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.     

Cryptococcus neoformans var. gattii isolates from two Peruvian patients.

The ecology of Cryptococcus neoformans var. gatti is restricted to tropical and subtropical areas whereas C. neoformans var. neoformans is cosmopolitan. Perú is a country with three different well defined geographic areas, some of them have the conditions for the presence of the variety gattii. In order to determine the presence of the two varieties of C. neoformans in Perú, we made the C. neoformans differentiation from the clinical isolates. C. neoformans var. gattii was identified by their ability to assimilate D-proline. We tested 68 strains; only two of them were recognized as the variety gattii and were recovered from two patients without any predisposing factor. We described the clinical spectrum of these two patients, who were diagnosed with disseminated cryptococcosis and cryptococcal meningitis. Neither the presence of Eucalyptus camaldulensis nor Eucalyptus tereticornis has been reported in Perú. So there should exist other ecologic niches for the presence of C. neoformans var. gattii in our country, different from those mentioned.     

Experimental inoculation of Terminalia catappa seedlings with an environmental isolate of Cryptococcus neoformans var. gattii serotype C

In 1997, our laboratory reported for the first time the isolation of Cryptococcus neoformans var. gattii serotype C associated with almond tree (Terminalia catappa) detritus. This finding led to a more detailed follow up of the association between the plant and the yeast. Preliminary data have shown that survival of the yeast in almond trees seedlings goes beyond 100 days. The aim of the present study was to establish if under the conditions previously studied, C. neoformans var. gattii would remain viable for longer periods. A total of 83 almond tree seedings, 20-40 cm high, were inoculated with C. neoformans var. gattii serotype C (INS-755). Assays were carried out inoculating the stem or the soil where the seedlings were planted. Observations were undertaken for a period of up to 12 months. As processing techniques we employed the endophytic fungi procedure (stems), maceration (roots, leaves) and standard suspension method (soils). Additionally, microscopic visualization of the yeast in plant tissues was done with trypan blue plus lactophenol. C. neoformans var. gattii was recovered from the inoculated plants for a period of up to 12 months post-inoculation; additionally, the fungus had the capacity to migrate from the stem to the soil and viceversa, without causing macroscopic or microscopic alterations in the plant tissues. This finding suggests that there appears to be an association between the host plant and C. neoformans var. gattii in the environment.     

Isolation of Cryptococcus neoformans from environmental samples in Alicante

A study of the incidence of Cryptococcus neoformans in Alicante was carried out in environmental samples from the cities of Alicante and Santa Pola. The samples were pigeons faeces and Eucalyptus camaldulensis tissues. This study shows that the prevalence of the yeast in faeces from captive pigeons is higher (81.5%) than in the same samples from urban pigeons (16.3%). Regarding the biotype and varieties, a 79.3% of the isolates belonged to C. neoformans, and all of them were C. neoformans var. neoformans. None of the E. camaldulensis samples showed C. neoformans growth, although some Cryptococcus laurentii strains were isolated from flowers.     

Chromosome length polymorphism in Cryptococcus neoformans clinical and environmental isolates

A protocol for intact DNA preparation from the basidiomycetous yeast Cryptococcus neoformans has been developed and applied to karyotyping C. neoformans isolates displaying different degrees of capsule formation. A total of 46 strains have been analyzed: 23 (50%) isolated from environmental samples (pigeon droppings), all of them belonging to C. neoformans var. neoformans; and 23 (50%) from clinical samples (human and veterinarian) including 10 isolates of C. neoformans var. neoformans and 13 isolates of C. neoformans var. gattii. Our results showed a global genome size ranging from 14.2 to 20.9 Mb for variety neoformans and from 7.9 to 16.8 Mb for variety gattii. The karyotype diversity was very high for variety neoformans (29 different patterns for the 33 analyzed strains) and lower for variety gattii (six different patterns for 13 strains). No grouping among variety neoformans strains from the same origin was found indicating very high genome diversity for this variety, irrespectively of the origin of the strains.     

Experimental infection of almond trees seedlings (Terminalia catappa) with an environmental isolate of Cryptococcus neoformans var. gattii, serotype C

Recently, our laboratory reported the isolation of Cryptococcus neoformans var. gattii, serotype C for the first time from almond trees (Terminalia catappa) detritus. The aim of the present study was to establish the survival of C. neoformans in almond trees seedlings. Thirty seedlings were infected in the stems and samples were taken and processed at different times and by different techniques. No morphological alterations (macro or microscopic) were observed in the infected plants. However, C. neoformans was found to be viable for at least 100 days after infection. These data constitute our first approach towards the understanding of the yeast interactions with a host-plant.     

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiania, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.     

Evidence of recombination in mixed-mating-type and alpha-only populations of Cryptococcus gattii sourced from single eucalyptus tree hollows

Disease caused by the pathogenic yeast Cryptococcus gattii begins with the inhalation of an infectious propagule. As C. gattii is heavily encapsulated, this propagule is most likely to be a basidiospore. However, most C. gattii strains are infertile in laboratory crosses, and population studies indicate that recombination and dispersal are very restricted. In addition, strains of the alpha mating type predominate, which would not be expected in a mating population. C. gattii comprises four genetically distinct molecular genotypes, designated VGI to VGIV. C. gattii molecular type VGI has a strong association with Eucalyptus camaldulensis and can be found in high numbers in E. camaldulensis hollows. Previous work on isolates obtained from E. camaldulensis suggested that environmental populations of C. gattii are highly fragmented, have limited ability to disperse, and are confined to individual tree hollows. In the current study, we examined large numbers of isolates from three separate hollows for evidence of recombination. In two hollows, the alpha and a mating types were present in approximately equal numbers. The third hollow had alpha cells only and was from a region where a isolates have never been found. Statistical analysis of multilocus genotypes revealed recombining subpopulations in the three Eucalyptus hollows. Recombination was equally present in the alpha-a and alpha-only populations. This is consistent with recent studies that have found evidence suggestive of alpha-alpha mating in C. gattii and Cryptococcus neoformans and raises the possibility this may be a widespread phenomenon, allowing these fungi to recombine despite a paucity of a mating partners.     

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.     

Cutaneous cryptococcosis due to Cryptococcus neoformans var. gattii

A case of cutaneous cryptococcosis due to Cryptococcus neoformans var. gattii in an immunocompetent host is presented. In addition a review of the literature on this subject was carried out and a brief comment made on occurrence of the variety gattii in Brazil.     

<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> Isolated from Passerine and Psittacine Bird Excreta in the State of Paraná, Brazil

Cryptococcus neoformans is an opportunistic basidiomycete yeast that causes life-threatening infections as meningoencephalitis primarily in immunocompromised hosts, generally associated with AIDS. The source of this organism is mainly pigeon excreta; however, other avian species' excreta are implicated as a source of this yeast. The occurrence of C. neoformans and Cryptococcus gattii in bird excreta in the state of Paraná in Brazil was determined in this study. A total of 141 samples of Passerine and Psittacine excreta from captive birds were collected. Additionally, 25 clinical samples from Hospital de Clínicas, in the state of Paraná were also analyzed. The determination of molecular and mating type of the isolates was performed by PCR fingerprinting, multiplex PCR, and mating type PCR. Cryptococcus neoformans var. grubii (VNI) was isolated from 36 (25.53%) of Passerine and Psittacine excreta samples. Almost all clinical samples, except one (C. gattii VGI), were classified as C. neoformans var. grubii (VNI). All environmental and clinical isolates were mating type alpha. These findings reinforce that, besides pigeon excreta, the excreta of these birds can also be a reservoir of C. neoformans in domestic and public environments and is of zoonotic importance to immunocompromised patients.     

Diversity among strains of Cryptococcus neoformans var. gattii as revealed by a sequence analysis of multiple genes and a chemotype analysis of capsular polysaccharide.

We demonstrated the diversity of Cryptococcus neoformans var. gattii strains by a sequence analysis of multiple genes: (i) the intergenic spacer (IGS) 1 and 2 regions of the rRNA gene; (ii) the internal transcribed spacer (ITS) region, including 5.8S of the rRNA gene; (iii) TOP1 (topoisomerase); and (iv) CAP59. In these studies, we compared C. neoformans var. gattii with varieties grubii, and neoformans of C. neoformans. Phylogenetic analysis indicated that both C. neoformans var. grubii and C neoformans var. neoformans are monophyletic, but C. neoformans var. gattii showed polyphyletic. C. neoformans var. gattii can be divided into three phylogenetic groups, I, II, and III, with high bootstrap support. Phylogenetic group I contains serotype B and C strains, and groups II and III include serotype B strains. Because the serotype B strains of C. neoformans var. gattii exhibited more genetic divergence, the serological characteristics and chemotypes of their capsular polysaccharide were further investigated. No remarkable difference among the serotype B strains was found in the reactivities to factor serum 5, which is specific for serotype B. The NMR spectra of the capsular polysaccharide from serotype B strains could be divided into three characteristic patterns, but the chemical shifts were very similar. These results suggested that the serotype B strain of C. neoformans var. gattii has more genetic diversity than the serotype C strain of C. neoformans var. gattii or the varieties grubii and neoformans of C. neoformans, but there was no correlation between genotype and chemotype.     

In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii

One of the differences observed between the two varieties of Cryptococcus neoformansis the greater difficulty to achieve an adequate therapeutical response in patients affected by C. neoformans var. gattii, an observation that has been validated in vitro only rarely. The aim of this work was to study the susceptibility patterns of 35 Colombian clinical isolates of C. neoformans, 20 of which belonged to the var. neoformans and 15 to the var. gattii. The minimal inhibitory concentration (MIC) was determined by broth microdilution, according to a modification of the methodology proposed by the National Committee for Clinical Laboratory Standards (NCCLS), using the breakpoints recently suggested by Nguyen et al. (Antimicrob Agents Chemother 1998; 42: 471-472). The antifungals tested were amphotericin B, fluconazole and itraconazole. Most of the isolates were susceptible to the three antimycotics tested regardless of the variety. Resistance to amphotericin B (MIC=2 microg/ml) was documented in two (10%) C. neoformans var. neoformans isolates; additionally, five (33%) C. neoformans var. gattii isolates felt in the category of fluconazole susceptible but dose dependent (MIC 16 microg/ml). In general, all C. neoformans var. gattii isolates proved susceptible only to the higher concentrations of the antifungals tested. For amphotericin B, seven (47%) isolates of this variety had MICs of 1 microg/ml, for fluconazole there were seven (47%) with MICs of 8 microg/ml and in the case of itraconazole, 10 isolates (66%) had MICs > 0.03 microg/ml. The data showed that although these isolates would be classified as susceptible, they actually require greater concentrations of the antifungals to be inhibited. This finding may well correlate both with the difficulty to attain therapeutic success in patients affected with C. neoformans var. gattii and with the need for more prolonged treatment courses in such cases.     

Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii

Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.     

Antibody action after phagocytosis promotes Cryptococcus neoformans and Cryptococcus gattii macrophage exocytosis with biofilm-like microcolony formation

Antibody-mediated phagocytosis was discovered over a century ago but little is known about antibody effects in phagolysosomes. We explored the consequences of antibody-mediated phagocytosis for two closely related human pathogenic fungal species, Cryptococcus neoformans and Cryptococcus gattii , of which C. neoformans encompasses two varieties: neoformans and grubii. The interaction between C. neoformans varieties grubii and neoformans and host cells has been extensively studied, but that of C. gattii and macrophages remains largely unexplored. Like C. neoformans , antibody-mediated phagocytosis of C. gattii cells was followed by intracellular replication, host cell cytoplasmic polysaccharide accumulation and phagosomal extrusion. Both C. gattii and C. neoformans cells exited macrophages in biofilm-like microcolonies where the yeast cells were aggregated in a polysaccharide matrix that contained bound antibody. In contrast, complement-opsonized C. neoformans variety grubii cells were released from macrophages dispersed as individual cells. Hence, both antibody- and complement-mediated phagocytosis resulted in intracellular replication but the mode of opsonization affected the outcome of exocytosis. The biofilm-like microcolony exit strategy of C. neoformans and C. gattii following antibody opsonization reduced fungal cell dispersion. This finding suggests that antibody agglutination effects persist in the phagosome to entangle nascent daughter cells and this phenomenon may contribute to antibody-mediated protection.     

The PRP8 inteins in Cryptococcus are a source of phylogenetic and epidemiological information

Only two nuclear encoded inteins have been described. The first, SceVMA, was found in a vacuolar ATPase gene of Saccharomyces cerevisiae and related yeasts. The second, CnePRP8, was found in the PRP8 gene of Cryptococcus neoformans. CnePRP8 contains protein sequences associated with intein splicing but no endonuclease domain. We compared allelic mini-inteins in both varieties of C. neoformans (var. neoformans and var. grubii) and in the related primary pathogen C. gattii to study the evolution of both the mini-intein and the host. We also describe a full-length, endonuclease-containing intein in Cryptococcus laurentii, a moderately distant relation of C. neoformans. We did not detect an intein in the PRP8 gene of other species of Cryptococcus including species closely related to the C. neoformans/C. gattii group. It is therefore probable that the C. neoformans/C. gattii mini-intein was derived from horizontal transfer in which C. laurentii or another intein-containing species was the source.     

Identification of Novel Hybrids Between Cryptococcus neoformans var. grubii VNI and Cryptococcus gattii VGII

Cryptococcus neoformans and Cryptococcus gattii are pathogenic yeasts causing meningoencephalitis in immunocompromised and immunocompetent hosts. The fungus is typically haploid, and sexual reproduction occurs normally between individuals with opposite mating types, α and a. C. neoformans var. grubii (serotype A) is comprised of molecular types VNI, VNII, and VNB, and C. neoformans var. neoformans (serotype D) contains the molecular type VNIV. Additionally, diploid or aneuploid AD hybrids (VNIII) have been reported. C. gattii contains the molecular types VGI, VGII, VGIII, and VGIV, which encompass both serotypes B and C. To identify possible hybrid strains, URA5-RFLP analysis was performed on 350 globally obtained clinical, environmental, and veterinary isolates. Four clinical isolates from cerebrospinal fluid showed combination patterns of C. neoformans var. grubii and C. gattii: Brazil (n = 2), Colombia (n = 1), and India (n = 1). These strains were monokaryotic and diploid or aneuploid. M13 PCR fingerprinting showed that they contained fragments of both proposed parental groups. Luminex IGS genotyping identified these isolates as hybrids with two different molecular type combinations: three VNI/VGII and one VNI/VGI. Blue color development on CGB agar was delayed in three isolates and absent in one. C. gattii-specific PCR confirmed the presence of C. gattii in the hybrids. CAP59 allele-specific PCR revealed that all the hybrids contained both serotype A and B alleles. Determination of mating-type allelic patterns by PCR revealed that the isolates were αA aB. This is the first study discovering novel natural hybrids between C. neoformans molecular type VNI and C. gattii molecular type VGII.     

Six monophyletic lineages identified within Cryptococcus neoformans and Cryptococcus gattii by multi-locus sequence typing.

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic basidiomycetous yeasts in which six haploid genotypic groups have been distinguished. The two haploid genotypic groups of C. neoformans have been described as variety grubii and variety neoformans. The four C. gattii genotypic groups have, however, not been described as separate taxa. One hundred and seventeen isolates representing all six haploid genotypic groups were selected for multi-locus sequence typing using six loci to investigate if the isolates consistently formed monophyletic lineages. Two monophyletic lineages, corresponding to varieties grubii and neoformans, were consistently present within C. neoformans, supporting the current classification. In addition, four monophyletic lineages corresponding to the previously described genotypic groups were consistently found within C. gattii, indicating that these lineages should be considered different taxa as well.     

Cryptococcus neoformans mates on pigeon guano: implications for the realized ecological niche and globalization

The ecological niche that a species can occupy is determined by its resource requirements and the physical conditions necessary for survival. The niche to which an organism is most highly adapted is the realized niche, whereas the complete range of habitats that an organism can occupy represents the fundamental niche. The growth and development of Cryptococcus neoformans and Cryptococcus gattii on pigeon guano were examined to determine whether these two species occupy the same or different ecological niches. C. neoformans is a cosmopolitan pathogenic yeast that infects predominantly immunocompromised individuals, exists in two varieties (grubii [serotype A] and neoformans [serotype D]), and is commonly isolated from pigeon guano worldwide. By contrast, C. gattii often infects immunocompetent individuals and is associated with geographically restricted environments, most notably, eucalyptus trees. Pigeon guano supported the growth of both species, and a brown pigment related to melanin, a key virulence factor, was produced. C. neoformans exhibited prolific mating on pigeon guano, whereas C. gattii did not. The observations that C. neoformans completes the life cycle on pigeon guano but that C. gattii does not indicates that pigeon guano could represent the realized ecological niche for C. neoformans. Because C. gattii grows on pigeon guano but cannot sexually reproduce, pigeon guano represents a fundamental but not a realized niche for C. gattii. Based on these studies, we hypothesize that an ancestral Cryptococcus strain gained the ability to sexually reproduce in pigeon guano and then swept the globe.