Paracoccidioides brasilienses isolates obtained from patients with acute and chronic disease exhibit morphological differences after animal passage

The basis for virulence in Paracoccidioides brasiliensis is not completely understood. There is a consensus that the sequencial 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 42.1mm in contrast with 32.9mm 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 16.0mm (SD ± 4.0), a value significantly higher than the 14.1mm (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.

Inoculation of BALB/c mice with Lacazia loboi

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.

Opportunistic infections in patients with aids admitted to an university hospital of the Southeast of Brazil

Opportunistic diseases in HIV-infected patients have changed since the introduction of highly active anti-retroviral therapy (HAART). This study aims at evaluating the frequency of associated diseases in patients with AIDS admitted to an university hospital of Brazil, before and after HAART. The medical records of 342 HIV-infected patients were reviewed and divided into two groups: group 1 comprised 247 patients before HAART and, group 2, 95 patients after HAART. The male-to-female rate dropped from 5:1 to 2:1for HIV infection. There was an increase in the prevalence of tuberculosis and toxoplasmosis, with a decrease in Kaposi's sarcoma, histoplasmosis and cryptococcosis. A reduction of in-hospital mortality (42.0% vs. 16.9%; p = 0.00002) has also occurred. An agreement between the main clinical diagnoses and autopsy findings was observed in 10 out of 20 cases (50%). Two patients with disseminated schistosomiasis and 2 with paracoccidioidomycosis are reported. Overall, except for cerebral toxoplasmosis, it has been noticed a smaller proportion of opportunistic conditions related to severe immunosuppression in the post HAART group. There was also a significant reduction in the in-hospital mortality, possibly reflecting improvement in the treatment of the HIV infection.

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

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

Rhinoscleroma: eight Peruvian cases

Rhinoscleroma is a rare infection in developed countries; although, it is reported with some frequency in poorer regions such as Central Africa, Central and South America, Eastern and Central Europe, Middle East, India and Indonesia. Nowadays, rhinoscleroma may be erroneously diagnosed as mucocutaneos leishmaniasis, leprosy, paracoccidioidomycosis, rhinosporidiasis, late syphilis, neoplasic diseases or other upper airway diseases. From 1996 to 2003, we diagnosed rhinoscleroma in eight patients attended in the Dermatologic and Transmitted Diseases service of "Cayetano Heredia" National Hospital, in Lima, Peru. The patients presented airway structural alterations producing nasopharyngeal, oropharyngeal and, in one patient, laryngeal stenosis. Biopsy samples revealed large vacuolated macrophages (Mikulicz cells) in all patients. Ciprofloxacin 500 mg bid for four to 12 weeks was used in seven patients and oxytetracycline 500 mg qid for six weeks in one patient. After follow-up for six to 12 months the patients did not show active infection or relapse, however, all of them presented some degree of upper airway stenosis. These cases are reported because of the difficulty diagnosing the disease and the success of antibiotic treatment.

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

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

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

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

DOSE RESPONSE EFFECT OF Paracoccidioides brasiliensis IN AN EXPERIMENTAL MODEL OF ARTHRITIS

Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb) and corresponds to prevalent systemic mycosis in Latin America. The aim of the present work was to evaluate the dose response effect of the fungal yeast phase for the standardization of an experimental model of septic arthritis. The experiments were performed with groups of 14 rats that received doses of 103, 104 or 105 P. brasiliensis (Pb18) cells. The fungi were injected in 50 µL of phosphate-buffered saline (PBS) directly into the knee joints of the animals. The following parameters were analyzed in this work: the formation of swelling in knees infused with yeast cells and the radiological and anatomopathological alterations, besides antibody titer by ELISA. After 15 days of infection, signs of inflammation were evident. At 45 days, some features of damage and necrosis were observed in the articular cartilage. The systemic dissemination of the fungus was observed in 11% of the inoculated animals, and it was concluded that the experimental model is able to mimic articular PCM in humans and that the dose of 105 yeast cells can be used as standard in this model.

DISSEMINATED FUNGAL INFECTION WITH ADRENAL INVOLVEMENT: REPORT OF TWO HIV NEGATIVE BRAZILIAN PATIENTS

Paracoccidioidomycosis and histoplasmosis are systemic fungal infections endemic in Brazil. Disseminated clinical forms are uncommon in immunocompetent individuals. We describe two HIV-negative patients with disseminated fungal infections, paracoccidioidomycosis and histoplasmosis, who were diagnosed by biopsies of suprarenal lesions. Both were treated for a prolonged period with oral antifungal agents, and both showed favorable outcomes.

THE POWER OF THE SMALL: THE EXAMPLE OF Paracoccidioides brasiliensis CONIDIA

SUMMARYResearch on Paracoccidioides brasiliensis has centered in the yeast cell probably because of the lack of distinctive features in the mycelium. In 1942 and for the first time, lateral conidia were noticed in the fungus' hyphae. Later on, Brazilian, Venezuelan and Argentinean researchers described "aleurias" when the fungus was grown in natural substrates. In 1970 authors became interested in the conidia and were able to obtain them in large numbers and treat them as individual units. Their shape and size were defined and the presence of all the elements of a competent eukaryotic cell were demonstrated. Conidia exhibited thermal dimorphism and, additionally, when given intranasally to BALB/c male mice, they converted into yeasts in the lungs and produce progressive pulmonary lesions with further dissemination to other organs. Studies on the phagocyte-conidia interaction were revealing and showed that these versatile structures allow a better understanding of the host- P. brasiliensisinteractions.

Paracoccidioides brasiliensis AND Paracoccidioides lutzii, A SECRET LOVE AFFAIR

SUMMARYTo commemorate Prof. Carlos da Silva Lacaz's centennial anniversary, the authors have written a brief account of a few, out of hundreds, biological, ecological, molecular and phylogenetic studies that led to the arrival of Paracoccidioides lutzii, hidden for more than a century within Paracoccidioides brasiliensis. Lacaz's permanent interest in this fungus, and particularly his conviction on the benefits that research on paracoccidioidomycosis would bring to patients, were pivotal in the development of the field.

Human serum antibody reactivity towards Paracoccidioides brasiliensis antigens treated with sodium metaperiodate

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

High molecular mass fraction in clinical isolates of Paracoccidioides brasiliensis

INTRODUCTION: Different serum levels of the IgG/IgE for Paracoccidioides brasiliensis high mass molecular (hMM) fraction (~366kDa) in the acute and chronic forms of the disease have been reported. Considering the nonexistence of hMM fraction investigation involving clinical isolates of P. brasiliensis, the present study aimed to investigate the presence of the hMM fraction (~366kDa) in cell free antigens (CFA) from P. brasiliensis clinical isolates. METHODS: CFA from 10 clinical isolates and a reference strain (Pb18) were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by gel image capturing and densitometer analysis. Additionally, CFA from 20 isolates and Pb18 were analyzed by capture ELISA (cELISA) using polyclonal (polAb) or monoclonal (mAb) antibodies to the hMM fraction. RESULTS: The presence of the hMM component was observed in CFA of all samples analyzed by SDS-PAGE/densitometry and by cELISA. In addition, Pearson's correlation test demonstrated stronger coefficients between hMM fraction levels using pAb and mAb (R = 0.853) in cELISA. CONCLUSIONS: The soluble hMM fraction was present in all the P. brasiliensis clinical isolates analyzed and the reference strain Pb18, which could be used as a source of this antigen. The work also introduces for first time, the cELISA method for P. brasiliensis hMM fraction detection. Analysis also suggests that detection is viable using polAb or mAb and this methodology may be useful for future investigation of the soluble hMM fraction (~366kDa) in sera from PCM patients.

Pulmonary paracoccidoidomycosis: radiology and clinical-epidemiological evaluation

INTRODUCTION: The purpose of this study was to compare respiratory signs and symptoms between patients with and without chest X-ray abnormalities in order to establish the meaning of radiographic findings in pulmonary PCM diagnosis. METHODS: The epidemiological, clinical and radiological lung findings of 44 patients with paracoccidiodomycosis (PCM) were evaluated. Patients were divided into two groups of 23 and 21 individuals according to the presence (group 1) or absence (group 2) of chest X-ray abnormalities, respectively, and their clinical data was analyzed with the aid of statistical tools. RESULTS: As a general rule, patients were rural workers, young adult males and smokers - group 1 and 2, respectively: males (91.3% and 66.7%); mean age (44.4 and 27.9 year-old); smoking (34.7% and 71.4 %); acute/subacute presentation (38.1% and 21.7%); chronic presentation (61.9% and 78.3%). The most frequent respiratory manifestations were - group 1 and 2, respectively: cough (25% and 11.4%) and dyspnea (22.7% and 6.8%). No statistical difference was observed in pulmonary signs and symptoms between patients with or without radiographic abnormalities. The most frequent radiological finding was nodular (23.8%) or nodular-fibrous (19%), bilateral (90.5%) and diffuse infiltrates (85.7%). CONCLUSIONS: Absence of statistical difference in pulmonary signs and symptoms between these two groups of patients with PCM indicates clinical-radiological dissociation. A simplified classification of radiological lung PCM findings is suggested, based on correlation of these data and current literature review.

A semi-nested PCR assay for molecular detection of Paracoccidioides brasiliensis in tissue samples

INTRODUCTION: Paracoccidioidomycosis is a systemic infection caused by Paracoccidioides brasiliensis. METHODS: In this study, a semi-nested PCR for paracoccidioidomycosis diagnosis was developed. The primers ITS1 and ITS4 were used in the first reaction, while the primers MJ03 and ITS1 primer were used in the second reaction. The semi-nested PCR was used to investigate biopsies of five patients with oral lesions that resembled paracoccidioidomycosis. RESULTS: The semi-nested PCR was positive for four samples and negative for a sample from a patient later diagnosed with leishmaniasis. CONCLUSIONS: The new semi-nested PCR describe is useful for paracoccidioidomycosis diagnosis.