Comparative evaluation of mass spectrometry and multiplex real time polymerase chain reaction for dermatophyte identification

Dermatofiti so vlaknate glive, ki večinoma povzročajo površinske okužbe kože, las in nohtov. Laboratorijska diagnostika teh okužb temelji na neposrednem mikroskopskem pregledu kužnin, njihovem gojenju na mikoloških gojiščih in identifikaciji na podlagi morfoloških lastnosti. Klasična identifikacija zahteva izkušnje in čas, zato v diagnostiko uvajamo hitrejše in objektivnejše metode prepoznavanja dermatofitov. Namen magistrske naloge je bil primerjati masno spektrometrijo (MS) MALDI-TOF (angl. matrix-assisted laser desorption ionization time-of-flight) in multipleksne verižne reakcije s polimerazo v realnem času (qPCR) za identifikacijo dermatofitov ter opredeliti ustreznost obeh metod za prepoznavo najpogostejših povzročiteljev dermatofitoz. Poleg tega je bil cilj naloge tudi uvesti protokol, s katerim bi dopolnili morfološko prepoznavanje dermatofitov, skrajšali čas do izvida in zmanjšali možnost subjektivnih napak. Izboljšati smo želeli tudi predpripravo vzorcev in omogočiti zanesljivejšo identifikacijo dermatofitov z metodo MS MALDI-TOF tako, da smo primerjali rezultate analiziranih kultur, ki so zrasle na dveh različnih dermatofitnih gojiščih. Uporabili smo 103 pozitivne vzorce, ki so jih v Laboratoriju za diagnostiko glivičnih infekcij na Inštitutu za mikrobiologijo in imunologijo (IMI) Medicinske fakultete Univerze v Ljubljani prejeli v diagnostično testiranje. Vzorce smo najprej nacepili na dve različni trdni gojišči, in sicer sulfit-cikloserin-azidni agar (SCA) in ID-Fungi Plates ter v tekoče gojišče tioglikolatni bujon (TIO). Po 3–5 dneh smo s trdnih gojišč postrgali dermatofitne kulture in jih nanesli na ploščico za analizo z metodo MS MALDI-TOF. Iz tekočega gojišča TIO pa smo izolirali dermatofitno DNA in jo do analize z qPCR shranili v hladilniku pri –20 ␃. Multipleksno PCR v realnem času smo izvedli s komercialno dostopnim kompletom DermaGenius® 2.0. Po primerjavi in statistični analizi rezultatov analiz smo ugotovili, da je bila analizna specifičnost MS MALDI-TOF MS večja, če smo namesto gojišča SCA za gojenje dermatofitov uporabili gojišče ID-Fungi Plates. Poleg tega smo ugotovili, da je analitska specifičnost multipleksne metode qPCR v realnem času boljša kot pri MS MALDI-TOF, saj smo z njo lahko pravilno identificirali večje število dermatofitov z manjšim številom neuspešnih prepoznav. Zato menimo, da bi bila metoda multipleksne qPCR primerna za dopolnitev klasične diagnostike. Za ustrezno verifikacijo metode pa bi morali izvesti dodatno raziskavo, s katero bi lahko zanesljivo opredelili vse parametre, ki so ključni za vpeljavo novih metod v standardne operativne postopke rutinskega diagnostičnega laboratorija.Dermatophytes are filamentous fungi, which in the majority of cases cause superficial infections of the skin, hair and nails. In clinical laboratory, they are routinely identified with the combined use of cultivation, microscopy of the morphological characteristics and MALDI-TOF MS. However, because dermatophytes are slow growing fungi it is necessary to wait from one to four weeks for a reliable identification. The purpose of this master’s dissertation was firstly to compare two methods, MALDI-TOF MS and multiplex qPCR, and secondly to evaluate whether these two methods provide good enough dermatophyte identification. Moreover, we wanted to establish a better identification protocol for the most clinically common dermatophytes and introduce it into the standard operating procedures in the clinical laboratory as an addition to morphological identification. In this way we would be able to shorten the time needed for diagnosis and lower the chance of subjective errors. Finally, we wished to attain a better identification with MALDI-TOF MS, and henceforth compared two different solid growing media for dermatophyte cultivation. Keeping this in mind, we cultivated 103 dermatophyte positive samples, which were sent to the Laboratory for Diagnostics of Fungal Infections at the Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana as clinical samples for diagnostic testing, on two solid growth media i.e., SCA and ID-Fungi Plates, as well as in a liquid growing medium TIO. After 3-5 days we identified the cultures with MALDI-TOF MS. From the liquid medium TIO we isolated the dermatophyte DNA with MagnaCycler and kept it frozen at -20 ␃ until we processed the samples with the DermaGenius® 2.0 kit which uses the multiplex qPCR method as an identification technique. The results of our study indicate that the analytical specificity of MALDI-TOF MS is improved when the dermatophytes are cultured on ID-Fungi Plates instead of SCA. Secondly the results indicate that multiplex qPCR has a greater analytical specificity compared to MALDI-TOF MS. Therefore, the study points to the conclusion that with additional verification, multiplex qPCR is a promising method with which we could improve the classical identification method that is used in the routine laboratory today