Nejvíce citovaný článek - PubMed ID 15567230
McRAPD as a new approach to rapid and accurate identification of pathogenic yeasts
An increasing trend in non albicans infections and various susceptibility patterns to antifungal agents implies a requirement for the quick and reliable identification of a number of medically important Candida species. Real-time PCR followed by high resolution melting analysis (HRMA) was developed, tested on 25 reference Candida collection strains and validated on an additional 143 clinical isolates in this study. All reference strains and clinical isolates inconclusive when using phenotypic methods and/or HRMA were analysed using ITS2 sequencing. Considering reference and clinical strains together, 23 out of 27 Candida species could be clearly distinguished by HRMA, while the remaining 4 species were grouped in 2 pairs, when applying the mean Tm ± 3 SD values, the shape of the derivative melting curve (dMelt curve) and, in some cases, the normalized and temperature-shifted difference plot against C. krusei. HRMA as a simple, rapid and inexpensive tool was shown to be useful in identifying a wide spectrum of clinically important Candida species. It may complement the current clinical diagnostic approach based on commercially available biochemical kits.
- MeSH
- Candida genetika izolace a purifikace MeSH
- DNA fungální analýza MeSH
- fenotyp MeSH
- kandidóza mikrobiologie MeSH
- kvantitativní polymerázová řetězová reakce * MeSH
- lidé MeSH
- tranzitní teplota MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA fungální MeSH
A case report of ventriculoperitoneal shunt infection caused by Candida lusitaniae in a 6-year-old patient with cerebral astrocytoma and obstructive hydrocephalus is presented briefly with emphasis on the course of antifungal treatment. Seven isolates recovered subsequently from the cerebrospinal fluid were studied retrospectively. To confirm identity, isolates were typed using pulsed-field gel electrophoresis and melting curve of random amplified polymorphic DNA (McRAPD). Further, the ability to form biofilm and its susceptibility to systemic antifungals were evaluated. Using McRAPD, identity of C. lusitaniae isolates showing slight microevolutionary changes in karyotypes was undoubtedly confirmed; successful application of numerical interpretation of McRAPD for typing is demonstrated here for the first time. The strain was also recognized as a strong biofilm producer. Moreover, minimum biofilm inhibitory concentrations were very high, in contrast to low antifungal minimum inhibitory concentrations of isolates. It can be concluded that McRAPD seems to be a simple and reliable method not only for identification but also for typing of yeasts. A ventriculoperitoneal shunt colonized by C. lusitaniae was revealed as the source of this nosocomial infection, and the ability of the strain to form biofilm on its surface likely caused treatment failure.
- MeSH
- antifungální látky farmakologie MeSH
- astrocytom mozkomíšní mok komplikace farmakoterapie mikrobiologie patologie chirurgie MeSH
- biofilmy účinky léků růst a vývoj MeSH
- Candida účinky léků genetika izolace a purifikace MeSH
- denaturace nukleových kyselin MeSH
- dítě MeSH
- hydrocefalus mozkomíšní mok komplikace farmakoterapie mikrobiologie patologie chirurgie MeSH
- infekce spojené se zdravotní péčí mozkomíšní mok komplikace farmakoterapie mikrobiologie patologie chirurgie MeSH
- kandidóza mozkomíšní mok komplikace farmakoterapie mikrobiologie patologie chirurgie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mykologické určovací techniky MeSH
- nádory mozku mozkomíšní mok komplikace farmakoterapie mikrobiologie patologie chirurgie MeSH
- neúspěšná terapie MeSH
- pulzní gelová elektroforéza MeSH
- technika náhodné amplifikace polymorfní DNA * MeSH
- ventrikuloperitoneální zkrat škodlivé účinky MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Názvy látek
- antifungální látky MeSH
BACKGROUND: Rapid, easy, economical and accurate species identification of yeasts isolated from clinical samples remains an important challenge for routine microbiological laboratories, because susceptibility to antifungal agents, probability to develop resistance and ability to cause disease vary in different species. To overcome the drawbacks of the currently available techniques we have recently proposed an innovative approach to yeast species identification based on RAPD genotyping and termed McRAPD (Melting curve of RAPD). Here we have evaluated its performance on a broader spectrum of clinically relevant yeast species and also examined the potential of automated and semi-automated interpretation of McRAPD data for yeast species identification. RESULTS: A simple fully automated algorithm based on normalized melting data identified 80% of the isolates correctly. When this algorithm was supplemented by semi-automated matching of decisive peaks in first derivative plots, 87% of the isolates were identified correctly. However, a computer-aided visual matching of derivative plots showed the best performance with average 98.3% of the accurately identified isolates, almost matching the 99.4% performance of traditional RAPD fingerprinting. CONCLUSION: Since McRAPD technique omits gel electrophoresis and can be performed in a rapid, economical and convenient way, we believe that it can find its place in routine identification of medically important yeasts in advanced diagnostic laboratories that are able to adopt this technique. It can also serve as a broad-range high-throughput technique for epidemiological surveillance.
- MeSH
- algoritmy MeSH
- automatizace metody MeSH
- Candida klasifikace genetika izolace a purifikace MeSH
- DNA fungální genetika MeSH
- druhová specificita MeSH
- genotyp MeSH
- lidé MeSH
- mykologické určovací techniky metody MeSH
- reprodukovatelnost výsledků MeSH
- Saccharomyces cerevisiae klasifikace genetika izolace a purifikace MeSH
- shluková analýza MeSH
- technika náhodné amplifikace polymorfní DNA metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- DNA fungální MeSH
The yeasts, being favorite eukaryotic microorganisms used in food industry and biotechnologies for production of biomass and various substances, are also used as model organisms in genetic manipulation, molecular and biological research. In this respect, Saccharomyces cerevisiae is the best-known species but current situation in medicine and industry requires the use of other species. Here we summarize the basic taxonomic, morphological, physiological, genetic, etc. information about the pathogenic yeast Candida glabrata that is evolutionarily very closely related to baker's yeast.
