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.     

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.     

Isolation of saprophytic Cryptococcus neoformans from Puerto Rico: Distribution and variety

Until the present decade, no studies had been conducted in Puerto Rico on the saprophytic distribution and variety of Cryptococcus neoformans. Samples (522) of pigeon droppings from 14 western towns were tested for the presence of C. neoformans. The yeast was recovered from 24.7% (129 isolates) of the samples, representing 10 of the 14 towns studied. All environmental isolates were identified as C. neoformans var. neoformans using canavanine-glycine-bromthymol blue (CGB) agar. The yeast was isolated from 79.4% of the samples in one town, Isabela. The average number of yeast cells isolated from sites within this municipality was 5.1×10⁵ per gram of pigeon droppings. This was 2.6 times the average number of yeast cells of C. neoformans isolated from sites in other towns. In addition, the yeast was isolated from four patients with the acquired immune deficiency syndrome (AIDS), each of whom died of cryptococcal meningitis. Each of these poorly encapsulated isolates was identified as C. neoformans var. neoformans using CGB agar. The results of this investigation demonstrate that C. neoformans var. neoformans is prevalent in Puerto Rico.This paper was presented in part at the X^th Congress of the International Society for Human and Animal Mycology, Barcelona, Spain from June 27 to July 1, 1988.     

Ecological niche of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var. <Emphasis Type="Italic">grubii</Emphasis> and <Emphasis Type="Italic">Cryptococcus gattii</Emphasis> in decaying wood of trunk hollows of living trees in Jabalpur City of Central India

Cryptococcus neoformans var. grubii and C. gattii were repeatedly isolated from decaying wood of trunk hollows in living trees growing in Jabalpur City in Central India. The isolation of C.␣gattii has been reported from decayed wood inside trunk hollow of Tamarindus indica (15.6%), Mangifera indica (2.2%), Pithecolobium dulce (12.5%), Syzygium cumini (14%), and one from bark of S.␣cumini. C. n. var. grubii was isolated from decaying wood debris of T. indica (4.4%), M. indica (13.3%), Terminalia arjuna (25%), S. cumini (2%), Cassia fistula (4.5%), and two from bark of S. cumini. The two varieties never co-occurred in the same hollow. C. gattii and C. n. var. grubii isolates belonged to serotype B and serotype A respectively. The data strongly supported the colonization of the pathogen in␣decaying wood hollow of all six-tree species. Evidence of this was found by repeated isolation up to 820 days. P. dulce is being reported for the first time as natural habitat of C. gattii and T.␣arjuna and C. fistula as natural habitat for C. n. var. grubii. M. indica is being reported for the second time as the natural habitat of both varieties (C. n. var. grubii and C. gattii). The population density of these pathogens from decaying wood debris of various tree species ranged between 0.5 × 10³ cells/g and 6 × 10⁵ cells/g. The seasonal variation has been seen in isolation of this yeast. Our result further reinforce the recently emerging evidence that the natural habitat of C. n. var. grubii and C. gattii is more generalized.     

Cryptococcus neoformàns var. gattii among patients with cryptococcal meningitis in Mexico. First observations

A retrospective study of 20 patients with cryptococcal meningitis and their isolated strains was performed. Cryptococcus neoformans var. neoformans was recovered from 14 (70%) cases, and var. gattii was recovered from six (30%) patients. Twelve patients had AIDS (all carrying var. neoformans), two had other diseases (one with var. neoformans and one var. gattii) and there was no identifiable underlying disease in six (one var. neoformans and five var. gattii). Fourteen patients (11 var. neoformans and three var. gattii) resided in the Mexico City area, where a temperate climate is prevalent, and there were six cases (three var. neoformans and three var. gattii) from states with a tropical/subtropical climate. Although there was no significant statistical difference between the two varieties, the fatal outcome was higher in patients with var. neoformans. The disease caused by var. gattii strains was characterized by a higher opening pressure, more inflamatory changes of CSF and a longer clinical course (delayed clinical and mycological cure). Cryptococcus neoformans var. gattii is a significant cause of cryptococcal meningitis in patients without underlying diseases in Mexico.     

An efficient method for homologous gene reconstitution in Cryptococcus gattii using URA5 auxotrophic marker

Cryptococcus gattii (Cg) is an emerging pathogen of both healthy and immunocompromised patients worldwide. Understanding the molecular genetic basis of virulence and physiology of this pathogen will be critical for defining its pathogenic mechanisms. The purine biosynthetic gene, URA5 encoding orate phosphorybosyltransferase (OPRTase), has been successfully used as a selectable marker for gene disruption by transformation and homologous recombination in Cg. Here, we report the characterization of ura5 auxotrophy and URA5 reversion phenomenon at the molecular, genetic, and structural levels, and use of ura5→URA5 reversion as a tool for reconstitution of gene of interest and auxotrophic marker to their native loci. We identified a single mutation of GG₁₂₈T→GAT with substitution of glycine to aspartic acid at amino acid position 43 resulting in ura5 auxotrophy. The ura5→URA5 reversion on CSM lacking uracil (CSM-U) was found to be a rare phenomenon with a reversion frequency of 0.000002%, and sequence analysis of URA5 from all the reverted strains revealed mutation of GA₁₂₈T→GGT back to its ancestral state. The URA5 allele in the reverted strains was fully functional, as demonstrated by the excellent growth of these strains on medium lacking uracil, as well as by the ability of this allele to efficiently transform ura5 mutant to restore prototrophy. The deduced Cg URA5 protein modeled on the known crystal structures of OPRTase from Salmonella typhimurium (1LH0_A, 1STO) and from Escherichia coli (1ORO_A) indicated that the glycine 43 of Cg URA5 was situated on a conserved loop, and it’s substitution to more globose aspartic acid may have resulted in URA5 inactivation in auxotrophic strain. The advantages of this approach for the generation of a reconstituted strain are (1) that it restores the functionality of the native URA5, (2) that it eliminates an additional biolistic delivery of exogenous URA5, and (3) that it allows easy selection of reconstituted strains with homologous integration. This strategy was successfully used for the generation of Cg can2+CAN2/URA5 homologous reconstituted strains, which grew in ambient air to the wild-type level while can2 mutant exhibited severe growth defect under similar conditions. Srinivas D. Narasipura and Ping Ren contributed equally to this work.     

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.     

Persistence of infection in mice inoculated intranasally with Cryptococcus neoformans

Cryptococcus neoformans was instilled intranasally into mice which were periodically sacrificed to determine the course of infection. Cryptococci persisted within the nasal passages throughout the 90 day study. Extranasal dissemination began 14–28 days after instillation and was still demonstrable 90 days post-exposure. Ten percent mortality was observed in mice receiving 10⁶ cryptococci, while no mortality was observed in mice exposed to 10³ or 10⁴ cryptococci. Our research suggests that nasal colonization with C. neoformans can precede pulmonary and systemic cryptococcosis by weeks or months.     

Decaying wood in tree trunk hollows as a natural substrate for Cryptococcus neoformans and other yeast-like fungi of clinical interest

The occurrence of Cryptococcus neoformans var. neoformans and other yeast-like fungi of clinical interest in decaying wood inside tree trunk hollows, bark and other plant materials is reported. The var. neoformans was isolated from 3 of 45 (6.6%) wood and one of 390 Eucalyptus bark samples. Two of the positive wood samples came from a tree trunk hollow of Butea monosperma (Family: Papilionaceae) growing in Roshan Ara Garden, Old Delhi whereas the third was from a trunk hollow of Tamarindus indica (Family: Papilionaceae) growing outside of Talkatora Garden, New Delhi. The solitary positive Eucalyptus bark sample originated from Amritsar. The isolations of var. neoformans from decaying wood inside trunk hollows of B. monosperma and T. indica constitute the first record of the natural occurrence of this pathogen in association with these trees. The observation reinforces the recent evidence for decaying wood inside trunk hollows of some trees to be a new natural habitat of the variety neoformans. Besides, in consonance with their essentially saprobic character, a number of other yeast-like fungi were sporadically isolated. This includes, Cryptoccus laurentii Cryptococcus albidus Candida lusitaniae C. guilliermondii C. krusei C. tropicalis C. zeylanoides Trichosporon cutaneum Rhodotorula mucilaginosa R. glutinis Geotrichum capitatum G. klebahnii and Sporobolomyces salmonicolor. Cryptococcus neoformans var. gattii was not found in any of the 702 samples of plant materials, including the bark and detritus of Eucalyptus camaldulensis and E. tereticornis trees. A more extensive environmental survey, covering divergent climatic regions, is warranted to identify the natural reservoirs of var. gattii in India. This revised version was published online in August 2006 with corrections to the Cover Date.     

The isolation,characterization and antifungal susceptibilities of <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> from bird excreta in Klang Valley,Malaysia

The occurrence of Cryptococcus neoformans in bird excreta in Klang valley, Malaysia was determined in this study. Of 544 samples of bird excreta collected from a local zoo, pet shops and public areas, 20 strains of C. neoformans were isolated. All C. neoformans strains were serotype A and thus identified as C. neoformans variety grubii. All did not produce color changes on canavanine–glycine–bromothymol blue agar. All were of α-mating types, as determined by a pheromone-specific PCR assay. The antifungal susceptibility testing using agar diffusion method Neo-sensitabs showed that all were susceptible to amphotericin B, fluconazole and itraconazole.     

Perilla frutescens seed agar, a new medium for identification of the Cryptococcus species complex: evaluation for all major molecular types

Here we present a new and simple medium made by Perilla frutescens seed as a tool for identification of the Cryptococcus species complex, namely Cryptococcus neoformans and Cryptococcus gattii. Its usefulness was evaluated for all major molecular types within the Cryptococcus species complex. As a result, this medium is better for identification of the species complex compared with Guizotia abyssinica or Helianthus annuus seed medium.     

Cutaneous Phaeohyphomycosis due to Alternaria Infectoria

Here we report a case of cutaneous alternariosis in a 74-year-old man treated by corticotherapy for myasthenia, and presenting with papular, crusted lesions on the left elbow and the right knee. Histological examination of the biopsy specimens showed fungal hyphae associated with round-shaped cells which were highly suggestive of alternariosis. Mycological culture allowed the isolation of a dematiaceous fungus which was identified as a member of the Alternaria infectoria species-group. This was confirmed by PCR amplification and sequencing of the internal transcribed spacer domain of the gene encoding nuclear ribosomal DNA and of the mitochondrial small subunit ribosomal DNA domain. The fungus was therefore referred to the Scientific Institute of Public Health where it was identified as Alternaria infectoria, on the basis of its very small 1 or 2-celled conidia often arranged in long chains and presenting with very long secondary conidiophores. Corticotherapy was stopped and a local antifungal treatment with ketoconazole was initiated, allowing the stabilisation of the cutaneous lesions within 2 months.     

Differences in Mitochondrial Genome Organization of Cryptococcus Neoformans Strains

The organization of the mitochondrial genomes in two strains belonging in different varieties of Cryptococcus neoformans was analysed. Physical maps of the mtDNA of the IFM5844 (var. neoformans) and IFO410 (var. grubii) strains were constructed by using EcoRI and EcoRV restriction enzymes; functional maps were constructed by hybridization, cloning and sequencing. Most of the genes important in the mitochondrial function (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, ATP6, ATP9, COX1, COX2 and COB) and protein synthesis (SsrRNA and LsrRNA) were localized. We did not find any differences between the strains in the order of these genes. However, they differed significantly in the sizes of the mtDNAs: 32.6 kb for IFM5844, and 24.1 kb for IFO410. This can be attributed to two large regions of the mtDNA. In these regions, differences were found in the numbers of introns in COX1 (no intron in var. grubii, 5 introns in var. neoformans), COB (1 intron in var. grubii, 2 introns in var. neoformans), LsrRNA (no intron in var. grubii, 2 introns in var. neoformans), and ND5 (no intron in var. grubii, 1 intron in var. neoformans) genes. In several introns of the COB and COX1 genes LAGLIDADG motifs were found. Differences were also observed in the nucleotide sequences of some genes and in the sizes and sequences of intergenic regions. The nucleotide sequences of the genes of the IFM and IFO strains were compared with those of the H-99 and JEC 21 strains from the database. Surprisingly high similarities were found between the strains belonging in var. grubii (IFO 410 and H-99) and var. neoformans (IFM 5844 and JEC 21).     

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.     

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.     

Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an 2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-¹⁴C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat 2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi. Published in 2003.     

Female Genital Coccidioidomycosis (FGC), Addison's Disease and Sigmoid Loop Abscess Due to Coccidioides Immites; Case Report and Review of Literature on FGC

Nine fungal strains of Dichotomomyces cejpii were studied for their ability to produce antibiotic metabolites. It was found that they produced secondary exo- and endo metabolites with antimicrobial activity against Gram-negative and Gram-positive bacteria, yeasts and moulds and with a toxic effect against animal organs in vitro. Detailed chemical characterization of these active principles need to be carried out. This revised version was published online in June 2006 with corrections to the Cover Date.