Cryptococcus neoformans var. grubii: Separate Varietal Status for Cryptococcus neoformans Serotype A Isolates

Cryptococcus neoformans var. neoformans presently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the URA5 sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting C. neoformans var. neoformans to isolates which are serotype D and describing a new variety, C. neoformans var. grubii, for serotype A isolates.     

Genetic Relationship between Cryptococcus neoformans var. neoformans Strains of Serotypes A and D

Cryptococcus neoformans serotypes A and D are responsible for the overwhelming majority of infections in patients with AIDS. The genetic relationship between the serotypes is poorly understood, but there are significant differences in the epidemiology and clinical presentation of serotype A and D infections. We evaluated the genetic relationship between reference C. neoformans strains belonging to serotypes A and D by analyzing their URA5 sequences and restriction fragment length polymorphisms (RFLPs) with the C. neoformans repetitive element 1 (CNRE-1) probe. The results were compared to those previously obtained for isolates from Brazil and New York City by the same typing methods, and dendrograms were generated. Serotype A and D strains produced distinct RFLP patterns consistent with their separation into two major clusters in the dendrogram generated on the basis of RFLP data. Similarly, serotype A and D strains clustered independently on the basis of the nucleotide sequences of their URA5 genes. Pairwise comparisons revealed average numbers of nucleotide differences within serotypes A and D of 3.0 ± 1.7 and 7.2 ± 3.4, respectively (P < 0.0001), and between serotypes A and D of 41.9 ± 2.7. In summary, our results indicate phylogenetic differences between the two serotypes of C. neoformans var. neoformans and suggest that these serotypes could probably be considered different varieties of C. neoformans.     

First case of human cryptococcosis due to Cryptococcus neoformans var. gattii in Spain

We report the first case of human cryptococcosis due to Cryptococcus neoformans var. gattii described in our country, which was presented as brain cryptococcoma in an immunocompetent patient. An extensive sampling of the patient's environment was carried out to find the source of infection.     

Detection of Cryptococcus neoformans DNA in Tissue Samples by Nested and Real-Time PCR Assays

Two PCR protocols targeting the 18S rRNA gene of Cryptococcus neoformans were established, compared, and evaluated in murine cryptococcal meningitis. One protocol was designed as a nested PCR to be performed in conventional block thermal cyclers. The other protocol was designed as a quantitative single-round PCR adapted to LightCycler technology. One hundred brain homogenates and dilutions originating from 20 ICR mice treated with different azoles were examined. A fungal burden of 3 × 10¹ to 2.9 × 10⁴ CFU per mg of brain tissue was determined by quantitative culture. Specific PCR products were amplified by the conventional and the LightCycler methods in 86 and 87 samples, respectively, with products identified by DNA sequencing and real-time fluorescence detection. An analytical sensitivity of 1 CFU of C. neoformans per mg of brain tissue and less than 10 CFU per volume used for extraction was observed for both PCR protocols, while homogenates of 70 organs from mice infected with other fungi were PCR negative. Specificity testing was performed with genomic DNA from 31 hymenomycetous fungal species and from the ustilaginomycetous yeast Malassezia furfur, which are phylogenetically related to C. neoformans. Twenty-four strains, including species of human skin flora like M. furfur and Trichosporon spp., were PCR negative. Amplification was observed with Cryptococcus amylolentus, Filobasidiella depauperata, Cryptococcus laurentii, and five species unrelated to clinical specimens. LightCycler PCR products from F. depauperata and Trichosporon faecale could be clearly discriminated by melting curve analysis. The sensitive and specific nested PCR assay as well as the rapid and quantitative LightCycler PCR assay might be useful for the diagnosis and monitoring of human cryptococcal infections.     

Microbiological characteristics of clinical isolates of Cryptococcus neoformans in Taiwan: serotypes,mating types,molecular types,virulence factors,and antifungal susceptibility

This study investigated the microbiological characteristics of 100 clinical isolates of Cryptococcus neoformans species complex, including serotypes, mating types, molecular types, antifungal susceptibility and virulence. The isolates were collected at National Taiwan University Hospital from 1999 to 2004. Eight isolates of C. neoformans from pigeon droppings were also evaluated. Among these isolates, 99 were C. neoformans var. grubii serotype A and one was C. neoformans var. gattii serotype B. All of these isolates were α mating types. PCR fingerprinting, generated by primers M13 and (GACA)₄, and URA5 gene restriction fragment length polymorphism analysis revealed that C. neoformans var. grubii isolates belonged to the VNI (98 isolates) and the VNII (one isolate) types, and the single C. neoformans var. gattii was VGI type. The similar profiles of clinical and environmental isolates suggest that patients might acquire these yeasts from the environment. The MIC₉₀ for fluconazole, itraconazole, 5-flucytosine, voriconazole and amphotericin B against all C. neoformans isolates were 8, 0.5, 4, 0.125 and 0.5 mg/L, respectively. All clinical isolates produced urease, phospholipase, capsule and melanin, but these activities varied with individual isolates. Analysis of six clinical and two environmental isolates with various levels of phospholipase activity indicated a correlation between phospholipase activity and the ability to adhere to the lung epithelial cell line, A549. The extent of cell damage, as indicated by lactate dehydrogenase release, also paralleled the phospholipase activity of these isolates. In addition, production of melanin contributed significant protection against amphotericin B killing of the isolates tested.     

Molecular typing of clinical Cryptococcus neoformans isolates collected in Germany from 2004 to 2010

Cryptococcosis is a fungal infection mostly caused by Cryptococcus neoformans. We identified agents of cryptococcosis diagnosed in Germany from 2004 to 2010. We used multi-locus sequence typing (MLST) to understand the molecular epidemiology of cryptococcosis. Sero- and mating types of individual patient isolates were determined by PCR. MLST was performed using the seven-locus scheme. Allele and nucleotide diversity was calculated for each locus of C. neoformans var. grubii and C. neoformans var. neoformans. Phylogenetic relations were assessed by dendrograms. Clinical data were compared between infections caused by the two variants. We studied 101 isolates. Eight were identified as hybrids (8 %). All non-hybrids were of the α mating type. Among 78 C. neoformans var. grubii (77 %), 16 sequence types (STs) were identified including three novel STs. They clustered in four groups, previously isolated in Asia, Europe or worldwide. Among 15 C. neoformans var. neoformans (15 %), 10 STs were identified, without clustering. These isolates showed higher allele, and nucleotide diversity compared with C. neoformans var. grubii. C. neoformans var. neoformans was more likely to cause soft-tissue infections (3/9, 33 vs. 1/63, 2 %, p = 0.005) and to affect non-AIDS patients (7/14, 50 vs. 15/76, 20 %, p = 0.036). C. neoformans var. grubii is the predominant agent of cryptococcosis in Germany. MLST suggests that a part of these cases are acquired abroad by immigrants or tourists. C. neoformans var. neoformans isolates represent a greater genetic diversity and are associated with more variable clinical presentations.     

A Multiplex Real-Time PCR Assay for Identification of Pneumocystis jirovecii,Histoplasma capsulatum,and Cryptococcus neoformans/Cryptococcus gattii in Samples from AIDS Patients with Opportunistic Pneumonia

A molecular diagnostic technique based on real-time PCR was developed for the simultaneous detection of three of the most frequent causative agents of fungal opportunistic pneumonia in AIDS patients: Pneumocystis jirovecii, Histoplasma capsulatum, and Cryptococcus neoformans/Cryptococcus gattii. This technique was tested in cultured strains and in clinical samples from HIV-positive patients. The methodology used involved species-specific molecular beacon probes targeted to the internal transcribed spacer regions of the rDNA. An internal control was also included in each assay. The multiplex real-time PCR assay was tested in 24 clinical strains and 43 clinical samples from AIDS patients with proven fungal infection. The technique developed showed high reproducibility (r² of >0.98) and specificity (100%). For H. capsulatum and Cryptococcus spp., the detection limits of the method were 20 and 2 fg of genomic DNA/20 μl reaction mixture, respectively, while for P. jirovecii the detection limit was 2.92 log₁₀ copies/20 μl reaction mixture. The sensitivity in vitro was 100% for clinical strains and 90.7% for clinical samples. The assay was positive for 92.5% of the patients. For one of the patients with proven histoplasmosis, P. jirovecii was also detected in a bronchoalveolar lavage sample. No PCR inhibition was detected. This multiplex real-time PCR technique is fast, sensitive, and specific and may have clinical applications.     

Differentiation of Cryptococcus neoformans varieties and Cryptococcus gattii using CAP59-based loop-mediated isothermal DNA amplification

Members of the Cryptococcus species complex (C. neoformans and C. gattii) are opportunistic pathogens responsible for frequently fatal cases of meningoencephalitis. These yeasts have been classified into five serotypes. Serotypes A andDare assigned to C. neoformans var. grubii and C. neoformans var. neoformans, respectively, Serotype AD strains are hybrids and serotype B and C strains are considered to belong to the related but distinct species C. gattii. Previous studies have identified ‘serotype-associated' alleles of several genes in the Cryptococcus species complex. We developed a loop-mediated isothermal DNA amplification method using CAP59 allele-specific primers to identify the serotypes A, D and B/C of the Cryptococcus species complex.     

Elucidating the Pathogenesis of Spores from the Human Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans was first described as a human fungal pathogen more than a century ago. One aspect of the C. neoformans infectious life cycle that has been the subject of earnest debate is whether the spores are pathogenic. Despite much speculation, no direct evidence has been presented to resolve this outstanding question. We present evidence that C. neoformans spores are pathogenic in a mouse intranasal inhalation model of infection. In addition, we provide mechanistic insights into spore-host interactions. We found that C. neoformans spores were phagocytosed by alveolar macrophages via interactions between fungal β-(1,3)-glucan and the host receptors Dectin-1 and CD11b. Moreover, we discovered an important link between spore survival and macrophage activation state: intracellular spores were susceptible to reactive oxygen-nitrogen species. We anticipate these results will serve as the basis for a model to further investigate the pathogenic implications of infections caused by fungal spores.Cryptococcus neoformans is an opportunistic fungal pathogen that has emerged as an important cause of morbidity and mortality in people with underlying immune deficiencies. During the last three decades, the incidence of C. neoformans cases has dramatically increased, due in large part to the global human immunodeficiency virus pandemic. C. neoformans is environmentally ubiquitous and can be found in a variety of soils contaminated with avian guano (11). Cryptococcal infection occurs as a result of environmental exposure and inhalation of aerosolized C. neoformans cells. In healthy people, initiation of innate and adaptive cellular immune responses limits the severity of the infection to an asymptomatic and often self-resolving pulmonary infection (11, 35). In contrast, hematogenous dissemination of C. neoformans from the lungs to the central nervous system in immunocompromised people can lead to cryptococcal meningoencephalitis, a life-threatening complication requiring aggressive chemotherapeutic intervention (11, 35).Prior to the discovery in 1975 that C. neoformans could produce spores, only the yeast form was considered to be an infectious propagule (29). Numerous studies showed that small, desiccated, encapsulated C. neoformans yeast could be recovered from soil, and these cells were readily aerosolized and could cause disease in animal models similar to human disease (10, 11, 15, 32, 36, 39). The discovery that C. neoformans had the potential to produce spores led Cohen et al. (12) to hypothesize that spores might also be infectious propagules. This hypothesis was consistent with the infectious life cycles of other human pathogenic fungi; inhalation of spores from Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides spp., and Paracoccidioides brasiliensis can cause pulmonary or disseminated disease in healthy people (38).For the last three decades, efforts to elucidate the role of spores in the pathogenesis of C. neoformans have been severely hampered because of the difficulty of isolating large numbers of pure spores (13, 42, 45). Two previous studies that succeeded in isolating spores from C. neoformans var. neoformans strains found that spores were infectious in mouse models of cryptococcosis, which suggested that they might contribute to the pathogenesis of C. neoformans (42, 45). Although mice inoculated with C. neoformans var. neoformans spores did develop infections, the animals did not exhibit signs of morbidity or mortality (42, 45), likely because the C. neoformans var. neoformans strains used in these studies possessed limited virulence potential. Because the spores used in these studies did not kill mice, the specific role of spores in the pathogenesis of C. neoformans was not addressed (42, 45).Our laboratory recently developed a technique to isolate large numbers of pure spores from the C. neoformans var. neoformans strains, and smaller numbers of pure spores from the virulent C. neoformans var. grubii strains (4). Our initial biochemical characterization of C. neoformans spores revealed that they possessed a cell surface composition different from yeast but physical properties similar to other fungal spores (4). These observations led us to speculate that, like other pathogenic fungi, the infectious life cycle of C. neoformans might include an infectious spore form that transitions into a pathogenic form in the lungs. Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides spp., and Paracoccidioides brasiliensis spores undergo a rapid transition to a parasitic form in the lungs, which is essential for establishment of infection and disease progression (30, 33). Pulmonary host defense against these and other pathogens is mediated by the recognition of pathogen-associated molecular patterns (PAMPS) by host pattern recognition receptors (PRRs). These interactions facilitate the phagocytosis and killing of fungi and other pathogenic microorganisms by activated alveolar macrophages.In the present study, we used spores from virulent C. neoformans var. grubii parental strains to test the hypothesis that spores are infectious propagules. In addition, we used spores from C. neoformans var. neoformans strains to identify mechanisms that contribute to host-fungal interactions. The rationale for using spores from two different varieties of C. neoformans was based on necessity; only a small number of spores could be isolated from the virulent C. neoformans var. grubii strains, which do not produce abundant spores. Thus, these spores were used for our in vivo animal studies. In contrast, C. neoformans var. neoformans strains produced abundant spores, which were required in large numbers for our in vitro assays. Overall, the results of the present study (i) provide evidence that spores are infectious propagules, (ii) that spores contribute to the pathogenesis of C. neoformans, (iii) provide mechanistic insights into the host-pathogen interactions that contribute to innate pulmonary defense against spores, (iv) and reveal the importance of alveolar macrophage activation state for defense against infection by spores.     

Hypersensitivity pneumonitis with antibodies to Cryptococcus neoformans

Clinical and immunological studies were made in forty-two patients diagnosed as suffering from hypersensitivity pneumonitis at Osaka Prefectural Habikino Hospital between 1973 and 1977. All the sera from forty-one patients tested had high litres of antibody against Cryptococcus neoformans in indirect fluorescent antibody tests, and twelve also had precipitins against Cryptococcus neoformans polysaccharide. Only about 10% of control sera from patients with otherlung diseases had low titresof antibody against Cryptococcus neoformans. Antibody against Cryptococcus neoformans was also found frequently in the sera of asymptomatic members of the families of the patients. A possible relationship of Cryptococcus neoformans to hypersensitivity pneumonitis is suggested.     

Spores as Infectious Propagules of Cryptococcus neoformans

Cryptococcus neoformans and Cryptococcus gattii are closely related pathogenic fungi that cause pneumonia and meningitis in both immunocompromised and immunocompetent hosts and are a significant global infectious disease risk. Both species are found in the environment and are acquired via inhalation, leading to an initial pulmonary infection. The infectious propagule is unknown but is hypothesized to be small desiccated yeast cells or spores produced by sexual reproduction (opposite- or same-sex mating). Here we characterize the morphology, germination properties, and virulence of spores. A comparative morphological analysis of hyphae and spores produced by opposite-sex mating, same-sex mating, and self-fertile diploid strains was conducted by scanning electron microscopy, yielding insight into hyphal/basidial morphology and spore size, structure, and surface properties. Spores isolated by microdissection were found to readily germinate even on water agarose medium. Thus, nutritional signals do not appear to be required to stimulate spore germination, and as-yet-unknown environmental factors may normally constrain germination in nature. As few as 500 CFU of a spore-enriched infectious inoculum (∼95% spores) of serotype A C. neoformans var. grubii were fully virulent (100% lethal infection) in both a murine inhalation virulence model and the invertebrate model host Galleria mellonella. In contrast to a previous report on C. neoformans var. neoformans, spores of C. neoformans var. grubii were not more infectious than yeast cells. Molecular analysis of isolates recovered from tissues of infected mice (lung, spleen, and brain) provides evidence for infection and dissemination by recombinant spore products. These studies provide a detailed morphological and physiological analysis of the spore and document that spores can serve as infectious propagules.Humans are exposed to infectious agents via inhalation and cutaneous exposure and from the microbiota. Transmission can involve direct human-human transmission, intermediate animal or insect vectors, or exposure to environmental sources. Animals are exposed to pathogenic fungi via direct/fomite transmission of dermatophytes that infect skin and/or nails, animal-animal transmission by the inhalation of the obligate pathogen Pneumocystis, and bloodstream penetration by Candida species from the gastrointestinal microbiota (46).Many pathogenic fungi (dimorphic fungal pathogens, molds such as Aspergillus fumigatus, and the basidiomycete Cryptococcus) are environmental, and exposure occurs via the inhalation of conidia and/or spores, hyphal fragments, or yeast cells. Particles of >5 μm are subject to efficient mucociliary airway clearance, and smaller infectious propagules more readily deposit deep in the lungs and alveoli (18). As such, spores and conidia represent known or suspected infectious propagules for many pathogenic fungi. Moreover, as spores are often stress resistant, abundant, and readily aerially dispersed, animals may encounter spores more often than other infectious forms. Studies of Schizophyllum commune revealed abundant spores present in air above the ocean at distances of up to a mile from shore (24).Cryptococcus neoformans is a common, opportunistic human fungal pathogen that causes meningoencephalitis in immunocompromised individuals and, if untreated, is uniformly fatal (6, 40). Cryptococcosis is caused by three varieties or sibling species that diverged 10 to 40 million years ago and that exhibit different environmental distributions and virulence properties (32, 43). C. neoformans var. grubii (serotype A) is the major cause (95%) of infections worldwide and >99% of infections in AIDS patients (6). C. neoformans var. neoformans (serotype D) strains account for <5% of infections worldwide, but serotype AD hybrids cause up to 20% of clinical infections in Europe (2, 9, 31). The serotype A and D lineages are globally distributed, associated with pigeon guano, and typically infect immunocompromised hosts.The closely related species Cryptococcus gattii (serotypes B and C) is more geographically restricted to tropical and/or subtropical regions, associated with trees, and commonly infects immunocompetent hosts and less frequently AIDS patients and other immunocompromised hosts (6, 29). A C. gattii outbreak has been occurring on Vancouver Island since 1999 and recently expanded to the Canadian mainland and the United States (5, 12, 25, 38, 52).The infectious propagules for many human fungal pathogens are thought to be spores. Humans are exposed to Cryptococcus by inhalation, leading to an initial pulmonary infection that can be asymptomatic or limited or can disseminate. For many individuals, the initial pulmonary infection is cleared; in others, the organism establishes a dormant latent granulomatous form in the hilar lymph nodes (10, 17). Individuals can die harboring granulomas without overt disease (1, 19, 48). In response to immunosuppression, either primary infection or recrudescence of latent infection results in dissemination via the bloodstream. The organism can infect many organs and tissues but exhibits a predilection to infect the central nervous system (45). The suspected infectious propagules for Cryptococcus are spores or small, desiccated, less encapsulated yeast cells that are an ideal size for alveolar deposition. C. neoformans var. neoformans serotype D spores have been reported to be up to 100 times more infectious than yeast cells in an immunocompromised murine inhalation model (50). Studies to purify spores and analyze their pathogenicity in animal models using different infection methods were previously reported, documenting that spores can serve as infectious propagules under certain conditions, including when directly inoculated intracerebrally, intravenously, or by inhalation (7, 50, 59).Cryptococcus neoformans and C. gattii have defined sexual cycles that produce abundant basidiospores, which could represent infectious propagules (13, 23, 27, 28, 39, 42). However, if spores are the infectious propagule, and spores are produced by mating between a and α cells, how and where would this occur in the largely α unisexual population? A potential resolution was the discovery that α isolates can produce hyphae, basidia, and basidiospores via monokaryotic fruiting (11, 54), a modified sexual cycle that requires only one of the two mating types (4, 12, 21, 33-36, 49). This might be a route by which infectious propagules are produced in nature; however, serotype A C. neoformans var. grubii and C. gattii strains do not reproducibly undergo monokaryotic fruiting under laboratory conditions. Population genetic studies provided evidence that they do so in nature (4, 21, 36, 49), but this remains to be established under defined laboratory conditions.As most previous studies of spore infectivity focused on serotype D, we focused our investigations on serotype A spores and virulence properties. Our analysis examined spore size, shape, and surface properties and conditions supporting spore germination. Importantly, we demonstrate that infectious inocula highly enriched for spores serve as efficient infectious propagules in both a murine inhalation model and an invertebrate host. Analyses of isolates recovered from infected animals provide evidence that recombinant spore progeny are infectious. Spores were not more virulent than yeast cells in either virulence assay. Taken together, our studies provide electron microscopic views of the spores and demonstrate that both spores and yeast cells can serve as infectious propagules.     

Development of a Nested PCR for Detection of Cryptococcus neoformans in Cerebrospinal Fluid

We report the development of a nested-PCR-based assay for the detection of Cryptococcus neoformans in cerebrospinal fluid. The specificity and sensitivity of the test were assessed. The technique was then applied to 40 cerebrospinal fluid samples. We obtained positive reactions for all 21 clinical samples from patients who had been previously diagnosed as having cryptococcal meningitis by conventional techniques and negative reactions for all 19 negative controls. Nested PCR is here compared with other diagnostic methods currently used in patients’ follow-up exams during anticryptococcal therapy.     

Origin of Cryptococcus neoformans var. neoformans Diploid Strains

The basidiomycetous yeast Cryptococcus neoformans is an important human fungal pathogen. Two varieties, C. neoformans var. neoformans and C. neoformans var. gattii, have been identified. Both are heterothallic with two mating types, MATa and MATalpha. Some rare isolates are self-fertile and are considered occasional diploid or aneuploid strains. In the present study, 133 isolates, mostly from Italian patients, were investigated to detect the presence of diploid strains in the Igiene Università Milano culture collection. All of the diploid isolates were further investigated by different methods to elucidate their origins. Forty-nine diploid strains were identified by flow cytometry. PCR fingerprinting using the (GACA)(4) primer showed that the diploid state was associated with two specific genotypes identified as VN3 and VN4. Determination of mating type on V8 juice medium confirmed that the majority of the strains were sterile. PCR and dot blotting using the two pheromone genes (MFa and MFalpha) as probes identified 36 of the 49 diploid isolates as MATa/alpha. The results of pheromone gene sequencing showed that two allelic MFalpha genes exist and are distinct for serotypes A and D. In contrast, the MFa gene sequence was conserved in both serotype alleles. Amplification of serotype-specific STE20 alleles demonstrated that the diploid strains contained one mating locus inherited from a serotype A parent and one inherited from a serotype D parent. The present results suggest that diploid isolates may be common among the C. neoformans population and that in Italy and other European countries serotype A and D populations are not genetically isolated but are able to recombine by sexual reproduction.     

Disseminated Infection due to Nocardia transvalensis Coincident with Cryptococcus neoformans variety gattii Meningitis

 A case of meningitis due to Cryptococcus neoformans var. gattii coincident with disseminated Nocardia transvalensis infection is reported. Nocardia infection initially progressed despite high-dose antimicrobial therapy. Although a specific immunologic defect could not be defined, in vitro lymphocyte proliferation in response to stimulation with the Nocardia isolate was reduced. It is proposed that coinfection with Cryptococcus neoformans may have contributed to the observed impairment of lymphocyte function, leading to disseminated Nocardia disease and a suboptimal treatment response.     

Mannoproteins of Cryptococcus neoformans induce proliferative response in human peripheral blood mononuclear cells (PBMC) and enhance HIV-1 replication

To investigate the possible role of Cryptococcus neoformans var. neoformans in HIV disease progression, and to identify the responsible cryptococcal components, an in vitro cell culture model was set up to study the C. neoformans-induced enhancement of HIV replication in HIV-1-infected PBMC. Similar to whole C. neoformans, cell-wall membrane fraction and mannoproteins induced proliferation of PBMC and enhancement of lymphotropic HIV replication in HIV-infected PBMC, while galactoxylomannan did not. MoAbs capable of interfering with MHC class II-mediated antigen presentation prevented the induction of cell proliferation by whole C. neoformans or cryptococcal mannoproteins. MoAb binding to adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) also inhibited C. neoformans-induced cell proliferation. In addition, anti-MHC class II MoAb inhibited the enhancement of HIV replication by C. neoformans. The results suggest that: (i) C. neoformans may accelerate HIV disease progression by stimulation of HIV replication through MHC class II-mediated antigen presentation; and (ii) cryptococcal mannoprotein may be one of the responsible components. The ability to enhance HIV replication in PBMC in vitro is not unique for C. neoformans. However, this is the first report to study in detail a yeast-induced enhancement of HIV replication in PBMC.     

Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients’ samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.     

Cryptococcus neoformans Resides in an Acidic Phagolysosome of Human Macrophages

Recently, we demonstrated that human monocyte-derived macrophages (MDM) treated with chloroquine or ammonium chloride had markedly increased antifungal activity against the AIDS-related pathogen Cryptococcus neoformans. Both of these agents raise the lysosomal pH, which suggested that the increased antifungal activity was a function of alkalinizing the phagolysosome. Moreover, there was an inverse correlation between growth of C. neoformans in cell-free media and pH. These data suggested that C. neoformans was well adapted to survive within acidic compartments. To test this hypothesis, we performed studies to determine the pH of human MDM and neutrophil phagosomes containing C. neoformans. Fungi were labeled with the isothiocyanate derivatives of two pH-sensitive probes: fluorescein and 2′,7′-difluorofluorescein (Oregon Green). These probes have pK_as of 6.4 and 4.7, respectively, allowing sensitive pH detection over a broad range. The phagosomal pH averaged approximately 5 after ingestion of either live or heat-killed fungi and remained relatively constant over time, which suggested that C. neoformans does not actively regulate the pH of its phagosome. The addition of 10 and 100 μM chloroquine resulted in increases in the phagosomal pH from a baseline of 5.1 up to 6.5 and 7.3, respectively. Finally, by immunofluorescence, colocalization of C. neoformans and the MDM lysosomal membrane protein LAMP-1 was demonstrated, establishing that fusion of C. neoformans-laden phagosomes with lysosomal compartments takes place. Thus, unlike many other intracellular pathogens, C. neoformans does not avoid fusion with macrophage lysosomal compartments but rather resides and survives in an acidic phagolysosome.     

Most Environmental Isolates of Cryptococcus neoformans var. grubii (Serotype A) Are Not Lethal for Mice

Most cases of cryptococcosis are caused by Cryptococcus neoformans var. grubii (serotype A), which is widespread in the environment, where it is primarily associated with pigeon excreta. A number of molecular epidemiological studies indicate that many environmental and clinical isolates of serotype A are indistinguishable. However, the murine virulence of environmental strains of C. neoformans has not been thoroughly evaluated. We used the murine intranasal model of cryptococcosis to compare the lethality of clinical and environmental strains of serotype A that possessed identical genotypes as determined by amplified fragment length polymorphisms (AFLP) and multilocus sequence typing (MLST). Eleven environmental strains were tested, and only one caused disease within 60 days postinfection, at which time the experiments were terminated. Conversely, 7 of 10 clinical isolates were lethal for mice at median times of 19 to 40 days. Passing environmental isolates in mice (up to three times) did not significantly increase their lethality. In follow-up studies, we developed a new genotyping technique based on hybridization with TCN2 and TCN4 retrotransposon-specific probes. Although the retrotransposon banding patterns were unstable after prolonged incubation in the laboratory, this method was able to differentiate clinical and environmental strains that had the same AFLP/MLST genotypes.Cryptococcus neoformans var. grubii (serotype A) is a ubiquitous saprobic yeast. In nature, it can be routinely isolated from avian, especially pigeon, habitats, soil contaminated with avian excreta, and decayed wood (10, 18, 20, 25, 35, 36). Mammalian infection with C. neoformans is acquired by the inhalation of airborne yeasts or spores. The ensuing pulmonary infection is often asymptomatic. Alternatively, this exposure may lead to subacute, invasive disease. The yeast cells may disseminate to any part of the body, but C. neoformans is neurotropic, and the typical, life-threatening clinical manifestation of cryptococcosis is meningoencephalitis. Cryptococcal disease occurs predominantly in people with impaired immunity, but individuals with apparently normal host defenses may also develop grave infections (5). With rare exceptions, cryptococcosis is not transmissible among humans or other animals (5).Although cryptococcosis can be caused by other species of Cryptococcus or any of the three serotypes of C. neoformans (A, D, or AD), most clinical and veterinary cases worldwide are caused by isolates of serotype A, which is also the most prevalent serotype among environmental samples. Genotypic analyses of isolates of serotype A have identified three genetically isolated subpopulations, designated VNI, VNII, and VNB (26-28). Representative isolates of all three populations have been cultured from patients and the environment. However, VNI strains of serotype A are globally dominant in patients, veterinary cases, and the environment. For example, using amplified fragment length polymorphisms (AFLP) and multilocus sequence typing (MLST), we genotyped numerous environmental VNI isolates from North America and found identical genotypes in clinical and environmental samples (25, 28). In contradistinction, strains of VNII are much less common in patients, and they are exceptionally rare in the environment (3, 28, 31). Regarding the VNB population, both clinical and environmental isolates appear to be confined to southern Africa (28).Since VNI strains with identical genotypes can be isolated from clinical specimens and environmental samples, it is widely accepted that fully virulent strains are present in the environment (5, 6, 21). However, few studies have experimentally tested the pathogenicity of environmental isolates of C. neoformans, and none have compared the virulence of clinical and environmental isolates with the same genotypes. In 2006, Silva et al. compared the virulence of 62 strains of serotype A that were isolated from pigeon excreta in Brazil (39). Using an intravenous mouse model of experimental cryptococcosis, they observed that the environmental strains varied in their abilities to cause disease; some mice developed symptoms of cryptococcosis, but others remained asymptomatic (39). In 1989, Fromtling et al. evaluated the murine virulence of environmental and clinical isolates of C. neoformans from Puerto Rico (14). They also used an intravenous model of infection, and they determined that the mean lethal dose resulting in the death of 50% of the infected mice was significantly higher for environmental strains than clinical strains (14). In the next-most-recent report of the virulence of environmental strains, published in 1963, the authors compared 21 clinical isolates of C. neoformans with 47 isolates from soil contaminated with pigeon feces (19). The isolates were tested for virulence in a murine intracerebral model of cryptococcosis, in which suspensions of the yeast cells were injected directly into the cerebra of the mice. They concluded that “the strains isolated from [human] cases of cryptococcosis were more virulent than the soil strains; however, almost half [<50%] of the soil strains demonstrated virulence within the range shown by the isolates from human disease…” (19). The murine virulence of C. neoformans was also studied in the 1950s (11, 12, 23). However, at that time, selective media for the in vitro isolation of C. neoformans from the environment were not available, and in these reports, mice were used for the primary isolation of environmental strains by injecting suspensions of pigeon guano into mice and, after several weeks, culturing their livers and spleens for C. neoformans. Obviously, the environmental strains they recovered were preselected for their ability to cause infections. Here, we selected clinical and environmental strains of serotype A with identical AFLP and MLST genotypes and compared their pathogenicities in mice to determine whether there was any correlation between genotype and virulence for mice.     

Development of Positive Selectable Markers for the Fungal Pathogen Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningitis in ~10% of patients with AIDS. New selectable markers which confer resistance to G418 or phleomycin when transformed into C. neoformans were made. A hygromycin-selectable marker was modified to allow selection with a single copy of the marker.