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 the mating type and serotype of each strain, and Amplified Fragment Length Polymorphism was used for molecular typing of the strains. In vitro assays compared the proteinase and phospholipase activities of the strains, and the Clinical and Laboratory Standards Institute (CLSI) protocol was used to determine their minimal inhibitory concentrations (MICs) to amphotericin B (AMB), itraconazole, and fluconazole. All strains were identified as C. neoformans var. grubii (serotype A), possessed the alpha mating type, and belonged to molecular type VNII. Ten of the strains demonstrated strong proteolytic activity, and the remaining five were weakly proteolytic. Nine of the strains were positive for phospholipase. In vitro antifungal susceptibility tests, determined the MIC (µg/ml) values for AMB, itraconazole, and fluconazole to be 0.03-0.5, 0.002-03, and 2-4 µg/ml, respectively. Remarkedly, all 15 strains belonged to the relatively rare molecular type, VNII. This report is one of few studies to characterize clinical strains of C. neoformans from India.

Host-associated differentiation (HAD) has played a major role in insect diversification at both macroevolutionary and microevolutionary scales. This evolutionary process has been reported in insects associated with wild and domesticated plant species. In particular, domesticated species harbor large genetic and phenotypic diversity associated with traits of human interest, including variation in nutrition, phenology, fruit, and leaf shape. This diversity may alter selection regimes affecting insect evolution and host specialization. The genus Liriomyza includes highly polyphagous species that are characterized for living and feeding inside plant leaves. Ecological and genetic data suggest the presence of cryptic species within this genus. Moreover, there is evidence of HAD in a group of populations of Liriomyza trifolii (Burgess) associated with Capsicum annum L. (Solanaceae). In this work, we explored HAD in L. trifolii populations from southeastern Mexico, and inquire into differentiation specific to peppers based on cytochrome oxidase I. We also evaluated the relationship between the genetic structure of leafminers and the different types of C. annuum. Our main results did not support previous findings of specialization of L. trifolli on C. annuum. Nevertheless, we found a divergent group of haplotypes associated to Allium cepa (Aspargales: Amaryllidaceae) in sympatric condition to Physalis philadelphica Lam. (Solanales: Solanaceae) and C. annum, suggesting the presence of HAD, as well as significant genetic differentiation of L. trifolii associated to peppers from Oaxaca and Yucatán.

ABSTRACT 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 evidence of determinative, microevolutionary changes within the host. Here, we analyzed serial isolates cultured from cerebrospinal fluid specimens of 18 South African patients with recurrent cryptococcal meningitis. The time between collection of the incident isolates and collection of the relapse isolates ranged from 124 days to 290 days, and the analyses revealed that, during this period within the patients, the isolates underwent several genetic and phenotypic changes. Considering the vast genetic diversity of cryptococcal isolates in sub-Saharan Africa, it was not surprising to find that the relapse isolates had acquired different genetic and correlative phenotypic changes. They exhibited various mechanisms for enhancing virulence, such as growth at 39°C, adaptation to stress, and capsule production; a remarkable amplification of ERG11 at the native and unlinked locus may provide stable resistance to fluconazole. Our data provide a deeper understanding of the microevolution of Cryptococcus species under pressure from antifungal chemotherapy and host immune responses. This investigation clearly suggests a promising strategy to identify novel targets for improved diagnosis, therapy, and prognosis. IMPORTANCE Opportunistic infections caused by species of the pathogenic yeast Cryptococcus lead to chronic meningoencephalitis and continue to ravage thousands of patients with HIV/AIDS. Despite receiving antifungal treatment, over 10% of patients develop recurrent disease. In this study, we collected isolates of Cryptococcus from cerebrospinal fluid specimens of 18 patients at the time of their diagnosis and when they relapsed several months later. We then sequenced and compared the genomic DNAs of each pair of initial and relapse isolates. We also tested the isolates for several key properties related to cryptococcal virulence as well as for their susceptibility to the antifungal drug fluconazole. These analyses revealed that the relapsing isolates manifested multiple genetic and chromosomal changes that affected a variety of genes implicated in the pathogenicity of Cryptococcus or resistance to fluconazole. This application of comparative genomics to serial clinical isolates provides a blueprint for identifying the mechanisms whereby pathogenic microbes adapt within patients to prolong disease.

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 as individual strains or clones. MLST molecular types often correlate with important phenotypes. Conventional MLST involves the extraction of genomic DNA and the amplification by PCR of several conserved, unlinked gene sequences from a sample of isolates of the taxon under investigation. In some cases, as few as three loci are sufficient to yield definitive results. The amplicons are sequenced, aligned, and compared by phylogenetic methods to distinguish statistically significant differences among individuals and clades. Although MLST is simpler, faster, and less expensive than whole genome sequencing, it is more costly and time-consuming than less reliable genotyping methods (e.g. amplified fragment length polymorphisms). Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in single assay. We demonstrate this methodology by genotyping isolates of the well-characterized, human pathogenic yeast Cryptococcus neoformans.

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 structure of Cng in southern Africa, we analysed isolates from 77 environmental samples and 64 patients. We detected significant genetic diversity among isolates and strong evidence of geographic structure at the local level. High proportions of isolates with the rare MATa allele were observed in both clinical and environmental isolates; however, the mating‐type alleles were unevenly distributed among different subpopulations. Nearly equal proportions of the MATa and MATα mating types were observed among all clinical isolates and in one environmental subpopulation from the eastern part of Botswana. As previously reported, there was evidence of both clonality and recombination in different geographic areas. These results provide a foundation for subsequent genomewide association studies to identify genes and genotypes linked to pathogenicity in humans.

ABSTRACT 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 Cryptococcus gattii isolated from serial episodes of cryptococcal meningitis that were separated by at least 110 days. The most common MLST genotypes among the isolates were the dominant global clinical genotypes (M5 and M4) of molecular type VNI, as well as the VNI genotypes apparently restricted to southern Africa. In addition, there was considerable genetic diversity among these South African isolates, as 15% of the patients had unique genotypes. Eleven percent of the patients were reinfected with a genetically different strain following their initial diagnosis and treatment. However, the majority of serial episodes (89%) were caused by strains with the same genotype as the original strain. These results indicate that serial episodes of cryptococcosis in South Africa are frequently associated with persistence or relapse of the original infection. Using a reference broth microdilution method, we found that the serial isolates of 11% of the patients infected with strains of C. neoformans var. grubii with identical genotypes exhibited ≥4-fold increases in the MICs to fluconazole. Therefore, these recurrent episodes may have been precipitated by inadequate induction or consolidation of antifungal treatment and occasionally may have been due to increased resistance to fluconazole, which may have developed during the chronic infection.

ABSTRACT 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 from the cerebrospinal fluid (CSF) of two AIDS patients with cryptococcal meningitis prior to antifungal therapy. The patients were infected with strains of C. neoformans var. grubii of molecular type VNI and VNII. Using RNA-seq, we compared the transcriptional profiles of these strains under three environmental conditions (in vivo CSF, ex vivo CSF, and yeast extract-peptone-dextrose [YPD]). Although we identified a number of differentially expressed genes, single nucleotide variants, and novel genes that were unique to each strain, the overall expression patterns of the two strains were similar under the same environmental conditions. Specifically, yeast cells obtained directly from each patient’s CSF were more metabolically active than cells that were incubated ex vivo in CSF. Compared with growth in YPD, some genes were identified as significantly upregulated in both in vivo and ex vivo CSF, and they were associated with genes previously recognized for contributing to pathogenicity. For example, genes with known stress response functions, such as RIM101, ENA1, and CFO1, were regulated similarly in the two clinical strains. Conversely, many genes that were differentially regulated between the two strains appeared to be transporters. These findings establish a platform for further studies of how this yeast survives and produces disease. IMPORTANCE Cryptococcus neoformans, an environmental, opportunistic yeast, is annually responsible for an estimated million cases of meningitis and over 600,000 deaths, mostly among HIV-infected patients in sub-Saharan Africa and Asia. Using RNA-seq, we analyzed the gene expression of two strains of C. neoformans obtained from the cerebrospinal fluid (CSF) of infected patients, thus creating a comprehensive snapshot of the yeasts’ genetic responses within the human body. By comparing the gene expression of each clinical strain under three conditions (in vivo CSF, ex vivo CSF, and laboratory culture), we identified genes and pathways that were uniquely regulated by exposure to CSF and likely crucial for the survival of C. neoformans in the central nervous system. Further analyses revealed genetic diversity between the strains, providing evidence for cryptococcal evolution and strain specificity. This ability to characterize transcription in vivo enables the elucidation of specific genetic responses that promote disease production and progression.

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 cause disease.However, in immunocompromised people, such as HIV/AIDS patients, and less often in healthy hosts, the yeasts can disseminate to almost any part of the body; however, they are neurotropic, and meningoencephalitis is the most frequent and deadliest clinical manifestation [1-3].An estimated 1 million new infections are acquired each year, and the majority of these cases occur in sub-Saharan Africa, which has the highest prevalence of patients with HIV/ AIDS [4].In this region, C. neoformans is the most common cause of meningitis, and mortality hovers around 50%.Others who succumb to cryptococcosis are apparently immunocompetent and exhibit no evidence of underlying disease.For example, 71% of cryptococcal infections in China occur in people without pre-existing conditions [5].There are two varieties, C. neoformans var.grubii (Cng) and C. neoformans var.neoformans (Cnn), which are distinguishable by molecular markers or their capsular serotypes, A or D, respectively.Diploid AD hybrids also occur in the environment and patients [6][7][8].In addition, a sibling species, Cryptococcus gattii, causes similar infections.However, isolates of both serotype D and AD hybrids, as well as C. gattii, are much less common.At least 90% of human cryptococcal disease and fatalities are caused by Cng (serotype A) [9][10][11].

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 than half of the patients are positive. These limitations may be eliminated by using real-time polymerase chain reaction (PCR) to detect Candida DNA in the blood specimens of patients at risk. Here, we optimized a PCR protocol to detect 5–10 yeasts in low volumes of simulated and clinical specimens. We also used a mouse model of systemic candidiasis and determined that candidemia is optimally detectable during the first few days after infection. However, PCR tests are often costly, labor-intensive, and inconvenient for routine use. To address these obstacles, we evaluated the innovative microfluidic real-time PCR platform (Advanced Liquid Logic, Inc.), which has the potential for full automation and rapid turnaround. Eleven and nine of 16 specimens from individual patients with culture-proven candidemia tested positive for C. albicans DNA by conventional and microfluidic real-time PCR, respectively, for a combined sensitivity of 94%. The microfluidic platform offers a significant technical advance in the detection of microbial DNA in clinical specimens.

In Fundamental Medical Mycology, Errol Reiss, Jean Shadomy, and Marshall Lyon have produced a valuable new text. Drs Reiss and Shadomy are medical mycologists who have extensive research and educational experience with the increasing spectrum of pathogenic fungi, including diagnosis of infections they cause and host defenses. Dr Lyon is an infectious diseases physician with clinical expertise in the epidemiology and management of opportunistic mycoses. This complementary team has produced a highly readable and comprehensive book, which they intend to be a text for medical and graduate students, a resource for microbiology technologists, and a reference for physicians and researchers. The book has been carefully organized, and the extensive table of contents enables readers to quickly identify specific areas of interest. The first 3 chapters contain basic information describing fungi, diagnostic methods, and antifungal chemotherapy. The succeeding 19 chapters review specific mycoses, using a similar format that addresses the following topics: etiology, clinical manifestations, ecology of the fungi, epidemiology of the infections, pathogenicity, animal infections, treatment, and laboratory diagnosis. Each chapter provides instructive case histories and ends with references and review questions. The book also includes a helpful glossary. A book of this nature can be judged by its completeness, accuracy and timeliness of coverage, clarity of the writing, and ease with which information can be accessed. By all of these criteria, Fundamental Medical Mycology merits high marks. The book does a superlative job in addressing recent advances in medical mycology, which include identifying emerging pathogens, new antifungal drugs and strategies for their use; progress in molecular diagnostics; and up-to-date knowledge about host defenses against fungi, especially opportunistic pathogens. For a 1-volume text, this book provides excellent coverage of several critical areas: detailed methods of identifying fungi; descriptions of common and rare mycoses; the nuances of interpreting serologic tests; and ongoing progress in detecting diagnostic fungal antigens, nucleic acids, and signature proteins in clinical specimens. In addition, the authors provide superb, concise descriptions of the strain diversity of the major pathogenic species and the clinical and epidemiologic relevance of certain phylogenetic clades. Currently unresolved or controversial topics are clearly explained, such as fungal sinusitis and the etiology of Malassezia spp. infections. When information is available, each chapter summarizes the mechanisms of pathogenicity and confirmed virulence factors. The authors discuss advances in understanding the innate and adaptive immune responses to fungi at the tissue, cellular, and molecular levels (e.g., the role of Th17 immune responses in candidiasis). Another asset is the frequent but unobtrusive inclusion of key citations to assist anyone seeking additional information. The illustrations include diagrams, clinical photographs, and photomicrographs from a variety of sources as well as original figures. Rather than attempt pictorial consistency throughout, the authors have gleaned images for their relevance to the text. This book will serve medical and graduate students who will value the book’s succinct, lucid coverage of key fungal infections, as well as instructive case vignettes and review questions. Clinical fellows and physicians will appreciate the readable summaries of specific mycoses, diagnostic procedures, common symptoms, and appropriate antifungal drugs. Biomedical scientists and educators in related fields will use this text as a resource for a quick review of specific topics.

Background: 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 plasticity and phenotypes of monosomic and disomic strains, and compared their virulence in a mouse model of cryptococcosisResults: In an initial set of strains, melanin production was correlated with monosomy at chromosome 13, and disomic variants were less melanized and attenuated for virulence in mice. After growth in culture or passage through mice, subsequent strains were identified that varied in melanin formation and exhibited copy number changes for other chromosomes. The correlation between melanin and disomy at chromosome 13 was observed for some but not all strains. A survey of environmental and clinical isolates maintained in culture revealed few occurrences of disomic chromosomes. However, an examination of isolates that were freshly collected from the cerebrospinal fluid of AIDS patients and minimally cultured provided evidence for infections with multiple strains and copy number variation.Conclusions: Overall, these results suggest that the genome of C. neoformans exhibits a greater degree of plasticity than previously appreciated. Furthermore, the expression of an essential virulence factor and the severity of disease are associated with genome variation. The occurrence of chromosomal variation in isolates from AIDS patients, combined with the observed influence of disomy on virulence, indicates that genome plasticity may have clinical relevance.

This chapter reviews the epidemiology, diagnosis, clinical manifestations, treatment, and prognosis of cryptococcosis in Africa and the ecology and population genetics of African isolates of Cryptococcus. In the pre-highly active antiretroviral therapy (HAART) era in sub-Saharan Africa, cryptococcosis was often a sentinel opportunistic infection among HIV-infected adults, heralding the diagnosis of AIDS in more than 88% of cryptococcal cases. The initial diagnosis of cryptococcal meningitis in HAART naive, HIV-infected patients is relatively straightforward and involves microscopy and culture of clinical specimens, as well as serology. Maintenance therapy with fluconazole is usually prescribed for those patients who survive their initial episode of disease. The diagnosis of cryptococcal disease in HAART-treated patients with paradoxical immune reconstitution inflammatory syndrome (IRIS) is slightly more difficult. The formidable challenges of cryptococcosis in sub-Saharan Africa involve significant problems with diagnosis, management, and prevention. In sub-Saharan Africa, cases of cryptococcosis due to C. neoformans far exceed the number of infections caused by C. gattii. Sub-Saharan Africa is the global epicenter of cryptococcosis and the HIV/AIDS pandemic. It is possible to elucidate the evolutionary relationships among African strains of Cryptococcus and their global ancestors.

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 meningitis. Patients with HIV/AIDS have been ravaged by cryptococcosis--an estimated one million new cases occur each year, and mortality approaches 50%. Using phylogenetic and population genetic analyses, we present evidence that C. neoformans var. grubii may have evolved from a diverse population in southern Africa. Our ecological studies support the hypothesis that a few of these strains acquired a new environmental reservoir, the excreta of feral pigeons (Columba livia), and were globally dispersed by the migration of birds and humans. This investigation also discovered a novel arboreal reservoir for highly diverse strains of C. neoformans var. grubii that are restricted to southern Africa, the mopane tree (Colophospermum mopane). This finding may have significant public health implications because these primal strains have optimal potential for evolution and because mopane trees contribute to the local economy as a source of timber, folkloric remedies and the edible mopane worm.

Cryptococcus neoformans and Cryptococcus gattii comprise the pathogenic Cryptococcus species complex. This chapter focuses on the population structure and ecology of C. neoformans and C. gattii. It summarizes the current knowledge of the population structure of C. neoformans and C. gattii and discusses the associations of the genetically isolated subpopulations with their ecological niches. The most reliable methods developed to genotype individuals use markers that detect polymorphisms among microsatellites and similar repetitive DNA elements, restriction fragment length polymorphisms (RFLP), amplified fragment length polymorphisms (AFLP), and direct DNA sequencing, such as multilocus sequence typing (MLST). Most studies of the population genetics of the pathogenic species of Cryptococcus use reference strains to recognize the major clades or subpopulations. These are designated as VNI through VNIV and VNB for C. neoformans and VGI through VGIV for C. gattii. A table shows the current designation of the nine major subpopulations of the C. neoformans/C. gattii complex and their relationships to the conventional serotypes. This method of serotyping served for years to separate C. neoformans (serotypes A, D, and AD) from C. gattii (serotypes B and C). Analyses of the population structure presented in the chapter are concerned with (i) genetic diversity, (ii) mode of reproduction (clonality versus recombination), and (iii) population subdivision. The chapter also talks about the advances in the understanding of the intertwining effects of ecology and genetics on the lifestyle of C. neoformans and C. gattii.

ABSTRACT 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 recovered from pediatric and adult patients during a 2-year period in South Africa. From reports to the Group for Enteric, Respiratory, and Meningeal Disease Surveillance in South Africa (GERMS-SA), we reviewed all cases of cryptococcosis in 2005 and 2006. We analyzed one isolate of C. neoformans from each of 82 pediatric patients (<15 years of age) and determined the multilocus sequence type (ST), mating type, ploidy, and allelic profile. This sample included isolates of all three molecular types of serotype A or C. neoformans var. grubii (molecular types VNI, VNII, and VNB) and one AD hybrid. Seventy-seven (94%) of the strains possessed the MATα mating type allele, and five were MAT a. Seventy-five (91%) were haploid, and seven were diploid. A total of 24 different STs were identified. The ratios of each mating type and the proportion of haploids were comparable to those for the isolates that were obtained from 86 adult patients during the same period. Notably, the most prevalent pediatric ST was significantly associated with male patients. Overall, these pediatric isolates exhibited high genotypic diversity. They included a relatively large percentage of diploids and the rarely reported MAT a mating type.

This chapter focuses on the environmental habitats of the dominant human-pathogenic species, Cryptococcus neoformans and Cryptococcus gattii, and their varieties and subgroups. The procedures for isolating C. gattii from any environmental sample are the same as those for C. neoformans. Several researchers have sampled specific sites temporally and spatially. Many have included a variety of telluric, arboreal, aquatic, and aerial niches. C. neoformans was recovered from 62 samples of decayed wood from the hollows of five pink shower trees, two fig trees, and one November shower tree. The majority of patients diagnosed with cryptococcal meningitis in Teresina resided in Piauḭ or the adjacent state of Maranhão. Strains of VNI can readily be isolated from pigeon feces, and they are able to grow and mate on media containing pigeon feces. As cited in the chapter, the rare environmental strains of VNII have included isolations from pigeon and arboreal habitats, but their numbers have been too low to define the ecological niche of this molecular type. C. gattii is rarely isolated from pigeon feces but is associated with various tree species in Australia, Asia, North America, and South America. These different environmental niches have led to the hypothesis that C. neoformans is ubiquitous in the environment due to dissemination by pigeons following migratory and trade routes and that C. gattii is restricted to tropical and subtropical regions because it is not associated with pigeons.

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 and exemplified. In addition, the epidemiology, ecology and geographic distribution of Cryptococcus species and subpopulations are reviewed.