Environmental isolation and characterisation of Cryptococcus species from living trees in Havana city, Cuba.

Cryptococcus isolates from Cuban patients were identified as C. neoformans var. grubii. Although this species has since long been associated with bird droppings, a recent genotyping study provided strong evidence for additional origins of exposure. We sampled different species of trees in Havana, Cuba to identify other potential sources of exposure to this fungus. A total of 662 samples were collected from 331 trees and cacti from Havana, Cuba. Initial selection of the isolates was carried out by conventional techniques. Isolates were further characterised using a combination of AFLP analysis and DNA sequence analysis. Identification by conventional methods yielded 121 C. neoformans and 61 C. gattii isolates. Molecular analyses showed that none of these isolates was C. gattii and only one isolate proved to be C. neoformans var. grubii. A total of 27 different other species were identified. The most prevalent species was C. heveanensis (33%). Sixty-five unidentifiable isolates segregated into ten potentially novel species. Conventional cultivation methods have a low specificity for C. neoformans complex and molecular analyses need to be applied to confirm identification of isolates from environmental sources. Environmental niches responsible for most of human cryptococcal infections in Cuba remain to be identified.

[Preservation of high risk fungal cultures of Histoplasma and Cryptococcus]. / Conservación de cultivos fúngicos de alto riesgo de Histoplasma y Cryptococcus.

INTRODUCTION: culture collections are responsible for providing the microbial resources for development of biological sciences. Storage in distilled water is one of the easiest and least expensive method for long-term fungal preservation. OBJECTIVE: to evaluate the usefulness of this preservation method in fungal culture of Histoplasma and Cryptococcus. METHODS: the preservation condition of the highest biological risk species from Histoplasma y Cryptococcus genera, included in the fungal culture collection of "Pedro Kouri" Institute of Tropical Medicine in Havana, was evaluated in this study. One hundred and two strains stored in distilled water, 92% of which had been preserved for more than 10 years, were analyzed. RESULTS: the percentages of recovered strains from H. capsulatum, C. neoformans and C. gattii were 64.3%; 79.1% and 100% respectively. This method of preservation proved to be satisfactory for fungal culture in labs with limited financial resources. A web-based database with interesting information about the collection was made. The importance of strict compliance with the biosafety measures in these collections, particularly with high risk pathogens. CONCLUSIONS: preservation of fungal cultures in distilled water is a very useful method for laboratories with limited resources. Culture collections should be assumed as an essential activity in order to solve increasing challenges in the development of biomedical sciences.

[Histoplasmosis updating]. / Una actualización acerca de histoplasmosis.

Histoplasmosis, an infection caused by the fungus Histoplasma capsulatum, has been reported all over the world and is considered endemic in the American continent, including Cuba. This fungus grows on the soils contaminated with bird and bat excreta, where it produces a great number of microconidia that could cause the infection when they are inhaled. The clinical spectrum varies from asymptomatic infections to serious disseminated diseases involving one or many organ systems and affects mainly AIDS patients, patients with hematological neoplasias, transplant recipients or other immunosuppressed patients. The main risk groups include those individuals whose working activities make them be exposed to aerosols contaminated with H. capsulatum. Lab diagnosis is based on the microscopic observation, isolation and identification of the fungus in fluid or tissue samples of patients, and on specific antigen and antibodies detection. The molecular methods based on polymerase chain reaction have not been sufficiently defined, and they could be an important advance in the early diagnosis of this mycosis. Itraconazole is recommended for treatment of moderate, localized and chronic infection whereas amphotericin B is the drug of choice for disseminated and serious manifestations, particularly in its lipidic formulations. At present, histoplasmosis is considered one of the most important systemic mycoses in the Americas, and it is widely spread over all regions of Cuba.

[Sensitivity of clinical isolates of Candida albicans to 5-fluorocytosine]. / Sensibilidad de aislamientos clínicos de Candida albicans frente a la 5-fluorocitosina.

5-fluorocytosine has been one of the most used antimycotics in the therapeutics of mycosis, however the appearance of resistant strains may be an important limitant for its use. That's why it is necessary to know the sensitivity to this drug. To this end, it was determined the minimum inhibitory concentration (MIC) of 5-fluorocytosine against 50 clinical isolates of Candida albicans by a micromethod of dilution in RPMI 1640 media, comparing 2 reading methods. The values of the geometrical mean obtained were 0.95 mg/mL for the visual reading and 0.75 mg/mL for the automated reading. There were no significant differences between them (p < 0.05). Pearson's lineal correlation coefficient was r = 0.81. It indicates that although there was a good correlation, there was also discrepancy between both readings. The IMC50 was of 0.5 microgram/mL and the IMC90 of 4 mg/mL. 90 and 92% of sensitive strains, between 6 and 8% of resistant strains and 2% of strains with intermediate sensitivity were found depending on the reading method used. These values show a low resistance against this drug, which may have a direct repercussion on the therapeutic behaviour to be followed in candidiasis.

[Determination of the minimum inhibitory concentration of fluconazole against Cryptococcus neoformans]. / Determinación de la concentración mínima inhibitoria de fluconazol frente a Cryptococcus neoformans.

The minimum inhibitory concentration of fluconozole was determined in 36 strains of Cryptococcus neoformans var. neoformans by a casitone broth microdilution method, taking into account that it is one of the most used antifungal agents in our country for the treatment of cryptococcal meningitis, the second opportunistic mycosis in order of frequency and that with the highest lethality among AIDS patients. The minimum inhibitory concentration of the studied strains ranged 0.125 to more than 64 micrograms/mL for a geometrical mean of 2.38 micrograms/mL. 50% of the strains were inhibited with 4 micrograms/mL and the least concentration that inhibited 90% was 16 micrograms/mL. It is stressed the importance of continuing this type of in vitro studies to detect the appearance of resistance of C. neoformans to antifungal agents.

[Isolation of anti-Cryptococcus neoformans immunoglobulin G]. / Obtención de inmunoglobulina G anti-Cryptococcus neoformans.

Two immunization schemes were applied to rabbits with an autoctonous strain aimed at obtaining an anti-Crytococcus neoformans IgG. Whole cells treated with formalin were used. Dosages and immunization routes were different. The sera obtained were titered and that with the highest titer was selected (1:1 024 by the laminar agglutination method) and purified by saline precipitation and ion exchange chromatography. An IgG of high purity and biological activity was obtained.

[Minimum inhibitory concentration of amphotericin B in yeasts of medical interest]. / Determinación de la concentración mínima inhibitoria de anfotericina B en levaduras de interés médico.

In order to know the sensitivity of Candida and Crytococcus to amphotericin B, main drug for the treatment od systemic mycosis, it was determined the minimum inhibitory concentration (MIC) in 90 clinical isolates of Candida and Crytococcus by a micromethod for the broth dilution. According to the results, Crytococcus neoformas was more sensitive then Candida albicans (geometrical means 0.24 and 0.41 respectively). Only one resistant strain was found (CMI = 16 micrograms/mL), corresponding to the Candida krusei species. The introduction of this technique in the Mycology Laboratory of the "Pedro Kourí" Institute of Tropical Medicine will allow to establish the sensitivity patterns and to detect the possible appearance of resistance in the main species of pathogenic fungus for men in our environment.

[Esophageal candidiasis in AIDS patients. A clinical and microbiological study]. / Candidiasis esofágica en pacientes con SIDA. Estudio clínico y microbiológico.

18 AIDS patients who presented high gastrointestinal symptoms or lesions in the oral cavity suggestive of candidiasis were studied. Clinical data, specimen of the oral cavity, biopsies and esophageal brushing, as well as serum from all patients were obtained. Dysphagia was the main symptom observed in the esophageal candidiasis. Candida albicans was the most isolated species with a predominance of serotype A. The protein profiles of 12 strains belonging to this species were analyzed by the polyacrylamide gel electrophoresis, and it was found that their molecular weight patterns were identical, which indicates that the same strain is in the oral cavity and in the esophagus. The response of the anti-C. albicans antibodies was evaluated. The double immunodiffusion was not useful for the diagnosis of esophageal candidiasis.

[The identification of Cryptococcus neoformans var. neoformans in Cuban clinical isolates]. / Identificación de Cryptococcus neoformans var. neoformans en aislamientos clínicos cubanos.

50 strains of Cryptococcus neoformans of clinical origin were studied by using the canavanine-glycine-bromothymol blue (CGB) medium. 56% of the strains were isolated from AIDS patients, and 16% belonged to patients with kidney transplantation. 90% of the samples were obtained from the samples of cerebrospinal fluid, which corresponded to the classical form of presentation of cryptococcosis. All the strains were identified as C. neoformans var. neoformans, coinciding with previous reports made in Cuba. Knowing the varieties of C. neoformans, inferences can be drawn on the epidemiology, clinics and response to the treatment of cryptococcosis.

[Isolation, identification, and typing of yeasts from HIV-positive patients with oral candidiasis]. / Aislamiento, identificación y tipificación de levaduras en pacientes VIH positivos con candidiasis oral.

A study of oral candidiasis was conducted among 25 HIV-positive and AIDS patients. The predominant clinical form of presentation was the pseudomembranous one, whereas the most frequently yeast species found in the isolations were: Candida albicans (54.1%), Candida tropicalis (8.1%), and Torulopsis glabrata (8.1%). It was demonstrated that patients with recurrent infections have colonization of the oral cavity by multiple strains and/or yeast species more often (30.8%) than those through their first episode of oral candiadisis (12.5%). Of the 3 culture media used for the initial isolation, the combination of the potatodextrose agar (PDA) with Sabouraud triphenytletrazolium agar (STA) allowed to obtain the maximum isolation and differentiation among strains. The Sabouraud agar (SA), the most used medium to these ends at the international level, proved to be less useful than the previous ones. The STA was very efficient in the studies of phenotypic typing of most of the isolated yeast species and specially of C. albicans, which will make possible to approach epidemiological studies.

[Progressive disseminated histoplasmosis in AIDS patients]. / Histoplasmosis diseminada progresiva en pacientes con SIDA.

The clinical histories of 12 AIDS patients whose histoplasmosis diagnosis has been confirmed by histopathological studies were reviewed aimed at knowing the main clinical and microbiological aspects of the disseminated progressive histoplasmosis. In every case it appeared as a febrile and waste disease with an elevated hepatomegaly frequency and generalized adenopathies. More than 50% of the patients had anemia and an accelerated erythrocyte sedimentation. The chest X-ray showed pulmonary lesions in 50% of the patients, whereas, the abdominal ultrasound allowed to observe hepatomegaly and splenomegaly in 58.3 and 41.6% of the patients, respectively. A low sensitivity was found in the culture of different clinical specimens as well as in the antibody detection techniques. It is discussed the need to improve tha laboratory diagnosis methods, particularly those which make possible the antigen detection in a fast and specific way.

[The evaluation of antigens and antisera for their use in the serodiagnosis of aspergillosis]. / Evaluación de antígenos y antisueros para su utilización en el serodiagnóstico de aspergilosis.

Aspergillosis became an important opportunistic mycosis during the last years, with a great variety of clinical manifestations. To contribute to the replenishing of this mycosis serodiagnosis, biologic reactives (antigens and antisera) were prepared from strains of the species Aspergillus niger, Aspergillus flavus and Aspergillus terreus for their use in immunoprecipitation assays. The reactives were assessed by double immunospreading versus a reference commercial system; satisfactory results were obtained, and this guarantees the widening of the aspergillosis serodiagnosis in the Mycology Laboratory of the "Pedro Kourí" the Tropical Medicine Institute (IPK) with important imports savings.

[Relationship of histoplasmin intradermal tests and antibodies levels detected by ELISA and immunodiffusion]. / Relación entre la prueba intradérmica de histoplasmina y los niveles de anticuerpos detectables por ELISA e inmunodifusión.

A prospective study was carried out in two groups of individuals: a group 1 (n = 40) included workers from a poultry farm, with potential occupational risk of exposure to Histoplasma capsulatum, etiologic agent of histoplasmosis, and a group 2 (n = 16), persons without occupational risk of exposure to the agent. Histoplasmin skin test was performed in both groups, and three sera were obtained from each individual: 1) before skin test was done, 2) 30 days after, and 3) 180 days after it. In both groups the histoplasmin skin test, even when the test was positive, was not a sufficient antigenic booster to provoke an increase in the H. capsulatum antibody levels capable to be detected by the serologic tests used (ELISA and Double Immunodiffusion). These results contribute to improve the interpretation of ELISA test values in the diagnosis of histoplasmosis.

[Clinico-mycological study of onychomycosis in elderly patients]. / Estudio clínico micológico de onicomicosis en ancianos.

Physical examination of nails was carried out in 210 elderly patients and nail scrapings were obtained from onychomycosis suggested lesions in order to determine their causative agents, incidence and clinical characteristics. Diagnostic was confirmed by the isolation of the agents from 74 patients, mainly from toe-nails (incidence 35.2). Tinea pedis occurred in 25% of the cases and Diabetes mellitus was the most prevalent associated disease and the most frequent clinical characteristics were the thickening, the opacity and the presence of longitudinal strias in the surface of the nails. It was compared the results obtained by microscopic examination and by culture. Trichophyton rubrum was the most common dermatophyte isolated; Candida parapsilosis was dominant among Candida species.

[The sources of histoplasmosis infection on the Isla de la Juventud, Cuba]. / Fuentes de infección de histoplasmosis en la Isla de la Juventud, Cuba.

The purpose of this work is to report the isolation of Histoplasma capsulatum, etiologic agent of histoplasmosis, from soil in sites inhabited by bats and chicken in the Island of Youth, Cuba. The fungus was cultured from four species of cave dwelling bats too. The identification of H. capsulatum was done by mycelial to yeast conversion and exoantigen test. It is pointed out the epidemiological value of some of these isolations in caves of great importance from the archaeological, speleological or tourist point of view, and the potential risk that they represent to human health. The authors conclude with some recommendation to prevent the infection with H. capsulatum in people who have to keep in contact with those environments.

[Histoplasmin reactivity in poultry farm workers in the province of Ciego de Avila, Cuba]. / Reactividad a la histoplasmina en trabajadores de granjas avícolas en la provincia de Ciego de Avila, Cuba.

An epidemiological survey with histoplasmin skin test was performed in 392 poultry farmers and 265 workers considered without occupational risk of exposition to Histoplasma capsulatum, etiologic agent of histoplasmosis. The results were positive in 28.8% and 13.2% in both groups respectively. Statistically, there was a significant difference between the two groups, so it can be considered that poultry farmers are in occupational risk of infection with H. capsulatum. In the first group, the workers which are more closely related with chicken manure showed a higher reactivity to histoplasmin skin test. The working time in the farms seems to influence in the test reactivity too.

[The diagnosis of cryptococcosis. A comparison of 2 latex systems for antigen detection]. / Diagnóstico de criptococosis. Comparación de 2 sistemas de látex para la detección de antígeno.

A latex reactive was designed for detecting Cryptococcus neoformans antigen. It was evaluated by a comparative study with a commercial system (Meridian Diagnostic, Inc.). Total coincidence was observed with both latex systems after studying 3 sample groups: patients with cryptococcosis diagnosis, blood bank donors and patients with clinical signs of the disease. Sensitivity, specificity and stability of the latex reactives prepared were assessed. This diagnostic technique opens new perspectives for quick diagnosis in Cuba, especially for immunosuppressed patients. It will allow for timely treatment and at the same time will contribute to saving expenses in imports.

[Detection of anti-Histoplasma capsulatum antibodies by the ELISA technique. Preliminary study]. / Detección de anticuerpos anti-Histoplasma capsulatum mediante la técnica de ELISA. Estudio preliminar.

An indirect micro-ELISA system is presented for diagnosing histoplasmosis. The diagnostic criteria are defined by using sera from 12 patients who are histoplasmosis carriers. For this group, the optical density values were superior to 1,000; use was made of 43 sera from blood bank donors and 9 sera from children without a history of exposure. The optical density values in these cases were inferior to 0,200. The significant difference found led to the diagnostic criterion for confirming 3 histoplasmosis carriers who showed clinical manifestations but had been negative to double immunodiffusion. Thus, the usefulness of the proposed micro-ELISA system for early diagnosis was proved.