Performance of optimized McRAPD in identification of 9 yeast species frequently isolated from patient samples: potential for automation.

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.

Molecular-genetic approaches to identification and typing of pathogenic Candida yeasts.

Currently, invasive candidal infections represent an increasing cause of morbidity and mortality in seriously ill hospitalised patients. Because the accurate diagnosis of candidiasis remains difficult, a fast and reliable assay for characterization of fungal pathogens is critical for the early initiation of adequate antifungal therapy and/or for introduction of preventive measures. As novel molecular genetic techniques are continuously introduced, their role in the management of infectious diseases has also been growing. Today, molecular strategies complement conventional methods and provide more accurate and detailed insight. It can be expected that future technical development will improve their potential furthermore. In this article, we provide a critical review on the value and limitations of molecular tools in pathogenic Candida species identification and strain typing regarding their sensitivity, discriminatory power, reproducibility, cost and ease of performance.

Usefulness of McRAPD for typing and importance of biofilm production in a case of nosocomial ventriculoperitoneal shunt infection caused by Candida lusitaniae.

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.