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Development of a singleplex PCR assay for rapid identification and differentiation of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, Cryptococcus gattii, and hybrids.

ABSTRACT: A singleplex PCR assay using a single primer pair targeting the putative sugar transporter gene was developed here to distinguish Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii according to the distinct size of the amplicon. The interspecies and intravarietal hybrids were also characterized on the basis of distinct combined profiles of amplicons. This PCR assay is a rapid, simple, and reliable approach suitable for laboratory diagnoses and large-scale epidemiologic studies.

SUBMITTER: Feng X

PROVIDER: S-EPMC3716086 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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Development of a singleplex PCR assay for rapid identification and differentiation of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, Cryptococcus gattii, and hybrids.

Feng Xiaobo X Fu Xiaohua X Ling Bo B Wang Lei L Liao Wanqing W Yao Zhirong Z

Journal of clinical microbiology 20130327 6

A singleplex PCR assay using a single primer pair targeting the putative sugar transporter gene was developed here to distinguish Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii according to the distinct size of the amplicon. The interspecies and intravarietal hybrids were also characterized on the basis of distinct combined profiles of amplicons. This PCR assay is a rapid, simple, and reliable approach suitable for laboratory diagnoses and l ...[more]

PMID: 23536400

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Project description: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.

Dataset Information

Development of a singleplex PCR assay for rapid identification and differentiation of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, Cryptococcus gattii, and hybrids.

Publications

Development of a singleplex PCR assay for rapid identification and differentiation of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, Cryptococcus gattii, and hybrids.

Similar Datasets

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