Thomas G Mitchell

• Ph.D.
• Duke University Medical Center

This is the first study on detailed characterization of strain of Cryptococcus neoformans from cases of Cryptococcal meningitis in India

Reports of clinical isolates of Cryptococcus neoformans often lack information on their mating types, molecular types, and in vitro antimycotic susceptibilities. This study compares these and other related characteristics of fifteen strains of C. neoformans obtained from cases of meningitis in different regions of India. PCR was used to determine t...

The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal ev...

The pathogenic species of Cryptococcus are a major cause of mortality owing to severe infections in immunocompromised as well as immunocompetent individuals. Although antifungal treatment is usually effective, many patients relapse after treatment, and in such cases, comparative analyses of the genomes of incident and relapse isolates may reveal ev...

Fig. S2 eburst analysis of the Cng isolates from Botswana.

Fig. S1 The genetic relationships among 141 Botswana Cng isolates are visualized by Principle Components Analysis.

Table S1 Summary information of 614 environmental sampling sites in Botswana. Table S2 Mating types of 304 Cng isolates. City information for clinical strains is based on the patients’ residence areas. Table S3 The MLST allelic profiles of 141 Cng isolates from Botswana. Table S4 Summary of polymorphism among environmental isolates from differen...

Cryptococcus neoformans var. grubii (Cng) is the most common cause of fungal meningitis and its prevalence is highest in sub-Saharan Africa. Patients become infected by inhaling airborne spores or desiccated yeast cells from the environment, where the fungus thrives in avian droppings, trees, and soil. To investigate the prevalence and population s...

Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species, and it is especially useful for analyzing haploid eukaryotes. MLST is rigorous, reproducible, and informative, and MLST genotyping has been shown to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well a...

Unlabelled: Cryptococcus neoformans is the leading cause of fungal meningitis worldwide. Previous studies have characterized the cryptococcal transcriptome under various stress conditions, but a comprehensive profile of the C. neoformans transcriptome in the human host has not been attempted. Here, we extracted RNA from yeast cells taken directly...

Patients with cryptococcal meningitis in sub-Saharan Africa frequently relapse following treatment. The natural history and etiology of these recurrent episodes warrant investigation. Here we used multilocus sequence typing (MLST) to compare the molecular genotypes of strains of Cryptococcus neoformans and C. gattii isolated from serial episodes of...

Species of Candida frequently cause life-threatening infections in neonates, transplant and intensive care unit (ICU) patients, and others with compromised host defenses. The successful management of systemic candidiasis depends upon early, rapid diagnosis. Blood cultures are the standard diagnostic method, but identification requires days and less...

Cryptococcus neoformans is a ubiquitous, saprobic yeast and the cause of life-threatening infections. Humans acquire the infection by inhaling airborne cells from the environment. In the lungs, these cells become encapsulated yeasts and proliferate. In people with healthy immune responses, the infection may resolve or remain latent and subsequently...

CGH analysis of 18 clinical and environmental strains (Figure S5). The strains were analyzed along with strains A5-35-17 and JP1086 (Figure 8) using the array for the reference strain H99.

Primer list for strain construction (Table S4). The primers employed to insert a selectable marker on chr 13 of strain H99 are listed in Table S4.

Analysis of three HC-6 isolates by fluorescence-activated flow cytometry (Figure S7). The ploidy of the three isolates from patient HC-6 were examined and the haploid strain H99 and the diploid strain KW5 were included as controls.

Genes up-regulated in the black strain B4 (Table S1). A microarray comparison of the transcriptomes of the black B4 isolate and the white W2 isolate identified genes with greater than two-fold elevated transcript levels in the B4 isolate.

Genes up-regulated in the white strain W2 (Table S2). A microarray comparison of the transcriptomes of the black B4 isolate and the white W2 isolate identified genes with greater than two-fold elevated transcript levels in the W2 isolate.

CGH analysis of additional strains obtained from mice (Figure S3). Passage of the CBS7779 variants W2, W3 and B6 in mice lead to variation at chr 4 and chr 13. As described in the text and presented in Figure 6, white and black strains collected from the lungs and brains of mice infected with CBS7779 variants were compared to the reference genome o...

Phenotypic characterization of variants of the laboratory strain H99 (Figure S4). (A) The screen for variants with reduced melanin production is shown for tagged strains on L-DOPA medium containing neomycin. This procedure yielded the strains analyzed in Figure 7. (B) The capsule size and cell morphology of the variants were examined with india ink...

CGH analysis of isolates from HIV/AIDS patients (Figure S6). Three isolated colonies were tested for patients HC-2, HC-3 and HC-5, and a representative colony is shown for the three colonies from patient HC-6. The analysis of another HC-6 colony is shown in Figure 8 (all three colonies had the same chromosome complement).

Comparison of gene expression for a second-generation black strain (Figure S1). Gene expression was compared for the CBS7779-B4 strain and the "second generation" black strain CBS7779-W2BA by microarray analysis. Strain CBS7779-W2BA was obtained from the white strain CBS7779-W2 (Figure 1). Two arrays were employed and two biological repeats were pe...

CGH analysis of additional strains with chromosome copy number variation (Figure S2). Passage of the white variant CBS7779-W2 (disomic for chr 13) in culture and analysis of black or white isolates by CGH revealed changes at additional chromosomes. The black variant W2-BB showed copy number increase for a segment of chr 12, and the white isolate W2...

Strain list (Table S3). The strains employed in the study are listed in Table S3.

Primer list for qPCR (Table S5). The primers employed for quantitative real time PCR to confirm the copy number of chromosomes 4 and 13 are listed in Table S5.

The adaptation of pathogenic fungi to the host environment via large-scale genomic changes is a poorly characterized phenomenon. Cryptococcus neoformans is the leading cause of fungal meningoencephalitis in HIV/AIDS patients, and we recently discovered clinical strains of the fungus that are disomic for chromosome 13. Here, we examined the genome p...

General geographical regions of Botswana (BW) and the Republic of South Africa (RSA) where environmental (red triangles) and clinical (blue triangles) isolates were obtained. (PDF)

Environmental samples yielding isolates of Cryptococcus neoformans var. grubii. (PDF)

Descriptions of the 142 strains of Cryptococcus neoformans var. grubii used in this study. (PDF)

Indices of association (IA) among the loci in subpopulations of C. neoformans var. grubii. (PDF)

The growth of African arboreal and avian strains on mopane bark and pigeon excreta media. C. neoformans var. grubii are able to grow in the laboratory on media containing 10% boiled mopane bark (left) or 12% pigeon excreta (right) as sole nutrients. All strains except the Botswanan isolate of Cryptococcus gattii (a sibling species of C. neoformans...

Ancestral recombination graphs (ARGs) of the eight MLST loci. Each ARG is rooted with serotype D sequence (H1). Blue ellipses designate the recombination nodes, and the numbers inside them indicate the SNP immediately to the left of the recombination breakpoint. The paths leading to the recombination nodes are labeled with a P (prefix) or S (suffix...

Genealogies of CAP59, PLB1 and TEF1 loci obtained using maximum likelihood method. Strains are color-coded based on their assignment to different subpopulations: VNII strains are green, VNB strains are red, VNI strains are blue. Strains are assigned to subpopulations based on NJ analysis of the concatenated loci (Fig. 2) and Bayesian algorithm impl...

Results of Templeton (T) and Kishino-Hasegawa (K-H) tests for conflict among phylogenetic topologies of the gene genealogies at each locus. (PDF)

MLST primers and PCR conditions used in this study. (PDF)

Most of the species of fungi that cause disease in mammals, including Cryptococcus neoformans var. grubii (serotype A), are exogenous and non-contagious. Cryptococcus neoformans var. grubii is associated worldwide with avian and arboreal habitats. This airborne, opportunistic pathogen is profoundly neurotropic and the leading cause of fungal mening...

Compared to the incidence in adults, cryptococcosis is inexplicably rare among children, even in sub-Saharan Africa, which has the highest prevalence of coinfection with HIV and Cryptococcus neoformans. To explore any mycological basis for this age-related difference in the incidence of cryptococcosis, we investigated isolates of C. neoformans reco...

Origin of the Fungi2Concepts and Terms3Phenotypes and Molecular Markers to Identify Fungi4Phylogenetic Methods5Genomics6Classification7Phylum Chytridiomycota8Phylum Zygomycota9Phylum Ascomycota10Phylum Basidiomycota11Clinical Applications12Acknowledgments Kingdom fungi, fungal phylogeny and systematics;phenotypes and molecular markers in identifyin...

Rapid, accurate diagnosis of community-acquired pneumonia (CAP) due to Mycoplasma pneumoniae is compromised by low sensitivity of culture and serology. Polymerase chain reaction (PCR) has emerged as a sensitive method to detect M. pneumoniae DNA in clinical specimens. However, conventional real-time PCR is not cost-effective for routine or outpatie...

This paper details the development of a digital microfluidic platform for multiplexed real-time polymerase chain reactions (PCR). Liquid samples in discrete droplet format are programmably manipulated upon an electrode array by the use of electrowetting. Rapid PCR thermocycling is performed in a closed-loop flow-through format where for each cycle...

This article reviews the more common DNA-based molecular markers used to genotype species and strains of Cryptococcus. The current schemata for molecular epidemiological typing of C. neoformans and C. gattii are defined and summarized. Common methods of assessing the population genetics of the C. neoformans–C. gattii species complex are described a...

This communication describes the consensus multi-locus typing scheme established by the Cryptococcal Working Group I (Genotyping of Cryptococcus neoformans and C. gattii) of the International Society for Human and Animal Mycology (ISHAM) using seven unlinked genetic loci for global strain genotyping. These genetic loci include the housekeeping gene...

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 virul...

Cryptococcus neoformans frequently causes fungal meningitis in immunocompromised patients, whereas the related species C. gattii is restricted to tropical and subtropical regions, where it usually infects immunocompetent individuals. An outbreak of C. gattii infection that began in 1999 on Vancouver Island has resulted in endemic C. gattii infectio...

The ubiquitous environmental human pathogen Cryptococcus neoformans is traditionally considered a haploid fungus with a bipolar mating system. In nature, the alpha mating type is overwhelmingly predominant over a. How genetic diversity is generated and maintained by this heterothallic fungus in a largely unisexual alpha population is unclear. Recen...

The Cryptococcus species complex contains two sibling taxa, Cryptococcus neoformans and Cryptococcus gattii. Both species are basidiomycetous yeasts and major pathogens of humans and other mammals. Genotyping methods have identified major haploid molecular types of C. neoformans (VNI, VNII, VNB and VNIV) and of C. gattii (VGI, VGII, VGIII and VGIV)...

[This corrects the article on p. e5862 in vol. 4, PMID: 19517012.].

Cryptococcus neoformans, a model pathogenic fungus, exemplifies the application of several genome-wide approaches to investigate fungal pathogenicity. This review focuses on the application of genome-wide approaches to large populations of clinical and environmental isolates rather than to a small number of well-defined laboratory strains. Specific...

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, alpha and a. However, the overwhelming predominance of mating type (MAT) alpha over a in C. neofor...

Supplemental Methods and Materials (30 KB DOC)

Aa and Da MAT Locus Probes Are Functional Fragmented genomic DNA from strains H99 (Aα), KN99a (Aa), JEC20 (Da), and JEC21 (Dα) was labeled with fluorescent dyes and competitively hybridized to a 70-mer genome array. The fluorescent signal level was normalized across the genome, and the average of at least two independent replicates of the fluoresce...

CGH of the Mating Type Locus of an Unrecognized αADα Hybrid Fragmented genomic DNA from isolate 713 and a mixture of genomic DNA from strains H99 (Aα) and JEC21 (Dα) was labeled with fluorescent dyes and competitively hybridized to a 70-mer genome array. The fluorescent signal level was normalized across the genome, and the average of three indepen...

A Transgenic Dα Strain Bearing the SXI1Dα C-Terminal Deletion in the JEC21 Background Is Fertile Wild-type strain JEC21, the sxi1Δ mutant CHY618 [6], and the transgenic sxi1ΔC strain XL1620 mated with Aa reference strain KN99a on V8 medium (pH 7.0) in the dark at 22 °C for 2 wk. The upper row shows mating hyphae with basidiospores by microscopy (×2...

αADα Hybrid Is More Resistant to UV Irradiation Than Isogenic Haploid or Diploid Isolates Aα strain H99, αAAα strain XL1501, Dα strain JEC21, αDDα strain XL143, and αADα strain XL1462 were grown in liquid YPD medium overnight and washed three times with distilled water. Cell concentration was determined by counting with hemacytometer. Three microli...

Multiple Alignment of the Predicted Amino Acid Sequences Encoded by SXI1α Genes The premature stop codon present in the majority of VGIII C. gattii strains is located in the C-terminal encoding region of the SXI1α gene, similar to the deletion site found in the SXI1Dα allele in the aADα and αADα populations. The C-terminal truncated Sxi1α found in...

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var....

Cryptococcus neoformans is a fungal human pathogen with a bipolar mating system. It undergoes a dimorphic transition from a unicellular yeast to hyphal filamentous growth during mating and monokaryotic fruiting. The traditional sexual cycle that leads to the production of infectious basidiospores involves cells of both alpha and a mating type. Mono...

Strains (34 KB XLS)

Marker Location, Genotypic, and Phenotypic Information (185 KB XLS)

Primer Sequences (27 KB XLS)

Association of MAC1 Allele with Trait Values Association of MAC1 alleles with filamentation (A), melanin production (B), and high temperature growth (C). The progeny percentile was plotted against the trait values to analyze the marker-trait association. Isolates possessing the B3501/XL304 allele are depicted as open bars and isolates possessing th...

We applied multilocus sequence typing (MLST) to investigate the population structure and mode of reproduction of Cryptococcus neoformans var. grubii (serotype A). This MLST system utilizes 12 unlinked polymorphic loci, which are dispersed on nine different chromosomes, and allows the unambiguous identification of closely related strains of serotype...

The study of quantitative traits provides a window on the interactions between multiple unlinked genetic loci. The interaction between hosts and pathogenic microbes, such as fungi, involves aspects of quantitative genetics for both partners in this dynamic equilibrium. One important pathogenic fungus is Cryptococcus neoformans, a basidiomycete yeas...

The association of Cryptococcus gattii with Eucalyptus trees has been well established. Here we report the isolation of both C. gattii and Cryptococcus neoformans var. grubii from the flowers and bark of Eucalyptus trees in India. We investigated a total of 233 samples of Eucalyptus trees: 120 flowers, 81 fragments of bark, and 32 leaves. C. gattii...

Cryptococcus gattii is a group of exogenous, neurotropic yeasts that possess the capsular serotype B or C. Isolates of serotype C are extremely rare and, until recently, were known to infect only immunocompetent individuals. We genotyped 176 isolates of Cryptococcus from patients in sub-Saharan Africa who had AIDS; 22 (13.7%) of 161 isolates from B...

Cryptococcus neoformans is a fungal pathogen that has evolved over the past 40 million years into three distinct varieties or sibling species (gattii, grubii, and neoformans). Each variety manifests differences in epidemiology and disease, and var. grubii strains are responsible for the vast majority of human disease. In previous studies, α strains...

Large chromosomal events such as translocations and segmental duplications enable rapid adaptation to new environments. Here we marshal genomic, genetic, meiotic mapping, and physical evidence to demonstrate that a chromosomal translocation and segmental duplication occurred during construction of a congenic strain pair in the fungal human pathogen...

Cryptococcus neoformans is a major, global cause of meningoencephalitis in immunocompromised patients. Despite advances in the molecular epidemiology of C. neoformans, its population structure and mode of reproduction are not well understood. In the environment, it is associated with avian guano or vegetation. We collected nearly 800 environmental...

Cryptococcus neoformans is a basidiomycetous yeast ubiquitous in the environment, a model for fungal pathogenesis, and an opportunistic human pathogen of global importance. We have sequenced its approximately 20-megabase genome, which contains approximately 6500 intron-rich gene structures and encodes a transcriptome abundant in alternatively splic...

Hemiascomycetes are species of yeasts within the order Saccharomycetales. The order encompasses disparate genera with a variety of life styles, including opportunistic human pathogens (e.g., Candida albicans), plant pathogens (e.g., Eremothecium gossypii), and cosmopolitan yeasts associated with water and decaying vegetation. To analyze the phyloge...

To construct a genetic linkage map of the heterothallic yeast, Cryptococcus neoformans (Filobasidiella neoformans), we crossed two mating-compatible strains and analyzed 94 progeny for the segregation of 301 polymorphic markers, consisting of 228 restriction site polymorphisms, 63 microsatellites, two indels, and eight mating-type (MAT)-associated...

The number of immunocompromised patients and subsequent invasive fungal infections continues to rise. However, the education of future medical mycologists to engage this growing problem is diminishing. While there are an increasing number of publications and grants awarded in mycology, the time and detail devoted to teaching medical mycology in Uni...

The most common cause of fungal meningitis in humans, Cryptococcus neoformans serotype A, is a basidiomycetous yeast with a bipolar mating system. However, the vast majority (>99.9%) of C. neoformans serotype A isolates possess only one of the two mating type alleles (MATα). Isolates with the other allele (MATa) were recently discovered and proven...

Cryptococcus neoformans is a major pathogen of humans throughout the world. Using commercial monoclonal antibodies to capsular epitopes, strains of C. neoformans manifest five serotypes: A, B, C, D and AD. Previous studies demonstrated significant divergence among serotypes A, B, C and D, which are typically haploid. In contrast, most strains of se...

The oral yeast flora of healthy humans from eastern North America and China were sampled and compared. Chinese persons harbored a greater number and diversity of yeast species in the mouth. Furthermore, Candida albicans, which is the predominant commensal and etiologic species of candidiasis in Europe and the Western Hemisphere, was relatively rare...

Fungi may cause human disease by establishing an infection, inducing an allergic response or producing a toxin. Most pathogenic fungi are free-living saprophytes and are only incidental pathogens of humans. The mycoses or fungal infections with the highest prevalence are caused by the few fungi that are part of the normal microbial flora or that ar...

A multiplex PCR method was developed to identify simultaneously multiple fungal pathogens in a single reaction. Five sets of species-specific primers were designed from the internal transcribed spacer (ITS) regions, ITS1 and ITS2, of the rRNA gene to identify Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Aspergil...

Cryptococcus neoformans is a major pathogen of humans throughout the world. Using commercial mAbs to capsular epitopes, strains of C. neoformans manifest five distinct serotypes--A, B, C, D and AD. Previous studies demonstrated significant divergence among serotypes A, B, C and D, which are thought to be haploid. In this study the origins and evolu...

Infections with the human pathogenic basidiomycetous yeastCryptococcus neoformans are often treated with fluconazole. Resistance to this antifungal agent has been reported. This study investigated the patterns of mutation to fluconazole resistance inC. neoformans in vitro. The MIC of fluconazole was measured for 21 strains of C. neoformans. The MIC...

A segregating population of single basidiospore isolates from a sexual cross was used to generate the first moderately dense genetic linkage map of Cryptococcus neoformans var. neoformans (Serotype D). Polymorphic DNA markers were developed using amplified fragment length polymorphisms, random amplified polymorphic DNA, and gene-encoding sequences....

Cryptococcus neoformans (= Filobasidiella neoformans) is a significant emerging fungal pathogen of humans. To understand the evolution of this pathogen, 34 strains were obtained from various locations around the world and fragments of four genes were sequenced from each. These strains represented all three varieties and five serotypes. The four seq...

To introduce the general mycologic aspects of fungal rhinosinusitis, this article reviews, in brief, the biology of fungi and the principles of fungal pathogenesis. A glossary of frequently used mycologic terms is provided. The basis of fungal classification and strategies for the diagnosis of mycotic infections are summarized. The morphologic crit...

PCR fingerprinting with single non-specific primers was used to type vaginal isolates of C. albicans from Portugal, Angola, Madagascar, and two regions of Germany (Berlin and Munich). In addition to analysing isolates that exhibited the normal biotype of C. albicans, the study included atypical strains that failed to assimilate glucosamine and N-ac...

The genotypes and susceptibilities to fluconazole of 78 strains of the human pathogenic yeast Candida albicans were compared. The strains comprised two sets of samples from Durham, N.C.: one from patients infected with the human immunodeficiency virus (HIV) and the other from healthy volunteers. For each strain, the MIC of fluconazole was determine...

Restriction fragment length polymorphism (RFLP) in the large ribosomal RNA region of the mitochondrial DNA (mtDNA) was developed as a genetic marker for investigating mitochondrial transmission in sexual crosses of the human pathogenic basidiomycetous yeast Cryptococcus neoformans. Strain JEC20 of C. neoformans var. neoformans (mat a) was mated wit...

Codominant single-locus markers were developed by amplifying genomic DNA of C. albicans with pairs of random primers. Monomorphic PCR products were screened for polymorphisms by the SSCP technique. Sequencing confirmed that SSCP's were mostly due to single nucleotide substitutions in the polymorphic fragments. A total of 85 polymorphic loci were ob...

In this Round Table, the application of several methods of molecular typing were discussed in reference to four important pathogenic fungi: Coccidioides immitis, Histoplasma capsulatum, Candida albicans and Paracoccidioides brasiliensis. Among the different methods the following were discussed: restriction fragment length polymorphisms (RFLP), sing...

We examined the patterns of strain relatedness among pathogenic yeasts from within and among groups of women to determine whether there were significant associations between genotype and host condition or body site. A total of 80 yeast strains were isolated, identified, and genotyped from 49 female volunteers, who were placed in three groups: (i) 1...

Atypical isolates of the pathogenic yeast Candida albicans have been reported with increasing frequency. To investigate the origin of a set of atypical isolates and their relationship to typical isolates, we employed a combination of molecular phylogenetic and population genetic analyses using rDNA sequencing, PCR fingerprinting, and analysis of co...