, 7(4), 463 - 8
A monoclonal antibody directed to terminal residue of beta-galactofuranose of a glycolipid antigen isolated from Paracoccidioides brasiliensis: cross-reactivity with Leishmania major and Trypanosoma cruzi; Suzuki E et al.; A mouse monoclonal antibody, MEST-1, was produced against Band 1 glycolipid antigen of Paracoccidioides brasiliensis . The glycan structure of Band 1 antigen was recently elucidated and the monosaccharides sequence was defined as: Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins . The reactivity of MEST-1 MAb was determined by solid-phase radioimmunoassay and high performance thin layer chromatography immunostaining . Selective oxidation of galactofuranose residues and inhibition assays with different methyl-glycosides, revealed that MAb MEST-1 is directed against the terminal residue of beta-D-galactofuranose of Band 1, a phosphoglyceroglycolipid antigen of P . brasiliensis . By indirect immunofluorescence, it was observed that the epitope recognized by MEST-1 is accessible to the antibody in yeast forms of this fungus . Reactivity of MEST-1 with parasites known to express galactofuranose containing glycoconjugates was also analyzed by indirect immunofluorescence . A positive fluorescence was observed with promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi . GIPL-1 was identified as the antigen recognized by MEST-1 in Leishmania major, indicating that the MAb MEST-1 recognizes terminal galactofuranose residue in either beta 1-->6 or beta 1-->3 linkage to the mannose.

J Med Vet Mycol, 1997 May-Jun, 35(3), 213 - 7
IgG, IgM and IgA antibody response for the diagnosis and follow-up of paracoccidioidomycosis: comparison of counterimmunoelectrophoresis and complement fixation; Bueno JP et al.; IgG, IgM and IgA antibodies to GP43 (glycoprotein fraction of Paracoccidioides brasiliensis) were measured by ELISA in 63 samples from 23 patients with paracoccidioidomycosis before and twice after chemotherapy was started . Antibodies against P . brasiliensis were detected by indirect immunofluorescence (IF) (IgG, IgM and IgA isotypes), counterimmunoelectrophoresis (CIE) and complement fixation . Two control groups composed of 19 healthy individuals and 12 patients with other diseases (six with histoplasmosis, three with tuberculosis and three with other mycoses) . The highest efficiency percentages were found with IgG and IgA-ELISA (100%), IgG-IF (96.2%), CIE (94.4%) and the lowest with CF (75.9%) . Highest positive and negative predictive values (100%) were observed for IgG and IgA ELISA . IgG and IgM-ELISA antibodies are more often found in patients with acute than chronic disease (P = 0.01) . Four to six months after treatment follow-up showed decreased levels of IgG and IgM-ELISA for acute cases and decreased titres of CIE for chronic cases in relation to pretreatment levels . This study suggests that IgG-ELISA anti-GP43 represents a good marker to monitor clinical response to therapy.

J Med Vet Mycol, 1997 May-Jun, 35(3), 205 - 11
Protein synthesis patterns of Paracoccidiodes brasiliensis isolates in stage-specific forms and during cellular differentiation; Salem-Izacc SM et al.; In this paper we compared the protein synthesis patterns of Paracoccidioides brasiliensis isolates . The protein profiles were compared for both yeast and mycelial forms and similarity analysis among them was performed by calculating similarity matrices and grouping the isolates in dendrograms . The examined isolates exhibited highly variable cellular morphology at 36 degrees C, when typical yeast cells were expected . On the other hand, at 26 degrees C all the isolates showed mycelial morphology . The analysis of protein synthesis profiles made it possible to cluster the P . brasiliensis isolates into groups that correlated with the morphological data . Interestingly, growth at 36 degrees C strongly decreased the heterogeneity of protein synthesis patterns seen in mycelial isolates . It was possible to cluster the isolates grown at 36 degrees C in three groups based on their two-dimensional protein synthesis analysis . The similarity index observed among the mycelial isolates was lower than that obtained with yeast cells, suggesting a more homogenous gene expression pattern in the host-adapted form than in the saprobic phase.

Microbiology, 1997 May, 143 ( Pt 5), 1583 - 6
Inhibition of Paracoccidioides brasiliensis by ajoene is associated with blockade of phosphatidylcholine biosynthesis; San-Blas G et al.; In Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans, no significant differences were observed in the phospholipid species of both morphological phases . The species observed were phosphatidylcholine (PC, 30-40%), phosphatidylethanolamine (PE, 27-28%), phosphatidylserine (16-19%), phosphatidylinositol (13-17%) and sphingomyelin (3-5%) . The main fatty acids found in the yeast (Y) phase were palmitate (56%), linoleate (18%) and oleate (15%), while linoleate predominated (61%) in the mycelial (M) phase, followed by palmitate (27%) and oleate (7%) . In the Y phase the main free sterol was ergosta-5,22-dien-3 beta-ol (82%) plus some lanosterol (12%) and ergosterol (6%), while in the M phase, the latter predominated (88%), followed by low levels of ergosta-5,22-dien-3 beta-ol (12%) . Ajoene {(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide}, a platelet aggregation inhibitor derived from garlic, induced alterations in phospholipid and fatty acid proportions such that PC was reduced to about 18% in both phases and PE increased to 38% (Y phase) or 44% (M phase), suggesting inhibition of PC synthesis . Ajoene also reduced saturated fatty acids (16:0 and 18:0) from 67 to 35% in the Y phase, with a corresponding increase in the unsaturated components . This effect was not seen in the M phase.

J Infect Dis, 1997 May, 175(5), 1263 - 7
Immunosuppression in paracoccidioidomycosis: T cell hyporesponsiveness to two Paracoccidioides brasiliensis glycoproteins that elicit strong humoral immune response; Benard G et al.; To assess human cellular immune response to paracoccidioidomycosis (PCM), lymphocyte proliferative responses to purified antigens from Paracoccidioides brasiliensis were determined in healthy persons previously infected by the fungus (positive donors), in healthy noninfected persons (controls), and in PCM patients . Affinity-purified gp70 and gp43, the two major antigens in humoral immune responses, were used . Both induced lymphocyte proliferation (gp43 species-specific) in positive donors but not in controls; healthy persons previously infected by Histoplasma capsulatum reacted to gp70 and not to gp43 . A similar cross-reactivity in antibody response to gp70 was previously reported; however, antibody response to gp43 has been considered specific . Lymphocytes from PCM patients, who, unlike positive donors, have high levels of anti-gp43 and anti-gp70 antibodies, proliferated poorly with gp70 and gp43 but better with other stimuli . This dichotomy between humoral and cellular antigen-specific responses suggests a Th2 immune response in PCM, which may be related to failure to control the infection.

Mutat Res, 1997 Apr 24, 390(1-2), 5 - 10
Human chromosome aberrations induced in vitro by Paracoccidioides brasiliensis glycoproteic component (GP 43); Freire-Maia DV et al.; The in vitro cytogenetic effects of the 43-kDa molecular mass exocellular glycoproteic component (GP 43) from Paracoccidioides brasiliensis were studied in cultures from human lymphocytes . The sample included 10 healthy, white, non-smoking, non-related males (mean age of 31.3 +/- 8.2 years) . Besides the control, three concentrations of GP 43 (0.125, 1.25 and 5 micrograms/ml) were used . In each group, around 1000 cells were examined in search of chromosome aberrations, and 30000 metaphases were analysed for the determination of the Mitotic Index . The authors conclude that GP 43 most probably causes inhibition of the cell cycle and aneugenic and clastogenic effects.

Fungal Genet Biol, 1997 Apr, 21(2), 223 - 7
Electrophoretic karyotype of clinical isolates of Paracoccidioides brasiliensis; Montoya AE et al.; Chromosomal DNA molecules were obtained from eight clinical isolates of Paracoccidioides brasiliensis during the early staged of the yeast to mycelial transformation process and were separated by contour-clamped homogeneous electric field gel electrophoresis . In seven of the isolates, it was possible to separate five bands, albeit with two different migration patterns (A and B) . In the remaining isolate only four bands were observed (pattern C) . The isolated chromosomes showed molecular sizes ranging from 3.2 to 10 Mb according to Schizosaccharomyces pombe molecular size standards . The three different patterns observed had bands of 3.2, 3.8, 4.1, 5.2, 6.7, 7.2, 8.8, and 10 Mb . These data imply that at least 30 Mb of the genome are organized as chromosomal macromolecules . Due to the fact that P . brasiliensis spheroplasts are difficult to obtain, technical details are provided.

Microbiol Immunol, 1997, 41(2), 93 - 9
In vitro amphotericin B effects on growth, viability and dimorphism of Paracoccidioides brasiliensis: reversal of the treatment; Jesuino RS et al.; The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated . We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB . This drug, at 30 micrograms/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation . The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed . The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug) . On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control . In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment . The two-dimensional gels of 35S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.

J Med Vet Mycol, 1997 Jan-Feb, 35(1), 37 - 43
Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor; Vicentini AP et al.; Adhesion is regarded as an important step in the pathogenesis of several microorganisms . Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness . Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein . By immunoblot analysis we show that MAb 1.H12 recognizes gp43 . This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P . brasiliensis infection in vivo . Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs . These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells . These findings reinforce the possibility of evolutionary conservation of such epitopes.

J Med Vet Mycol, 1997 Jan-Feb, 35(1), 13 - 8
Differences in reactivity of paracoccidioidomycosis sera with gp43 isoforms; Souza MC et al.; The glycoprotein gp43 from Paracoccidioides brasiliensis is the main antigenic component in paracoccidioidomycosis (PCM) because it is recognized by 100% of PCM patients . It has also been shown that different fungal strains produce gp43 with at least four isoform profiles . In this study, different isoform profiles from gp43, with pIs ranging from 5.8 to 8.5, were affinity purified from various P . brasiliensis (B-339, S.S., 1925 and I9) exoantigens . Because of the isoform heterogeneity, we questioned whether those isoform profiles could be similarly recognized by acute or chronic PCM patients . By using a specific and sensitive method for detection of human IgG anti-gp43 antibodies, the monoclonal antibody capture immunoassay, we report that not all gp43 isoform profiles are equally recognized in PCM sera when anti-gp43 MAb 17c was employed as capturing antibody . Our results showed that recognition of pI8.5 gp43 isoform was significantly lower for both acute (56%) and chronic patients (71%), compared with gp43 isoforms from the standard strain B-339 . On the other hand, when anti-gp43 MAb 8a, which recognizes a different antigenic epitope was used to capture the different gp43 isoform profiles, all patient's sera reacted similarly . The results described suggest that not all the antigenic epitopes expressed by gp43 are equally present in all P . brasiliensis strains.

Microbiology, 1997 Jan, 143 ( Pt 1), 197 - 202
Use of morphology index histograms to quantify populations of the fungal pathogen Paracoccidioides brasiliensis; San-Blas G et al.; To quantify the dimorphic process in wild and mutant strains of Paracoccidioides brasiliensis, we defined a morphology index (Mi) in terms of the maximum cell length (l), maximum cell diameter (d), and septal diameter (s), according to the equation Mi = 2.13 + 1.13 log10 (ls/d2), whose intercept and slope were such that Mi was around 1 for yeast (spherical) cells or 4 for hyphal (elongated) cells . This discriminatory power was used to quantify morphological population mixtures through Mi histograms . During the temperature-induced dimorphic transition (either way), mean Mi (Mi) varied linearly with time, suggesting a continuity in the process . Also, in wild strains and mutants thereof we found an inverse relationship between Mi and content of both cell wall chitin and 1,3-alpha-glucan.

Pediatr Radiol, 1997 Jan, 27(1), 67 - 9
Bony involvement in paracoccidioidomycosis; Doria AS et al.; We report a rare case of bony involvement by paracoccidioidomycosis in a 4-year-old boy . Extremity radiographs show a diffuse permeative pattern of metaphyseal bony destruction . Although these findings are nonspecific, they may suggest the disease in an appropriate clinical setting . Definitive diagnosis, however, depends upon detection of Paracoccidioides braziliensis on laboratory analysis.

Arch Microbiol, 1996 Dec, 166(6), 411 - 3
Polyamines in growth and dimorphism of Paracoccidioides brasiliensis; San-Blas G et al.; Putrescine and spermidine were the only polyamines found in Paracoccidioides brasiliensis, a dimorphic fungus pathogenic for humans . Free polyamines (putrescine > spermidine) increased during the first 24 h of yeast growth, with a second peak at 42 h, and also during the first 12 h of mycelium-to-yeast transition (spermidine > putrescine) . Conjugated and bound polyamines were also quantified . 1, 4-Diamino-2-butanone decreased free putrescine and spermidine accumulation by inhibiting the activity of ornithine decarboxylase . The increase in free polyamines corresponds to bud emergence in yeast growth and to the mycelium-to-yeast transition of P . brasiliensis.

Diagn Cytopathol, 1996 Dec, 15(5), 442 - 6
Osteomyelitis by Paracoccidioides brasiliensis (South American blastomycosis): cytologic diagnosis on fine-needle aspiration biopsy smears: a case report; Fulciniti F et al.; A case of osteomyelitis caused by Paracoccidioides brasiliensis primarily diagnosed by means of fine-needle aspiration biopsy is reported here in a 60-yr-old Italian patient who had lived in Venezuela for 40 yr . The cytologic and electron microscopic features of the exudate aspirated from a left femoral osteolytic area are described, and the differential diagnosis of this mycotic infection is discussed briefly.

Hybridoma, 1996 Dec, 15(6), 415 - 22
Monoclonal antibodies against the 43,000 Da glycoprotein from Paracoccidioides brasiliensis modulate laminin-mediated fungal adhesion to epithelial cells and pathogenesis; Gesztesi JL et al.; The surface glycoprotein gp43, a highly immunogenic component of Paracoccidioides brasiliensis, is used in the serodiagnosis of paracoccidioidomycosis (PCM) and has recently been shown to specifically bind the extracellular matrix protein laminin . Binding to laminin induces the increased adhesion of the fungus to epithelial cells; a hamster testicle infection model has shown that the gp43-dependent binding of fungal cells to laminin enhances their pathogenicity in vivo . We report on the production and characterization of 12 monoclonal antibodies against the gp43 that recognize peptide sequences in the molecule detecting at least three different epitopes as well as different isoforms of this antigen . MAbs interfered in the fungal pathogenicity in vivo either by inhibiting or enhancing granuloma formation and tissue destruction . Results suggest that P . brasiliensis propagules may start infection in man by strongly adhering to human lung cells . Thus, laminin-mediated fungal adhesion to human lung carcinoma (A549) cells was much more intense than to Madin-Darby canine kidney cells (MDCK), indicating differences in binding affinity . Subsequent growth of fungi bound to the lung cells could induce the granulomatous inflammatory reaction characteristic of PCM . Both steps are greatly stimulated by laminin binding in infective cells expressing gp43.

Mem Inst Oswaldo Cruz, 1996 Nov-Dec, 91(6), 665 - 70
Isolation of fungi from nature in the region of Botucatu, state of São Paulo, Brazil, an endemic area of paracoccidioidomycosis; Montenegro MR et al.; In an attempt to isolate Paracoccidioides brasiliensis from nature 887 samples of soil from Botucatu, SP, Brazil, were collected cultured in brain heart infusion agar supplemented with dextrose, in potato dextrose agar and in yeast extract starch dextrose agar, all with antibiotics, at 25 degrees and 37 degrees C . Five thermo-dependent dimorphic fungi morphologically resembling P . brasiliensis were isolated; two from armadillo holes; further studies of the biology, antigenicity and genetic features of the five dimorphic fungi are necessary to clarify their taxonomy and their possible relation to P . brasiliensis . In addition, 98 dematiaceous fungi and 581 different species of Aspergillus spp . were also isolated . Our findings emphasize that armadillos and their environment are associated with thermo-dimorphic fungi and confirm the ubiquity of pathogenic dematiaceous fungi and Aspergillus spp.

Braz J Med Biol Res, 1996 Nov, 29(11), 1441 - 4
Isolation and possible composition of glucosylceramides from Paracoccidioides brasiliensis; Takahashi HK et al.; Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated from mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC . The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM) . Carbohydrate analysis indicated that CMHs contain glucose . Analysis of 1H-NMR and mass spectrometry data suggest that the structure of the CMHs is Glcp beta 1-->Cer (mycelium forms present 12 different ceramides and yeast forms present 2 different ceramides) . The composition of the lipid moieties was analyzed by negative fast atom bombardment mass spectrometry . No glycosphingolipid other than glucosylceramide was detected in P . brasiliensis.

Am J Trop Med Hyg, 1996 Nov, 55(5), 496 - 503
The use of glucan as immunostimulant in the treatment of paracoccidioidomycosis; Meira DA et al.; A group of 10 patients, nine of them seriously infected with Paracoccidioides brasiliensis (G1), received glucan (beta-1,3 polyglucose) as an immunostimulant intravenously once a week for one month, followed by monthly doses (10 mg) over an 11-month period, together with a specific anti-fungal agent as an immunostimulant . A second group of eight moderately infected patients (G2) was treated with only the anti-fungal agent . Among the patients in G1, there was only one case of relapse compared with five in G2 . Values for the erythrocyte sedimentation rate (ESR) showed a significant difference (P < 0.001) post-treatment in G1 patients, when compared with the pretreatment levels . There was also a significant reduction (P < 0.001) in the level of serum antibodies to P . brasiliensis in the G1 patients in post-treatment examinations . The phytohemagglutinin (PHA) skin test showed a positive reaction among the patients in G1 (P < 0.01) post-treatment and there was a tendency towards an increase in the number of CD4+ T lymphocytes in both groups after treatment . The serum level of tumor necrosis factor (TNF) proved to be significantly higher (P < 0.02) in the G1 patients during treatment . In the G1 patients, the correlation between ESR and TNF tended to be negative whereas that between ESR and serum antibodies was positive . The present results indicate that the patients who received glucan, in spite of being more seriously ill, had a stronger and more favorable response to therapy.

Clin Infect Dis, 1996 Nov, 23(5), 1026 - 32
Paracoccidioidomycosis: case report and review; Manns BJ et al.; A previously well 59-year-old man presented with paracoccidioidomycosis, more than 15 years after leaving South America . He failed to respond to conventional therapies, first with oral itraconazole and then with amphotericin B plus sulfadiazine, and eventually died of recurrent arterial emboli possibly due to paracoccidioidomycotic aortitis . This patient's presentation demonstrates the difficulties that may be encountered in diagnosing and managing this disease . Paracoccidioidomycosis should be suspected in patients with an appropriate travel history who experience weight loss and have pulmonary, mucosal, and cutaneous lesions . This article comprehensively reviews the literature, with emphasis on epidemiology, clinical presentation, diagnosis, and therapy with imidazole antifungal medications.

Am J Trop Med Hyg, 1996 Oct, 55(4), 391 - 8
Paracoccidioides brasiliensis infection in nude mice: studies with isolates differing in virulence and definition of their T cell-dependent and T cell-independent components; Burger E et al.; Athymic and euthymic BALB/c mice infected with highly (Pb18) or slightly (Pb265) virulent Paracoccidioides brasiliensis isolates were compared regarding mortality, presence of viable yeasts, specific immunoglobulin M (IgM) and IgG titers, and the antigen recognition patterns of these antibodies . Isolate Pb18 caused a more severe disease in athymic mice, as supported by higher number of infected organs and shorter survival times . These animals, however, were resistant to Pb265 infection . High titers of antibodies were found only in euthymic mice, seven weeks after Pb18 infection . At this time, euthymic animals presented IgG antibodies to numerous protein bands that were not detected at four weeks postinfection or after Pb265 inoculation . In contrast, antibodies from athymic mice always reacted with few antigen bands . Although the majority of P . brasiliensis antigens are T cell-dependent, the immunodominant gp43 and also the 41.5- and 27.5-kD antigens are here, for the first time, characterized as T cell-independent antigens of P . brasiliensis.

Int J Syst Bacteriol, 1996 Oct, 46(4), 1125 - 30
Isolation and characterization of a new gram-negative, acetone-degrading, nitrate-reducing bacterium from soil, Paracoccus solventivorans sp . nov; Siller H et al.; An acetone-degrading, nitrate-reducing, coccoid to rod-shaped bacterium, strain L1, was isolated from soil on the site of a natural gas company . Cells of the logarithmic growth phase reacted gram positive, while those of the stationary growth phase were gram negative . Single organisms were 0.4 to 0.5 by 0.9 to 1.5 microns in size, nonmotile, and non-spore forming and had poly-beta-hydroxybutyrate inclusions . The doubling time of strain L1 on acetone-CO2-nitrate at the optimal pH of 7 to 8 and the optimal temperature of 30 to 37 degrees C was 12 h . More than 0.2% NaCl or 10 mM thiosulfate inhibited growth . For oxygen or nitrate respiration, acetone and a few organic chemicals were utilized as carbon sources whereas many others could not be used (for details, see Results) . Bicarbonate (or CO2) was essential for growth on acetone but not for growth on acetoacetate . The growth yields for acetone-CO2 and acetoacetate were 28.3 and 27.3 g/mol, respectively . With acetone as the carbon source, poly-beta-hydroxybutyrate accounted for up to 40% of the cellular dry weight . The DNA of strain L1 had a G + C content of 68.5 mol% (as determined by high-performance liquid chromatography of nucleotides) or 70 mol% (as determined by the TM method) . The sequence of the gene coding for the 16S rRNA led to the classification of strain L1 in the paracoccus group of the alpha subclass of the Proteobacteria . The new isolate is named Paracoccus solventivorans sp . nov . DSM 6637.

Arq Neuropsiquiatr, 1996 Sep, 54(3), 474 - 8
Thoracic intraspinal Paracoccidioidomycosis . Case report; Pacheco RA et al.; Intramedullary lesions caused by Paracoccidioides brasiliensis have been rarely described . Its diagnosis may be challenging and surgical approach is indicated for diagnostic and therapeutic purposes . We hereby report a case with MRI and surgical findings in a 45 year-old woman with intramedullary paracoccidioidomycosis, and make a review of other cases presented in the literature.

Arq Neuropsiquiatr, 1996 Sep, 54(3), 466 - 73
Intramedullary spinal cord paracoccidioidomycosis . Report of two cases; Colli BO et al.; Two cases of intramedullary paracoccidioidomycosis are reported . Paracoccidioidomycosis is a systemic disease that involves the buccopharyngeal mucosa, lungs lymph nodes and viscera and infrequently the central nervous system . Localization in the spinal cord is rare . Case 1: a 55-year old male admitted with crural pararesis, tactile/painful hypesthesia and sphincter disturbances of 15 days duration . Cutaneous-pulmonary blastomycosis was diagnosed 17 years ago . Myelotomography showed a blockade of T3-T4 (intramedullary lesion) . The lesion surgically removed was a Paracoccidioides brasiliensis granuloma . Treatment with sulfadiazine was started after the surgery . Follow-up of 15 month showed an improvement of the clinical signs . Case 2: a 57-year old male was admitted elsewhere 6 months ago and, with a radiologic diagnosis of pulmonary paracoccidioidomycosis, was treated with amphotericin B . He progressively developer paresthesia and tactile/pain anaesthesia on the left side, sphincter disturbances and tetraparesis with bilateral extensor plantar response and clonus of the feet . Myelotomography showed a blockade of C4-C6 (intramedullary lesion) . The lesion was not found during surgical exploration and the patient deteriorated and died . Post-mortem examination revealed an intramedullary tumor above the site of the mielotomy (Paracoccidioides brasiliensis granuloma) . The preoperative diagnosis of intramedullary paracoccidioidomycotic granulomas is difficult because the clinical and radiologic manifestations are uncharacteristic . Clinical suspicion was possible in our cases based on the history of previous systemic disease . Contrary to intracranial localizations, paracoccidioidomycotic granulomas causing progressive spinal cord compression may require early surgery because response to clinical treatment is slow and the reversibility of neurological deficits depends on the promptness of the decompression.

J Clin Microbiol, 1996 Sep, 34(9), 2328 - 30
Pulmonary paracoccidioidomycosis misdiagnosed as Pneumocystis pneumonia in an immunocompromised host; Silletti RP et al.; Yeast cells of Paracoccidioides brasiliensis can resemble the cysts of Pneumocystis carinii in smears stained with Grocott's modification of the Gomori methanamine silver stain . Furthermore, P . brasiliensis can cross-react in material stained with a widely used P . carinii immunofluorescent stain which uses monoclonal antibodies . The need to differentiate P . brasiliensis and P . carinii will become more important as the increasing incidence of immunosuppression results in the reactivation of latent P . brasiliensis infections.

Nihon Kyobu Shikkan Gakkai Zasshi, 1996 Aug, 34(8), 911 - 5
{Chronic pulmonary paracoccidioidomyosis in a Japanese Adult}; Kawayama T et al.; We encountered a case of chronic pulmonary paracoccidiodomycosis in Japan . A 53-year-old Japanese man, who had worked in Brazil from 1964 to 1969 . Came to our hospital because of abnormal shadows on a screening chest roentgenogram . In 1989, he had been treated with fluconazole for mucocutaneous-lymphangitic paracoccidioidomycosis with oral ulceration and neak hymphadenitis . Chest roentgenograms and computed tomograms showed diffuse small nodular and emphysematous shadows . Microscopical examination of specimens obtained by transbronchial lung biopsy showed no abnormality . He was treated with oral fluconazole, and the abnormal radiographic shadows regressed . We believe that this was the first case of chronic pulmonary paracoccidiodomycosis in Japan.

Am J Trop Med Hyg, 1996 Aug, 55(2), 235 - 42
Histopathology of paracoccidioidomycotic infection in athymic and euthymic mice: a sequential study; Burger E et al.; We report the comparative and sequential histologic analysis of lesions developed by Paracoccidioides brasiliensis-infected athymic and euthymic BALB/c mice, using conventional and specific stain to characterize the morphology of fungi and inflammatory cells as well as the extracellular matrix patterns . In both groups, neutrophils and macrophages were the predominant cells; macrophage transition to giant and epithelioid cells was observed . Degenerated fungi and fungal dust were associated with local neutropil infiltration and the presence of pseudoxantomatous macrophages . Paracoccidioides brasiliensis are always found within an extracellular matrix microenvironment . The main differences between lesions developed by nu/+ and nude (nu/nu) mice resided in the more pronounced encapsulating tendency in the former ones . In both groups of animals, lesions in several organs were associated with extensive hemopoiesis . At the first week of infection, there was evidence of fungal destruction and control of its proliferation in both mouse groups . At the fourth and seventh weeks, nu/+ mice maintained the control of the infection, as shown by the scarce numbers of budding yeast cells, while experimental nu/nu mice showed a tumor-like progression of the disease with a striking increase in fungal load in many organs (e.g., omental and mesenteric milky spots, lymph nodes, spleen), some of which present their structure replaced by granulomatous lesions.

Glycobiology, 1996 Jul, 6(5), 507 - 15
Structure of the N-linked oligosaccharide of the main diagnostic antigen of the pathogenic fungus Paracoccidioides brasiliensis; Almeida IC et al.; The major diagnostic antigen of Paracoccidioides brasiliensis is the exocellularly secreted 43,000 Da glycoprotein (gp43) which contains a single N-linked oligosaccharide chain . This oligosaccharide, although poorly immunogenic in man, is responsible for the cross-reactivity of the gp43 with sera from patients with histoplasmosis, and may have a role in fungal virulence . It contains a neutral high-mannose core (Man7GlcNAc2) to which a (1-->6)-linked alpha-D-Manp chain of variable length, substituted at the 2-O positions by single alpha-D-Manp residues, is attached . A terminal unit of beta-D-galactofuranose is (1-->6)-linked to one of the (1-->2)-linked mannosyl residues, either in the C or in the A arm of the oligosaccharide . The heterogeneity of the oligosaccharide is determined by the different sizes of the A arm and the sites of insertion of the beta-galactofuranosyl unit . The complete structure was determined by methylation analysis, 1H-NMR, mass spectrometry, acetolysis and mannosidase degradation . Electrospray mass spectrometry showed that the oligosaccharide comprises several subtypes ranging from Hex18GlcNAc2 to Hex10GlcNAc2 which accounts for the diffuse migration of the gp43 in polyacrylamide gels . The average size of the most frequent subtype is Hex13.6GlcNAc2 . Dilute acid treatment to remove beta-D-Galf reduced the molecular masses of the majority of the subtypes by a single sugar unit.

J Med Vet Mycol, 1996 Jul-Aug, 34(4), 273 - 7
Specific components found in circulating immune complexes (CIC) in paracoccidioidomycosis; Unterkircher CS et al.; Circulating immune complexes (CIC) from 15 paracoccidioidomycosis (PCM) patient sera and from 20 healthy control sera were analysed . After CIC precipitation, solubilization and acid treatment, only a little reactivity to P . brasiliensis antigens was found in the free antibodies from PCM-CIC . This result has suggested that there were antibodies with a high affinity bound to fungus components . Dissociated CIC were fractionated in a column of Sephacryl S300 and the fractions that probably contained antigens were pooled and applied to an affinity column, prepared with mouse anti-gp43 monoclonal antibody . Using ECL-Western blotting assay two polypeptide with apparent mass of 43 and 62 kDa were found.

J Med Vet Mycol, 1996 Jul-Aug, 34(4), 265 - 72
Evaluation of Paracoccidioides brasiliensis exoantigen in the detection of delayed dermal hypersensitivity in experimental and human paracoccidioidomycosis; Marques M et al.; The exoantigen of Paracoccidioides brasiliensis standardized by Camargo et al . (AgR) was used to evaluate the in vivo and in vitro cell immune response of experimental animals and of patients with paracoccidioidomycosis (PBM) . Fava Netto antigen (AgF) was tested in parallel as a control antigen . The study was conducted with mice and guinea pigs infected with P . brasiliensis or immunized with its fungal antigens, on patients with PBM and on their respective control groups . The cell immune response was analysed by skin tests, and by the macrophage and leucocyte migration inhibition tests (MMIT and LMIT) in the animals and in the patients, respectively . The skin test with AgR as paracoccidioidin was positive in infected or immunized mice and guinea pigs and negative in control animals . The skin tests with AgR (24 h) showed 96.7% positivity in patients with PBM and were negative in control individuals . Histopathological study of the in vivo tests in the different experimental models was consistent with a delayed hypersensitivity response (DHR) . Immunohistochemical study of the skin tests of PBM patients demonstrated a predominance of T lymphocytes, confirming the nature of a DHR to the fungal antigens . The in vitro cell immune response showed variable results for the various experimental models, i.e . significant rates of MMIT in immunized mice, a tendency to positivity in infected guinea pigs, and the absence of migration inhibition in PBM patients . Taken together, the data indicate that the AgR is efficient as paracoccidioidin in the evaluation of DHR in PBM, with an optimum time of reading the test of 24 h.

J Med Vet Mycol, 1996 Jul-Aug, 34(4), 247 - 50
Cycloactive, aneugenic and clastogenic effects of Paracoccidioides brasiliensis exoantigen in human lymphocyte cultures; Balarin MA et al.; The in vitro effect of Paracoccidioides brasiliensis exoantigen on the human lymphocytes cell cycle and chromosomes was studied . Human peripheral blood lymphocyte cultures from ten healthy, white, non-smoking, non-related adult males (mean age 31.3 +/- 8.2 years) were studied . Blood cultures were treated with three exoantigen concentrations (0.25, 2.50 and 10.00 micrograms ml-1) . At least 1000 metaphases were analysed at each concentration, for evaluation of numerical and structural chromosome aberrations (CA) and 30,000 for mitotic index (MI) . Among the treated cultures, statistically significant differences in the frequencies of MI and CA were not observed . Nevertheless, when compared with control cultures, they all showed a significantly lower frequency of MI and higher frequency of CA . It is suggested that the detected alterations were caused by the exoantigen, its fractions or its metabolites.

Rev Soc Bras Med Trop, 1996 Jul-Aug, 29(4), 363 - 6
{The diagnosis of severe disseminated paracoccidioidomycosis in a bone marrow aspirate: a case report}; Ozaki KS et al.; Acute or chronic disseminated paracoccidioidomycosis can be associated with changes in blood and bone marrow cell counts, mainly in the severe forms of this disease . However, there are few reports about the microbiological confirmation of the mycosis in bone marrow tissue . The present report describes a case of an adult patient with severe chronic multifocal paracoccidioidomycosis, whose etiological diagnosis has been done by the microscopical exam and culture fo the bone marrow aspirate . The authors emphasize the importance of these exams as an alternative way for the diagnosis of suspected cases of severe paracoccidioidomycosis.

Medicine (Baltimore), 1996 Jul, 75(4), 213 - 25
Paracoccidioidomycosis of bones and joints . A clinical, radiologic, and pathologic study of 9 cases; Amstalden EM et al.; Paracoccidioidomycosis is a systemic fungal infection endemic to Central and South America . It is associated with a broad spectrum of clinical manifestations and has been classified into acute and chronic forms . The latter is the most common type and usually affects male agricultural workers in rural communities . The disease typically begins in the lungs producing varying degrees of parenchymal damage, and in a significant number of cases the organism spreads through bronchogenic, lymphatic, or hematogenous routes to involve 1 or more organs . Bone and joint infection is relatively uncommon and has not been well described in the English literature . Much of the information on this form of the disease has been derived from radiographic and autopsy studies on patients with severe or fatal infections in whom skeletal involvement was a minor or incidental finding . We describe our experience with 9 cases in which osteoarticular manifestations were the sole or 1 of the few major complications of the disease . All 9 patients were male, from 9 to 49 years of age (mean, 36.6 yr) . Six were farmers . Evidence of visceral infection was present in 6 patients, in all of whom the lungs were considered the primary site of disease . The osteoarticular lesions were symptomatic in all cases, with the duration of symptoms ranging from 1 week to 2 years . The lesions were centered in bone in 2 cases: they manifested radiographically as circumscribed areas of lysis with or without a rim of sclerosis . The disease was centered in joints in 7 cases; the associated radiographic changes included evidence of joint effusion, periarticular bony erosions, and narrowing of the joint space . Biopsy of the involved skeletal site revealed compact or loose granulomas containing variable numbers of fungi . Although follow-up information was not available for all patients, those treated with TMP-SMX (and 1 patient who also received amphotericin B) and who were followed had prompt resolution of their signs and symptoms . Our findings substantiate some of what is known about the epidemiology of bone and joint involvement in chronic paracoccidioidomycosis . Our patients exhibited features of skeletal infection that have not been emphasized previously, however, including 1) frequent involvement of large joints or long bones of the extremities, 2) presentation with relatively rapidly developing musculoskeletal symptoms, and 3) manifestation as a solitary joint or bone lesion with or without concurrent clinically detectable pulmonary disease . Bone and joint paracoccidioidomycosis should be considered in the differential diagnosis in patients who have skeletal lesions with or without pulmonary involvement and have either lived in or traveled through endemic areas . Early diagnosis and treatment with antifungal medications can achieve an excellent outcome with limited local sequelae.

Fungal Genet Biol, 1996 Jun, 20(2), 125 - 31
Molecular cloning, nucleotide sequencing, and characterization of a 27-kDa antigenic protein from Paracoccidioides brasiliensis; McEwen JG et al.; A gene encoding a 27-kDa antigenic protein from Paracoccidioides brasiliensis was cloned, sequenced, and characterized . A cDNA library of the mycelial phase was produced and packed in Uni-Zap-XR vector, lambda Zap II synthesis kit (Stratagene, La Jolla, Ca) . The screening of the library was carried out using a pool of sera from paracoccidioidomycosis patients that had proven reactive in serological testing . Among 44,000 immuno-screened clones from the library, 2 were positive (clones 2 and 3) . The former was not characterized further . The latter has a 1-kb DNA insert with an open reading frame encoding a protein of 259 amino acids with a predicted molecular mass of 28.6 kDa (27 kDa by SDS-PAGE) . This protein corresponds to a 25-kDa protein in antigenic preparations of P . brasiliensis as determined by Western blot analysis . Comparison of the transcribed sequence with different gene banks failed to reveal a high degree of homology with other proteins . The cloned DNA fragment was easily expressed in Escherichia coli without the need of induction by isopropyl-beta-D-thiogalactopyranoside . These findings suggest that the gene encodes a P . brasiliensis-specific protein.

J Med Vet Mycol, 1996 Jun-Jul, 34(3), 181 - 6
Immunochemical and subcellular localization of the 43 kDa glycoprotein antigen of Paracoccidioides brasiliensis with monoclonal antibodies; Straus AH et al.; Two monoclonal antibodies ST-7 (IgG1) and ST-8 (IgG2b), directed against a 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis were produced . It was possible to detect the gp43 by ELISA in amounts as low as 100 ng per well, and by Western blot about 300 ng were detected . Mild treatment of the gp43 with sodium metaperiodate did not alter its reactivity with ST-7 and ST-8, which suggests that these MAbs recognize peptide epitopes . Confirming the periodate oxidation data, the 38 kDa protein resulting from deglycosylation of the gp43 with trifluoromethanesulphonic acid (TMFS), was reactive with ST-7 and ST-8 . Immunoelectron microscopy showed that the gp43 is stored inside large dense core vesicles, which flowed into the plasma membrane and extruded from cell membrane into the cell wall . Finally the antigen was secreted into the extracellular space as dense membrane-free material . Secretion of the gp43 occurred at scattered sites interspersed along the cell surface.

J Med Vet Mycol, 1996 Jun-Jul, 34(3), 155 - 61
Paracoccidioides brasiliensis-gp43 used as paracoccidioidin; Saraiva EC et al.; A purified glycoprotein of 43,000 daltons from Paracoccidioides brasiliensis (gp43) was tested as paracoccidioidin in delayed-type hypersensitivity (DTH) tests in both experimental animals (guinea pig and mice) and patients with paracoccidioidomycosis (PCM) . The gp43 paracoccidioidin was compared with the traditional Fava Netto antigen (AgFN) . In guinea pigs, the intradermal injection of 2 micrograms of gp43 showed a similar response to those obtained with AgFN, showing in histological sections a population of lymphoid cells that participate in DTH . In mice, gp43 at a dose of 3.75 micrograms showed positive DTH response . The use of gp43 as paracoccidioidin in humans showed that this molecule can be used to evaluate the DTH response in patients with PCM . Of 25 PCM patients studied, 48% were positive to gp43 while only 28% were positive to AgFN; 12 PCM patients were completely anergic to both antigens . Considering only those 13 PCM patients who were responsive to gp43 and/or to AgFN, 92.3% reacted against gp43 and 53.8% reacted against AgFN (P < 0.05) . Gp43 skin test responses (13.67 +/- 9.56 mm) were significantly larger than those obtained with AgFN (8.43 +/- 3.69 mm) . Immunohistochemical study of the human skin showed a perivascular inflammatory response constituted predominantly by T lymphocytes, macrophages and polymorphonuclear leukocytes.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 639 - 45
Structural characterization of a new galactofuranose-containing glycolipid antigen of Paracoccidioides brasiliensis; Levery SB et al.; An acidic glycolipid (Band 1), purified from P . brasiliensis by a combination of ion exchange chromatography, HPLC, and HPTLC, was found to be reactive with sera of all patients with paracoccidioidomycosis (PCM) . Monosaccharide analysis of Band 1 yielded mannose and galactose in a 2:1 ratio, while mild acid hydrolysis and mild periodate oxidation/NaB3H4 reduction indicated the presence of a terminal galactofuranose . Preliminary analysis of 1H-NMR and MS data suggests that the structure of the glycan is Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins (Ins = myo-inositol) . Removal of the galacto-furanose decreased by 60-80% the reactivity of sera from PCM patients with Band 1, suggesting that this residue is immunodominant . With the presumed absence of galactofuranose in mammalian hosts, compounds containing this residue may be useful targets for therapy of several parasitic and fungal diseases.

Allergol Immunopathol (Madr), 1996 May-Jun, 24(3), 98 - 105
Cellular immunity and immunostimulation in paracoccidioidomycosis; Oliveira ZN et al.; We have evaluated clinical and laboratorial effects of immunostimulation scarification with antigen of Paracoccidioides brasiliensis in patients with paracoccidioidomycosis, who often show depressed immunity . We have studied 62 Brazilian patients with paracoccidioidomycosis, 46 with active disease and 16 cured, in a 10-year period between 1982 and 1992 . Among the 46 patients with active disease: 10 had acute form, 12 chronic unifocal form and 24 the chronic multifocal form of the disease; 16 showed positive paracoccidioidin intradermal reaction and 30 negative reaction . Amongst 16 cured patients, 5 showed negative paracoccidioidin intradermal reaction and 11 positive reaction . We immunostimulated 20 patients by scarification with polysaccharide antigen of Paracoccidioides brasiliensis . All 20 immunostimulated patients had negative paracoccidioidin intradermal reaction, and 18 patients had active disease and 2 were cured . Out of 18 immunostimulated patients with active disease, 3 had active form, 10 chronic multifocal form and 5 chronic unifocal form of the disease . Before and after immunostimulation, each patients was inmunologically evaluated by in vivo and in vitro methods; in vivo methods consisted of intradermal reactions and DNCB sensitization tests; and in vitro methods consisted of total sub-set lymphocyte counting, leukocytes migration inhibition test, chemotaxix of total leukocytes and monuclear leukocyte phagocytosis . Remaining 42 patients served as a non-immunostimulated control group who has followed only clinically . We have observed significant clinical and immunological improvement in immunostimulated patients . We conclude that the immunostimulation by scarification with antigen of P . brasiliensis can be a helpful adjuvant therapy for paracoccidioidomycosis.

Rev Soc Bras Med Trop, 1996 May-Jun, 29(3), 241 - 4
{Bone involvement in chronic disseminated paracoccidioidomycosis . A report on the first cases in Rio Grande do Sul}; Severo LC et al.; The first cases of bone involvement in paracoccidioidomycosis observed in the state of Rio Grande do Sul (Brazil) are reported . Clinical and radiological findings are commented and some peculiarities are pointed out.

Arch Microbiol, 1996 May, 165(5), 311 - 6
Ornithine decarboxylase in Paracoccidioides brasiliensis; San-Blas G et al.; Ornithine decarboxylase in Paracoccidioides brasiliensis, a dimorphic human pathogenic fungus, was more active at 37 degrees C in the yeast phase and at 30 degrees C in the mycelial phase . In contrast to other fungal systems, yeast growth and mycelium-to-yeast transition in P . brasiliensis were accompanied by a high activity of ornithine decarboxylase at the onset of the budding process, the activity of which was inhibited by 1,4-diamino-2-butanone . The activity of ornithine decarboxylase remained at a basal level during vegetative growth of both the mycelial phase and the late stage of yeast phase, and also through the yeast-to-mycelium transition.

J Leukoc Biol, 1996 Apr, 59(4), 526 - 33
Neutrophil oxidative metabolism and killing of P . brasiliensis after air pouch infection of susceptible and resistant mice; Meloni-Bruneri LH et al.; The oxidative burst of polymorphonuclear neutrophils (PMN) and their ability to inhibit Paracoccidioides brasiliensis growth was studied in susceptible (B10.A) and resistant (A/J) mice . The cells were obtained after subcutaneous inoculation in air pouches, yielding highly pure PMN preparations; the number of cells was similar for both strains at 24 h and five times higher in the resistant strain at 15 days . The oxidative metabolism of these PMN was evaluated by the luminol and lucigen-enhanced chemiluminescence upon stimulation with PMA or killed P . brasiliensis (Pb) . At 24 h of infection PMN from both strains showed similar responses . However, at 15 days a great enhancement of the Pb-stimulated luminol-enhanced chemiluminescence was observed only in PMN from resistant mice . Such increase was markedly inhibited by the addition of catalase . Independent of the mouse strain or time of infection of lucigen-enhanced chemiluminescence showed the same intensity . The lucigen-enhanced chemiluminescence of PMN without stimuli from resistant mice did not change with the time of infection, however, after 15 days of infection a significantly lower chemiluminescence was detected with PMN from susceptible mice . At 15 days of infection the PMN from B10.A were unable to kill P . brasiliensis yeast cells in vitro . Because the lucigenin- and luminol-enhanced chemiluminescence detects, respectively, the O2- production and the myeloperoxidase/hydrogen peroxide halide system, the present data show parallels between deficiency in the production of oxygen-reactive species by PMN and lower fungicidal activity.

Mycoses, 1996 Mar-Apr, 39(3-4), 95 - 8
Evaluation and comparison of the growth and viability of yeast-like cells of Paracoccidioides brasiliensis; de Moraes Borba C et al.; The growth and viability of two Paracoccidioides brasiliensis strains were evaluated in PYG and MMcM media containing no boric acid . The two P . brasiliensis strains exhibited a similar growth pattern in PYG medium, which was considered to be good compared with the growth curves in defined MMcM medium without boric acid . Calculation of cell generation time in the media studied demonstrated that the cells had greater difficulty in forming new generations in MMcM medium containing no boric acid . The present data confirm the literature view that PYG medium is the most complete in terms of nutritive substances for the growth and development of P . brasiliensis.

J Biol Chem, 1996 Feb 23, 271(8), 4553 - 60
Cloning, characterization, and epitope expression of the major diagnostic antigen of Paracoccidioides brasiliensis; Cisalpino PS et al.; The 43,000-Da glycoprotein (gp43) of Paracoccidioides brasiliensis is an immunodominant antigen for antibody-dependent and immune cellular responses in patients with paracoccidioidomycosis . In order to identify the peptide epitopes involved in the immunological reactivities of the gp43 and to obtain highly specific recombinant molecules for diagnosis of the infection, genomic and cDNA clones representing the entire coding region of the antigen were sequenced . The gp43 open reading frame was found in a 1,329-base pair fragment with 2 exons interrupted by an intron of 78 nucleotides . The gene is present in very few copies per genome, as indicated by Southern blotting and chromosomal megarestriction analysis . A single transcript of 1.5 kilobase pairs was verified in the yeast phase . The gene encodes a polypeptide of 416 amino acids (Mr 45,947) with a leader peptide of 35 residues; the mature protein has a single N-glycosylation site . The deduced amino acid sequence showed similarities of 56-58% with exo-1,3- beta-D-glucanases from Saccharomyces cerevisiae and Candida albicans . However, the gp43 is devoid of hydrolase activity and does not cross-react immunologically with the fungal glucanases . Internal and COOH-terminal gene fragments of the gp43 were expressed as recombinant fusion proteins, which reacted with antibodies elicited against the native antigen.

Mycopathologia, 1996, 135(2), 89 - 98
Epidemiologic skin test survey of sensitivity to paracoccidioidin, histoplasmin and sporotrichin among gold mine workers of Morro Velho Mining, Brazil; Rodrigues MT et al.; Skin tests with paracoccidioidin, histoplasmin and sporotrichin were applied to 417 workers of Morro Velho Mining in the State of Minas Gerais, Brazil, with the main purpose of detecting the prevalence of paracoccidioidomycosis-infection, histoplasmosis capsulate-infection and sporotrichosis-infection . The rates of positivity to the skin tests were 13.43% for paracoccidioidin, 17.50% for histoplasmin and 13.67% for sporotrichin . Several epidemiological factors were investigated for a better interpretation of the results . Paracoccidioides brasiliensis, Histoplasma capsulatum var . capsulatum and Sporothrix schenkii were not isolated from the soil samples from the mines investigated.

Mycopathologia, 1996, 135(1), 13 - 9
Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences; Franco M et al.; We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol . Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (immunodiffusion-ID and footpad swelling test-FPT) . Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P . brasiliensis antigens . In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media . The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT . In experiment #2, the reference system for P . brasiliensis showed 26 antigen peaks . There were important differences between batches prepared from the same isolate and between batches from different isolates . Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P . brasiliensis isolate, under controlled incubation conditions.

Rev Soc Bras Med Trop, 1996 Jan-Feb, 29(1), 1 - 3
{An intradermal test with histoplasmin and paracoccidioidin in 2 regions of Rio Grande do Sul}; Zembrzuski MM et al.; From August to October 1992 a study of prevalence of cutaneous positivity to histoplasmin and paracoccidioidin was carried out in the cities of Cachoeira do Sul (Valley of Jacui River) and Santo Angelo (western hillside of the PlateAn), Rio Grande do Sul, Brazil . Skin tests were made in 193 soldiers from Cachoeira do Sul and 161 soldiers from Santo Angelo, both groups of 17 to 19 years of age . In Cachoeira do Sul the prevalence of positive tests to histoplasmin was 89% and to paracoccidioidin 82% . In Santo Angelo 48% of the intradermical reactions were positive to histoplasmin and 39% to paracoccidioidin.

Dermatol Clin, 1996 Jan, 14(1), 125 - 35
Cutaneous manifestations of systemic mycoses; Body BA; Systemic fungal diseases are primary pulmonary diseases caused by the dimorphic fungal pathogens, Blastomyces dermatitides, Coccidioides immitis . Histoplasma capsulatum, or Paracoccidioides brasiliensis . Infection occurs after inhalation of the infectious form of the fungus and may be acute, self-limited, or subclinical . Primary cutaneous infection occurs only after traumatic implantation of the fungus and is unusual . Erythema nodosum or erythema multiforme may accompany the acute form of the disease . Other cutaneous manifestations represent disseminated disease and, as such, require systemic antifungal therapy . Because cutaneous lesions have occurred coincidentally with other cutaneous pathologies, emphasis should be placed on a complete clinical history, physical examination, and diagnosis by histopathology and culture.

Mycopathologia, 1996, 133(3), 135 - 8
Inoculation experimental animals with Paracoccidioides brasiliensis strains: an attempt to reestablish the dimorphic process and variation in pathogenicity as a function of time of preservation under mineral oil; Mendes da Silva AM et al.; In an attempt to reestablish the dimorphic process in strains of Paracoccidioides brasiliensis in the transition phase (Y reversible M) and to reisolate them, five strains in the transitional phase due to the long time of preservation under mineral oil and two strains in the yeast-like phase were inoculated into male albino rats . The animals were then studied for the presence of paracoccidioidomycotic granulomata . Of the seven strains inoculated, five caused granulomatous nodules in several organs of the animals and only two of these five strains, which had been preserved for the shortest period of time (9 years) were reisolated in culture . Two strains were unable to provoke infection, with no lesions detected in any organ . It is assumed that the long period of time during which the strains were left under oil favored the alteration of celt wall contents, leading to differences in pathogenicity.

Am J Trop Med Hyg, 1996 Jan, 54(1), 7 - 12
Antigen-specific immunosuppression in paracoccidioidomycosis; Benard G et al.; To characterize the immune dysfunction associated with paracoccidioidomycosis, we studied the in vitro lymphocyte reactivity to phytohemagglutinin (PHA), pokeweed mitogen (PWM), a Candida albicans antigen (CMA), and a Paracoccidioides brasiliensis antigen (PbAg) in 32 patients with the acute and the chronic form of the disease before or during the initial phase of treatment and after clinical cure . We also studied, as controls, 30 healthy individuals, 15 of them immune to P . brasiliensis . Results showed a strong hyporesponsiveness to the PbAg while responses to mitogens and CMA were comparable with those of controls . Patients with the acute form of the disease (usually more severe) had more marked PbAg hyporesponsiveness than those with the chronic form . After patients' clinical cure, PbAg proliferative responses were similar to controls and greater than those seen before pretreatment . Changes in other parameters were also seen in the treated patients; skin test anergy to paracoccidioidin, high levels of anti-P . brasiliensis antibodies, leukocytosis, and eosinophilia . These changes were usually more intense in patients with the acute form of the disease . The post-treatment CD4+, CD8+, and total lymphocyte counts were similar to those of controls . Correlation between these parameters and the lymphoproliferative responses to the various stimuli was only found with PbAg: PbAg responses correlated inversely with eosinophil and anti P . brasiliensis antibody levels . Overall, our results demonstrate an antigen-specific-cellular immunity defect, which is reversible with treatment and possibly related to a T helper cell-2 pattern of immune response during active disease.

Neuroradiology, 1995 Nov, 37(8), 636 - 41
Paracoccidioidomycosis of the central nervous system: CT findings; Rodacki MA et al.; A retrospective analysis of six cases of central nervous system paracoccidioidomycosis, all but one proven by biopsy and surgery, was carried out to study the CT and clinical data and pathological correlation . Most of the patients were from the country . Headache, vomiting, seizures and hemiparesis were the most frequent symptoms . Papilloedema was present in four patients with raised intracranial pressure . Five patients had chronic lung disease and two with advanced systemic disease, skin and mucous membrane lesions were also observed . The neurological disturbance was sometimes the presenting features and the diagnosis was discovered incidentally after surgery . Both solitary and multiple parenchymal lesions were observed and the cerebral hemispheres were more commonly involved in four patients . Local meningeal involvement was observed in one with a single cortical granuloma . We emphasise the usefulness of CT, showing a rounded or lobulated mass with an isodense or radiolucent centre after contrast enhancement, surrounded by an irregular wall of varying thickness . There was always moderate oedema, extending peripherally . Other infections or neoplastic diseases may present similar findings . Preoperative diagnosis should rest on integration of clinical data, chest films, laboratory and neuroimaging studies.

Mycoses, 1995 Nov-Dec, 38(11-12), 481 - 4
An atypical isolate of Paracoccidioides brasiliensis; Hamdan JS et al.; An atypical strain of Paracoccidioides brasiliensis was obtained from a patient with disseminated paracoccidioidomycosis . This isolate, which has been maintained in the laboratory for 4 years by successive subcultures (30-40 days) in Fava Netto medium, produces cerebriform colonies characteristic of the yeast phase of the fungus when left at room temperature . Microscopy shows a predominance of yeasts with multiple buds, side by side with some branching filamentous structures presenting intercalary chlamydospores . The strain does not develop well at 37 degrees C, suggesting heat sensitivity . Storage at 4 degrees C does not change its macro- or microscopic characteristics . These findings support the hypothesis raised by others that the dimorphism of P . brasiliensis may not be exclusively heat dependent . Studies are currently being developed in our laboratory to better characterize this isolate in terms of biochemical peculiarities, degree of virulence and susceptibility/resistance to antifungal drugsPublication Types:

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