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

Abstract

BackgroundRapid, 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.ResultsA 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.ConclusionSince 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.

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

“…An innovative approach to the latter method, termed melting curve of random amplified polymorphic DNA (McRAPD), is based on melting analysis of RAPD products and was successfully used for identification of yeasts and bacteria (Plachy et al 2005;Trtkova et al 2009;Ruskova and Raclavsky 2011).…”

The Potential of High Resolution Melting Analysis (Hrma) to Streamline, Facilitate and Enrich Routine Diagnostics in Medical Microbiology

“…It performs well in identification of pure culture of pathogenic yeast species and has its strong point in partial strain subtyping 5 , that can be particularly useful for epidemiological surveillance and that also has the potential to discover new cryptic yeast species in future as happened with RAPD in the recent past (Candida dubliniensis vs. C. albicans, Candida orthopsilosis and metapsilosis vs. C. parapsilosis). Owing to the principle of RAPD, its interlaboratory reproducibility will always be limited however although it can outdo conventional techniques in several aspects when optimized carefully 5 . It should also be noted, that because of the digital data obtained in melting analysis the McRAPD approach can be used for automated strain typing, as demonstrated by Hamal et al 26 .…”

“…by Santos 5,20,21 . In the field of broad-range bacterial species identification, Klaschik et al 22 were the first to develop a PCR and melting analysis assay using 2 probes for identification of 17 common bacterial species.…”

“…4). This approach was first used in processing of McRAPD data 5 , which can be useful both for species identification and strain typing. Briefly, normalized fluorescence values measured at each temperature point during melting analysis are subtracted in each pair of compared curves.…”

McRAPD unlike MALDI-TOF MS is a suitable candidate for routine discrimination of new Haemophilus influenzae strain acquisition in chronic obstructive pulmonary disease (COPD) and cystic fibrosis

“…to our previous experience. Melting analysis of RAPD, termed McRAPD, was first used by our group for purposes of yeast species identification 19 , but later proved to be useful for strain typing in several other settings 17,[20][21][22] . In this study, McRAPD provided excellent results that were in full accordance with the results of MLST, which is considered the gold standard for microbial typing.…”