Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for rapid identification of mold and yeast cultures of Penicillium marneffei

Abstract

BackgroundPenicillium marneffei is the most important thermal dimorphic fungus causing systemic mycosis in HIV-infected and other immunocompromised patients in Southeast Asia. However, laboratory diagnosis of penicilliosis, which relies on microscopic morphology and mycelial-to-yeast conversion, is time-consuming and expertise-dependent, thus delaying diagnosis and treatment. Although matrix -assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is useful for identification of various medically important fungi, its performance for identification of P. marneffei is less clear.ResultsWe evaluated the performance of the Bruker MALDI-TOF MS system for identification of mold and yeast cultures of 59 clinical strains and the type strain of P. marneffei using the direct transfer method, with results compared to four phylogenetically closely related species, P. brevi-compactum, P. chrysogenum, Talaromyces aurantiacus and T. stipitatus. Using the Bruker original database combined with BDAL v4.0.0.1 and Filamentous Fungi Library 1.0, MALDI-TOF MS failed to identify the 60 P. marneffei strains grown in mold and yeast phase (identified as P. funiculosum and P. purpurogenum with scores <1.7 respectively). However, when the combined database was expanded with inclusion of spectra from 21 P. marneffei strains in mold and/or yeast phase, all the remaining 39 P. marneffei strains grown in mold or phase were correctly identified to the species level with score >2.0. The MS spectra of P. marneffei exhibited significant difference to those of P. brevi-compactum, P. chrysogenum, T. aurantiacus and T. stipitatus. However, MALDI-TOF MS failed to identify these four fungi to the species level using the combined database with or without spectra from P. marneffei.ConclusionsMALDI-TOF MS is useful for rapid identification of both yeast and mold cultures of P. marneffei and differentiation from related species. However, accurate identification to the species level requires database expansion using P. marneffei strains.

In Vitro Activity of Posaconazole against Talaromyces marneffei by Broth Microdilution and Etest Methods and Comparison to Itraconazole, Voriconazole, and Anidulafungin

“…In recent decades, penicilliosis has emerged in HIV-infected and other immunocompromised patients in various countries (3)(4)(5)(6). Since the disease carries a high mortality rate and is associated with relapse, finding ways to improve diagnosis and treatment is crucial (7,8). Moderate or severe penicilliosis is often treated with amphotericin B for 2 weeks, followed by maintenance with prolonged itraconazole to prevent relapse.…”

“…All T. marneffei strains were identified by conventional phenotypic tests and sequencing of three housekeeping genes (mannose phosphate isomerase [MPI], plasma membrane H ϩ ATPase [PM-ATPase], and pyruvate kinase [PK]) and by matrix-assisted laser desorption ionization-time of flight mass spectrometry as described previously (7). Yeast and mycelial cultures of T. marneffei were grown on Sabouraud dextrose agar (SDA) (Oxoid, Cambridge, United Kingdom) as described previously with modifications (14).…”

PenicilliumSpecies Other ThanTalaromyces marneffeiProducing Red Pigment from Clinical Specimens: Isolation ofTalaromyces albobiverticillius

“…Recently, matrix-assisted laser desorption ionization time-of-ight mass spectrometry (MALDI-TOF MS) was used to identify various clinically important fungi, including T. marneffei and related species [12]. If rapid and accurate identi cation is required, DNA sequencing or MALDI-TOF MS should be used.…”

Identification of Molds by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

“…Although these methods can provide accurate results, they are expensive, require specialized equipment or expertise, and are not commonly available in clinical laboratories. In the present review, findings from the 10 studies (23)(24)(25)(27)(28)(29)(30)(31)(32)(33) shown in Table 1 indicate that before MALDI-TOF MS testing, only one study identified isolates via conventional phenotypical analyses (33), whereas almost all the remaining studies also sequenced all their isolates, and in two studies, only molecular biology analyses of the isolates were undertaken (27,28). This supports the idea that the overall high level of accuracy of MALDI-TOF MS-based mold identification shown in these studies might not be overestimated.…”

“…Nonetheless, demonstration of mycelium-to-yeast conversion, which is necessary for the definitive identification of the fungus, takes approximately 2 weeks, thereby delaying diagnosis and treatment. To our knowledge, a specific study focusing on the use of MALDI-TOF MS for the identification of T. marneffei appeared in the literature only very recently (28). In that study, spectra from mold and yeast cultures of 60 strains (59 clinical strains and 1 reference strain) of T. marneffei were analyzed with MALDI Biotyper 3.0 software against the Bruker database (BDAL version 4.0.0.1 and Filamentous Fungi Library 1.0), with or without the inclusion of additional spectra from T. marneffei strains selected from the set of test strains.…”