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PubMed2006

Determination of molecular types and genetic heterogeneity of Cryptococcus neoformans and C. gattii in Malaysia.

The molecular types and genetic heterogeneity of Cryptococcus neoformans and C. gattii clinical isolates in Malaysia were determined in this study. Of 44 C. neoformans collected between 1980 and 2003, 42 (95.5%) were molecular type VNI, 2 (4.5%) were molecular type VNII. Of 17 C.gattii isolates, 13 (76.5%) were molecular type VGI, and 4 (23.5%) were molecular type VGII. A difference was noted when comparing the molecular types of cryptococcal isolates in the earlier and recent cases of cryptococcosis. While both molecular types VNI and VGI were equally predominant in the earlier cases of cryptococcosis, VNI was the most predominant molecular type isolated from the recent cases. VNII was a new molecular type, isolated from 5.1% of the recent cases. All the bird dropping isolates were molecular type VNI. The genetic heterogeneity of the two predominant molecular types, i.e., VNI, VGI clinical isolates and bird dropping isolates of C. neoformans were further determined by polymerase chain reaction (PCR) fingerprinting method, using (GTG)5 as single primer. Two clusters of cryptococcal isolates were distinguished at 68.5% of similarity, with cluster I consisting of VNI isolates and cluster II consisting of VGI isolates. Each cluster was further subdivided into three subtypes at >/=80% of similarity. Fourteen bird dropping isolates were grouped into a subtype within VN1, sharing 82.7% of similarity with the clinical isolates. A higher degree of similarities, ranging from 93.4-97.6% was noted between 3 bird dropping isolates with the clinical isolates in another subtype. This study demonstrated the existence of various molecular types of C. neoformans isolates in Malaysia and the genetic heterogeneity within the predominant molecular types. The study also provides evidence for genetic relatedness of clinical isolates with bird dropping isolates in the environment.

Cryptococcosis↗
PubMed2006

Isolation of Cryptococcus neoformans var. neoformans serotype D from Eucalypts in South Brazil.

Cryptococcus neoformans causes the second most common opportunistic infection in patients with AIDS. In Brazil, 4.5% of the AIDS-related opportunistic infections are caused by C. neoformans and all varieties are recognized as etiological agents of cryptococcosis. This pathogen is a ubiquitous environmental yeast, commonly associated with avian excreta and decaying wood, especially Eucalypt species. The aim of the present study was to search for C. neoformans in Eucalypts and analyze the genotypic diversity of the obtained isolates by RAPD and PCR fingerprinting. All obtained isolates have been C. neoformans var. neoformans, serotype D molecular type VNIV. Serotype D, was isolated from 3 (37.5%) out of 8 cities surveyed in the South Brazilian state Rio Grande do Sul. Nine (9%) out of 99 environmental samples were obtained from Eucalypt species, Eucalyptus calmadulensis and Eucalyptus tereticornis. Molecular analysis using RAPD and PCR-fingerprinting revealed very little genetic diversity in the obtained cryptococcal serotype D isolates. To our knowledge this is the first report of the isolation of serotype D from Eucalyptus trees in Brazil. More studies are required in order to establish the ecological significance of this finding.

Brazil↗
PubMed2004

Cryptococcus neoformans var. gattii can exploit Acanthamoeba castellanii for growth.

It has recently been proposed that the origin and maintenance of virulence in certain environmental fungi is influenced by their interactions with non-vertebrate hosts such as amoebae and nematodes. In prior studies we have shown that the interactions of the soil amoebae Acanthamoeba castellanii with Cryptococcus neoformans varieties neoformans and grubii resemble those with macrophages. Here we extend those studies to C. neoformans variety gattii and describe quantitative differences in the type and outcome of the interactions observed relative to the other varieties. C. neoformans var. gattii proliferated in the presence of A. castellanii but the interaction was primarily extracellular with a paucity of phagocytic events. Experiments with acapsular cells coated with polysaccharide suggest that differences in the capsule structure may be responsible for the different interactions between cells of varieties neoformans, grubii, and gattii with amoebae. The ability of C. neoformans var. gattii to exploit amoebae indicates that despite major biological differences between C. neoformans varieties, all retain the ability to be pathogenic for A. castellanii.

Acanthamoeba↗
PubMed1993

Fluconazole-resistant Cryptococcus neoformans var gattii in an AIDS patient.

We describe a case of meningitis due to Cryptococcus neoformans var gattii in an AIDS patient. The reasons for the observed predominance of C. neoformans var neoformans infections in AIDS patients living in areas endemic for the gattii variety are not understood thus far. Treatment with fluconazole resulted in poor clinical and mycological response. In vitro sensitivity testing of the fungus revealed high minimal inhibitory concentrations, suggesting fluconazole resistance. Treatment with amphotericin B was successful.

AIDS-Related Opportunistic Infections↗
PubMed2003

Cryptococcus neoformans var. gattii in the koala (Phascolarctos cinereus): a review of 43 cases of cryptococcosis.

Details of 11 previously reported cases and 32 new cases of cryptococcosis in captive and wild koalas were analysed. Cryptococcus neoformans var. gattii accounted for all 29 cases in which varietal status was determined. No age or sex predisposition was observed. The respiratory tract was the primary focus of disease in 77% of cases. Although the lower respiratory tract was affected most commonly (60% of cases), 30% of cases had upper respiratory tract lesions and 14% had both. Dissemination was common, especially to the central nervous system (37% cases). Local extension to surrounding tissues was a feature of upper respiratory tract disease. Other tissues showing cryptococcal invasion included lymph nodes (19%), gastrointestinal tract (12%), kidneys (12%), spleen (9%) and skin (7%). Only three cases (7%) had no respiratory tract or central nervous system involvement, two cases of primary skin inoculation and one case of primary lymphadenopathy. Late presentation was a likely factor in the high proportion of cases with disseminated disease (40%). The proportion of koala cases with involvement of the central nervous system, lower respiratory tract and skin, parallels what has been reported for immunocompetent people. Cryptococcosis in the koala appears to be an excellent naturally occurring model for examination of the cryptococcal host-parasite relationship in all species.

Animals↗
PubMed2003

Decayed wood of Syzygium cumini and Ficus religiosa living trees in Delhi/New Delhi metropolitan area as natural habitat of Cryptococcus neoformans.

The isolation is reported of Cryptococcus neoformans var. gattii and C. n. var. neoformans from decayed wood inside trunk hollows of Syzygium cumini and of C. n. var. neoformans from Ficus religiosa trees in the Delhi/New Delhi metropolitan area. Fourteen of sixty-six (21%) S. cumini trees investigated proved to be positive, seven for each variety. The two varieties never co-occurred in the same hollow. C. n. var. neoformans was also isolated from three of seventeen Ficus religiosa-trees. Two of these isolates originated from decayed wood and one from bark. The C. n. var. gattii and C. n. var. neoformans isolates belonged to serotype B and serotype A, respectively. The data strongly supported colonization of S. cumini by both varieties and of F. religiosa trees by C. n. var. neoformans. Evidence of this was found by repeated isolations. For example, in 36/44 (82%) samples for C. n. var. gattii and 22/27 (81%) samples for C. n. var. neoformans, and by a high population density in the tested wood debris (maximally 6 x 10(5) colony-forming units per gram [c.f.u./g] for C. n. var. gattii and 8 x 10(4) c.f.u./g for C. n. var. neoformans). No eucalypt trees were seen near the positive S. cumini and F. religiosa trees. The densities of C. neoformans in these trees exceeded those found previously in Eucalyptus camaldulensis and in other tree species more rarely reported to be sources of C. neoformans in India. S. cumini and F. religiosa appear not to have been reported to date as sources for either C. n. var. gattii or C n. var. neoformans. Our results add to the recently emerging evidence that the natural habitat of C. n. var. gattii and C. n. var. neoformans is not specific to woody or other debris of particular tree species, but instead is more generalized.

Colony Count, Microbial↗
PubMed2000

Genotyping Cryptococcus neoformans var. neoformans with specific primers designed from PCR-fingerprinting bands sequenced using a modified PCR-based strategy.

Cryptococcus neoformans var. neoformans can be subdivided into six genotypes (VN1-VN6) based on different combinations of four major bands (420, 475, 540 and 800 bp) obtained by polymerase chain reaction (PCR) fingerprinting using the (GACA)4 primer. The aim of this study was to identify specific primers able to amplify these bands. A modified PCR-based sequencing strategy was adopted to overcome the limitations of using (GACA)4 as a single cycle sequencing primer. The original bands, made up of amplicons with two terminal (GACA)4 sequences, were digested with five restriction enzymes. Digestion products yielding two or three fragments were selected. Each fragment was expected to have no more than one terminal (GACA)4 sequence, making cycle sequencing possible. Fragments were purified and sequenced with the (GACA)4 primer. New primers specific for each of the four major bands were then designed and the remaining regions were sequenced using both purified bands and PCR-fingerprinting products as template. These primers were used to amplify the genomic DNA of 12 C. neoformans strains and five strains of other yeast species. The new primers, used as separate pairs or in a mixture of all pairs, amplified the expected bands only in C. neoformans var. neoformans strains, confirming the species specificity of the bands selected for molecular typing of this yeast.

Base Sequence↗
PubMed2000

Timed-kill curves for Cryptococcus neoformans isolated from patients with AIDS.

Infection with Cryptococcus neoformans is an increasing problem in immunocompromised patients, particularly those with acquired immune deficiency syndrome (AIDS). Amphotericin B and fluconazole are currently acceptable therapies for cryptococcal meningitis; however, their effects remain suboptimal and recurrence or treatment failure is still a problem. Antifungal susceptibility testing may be an important tool for guiding therapy, but for C. neoformans, a reliable method is still not available. This retrospective study evaluated minimal inhibitory concentration (MIC) for amphotericin B and fluconazole, and minimal fungicidal concentration (MFC) and timed-kill curves for amphotericin B against 16 clinical isolates of C. neoformans obtained from AIDS patients with cryptococcal meningitis. No correlation between clinical outcome and MIC was observed for amphotericin B. In selected cases, the MFC seemed to be a better predictor of outcome than MIC. In this study, amphotericin B timed-kill curves appeared to show a correlation with clinical outcome of the 16 patients with AIDS-associated cryptococcal meningitis. These in vitro tests must be further evaluated in prospective studies to confirm their potential usefulness for guiding cryptococcal meningitis therapy.

AIDS-Related Opportunistic Infections↗
PubMed2000

Differing requirement for inducible nitric oxide synthase activity in clearance of primary and secondary Cryptococcus neoformans infection.

The role of nitric oxide in resistance to cryptococcal infection was investigated. Mice deficient in inducible nitric oxide synthase (INOS) did not survive a primary intratracheal infection as did INOS-replete control mice. Despite adequate recruitment of host cells and generation of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha at the site of infection, INOS-deficient mice failed to clear yeast from their lungs by five weeks of infection, in contrast to wild-type mice. INOS-deficient mice also had higher yeast brain burdens than did control mice after a primary intracerebral infection. Therefore, generation of nitric oxide is required for resistance to primary cryptococcal infection. However, INOS-deficient mice vaccinated subcutaneously and rechallenged intravenously had lung and brain yeast burdens equivalent to those of vaccinated controls, and therefore expressed effective acquired immunity to Cryptococcus neoformans. Cells harvested from infected INOS-deficient mice by bronchoalveolar lavage acted as anti-cryptococcal effectors in vitro at an effector:target ratio of 100:1, provided IFN-gamma was present, but did not inhibit yeast proliferation at a 10:1 effector:target ratio as cells from wild-type mice did. Therefore, INOS activity is important for anti-cryptococcal function of effectors of immunity during the primary response, but not for the generation or expression of secondary immunity to C. neoformans.

Animals↗
PubMed2000

Possible primary ecological niche of Cryptococcus neoformans.

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

Colony Count, Microbial↗
PubMed2000

Environmental isolation of Cryptococcus neoformans var. gattii and C. neoformans var. neoformans in the city of São Paulo, Brazil.

In order to determine the environmental occurrence of both varieties of Cryptococcus neoformans in the city of São Paulo, pigeon droppings and vegetable material from trees of the genus Eucalyptus were collected at typically crowded places. A total of 38 sites downtown where large heaps of pigeon droppings could be found were selected for sampling. Pigeon droppings from 10 (26.3%) of these sites were positive for C. neoformans var. neoformans in at least one sample. Twelve eucalyptus woods located within four municipal parks were also surveyed; vegetable material from Eucalyptus spp. trees were collected monthly over a 2-year period. C. neoformans var. gattii was recovered from a wood in Ibirapuera Park during the same season on two different occasions (November 1996 and November 1997); this park contained specimens of Eucalyptus camaldulensis, a natural habitat of C. n. var. gattii. C. n. var. neoformans was detected in a wood in Ibirapuera Park and Aclimação Park. The results show that both C. n. var. neoformans and C. n. var. gattii are present in the urban environment of São Paulo city at sites where large numbers of people normally gather.

Animals↗
PubMed2000

Immunoregulation by capsular components of Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated yeast that is pathogenic for humans. The capsule is a major virulence factor composed mainly of glucuronoxylomannan (GXM) and two minor constituents, galactoxylomannan, and mannoprotein (MP). A hallmark of disseminated cryptococcosis is the presence of high concentrations of GXM in body fluids of infected hosts. GXM provides a critical negative signal for T cell activation and neutrophil migration at the site of the inflammatory process. There is also strong evidence that MP promotes critical events associated with protective responses such as delayed type hypersensitivity and presumably a T helper type 1 response. The contrasting roles of GXM and MP in regulation of the immune response to C. neoformans offer a promising template for a successful approach to intervention, by scavenging GXM to attenuate its negative signals, while preserving the positive effects of MP.

Animals↗
PubMed2000

Lipid peroxidation by alveolar macrophages challenged with Cryptococcus neoformans, Candida albicans or Aspergillus fumigatus.

Increased formation of oxygen radicals has previously been shown for alveolar macrophages (AM) challenged with Cryptococcus neoformans cells opsonized with fresh serum or polyclonal immunoglobulin G. AM show similar responses to Candida albicans or Aspergillus fumigatus. Oxygen radicals are capable of damaging various macromolecules, including lipids. In the present study, lipid peroxidation (LPO) caused by AM incubated with the fungi was examined in the presence and absence of lung surfactant. The level of malonaldehyde was used as an indicator of LPO. AM damage was examined by electron microscopy (EM), by trypan blue exclusion and by counting the AM loss from culture dish to supernatant. Stimulation of AM by each fungus increased cellular LPO but did not affect AM viability. A slight surfactant LPO induced by AM alone was shown with significantly increased values after addition of each fungus. EM studies showed that dense lipid droplets, presumably consisting of oxidized lipids, were ingested in high amounts together with C. neoformans cells that had been opsonized in fresh serum, and in low amounts in combination with C. albicans. These processes were accompanied by increased numbers of AM in the supernatants. LPO and detachment of AM were counteracted by vitamin E. In the lungs, AM exposed to one of these fungal pathogens might promote peroxidation of surfactant lipids.

Animals↗
PubMed2001

A new dominant selectable marker for use in Cryptococcus neoformans.

Cryptococcus neoformans is an excellent model system for studies on the molecular pathogenesis of fungal infections. There is only one dominant selectable market that can be used in the transformation of this organism, and we wanted to develop another. We found that various strains of C. neoformans are very sensitive to the aminoglycoside antibiotic nourseothricin, and that spontaneous resistance to this drug must be an extremely rare event. Resistance to nourseothricin is conferred by the product of the nourseothricin acetyltransferase gene (nat1) from Streptomyces noursei. In order to express this gene in C. neoformans, we created a fusion construct by driving expression of natl with the promoter sequence from a C. neoformans actin gene. Biolistic transformation of the serotype A C. neoformans strain H99 and the serotype D strain JEC21 with this construct resulted in transformation efficiencies of approximately 1,000 transformants microg(-1) of DNA and 20 transformants microg(-1) of DNA, respectively. Southern blots were performed using DNA from some of the H99 transformants, and this confirmed that all of the resistant isolates had the construct integrated in a random fashion within the genome. There was no cross-resistance of the nourseothricin-resistant transformants to hygromycin B, which is the other antibiotic used as a dominant selection marker in C. neoformans. The development of nourseothricin resistance as a second dominant selectable market will be helpful in future molecular studies on this important pathogenic fungus.

Acetyltransferases↗
PubMed2001

Melanization of Cryptococcus neoformans reduces its susceptibility to the antimicrobial effects of silver nitrate.

Cryptococcus neoformans is a human pathogenic fungus that is frequently found in avian feces and Eucalyptus trees. There is evidence that C. neoformans can make a melanin-like pigment in pigeon excreta, a major natural environmental niche. Silver nitrate, AgNO3, is a highly toxic compound for bacteria and fungi. In this study we investigated the effects of melanin production by C. neoformans on the susceptibility of this fungus to AgNO3. C. neoformans was grown in media with and without the melanin precursor, L-dopa, for various times and susceptibility to AgNO3 was determined by measuring percentage of survival after incubation in AgNO3. There was an inverse association between time allowed for melanization and susceptibility to Ag+. Addition of melanin particles to a suspension of non-melanized C. neoformans cells reduced their susceptibility to AgNO3, consistent with metal ion chelation by melanin. Binding of Ag+ to melanin particles was demonstrated by atomic absorption spectroscopy. The results indicate that melanization of C. neoformans reduces susceptibility to a toxic heavy metal. This suggests a role for melanin in environmental protection against heavy metal toxicity.

Anti-Bacterial Agents↗
PubMed2001

Colonization of a voice prosthesis by Cryptococcus neoformans.

Tracheoesophageal voice prostheses in laryngectomized patients commonly deteriorate due to the presence of yeasts, particularly Candida species. We describe the first case of colonization of such a device by Cryptococcus neoformans in a patient with a history of glottic carcinoma. The isolate showed an identical genomic pattern with C. neoformans from pigeon excreta in the patient's environment.

Animals↗
PubMed2001

Isolation of a Cryptococcus neoformans serotype A MATa strain from the Italian environment.

Cryptococcus neoformans is a heterothallic basidiomycete which possesses a bipolar mating system based on two mating type alleles, MATa and MATalpha. In the type variety, C. neoformans var. neoformans, both mating types have been found among strains of one serotype, serotype D, whereas only MATalpha was identified after extensive survey of serotype A strains. Serotype A MA Ta appeared to be extinct or to exist only in a vestigial, non-functional form. We report the isolation of a C. n. var. neoformans serotype A MATa strain from the Italian environment. The strain was serotyped by slide agglutination test, genotyped by polymerase chain reaction (PCR) fingerprinting using the (GACA)4 primer, and its haploid state was determined by flow cytometry. The mating type was identified by PCR amplification of the pheromone a gene. In addition, the amplification of the four STE20 alleles, specific for the mating type of serotypes A and D, showed that the strain contains only the MATa locus. By crossing experiments the strain was found to be fertile. The interest in the finding of this fertile isolate is related to the possibility to construct a congenic pair of serotype A MATa/MATalpha strains to be used in genetic and pathogenesis studies.

Cryptococcus neoformans↗
PubMed2001

Prospective use of RFLP analysis on amplified Cryptococcus neoformans URA5 gene sequences for rapid identification of varieties and serotypes in clinical samples.

Clinical isolates of Cryptococcus neoformans, whole blood, cerebrospinal fluid, bronchoalveolar lavage fluid from patients with positive cryptococcal antigen latex-agglutination test, and spiked clinical material from healthy individuals, were tested by polymerase chain reaction (PCR) with primers amplifying C. neoformans URA5 gene sequences. To test compatibility of different DNA extraction protocols with the PCR-restriction fragment length polymorphism (RFLP) assay, a commercial DNA extraction kit (XTRAX; Gull Laboratories, UT, USA) was used alongside with the hexadecyltrimethylammonium bromide (CTAB) method on spiked biological fluids. Both methods extracted DNA from spiked clinical samples containing C. neoformans (8 +/- 2 cells ml(-1)) and generated amplification products suitable for restriction enzyme analysis. Alu I digestion differentiated the two varieties of C. neoformans. Three distinct RFLP patterns were obtained upon restriction with MspI corresponding to serotypes A, AD and B, C and D. URA5 PCR followed by RFLP analysis, coupled with a sensitive in-house or commercially available DNA extraction method from clinical samples, could be successfully incorporated into rapid routine diagnostic strategies. It could also provide an expeditious tool for epidemiology-based population genetics studies.

Base Sequence↗