c Forty-two putative Cryptococcus laurentii isolates identified by the Vitek 2 system were collected in China. The gold standard, internal transcribed spacer (ITS) sequencing, confirmed that only two isolates were genuine C. laurentii. Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry was able to identify the C. laurentii isolates with an expanded custom database.
Cryptococcus laurentii is one of the very rare non-neoformans Cryptococcus species that cause human infections (1-3). The clinical presentation of C. laurentii is similar to that of C. neoformans, but the cryptococcal antigen test is often negative (4), and the organism exhibits decreased fluconazole susceptibility (5, 6). Therefore, accurate identification of the species is essential for treatment drug selection.China Hospital Invasive Fungal Surveillance Net (CHIF-NET) is a nationwide surveillance program for invasive fungal diseases (IFDs) in China (7). During a recent 5-year study period (2009 to 2014), 9,673 yeast isolates were collected, with 42 (0.4%) isolates initially identified as C. laurentii by Vitek 2 at participating hospitals. This unexpectedly high prevalence of C. laurentii than previously reported (e.g., ARTEMIS [1997 to 2007], 0.04%; SENTRY [2008 to 2012], 0%) (3, 8-10) prompted us to investigate further the identity of the isolates.The 42 putative C. laurentii isolates originated from 16 different hospitals, and the identity of the isolates was confirmed at the coordinating central laboratory by sequencing of the internal transcribed spacer (ITS) region, with results queried against the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center database (http://www.cbs.knaw.nl/Collections/Biolo MICSSequences.aspx) as previously described (7, 11). Furthermore, the isolates were reidentified by the Vitek 2 (bioMérieux, Marcy l'Etoile, France) yeast identification card and API 20C AUX method (bioMérieux) at the coordinating lab, with testing staff blinded to previous Vitek 2 and sequencing results.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis was performed on all isolates by both the Vitek MS system (IVD Knowledgebase version 2.0; bioMérieux) and the Bruker Autoflex Speed TOF/TOF MS system (Biotyper version 3.1 software; Bruker Daltonics, Billerica, MA, USA), according to the manufacturer's instructions. Mass spectral profiles of the two C. laurentii clinical isolates confirmed by ITS sequencing were used to construct a main spectrum profile (MSP) dendrogram along with reference spectra of C. laurentii and other Cryptococcus species provided in the Bruker database, for fingerprint relatedness analysis. These data were subsequently used to expand the Bruker MALDI-TOF MS database following the manufacturer's instructions (12).Among 42 putative C. laurentii isolates identified by the Vitek 2 system at local hospitals, only two isolates (4.8%) were confirmed as C. laurentii by sequencing of the ITS region. Of the remaining 40 isolat...