Immunoperoxidase technique using an anti-Leishmania (L.) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

Cutaneous sporotrichosis treatment with potassium iodide: a 24 year experience in São Paulo state, Brazil

BACKGROUND: Sporotrichosis is a subacute or chronic disease caused by a dimorphic fungus, Sporothrix schenckii. The first and most traditional treatment is potassium iodide in satured solution (SSKI) used by DE BEURMANN in 1907. For its effectiveness, it is still used for cutaneous sporotrichosis. OBJECTIVE: To evaluate the treatment of cutaneous sporotrichosis with SSKI in relation to clinical cure, side effects, length of treatment and reactivation. METHODS: We conducted a retrospective analysis of medical records over a 24-year period (1981-2005). Patients of all ages who were treated in the hospital´s division of dermatology were included in the study providing that they had a positive culture of S. schenckii. Satured solution of potassium iodide (3 to 6g per day) was the treatment prescribed. For children, half of the dose was prescribed. RESULTS: The lymphocutaneous disease was prevalent, the cure rate was 94.7%, side effects were described in 5.5% of the cases, mean length of treatment was 3.5 months and possible reactivation was observed in 11.1%. CONCLUSION: SSKI is an effective drug, with many side effects, but with low frequency. Resolution was for maximum six months of treatment. SSKI has been found to be a very effective drug in this retrospective study of culture-proven cases of cutaneous and lymphocutaneous sporotrichosis. It should be used as first drug of choice especially in resource-limited settings.

Host-induced changes in the cell surface N-linked glycoproteins, from Aspergillus fumigatus. Search for specific targets with potential for clinical therapy and/or diagnosis.

This dissertation is presented to obtain a Master degree in Structural and Functional Biochemistry

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

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.

ISOLATION OF Cryptococcus neoformans FROM ENVIRONMENTAL SAMPLES COLLECTED IN SOUTHEASTERN NIGERIA

SUMMARY Cryptococcosis caused by Cryptococcus neoformans is the second most common fungal opportunistic pathogen and a lifethreatening infection with serious clinical manifestations especially in HIV/AIDS and other immunocompromised patients. In Nigeria, HIV/AIDS infection has reached an alarming level. Despite this, information on the presence of this fungus in clinical and environmental samples is very scanty in Nigeria and many other parts of Africa. We set out to evaluate the presence of Cryptococcus neoformans or C. gattii in pigeon droppings obtained from Southeastern Nigeria. One hundred and seventy-seven samples of pigeon droppings from six sample types were collected. The area covered comprised of ten cities and other locations spanning across five States in Nigeria. Using established techniques, Cryptococcus neoformans was isolated from 39 of the 177 (22.0%) samples overall. No C. gattiiwas isolated. Most of the isolates (32.4%) were recovered from dovecotes (11 of 34) followed closely by samples taken from markets (31.8%; seven of 22) and least from the church (4.0%; one of 25). The highest isolation rate (38.9%) was found in samples from Enugu-Ezike(seven of 23) while the least came from Afikpoand the other locations each with 9.1% isolation rate. This is the first large-scale screening of Cryptococcus neoformans from pigeon droppings in Nigeria. The ecological and epidemiological significance of these findings are discussed.

ANTIFUNGAL ACTIVITY OF Cymbopogon nardus (L.) Rendle (CITRONELLA) AGAINST Microsporum canis FROM ANIMALS AND HOME ENVIRONMENT

Dermatophytosis is a common zoonosis in urban centers. Dogs and cats have played an important role as its disseminators. Environmental decontamination is essential for the prevention of its propagation to humans and animals. However, sanitizers or disinfectants with antifungal activity, currently available, have high toxicity. The present study evaluated the in vitro effects of an extract of citronella (Cymbopogon nardus) on 31 Microsporum canis isolates from animals and home environments. Susceptibility tests were performed based on document M38-A2 (2008) of the Clinical and Laboratory Standards Institute with modifications for natural products. Although susceptibility variation was observed between the fungus tested, the concentrations that inhibited the growth of 50 and 90% of the microorganisms were low (19.5 and 78 µg/mL, respectively). Thus, this citronella extract showed potent fungistatic and fungicide activities against M. canis isolated from animals and home environments. Therefore, it could be an alternative for dermatophytosis prophylaxis in the home environment.

EXPERIMENTAL PARACOCCIDIOIDOMYCOSIS IN PREGNANT RATS

Paracoccidioidomycosis (PCM), caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb), is the most prevalent systemic mycosis in Latin America. There are few reports in the literature about the disease damages during pregnancy and the consequences to the fetuses and breeding. This study evaluated the implications of PCM during pregnancy on offspring and mothers in Wistar rats. Groups of rats were submitted to systemic Pb infection, by intraperitoneal infusion, and mated 30 days after the infection date. Immediately after birth, rats and neonates were sacrificed to obtain organs for standard histological examination, morphometric analysis, fungi recovery by plating (CFU) and dosing of anti-Pb antibodies by ELISA. There were no stillbirths or miscarriages, however, the fetuses from infected pregnant rats had lower body and organ weight but the fertility rate was 100%. The largest number of CFU was recovered from the organ of pregnant rats, the pathological examination revealed more severe infection in the same group, further on the largest number of granulomas and fungal field. It can be concluded that the PCM was more severe in the group of pregnant rats, with implications to the weight of offspring.

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.

EPIDEMIOLOGY OF PARACOCCIDIOIDOMYCOSIS

SUMMARYThe epidemiological characteristics of paracoccidioidomycosis were reviewed and updated. The new endemic areas in Brazil were discussed in the section regarding the geographic distribution of the mycosis. Subclinical infection with Paracoccidioides brasiliensis was discussed on the basis of skin test surveys with antigens of the fungus, seroepidemiological studies, and disease cases outside Latin America. Large case series permitted a comparison of the prevalence of the mycosis in different regions, its estimated incidence and risk factors for the development of the disease. Aspects modulating the expression of the clinical forms of paracoccidioidomycosis are also presented. This review also deals with diseases associated with the mycosis, opportunistic paracoccidioidomycosis, lethality, mortality and infection and disease in animals.

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.

Molecular mechanisms of sexual development in basidiomycetes: exploring connections with lifestyles

Dissertação para obtenção do Grau de Doutor em Biologia

A modification of the indirect fluorescence test (IFT) for Paracoccidioidomycosis

A modification of the indirect fluorescence test (IFT) for serological diagnosis of Paracoccidioidomycosis is described in which formolized budding forms (yeast cells) of the fungus Paracoccidioides brasiliensis are used as antigen. A further modification introduced is a less eiaborate technical procedure of the test without lowering the sensitivity of the reaction. The test may be considered as adequateiy accurate and easy to perform in any laboratory with immunofluorescence facilities.

Evaluation of the phagocytic activity of peripheral blood monocytes of patients with Jorge Lobo's disease

Studies on host-parasite interaction in Jorge Lobo's disease are scarce, with no report in the literature on the phagocytosis of Lacazia loboi by phagocytic mononuclear cells. Thus, the objective of the present study was to assess the phagocytic activity of blood monocytes in the presence of L. loboi in patients with the disease and in healthy subjects (controls) over 3 and 24 hours of incubation. Statistical analyses of the results showed no significant difference in percent phagocytosis of the fungus between patient and control monocytes. With respect to incubation time, however, there was a significant difference, in that percent phagocytosis was higher at 3 hours than at 24 hours (p <0.01). These results suggest that monocytes from patients with the mycosis are able to phagocyte the fungus, as also observed in control individuals.

Cryptococcus gattii molecular type VGII as agent of meningitis in a healthy child in Rio de Janeiro, Brazil: report of an autochthonous case

Cryptococcus gattii causes meningoencephalitis in immunocompetent hosts, occurring endemically in some tropical and subtropical regions. Recently, this fungus was involved in an outbreak in Vancouver Island and British Columbia (Canada). In this temperate region, the VGII type is predominant. The paper describes an autochthonous case of meningoencephalitis by C. gattii VGII in a previously health child in Rio de Janeiro, considered nonendemic region of Brazil. The fungus was identified by biochemical tests and the molecular type was determined by URA5-RFLP. The present report highlights the need for clinical vigilance for primary cryptococcal meningitis in nonendemic areas.