Characterization ofCryptococcus neoformans var.neoformans serotype A and A/D in samples from Egypt (original) (raw)
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Mycoses, 1998
Schlusselworter. Cyptococcus neofomans, DNA-Extraktion Summary. The mucopolysaccharide capsule of Cgpococcus neOfOnnans and other pathogenic yeasts prevent the extraction of DNA from these important zoonotic agents. We report that the use of a lysis buffer containing a high concentration of urea is an easy, efficient and time-saving technique to obtain high yields of good-quality DNA for molecular diagnosis. The use of urea also prevents the degradation of DNA during storage of samples at room temperature for up to 6 months. Zusarnmenfassung. Die Mukopolysaccharid-Kapsel von Cgpococcus negormans und anderen pathogenen Hefepilzen verhindern die DNA-Extraktion dieser wichtigen Erreger. Wir belegen, da8 der Einsatz eines lytischen Puffers mit hoher Harnstoff-Konzentration eine einfache, effiziente und zeitsparende Technik darstellt, um eine hohe DNA-Ausbeute guter Qualitat fur die molekulare Diagnose zu erhalten. Der Einsatz von Harnstoff verhindert zudem die Degradierung der DNA wahrend der Proben-Lagerung bei Zimmertemperatur bis zu sechs Monaten.
Assessment of a PCR technique for the detection and identification ofCryptococcus neoformans
Medical Mycology, 1996
The 18S ribosomal RNA gene of Cryptococcus neoformans was amplified by polymerase chain reaction (PCR). The primers CPL1 and CPR4 were tested for their ability to amplify DNA from 30 strains of C. neoformans and 27 specimens of cerebrospinal fluid (CSF) from patients with cryptococcal meningitis. A 343 bp product was obtained and its specificity confirmed by Southern hybridization with an internal sequence (INSR4) probe. The sensitivity was 100 fg by Southern analysis and 1 pg using the PCR. Neither human nor a variety of other fungal and bacterial strains (n = 78) gave an amplified product. This PCR method can detect as few as 5 cells ml-l of C. neoformans in spiked-CSF following a simple processing procedure. The developed system of PCR was more sensitive than the culture method and revealed a very high specificity. The PCR was easy to perform and needed only 4 h for all processes from receiving the CSF to detection of a specific DNA band after agarose gel electrophoresis. This would provide another rapid laboratory method for the diagnosis of cryptococcal meningitis.
In the present work RAPD-PCR was used to study the relatedness among 10 strains of both Candida albicans and Cryptococcus neoformans previously isolated from man, animals and soil in El-Fayoum Governorate. The profile of DNA fragments for C. albicans strains showed the differentiation of the genomic DNA of C. albicanss isolates into numbers of DNA bands, which were different in molecular weight. The dendogram analysis of RAPD pattern of C. albicans isolates using specific primers divided the isolates into 3 groups of high similarity (92 -100 %) and a group of low similarity (21%). These results indicated that the strains that were isolated from the same source of samples gave a high degree of similarity between DNA fragments as in human vaginal and throat swabs. The dendogram analysis of RAPD pattern of C. neoformans isolates revealed a high similarity between most of the isolates that varied between 92 -100 %. It was observed, that the strains isolated from different sites in the same species gave a high degree of similarity. On the other hand, the similarity among animal and soil isolates was low (19-30 %), although it was 92-93 % among the vaginal isolates of buffaloes, cows and soil samples.
In the present work RAPD-PCR was used to study the relatedness among 10 strains of both Candida albicans and Cryptococcus neoformans previously isolated from man, animals and soil in El-Fayoum Governorate. The profile of DNA fragments for C. albicans strains showed the differentiation of the genomic DNA of C. albicanss isolates into numbers of DNA bands, which were different in molecular weight. The dendogram analysis of RAPD pattern of C. albicans isolates using specific primers divided the isolates into 3 groups of high similarity (92 -100 %) and a group of low similarity (21%). These results indicated that the strains that were isolated from the same source of samples gave a high degree of similarity between DNA fragments as in human vaginal and throat swabs. The dendogram analysis of RAPD pattern of C. neoformans isolates revealed a high similarity between most of the isolates that varied between 92 -100 %. It was observed, that the strains isolated from different sites in the same species gave a high degree of similarity. On the other hand, the similarity among animal and soil isolates was low (19-30 %), although it was 92-93 % among the vaginal isolates of buffaloes, cows and soil samples.
In the present work RAPD-PCR was used to study the relatedness among 10 strains of both Candida albicans and Cryptococcus neoformans previously isolated from man, animals and soil in El-Fayoum Governorate. The profile of DNA fragments for C. albicans strains showed the differentiation of the genomic DNA of C. albicanss isolates into numbers of DNA bands, which were different in molecular weight. The dendogram analysis of RAPD pattern of C. albicans isolates using specific primers divided the isolates into 3 groups of high similarity (92 -100 %) and a group of low similarity (21%). These results indicated that the strains that were isolated from the same source of samples gave a high degree of similarity between DNA fragments as in human vaginal and throat swabs. The dendogram analysis of RAPD pattern of C. neoformans isolates revealed a high similarity between most of the isolates that varied between 92 -100 %. It was observed, that the strains isolated from different sites in the same species gave a high degree of similarity. On the other hand, the similarity among animal and soil isolates was low (19-30 %), although it was 92-93 % among the vaginal isolates of buffaloes, cows and soil samples.
Identification by polymerase chain reaction fingerprinting of Cryptococcus neoformans serotype AD
Medical Mycology, 1997
Seventy-three Cryptococcus neoformans isolates and eight other yeast strains were studied. Fingerprints produced by priming with (GACA)4 differentiated C. neoformans from all other yeasts tested and identified the five C. neoformans serotypes. Four major bands of molecular size 800, 540, 475 and 410 bp were recognized for serotypes A, AD and D. Two of them were specific for serotype A and the other two for serotype D isolates. Serotype AD strains were identified by five different genotypic patterns in which at least one of the two bands specific for serotype A and D were present in different combinations. On repeated and simultaneously performed genotype and serotype testing of nine strains, the genotypic pattern did not change, whereas serotyping was unstable in three cases. PCR-fingerprinting using (GACA)4 as a primer proved more stable than serology in discriminating among C neoJormans serotypes A, D and AD and was able to distinguish among serotype AD strains.
FEMS yeast …, 2005
Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI + 5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.
Molecular methods for the diagnosis and characterization of Cryptococcus : a review
Canadian Journal of Microbiology, 2010
Cryptococcosis is a fungal infection caused by yeasts of the genus Cryptococcus, with Cryptococcus neoformans and Cryptococcus gattii as the primary pathogenic species. This disease is a threat to immunocompromised patients, especially those who have AIDS. However, the disease has also been described in healthy individuals. The tests used to identify these microorganisms have limitations that make final diagnosis difficult. However, currently there are specific gene sequences that can be used to detect C. neoformans and C. gattii from clinical specimens and cultures. These sequences can be used for identification, typing, and the study of population genetics. Among the main identification techniques are hybridization, which was the pioneer in molecular identification and development of specific probes for pathogen detection; PCR and other PCR-based methods, particularly nested PCR and multiplex PCR; and sequencing of specific genomic regions that are amplified through PCR, which is especially useful for diagnosis of cryptococcosis caused by unconventional Cryptococcus sp. Concerning microorganism typing, the following techniques have shown the best ability to differentiate between fungal serotypes and molecular types: PCR fingerprinting, PCR-RFLP, AFLP, and MLST. Thus, the accumulation of data generated by molecular methods can have a positive impact on monitoring resistant strains and treating diseases.
Electrophoretic Karyotype of the Pathogenic Yeast Cryptococcus neoformans
Microbiology, 1989
The electrokaryotype of the pathogenic yeast Cryptococcus neoformans is described for the first time. Three different patterns were seen: (a) serotypes B and C (variety gattii) are similar and consist of nine chromosome mobility groups of >580 kb; (b) serotype A (variety neoformans) revealed eight chromosome-like groups > 700 kb; (c) serotype D (the second serotype of variety neoformans) not only differs from those described above, but each D isolate tested showed a different distribution of bands. The discrepancy, and the importance of electrokaryotyping as a taxonomic tool, are discussed.