Yeast identification by sequencing, biochemical kits, MALDI–TOF MS and rep-PCR DNA fingerprinting

Yeast identification by sequencing, biochemical kits, MALDI–TOF MS and rep-PCR DNA fingerprinting Abstract No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI–TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI–TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI–TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI–TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species. yeasts, identification, DNA sequencing, MALDI–TOF MS, rep-PCR DNA fingerprinting INTRODUCTION Identification of yeasts in clinical microbiology laboratories is traditionally performed by phenotypic tests, such as microscopic appearance after Gram-smear, germ tube production, and biochemical tests. However, these phenotypic tests may result in ambiguous biochemical profiles and hence “difficult-to-identify” isolates. Since the advancement of molecular technologies, sequencing of conserved DNA regions has been used as the standard for identifying these “difficult-to-identify” yeasts. The most widely used and accepted molecular targets for yeast identification are the 28S nuclear ribosomal DNA (nrDNA) and internal transcribed spacer (ITS) region comprising the ITS1–5.8S nrDNA–ITS2 gene cluster.1–3 In the last few years, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI–TOF MS), which has been used for identification of bacteria successfully, has also been employed for rapid identification of yeasts.4,5 Recently, a new technology, named repetitive sequence-based polymerase chain reaction (rep-PCR) DNA fingerprinting, has emerged as a rapid method for the discrimination of various clinical yeasts.6 Although the performance of some commercial systems for yeast identification has been individually compared with sequence-based method,7–9 so far, no study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI–TOF MS platforms, and rep-PCR DNA fingerprinting altogether using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, we determined the concordance of 28S nrDNA and ITS sequencing and used them as the gold standard for identifying yeasts collected from a clinical microbiology laboratory. Using this standard, the performance of two commercial biochemical test kits, two MALDI–TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology were evaluated. Methods Yeast isolates A total of 1746 nonduplicate yeast isolates were recovered from various clinical specimens in the clinical microbiology laboratory of Peking Union Medical College Hospital, Beijing, China, over a 15-month (January 2009 to March 2010) period. Out of these 1746 isolates, the colonies of 1100 isolates showed typical colours and morphologies on CHROMagar Candida medium (CHROMagar Microbiology, France) as Candida albicans (n = 1067) or Candida krusei (Pichia kudriavzevii; n = 33). For the remaining 646 strains that were not readily identifiable by CHROMagar, 71 clinically important isolates (recovered from blood [n = 20], cerebrospinal fluid [n = 5], abdominal drainage fluid [n = 5], pleural fluid [n = 3], tissue [n = 6], pus [n = 10], central venous catheter tip [n = 1], urine [n = 15], bronchoalveolar lavage [n = 1], or high vaginal swab [n = 5]) were selected for further identification by gene sequencing, biochemical tests, rep-PCR, and MALDI–TOF MS (Fig. 1). The reference strains Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258T, Candida parapsilosis ATCC 22019T, and Meyerozyma (Candida) guilliermondii ATCC 6260T were obtained from the American Type Culture Collection (ATCC), USA, and were used as quality controls. Figure 1. View largeDownload slide Organization and flow of the current study. BAL, bronchoalveolar fluid; ITS, internal transcribed spacer region; MALDI–TOF MS, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry; nrDNA nuclear ribosomal DNA; rep-PCR, repetitive sequence-based polymerase chain reaction. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Organization and flow of the current study. BAL, bronchoalveolar fluid; ITS, internal transcribed spacer region; MALDI–TOF MS, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry; nrDNA nuclear ribosomal DNA; rep-PCR, repetitive sequence-based polymerase chain reaction. This Figure is reproduced in color in the online version of Medical Mycology. DNA extraction, PCR, as well as ITS and partial 28S nrDNA sequencing For each yeast isolate, DNA was extracted from the pure culture on Sabouraud glucose agar (SGA; Oxoid, UK) after 24–48 h of incubation at 28°C using the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's protocol. The extracted DNA was stored at −20°C until use. PCR amplification of the ITS region and partial 28S nrDNA was performed as described previously10 using the primer pairs ITS1/ITS411 and NL1/NL4,12 respectively. The PCR products were agarose gel-purified and sequenced in both directions using primers the same as those used for PCR amplification by Tsingke Biological Technology (China) with the 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The sequence electropherograms were checked manually to ensure the sequencing results were of high quality. Sequence-based species identification The ITS and partial 28S nrDNA sequences of each yeast isolate were queried against sequences in the Westerdijk Fungal Biodiversity Institute database by pairwise alignment (http://www.westerdijkinstitute.nl/Collections/BioloMICSSequences.aspx) for species identification. Biochemical profiling The biochemical profiles of the yeast isolates were characterized using the API 20C AUX system (bioMérieux, France) and the Vitek 2 YST ID Card system (bioMérieux) according to the manufacturer's instructions. For the API 20C AUX system, identifications of the isolates were only accepted if the per cent identities were greater than 90 with T indexes larger than 0.75; while for the Vitek 2 YST ID Card system, identifications of the isolates were only accepted if there was only one selected organism for each isolate with identification probability of greater than 93%. MALDI–TOF MS Matrix-assisted laser desorption ionisation–time of flight mass spectrometry (MALDI–TOF MS) was performed using both the Bruker Daltonics and the Vitek MS platforms, and all the chemical reagents used were of liquid chromatography/mass spectrometry (LC/MS) grade. Analysis by the Bruker Daltonics platform was performed by the direct smear method according to the manufacturer's instruction. Briefly, for each yeast isolate cells from a single colony on SGA were harvested after 24–48 h of incubation at 28°C and were smeared on three individual spots on the MSP 96 polished steel BC targets plate (Bruker Daltonics, Germany), using the IVD Bacterial Test Standard (BTS) (Bruker Daltonics) as a control. Each cell smear was then overlaid with 1 μl of 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA), and was air-dried. Each spot was then further overlaid with 1 μl of saturated α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Sigma-Aldrich), and was air-dried. The target plate was then analysed by the autoflex speed TOF/TOF system (Bruker Daltonics). Fungal identification was achieved using MALDI Biotyper 3.1 (Bruker Daltonics) with the reference database DB 5627 (Bruker Daltonics). Isolates with top match scores of ≥2.0 were considered to be successfully identified down to the species level. Isolates with top match scores of < 2.0 but ≥1.7 were considered to be successfully identified down to the genus level only. Isolates with top match scores of < 1.7 were considered to be unidentifiable by Bruker Daltonics MALDI–TOF MS. For isolates with multiple identifications where the top match score for each of the identifications was greater than 2.0, these isolates were considered to be identified to the genus level only. For the Vitek MS platform, the isolates were characterized according to our previous publication,10 and results with confidence values ≥60.0% were accepted for species level identification. DiversiLab automated repetitive sequence-based PCR (rep-PCR) DNA fingerprinting Isolates of the Candida parapsilosis species complex and the reference strain Candida parapsilosis ATCC 22019T were characterized by the DiversiLab automated rep-PCR DNA fingerprinting system to evaluate its capacity for species identification. Briefly, for each yeast isolate, genomic DNA was extracted using the UltraClean Microbial DNA Isolation Kit and repetitive sequences were amplified from the genomic DNA using the DiversiLab Yeast Fingerprint Kit (bioMérieux) following the manufacturer's protocol. Detection and analysis of the rep-PCR products were performed using the DiversiLab software version 3.4 (bioMérieux) implemented in the DiversiLab system (bioMérieux), where Pearson correlation coefficient and unweighted pair group method with arithmetic mean (UPGMA) were used to compare the rep-PCR DNA fingerprints of the yeast isolates. Species identification of an isolate was given if its rep-PCR DNA fingerprint possessed at least 90% similarities to one or more strains of a particular species in the Candida library. The rep-PCR DNA fingerprints of these yeast isolates were also visualised as electropherograms and analyzed by hierarchical cluster analysis (HCA) for generation of dendrogram using the DiversiLab software. Nucleotide sequence accession numbers The ITS and 28S nrDNA sequences of the 71 yeast isolates included in the present study have been deposited in the International Nucleotide Sequence Databases (INSD) with the accession numbers JN391273–JN391335, LC042127–LC042133, LC042135–LC042148, and LC088212–LC088214 (ITS), as well as JN391336–JN391406 (28S nrDNA). Results Concordance of molecular identification using 28S nrDNA and ITS sequences The identities of the 71 yeast isolates were confirmed by sequencing of the 28S nrDNA and ITS. The identification results based on 28S nrDNA sequencing were in complete agreement with those based on ITS sequencing. The 71 isolates belonged to 18 different yeast species (Cryptococcus neoformans [n = 17, 23.9%], Candida parapsilosis [n = 14, 19.7%], Candida glabrata [n = 10, 14.1%], Candida [Clavispora] lusitaniae [n = 5, 7.0%], Trichosporon asahii [n = 5, 7.0%], Candida krusei [n = 3, 4.2%], Candida tropicalis [n = 3, 4.2%], Candida [Pichia] norvegensis [n = 2, 2.8%], Candida orthopsilosis [n = 2, 2.8%], Exophiala dermatitidis [n = 2, 2.8%], Candida dubliniensis [n = 1, 1.4%], Candida intermedia [n = 1, 1.4%], Candida kefyr [Kluyveromyces marxianus; n = 1, 1.4%], Candida metapsilosis [n = 1, 1.4%], Candida nivariensis [n = 1, 1.4%], M. guilliermondii [n = 1, 1.4%], Saccharomyces cerevisiae [n = 1, 1.4%], and Trichosporon jirovecii [n = 1, 1.4%]) (Table 1). The DNA sequencing-based identities were then used as the reference standards to evaluate the performance of other commercially available identification systems. Table 1. Identification of 71 clinical yeast isolates (excluding Candida albicans) by CHROMagar, molecular method, biochemical profiling, and MALDI–TOF MS. Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  aThe ITS of these isolates were sequenced in our previously published, related study.16 bPICC, peripherally inserted central catheter. cCSF, cerebrospinal fluid. dCandida robusta, anamorphic synonym of Saccharomyces cerevisiae. View Large Table 1. Identification of 71 clinical yeast isolates (excluding Candida albicans) by CHROMagar, molecular method, biochemical profiling, and MALDI–TOF MS. Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  aThe ITS of these isolates were sequenced in our previously published, related study.16 bPICC, peripherally inserted central catheter. cCSF, cerebrospinal fluid. dCandida robusta, anamorphic synonym of Saccharomyces cerevisiae. View Large Biochemical profiling Out of the 71 isolates, 56 (78.9%) and 51 (71.8%) isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively (Table 1). Forty-nine isolates (69.0%) could be correctly identified by both systems. Seven isolates (9.9%) could only be correctly identified by the API 20C AUX system, while two isolates (2.8%) could only be correctly identified by the Vitek 2 YST ID Card system. For the API 20C AUX system, 11 isolates (15.5%) were misidentified as other yeast species, one isolate (1.4%) was identified as “unidentified organism”, and three isolates (4.2%) could not be identified due to low discrimination values; while for the Vitek 2 YST ID Card system, 13 isolates (18.3%) were misidentified as other yeast species, one isolate (1.4%) was identified as “unidentified organism,” and six isolates (8.5%) could not be identified due to low discrimination values. Generally, even using a combination of both systems, correct identification could not be achieved for one of the Cryptococcus neoformans isolate (PUMY046). Both Candida metapsilosis (PUMY018) and Candida orthopsilosis (PUMY022 and PUMY023) were misidentified as Candida parapsilosis. For some rarely encountered yeast species, including Candida dubliniensis (PUMY001), Candida intermedia (PUMY013), Candida nivariensis (PUMY019), Candida norvegensis (PUMY020 and PUMY021), E. dermatitidis (PUMY063 and PUMY064), M. guilliermondii (PUMY012), and Trichosporon jirovecii (PUMY071), correct identification was also not successful. MALDI–TOF MS Among the 71 isolates, 65 (91.5%) and 57 (80.3%) isolates were successfully identified to the correct species using the Bruker and Vitek platforms, respectively (Table 1). Fifty-four isolates (76.1%) could be correctly identified by both platforms. Eleven isolates (15.5%) could only be correctly identified by the Bruker platform, while three isolates (4.2%) could only be correctly identified by the Vitek platform. For the Bruker platform, five isolates (7.0%) were misidentified as other yeast species, and no identification was provided for one isolate (1.4%); while for the Vitek platform, two isolates (2.8%) were misidentified as other yeast species, multiple identifications were provided for three isolates (4.2%), and no identification was provided for nine isolates (12.7%). Although Candida metapsilosis (PUMY018) and Candida orthopsilosis (PUMY022 and PUMY023) could be correctly identified using the Bruker platform, these two species were not correctly identifiable using the Vitek platform. Correct identification for one Cryptococcus neoformans isolate (PUMY046), one E. dermatitidis isolate (PUMY064), and Trichosporon jirovecii (PUMY071) could not be achieved using both platforms. DiversiLab automated rep-PCR DNA fingerprinting Of the 17 clinical isolates and one reference strain (ATCC 22019T) belonging to the Candida parapsilosis species complex, all were identified by DiversiLab automated rep-PCR DNA fingerprinting only as Candida parapsilosis with similarities ≥93.2% (Table 2), indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, HCA of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters (Fig. 2). Figure 2. View largeDownload slide Hierarchical cluster analysis of the DiversiLab automated rep-PCR DNA fingerprints of the 17 clinical isolates and one reference strain (ATCC 22019T) belonging to the Candida parapsilosis species complex. Figure 2. View largeDownload slide Hierarchical cluster analysis of the DiversiLab automated rep-PCR DNA fingerprints of the 17 clinical isolates and one reference strain (ATCC 22019T) belonging to the Candida parapsilosis species complex. Table 2. Identification of Candida parapsilosis species complex isolates by DiversiLab rep-PCR fingerprinting system.       Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes        Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes  View Large Table 2. Identification of Candida parapsilosis species complex isolates by DiversiLab rep-PCR fingerprinting system.       Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes        Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes  View Large Discussion When DNA sequencing of conserved molecular targets was used for fungal identification, the variable domains 1 and 2 (D1/D2 regions) of the 28S nrDNA have been utilised for species identification and phylogenetic inference of yeasts.1 As a result, an abundant set of 28S nrDNA sequence data is available at public nucleotide repositories, such as the INSD. On the other hand, the ITS region has also been used as an alternative marker for yeast identification lately,3,13 and it was found that the ITS could provide an even better resolution for species identification.14 More recently, the ITS region has been adopted as the universal DNA barcode for fungi.15 In this study, we confirmed that there is 100% concordance between the identification results of 28S nrDNA and ITS sequencing for all the 71 clinically important yeast strains not readily identifiable by CHROMagar, although six of the 71 strains possessed multiple ITS copies and required cloning of the PCR products and subsequent sequencing for unambiguous identification.16 These identifications were used as the gold standard for subsequent evaluation of the other identification methods. It is important to note that one of the most critical steps for correct species identification by DNA sequencing is the accuracy of the sequence data in the databases. Therefore, curated databases, such as the Westerdijk Fungal Biodiversity Institute database, which we have used in this study, or the International Society for Human and Animal Mycology (ISHAM) ITS database,17 should be utilized in order to obtain reliable identification results. Both the API 20C AUX and Vitek 2 YST ID Card systems were suboptimal in identifying rarely encountered yeasts. In this study, both biochemical profiling systems only performed moderately well in identifying clinical yeast isolates (70–80% identification rates). The performance of the two systems were similar (P = .4363, Fisher's exact test). In general, more frequently encountered yeast species, such as Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis sensu stricto, Cryptococcus neoformans, and Trichosporon asahii, were more readily identified correctly by the two biochemical tests. On the other hand, for some less commonly encountered yeast species, including Candida dubliniensis, Candida norvegensis, M. guilliermondii, and for those uncommonly found in clinical specimens, including Candida metapsilosis, Candida nivariensis, Candida orthopsilosis, E. dermatitidis, Saccharomyces cerevisiae, and Trichosporon jirovecii, the performance of both biochemical identification systems was not satisfactory. This observation of the suboptimal performance of both systems in identifying uncommonly found yeasts in clinical specimens was in line with previous studies on commercial kits based on biochemical profiling.18,19 In general, for most commercial systems, around 56–89% of the studied yeast isolates were successfully identified, depending on the spectrum of yeast species that were included in the particular study, and the yeast species that were misidentified usually include those that were uncommonly encountered in clinical samples, such as Candida famata, Candida intermedia, Candida nivariensis, Candida pulcherrima, Candida zelanoides, and so forth.18,19 In this study, the uncommon clinical yeast E. dermatitidis (PUMY063 and PUMY064) could not be correctly identified by both commercial biochemical kits. This is because reference profiles for this species are not included in the databases of the API 20C AUX system nor the Vitek 2 YST ID Card system. This illustrates that the data volume of these biochemical profiling kits should be expanded to cover more yeast species so as to even allow the correct identification of uncommonly encountered clinical yeasts using these commercial biochemical kits. As for the particularly difficult-to-identify Cryptococcus neoformans isolate PUMY046 by multiple methods, it was recovered from the cerebrospinal fluid of a 64-year-old man with chronic meningitis. Cerebrospinal fluid for cryptococcal antigen was also positive. Review of the API 20C AUX and Vitek 2 YST ID card biochemical profiles of PUMY046 revealed that the abilities of this isolate to assimilate glycerol and l-arabinose as well as l-malate, d-amygdalin, and d-gluconate respectively might have led to the failure of identifying it correctly as Cryptococcus neoformans (Table 1). MALDI–TOF MS, coupled with hierarchical cluster analysis, is a promising technology for the rapid and accurate identification of microorganisms in clinical laboratories, provided that there is a sufficiently-sized reference database. The usefulness of MALDI–TOF MS has been greatly evaluated for the correct identification of clinical bacteria, and the application of this technology has been proven to be successful.20 Besides pathogenic bacteria, a number of studies have attempted to evaluate the performance of MALDI–TOF MS platforms for the identification of clinical yeast isolates.18,21,22 However, the use of MALDI–TOF MS for clinical mycology moves in a much slower pace than that for the identification of clinical bacteria,20 and more evaluation on this technology is required before it is widely adopted for clinical fungi identification. In this study, although the Bruker MALDI–TOF MS platform may have performed slightly better than the Vitek platform, the difference was not statistically significant (P = .0894, Fisher's exact test). Generally, both platforms performed well in identifying those clinically commonly encountered yeast species, such as Candida dubliniensis, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida norvegensis, Candida parapsilosis sensu stricto, Cryptococcus neoformans, M. guilliermondii, Saccharomyces cerevisiae, and Trichosporon asahii; although in occasional cases misidentification or unidentification was resulted for some strains. In contrast, for rarely encountered species the two platforms exhibited different discriminating powers. In particular, even though the Bruker platform successfully identified Candida metapsilosis, Candida nivariensis, Candida orthopsilosis, and one E. dermatitidis isolate, the Vitek platform failed to identify these species. The main reason for this should be the lack of reference mass spectra for these species in the Vitek database v2.0. On the other hand, although the Vitek platform successfully identified Candida intermedia, the Bruker platform was unsuccessful in doing so despite the presence of Candida intermedia reference mass spectra in the Bruker database. Similarly, although the Bruker database contains reference spectral data for E. dermatitidis and Trichosporon asahii, the strains PUMY064 (E. dermatitidis) and PUMY066 (Trichosporon asahii) were misidentified as Candida lusitaniae and Candida tropicalis, respectively, even after several attempts. The reason for the identification failure for these strains is not clear since these strains did not exhibit unusual phenotypic traits. Both Bruker and Vitek platforms were unable to identify Trichosporon jirovecii. The capability of MALDI–TOF MS platforms to identify more yeast strains may be improved if the database is expanded with more reference spectra, as demonstrated by previous studies.23–25 Similar to MALDI–TOF MS, the database of DiversiLab automated rep-PCR DNA fingerprinting needs further improvement. For rep-PCR, various noncoding, repetitive sequences of different lengths in the genome are amplified. Following microfluidics electrophoresis, the amplified products are separated to generate DNA fingerprints. Since different microorganisms possess different sets of repetitive sequences and so by comparing the DNA fingerprints with those in the database unknown isolates could be identified.26 So far, only seven studies have attempted to utilise the DiversiLab system to achieve strain differentiation or typing of yeasts, with an emphasis on Candida species,6,27–29Cryptococcus neoformans/Cryptococcus gatti,30,31 and Trichosporon asahii.32 Although it has already been demonstrated that Candida parapsilosis sensu stricto could be successfully differentiated from other commonly encountered clinical Candida species,6,28 the ability of this technology to differentiate the three member species of the Candida parapsilosis species complex has not been evaluated. Because of this and since DiversiLab automated rep-PCR DNA fingerprinting is expensive (about 90 USD per isolate), in this study, we have only characterized 17 clinical isolates and one reference strain of the Candida parapsilosis species complex for a preliminary evaluation of its performance. Results showed that rep-PCR DNA fingerprinting was not able to differentiate Candida parapsilosis, Candida metapsilosis, and Candida orthopsilosis alone. However, when a dendrogram using the rep-PCR DNA fingerprints of these three species was constructed, it was revealed that the three Candida species formed three distinct clusters (Fig. 2). This indicates that if the database of DiversiLab automated rep-PCR DNA fingerprinting is improved, especially when more reference DNA fingerprints of Candida metapsilosis and Candida orthopsilosis are included and used for further evaluation, this technology platform should possess a great potential for the correct identification of these three Candida species to the species level. Acknowledgments This work was partly supported by the Strategic Research Theme Fund and Dr Cheng Yu Tung Fellowship, The University of Hong Kong, Hong Kong; as well as Croucher Senior Medical Research Fellowship, Croucher Foundation, Hong Kong. Declaration of interest P.C.Y.W. has provided scientific advisory/laboratory services for Gilead Sciences, Incorporated and International Health Management Associates, Incorporated/Pfizer, Incorporated. 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Treviño M, García-Riestra C, Areses P et al.   Emerging Trichosporon asahii in elderly patients: epidemiological and molecular analysis by the DiversiLab system. Eur J Clin Microbiol Infect Dis . 2014; 33: 1497– 1503. Google Scholar CrossRef Search ADS PubMed  © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

Yeast identification by sequencing, biochemical kits, MALDI–TOF MS and rep-PCR DNA fingerprinting

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Abstract

Abstract No study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI–TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, using 71 clinically important yeast isolates (excluding Candida albicans) collected from a single centre, we determined the concordance of 28S nrDNA and ITS sequencing and evaluated the performance of two commercial test kits, two MALDI–TOF MS platforms, and rep-PCR DNA fingerprinting. 28S nrDNA and ITS sequencing showed complete agreement on the identities of the 71 isolates. Using sequencing results as the standard, 78.9% and 71.8% isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively; and 90.1% and 80.3% isolates were correctly identified using the Bruker and Vitek MALDI–TOF MS platforms, respectively. Of the 18 strains belonging to the Candida parapsilosis species complex tested by DiversiLab automated rep-PCR DNA fingerprinting, all were identified only as Candida parapsilosis with similarities ≥93.2%, indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, hierarchical cluster analysis of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters, indicating that this technology can potentially differentiate the three species. To achieve higher accuracies of identification, the databases of commercial biochemical test kits, MALDI–TOF MS platforms, and DiversiLab automated rep-PCR DNA fingerprinting needs further enrichment, particularly for uncommonly encountered yeast species. yeasts, identification, DNA sequencing, MALDI–TOF MS, rep-PCR DNA fingerprinting INTRODUCTION Identification of yeasts in clinical microbiology laboratories is traditionally performed by phenotypic tests, such as microscopic appearance after Gram-smear, germ tube production, and biochemical tests. However, these phenotypic tests may result in ambiguous biochemical profiles and hence “difficult-to-identify” isolates. Since the advancement of molecular technologies, sequencing of conserved DNA regions has been used as the standard for identifying these “difficult-to-identify” yeasts. The most widely used and accepted molecular targets for yeast identification are the 28S nuclear ribosomal DNA (nrDNA) and internal transcribed spacer (ITS) region comprising the ITS1–5.8S nrDNA–ITS2 gene cluster.1–3 In the last few years, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry (MALDI–TOF MS), which has been used for identification of bacteria successfully, has also been employed for rapid identification of yeasts.4,5 Recently, a new technology, named repetitive sequence-based polymerase chain reaction (rep-PCR) DNA fingerprinting, has emerged as a rapid method for the discrimination of various clinical yeasts.6 Although the performance of some commercial systems for yeast identification has been individually compared with sequence-based method,7–9 so far, no study has comprehensively evaluated the performance of 28S nrDNA and ITS sequencing, commercial biochemical test kits, MALDI–TOF MS platforms, and rep-PCR DNA fingerprinting altogether using a cohort of yeast strains collected from a clinical microbiology laboratory. In this study, we determined the concordance of 28S nrDNA and ITS sequencing and used them as the gold standard for identifying yeasts collected from a clinical microbiology laboratory. Using this standard, the performance of two commercial biochemical test kits, two MALDI–TOF MS platforms, and the emerging rep-PCR DNA fingerprinting technology were evaluated. Methods Yeast isolates A total of 1746 nonduplicate yeast isolates were recovered from various clinical specimens in the clinical microbiology laboratory of Peking Union Medical College Hospital, Beijing, China, over a 15-month (January 2009 to March 2010) period. Out of these 1746 isolates, the colonies of 1100 isolates showed typical colours and morphologies on CHROMagar Candida medium (CHROMagar Microbiology, France) as Candida albicans (n = 1067) or Candida krusei (Pichia kudriavzevii; n = 33). For the remaining 646 strains that were not readily identifiable by CHROMagar, 71 clinically important isolates (recovered from blood [n = 20], cerebrospinal fluid [n = 5], abdominal drainage fluid [n = 5], pleural fluid [n = 3], tissue [n = 6], pus [n = 10], central venous catheter tip [n = 1], urine [n = 15], bronchoalveolar lavage [n = 1], or high vaginal swab [n = 5]) were selected for further identification by gene sequencing, biochemical tests, rep-PCR, and MALDI–TOF MS (Fig. 1). The reference strains Candida albicans ATCC 90028, Candida glabrata ATCC 90030, Candida krusei ATCC 6258T, Candida parapsilosis ATCC 22019T, and Meyerozyma (Candida) guilliermondii ATCC 6260T were obtained from the American Type Culture Collection (ATCC), USA, and were used as quality controls. Figure 1. View largeDownload slide Organization and flow of the current study. BAL, bronchoalveolar fluid; ITS, internal transcribed spacer region; MALDI–TOF MS, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry; nrDNA nuclear ribosomal DNA; rep-PCR, repetitive sequence-based polymerase chain reaction. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide Organization and flow of the current study. BAL, bronchoalveolar fluid; ITS, internal transcribed spacer region; MALDI–TOF MS, matrix-assisted laser desorption/ionisation–time of flight mass spectrometry; nrDNA nuclear ribosomal DNA; rep-PCR, repetitive sequence-based polymerase chain reaction. This Figure is reproduced in color in the online version of Medical Mycology. DNA extraction, PCR, as well as ITS and partial 28S nrDNA sequencing For each yeast isolate, DNA was extracted from the pure culture on Sabouraud glucose agar (SGA; Oxoid, UK) after 24–48 h of incubation at 28°C using the UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) according to the manufacturer's protocol. The extracted DNA was stored at −20°C until use. PCR amplification of the ITS region and partial 28S nrDNA was performed as described previously10 using the primer pairs ITS1/ITS411 and NL1/NL4,12 respectively. The PCR products were agarose gel-purified and sequenced in both directions using primers the same as those used for PCR amplification by Tsingke Biological Technology (China) with the 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The sequence electropherograms were checked manually to ensure the sequencing results were of high quality. Sequence-based species identification The ITS and partial 28S nrDNA sequences of each yeast isolate were queried against sequences in the Westerdijk Fungal Biodiversity Institute database by pairwise alignment (http://www.westerdijkinstitute.nl/Collections/BioloMICSSequences.aspx) for species identification. Biochemical profiling The biochemical profiles of the yeast isolates were characterized using the API 20C AUX system (bioMérieux, France) and the Vitek 2 YST ID Card system (bioMérieux) according to the manufacturer's instructions. For the API 20C AUX system, identifications of the isolates were only accepted if the per cent identities were greater than 90 with T indexes larger than 0.75; while for the Vitek 2 YST ID Card system, identifications of the isolates were only accepted if there was only one selected organism for each isolate with identification probability of greater than 93%. MALDI–TOF MS Matrix-assisted laser desorption ionisation–time of flight mass spectrometry (MALDI–TOF MS) was performed using both the Bruker Daltonics and the Vitek MS platforms, and all the chemical reagents used were of liquid chromatography/mass spectrometry (LC/MS) grade. Analysis by the Bruker Daltonics platform was performed by the direct smear method according to the manufacturer's instruction. Briefly, for each yeast isolate cells from a single colony on SGA were harvested after 24–48 h of incubation at 28°C and were smeared on three individual spots on the MSP 96 polished steel BC targets plate (Bruker Daltonics, Germany), using the IVD Bacterial Test Standard (BTS) (Bruker Daltonics) as a control. Each cell smear was then overlaid with 1 μl of 70% formic acid (Sigma-Aldrich, St. Louis, MO, USA), and was air-dried. Each spot was then further overlaid with 1 μl of saturated α-cyano-4-hydroxycinnamic acid (HCCA) matrix (Sigma-Aldrich), and was air-dried. The target plate was then analysed by the autoflex speed TOF/TOF system (Bruker Daltonics). Fungal identification was achieved using MALDI Biotyper 3.1 (Bruker Daltonics) with the reference database DB 5627 (Bruker Daltonics). Isolates with top match scores of ≥2.0 were considered to be successfully identified down to the species level. Isolates with top match scores of < 2.0 but ≥1.7 were considered to be successfully identified down to the genus level only. Isolates with top match scores of < 1.7 were considered to be unidentifiable by Bruker Daltonics MALDI–TOF MS. For isolates with multiple identifications where the top match score for each of the identifications was greater than 2.0, these isolates were considered to be identified to the genus level only. For the Vitek MS platform, the isolates were characterized according to our previous publication,10 and results with confidence values ≥60.0% were accepted for species level identification. DiversiLab automated repetitive sequence-based PCR (rep-PCR) DNA fingerprinting Isolates of the Candida parapsilosis species complex and the reference strain Candida parapsilosis ATCC 22019T were characterized by the DiversiLab automated rep-PCR DNA fingerprinting system to evaluate its capacity for species identification. Briefly, for each yeast isolate, genomic DNA was extracted using the UltraClean Microbial DNA Isolation Kit and repetitive sequences were amplified from the genomic DNA using the DiversiLab Yeast Fingerprint Kit (bioMérieux) following the manufacturer's protocol. Detection and analysis of the rep-PCR products were performed using the DiversiLab software version 3.4 (bioMérieux) implemented in the DiversiLab system (bioMérieux), where Pearson correlation coefficient and unweighted pair group method with arithmetic mean (UPGMA) were used to compare the rep-PCR DNA fingerprints of the yeast isolates. Species identification of an isolate was given if its rep-PCR DNA fingerprint possessed at least 90% similarities to one or more strains of a particular species in the Candida library. The rep-PCR DNA fingerprints of these yeast isolates were also visualised as electropherograms and analyzed by hierarchical cluster analysis (HCA) for generation of dendrogram using the DiversiLab software. Nucleotide sequence accession numbers The ITS and 28S nrDNA sequences of the 71 yeast isolates included in the present study have been deposited in the International Nucleotide Sequence Databases (INSD) with the accession numbers JN391273–JN391335, LC042127–LC042133, LC042135–LC042148, and LC088212–LC088214 (ITS), as well as JN391336–JN391406 (28S nrDNA). Results Concordance of molecular identification using 28S nrDNA and ITS sequences The identities of the 71 yeast isolates were confirmed by sequencing of the 28S nrDNA and ITS. The identification results based on 28S nrDNA sequencing were in complete agreement with those based on ITS sequencing. The 71 isolates belonged to 18 different yeast species (Cryptococcus neoformans [n = 17, 23.9%], Candida parapsilosis [n = 14, 19.7%], Candida glabrata [n = 10, 14.1%], Candida [Clavispora] lusitaniae [n = 5, 7.0%], Trichosporon asahii [n = 5, 7.0%], Candida krusei [n = 3, 4.2%], Candida tropicalis [n = 3, 4.2%], Candida [Pichia] norvegensis [n = 2, 2.8%], Candida orthopsilosis [n = 2, 2.8%], Exophiala dermatitidis [n = 2, 2.8%], Candida dubliniensis [n = 1, 1.4%], Candida intermedia [n = 1, 1.4%], Candida kefyr [Kluyveromyces marxianus; n = 1, 1.4%], Candida metapsilosis [n = 1, 1.4%], Candida nivariensis [n = 1, 1.4%], M. guilliermondii [n = 1, 1.4%], Saccharomyces cerevisiae [n = 1, 1.4%], and Trichosporon jirovecii [n = 1, 1.4%]) (Table 1). The DNA sequencing-based identities were then used as the reference standards to evaluate the performance of other commercially available identification systems. Table 1. Identification of 71 clinical yeast isolates (excluding Candida albicans) by CHROMagar, molecular method, biochemical profiling, and MALDI–TOF MS. Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  aThe ITS of these isolates were sequenced in our previously published, related study.16 bPICC, peripherally inserted central catheter. cCSF, cerebrospinal fluid. dCandida robusta, anamorphic synonym of Saccharomyces cerevisiae. View Large Table 1. Identification of 71 clinical yeast isolates (excluding Candida albicans) by CHROMagar, molecular method, biochemical profiling, and MALDI–TOF MS. Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  Isolates  Isolation source  Colour on CHROMagar  Molecular identification  Biochemical profiling  MALDI–TOF MS          API 20C AUX  Vitek 2 YST ID Card  Vitek  Bruker  PUMY001  Peripheral blood  Bluish green  Candida dubliniensis  Candida albicans  Cryptococcus neoformans  Candida dubliniensis  Candida dubliniensis  PUMY002  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY003  Vaginal swab  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY004  Deep vein catheter blood  Violet  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY005  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY006  Peripheral blood  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY007  Clean catch midstream urine  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY008  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY009  Secretion  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY010a  Abdominal fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY011a  Abdominal drainage fluid  Light purple  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  Candida glabrata  PUMY012  Peripheral blood  Dark pink  Meyerozyma guilliermondii  Cryptococcus neoformans  Low discrimination  Meyerozyma guilliermondii  Meyerozyma guilliermondii  PUMY013  Clean catch midstream urine  Light purple  Candida intermedia  Meyerozyma guilliermondii  Low discrimination  Candida intermedia  Meyerozyma guilliermondii  PUMY014  Rhinal secretion  Pink  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  Candida kefyr  PUMY015  Abdominal pus  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida rugosa  Candida krusei  Candida krusei  PUMY016  Clean catch midstream urine  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida parapsilosis  Candida krusei  Candida parapsilosis  PUMY017  Peripheral blood  Pale pink (rough and spreading)  Candida krusei  Candida krusei  Candida krusei  Candida krusei  Candida krusei  PUMY018  Skin tissue  Pinkish white  Candida metapsilosis  Candida parapsilosis  Candida parapsilosis  Misidentification (multiple results)  Candida metapsilosis  PUMY019  Vaginal swab  Pinkish white  Candida nivariensis  Low discrimination  Candida glabrata  Misidentification (multiple results)  Candida nivariensis  PUMY020a  Pleural fluid  Pale pink (slightly rough and spreading)  Candida norvegensis  Candida krusei  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY021a  Peripheral blood  Pale pink (slightly rough and spreading)  Candida norvegensis  Low discrimination  Candida krusei  Candida norvegensis  Candida norvegensis  PUMY022  Clean catch midstream urine  Pinkish white  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  Candida lusitaniae  Candida orthopsilosis  PUMY023  Peripheral blood  Pink  Candida orthopsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida orthopsilosis  PUMY024  Pus  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY025  Central venous catheter blood  Pink  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY026  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY027  Central venous catheter tip  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY028  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY029  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY030  PICC catheter bloodb  Pinkish white  Candida parapsilosis  Candida parapsilosis  Low discrimination  Candida parapsilosis  Candida parapsilosis  PUMY031  Pustule  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY032  Clean catch midstream urine  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY033  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY034  Peripheral blood  Pink  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY035  Pleural pus  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY036  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  No identification  Candida parapsilosis  PUMY037  Peripheral blood  Pinkish white  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  Candida parapsilosis  PUMY038  Abscess drainage fluid  Bluish gray  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY039  Pustule  Dark blue  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY040a  Abdominal fluid  Blue (slightly rough)  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  Candida tropicalis  PUMY041  Abscess drainage fluid  Grayish purple  Candida lusitaniae  Meyerozyma guilliermondii  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY042  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY043  Vaginal swab  Grayish purple  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY044  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Multiple identifications  Candida lusitaniae  PUMY045  Catheter urine  Pink  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  Candida lusitaniae  PUMY046  CSFc  Pale pink  Cryptococcus neoformans  Low discrimination  Cryptococcus laurentii  Saccharomyces cerevisiae  Candida robustad  PUMY047  Peripheral blood  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Unidentified organism  Cryptococcus neoformans  Cryptococcus neoformans  PUMY048  CSFc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY049  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY050  Clean catch midstream urine  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY051  Csfc  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY052  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY053  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY054  Lung tissue  Dark green  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY055  Lung tissue  Pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY056  Abscess puncture fluid  Pale pink  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  No identification  Cryptococcus neoformans  PUMY057  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY058  Peripheral blood  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY059  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY060  Lung tissue  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Low discrimination  Cryptococcus neoformans  Cryptococcus neoformans  PUMY061  CSFc  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY062  Bronchoalveolar lavage fluid  Pinkish white  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  Cryptococcus neoformans  PUMY063  Peripheral blood  Grayish green  Exophiala dermatitidis  Rhodotorula mucilaginosa  Candida lipolytica  No identification  Exophiala dermatitidis  PUMY064  Lung tissue  Dark brown  Exophiala dermatitidis  Rhodotorula minuta  Trichomonascus ciferrii  No identification  Candida lusitaniae  PUMY065a  Pleural pus  White purple  Saccharomyces cerevisiae  Unidentified organism  Saccharomyces cerevisiae  Saccharomyces cerevisiae  Candida robustad  PUMY066  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Candida tropicalis  PUMY067  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY068  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY069  Clean catch midstream urine  Dark green (rough)  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  Trichosporon asahii  PUMY070  Catheter urine  Grayish green (small and rough)  Trichosporon asahii  Trichosporon asahii  Low discrimination  Trichosporon asahii  Trichosporon asahii  PUMY071  Clean catch midstream urine  Grayish green (small and rough)  Trichosporon jirovecii  Candida laurentii  Candida laurentii  No identification  No identification  aThe ITS of these isolates were sequenced in our previously published, related study.16 bPICC, peripherally inserted central catheter. cCSF, cerebrospinal fluid. dCandida robusta, anamorphic synonym of Saccharomyces cerevisiae. View Large Biochemical profiling Out of the 71 isolates, 56 (78.9%) and 51 (71.8%) isolates were correctly identified using the API 20C AUX and Vitek 2 YST ID Card systems, respectively (Table 1). Forty-nine isolates (69.0%) could be correctly identified by both systems. Seven isolates (9.9%) could only be correctly identified by the API 20C AUX system, while two isolates (2.8%) could only be correctly identified by the Vitek 2 YST ID Card system. For the API 20C AUX system, 11 isolates (15.5%) were misidentified as other yeast species, one isolate (1.4%) was identified as “unidentified organism”, and three isolates (4.2%) could not be identified due to low discrimination values; while for the Vitek 2 YST ID Card system, 13 isolates (18.3%) were misidentified as other yeast species, one isolate (1.4%) was identified as “unidentified organism,” and six isolates (8.5%) could not be identified due to low discrimination values. Generally, even using a combination of both systems, correct identification could not be achieved for one of the Cryptococcus neoformans isolate (PUMY046). Both Candida metapsilosis (PUMY018) and Candida orthopsilosis (PUMY022 and PUMY023) were misidentified as Candida parapsilosis. For some rarely encountered yeast species, including Candida dubliniensis (PUMY001), Candida intermedia (PUMY013), Candida nivariensis (PUMY019), Candida norvegensis (PUMY020 and PUMY021), E. dermatitidis (PUMY063 and PUMY064), M. guilliermondii (PUMY012), and Trichosporon jirovecii (PUMY071), correct identification was also not successful. MALDI–TOF MS Among the 71 isolates, 65 (91.5%) and 57 (80.3%) isolates were successfully identified to the correct species using the Bruker and Vitek platforms, respectively (Table 1). Fifty-four isolates (76.1%) could be correctly identified by both platforms. Eleven isolates (15.5%) could only be correctly identified by the Bruker platform, while three isolates (4.2%) could only be correctly identified by the Vitek platform. For the Bruker platform, five isolates (7.0%) were misidentified as other yeast species, and no identification was provided for one isolate (1.4%); while for the Vitek platform, two isolates (2.8%) were misidentified as other yeast species, multiple identifications were provided for three isolates (4.2%), and no identification was provided for nine isolates (12.7%). Although Candida metapsilosis (PUMY018) and Candida orthopsilosis (PUMY022 and PUMY023) could be correctly identified using the Bruker platform, these two species were not correctly identifiable using the Vitek platform. Correct identification for one Cryptococcus neoformans isolate (PUMY046), one E. dermatitidis isolate (PUMY064), and Trichosporon jirovecii (PUMY071) could not be achieved using both platforms. DiversiLab automated rep-PCR DNA fingerprinting Of the 17 clinical isolates and one reference strain (ATCC 22019T) belonging to the Candida parapsilosis species complex, all were identified by DiversiLab automated rep-PCR DNA fingerprinting only as Candida parapsilosis with similarities ≥93.2% (Table 2), indicating the misidentification of Candida metapsilosis and Candida orthopsilosis. However, HCA of the rep-PCR DNA fingerprints of these three species within this species complex formed three different discrete clusters (Fig. 2). Figure 2. View largeDownload slide Hierarchical cluster analysis of the DiversiLab automated rep-PCR DNA fingerprints of the 17 clinical isolates and one reference strain (ATCC 22019T) belonging to the Candida parapsilosis species complex. Figure 2. View largeDownload slide Hierarchical cluster analysis of the DiversiLab automated rep-PCR DNA fingerprints of the 17 clinical isolates and one reference strain (ATCC 22019T) belonging to the Candida parapsilosis species complex. Table 2. Identification of Candida parapsilosis species complex isolates by DiversiLab rep-PCR fingerprinting system.       Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes        Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes  View Large Table 2. Identification of Candida parapsilosis species complex isolates by DiversiLab rep-PCR fingerprinting system.       Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes        Consistency    Identification by  Similarity  with molecular  Isolate  DiversiLab system  (%)  identification  PUMY018  Candida parapsilosis  94.7  no  PUMY022  Candida parapsilosis  94.9  no  PUMY023  Candida parapsilosis  97.0  no  PUMY024  Candida parapsilosis  97.1  yes  PUMY025  Candida parapsilosis  96.7  yes  PUMY026  Candida parapsilosis  96.1  yes  PUMY027  Candida parapsilosis  96.3  yes  PUMY028  Candida parapsilosis  98.2  yes  PUMY029  Candida parapsilosis  97.4  yes  PUMY030  Candida parapsilosis  96.4  yes  PUMY031  Candida parapsilosis  95.9  yes  PUMY032  Candida parapsilosis  96.1  yes  PUMY033  Candida parapsilosis  98.2  yes  PUMY034  Candida parapsilosis  95.6  yes  PUMY035  Candida parapsilosis  95.8  yes  PUMY036  Candida parapsilosis  93.2  yes  PUMY037  Candida parapsilosis  96.5  yes  ATCC 22019T  Candida parapsilosis  98.1  yes  View Large Discussion When DNA sequencing of conserved molecular targets was used for fungal identification, the variable domains 1 and 2 (D1/D2 regions) of the 28S nrDNA have been utilised for species identification and phylogenetic inference of yeasts.1 As a result, an abundant set of 28S nrDNA sequence data is available at public nucleotide repositories, such as the INSD. On the other hand, the ITS region has also been used as an alternative marker for yeast identification lately,3,13 and it was found that the ITS could provide an even better resolution for species identification.14 More recently, the ITS region has been adopted as the universal DNA barcode for fungi.15 In this study, we confirmed that there is 100% concordance between the identification results of 28S nrDNA and ITS sequencing for all the 71 clinically important yeast strains not readily identifiable by CHROMagar, although six of the 71 strains possessed multiple ITS copies and required cloning of the PCR products and subsequent sequencing for unambiguous identification.16 These identifications were used as the gold standard for subsequent evaluation of the other identification methods. It is important to note that one of the most critical steps for correct species identification by DNA sequencing is the accuracy of the sequence data in the databases. Therefore, curated databases, such as the Westerdijk Fungal Biodiversity Institute database, which we have used in this study, or the International Society for Human and Animal Mycology (ISHAM) ITS database,17 should be utilized in order to obtain reliable identification results. Both the API 20C AUX and Vitek 2 YST ID Card systems were suboptimal in identifying rarely encountered yeasts. In this study, both biochemical profiling systems only performed moderately well in identifying clinical yeast isolates (70–80% identification rates). The performance of the two systems were similar (P = .4363, Fisher's exact test). In general, more frequently encountered yeast species, such as Candida glabrata, Candida kefyr, Candida krusei, Candida parapsilosis sensu stricto, Cryptococcus neoformans, and Trichosporon asahii, were more readily identified correctly by the two biochemical tests. On the other hand, for some less commonly encountered yeast species, including Candida dubliniensis, Candida norvegensis, M. guilliermondii, and for those uncommonly found in clinical specimens, including Candida metapsilosis, Candida nivariensis, Candida orthopsilosis, E. dermatitidis, Saccharomyces cerevisiae, and Trichosporon jirovecii, the performance of both biochemical identification systems was not satisfactory. This observation of the suboptimal performance of both systems in identifying uncommonly found yeasts in clinical specimens was in line with previous studies on commercial kits based on biochemical profiling.18,19 In general, for most commercial systems, around 56–89% of the studied yeast isolates were successfully identified, depending on the spectrum of yeast species that were included in the particular study, and the yeast species that were misidentified usually include those that were uncommonly encountered in clinical samples, such as Candida famata, Candida intermedia, Candida nivariensis, Candida pulcherrima, Candida zelanoides, and so forth.18,19 In this study, the uncommon clinical yeast E. dermatitidis (PUMY063 and PUMY064) could not be correctly identified by both commercial biochemical kits. This is because reference profiles for this species are not included in the databases of the API 20C AUX system nor the Vitek 2 YST ID Card system. This illustrates that the data volume of these biochemical profiling kits should be expanded to cover more yeast species so as to even allow the correct identification of uncommonly encountered clinical yeasts using these commercial biochemical kits. As for the particularly difficult-to-identify Cryptococcus neoformans isolate PUMY046 by multiple methods, it was recovered from the cerebrospinal fluid of a 64-year-old man with chronic meningitis. Cerebrospinal fluid for cryptococcal antigen was also positive. Review of the API 20C AUX and Vitek 2 YST ID card biochemical profiles of PUMY046 revealed that the abilities of this isolate to assimilate glycerol and l-arabinose as well as l-malate, d-amygdalin, and d-gluconate respectively might have led to the failure of identifying it correctly as Cryptococcus neoformans (Table 1). MALDI–TOF MS, coupled with hierarchical cluster analysis, is a promising technology for the rapid and accurate identification of microorganisms in clinical laboratories, provided that there is a sufficiently-sized reference database. The usefulness of MALDI–TOF MS has been greatly evaluated for the correct identification of clinical bacteria, and the application of this technology has been proven to be successful.20 Besides pathogenic bacteria, a number of studies have attempted to evaluate the performance of MALDI–TOF MS platforms for the identification of clinical yeast isolates.18,21,22 However, the use of MALDI–TOF MS for clinical mycology moves in a much slower pace than that for the identification of clinical bacteria,20 and more evaluation on this technology is required before it is widely adopted for clinical fungi identification. In this study, although the Bruker MALDI–TOF MS platform may have performed slightly better than the Vitek platform, the difference was not statistically significant (P = .0894, Fisher's exact test). Generally, both platforms performed well in identifying those clinically commonly encountered yeast species, such as Candida dubliniensis, Candida glabrata, Candida kefyr, Candida krusei, Candida lusitaniae, Candida norvegensis, Candida parapsilosis sensu stricto, Cryptococcus neoformans, M. guilliermondii, Saccharomyces cerevisiae, and Trichosporon asahii; although in occasional cases misidentification or unidentification was resulted for some strains. In contrast, for rarely encountered species the two platforms exhibited different discriminating powers. In particular, even though the Bruker platform successfully identified Candida metapsilosis, Candida nivariensis, Candida orthopsilosis, and one E. dermatitidis isolate, the Vitek platform failed to identify these species. The main reason for this should be the lack of reference mass spectra for these species in the Vitek database v2.0. On the other hand, although the Vitek platform successfully identified Candida intermedia, the Bruker platform was unsuccessful in doing so despite the presence of Candida intermedia reference mass spectra in the Bruker database. Similarly, although the Bruker database contains reference spectral data for E. dermatitidis and Trichosporon asahii, the strains PUMY064 (E. dermatitidis) and PUMY066 (Trichosporon asahii) were misidentified as Candida lusitaniae and Candida tropicalis, respectively, even after several attempts. The reason for the identification failure for these strains is not clear since these strains did not exhibit unusual phenotypic traits. Both Bruker and Vitek platforms were unable to identify Trichosporon jirovecii. The capability of MALDI–TOF MS platforms to identify more yeast strains may be improved if the database is expanded with more reference spectra, as demonstrated by previous studies.23–25 Similar to MALDI–TOF MS, the database of DiversiLab automated rep-PCR DNA fingerprinting needs further improvement. For rep-PCR, various noncoding, repetitive sequences of different lengths in the genome are amplified. Following microfluidics electrophoresis, the amplified products are separated to generate DNA fingerprints. Since different microorganisms possess different sets of repetitive sequences and so by comparing the DNA fingerprints with those in the database unknown isolates could be identified.26 So far, only seven studies have attempted to utilise the DiversiLab system to achieve strain differentiation or typing of yeasts, with an emphasis on Candida species,6,27–29Cryptococcus neoformans/Cryptococcus gatti,30,31 and Trichosporon asahii.32 Although it has already been demonstrated that Candida parapsilosis sensu stricto could be successfully differentiated from other commonly encountered clinical Candida species,6,28 the ability of this technology to differentiate the three member species of the Candida parapsilosis species complex has not been evaluated. Because of this and since DiversiLab automated rep-PCR DNA fingerprinting is expensive (about 90 USD per isolate), in this study, we have only characterized 17 clinical isolates and one reference strain of the Candida parapsilosis species complex for a preliminary evaluation of its performance. Results showed that rep-PCR DNA fingerprinting was not able to differentiate Candida parapsilosis, Candida metapsilosis, and Candida orthopsilosis alone. However, when a dendrogram using the rep-PCR DNA fingerprints of these three species was constructed, it was revealed that the three Candida species formed three distinct clusters (Fig. 2). This indicates that if the database of DiversiLab automated rep-PCR DNA fingerprinting is improved, especially when more reference DNA fingerprints of Candida metapsilosis and Candida orthopsilosis are included and used for further evaluation, this technology platform should possess a great potential for the correct identification of these three Candida species to the species level. Acknowledgments This work was partly supported by the Strategic Research Theme Fund and Dr Cheng Yu Tung Fellowship, The University of Hong Kong, Hong Kong; as well as Croucher Senior Medical Research Fellowship, Croucher Foundation, Hong Kong. Declaration of interest P.C.Y.W. has provided scientific advisory/laboratory services for Gilead Sciences, Incorporated and International Health Management Associates, Incorporated/Pfizer, Incorporated. J.F.W.C. has provided scientific advisory services for Gilead Sciences, Incorporated, and received travel grants from Pfizer, Incorporated and Astellas Pharma, Incorporated. All other authors report no conflicts of interest. The funding sources had no role in study design, data collection, analysis, interpretation, or writing of the report. The authors alone are responsible for the content and the writing of the paper. References 1. Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek . 1998; 73: 331– 371. Google Scholar CrossRef Search ADS PubMed  2. Scorzetti G, Fell JW, Fonseca A, Statzell-Tallman A. Systematics of basidiomycetous yeasts: a comparison of large subunit D1/D2 and internal transcribed spacer rDNA regions. FEMS Yeast Res . 2002; 2: 495– 517. Google Scholar CrossRef Search ADS PubMed  3. 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Medical MycologyOxford University Press

Published: Dec 8, 2017

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