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  Table of Contents  
BRIEF COMMUNICATION
Year : 2017  |  Volume : 35  |  Issue : 4  |  Page : 588-592
 

Clinically relevant yeast species identified by sequencing the internal transcribed spacer region of r-RNA gene and Vitek 2 compact (YST card) commercial identification system: Experience in a Tertiary Care Hospital in Assam, Northeast India


1 Department of Microbiology, Assam Medical College and Hospital, Dibrugarh, Assam, India
2 Department of Paediatrics, Assam Medical College and Hospital, Dibrugarh, Assam, India
3 Regional Medical Research Center, Dibrugarh, Assam, India

Date of Web Publication1-Feb-2018

Correspondence Address:
Dr. Reema Nath
Department of Microbiology, Assam Medical College and Hospital, Dibrugarh - 786 002, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_100

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 ~ Abstract 

In this retrospective study from 2012 to 2015, 333 clinical isolates of yeasts were identified using Vitek 2 Compact System YST ID card (Biomerieux, France) and internal transcribed spacer (ITS) sequencing. Eighteen species were identified by ITS sequencing. Candida albicans was the most common species (46.5%), followed by Candida tropicalis (27%). The total species supported by Vitek System was 11 (61.11%). The sensitivity of the system in identifying these 11 species was 66.66%–100%; specificity 98.37%–100%; positive predictive value 70%–100%, negative predictive value 96.05%–100%, and diagnostic accuracy 96.99%–100%. Diagnostic accuracy of ITS1 and ITS2 sequences individually was 98.49% and 100% using NCBI Genbank database.


Keywords: Assam, internal transcribed spacer sequencing, rare yeast, Vitek YST card, yeast


How to cite this article:
Nath R, Bora R, Borkakoty B, Saikia L, Parida P. Clinically relevant yeast species identified by sequencing the internal transcribed spacer region of r-RNA gene and Vitek 2 compact (YST card) commercial identification system: Experience in a Tertiary Care Hospital in Assam, Northeast India. Indian J Med Microbiol 2017;35:588-92

How to cite this URL:
Nath R, Bora R, Borkakoty B, Saikia L, Parida P. Clinically relevant yeast species identified by sequencing the internal transcribed spacer region of r-RNA gene and Vitek 2 compact (YST card) commercial identification system: Experience in a Tertiary Care Hospital in Assam, Northeast India. Indian J Med Microbiol [serial online] 2017 [cited 2019 Jun 26];35:588-92. Available from: http://www.ijmm.org/text.asp?2017/35/4/588/224417



 ~ Introduction Top


The incidence of fungal infections including yeasts has increased worldwide. Infections due to less common species are also increasingly being reported.[1] Clinical mycology laboratories mostly use commercial automated phenotypic methods for the species identification which is essential for proper antifungal therapy. Molecular methods including internal transcribed spacer (ITS) regions of r-RNA gene have been used extensively for fungal species delineation.[1],[2]

This retrospective laboratory-based study was done in the clinical mycology laboratory of a tertiary care hospital in Assam to identify the clinically relevant yeast species from 2012 to 2015. This study evaluated the Vitek 2 YST ID card commercial phenotypic yeast identification system (Biomerieux, France) with gold standard test of sequence comparison of ITS region. The feasibility of using ITS1 and ITS2 sequences alone in identification of these yeast species was studied.

Institutional ethics committee permission was taken for the study.


 ~ Materials and Methods Top


All consecutive significant yeast isolates from various samples received at the Department of Microbiology from patients attending Assam Medical College and Hospital were included in the study. The isolated strains were subcultured onto Sabouraud dextrose agar media (Hi-Media, India) for 48 h before testing.

Vitek 2 Compact YST card System

Yeast isolates were identified to the species level using the Vitek 2 YST card system (Biomerieux, France) following the manufacturer's instructions.

DNA preparation, amplification, sequencing and data analysis

Yeast isolates were subcultured on Sabouraud dextrose agar (Hi-Media, India) and incubated at 28°C for 24–48 h. Colonies of these strains were suspended in saline to obtain a turbidity of 0.5 McFarland standard at a 530-nm wavelength. One millilitre of the cell suspension was centrifuged at 5000 ×g for 3 min in a microcentrifuge. Genomic DNA was extracted from the strains using the Qiagen DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol.[3],[4] Previously described primers ITS1 (5'TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') are used for amplification of ITS 1, 5.8S rRNA, and ITS 2 in a cycle of initial denaturation at 94°C for 3 min, denaturation at 94°C for 60 s, annealing at 56°C for 60 s, extension at 72°C for 2 min and final extension at 72°C for 7 min. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with forward and reverse primers using Big Dye Terminator version 3.1 Cycle sequencing kit (Applied Biosystems) on ABI 3730 × l Genetic Analyzer (Applied BioSystems). Consensus sequence was generated using CodonCode aligner software (CodonCode Corporation). Species were identified by searching databases using the BLAST sequence analysis tool (http://www.ncbi.nlm.nih.gov/BLAST/), and comparative ITS sequence identification was done using International Society of Human and Animal Mycology (ISHAM)-ITS reference database available at Genbank and http://its.mycologylab.org/. If the query sequence of ITS1-5.8S rRNA-ITS2 matched >98.5%, it was taken as correct identification.[4] Any query sequence matching <98.5% with database was taken as uncertain identification.[4] If ISHAM database was not available for some strain sequence, then >99% match with Genbank sequences was taken as correct identification. After sequencing for further analysis, portions of the 18S, 5.8S, and 26S rRNA gene sequences of the PCR products were removed to obtain the exact ITS1 and ITS2 sequences for comparison along with the ITS1-5.8S-ITS2 sequences. For all yeasts, the sequences of the 3' ends of the 18S and 5.8S rRNA genes were GCGGAAGGATCATTA and GTTTGAGCGTCATTT, respectively, and the sequences of the 5' ends of the 5.8S and 26S rRNA genes were AAACTTTCAACAA and GACCTCAAATCAG, respectively.[1] The ITS1 or ITS2 sequences were compared using nucleotide-nucleotide BLAST (blastn) with default settings, except that sequences were not filtered for low complexity.[1] Species identification was carried out from the lowest expect value of the BLAST output.[1] Occasionally, the BLAST search with the query sequence hit sequences from two different species with 100% identity when using ITS1 or ITS2 sequences alone. Under these conditions, the whole ITS sequence comprising ITS1-5.8SrRNA-ITS2 was used to determine the species.[3],[4],[5],[6] For phylogenetic analysis, the sequences were aligned using CLUSTAL W [5] that is enclosed in MEGA version 6.0 after testing the best fit model.[6],[7],[8]


 ~ Results and Observation Top


[Table 1] shows the various samples and the species isolated.
Table 1: Different species isolated from various clinical samples

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Eighteen species were identified by the gold standard method of ITS sequencing, whereas total number of species identified by the Vitek 2 YST ID Card automated system was 15. Out of these 15 species, Trichosporon inkin, Candida rugosa and Candida famata were misidentifications. Candida metapsilosis, Candida palmioleophila, Cyberlindnera fabianii, Candida akabanensis, Cutaneotrichosporon debeurmannianum, Candida mesorugosa and Meyerozyma caribbica were the species identified by reference DNA sequencing method. These were either not identified or misidentified by the Vitek commercial system. These seven species comprised of 9% of the isolates. A total of 285 (85.58%) isolates were correctly identified by this commercial system. Three (0.9%) isolates remained unidentified, 19 (5.70%) were reported as low discriminatory and 26 (7.80%) isolates were misidentified.

This commercial system supported the identification of 11 species of yeasts in this study. [Table 2] shows performances sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of Vitek 2 (YST ID card) in identifying 11 species of yeast.
Table 2: Diagnostic performances (sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of Vitek 2 [YST ID card] in identifying 11 species of yeast)

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Besides using ITS1-5.8SrRNA–ITS2 as the gold standard for identification, our study also looked into the feasibility of using ITS1 and ITS2 sequences individually for the identification of these species using the same reference databases. ITS 1 sequence of all the species except M. caribbica was sufficient to differentiate these species. ITS2 sequences of all 18 species were sufficient for differentiation of species. [Figure 1] shows the phylogenetic tree made from ITS1-5.8SrRnA-ITS2 sequences of 18 species. In our study, two closely related species groups were found. They are Meyerozyma guilliermondii-M. caribbica and Candida parapsilosis-C. metapsilosis. ITS1 sequences of M. caribbica failed to differentiate from M. guilliermondii. The mean interpopulation diversity of ITS1-5.8S-ITS2, ITS1 and ITS2 sequences of the M. guilliermondii and M. caribbica is found to be 0.005, 0 and 0.01, respectively. The diversity of ITS1-5.8S-ITS2 sequences between closely related species C. parapsilosis and C. metapsilosis was found to be 0.02. In ITS1 sequences, this difference was 0.05 and in ITS2 sequences it was 0.01. Both the ITS1 and ITS2 sequences could differentiate these two species.
Figure 1: Phylogenetic tree constructed by the maximum likelihood method using ITS sequences of 79 of the studied sequences with 1000 bootstrap replications (Tamura 3 parameter method)

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The correct identification rates of ITS1 and ITS2 sequences individually were found to be 98.49% and 100%, respectively, in comparison to the gold standard of whole ITS sequences.


 ~ Discussion Top


The non-albicans non-tropicalis isolates consisted of 16 species. These species were relatively rare and correctly identified only by molecular method. Maximum number of isolates per species in this group was 10 and a minimum of 1 (mean 5.68 = 6 isolates). From blood samples, 14 species were isolated followed by urine (12 species) [Table 1]. Most common species in this group isolated from blood in our study was Pichia jadinii (15.87%). Majority of the blood isolates (93.65%) were from neonates with clinical suspicion of septicaemia. We could not find any other report of isolation of the rare species C. akabanensis and C. debeurmannianum from neonatal blood.

Limitation of the commercial identification system Vitek

Cryptococcus laurentii (n = 7, 26.9%), C. parapsilosis (n = 6, 23.07%), C. rugosa (n = 4, 15.38%) and C. famata (n = 4, 15.38%) were the common species misreported by the Vitek YST-ID card. C. laurentii and C. famata were not found in this study as confirmed by ITS sequencing. The organism misrepresented as C. laurentii was Cutaneotrichosporon debeurmannianum which is not supported by YST card. C. metapsilosis was misrepresented as closely related species C. parapsilosis. C. mesorugosa and C. akabanensis were misidentified as closely related species C. rugosa. M. guilliermondii was misrepresented by low discrimination C. famata/C. guilliermondii in 3 (33.33%) isolates. M. caribbica isolates were also misreported by Vitek as low discriminatory C. guilliermondii/famata and C. famata. Misidentification of M. guilliermondii as C. famata has already been reported.[9]M. caribbica is often misidentified as M. guilliermondii.[10] The low discrimination report of C. famata/C guilliermondii by Vitek in our study was found to be 100% specific for Meyerozyma species. These findings correlate well with other published studies.[11],[12] One isolate of C. akabanensis was misreported as C. rugosa in our study. C. debeurmannianum was misreported as C. laurentii and low discrimination C. laurentii/Trichosporon mucoides. Though we searched available literature, we could not find any other report of misidentification of this rare species as C. laurentii and C. laurentii/T. mucoides (unpublished report of the authors). C. famata was the commonly misreported organism by Vitek 2 compact representing various species. The species misrepresented by C. famata were M. guilliermondii, C. albicans, Trichosporon asahii, C. parapsilosis, C. tropicalis and Wickerhamomyces anomalus. No C. famata strain was found in our study. This finding is similar to another study which reported that identification of C. famata by certain phenotypic system was almost certainly incorrect.[10]

This commercial system was found to be 100% sensitive and specific in identifying four species of yeasts out of the 18 in this study. These are Cryptococcus neoformans, Candida krusei, Kodamaea ohmeri and Candida glabrata [Table 2].

Our study looked into the feasibility of using the ITS1 or ITS 2 sequences separately to identify the yeast isolates by using the DNA barcode of reference strains provided by ISHAM-ITS reference DNA database available at NCBI. Both the ITS1 and ITS2 sequences individually were found to be 97.20% and 100% accurate in identifying the isolates using ISHAM database excluding C. debeurmannianum isolates as this species was not found in the ISHAM database. This accuracy was 98.49% and 100% for both ITS1 and ITS2 sequences using NCBI Genbank database. With ITS1 sequencing alone, four strains of C. albicans failed to match with ISHAM reference strains available at http://www.ncbi.nlm.nih.gov/BLAST. The ITS1 sequences of these four strains had deletion of T at position 131. Multiple sequence analysis demonstrated that the closely related species M. guilliermondii and M. caribbica differ only by three nucleotides at positions 140, 141 and 143 in the ITS1 sequence. They are C, A and T in M. guilliermondii whereas T, T and A in M. caribbica. Hence, these three positions in ITS1 can be used as a signature sequence in the identification of these two species if ITS1 sequence alone is used. Leaw et al. in an earlier study using 41 Candida and 45 non-Candida reference strains had found diagnostic accuracy of 97% and 99.7% using ITS1 and ITS2 sequences individually. The same workers have reported an accuracy rate of 95.9% and 100% for clinical isolates in the same study.[1] Methods based on short ITS2 sequences can be cost-effective in comparison to whole ITS sequences. Validated methods based on ITS2 sequences without the sequencing or electrophoresis step has been published.[13] Innovations of this kind will help in accurate identification of yeast species which is important for accurate and effective antifungal therapy.

Representatives of the species in this study are submitted to Genbank (KT250730–KT250732, KU961974–KU962046).

Financial support and sponsorship

Authors acknowledge the financial grant from Department of Biotechnology, Ministry of Science and Technology, Government of India [No.BT/234/NE/TPB 2011].

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

1.
Leaw SN, Chang HC, Sun HF, Barton R, Bouchara JP, Chang TC, et al. Identification of medically important yeast species by sequence analysis of the internal transcribed spacer regions. J Clin Microbiol 2006;44:693-9.  Back to cited text no. 1
    
2.
Chen YC, Eisner JD, Kattar MM, Rassoulian-Barrett SL, LaFe K, Yarfitz SL, et al. Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes. J Clin Microbiol 2000;38:2302-10.  Back to cited text no. 2
    
3.
White TJ, Bruns T, Lee S, Taylor JW. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR Protocols: A Guide to Methods and Applications. New York: Academic Press, Inc.; 1990. p. 315-22.  Back to cited text no. 3
    
4.
Irinyi L, Serena C, Garcia-Hermoso D, Arabatzis M, Desnos-Ollivier M, Vu D, et al. International Society of Human and Animal Mycology (ISHAM)-ITS reference DNA barcoding database – The quality controlled standard tool for routine identification of human and animal pathogenic fungi. Med Mycol 2015;53:313-37.  Back to cited text no. 4
    
5.
Thompson JD, Higgins DG, Gibson TJ. Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673-80.  Back to cited text no. 5
    
6.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725-9.  Back to cited text no. 6
    
7.
Tamura K. Estimation of the number of nucleotide substitutions when there are strong transition-transversion and G+C-content biases. Mol Biol Evol 1992;9:678-87.  Back to cited text no. 7
    
8.
Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111-20.  Back to cited text no. 8
    
9.
Kim SH, Shin JH, Mok JH, Kim SY, Song SA, Kim HR, et al. Misidentification of Candida guilliermondii as C. famata among strains isolated from blood cultures by the Vitek 2 system. Biomed Res Int 2014;2014:250408.  Back to cited text no. 9
    
10.
Castanheira M, Woosley LN, Diekema DJ, Jones RN, Pfaller MA. Candida guilliermondii and other species of Candida misidentified as Candida famata: Assessment by Vitek 2, DNA sequencing analysis, and matrix-assisted laser desorption ionization-time of flight mass spectrometry in two global antifungal surveillance programs. J Clin Microbiol 2013;51:117-24.  Back to cited text no. 10
    
11.
Cendejas-Bueno E, Gomez-Lopez A, Mellado E, Rodriguez-Tudela JL, Cuenca-Estrella M. Identification of pathogenic rare yeast species in clinical samples: Comparison between phenotypical and molecular methods. J Clin Microbiol 2010;48:1895-9.  Back to cited text no. 11
    
12.
Chaves GM, Terçarioli GR, Padovan AC, Rosas RC, Ferreira RC, Melo AS, et al. Candida mesorugosa sp. nov. a novel yeast species similar to Candida rugosa, isolated from a tertiary hospital in Brazil. Med Mycol 2013;51:231-42.  Back to cited text no. 12
    
13.
Duyvejonck H, Cools P, Decruyenaere J, Roelens K, Noens L, Vermeulen S, et al. Validation of high resolution melting analysis (HRM) of the amplified ITS2 region for the detection and identification of yeasts from clinical samples: Comparison with culture and MALDI-TOF based identification. PLoS One 2015;10:e0132149.  Back to cited text no. 13
    


    Figures

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    Tables

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