Association of subgingival colonization of Candida albicans and other yeasts with severity of chronic periodontitis.

BACKGROUND AND OBJECTIVE: The aim of this study is to analyze the relationship between the subgingival colonization by Candida albicans and other yeasts with the severity of chronic periodontitis (CP). MATERIAL AND METHODS: After sample size calculation, 40 patients with CP and 20 healthy subjects (HS) were included in the study. Cases of slight-moderate (MCP, n = 23) and severe CP (SCP, n = 17) were defined according to the Centers for Disease Control/American Association of Periodontology classification. Subgingival samples were acquired using sterile paper-points from the sulcus or the deepest periodontal pocket of each healthy and subject with CP, respectively, and were cultured aerobically on three selective media. Yeast colonies that grew on the surface of plates were later identified by biochemical reactions. Statistical tests were used to analyze the association between subgingival yeast colonization (number of yeast-positive individuals and colony forming units (CFU) per subject) and periodontal disease status, considering statistical significance when P < 0.05. RESULTS: Although several yeast species were found (C. parapisilosis, Rhodotorula sp., C. dubliniensis and C. tropicalis), only C. albicans was present in all the patients with yeast-positive CP. Twelve patients (30%) with CP presented yeasts in the subgingival biofilm while only three patients (15%) in the HS group were positive for these microorganisms. No statistical difference was found between the CP and HS groups (P = 0.084). However, when the CP group was divided on the basis of severity, statistical differences were observed between the SCP and MCP groups (47% vs. 17%, P = 0.043), and between the SCP and HS groups (47% vs. 15%, P = 0.033). No statistical difference was observed between the MCP and HS groups (17% vs. 15%, P = 0.832). High densities of yeasts were found only in patients with MCP and SCP (mean and range 61.25 (0-100) CFU/plate and mean and range 51 (0-101) CFU/plate, respectively). CONCLUSION: In this group of patients, subgingival colonization of some yeasts, especially C. albicans, was associated with the severity of CP.

Entomophthoramycosis by Conidiobolus coronatus. Report of a case successfully treated with the combination of itraconazole and fluconazole.

Rhinoentomophthoramycosis caused by Conidiobolus coronatus in a 61-year old woman was unsuccessfully treated during 8 years with all the antifungals available in the Brazilian market, including potassium iodide for 1 month, sulfamethoxazole plus trimethoprim for 2 months, amphotericin B, total dose of 1130 mg, cetoconazole, 400 mg/day for 6 months, fluconazole, 200 mg/day, for at least 2 months and, itraconazole, 400 mg/day for 2 months, followed by 200 mg/day for 4 more months. Complete clinical and mycological cure was achieved using itraconazol 400 mg/day in association with fluconazol 200 mg/day during 24 months. After cure she was submitted to plastic surgery to repair her facial deformation. Today she remains clinically and mycologically cured after 59/60 months (5 years!) without any specific antifungal. We thus suggest the use of the combination of itraconazole and fluconazole as an additional option for the treatment of this mycosis.

Development of an enzyme-linked immunosorbent assay for the serodiagnosis of several clinical forms of sporotrichosis.

We performed a serological study with sera from 92 patients with confirmed sporotrichosis registered between 1999 and 2004 in two hospitals in Rio de Janeiro State, Brazil. The clinical presentation of sporotrichosis was distributed as follows: lymphocutaneous, 67%; fixed cutaneous, 23%; disseminated cutaneous, 8%; and extracutaneous, 2%. Sera were assayed by ELISA against a cell wall antigen of Sporothrix schenckii, SsCBF, that we have previously described. The cross-reactivity was determined with 77 heterologous sera. The serological test showed a sensitivity of 90% and a global efficiency of 86%. A group of 55 patients with several clinical presentations of sporotrichosis was clinically and serologically followed-up for at least 6 months. We observed by ELISA data a decrease in the antibody serum titers which correlated with the progress in healing. An HIV-positive patient with meningeal sporotrichosis was serologically followed-up for over 2 years. Serum and cerebrospinal fluid specimens were examined and significant antibodies levels against the antigen SsCBF were detected. Our results strongly suggest that this serological test is valuable for the differential diagnosis and follow-up of all clinical forms of sporotrichosis.

Fungal infections in the immunocompromised host.

In recent years many remarkable changes occurred in our way of life, producing opportunities for microbes. All these changes are related to the recent emergence of previously unrecognized diseases, or the resurgence of diseases that, at least in developed countries, were thought to be under control. This concept is reviewed regarding fungal infections and their agents in the immunocompromised host. The changing pattern of these infections, the portals of entry of fungi into the human host, fungal pathogenicity and the main predisposing factors are analyzed. Opportunistic fungal infections in cancer, organ transplant and acquired immunodeficiency syndrome patients are reviewed, specially candidiasis and aspergillosis.

First isolation of Cryptococcus neoformans var. gattii from a native jungle tree in the Brazilian Amazon rainforest.

Cryptococcus neoformans var. gattii was isolated for the first time from decaying wood in a hollow of a native jungle tree Guettarda acreana, in a wild area of an Amazon rainforest island, in Brazil. The presence of this variety in a virgin environment without either anthropic action or introduced vegetation is discussed with regard to the common knowledge of Cr. neoformans ecology.

Use of D-proline assimilation and CGB medium for screening Brazilian Cryptococcus neoformans isolates.

D-proline assimilation and CGB tests were performed on 233 Cryptococcus neoformans isolates from Brazil. The results disclosed a high sensitivity, D-proline 99-13% and CGB 99.57%, for screening of the varieties. D-proline assimilation test revealed two false negative results for 5.1% of 39 strains of var. gattii, and no false positive results were observed in 194 strains of var. neoformans. No false results and only one doubtful result were found on the CGB medium.

Cryptococcus neoformans var. gattii--evidence for a natural habitat related to decaying wood in a pottery tree hollow.

To study hollows of living trees as the natural habitat of Cryptococcus neoformans in an endemic area of cryptococcosis in the northeastern Brazilian region, samples of decaying wood were collected inside the hollows, plated on niger seed agar and inoculated into mice and hamsters. Identification of C. neoformans was based on morphological and physiological tests. Canavanine-glycine-bromothymol medium was used to screen the varieties and Crypto Check Iatron Kit to serotype the isolates. For a period of 29 months C. neoformans var. gattii serotype B was isolated repeatedly from the hollow of a pottery tree (Moquilea tomentosa), pointing to the natural occurrence of C. neoformans var. gatti in decaying wood forming hollows in living trees. Evidence for a natural habitat of the variety gattii other than that related to Eucalyptus camaldulensis are discussed.

Genetic characterization of environmental isolates of the Cryptococcus neoformans species complex from Brazil.

The genetic affiliation of a large number of isolates of the Cryptococcus neoformans species complex from environmental sources in Brazil has been investigated using amplified fragment length polymorphism (AFLP). The strains of C. neoformans isolated from a single tree, as well as from neighbouring trees, showed high similarity values (> 95%) of their AFLP patterns, thus suggesting considerable genetic homogeneity. The majority of isolates of C. neoformans belonged to AFLP genotype 1, and had serotype A and mating type alpha (= C. neoformans var. grubii). Three isolates belonged to AFLP genotype 2, with serotype D and mating type alpha (= C. neoformans var. neoformans). One isolate, obtained from a building in Rio de Janeiro inhabited by pigeons, belonged to the AD hybrid AFLP genotype 3. All isolates from trees of C. neoformans var. gattii (= C. gattii) belonged to AFLP genotype 6, and their banding patterns showed relatively low genetic homogeneity with a similarity value of about 76%. Isolates of this genotype occupy an environmental niche in the Americas, and they may cause disease in non-AIDS and AIDS patients as well.

Cryptococcus neoformans isolated from human dwellings in Rio de Janeiro, Brazil: an analysis of the domestic environment of AIDS patients with and without cryptococcosis.

One hundred and fifty-four human dwellings in the metropolitan area of Rio de Janeiro, Brazil were studied. A total of 824 samples of indoor dust, outdoor soil and avian droppings were collected. Cryptococcus neoformans var. neoformans was isolated from 20 (13%) dwellings, comprising five (15.6%) of 32 dwellings of patients with AIDS-associated cryptococcosis; four (8.9%) of 45 dwellings of patients with AIDS but without cryptococcosis; and 11 (14.3%) of 77 dwellings of apparently healthy individuals (P>0.05). The principal factor associated with domiciliar contamination by C. neoformans var. neoformans was the presence of avians in the domestic environment or nearby the home. Cryptococcosis was more frequent among AIDS patients residing in dwellings from which C. neoformans var. neoformans was isolated than among AIDS patients from whose domestic environment the fungus was not demonstrated by the methods used (odds ratio (OR)=2.05). These findings suggest that the distribution of C. neoformans var. neoformans in Rio de Janeiro is not restricted to the classically known biotopes as well as reinforce the possibility of exogenous infection in opportunistic cryptococcosis, including exogenous infection acquired in the domestic environment.

Coccidioides immitis isolated from armadillos (Dasypus novemcinctus) in the state of Piauí, northeast Brazil.

Natural infection of armadillos with Coccidioides immitis was studied in the state of Piauí, northeast of Brazil, endemic for coccidioidomycosis. In 1998, 26 nine-banded armadillos (Dasypus novemcinctus) were captured in 4 different counties. The animals were sacrificed under deep anesthesia with ether. At necropsy fragments of spleen, liver, lungs and heart were homogenized and seeded onto Sabouraud dextrose agar with and without cycloheximide (BBL, USA). Part of each organ was also processed for histological examination. Suspected colonies of filamentous fungi observed after the second week of incubation at room temperature, exhibiting barrel-shaped arthroconidia alternating with empty spaces, were inoculated intraperitoneally into mice. Three armadillos proved to be infected with C. immitis. Mice inoculated with suspected colonies obtained from homogenized spleen of three and liver of two armadillos developed disseminated coccidioidomycosis and immature and mature spherules of C. immitis were disclosed in several organs. For the first time armadillos (D. novemcinctus) were found naturally infected with C. immitis, adding new data on the ecology and on a possible role of these ancestral mammals in the evolutionary life cycle of this fungus.

Possible primary ecological niche of Cryptococcus neoformans.

To study hollows of living trees as natural habitats of Cryptococcus neoformans in an endemic area of cryptococcosis in the northeastern region of Brazil, samples of decaying wood were collected inside 32 hollows of living trees and plated on niger seed agar. Identification of C. neoformans was based upon morphological and physiological tests. Canavanine-glycine-bromothymol medium was used to screen the varieties and Crypto Check Iatron Kit to serotype the isolates. A total of 123 C. neoformans colonies were recovered from samples of six (18.5%) out of 32 hollow trees. C. neoformans var. neoformans and C. neoformans var. gattii were found occurring alone (pink shower tree, fig tree and pottery tree) or sharing the same hollow (pink shower tree). Long lasting positivity (19-36 months) and significant number of cfu of C. neoformans per gram of decaying wood (0.15-21.7 x 10(3) cfu g(-1)) inside hollows of pink shower tree, fig tree and pottery tree were observed, indicating colonization of these habitats by the fungus. For the first time, C. n. var. neoformans and C. n. var. gattii were found sharing the same natural biotope, thus establishing a possible link between them in their life cycle in nature and suggesting the primary natural niche for the species.

Molecular epidemiology of Cryptococcus neoformans isolates from AIDS patients of the Brazilian city, Rio de Janeiro.

A high biodiversity of Cryptococcus neoformans isolates is known to exist in some Brazilian urban areas, raising the possibility that patients may encounter multiple inoculum sources in their daily life. C. neoformans isolates from two groups of AIDS patients with cryptococcosis from Rio de Janeiro were studied by polymerase chain reaction (PCR) fingerprinting and randomly amplified polymorphic DNA (RAPD) analysis. The first group contained 60 serial isolates obtained from 19 patients over periods ranging from 18 to 461 days; the intent was to determine whether the original strain persisted or whether reinfection with a new strain occurred. The second group was made up of 22 isolates from 11 patients, and consisted of a pair of isolates collected from blood and cerebrospinal fluid from each patient either before or shortly after treatment was initiated. The aim was to determine if the patient was infected by different strains simultaneously. All isolates were subtyped by PCR fingerprinting, using minisatellite (M13), and microsatellite [(GACA)4 and (GTG)5] specific primers, and RAPD analysis employing the combined primers 5SOR and CN1. The majority of isolates were C. neoformans var. grubii, specifically, molecular types VNI or VNII, but numerous distinguishable subtypes were found. Only three isolates were C. n. var. gattii (molecular types VGI or VGII). Except in two cases, all isolates obtained from the same patient showed identical PCR profiles independent of time of isolation or body site. Almost all patients, however, carried unique genotypes not found in any other patient. Our results confirm that persistent cryptococcal infection is caused by relapse rather than reinfection, but they also show that in exceptional cases, patients may be infected with more than one C. neoformans strain.

Haploid fruiting in Cryptococcus neoformans is not mating type alpha-specific.

Under appropriate conditions, haploid Cryptococcus neoformans cells can undergo a morphological switch from a budding yeast form to develop hyphae and viable basidiospores, which resemble those produced by mating. This process, known as haploid fruiting, was previously thought to occur only in MATalpha strains. We identified two new strains of C. neoformans var. neoformans serotype D that are MATa type and are able to haploid fruit. Further, a MATa reference strain, B-3502, also produced hyphae and fruited after prolonged incubation on filament agar. Over-expression of STE12a dramatically enhanced the ability of all MATa strains tested to filament. Segregation analysis of haploid fruiting ability confirmed that haploid fruiting is not MATalpha-specific. Our results indicate that MATa cells are intrinsically able to haploid fruit and previous observations that they do not were probably biased by the examination of a small number of genetically related isolates that have been maintained in the laboratory for many years.

Cryptococcus neoformans varieties as agents of cryptococcosis in Brazil.

The study of the clinical isolates of Cryptococcus neoformans from 83 Brazilian patients with disseminated cryptococcosis showed that 75 were C. neoformans var. neoformans and 8 were var. gattii. Twenty-seven isolates were serotyped; all 19 var. neoformans were serotype A and all 8 var. gattii were serotype B. The correlation of the varieties of C. neoformans with the presence or not of hosts predisposing conditions to the mycosis showed that: (1) cryptococcosis caused by gattii variety occurred in 7 (58.3%) of the 12 nonimmunosuppressed patients, and (2) cryptococcosis caused by neoformans variety occurred in 65 (98.5%) of the 66 AIDS patients and in all 5 patients with other immunosuppressive conditions. The comparison of the distribution of the gattii and neoformans varieties between the nonimmunosuppressed and immunosuppressed patients showed a significant statistical difference (p < 0.01).

PIP: 8 mycologists reviewed the medical records of 83 patients with disseminated crypttococcosis living in Brazil to examine the varieties of Cryptococcus neoformans. 12 patients were not immunosuppressed (group 1). 65 patients had AIDS (group 2). 5 patients either also had another disease and/or used immunosuppressive drugs (group 3). Almost all the clinical isolates were from the cerebrospinal fluid and/or blood (80 isolates [96.4%]. C. neoformans var. gattii caused cryotococcosis in 58.3% (7) of the nonimmunosuppressed patients. C. neoformans var. neoformans was responsible for crypttococcosis in all but 1 of the AIDS patients (98.5%) and in all 5 patients who had another disease and/or used immunosuppressive drugs. 6 of the 8 cases with crypttococcosis caused by C. neoformans var. gatti lived in rural areas during their first 10 years. A statistically significant difference existed between the nonimmunosuppressed group (group 1) and the immunosuppressed patients (groups 2 and 3) in the comparison of the distribution of the gattii and neoformans varieties (p.01). The omnipresence of C. neoformans var. neoformans, the urban population's regular exposure to C. neoformans var. neoformans, and predisposing conditions (i.e., immunosuppression) to crypttococcosis likely accounted for the neoformans variety mainly being the cause of crypttococcosis in areas in Brazil where C. neoformans var. gattii is indigenous.

Natural habitat of Cryptococcus neoformans var. neoformans in decaying wood forming hollows in living trees.

Cryptococcus neoformans var. neoformans was repeatedly isolated from decaying wood forming hollows in living trees growing in urban areas of Rio de Janeiro, Brazil. A new natural habitat for C. neoformans var. neoformans has been found that is not associated with specific trees.

Chronic pulmonary histoplasmosis in the State of Rio de Janeiro, Brazil.

Three cases of chronic pulmonary histoplasmosis affecting aged patients with chronic obstructive pulmonary disease are reported. They had a history of recurrent episodes of respiratory infection and presented radiological lung lesions inducing a misdiagnosis of chronic pulmonary tuberculosis of the adults. The diagnosis of histoplasmosis, suggested by the immunodiffusion test and the detection of yeastlike cells in smeared and stained sputum, was confirmed by the isolation and identification of Histoplasma capsulatum var. capsulatum in selective media. The treatment was carried out with amphothericin B and ketoconazole or itraconazole. Clinical, radiologic, mycologic and serologic improvement was obtained in all the patients. However, relapses occurred within a period of 1 to 18 months after the interruption of the treatment. Mycological diagnosis and the difficulties observed in the treatment were discussed. In addition data on the epidemiology of histoplasmosis in the state of Rio de Janeiro, Brazil, were presented.

Investigation of an outbreak of endemic coccidioidomycosis in Brazil's northeastern state of Piauí with a review of the occurrence and distribution of Coccidioides immitis in three other Brazilian states.

An outbreak of coccidioidomycosis is described that involved three individuals and eight of their dogs, who had engaged in a successful hunt for nine-banded armadillos (Dasypus novemcinctus) in the environs of Oeiras, a community in Brazil's north eastern state of Piauí. Diagnosis was based on clinical, serological and cultural findings. Four of 24 soil samples collected in and around the burrow of an armadillo yielded cultures of Coccidioides immitis, thus establishing the endemicity of that mould in the state of Piauí. A literature review revealed that C. immitis, aside from that state, is endemic in three other Brazilian states--Bahia, Ceará and Maranhão. These four contiguous states have semi-arid regions where climatic conditions and their flora are similar to those that exist in C. immitis's endemic regions in North, Central and South America.

Antifungal susceptibilities, varieties, and electrophoretic karyotypes of clinical isolates of Cryptococcus neoformans from Brazil, Chile, and Venezuela.

One hundred clinical isolates of Cryptococcus neoformans from human immunodeficiency virus (HIV)-infected and non-HIV-infected patients from Brazil, Chile, and Venezuela were separated according to varieties and tested for antifungal susceptibility. A high susceptibility to antifungal agents was observed among all the isolates. The electrophoretic karyotyping of 51 strains revealed good discrimination among Cryptococcus neoformans var. neoformans strains.

Cryptococcus neoformans shows a remarkable genotypic diversity in Brazil.

The genotypic diversity of Brazilian Cryptococcus neoformans strains was analyzed. The majority of the samples were alphaA (65%), followed by alphaB (17.5%), alphaD (9%), alphaAaD hybrids (5%), and alphaC (3.5%). A considerable genotypic diversity occurred within C. neoformans var. grubii, and a new amplified fragment length polymorphism genotype, 1B, was recognized.