Intra-abdominal Cryptococcosis by Cryptococcus gattii: Case Report and Review

Although abdominal cryptococcomas and visceral cryptococcal lymphadenitis as part of disseminated fungal infection have been reported mostly in HIV-infected patients, localized intra-abdominal involvement due to Cryptococcus gattii has not been previously described in non-HIV-infected patients. In general, a smaller proportion of cryptococcosis is caused by C. gattii. We report here on a type II diabetic HIV-negative patient who presented with a localized intra-abdominal cryptococcal mass due to C. gattii. In addition, we review the general aspects of intra-abdominal and gastrointestinal involvement by Cryptococcus neoformans in the literature and discuss the importance of identifying the C. neoformans varieties and C. gattii in routine laboratories.     

Characterization of environmental sources of the human and animal pathogen Cryptococcus gattii in British Columbia, Canada, and the Pacific Northwest of the United States

Cryptococcus gattii has recently emerged as a primary pathogen of humans and wild and domesticated animals in British Columbia, particularly on Vancouver Island. C. gattii infections are typically infections of the pulmonary and/or the central nervous system, and the incidence of infection in British Columbia is currently the highest reported globally. Prior to this emergence, the environmental distribution of and the extent of colonization by C. gattii in British Columbia were unknown. We characterized the environmental sources and potential determinants of colonization in British Columbia. C. gattii was isolated from tree surfaces, soil, air, freshwater, and seawater, and no seasonal prevalence was observed. The C. gattii concentrations in air samples were significantly higher during the warm, dry summer months, although potentially infectious propagules (<3.3 microm in diameter) were present throughout the year. Positive samples were obtained from many different areas of British Columbia, and some locations were colonization "hot spots." C. gattii was generally isolated from acidic soil, and geographic differences in soil pH may influence the extent of colonization. C. gattii soil colonization also was associated with low moisture and low organic carbon contents. Most of the C. gattii isolates recovered belonged to the VGIIa genetic subtype; however, sympatric colonization by the VGIIb strain was observed at most locations. At one sampling site, VGIIa, VGIIb, VGI, and the Cryptococcus neoformans serotype AD hybrid all were coisolated. Our findings indicate extensive colonization by C. gattii within British Columbia and highlight an expansion of the ecological niche of this pathogen.     

Cryptococcus gattii Infection in an Immunocompetent Patient from Southern Italy

Cryptococcus gattii has becoming more prevalent in temperate climate zones, during the past decades. We describe a C. gattii serotype B infection in an immunocompetent Italian patient with sclerosing cholangitis. The patient traveled once to Eastern Canada and otherwise no other countries than Italy were visited. Molecular analysis revealed that the C. gattii isolate belong to genotype AFLP4/VGI and has mating-type α which is the most common genotype in the Mediterranean environment. The C. gattii strain was found to be closely related, but not identical, to other C. gattii strains from the Mediterranean area.     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

格特隐球菌病暴发流行机制的研究进展

目前已证实, 格特隐球菌病可暴发流行。近10 年来, 在加拿大温哥华岛暴发的格特隐球菌病发病率显著高于其他地区, 且大部分感染者免疫力正常, 该致病菌已蔓延至周边地区。本文就格特隐球菌病暴发流行机制, 包括致病菌的分子流行病学分析、可能来源、毒力分析和播散机制进行综述。     

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.     

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.     

A Decade of Experience: Cryptococcus gattii in British Columbia

It has been over a decade since Cryptococcus gattii was first recognized as the causative organism of an outbreak of cryptococcosis on Vancouver Island, British Columbia. A number of novel observations have been associated with the study of this emergent pathogen. A novel genotype of C. gattii, VGIIa was described as the major genotype associated with clinical disease. Minor genotypes, VGIIb and VGI, are also responsible for disease in British Columbians, in both human and animal populations. The clinical major genotype VGIIa and minor genotype VGIIb are identical to C. gattii isolated from the environment of Vancouver Island. There is more heterogeneity in VGI, and a clear association with the environment is not apparent. Between 1999 and 2010, there have been 281 cases of C. gattii cryptococcosis. Risk factors for infection are reported to be age greater than 50 years, history of smoking, corticosteroid use, HIV infection, and history of cancer or chronic lung disease. The major C. gattii genotype VGIIa is as virulent in mice as the model Cryptococcus, H99 C. neoformans, although the outbreak strain produces a less protective inflammatory response in C57BL/6 mice. The minor genotype VGIIb is significantly less virulent in mouse models. Cryptococcus gattii is found associated with native trees and soil on Vancouver Island. Transiently positive isolations have been made from air and water. An ecological niche for this organism is associated within a limited biogeoclimatic zone characterized by daily average winter temperatures above freezing.     

Fatal disseminated Cryptococcus gattii infection in New Mexico

We report a case of fatal disseminated infection with Cryptococcus gattii in a patient from New Mexico. The patient had no history of recent travel to known C. gattii-endemic areas. Multilocus sequence typing revealed that the isolate belonged to the major molecular type VGIII. Virulence studies in a mouse pulmonary model of infection demonstrated that the strain was less virulent than other C. gattii strains. This represents the first documented case of C. gattii likely acquired in New Mexico.     

New ITS genotype of Cryptococcus gattii isolated from an AIDS patient in Brazil

Based on combinations of nine variable nucleotides at nine different base positions in the ITS1-5.8S-ITS2 region, Cryptococcus gattii strains were classified into six genotypes. A new genotype of C. gattii , designated as ITS type 8, was isolated from an AIDS patient in Brazil. The ITS type 8 strain is closely related to the ITS type 4 strain, which has been frequently isolated in Brazil and the USA, but which shows ITS-signatured nucleotide difference at each nucleotide position. The ITS type 8 strain is also differentiated from all heretofore reported ITS types of C. gattii strains in the RAPD band patterns and IGS sequence information.     

Genetic differentiation, recombination and clonal expansion in environmental populations of Cryptococcus gattii in India

Cryptococcus gattii is a ubiquitous eukaryotic pathogen capable of causing life-threatening infections in a wide variety of hosts, including both immunocompromised and immunocompetent humans. Since infections by C. gattii are predominantly obtained from environmental exposures, understanding environmental populations of this pathogen is critical, especially in countries like India with a large population and with environmental conditions conducive for the growth of C. gattii. In this study, we analysed 109 isolates of C. gattii obtained from hollows of nine tree species from eight geographic locations in India. Multilocus sequence typing was conducted for all isolates using nine gene fragments. All 109 isolates belonged to the VGI group and were mating type α. Population genetic analyses revealed limited evidence of recombination but unambiguous evidence for clonal reproduction and expansion. However, the observed clonal expansion has not obscured the significant genetic differentiation among populations from either different geographic areas or different host tree species. A positive correlation was observed between genetic distance and geographic distance. The results obtained here for environmental populations of C. gattii showed both similarities and differences with those of the closely related Cryptococcus neoformans var. grubii from similar locations and host tree species in India.     

Laccase activity in Cryptococcus gattii strains isolated from goats

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.     

Anticryptococcal activity of Voriconazole against Cryptococcus neoformans var. gatti vs var. neoformans: Comparison with Fluconazole and effect of human serum

Voriconazole (VCZ), a new wide-spectrum antifungal triazole currently in development, was tested for activity against Cryptococcus neoformans (CN) var. gattii and var. neoformans in RPMI-1640 (RPMI) or RPMI plus human serum. In RPMI VCZ was 10-fold more inhibitory than FCZ for both varieties of CN. In the presence of human serum neither VCZ nor FCZ had enhanced activity against CN var. gattii. By contrast, both VCZ and FCZ had significantly increased activity in the presence of serum against CN var. neoformans. The lack of serum-enhancing activity for VCZ or FCZ against CN var. gattii may reflect the in vivo situation and predict less efficacy in CN var. gattii infections. This revised version was published online in June 2006 with corrections to the Cover Date.     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6%) of the HIV-positive subjects and in 13/26 (50%) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.     

Cryptococcus gattii: a resurgent fungal pathogen

Cryptococcus gattii and Cryptococcus neoformans are causal agents of cryptococcosis, which manifests as pneumonia and meningitis. C. gattii has recently received widespread attention owing to outbreaks in British Columbia, Canada and the US Pacific Northwest. The biology of this tree-dwelling yeast is relatively unexplored, and there are few clues about how it causes infections in humans and animals. In this review, we summarize recent discoveries about C. gattii genetics and its ecological niche and highlight areas ripe for future exploration. Increased focus on epidemiology, ecological modeling and host-pathogen interactions is expected to yield a better understanding of this enigmatic yeast, and ultimately lead to better measures for its control.     

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.