Valerio Vidotto

Extracellular proteolytic activity of Cryptococcus neoformans.

Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.

Phospholipase activity in Cryptococcus neoformans

Phospholipases have only been detected in a few fungi and yeasts, in particular in Candida albicans. Secreted phospholipases are considered by some researchers to be a potential factor of virulence and pathogenicity in C. albicans. Twenty-three Cryptococcus neoformans strains were tested in order to observe phospholipase production. Twenty-two of the 23 strains tested were able to produce phospholipases, and the ratio diameter of the colony to total diameter of the colony plus zone of precipitation (Pz) ranged between 0.271 and 0.949. C. neoformans, just like C. albicans, can be divided on the basis of the Pz into different strains according to their virulence and pathogenicity. There also appeared to be a correlation between the phospholipase production and the size of the capsule in the strains isolated from AIDS patients. For this reason, further studies on C. neoformans phospholipase activity would be useful in evaluating the virulence of different strains.

Serotype identification of Cryptococcus neoformans by multiplex PCR

The pathogenic yeast Cryptococcus neoformans is traditionally classified into three varieties with five serotypes: var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C) and serotype AD (hybrid of serotypes A and D). A commercial kit, Crypto Check (Iatron Laboratories, Tokyo, Japan), has been used worldwide for serotyping isolated strains. However, its production was discontinued in 2004, and hence the present study aimed to develop a simple polymerase chain reaction (PCR) method for serotyping C. neoformans strains. Subjecting genomic DNA of 59 strains of the five serotypes to multiplex PCR amplification using a set of four primers designed for the laccase gene (LAC1) differentiated serotypes A, D, B and C, but could not separate serotype AD from serotype D. However, a primer pair designed for the capsule gene (CAP64) allowed serotypes D and AD to be differentiated. When PCR amplification was performed in the simultaneous presence of the above six primers, the five serotypes produced two to five DNA fragments that could be used to distinguish them. This multiplex PCR method is useful for serotyping C. neoformans isolates, and represents an effective replacement for the Crypto Check kit.

Biofilm development by clinical isolates of Malassezia pachydermatis.

Malassezia pachydermatis fungemia has been reported in patients receiving parenteral nutrition. Biofilm formation on catheters may be related to the pathogenesis of this mycosis. We investigated the biofilm-forming ability of 12 M. pachydermatis strains using a metabolic activity plate-based model and electronic microscopic evaluation of catheter surfaces. All M. pachydermatis strains developed biofilms but biofilm formation showed variability among the different strains unrelated to their clinical origin. This study demonstrates the ability of M. pachydermatis to adhere to and form biofilms on the surfaces of different materials, such as polystyrene and polyurethane.

Phagocytic activity in human immunodeficiency virus type 1 infection.

Macrophages and neutrophils constitutes one of the main effectors of nonspecific immunity, phagocytosing conventional and opportunistic microorganisms and presenting their antigens to T lymphocytes. This consequently induces specific immune functions ([13][1], [91][2]). Moreover, activated

Dermatophytoses in Cusco (Peru).

Summary. A total of 90 cases of suspected dermatophytoses was observed over a period of 9 months in the city of Cusco (Peru) and in its environs. The age groups most commonly infected were 1–10 and 11–20 year old. The most common tineae diagnosed were tinea capitis (13.3%) and tinea unguium (11.1%). Only four species of dermatophytes were isolated: Microsporum canis (52.4%), Trichophyton mentagrophytes (35.7%), Tr. rubrum (9.5%) and Epidermophyton floccosum (2.4%). Dry weather, low temperature and high altitude do not seem to affect proliferation and infections by dermatophytes.

Actividad enzimática extracelular en Cryptococcus neoformans en diferentes países

Three hundred and ten Cryptococcus neoformans strains isolated from AIDS patients in five different countries (151 from Brazil, 23 from Italy, 28 from Spain, 104 from Thailand and four from Turkey) were tested by the API-ZYM kit to detect their extracellular enzymatic activity. The enzymes esterase (C4) (no 3), esterase lipase (C8) (no 4), leucine arylamidase (no 6) and acid phosphatase (no 11) were commonly positive in most of the strains (more than 95%). These enzymes could be considered a useful tool not only for C. neoformans identification, but in particular for their possible relationship to new C. neoformans virulence factors and also for epidemiological research. Interestingly, it is also the high positive percentage of alpha-glucosidase and beta-glucosidase detected in all isolates. The serotype A was the most predominant serotype in all countries, except for Italy where the serotype D was predominant. Further studies are needed to draw a clear correlation between the API-ZYM profile and serotype.Three hundred and ten Cryptococcus neoformans strains isolated from AIDS patients in five different countries (151 from Brazil, 23 from Italy, 28 from Spain, 104 from Thailand and four from Turkey) were tested by the API-ZYM kit to detect their extracellular enzymatic activity. The enzymes esterase (C4) (no 3), esterase lipase (C8) (no 4), leucine arylamidase (no 6) and acid phosphatase (no 11) were commonly positive in most of the strains (more than 95%). These enzymes could be considered a useful tool not only for C. neoformans identification, but in particular for their possible relationship to new C. neoformans virulence factors and also for epidemiological research. Interestingly, it is also the high positive percentage of alpha-glucosidase and beta-glucosidase detected in all isolates. The serotype A was the most predominant serotype in all countries, except for Italy where the serotype D was predominant. Further studies are needed to draw a clear correlation between the API-ZYM profile and serotype.

Actividad enzimática extracelular y serotipo en cepas de Cryptococcus neoformans de pacientes con sida en Brasil

Resumen Se estudiaron las actividades fosfolipasa, proteasa y otras actividades enzimaticas extracelulares de 151 cepas de Cryptococcus neoformans aisladas de pacientes con sida y mantenidas en el Instituto Adolfo Lutz (Sao Paulo, Brasil). La actividad enzimatica extracelular se determino por medio del test API-ZYM, que evalua 19 enzimas extracelulares, y el serotipo mediante aglutinacion celular (Crypto-check, Patron, Japon). De las 151 cepas de C. neoformans ensayadas por aglutinacion 147 resultaron identificadas como serotipo A y solamente cuatro como serotipo AD. La produccion de fosfolipasa y proteasa resulto muy abundante, sobre todo en los primeros dias de incubacion. Es importante anadir que todas las cepas presentaron actividades fosfolipasa y proteasa. Por medio del test API-ZYM mas del 90% de las cepas de C. neoformans resultaron positivas para los siguientes enzimas: esterasa C4, esterasa-lipasa C8, leucina-arilamidasa, fosfatasa acida, naftol-AS-BI-fosfohidrolasa, α-glucosidasa y s-glucosidasa. Se han observado diferencias entre las actividades enzimaticas de las cepas brasilenas y las aisladas en otras naciones. Las actividades fosfolipasa, proteasa y de otros enzimas extracelulares pueden facilitar la invasion de C. neoformans en el tejido del huesped.

First reported Nocardia otitidiscaviarum infection in an AIDS patient in Italy

Nocardiosis is a well-described infection in immunocompromised patients, and has been rarely documented in patients with AIDS.Nocardia asteroides is the most frequently isolated etiologic agent. Rare cases are due toN. brasiliensis andN. otitidiscaviarum. This work describes the first case of nocardiosis in Italy caused byN. otitidiscaviarum in an AIDS patient. A 31 year-old intravenous drug abuser with a diagnosis of full-blown AIDS, presented with high fever and lymphadenitis with a fistula draining copious purulent discharge. Broad-spectrum antibiotic therapy was initiated, but the patient did not show any improvement. Direct examination of the pus revealed numerous gram positive rods. When culturedN. otitidiscavarium was isolated and identified by morphological and biochemical tests.

Glucose influence on germ tube production in Candida albicans

The influence of different glucose concentrations was tested in a minimal synthetic medium onCandida albicans strain. After 18 hours of starvation, germ-tube (GT) production, amount of consumed glucose, oxygen and the pH of the medium were checked every hour from the beginning through the end of the experiment. Optimal GT production was obtained with 1 g/l of glucose. At this concentration the greatest glucose and oxygen consumption were also noted. No pH variations in the medium were observed in all of the glucose concentrations used. At 3 and 5 g/l glucose concentrations a lower GT production were obtained. The Crab-tree effect might interfere with GT production when glucose concentration is higher than 1 g/l. This data may support the hypothesis that GT production is strictly glucose dependent.