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Typing of Histoplasma capsulatum isolates based on nucleotide sequence variation in the internal transcribed spacer regions of rRNA genes.

ABSTRACT: The nucleotide sequences of internal transcribed spacer (ITS) regions of rRNA genes of 24 isolates of Histoplasma capsulatum were examined. The results indicate that the sequences of ITS regions in different isolates are not identical. Sequence variations were found at 20 positions in the 496 bp that were sequenced. Ten different sequence patterns, designated types A through H, were observed when the sequences from the 24 isolates were aligned. Twelve isolates from Indianapolis were classified into four different types. Two isolates from New York belonged to type G. Three isolates from different cities were type F. The remaining six isolates were of different types.

SUBMITTER: Jiang B

PROVIDER: S-EPMC88702 | biostudies-literature | 2000 Jan

REPOSITORIES: biostudies-literature

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Typing of Histoplasma capsulatum isolates based on nucleotide sequence variation in the internal transcribed spacer regions of rRNA genes.

Jiang B B Bartlett M S MS Allen S D SD Smith J W JW Wheat L J LJ Connolly P A PA Lee C H CH

Journal of clinical microbiology 20000101 1

The nucleotide sequences of internal transcribed spacer (ITS) regions of rRNA genes of 24 isolates of Histoplasma capsulatum were examined. The results indicate that the sequences of ITS regions in different isolates are not identical. Sequence variations were found at 20 positions in the 496 bp that were sequenced. Ten different sequence patterns, designated types A through H, were observed when the sequences from the 24 isolates were aligned. Twelve isolates from Indianapolis were classified i ...[more]

PMID: 10618094

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Project description:BackgroundHistoplasmosis is a neglected disease that affects mainly immunocompromised patients, presenting a progressive dissemination pattern and a high mortality rate, mainly due to delayed diagnosis, caused by slow fungal growth in culture. Therefore, a fast, suitable and cost-effective assay is required for the diagnosis of histoplasmosis in resource-limited laboratories. This study aimed to develop and evaluate two new molecular approaches for a more cost-effective diagnosis of histoplasmosis.MethodologySeeking a fast, suitable, sensitive, specific and low-cost molecular detection technique, we developed a new Loop-mediated Isothermal Amplification (LAMP) assay and nested PCR, both targeting the Internal Transcribed Spacer (ITS) multicopy region of Histoplasma capsulatum. The sensitivity was evaluated using 26 bone marrow and 1 whole blood specimens from patients suspected to have histoplasmosis and 5 whole blood samples from healthy subjects. All specimens were evaluated in culture, as a reference standard test, and Hcp100 nPCR, as a molecular reference test. A heparin-containing whole blood sample from a heathy subject was spiked with H. capsulatum cells and directly assayed with no previous DNA extraction.ResultsBoth assays were able to detect down to 1 fg/μL of H. capsulatum DNA, and ITS LAMP results could also be revealed to the naked-eye by adding SYBR green to the reaction tube. In addition, both assays were able to detect all clades of Histoplasma capsulatum cryptic species complex. No cross-reaction with other fungal pathogens was presented. In comparison with Hcp100 nPCR, both assays reached 83% sensitivity and 92% specificity. Furthermore, ITS LAMP assay showed no need for DNA extraction, since it could be directly applied to crude whole blood specimens, with a limit of detection of 10 yeasts/μL.ConclusionITS LAMP and nPCR assays have the potential to be used in conjunction with culture for early diagnosis of progressive disseminated histoplasmosis, allowing earlier, appropriate treatment of the patient. The possibility of applying ITS LAMP, as a direct assay, with no DNA extraction and purification steps, makes it suitable for resource-limited laboratories. However, more studies are necessary to validate ITS LAMP and nPCR as direct assay in other types of clinical specimens.

Project description:The fungal pathogen Histoplasma capsulatum is thought to be the most common cause of fungal respiratory infections in immunocompetent humans, yet little is known about its biology. Here we provide the first genome-wide studies to experimentally validate its genome annotation. A functional interrogation of the Histoplasma genome provides critical support for continued investigation into the biology and pathogenesis of H. capsulatum and related fungi. We employed a three-pronged approach to provide a functional annotation for the H. capsulatum G217B strain. First, we probed high-density tiling arrays with labeled cDNAs from cells grown under diverse conditions. These data defined 6,172 transcriptionally active regions (TARs), providing validation of 6,008 gene predictions. Interestingly, 22% of these predictions showed evidence of anti-sense transcription. Additionally, we detected transcription of 264 novel genes not present in the original gene predictions. To further enrich our analysis, we incorporated expression data from whole-genome oligonucleotide microarrays. These expression data included profiling under growth conditions that were not represented in the tiling experiment, and validated an additional 2,249 gene predictions. Finally, we compared the G217B gene predictions to other available fungal genomes, and observed that an additional 254 gene predictions had an ortholog in a different fungal species, suggesting that they represent genuine coding sequences. These analyses yielded a high confidence set of validated gene predictions for H. capsulatum. The transcript sets resulting from this study are a valuable resource for further experimental characterization of this ubiquitous fungal pathogen. The data is available for interactive exploration at http://histo.ucsf.edu. The non-redundant genome sequence of Histoplasma capsulatum G217B was tiled over a set of 93 CombiMatrix arrays, which were then hybridized with labeled cDNA samples from yeast-form (red channel) or mycelial-form (green channel) Histoplasma. Due to low yields from the mycelial samples, only the red channel intensities were analyzed (and the red foreground intensities are, therefore, reported in the VALUE column for each sample). Rather than normalizing intensities across arrays, each probe was evaluated as detected or undetected relative to the negative control intensities on the corresponding array, and the density of detected probes as a function of genome position was used for the remaining analysis.

Dataset Information

Typing of Histoplasma capsulatum isolates based on nucleotide sequence variation in the internal transcribed spacer regions of rRNA genes.

Publications

Typing of Histoplasma capsulatum isolates based on nucleotide sequence variation in the internal transcribed spacer regions of rRNA genes.

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