Cryptococcus neoformans and Cryptococcus gattii Isolated from the Excreta of Psittaciformes in a Southern Brazilian Zoological Garden

Cryptococcus neoformans, a major pathogen in immunocompromised patients, is a ubiquitous free-living fungus that can be isolated from soils, avian excreta and plant material. To further study potential saprophytic sources of this yeast in the Southern Brazilian State Rio Grande do Sul, we analyzed fecal samples from 59 species of captive birds kept in cages at a local Zoological Garden, belonging to 12 different orders. Thirty-eight environmental isolates of C. neoformans were obtained only from Psittaciformes (Psittacidae, Cacatuidae and Psittacula). Their variety and serotype were determined, and the genetic structure of the isolates was analyzed by use of the simple repetitive microsatellite specific primer M13 and the minisatellite specific primer (GACA)₄ as single primers in the PCR. The varieties were confirmed by pulsed-field gel electrophoresis (PFGE). Thirty-three isolates (87%) were from the var. grubii, serotype A, molecular type VNI and five (13%) were Cryptococcus gattii, serotype B, molecular type VGI. All the isolates were mating type α. Isolates were screened for some potential virulence factors. Quantitative urease production by the environmental isolates belonging to the C. gattii was similar to the values usually obtained for clinical ones.     

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.     

Serotypes of Cryptococcus neoformans isolated from patients prior to and during the AIDS era in Thailand

One hundred and eighty-seven strains of Cryptococcus neoformans isolated from patients in Thailand were charcterized by biochemical varieties relating to serogroups. Canavanine-glycine-bromothymol blue (CGB) agar was used for differentiating the varieties of C. neoformans. Slide agglutination tests were performed with Crypto Check (Iatron, Inc., Tokyo) to determine their serotypes. Fifty-five percent (10 out of 18) of the pre-AIDS isolates were serotype B, 28% were serotype A, 5% were serotype D, and an unexpected 11% (2 out of 18) were serotype C. These are the first to be recorded in Asia. In contrast, among the 169 clinical isolates obtained between January 1993 and March 1995 (AIDS epidemic), serotype A was outstandingly predominant-93% (157 out of 169), serotype B was relatively low (3.6%) and both serotypes D and AD were 1.8%. The pattern of serotypes of the 59 isolates from known HIV-positive patients was closely similar to the total isolates during the AIDS epidemic. In determining the varieties of C. neoformans by CGB, only 1 of the 187 isolates gave a false reaction. On the basis of our findings, we believe that in the pre-AIDS era either C. neoformans var. gattii serorype B or serotype C were the common causative agents of cryptococcosis in Thailand. The advent of AIDS changed the pattern of serotypes with serotype A becoming predominant as has been reported world wide.     

Comparative analysis of Cryptococcus neoformans acid-resistant particles generated from pigmented cells grown in different laccase substrates

Cryptococcus neoformans produces pigments in vitro in the presence of exogenous substrate. We characterized acid-resistant particles isolated from pigmented cells grown in L-dopa, methyl-dopa, (-)-epinephrine or (-)-norepinephrine. The goals of this study were to determine whether pigments made from each of these substrates were melanins and the consequences of pigmentation on related cell characteristics. The greatest yield of acid-resistant particles occurred with methyl-dopa followed by L-dopa. Electron microscopy indicated that L-dopa and methyl-dopa produced particles with thicker shells. The mAb 6D2 reacted with all particles, but a lower reactivity was observed with epinephrine-derived particles. ESR analysis revealed that epinephrine-derived particles failed to produce a stable free radical signal typical of melanins. Growth of C. neoformans in different substrates affected cell and capsule size but not capsule induction. Hence, the type of pigment produced by C. neoformans is dependent on the substrate and not all pigments meet the criteria for melanins.     

Microreview: Capsule-associated genes of Cryptococcus neoformans

Cryptococcosis, caused by Cryptococcus neoformans is a common systemic mycosis in man and animals, particularly immunocompromised patients. This pathogenic fungus produces a thick extracellular polysaccharide capsule. Four capsule-associated genes (CAP10, CAP59, CAP60, CAP64) were cloned and sequenced, and proved to be essential for capsule synthesis. However biochemical functions of CAP gene products have not been clarified yet. Recently, the relatedness of the polysaccharide capsule and four capsule-associated genes has partly been elucidated. Nucleotide sequence of four CAP gene fragments was analyzed for phylogenetic relationships, and they were in agreement with the conventional classification of varieties and serotypes within C. neoformans. Expression of four CAP genes and capsule size were examined using two media containing different amount of glucose, and the results indicated that CAP genes might play important roles in elaboration of extracellular polysaccharide capsule. Furthermore, analyses of CAP genes in various clinical samples would give the useful information to diagnose cryptococcosis in human and animals.     

A rapid urease test for presumptive identification of Cryptococcus neoformans

A rapid method to evidence urease activity is described. Urea hydrolysis and consequent production ammonia are detected by a chemical reaction producing a blue phenol compound (indophenol blue). Three hundred and three yeast were tested. Out of 107 urease-positive organisms detected by Christensen's Urea Agar Test (CUAT) 102 were positive by our method. No false negatives were observed by this method when testing 87 Cryptococcus strains. Ths practical screening test for presumptive identification of Cryptococcus neoformans is simple, unaffected by pH changes and requires 15 minutes to be performed.     

A new species of Lepanthes (Orchidaceae) from Puerto Rico

A new species,Lepanthes caritensis, is described and illustrated. There are now nine species ofLepanthes known to occur in Puerto Rico. Plant habitat and floral morphology ofL. caritensis is most similar to that ofL. sanguinea. A survey of the area where the new species is located suggests that it is rare and should be protected under the Federal Endangered Species Act. Una especic nueva,Lepanthes caritensis, es descrita e ilustrada para Puerto Rico. Con ésta hay ahora nueve especies deLepanthes en Puerto Rico. El hábito de la planta y la morfología de la flor deL. caritensis es más similar aL. sanguinea. Un muestreo del área donde se encuentra la nueva especie sugiere que ésta es rara y que debería ser protegida por el Federal Endangered Species Act.     

First environmental isolation of Cryptococcus neoformans var. neoformans and var. gatti from the Gharbia Governorate,Egypt

Flowers from two Eucalyptus camaldulensis trees in the Qutur area and one tree from the Tanta area yielded three isolates of Cryptococcus neoformans var. gattii. Pigeon and sparrow droppings were also investigated for the occurrence of C. neoformans within the study area. Ninety five isolates of the neoformans variety of C. neoformans were recovered from 550 samples of avian droppings. This revised version was published online in October 2005 with corrections to the Cover Date.     

Jacquinia pauciflora (Theophrastaceae), a new species from Puerto Rico

A new species,Jacquinia pauciflora, is described from subtropical moist forest on karst hills (mogotes) in northwestern Puerto Rico.Jacquinia pauciflora is similar toJ. stenophylla Urban, from which it differs in several floral and leaf characters, but is possibly most closely related toJ. umbellata A. DC. A key to the species ofJacquinia occurring in Puerto Rico is provided.     

DNA fingerprinting pattern and susceptibility to antifungal drugs in Cryptococcus neoformans variety grubii isolates from Barcelona city and rural environmental samples

Cryptococcus neoformans var. grubii (serotype A) was isolated from 12 soil samples mixed with pigeon droppings (16.9%) from 71 soil samples in Barcelona and rural areas of Catalonia. C. neoformans was not isolated from indoor dust and Eucalyptus debris. PCR fingerprinting was performed in 22 representative isolates and all of them corresponded to the VNI pattern. Susceptibility testing for the 22 isolates of C. neoformans var. grubii showed that all of them were susceptible to amphotericin B. Three isolates presented MICs (Minimal Inhibitory Concentrations) ≥ 1 μg/ml to Itraconazole, five MICs ≥ 1 μg/ml to ketoconazole and four were fluconazole resistant, (MICs ≥ 64 μg/ml), while three of them were shown to have MICs ≥ 1 μg/ml to voriconazole. In spite that all isolates presented the same DNA fingerprinting pattern, the susceptibility to antifungals is very variable. The possibility of acquiring cryptococcosis infection with primarily resistant environment strains is feasible.     

Expression of capsule-associated genes of Cryptococcus neoformans

Cryptococcus neoformans produces an extracellular polysaccharide capsule that is related to its virulence. The production of capsular components was reported to be accelerated when cultured on media with lower amount of glucose. In this study, relationship between capsule synthesis and expression of capsule-associated genes (CAP genes) was investigated by quantitative real-time PCR analysis. Normally encapsulated strains and a stable acapsular strain were cultured in 1% polypepton medium with 0.1% or 15% glucose. The results of assessment of the capsule size showed that the capsule of yeast cells cultured in the medium with low amount of glucose was thicker than that with high amount of glucose. The CAP gene expressions of normally encapsulated strains were higher in the medium with 0.1% glucose than in the medium with 15% glucose. Furthermore, CAP10, CAP59 and CAP60 genes were expressed very low in a stable acapsular strain, and CAP64 gene was not expressed. Results of assessment of capsule size and CAP gene expressions by quantitative real-time PCR analysis indicated that CAP gene expressions might be related to the production of capsule, and that glucose concentration in culture media might be related to the expression of CAP genes.     

Biofilm Formation by Cryptococcus neoformans Under Distinct Environmental Conditions

Cryptococcus neoformans is an opportunistic fungal pathogen with a propensity to infect the central nervous system of immune compromised individuals causing life-threatening meningoencephalitis. Cryptococcal biofilms have been described as a protective niche against microbial predators in nature and shown to enhance resistance against antifungal agents and specific mediators of host immune responses. Based on the potential importance of cryptococcal biofilms to its survival in the human host and in nature, these studies were designed to investigate those factors that mediate biofilm formation by C. neoformans. We observed that C. neoformans preferentially grew as planktonic cells when cultured under specific conditions designed to mimic growth within host tissues (37°C, neutral pH, and ~5% CO₂) or phagocytes (37°C, acidic pH, and ~5% CO₂) and as biofilms when cultured under conditions such as those encountered in the external environment (25–37°C, neutral pH, and ambient CO₂). Altogether, our studies suggest that conditions similar to those observed in its natural habitat may be conducive to biofilm formation by C. neoformans.     

Melanization decreases the susceptibility of Cryptococcus neoformans to enzymatic degradation

Cryptococcus neoformans is a free-living fungus that is primarily found in soils contaminated with avian excreta. Recent studies have shown that C. neoformans can synthesize melanins or melanin-like compounds in avian excreta. Melanization has been associated with protection of C. neoformans against harsh environmental conditions, such as ultraviolet radiation and extremes of temperature. In this study we examined whether melanization can protect C. neoformans against enzymatic degradation. Our results demonstrated that in vitro melanization decreases the susceptibility of C. neoformans to hydrolytic enzymes. This suggests a role for melanin in protection of C. neoformans against enzymatic degradation by antagonistic microbes in the environment. This revised version was published online in August 2006 with corrections to the Cover Date.     

Efficacy of voriconazole in experimental Cryptococcus neoformans infection

Voriconazole is a third generation triazole with improved activity against many fungal pathogens. We examined the efficacy of voriconazole in a murine infection model and evaluated the drug’s effect on cellular characteristics and serum polysaccharide levels. The antifungal reduced serum polysaccharide and significantly prolonged the survival of lethally infected animals.     

Growth of Cryptococcus neoformans in a thiamine-free medium

The growth of Cryptococcus neoformans in a minimal liquid synthetic medium with or without thiamine (10 g/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.     

Clinical and environmental isolates of Cryptococcus neoformans in Bangkok (Thailand)

Cryptococcus neoformans was isolated from 13 patients (7 females and 6 males) suffering from systemic cryptococcosis. Eight patients were suffering from central nervous system cryptococcosis and 5 were suffering from disseminated cryptococcosis. Systemic lupus erythematosus was found to be the common underlying disease in 3 patients. The results of treatment depended on the underlying diseases (7 improved, 6 died). Also, 13 isolates of C. neoformans were obtained from feces of 30 pet birds. All 26 isolates of C. neoformans were cultured in glycine cycloheximide medium and were found to be of serotypes A and D.     

Cryptococcus neoformans varieties from material under the canopies of eucalyptus trees and pigeon dropping samples from four major cities in Jordan

To our best knowledge, any study related to the ecological distribution of Cryptococcus neoformans in Jordan does not exist in the medical literature. In order to determine the environmental occurrence of both varieties of Cryptococcus neoformans in Jordan, pigeon droppings and material under the canopies of eucalyptus trees were collected from four major cities of this country. For the isolation of Cryptococcus neoformans variety gattii from environmental sources, 500 samples of the mixed soil debris, including tree materials, under the eucalyptus trees from cities of Amman, Irbid, Jerash, and Ajlun were collected. Also, 509 samples of pigeon droppings were collected from the same cities for the isolation of Cryptococcus neoformans variety neoformans. After inoculating the samples onto modified Staib agar medium in Petri dishes, a total of 336 melanoid yeast colonies were picked up during screening process. At the end of serial mycological studies, none of these isolates was identified as Cryptococcus neoformans, but all were Cryptococcus species other than C. neoformans. For determining the exact status, more extensive environmental studies need to be done in the future.     

Virulence and antifungal susceptibility of environmental and clinical isolates of Cryptococcus neoformans from Puerto Rico

A case of cerebral cladosporiosis caused by Cladosporium trichoides (bantianum) now known as Xylohypha bantiana is described and illustrated. Predisposing debilitating diseases were not detectable. The Cladosporiosis diagnosis was based on visualisation of hyphal element in direct Gram's stain, direct KOH preparate of pus from brain abscess and on repeated successful cultivation of Cladosporium trichoides from specimen and by histopathology. Following surgery and anti-fungal chemotherapy the patient was cured.