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A possible correlation between the presence of discontinuous fringes and high virulence has been previously suggested. The aim of this study was to compare the pathogenicity of Candida albicans and Candida dubliniensis with continuous and discontinuous fringes morphotypes on mice. For C. albicans, two discontinuous fringe morphotype isolates (PN 69, PN 74), two continuous fringe morphotype isolates (N 60, N 33) and one reference strain were used. For C. dubliniensis, three discontinuous fringe morphotype isolates (97487, 97464, 97519), two continuous fringe morphotype isolates (97040, 98026) and one reference strain were used. Swiss male mice were inoculated with a standardised suspension of the microorganisms and observed for 35 days. The pathogenicity of the isolates was analysed according to parameters proposed previously. Three isolates were considered pathogenic: PN 74, N 60 and 98026. Strain N 60 killed the highest amount of mice (80%). Animals inoculated with C. albicans did not show differences on survival estimate. Candida dubliniensis 98026 was more pathogenic than samples 97464 and 97519. On the other hand, the sample 97487 showed a higher pathogenicity when compared with 97040 (Kaplan-Meier test, P = 0.008). Strains with continuous fringe morphotypes were also associated with Candida sp. virulence in vivo., (© 2010 Blackwell Verlag GmbH.)

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.

Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.

A stray, young male, wire-haired pointing griffon dog, found in a street of Perugia (Italy), was examined in order to check his health status. Two oropharyngeal swabs were collected in 24 h and streaked onto Sabouraud agar and after 6 days the yeasts colonies were transferred onto Malt agar. Ascospores were observed on Potato Dextrose Agar medium. The major ubiquinone of an isolated yeast was identified as ubiquinone-9 (Q-9), and genetical analyses were performed together with the type strains of Debaryomyces hansenii (var. hansenii and var. fabry), C. psychrophila and D. nepalensis type strain. The base sequences of ITS1 and ITS2, and D1/D2 domains of LSU rDNA completely coincided with those of D. nepalensis. From these results, the isolated yeast was identified as D. nepalensis. RAPD patterns between the two strains were found to be identical. The results indicate the first colonization of D. nepalensis in a dog.

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.

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.

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.

In screening for antifungal substances, a new macrolide, eushearilide (1), was isolated from Eupenicillium shearii IFM54447. The structure of 1 was established to be 24-membered macrolide having a non-conjugated diene and a choline phosphate ester moetiy on the basis of detailed investigation of NMR, UV, IR and MS spectral data. Compound 1 showed antifungal activity against various fungi and yeasts, including human pathogens Aspergillus fumigatus, Trichophyton spp. and Candida spp.

Sixty isolates of Candida albicans, 30 obtained from the oral cavity of denture wearers presenting signs of candidosis and 30 obtained from the oral cavity of denture wearers with normal palatal mucosa were assayed for phospholipase and proteinase production, as well as for adherence to buccal epithelial cells. Likewise, susceptibility of the isolates to antifungals was determined by the NCCLS reference method and the E-test method. Proteinase activity was increased among the strains obtained from oral candidosis patients. In contrast, no significant differences between the two groups of isolates were observed in their adherence ability in vitro, in phospholipase production, and susceptibility to antifungal drugs.

Interleukin (IL)-18 is a proinflammatory cytokine that plays an important role in both innate and adaptive immune responses against several infectious pathogens. Relatively little is known about its production in HIV-1 infection, and there are controversial data on the influence of IL-18 on HIV-1 replication in vitro. In this study, we investigated the effect of HIV-1 infection, and challenge with recombinant HIV-1 proteins, on IL-18 production by THP-1 cells. This is a monocytoid cell line spontaneously producing IL-18, and consequently is particularly suitable for the study of HIV-1 effects on this type of cytokine regulation. The results reported here demonstrate a significant reduction in IL-18 secretion during HIV-infection. In fact, low levels of IL-18 were released until 120 h from viral challenge (15 +/- 11 pg/mL at 24 h and 17 +/- 13 at 96 h and < 12.5 at 120 h), whereas IL-18 production by uninfected control cells was 193 +/- 104 pg/mL and 214 +/- 114 pg/mL at 24 h and 120 h respectively. At 168 h of incubation, IL-18 production by infected and uninfected cells was found to be 164 +/- 88 pg/mL and 325 +/- 101 pg/mL respectively (p = 0.001). Of the following viral proteins: gp 120, p24 and Nef, only the last one induced decreased IL-18 secretion in the supernatants of THP-1 cells. This effect is more evident with the concentrations of 5 -1.25 microg/mL of Nef protein (p < 0.0001). In conclusion, our data show that HIV-1 and its regulatory protein, Nef, are able to down-regulate the release of IL-18, in vitro. These results confirm that a variety of modulating effects on the immune response, induced by HIV-infection, may facilitate progression of HIV-1 infection.

The authors have reviewed some biological properties of HIV-1 Tat protein, and have also reported some personal data. This viral regulatory protein is endowed with multifunctional activities, acting as an endogenous factor in the infected cells and exogenously, on those uninfected. In particular, Tat-induced proliferation and differentiation of HIV target cells which promotes viral infection, is discussed in this review. However, exogenous Tat protein can sometimes also produce, directly or indirectly, damaging effects in different organs and host systems, such as myocardium, kidney, liver and central nervous system (CNS). For example some data also demonstrate an increase in the apoptotic index induced by Tat at various levels, including the immune system. The effective role of HIV-1 Tat protein in promoting viral replication and its high immunogenicity suggest useful employment of this protein for therapeutic or preventive vaccine preparations., (Copyright 2004 John Wiley & Sons, Ltd.)

One hundred and fifty-one Cryptococcus neoformans strains isolated from AIDS patients in Brazil and maintained in the Adolfo Lutz Institute (São Paulo, Brazil) were tested for phospholipase, protease and other extracellular enzymatic activities and their serotypes determined. Production of extracellular phospholipase and protease was tested by the agar plate methods. Determination of extracellular enzyme profile of the strains was performed by using the API-ZYM kit system, which can test 19 different enzymes. The serotypes were determined by cell agglutination using the Crypto-check method. Among the 151 strains, 147 were identified as serotype A and four strains were serotype AD. Production of extracellular phospholipase and protease was extensive and observable at early stages of incubation. All of the tested strains were positive for the production of both enzymes. In the API-ZYM tests, more than 90 % of the 151 tested strains were positive for esterase C4 (No. 3), esterase lipase C8 (No. 4), leucine arylamidase (No. 6), phosphatase acid (No. 11), naphtol-AS-BI-phosphohydrolase (No. 12), alpha-glucosidase (No. 16) and beta-glucosidase (No. 17). Differences in enzymatic activities between the Brazilian strains and strains isolated in other countries were observed. The phospholipase, protease and other enzyme activities may play a role in host tissue invasion by C. neoformans.

Cryptococcus neoformans is a fungus causing life-threatening infections in immunocompromised hosts. Melanin production is a major virulence factor of this fungus and the initial steps of dihydroxyphenylalanine (DOPA)-melanin biosynthesis pathways are catalyzed by laccase. To understand phylogenetic relationships among serotypes of three varieties, partial sequences (about 600 bases) of the laccase gene (CNLAC1) were determined in a total of 64 strains, including 10 melanin-deficient variants. The phylogenetic tree constructed from the nucleotide sequence grouped the 64 strains into the clusters corresponding to the three varieties. The diversity of the fragment sequences was very minor among strains of each of var. grubii and var. neoformans. Strains in var. gattii, however, were subdivided into two groups, although differences between serotypes B and C were not large. The sequences of the melanin-deficient variants were almost completely homologous to those of the melanin-producing strains in the same serotype. Results of laccase assay and northern blot analysis suggested that the lower melanin production in the variants was associated with lower transcription of the laccase gene.

Melanin production is one of the most important criteria for rapid identification of Cryptococcus neoformans. Most of the media described in the literature for identifying C. neoformans are very complex; they contain many organic or inorganic compounds and are difficult to prepare and store. The new minimal synthetic caffeic acid medium described in this paper is simpler to prepare, convenient and constitutes an interesting new medium for the rapid identification of C. neoformans isolates.

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.

Manifestations of cardiovascular system involvement are not uncommon complications of HIV infection, especially in AIDS patients. However, the frequency of these manifestations is influenced by different variables including: survival prolongation in HIV-infected patients, because of advances in antiretroviral treatment; improvement of immunodepression and reduction in the occurrence of opportunistic infections; adverse effects of some drugs. At present, on the whole cardiovascular complications that are HIV correlated in the western world, including Italy, occur less frequently than in the past. However complications associated with alterations in lipometabolism prevail because they can be promoted by some protease inhibitors in predisposed subjects. The most frequently reported questions and a careful analysis of recent data in the medical literature regarding the most common HIV-correlated cardiovascular complications are discussed in this review., (Copyright 2004 John Wiley & Sons, Ltd.)

Twenty-seven Candida albicans strains and 26 Candida dubliniensis strains, isolated from HIV patients, were tested for their adherence to buccal and vaginal epithelial cells. Both species showed important levels of adhesion to buccal and vaginal epithelial cells, although C. albicans showed the highest levels of adhesion. These results suggest that both Candida species are well adapted, in terms of adhesion capability, to the oral and vaginal environment.

The aim of this study was to compare microbiological and salivary variables possibly related to caries risk in treated and untreated mouthbreathing syndrome (MBS) children and control children. Thirty control children, 30 mouthbreathers and 25 treated mouthbreathers were studied for the numbers of lactobacilli, mutans streptococci and yeasts in their saliva. Snyder's test, salivary flow and buffering capacity were also evaluated. Levels of immunoglobulins to Candida albicans and Streptococcus mutans in the saliva were quantified using ELISA. Considering the results obtained for the microbiological and salivary caries risk tests, no significant differences were observed among the proportions of patients with small/negative and high/moderate caries risk in the studied groups. The level of IgG to S. mutans was significantly higher in the treated MBS group in relation to MBS patients. On the other hand, the median anti-S. mutans IgM level was lower in the treated MBS patients than in the other groups. For the studied anti-Candida immunoglobulins, IgM level was significantly lower in the treated MBS group than in the other groups. No differences were observed for anti-S. mutans and anti-Candida IgA levels among the groups. The findings suggest that mouthbreathing cannot be considered a risk factor for dental caries., (Copyright 2003 S. Karger AG, Basel)

Interleukin-18 (IL-18) is a recently identified immunoregulatory cytokine expressed by activated macrophages, that induces production of interferon-gamma (IFN-gamma) and Th-1 development. Recently some investigators reported controversial in vitro data on IL-18 stimulation of HIV-1 replication in several cell lines. In the present study the effect of IL-18 on HIV replication in a human chronically HIV-1-infected lymphocytic T cell line (H9-V) was investigated. HIV-1 replication was determined by an immunoassay method in order to evaluate the content of p24 antigen in the cell culture supernatants. Stimulation of H9-V cells with IL-18 resulted in increased production of p24, especially at concentrations of 0.01 microg ml(-1) and 0.10 microg ml(-1). Moreover a significant and persistent IL-18 stimulation of HIV-1 replication was observed at a concentration of 0.01 microg ml(-1) during a 7-day period. Pre-treatment of IL-18 with a specific neutralizing monoclonal antibody significantly reduced HIV-1 replication. These experiments show that IL-18 promotes the increase of HIV-1 replication in human chronically-infected lymphocytic T cells and confirm the role of IL-18 as a proimflammatory cytokine in stimulating and maintaining HIV-1 replication during the course of the disease. In a successive set of experiments, since one of the main activities of IL-18 is the induction of IFN-gamma, we evaluated the effect of this biological modifier on H9-V cells. In particular, IFN-gamma shows a significant effect on cell replication and on reduction of CD4 and CD71 surface expression., (Copyright 2002 John Wiley & Sons, Ltd.)

Background: Tick borne encephalitis (TBE) virus is diffused in some European countries and it is transmitted by tick bites. In Italy Isodex ricinus represents the main vector of the infection, that rarely produces the neurologic manifestations, characterising the secondary phase of the same., Methods: In Italy TBE has been little studied and this only in the Middle and Northern regions of the country. Seroepidemiological researches were done prevalently on subjects at high risk of tick bite, such as hunters or forest guards and especially in Trentino and Tuscany. No precise information about TBE virus diffusion was disposable in the Piedmont region and particularly in the Susa valley where, before our investigation failed the data about it., Results: We found that usual hunters and wild boar breeders seem to be particularly exposed to the risk of TBE virus infection, but none neurologic involvement was detected in the anamnesis of the significantly seropositive subjects and also of the borderline ones, that we have studied, despite the limited number of these subjects., Conclusions: Nevertheless we hope for a following extension of our case report, also in consideration that rare cases of encephalitis of unknown etiology, are signalled in Piedmont.

We previously reported that a respiration-competent parent strain (K) of Candida albicans was more susceptible to the intracellular superoxide radical (O2-) generator paraquat (PQ) than was a respiration-deficient mutant (KRD-19), although both showed a similar sensitivity to extracellularly generated O2-. To clarify the cause of the differential PQ lethality, we developed a chemiluminescence method for measuring O2- generated by C. albicans cells by using the probe methyl-Cypridina-luciferin analogue (MCLA), and examined the effects of PQ on O2- generation in both parent and mutant strains. Endogenous O2- generation without stimulation by PQ was unexpectedly low in both strains. PQ-induced O2- generation in the parent strain was maximal in logarithmic phase cells and lowered in stationary phase cells. In contrast, O2- generation in the mutant remained low throughout the growth phase, even when stimulated by PQ. The extent of PQ-induced O2- generation in the parent strain depended on the carbon source added to the assay mixture; in decreasing order, glucose, glycerol, no carbon source. The inhibitor of the cytochrome respiratory chain, antimycin A, suppressed almost completely the PQ-induced O2- generation in the parent strain. It has been established that PQ is converted to its radical form (PQ+) by receiving a single electron in cells. PQ+ then reduces molecular oxygen to O2- by redox cycling. Thus, the high tolerance to PQ of the respiration-deficient mutant can be explained by minimal PQ+/O2- production due to the limited supply of electrons from the impaired respiratory system.

Melanin synthesis in Cryptococcus neoformans, catalyzed by phenoloxidase activity, is one of the oldest virulence factors known. However, until now, the relationship between melanin production in C. neoformans and its virulence has been poorly understood. Among different chemical compounds only Fe3+ and Cu2+ cations enhance the phenoloxidase activity in C. neoformans. A few reports in the literature describe the influence of different cations on C. neoformans phenoloxidase activity, excluding iron. In this study, 13 C. neoformans strains isolated from AIDS patients and 7 from bird droppings (B.D.), were examined in order to clarify the effect of different K+ concentrations on phenoloxidase activity. A new solid and liquid caffeic acid minimal synthetic medium (MSM-CAF) containing only caffeic acid and ferric citrate with different potassium concentrations was used to evaluate C. neoformans phenoloxidase activity. In the MSM-CAF solid medium the degree of brown pigmentation on the agar plates was read on days 1, 2 and 3 of incubation, and the pigmentation of the C. neoformans strains was classed into 5 categories. The brown pigment of the liquid MSM-CAF test tubes were checked after 24 hours of incubation by measuring the optical density (O.D.) at 480 nm. Three C. neoformans AIDS and B.D. strains, randomly chosen, were tested for phenoloxidase activity, according to the modified protocols of Polacheck et al., Torres-Guerrero et al. and Rhodes. According to the results obtained, it has been observed that K+ does not activate the phenoloxidase activity in the C. neoformans AIDS and B.D. strains. In particular, with an increase in potassium concentrations in the MSM-CAF solid and liquid medium, there was a corresponding inhibition of the phenoloxidase activity on both the C. neoformans AIDS and B.D. strains.

The aim of this study was to examine three serial isolates of Cryptococcus neoformans from a patient with AIDS for genotypical and phenotypical characteristics. The isolates were obtained during a first episode of cryptococcosis (simultaneous sampling of blood and cerebrospinal fluid) and after a relapse 3 years later (sampling of cerebrospinal fluid). Pulsed-field gel electrophoresis and random amplification of polymorphic DNA revealed that the blood isolate 1525 (first episode) was different from the two cerebrospinal fluid isolates (1526, first episode; 1782, relapse), yet the cerebrospinal fluid isolates were indistinguishable from each other regardless of the analysis performed. Phenotypical studies showed that isolate 1782 had significantly improved resistance to phagocytosis and killing by monocytes and polymorphonuclear cells and an altered efficacy in evoking cytokine response (augmentation of tumour necrosis factor-alpha, interleukin [IL]-1beta, IL-10, and interferon-gamma, decrease of IL-12). Interestingly, capsule size and antifungal drug resistance remained unchanged, while production of phospholipase and protease was consistently enhanced in the 1782 isolate with respect to the 1525 and 1526 isolates. In conclusion, in serial Cryptococcus neoformans isolates from a patient with AIDS, phenotypical changes but not molecular changes were documented, thus supporting the role of microevolution as a pathogenetic mechanism(s) for persistence/reactivation of fungal organisms.

Candida albicans virulence is in part mediated by fibronectin (FN) interaction. We compared the adherence level to FN (using Becton Dickinson FN-coated plates) of several strains of yeast isolated from HIV-1 infected or uninfected subjects affected by candidiasis (30 strains from HIV+ subjects and 18 from HIV- subjects). More adhesive strains were found in HIV+ patients than in HIV- subjects. In particular a mean increase of 120 per cent as regards the total number of adhesive cells and 230 per cent as regards the adhesive cells producing germ tubes was detected in the former group of strains as compared to the latter ( p < 0.001 in both cases). The enhancement of FN expression induced by HIV-1 infection, as we have previously demonstrated, can increase interest in the adherence to FN of C. albicans strains isolated from AIDS-affected patients. Moreover, we also underline the important role played by HIV Nef protein in increasing the C. albicans aggressiveness. In fact a significant inhibitory effect of Nef on the phagocytosis of this yeast by macrophages has been demonstrated and the oxidative processes of these cells seem to be down-regulated by this protein., (Copyright 2000 John Wiley & Sons, Ltd.)

Nineteen Cryptococcus neoformans strains isolated from AIDS patients and 16 from bird droppings were tested for their extracellular activity. Typical enzymatic activity that was different from other medically important yeasts was found. The results obtained may indicate that there are new extracellular enzymatic activities that imply a relationship between C. neoformans and its virulence. A correlation among the different enzymatic activities was also investigated and according to the results obtained no relationship was observed among any of the recorded extracellular enzymatic activities. Research on C. neoformans extracellular enzymatic activity is useful not only to better understand its metabolism but in particular to establish a possible relationship between its virulence and pathogenicity.

One-hundred and thirteen Candida albicans strains isolated from patients infected by the human immunodeficiency virus (HIV) and twenty five from HIV-negative individuals were studied. The C. albicans strains isolated from different sites of the body were tested for germ-tube (GT), phospholipase production and serotype. The results obtained indicate that the serotype A was predominant in all the groups except for the vaginal strains. No correlation was observed between phospholipase activity and serotype distribution. Germ tube (GT) production was higher among the serotype B strains. A positive correlation between GT induction and phospholipase activity was observed only for the isolates from the oral cavity. It is possible that the correlation between phospholipase activity and high GT production in C. albicans strains can facilitate the penetration through the mucosa.

It is important to know responses of the pathogenic fungi to reactive oxygen species by which hosts protect themselves against fungal infection. In the present study, sensitivities to the superoxide radical (O2-) and superoxide dismutase (SOD) were compared between a wild-type parent strain and a respiration-deficient mutant of Candida albicans. When their survival was examined on an agar medium containing an intracellular O2- generator, paraquat (PQ), the parent strain was selectively killed by increasing the PQ concentration. In contrast, when cells of both strains were illuminated in a riboflavin solution, they exhibited similar sensitivity to O2- generated extracellularly by photo-reduced riboflavin. There were no large differences in sensitivity to hydrogen peroxide in the two strains. Thus, the high tolerance of the mutant to PQ was suggested to result from low intracellular O2- generation by PQ due to the respiratory deficiency. It is generally accepted that fungal cells contain manganese (Mn)-SOD in the mitochondria and copper and zinc (CuZn)-SOD in the cytoplasm. Cyanide-insensitive SOD activity (attributable to Mn-SOD) was dominant in the parent strain throughout growth phases, whereas cyanide-sensitive activity (attributable to CuZn-SOD) occurred in the mutant. The activity bands of Mn- and CuZn-SODs were clearly separated by electrophoresis of the cell extracts of both strains on non-denaturing polyacrylamide gels. The electrophoretic profiles obtained were consistent with the results of the activity assay. These results showed that the respiratory deficiency affected oxidative stress sensitivity and SOD in C. albicans.

Because several stimuli of microbial origin enhance the activity of nitric oxide synthase (NOS) in human cells of the myeloid lineage, we decided to investigate whether cellular damage induced by Aspergillus terreus mycotoxins could be associated with an increase in NOS activity. A pool of mycotoxins rather than individual toxins was tested so that the natural conditions could be mimicked. In the present study, we report that a crude extract of A. terreus induces cellular damage and increases NOS activity in K-562 cells, an erythroleukaemic cell line in which NOS is particularly active. The specificity of this association was further investigated by using NOS inhibitors and by comparing, in the same cellular model, the effects of the extract with the activity of other microbial toxins of a defined mechanism of action. Canavanine, an inhibitor of NOS, significantly reduced cell death in the presence of the extract, suggesting that cellular damage, induced by the mycotoxins of A. terreus is at least in part mediated by NOS activity. Moreover, Escherichia coli lipopolysaccharide (LPS), known to be a potent NOS inducer, increased NOS activity in our experimental model as well. In contrast, Bordetella pertussis toxin did not show any effect on NOS activity. The results of this study suggest that NOS may be involved in mycotoxicoses.

Determination of the enzymatic profile of 41 Cryptococcus neoformans strains, 20 isolated from AIDS patients and 21 from bird droppings, was performed by using the API-ZYM commercial kit system (Bio-Mérieux, France), which tests 19 different kinds of enzymes. All the strains showed positive enzymatic activity to the esterase (C4) (n. 3). On the contrary, alkaline phosphatase (n. 2), cystine arylamidase (n. 8), trypsin (n. 9), chymotripsin (n. 10), alpha-galactosidase (n. 13), beta-glucuronidase (n. 15), alpha-mannosidase (n. 19), alpha-fucosidase (n. 20) were negative in all the strains. The other 10 enzymes (n. 4,5,6,7,11,12,14,16,17,18) were distributed among the strains in different positive percentages. From the results of each enzymatic profile obtained, the 20 AIDS strains were grouped into 15 types, while the 21 bird dropping strains were grouped into 14 types. Interestingly, only one enzyme profile type occurred in the strains isolated from the AIDS patients and from the bird droppings. These results suggest that the API ZYM system is useful in discriminating between the AIDS strains and the bird dropping strains.

Sixty-six oral strains of Candida albicans, which had been consecutively isolated from 22 normal, young females in three isolation trials at intervals of one to three weeks, were biotyped by their susceptibility to boric acid, cetrimide, silver nitrate, sodium periodate and sodium selenite. The 66 isolates were grouped into 13 resistogram types. An identical biotype strain was found three times and twice in seven and six each of the 22 subjects in the three isolation trials, respectively. In the remaining nine subjects, different strains were found at the three trials. These results suggested that certain strains tended to persist in the oral cavity of the normal subjects although changes in the biotype of oral C. albicans strains occurred to a certain extent.

We have examined the influence of subinhibitory concentrations of several antifungals, the different glucose and ammonium sulphate concentrations in the culture medium as well as the strain variability on the expression in vitro of stress mannoproteins reactive with salivary sIgA in C. albicans and other Candida spp isolates. Irrespective of the conditions used, no reactivity with salivary sIgA was observed in yeast cells grown at 25 degrees C. However, when grown at 37 degrees C, all of the 10 C. albicans strains, but only 9 out of 28 non-C. albicans isolates studied showed reactivity with salivary sIgA. Cells grown at 37 degrees C in medium containing maximum concentrations of glucose and ammonium sulphate expressed the antigens reactive with sIgA during longer periods of time than the cells grown in medium with minimal concentrations of the same compounds. The regulatory role showed by the concentration of glucose and ammonium sulphate on the antigenic expression was subordinated, nevertheless, to the most important factor, the temperature of incubation. Only isolates showing low susceptibility expressed the antigens reactive with sIgA under the influence of subinhibitory concentration of antifungals. However, induced resistance to one of the antifungals tested (5 fluorocytosine) allowed the antigenic expression at elevated subinhibitory concentrations even in previous susceptible strains. In conclusion, in addition to the temperature, factors such as characteristics of the strain, the concentration of glucose and ammonium sulphate in the culture medium and the resistance to antifungals played a role on the expression of C. albicans antigens reactive with sIgA, which could be of clinical relevance in the course of infection.

Secreted phospholipase has been recently proposed as a virulence determinant in Cryptococcus neoformans as well as Candida albicans. This issue of cryptococcal phospholipase requires screening of phospholipase production in a larger number of isolates from clinical and environmental sources. In this study we examined phospholipase production in a total of 67 C. neoformans isolates from AIDS patients and bird droppings by using the egg-yolk plate method. Phenoloxidase activity, capsule size and growth at 37 degrees C were also measured in these strains in order to observe a possible relationship between phospholipase production of different C. neoformans strains and its virulence. Four of the 21 AIDS strains at 28 degrees C and 1 at 37 degrees C did not produce phospholipase, respectively. In contrast, 38 and 34 of the 46 bird dropping strains were negative for phospholipase production at 28, and 37 degrees C, respectively. Statistical analysis revealed a significant difference in phospholipase production, capsule size and growth ability at 37 degrees C, but not phenoloxidase activity, between the AIDS and the bird dropping strains. The highly prevalent distribution of phospholipase activity in the AIDS strains suggests a role of the enzyme in invading the host.

The presence of red (RNA) and green (DNA) fluorescent material identifying nucleic acids in the chlamydospores of Candida albicans has been well documented. Red fluorescence in chlamydospores is relatively diffused and ranges from small spots, observed in hyphal cells, to the entire protoplasmic content. Green fluorescence is rarely visible in these structures and, when present it can be observed next to the plasmalemma. The initial percentage values of the two curves related to the cell counts of red fluorescence of the suspensor cells and chlamydospores showed remarkable differences between the two structures. About 54% of the chlamydospores showed red fluorescence compared to about 28% of the suspensor cells. It seems from the results obtained in this study that much RNA was produced and/or accumulated in the chlamydospores and suspensor cells, rather than in mycelium where red fluorescence was not observed. The results obtained sustain the hypothesis that a chlamydospore should he considered a fully functional cell that is morphologically and physiologically active and independent from mycelium.

Citreoviridin, a mycotoxin produced by some molds of the genera Penicillium and Aspergillus, inhibits the growth of bacteria of the Bacillus genus. Since significant information was not available on the effects of citreoviridin on eukaryotic cells and viruses, this molecule was tested on CD4+ T-lymphoid cell lines, on HIV-1 and on Candida albicans, which sometimes complicates HIV-infection. Antiviral activity was detected in H9 HTLV IIIB cells, a clone chronically infected by HIV-1. Citreoviridin reduced p24 in the supernatant of H9 HTLV IIIB in a dose-dependent manner with a pharmacological selectivity index of 2.6. In C. albicans, the effects of the mold-derivative were evaluated on some parameters associated with pathogenicity and virulence: cellular proliferation, germ tube production, expression of heat shock mannoproteins, release of proteases and phospholipases. At a 12.5 microM dose, citreoviridin showed a marked inhibitory effect on all parameters analyzed. As regards the mechanism of action, it is possible to hypothesize that the effects of citreoviridin may be due to a reduction of protein synthesis, since it inhibited the replication of HIV-1 at post-integrational stages and reduced the intracellular RNA and protein content in C. albicans.

Many studies have evaluated the toxicity of mycotoxins to mammals, but there is little information on their action against fungal cells, even although mycotoxins are frequently active against fungi in nature. A crude extract of Aspergillus sulphureus was tested for its growth-inhibitory effect on Cryptococcus neoformans. The reduction in cell growth of Cr. neoformans caused by the extract was dose dependent. Using a liquid medium containing 2% A. sulphureus extract, the RNA content of Cryptococcus amounted to about 60% of that of non-treated cells. Capsule thickening, demonstrated biochemically and with cytological stains, occurred at doses that had minimal effect on cell growth and RNA content. Our results suggest that the virulence of Cr. neoformans may increase in cases of coenobiosis with A. sulphureus, which is theoretically possible in places where corn-fed pigeons are numerous.

The influence of different glucose concentrations was tested in minimal synthetic medium on Candida 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.

The influence of different fructose concentrations (5, 3, 1 and 0 g/l) was tested on Germ Tube (GT) production by Candida albicans strain AS3P, using a Minimal Synthetic Medium (MSM) without (NH4)2SO4. The results obtained showed good GT production in the presence of all the different fructose concentrations and in the absence of any nitrogen source. The greatest GT production was obtained with 3 g/l of fructose vs 1 g/l of glucose, after 4 hr of incubation. On the other hand fructose consumption was lower than that of glucose at all concentrations over the 4 hour period. The data obtained may suggest that fructose is metabolized in a different way from glucose for GT production by C. albicans.

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.

The use of a simple synthetic medium is essential for study on the growth and physiology of Cryptococcus neoformans. In the present study, a minimal synthetic liquid medium (MSM) was tested for the growth of 23 C. neoformans strains. This medium contained a low concentration of glucose, ammonium sulphate and inorganic salts with a pH value of 4.5, but no amino acids or vitamins. The strains were starved for 4 days to eliminate nutrients which might have been carried over from their pre-culture medium. Then, they were inoculated in the MSM as an initial OD of 0.020 at 550 nm and incubated at 37 degrees C for 20 days. Cell growth was generally monitored daily by measuring the absorbance at 550 nm. The medium supported the growth of the strains tested and gave an average final OD of 0.500. The results obtained indicate that C. neoformans may be autotrophic with respect to vitamins and in particular to thiamine. The MSM medium is easy to prepare and store. It is highly reproducible and useful for studies on the growth and physiology of C. neoformans.

In this study we reported about the antiviral activity of Aspergillus terreus alcohol extract against Herpes simplex type 1 virus. This activity is dose-dependent, is related to the concentration of the challenging virus and depends particularly on a proteic fraction of 5-10 Kda. Optimal effects were observed with doses ranging from 25 to 6.25 micrograms/ml for crude alcohol extract and up to 3 micrograms/ml for 5-10 Kda fraction. Moreover, antiviral activity was evident in viral replication, but not on virus out of the cells. An increased 3H thymidine incorporation was noted on infected cells treated with the extract and this effect may suggest an intracellular accumulation of viral DNA in the absence or reduction of virion production.

Eight strains of Cryptococcus 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 that Cr. neoformans var. neoformans is able to secrete protease and to utilize protein as a source of nitrogen.

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 to N. brasiliensis and N. otitidiscaviarum. This work describes the first case of nocardiosis in Italy caused by N. 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 shown any improvement. Direct examination of the pus revealed numerous gram positive rods. When cultured N. otitidiscavarium was isolated and identified by morphological and biochemical tests.

There are very few and unconfirmed data regarding the antineoplastic activity of mycotic derivates in human cells. The effects on neoplastic and non-neoplastic cell replication of Aspergillus terreus extracts have been tested. In fact, among different species of Aspergillus mycotoxin producers. A terreus seems to be more suitable for hypothetical therapeutic purposes because of its low mycotoxin toxicity. Very evident antiblastic activity of alcoholic crude extract of A. terreus on tumor cells has been demonstrated. Doses between 3.1 and 6.2 inhibited more than 50% of tumor cells; the same effect was obtained with doses > 25 micrograms/ml on non-neoplastic cells. The action of the crude extract does not influence cellular cAMP in either neoplastic or non-neoplastic cells. The antiblastic action seems to depend primarily on the inhibitory effect of DNA duplication. Some chromatographed fractions of the mycotic extract showed inhibiting or enhancing effects on cell growth.

Candida albicans starved cells were incubated in minimal synthetic liquid media containing different concentrations of ammonium sulphate (0.00, 0.02, 0.05, 0.10, 0.03, 0.50 g/L). Culture growth was monitored by measuring daily the optical density and by evaluating RNA and protein cellular content after 48 and 96 hours from the inoculum. The environmental availability of ammonium ion influenced the biomass production, that was maximum when its concentration was 0.10 and 0.30 g/L. In addition, an effect on cell duplication time was observed, this was particularly evident when the (NH4)2SO4 concentration was 0.10 g/L. The protein content increased in relation to the increase of ammonium ion availability, with a peak in correspondence to 0.30 g/L and a drop when the greatest concentrations were employed. RNA production was inversely proportional in respect to protein production. The optimal range of ammonium sulphate concentration for C. albicans growth was 0.10-0.30 g/L; over these concentrations there was an inhibitory effect. The rate of the protein and RNA syntheses seems to indicate the growth phase and the nitrogen nutritional conditions of the cultures, respectively.

A study of the vaginal yeast flora in pregnant women living in Cusco and in its region (Peru), located approximately 3000 m above sea level, is reported. We observed 300 pregnant, healthy and non-diabetic women who attended a gynaecological clinic in the Lorena, Regional or IPSS (Instituto Peruano de Seguridad Social) hospitals in Cusco. A comprehensive clinical history was obtained from each patient. It included age, work, parity, time of pregnancy, use of contraceptives or antibiotics, type of vaginal symptoms, type and amount of vaginal secretion. The yeasts were isolated from 44.3% of the cases. The positive cases were more frequently found in the following categories: 20-30 year-old patients (69.2%), ninth month of pregnancy (49.6%), first pregnancy (41.4%), no delivery (58.7%) and no abortion (66.9%). Most women complained of leukorrhea (75.9%) with mucus, scarce without odour (18.8%) or lumpy, regular, without odour (15.8%) secretion. The yeasts isolated were Candida albicans (66.2%), other Candida species (12.8%), Torulopsis glabrata (8.3%) or other Torulopsis species (2.2%), Saccharomyces cerevisiae (7.5%), Rhodotorula sp. (1.5%) and Trichosporon cutaneum (1.5%).

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 micrograms/ml) was investigated. In these media the presence or absence of thiamine had no effect on the development of C. neoformans. To check these results, we performed a series of experiments on a solid form of the minimal synthetic medium. In this study a series of six serial transfers were carried out to starve the cells of nutrients that may have been carried over from their growth on rich media. In each of the transfers on the solid synthetic medium, C. neoformans showed a similar and scarce growth. This finding indicates that C. neoformans could be autotrophic in respect to thiamine.

A new minimal synthetic medium, with low amount of glucose, without aminoacids, vitamins and neutral pH, which induces germ-tubes production in Candida albicans, is reported in this work. The results indicate a perfect agreement between the germ-tube test performed with the standard method in human or animal serum and this test performed in minimal synthetic medium. In this medium the germ-tube test for the presumptive identification of Candida albicans can be performed with the same formality, time and reproducibility as those in human or animal serum. This constitutes an interesting finding because it is easy to prepare, to store and is highly reproducible.