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

Adequate treatment of microbial infections requires rapid and accurate identification of the etiological agent. In routine diagnostics, identification of bacteria conventionally relies on phenotypic testing, which can be hindered by phenotypic variations. Therefore, genotyping techniques should perform faster and more accurately. Recently, the technique of high-resolution melting analysis (HRMA) of PCR amplicons promises to provide a convenient and economic tool of genotypic identification. In our study, we performed prospective routine testing of a PCR-HRMA system that was recently published in a proof-of-the-principle study. The system was evaluated by analysing 275 clinical isolates of bacteria acquired from 65 patients suffering from cystic fibrosis or chronic obstructive pulmonary disease. Our results show that its routine use may result in partial worsening of its discriminatory power; however, it still outmatched conventional phenotyping in the group of non-fermentative Gram-negative rods. Moreover, when supplemented by rapid, simple and economic oxidase test, it can be even simplified for more economic performance.

• MeSH
• bakteriologické techniky metody   MeSH
• chronická obstrukční plicní nemoc komplikace   MeSH
• cystická fibróza komplikace   MeSH
• diagnostické techniky molekulární metody   MeSH
• DNA bakterií chemie   genetika   MeSH
• gramnegativní aerobní tyčinky a koky genetika   izolace a purifikace   MeSH
• gramnegativní bakteriální infekce diagnóza   mikrobiologie   MeSH
• infekce dýchací soustavy mikrobiologie   MeSH
• lidé MeSH
• polymerázová řetězová reakce metody   MeSH
• prospektivní studie MeSH
• tranzitní teplota MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• DNA bakterií 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