Evaluation of VITEK 2 for discriminating Trichosporon species: misidentification of Trichosporon non-T. asahii Dulce Sachiko Yamamoto de Figueiredo, Jo˜ao N´obrega de Almeida Jr., Adriana Lopes Motta, Dulcilena Mattos Castro e Silva, Maria Walderez Szeszs, Gilda Maria Barbaro Del Negro PII: DOI: Reference:
S0732-8893(14)00211-9 doi: 10.1016/j.diagmicrobio.2014.05.017 DMB 13622
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
12 March 2014 12 May 2014 14 May 2014
Please cite this article as: de Figueiredo Dulce Sachiko Yamamoto, de Almeida Jr. Jo˜ao N´obrega, Motta Adriana Lopes, Castro e Silva Dulcilena Mattos, Szeszs Maria Walderez, Del Negro Gilda Maria Barbaro, Evaluation of VITEK 2 for discriminating Trichosporon species: misidentification of Trichosporon non-T. asahii, Diagnostic Microbiology and Infectious Disease (2014), doi: 10.1016/j.diagmicrobio.2014.05.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of VITEK 2 for discriminating Trichosporon species: misidentification
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Running title: Trichosporon misidentification by VITEK 2
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of Trichosporon non-T. asahii
Dulce Sachiko Yamamoto de Figueiredo1, João Nóbrega de Almeida Junior2, Adriana
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Lopes Motta2, Dulcilena Mattos Castro e Silva3, Maria Walderez Szeszs3, Gilda Maria
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Barbaro Del Negro1*
1. Laboratory of Medical Mycology (LIM-53) – Clinical Dermatology Division, Hospital das Clínicas da FMUSP and Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, Brazil. 2. Microbiology Section, Central Laboratory Division (LIM-03), Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Brazil.
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3. Mycology Unit, Adolfo Lutz Institute, Public Health Reference Laboratory, Secretary of Health, São Paulo State, Brazil
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* Corresponding author: Gilda M. B. Del Negro Laboratory of Medical Mycology (LIM-53) Instituto de Medicina Tropical de São Paulo/USP Av. Dr. Enéas Carvalho de Aguiar, 500 Andar térreo – CEP 05403-900 São Paulo – SP – Brazil Tel: 55-11-3061.7045 FAX: 55-11-3088.5237 E-mail: [email protected]
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ABSTRACT The VITEK 2 system was evaluated for the identification of 74 Trichosporon
T
invasive and non-invasive clinical isolates, comparing its results with the IGS1
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sequencing. The system correctly identified T. asahii but not non-T. asahii isolates,
SC
which represented nearly 50% of the invasive infections in our nosocomial setting.
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PT
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Key words: Trichosporon, VITEK 2, sequencing, misidentification
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As the second most common genus isolated during fungemia episodes in patients with onco-hematological diseases, Trichosporon spp. have emerged as important
T
pathogens in recent decades (Walsh et al., 2004; Miceli et al., 2011). The mortality rates
RI P
of invasive infections caused by Trichosporon remain high (50-80% of cases), reaching nearly 100% in patients with persistent neutropenia (Suzuki et al., 2010; Colombo et al.,
SC
2011). These elevated indices of mortality are related to the difficulty in establishing an
MA NU
early diagnosis and to the relative resistance of Trichosporon to conventional antifungal drugs (Liao et al., 2012).
The genus and species of Trichosporon are routinely identified by macro and micro-morphological characteristics, as well by commercially available assimilation
ED
tests, such as the API 20 C AUX™ (BioMérieux, France), ID 32C™ (BioMérieux), and automated VITEK 2™ system (BioMérieux) (Massonet et al., 2004; Colombo et al.,
PT
2011). In the last, the tests are performed within 18-24 hours, but the current database
CE
comprises only three Trichosporon species: T. asahii, T. inkin, and T. mucoides (Colombo et al., 2011). Consequently, identification at the species level may be
AC
unreliable, and hinder the appropriate choice and application of antifungal therapy as there are differences in the antifungal susceptibility profiles among Trichosporon species (Chagas-Neto, 2008). Pfaller et al. (2009) found higher minimal inhibitory concentrations (MICs) of fluconazole and voriconazole against 14% and 5.5% of the T. asahii isolates respectively, while most T. mucoides and T. inkin isolates were susceptible to these azole drugs. Other authors reported that some T. dermatis clinical isolates exhibited high MICs for voriconazole and fluconazole (Rodriguez-Tudela et al., 2005).
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DNA-based methods have been extensively employed for the accurate identification of Trichosporon species (Guo et al., 2011). Analyses of both the IGS1 and
T
D1/D2 regions of the ribosomal RNA gene have largely been used to distinguish closely
RI P
related emergent species (Zeng et al., 2009).
This study was aimed to evaluate the VITEK 2 system for discriminating
SC
Trichosporon species isolated from clinical specimens by comparing the results with the
MA NU
gold standard sequencing method.
The 74 isolates analyzed were obtained from clinical samples of patients hospitalized in São Paulo, Brazil, from 2003 to 2011, comprising 19 invasive (blood and other sterile sites) and 55 non-invasive isolates (urine and catheter), as described in
ED
Table 1. Reference strains Trichosporon asahii (ATCC90039), T. inkin (ATCC18020), and T. mucoides (ATCC204094) were employed as controls of the tests.
PT
Morphological features, as assessed by microscopy on cornmeal agar-Tween 80
CE
(Difco, USA), and urease tests were performed to confirm genus Trichosporon. The species identifications were performed by the VITEK 2 system using ID YST cards
AC
(BioMérieux) according to the manufacturer’s instructions. Molecular identifications were carried out with previously described primers targeting the IGS1 region of the rDNA gene (Sugita et al., 2002). The amplification products were purified and then sequenced in both the forward and reverse directions using the ABI PRISM 3730 analyzer (Applied Biosystems, USA). For all isolates, sequencing was repeated twice before the analysis. The sequences were analyzed by the software Sequence Scanner v1.0 (Life Technologies, USA) and aligned using the ClustalW2 program. The consensus nucleotide sequences were compared with the Trichosporon reference strains available in the Fungal Biodiversity Center of the Centraal Bureau voor
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5
Schimmelcultures (CBS) and GenBank databases. Species identification was determined when the sequence identity was ≥ 99.5% (Pfaller et al., 2012). A
T
phylogenetic tree was constructed using IGS1 region sequences from 10 invasive T.
RI P
asahii and all Trichosporon non-T. asahii isolates by the unweighted-pair group method (UPGMA) and 1,000 bootstrap simulations with MEGA 4 (Molecular Evolutionary
SC
Genetic Analysis software, http://www.megasoftware.net).
MA NU
The identification results obtained by both the VITEK 2 system and molecular sequencing are summarized in Table 1. The phylogenetic tree is represented in Figure 1. The VITEK 2 system correctly identified 98.4% of the T. asahii isolates, demonstrating its good performance in identifying this species. However, eight T. inkin
ED
isolates were misidentified as T. asahii, though assimilation of rhamnose and Larabinose provided by VITEK 2 was compatible with the T. inkin profile (Sugita, 2011).
PT
Guo et al. (2011) also verified elevated percentages of correct identification of T. asahii
misidentified.
CE
by the automated system, whereas the non-T. asahii species were consistently
AC
The inability of the VITEK 2 in identifying T. inkin isolates in our study is of
clinical relevance once 31.5% of the episodes of invasive infections were caused by this species (Table 1). In fact, recently this species has been documented as a pathogen of deep-seated infections. The rate found in the current study was higher than that previously showed in other Brazilian reports and may represent an increase of trichosporonosis episodes caused by this species (Moretti-Branchini el al, 2001; Chagas Neto et al., 2009; Macêdo et al., 2011). Two isolates (IAL03 and 8G) of T. faecale were misidentified as T. asahii by VITEK 2’s database, and a T. dermatis isolate (DLC10) was identified as T. mucoides,
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with a high percentage of probability (Table 1). As the carbohydrate assimilation profiles between these closely related species (T. faecale/T. asahii and T. dermatis/T.
T
mucoides) are very similar (Sugita, 2011), the discrimination is difficult even with the
RI P
automated system (Gunn et al., 2006).
The VITEK 2 failed in identifying all Trichosporon non-T. asahii isolates which
SC
represented nearly 50% of the invasive infections in our nosocomial settings. Only the
MA NU
IGS1 sequencing analysis was a reliable tool to discriminate those species: of the 74 isolates, 98.6% were appropriately identified by sequencing this rDNA region. One exception was an invasive isolate unidentified by VITEK 2 (IAL 04, Table 1) for which IGS1 sequencing achieved 97.7% identity. However, in the present study, the
ED
phylogenetic tree generated by the alignment of the IGS1 sequences showed that the IAL 04 clustered with the other two T. faecale isolates (Figure 1).
PT
In conclusion, the study showed that the VITEK 2 system provides accurate
CE
results for T. asahii isolates but is not reliable to identify the non-T.asahii species. Correction of inconsistencies of the assimilation profiles analyses and expansion of its
AC
database are required. However the identification of closely related species may remain unsolved due to the similarity of their biochemical profiles. Thus, for the clinically relevant Trichosporon non-T. asahii the molecular method still remains the tool that best provides correct identification, and consequently contributes to the management of invasive infections caused by this emergent genus.
Acknowledgements We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2010/20187-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support.
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REFERENCES Chagas-Neto TC, Chaves GM, Colombo AL. Update on the genus Trichosporon.
RI P
T
Mycopathologia. 2008;166(3):121-32.
Chagas-Neto TC, Chaves GM, Melo AS, Colombo AL. Bloodstream infections due to
SC
Trichosporon spp.: species distribution, Trichosporon asahii genotypes determined on
MA NU
the basis of ribosomal DNA intergenic spacer 1 sequencing, and antifungal susceptibility testing. J Clin Microbiol. 2009; 47(4):1074-81.
Colombo AL, Padovan AC, Chaves GM. Current knowledge of Trichosporon spp. and
ED
Trichosporonosis. Clin Microbiol Rev. 2011;24(4):682-700.
PT
Gunn SR, Reveles XT, Hamlington JD, Sadkowski LC, Johnson-Pais TL, Jorgensen JH.
CE
Use of DNA sequencing analysis to confirm fungemia due to Trichosporon dermatis in
AC
a pediatric patient. J Clin Microbiol. 2006;44(3):1175-7.
Guo LN, Xiao M, Kong F, Chen SC, Wang H, Sorrell TC, et al. Three-locus identification, genotyping, and antifungal susceptibilities of medically important Trichosporon species from China. J Clin Microbiol. 2011;49(11):3805-11.
Liao Y, Hartmann T, Zheng T, Yang RY, Ao JH, Wang WL. Breakthrough trichosporonosis in patients receiving echinocandins: case report and literature review. Chin Med J (Engl). 2012;125(14):2632-5.
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Macêdo DP, de Oliveira NT, da Silva VK, de Almeida Farias AM, de Lima Neto RG,
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with Pulmonary Cancer. Mycopathologia. 2011;171(4):279-83.
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Wilheim AB, et al. Trichosporon inkin Esophagitis: An Uncommon Disease in a Patient
Massonet C, Van Eldere J, Vaneechoutte M, De Baere T, Verhaegen J, Lagrou K.
SC
Comparison of VITEK 2 with ITS2-fragment length polymorphism analysis for
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identification of yeast species. J Clin Microbiol. 2004;42(5):2209-11.
Miceli MH, Díaz JA, Lee SA. Emerging opportunistic yeast infections. Lancet Infect
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Dis. 2011;11(2):142-51.
Moretti-Branchini ML, Fukushima K, Schreiber AZ, Nishimura K, Papaiordanou PM,
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Trabasso P, et al. Trichosporon species infection in bone marrow transplanted patients.
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Diagn Microbiol Infect Dis. 2001;39(3):161-4.
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Pfaller MA, Diekema DJ, Gibbs DL, Newell VA, Bijie H, Dzierzanowska D, Klimko NN, Letscher-Bru V, Lisalova M, Muehlethaler K, Rennison C, Zaidi M; Global Antifungal Surveillance Group. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: 10.5-year analysis of susceptibilities of noncandidal yeast species to fluconazole and voriconazole determined by CLSI standardized disk diffusion testing. J Clin Microbiol. 2009;47(1):117-23.
Pfaller MA, Woosley LN, Messer SA, Jones RN, Castanheira M. Significance of molecular identification and antifungal susceptibility of clinically significant yeasts and
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in
a
global
antifungal
surveillance
programme.
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Mycopathologia.
Rodriguez-Tudela JL, Diaz-Guerra
TM, Mellado
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2012;174(4):259-71.
E, Cano
V, Tapia
C, Perkins
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A, Gomez-Lopez A, Rodero L, Cuenca-Estrella M. Susceptibility patterns and molecular identification of Trichosporon species. Antimicrob Agents Chemother. 2005;
MA NU
49(10):4026-34.
Sugita T. Trichosporon Behrend (1890). In: Kurtzman CP, Fell JW, Boekhout T, editors. The yeasts: a taxonomic study. 5th ed. Amsterdam: Elsevier Science; 2011. p.
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2015-61.
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Sugita T, Nakajima M, Ikeda R, Matsushima T, Shinoda T. Sequence analysis of the
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ribosomal DNA intergenic spacer 1 regions of Trichosporon species. J Clin Microbiol.
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2002;40(5):1826-30.
Suzuki K, Nakase K, Kyo T, Kohara T, Sugawara Y, Shibazaki T, et al. Fatal Trichosporon fungemia in patients with hematologic malignancies. Eur J Haematol. 2010;84(5):441-7.
Walsh TJ, Groll A, Hiemenz J, Fleming R, Roilides E, Anaissie E. Infections due to emerging and uncommon medically important fungal pathogens. Clin Microbiol Infect. 2004;10 Suppl 1:48-66.
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Zeng J, de Souza Motta CM, Fukushima K, Takizawa K, Magalhes OM, Neves RP, et al. Identification of Trichosporon spp. strains by sequencing D1/D2 region and sub-
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SC
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Huazhong Univ Sci Technolog Med Sci. 2009;29(5):655-8.
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typing by sequencing ribosomal intergenic spacer region of ribosomal DNA. J
Legend of Figure 1.
Rooted phylogenetic tree based on IGS1 rDNA sequences of Trichosporon clinical isolates, four Trichosporon reference strains (GenBank) and the outgroup species
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1,000 bootstrap simulation.
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Cryptococcus gatti and C. neoformans (GenBank), obtained by UPGMA analysis and
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T
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Figure 1
11
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Site of isolation (number of isolates)
Species identification by VITEK 2a
Species identification by sequence of IGS1 regionb
10 isolates
Blood (9) / Subcutaneous tissue (1)
T. asahii (≥91%) T. asahii CBS 2479 (100%)
DLC 02
Peritoneal fluid
T. asahii (97%)
T. inkin CNM-CL6516 (100%)
DLC 03
Blood
T. asahii (95%)
T. inkin CNM-CL6516 (100%)
DLC 06
Bone
DLC 10
T. inkin CNM-CL6516 (100%)
Peritoneal fluid
T. mucoides (98%)
T. dermatis CBS 2043 (100%)
DLC 11
Bone
T. asahii (97%)
T. inkin CNM-CL6516 (100%)
DLC 15
Blood
T. asahii (95%)
T. inkin CNM-CL6516 (99.6%)
DLC 16
Blood
T. asahii (99%)
T. inkin CNM-CL6516 (99.5%)
IAL 03
Blood
T. asahii (94%)
T. faecale CNM-CL4327 (100%)
IAL 04
Blood
Low discrimination
T. faecale CNM-CL6515 (97.7%)
51 isolates
Urine
T. asahii (≥91%)
T. asahii CBS 2479 (≥99.5%)
3B
Urine
Low discrimination
T. asahii CBS 2479 (100%)
8C
Catheter
T. asahii (94%)
T. inkin CNM-CL6099 (100%)
9E
Urine
T. asahii (97%)
T. inkin CNM-CL6516 (100%)
8G
Urine
T. asahii (97%)
T. faecale CBS 4828 (100%)
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T. asahii (97%)
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Isolates
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NO N- IN VASI VE
INVASIVE
Table 1. Trichosporon species identification by the VITEK 2 system and IGS1 sequencing analyses
CNM-CL = Yeast Collection Spanish National Center of Microbiology a (%) Probability values provided by the VITEK 2 database b (%) Identity with sequences available in GenBank and CBS (Centraal Bureau voor Schimmelcultures) databases
S0732-8893(14)00211-9 doi: 10.1016/j.diagmicrobio.2014.05.017 DMB 13622
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
12 March 2014 12 May 2014 14 May 2014
Please cite this article as: de Figueiredo Dulce Sachiko Yamamoto, de Almeida Jr. Jo˜ao N´obrega, Motta Adriana Lopes, Castro e Silva Dulcilena Mattos, Szeszs Maria Walderez, Del Negro Gilda Maria Barbaro, Evaluation of VITEK 2 for discriminating Trichosporon species: misidentification of Trichosporon non-T. asahii, Diagnostic Microbiology and Infectious Disease (2014), doi: 10.1016/j.diagmicrobio.2014.05.017
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Evaluation of VITEK 2 for discriminating Trichosporon species: misidentification
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Running title: Trichosporon misidentification by VITEK 2
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of Trichosporon non-T. asahii
Dulce Sachiko Yamamoto de Figueiredo1, João Nóbrega de Almeida Junior2, Adriana
SC
Lopes Motta2, Dulcilena Mattos Castro e Silva3, Maria Walderez Szeszs3, Gilda Maria
MA NU
Barbaro Del Negro1*
1. Laboratory of Medical Mycology (LIM-53) – Clinical Dermatology Division, Hospital das Clínicas da FMUSP and Instituto de Medicina Tropical de São Paulo, Universidade de São Paulo, Brazil. 2. Microbiology Section, Central Laboratory Division (LIM-03), Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Brazil.
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3. Mycology Unit, Adolfo Lutz Institute, Public Health Reference Laboratory, Secretary of Health, São Paulo State, Brazil
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* Corresponding author: Gilda M. B. Del Negro Laboratory of Medical Mycology (LIM-53) Instituto de Medicina Tropical de São Paulo/USP Av. Dr. Enéas Carvalho de Aguiar, 500 Andar térreo – CEP 05403-900 São Paulo – SP – Brazil Tel: 55-11-3061.7045 FAX: 55-11-3088.5237 E-mail: [email protected]
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ABSTRACT The VITEK 2 system was evaluated for the identification of 74 Trichosporon
T
invasive and non-invasive clinical isolates, comparing its results with the IGS1
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sequencing. The system correctly identified T. asahii but not non-T. asahii isolates,
SC
which represented nearly 50% of the invasive infections in our nosocomial setting.
AC
CE
PT
ED
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Key words: Trichosporon, VITEK 2, sequencing, misidentification
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As the second most common genus isolated during fungemia episodes in patients with onco-hematological diseases, Trichosporon spp. have emerged as important
T
pathogens in recent decades (Walsh et al., 2004; Miceli et al., 2011). The mortality rates
RI P
of invasive infections caused by Trichosporon remain high (50-80% of cases), reaching nearly 100% in patients with persistent neutropenia (Suzuki et al., 2010; Colombo et al.,
SC
2011). These elevated indices of mortality are related to the difficulty in establishing an
MA NU
early diagnosis and to the relative resistance of Trichosporon to conventional antifungal drugs (Liao et al., 2012).
The genus and species of Trichosporon are routinely identified by macro and micro-morphological characteristics, as well by commercially available assimilation
ED
tests, such as the API 20 C AUX™ (BioMérieux, France), ID 32C™ (BioMérieux), and automated VITEK 2™ system (BioMérieux) (Massonet et al., 2004; Colombo et al.,
PT
2011). In the last, the tests are performed within 18-24 hours, but the current database
CE
comprises only three Trichosporon species: T. asahii, T. inkin, and T. mucoides (Colombo et al., 2011). Consequently, identification at the species level may be
AC
unreliable, and hinder the appropriate choice and application of antifungal therapy as there are differences in the antifungal susceptibility profiles among Trichosporon species (Chagas-Neto, 2008). Pfaller et al. (2009) found higher minimal inhibitory concentrations (MICs) of fluconazole and voriconazole against 14% and 5.5% of the T. asahii isolates respectively, while most T. mucoides and T. inkin isolates were susceptible to these azole drugs. Other authors reported that some T. dermatis clinical isolates exhibited high MICs for voriconazole and fluconazole (Rodriguez-Tudela et al., 2005).
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4
DNA-based methods have been extensively employed for the accurate identification of Trichosporon species (Guo et al., 2011). Analyses of both the IGS1 and
T
D1/D2 regions of the ribosomal RNA gene have largely been used to distinguish closely
RI P
related emergent species (Zeng et al., 2009).
This study was aimed to evaluate the VITEK 2 system for discriminating
SC
Trichosporon species isolated from clinical specimens by comparing the results with the
MA NU
gold standard sequencing method.
The 74 isolates analyzed were obtained from clinical samples of patients hospitalized in São Paulo, Brazil, from 2003 to 2011, comprising 19 invasive (blood and other sterile sites) and 55 non-invasive isolates (urine and catheter), as described in
ED
Table 1. Reference strains Trichosporon asahii (ATCC90039), T. inkin (ATCC18020), and T. mucoides (ATCC204094) were employed as controls of the tests.
PT
Morphological features, as assessed by microscopy on cornmeal agar-Tween 80
CE
(Difco, USA), and urease tests were performed to confirm genus Trichosporon. The species identifications were performed by the VITEK 2 system using ID YST cards
AC
(BioMérieux) according to the manufacturer’s instructions. Molecular identifications were carried out with previously described primers targeting the IGS1 region of the rDNA gene (Sugita et al., 2002). The amplification products were purified and then sequenced in both the forward and reverse directions using the ABI PRISM 3730 analyzer (Applied Biosystems, USA). For all isolates, sequencing was repeated twice before the analysis. The sequences were analyzed by the software Sequence Scanner v1.0 (Life Technologies, USA) and aligned using the ClustalW2 program. The consensus nucleotide sequences were compared with the Trichosporon reference strains available in the Fungal Biodiversity Center of the Centraal Bureau voor
ACCEPTED MANUSCRIPT
5
Schimmelcultures (CBS) and GenBank databases. Species identification was determined when the sequence identity was ≥ 99.5% (Pfaller et al., 2012). A
T
phylogenetic tree was constructed using IGS1 region sequences from 10 invasive T.
RI P
asahii and all Trichosporon non-T. asahii isolates by the unweighted-pair group method (UPGMA) and 1,000 bootstrap simulations with MEGA 4 (Molecular Evolutionary
SC
Genetic Analysis software, http://www.megasoftware.net).
MA NU
The identification results obtained by both the VITEK 2 system and molecular sequencing are summarized in Table 1. The phylogenetic tree is represented in Figure 1. The VITEK 2 system correctly identified 98.4% of the T. asahii isolates, demonstrating its good performance in identifying this species. However, eight T. inkin
ED
isolates were misidentified as T. asahii, though assimilation of rhamnose and Larabinose provided by VITEK 2 was compatible with the T. inkin profile (Sugita, 2011).
PT
Guo et al. (2011) also verified elevated percentages of correct identification of T. asahii
misidentified.
CE
by the automated system, whereas the non-T. asahii species were consistently
AC
The inability of the VITEK 2 in identifying T. inkin isolates in our study is of
clinical relevance once 31.5% of the episodes of invasive infections were caused by this species (Table 1). In fact, recently this species has been documented as a pathogen of deep-seated infections. The rate found in the current study was higher than that previously showed in other Brazilian reports and may represent an increase of trichosporonosis episodes caused by this species (Moretti-Branchini el al, 2001; Chagas Neto et al., 2009; Macêdo et al., 2011). Two isolates (IAL03 and 8G) of T. faecale were misidentified as T. asahii by VITEK 2’s database, and a T. dermatis isolate (DLC10) was identified as T. mucoides,
ACCEPTED MANUSCRIPT
6
with a high percentage of probability (Table 1). As the carbohydrate assimilation profiles between these closely related species (T. faecale/T. asahii and T. dermatis/T.
T
mucoides) are very similar (Sugita, 2011), the discrimination is difficult even with the
RI P
automated system (Gunn et al., 2006).
The VITEK 2 failed in identifying all Trichosporon non-T. asahii isolates which
SC
represented nearly 50% of the invasive infections in our nosocomial settings. Only the
MA NU
IGS1 sequencing analysis was a reliable tool to discriminate those species: of the 74 isolates, 98.6% were appropriately identified by sequencing this rDNA region. One exception was an invasive isolate unidentified by VITEK 2 (IAL 04, Table 1) for which IGS1 sequencing achieved 97.7% identity. However, in the present study, the
ED
phylogenetic tree generated by the alignment of the IGS1 sequences showed that the IAL 04 clustered with the other two T. faecale isolates (Figure 1).
PT
In conclusion, the study showed that the VITEK 2 system provides accurate
CE
results for T. asahii isolates but is not reliable to identify the non-T.asahii species. Correction of inconsistencies of the assimilation profiles analyses and expansion of its
AC
database are required. However the identification of closely related species may remain unsolved due to the similarity of their biochemical profiles. Thus, for the clinically relevant Trichosporon non-T. asahii the molecular method still remains the tool that best provides correct identification, and consequently contributes to the management of invasive infections caused by this emergent genus.
Acknowledgements We thank the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2010/20187-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support.
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7
REFERENCES Chagas-Neto TC, Chaves GM, Colombo AL. Update on the genus Trichosporon.
RI P
T
Mycopathologia. 2008;166(3):121-32.
Chagas-Neto TC, Chaves GM, Melo AS, Colombo AL. Bloodstream infections due to
SC
Trichosporon spp.: species distribution, Trichosporon asahii genotypes determined on
MA NU
the basis of ribosomal DNA intergenic spacer 1 sequencing, and antifungal susceptibility testing. J Clin Microbiol. 2009; 47(4):1074-81.
Colombo AL, Padovan AC, Chaves GM. Current knowledge of Trichosporon spp. and
ED
Trichosporonosis. Clin Microbiol Rev. 2011;24(4):682-700.
PT
Gunn SR, Reveles XT, Hamlington JD, Sadkowski LC, Johnson-Pais TL, Jorgensen JH.
CE
Use of DNA sequencing analysis to confirm fungemia due to Trichosporon dermatis in
AC
a pediatric patient. J Clin Microbiol. 2006;44(3):1175-7.
Guo LN, Xiao M, Kong F, Chen SC, Wang H, Sorrell TC, et al. Three-locus identification, genotyping, and antifungal susceptibilities of medically important Trichosporon species from China. J Clin Microbiol. 2011;49(11):3805-11.
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Legend of Figure 1.
Rooted phylogenetic tree based on IGS1 rDNA sequences of Trichosporon clinical isolates, four Trichosporon reference strains (GenBank) and the outgroup species
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1,000 bootstrap simulation.
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Cryptococcus gatti and C. neoformans (GenBank), obtained by UPGMA analysis and
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Figure 1
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Site of isolation (number of isolates)
Species identification by VITEK 2a
Species identification by sequence of IGS1 regionb
10 isolates
Blood (9) / Subcutaneous tissue (1)
T. asahii (≥91%) T. asahii CBS 2479 (100%)
DLC 02
Peritoneal fluid
T. asahii (97%)
T. inkin CNM-CL6516 (100%)
DLC 03
Blood
T. asahii (95%)
T. inkin CNM-CL6516 (100%)
DLC 06
Bone
DLC 10
T. inkin CNM-CL6516 (100%)
Peritoneal fluid
T. mucoides (98%)
T. dermatis CBS 2043 (100%)
DLC 11
Bone
T. asahii (97%)
T. inkin CNM-CL6516 (100%)
DLC 15
Blood
T. asahii (95%)
T. inkin CNM-CL6516 (99.6%)
DLC 16
Blood
T. asahii (99%)
T. inkin CNM-CL6516 (99.5%)
IAL 03
Blood
T. asahii (94%)
T. faecale CNM-CL4327 (100%)
IAL 04
Blood
Low discrimination
T. faecale CNM-CL6515 (97.7%)
51 isolates
Urine
T. asahii (≥91%)
T. asahii CBS 2479 (≥99.5%)
3B
Urine
Low discrimination
T. asahii CBS 2479 (100%)
8C
Catheter
T. asahii (94%)
T. inkin CNM-CL6099 (100%)
9E
Urine
T. asahii (97%)
T. inkin CNM-CL6516 (100%)
8G
Urine
T. asahii (97%)
T. faecale CBS 4828 (100%)
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T. asahii (97%)
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Isolates
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NO N- IN VASI VE
INVASIVE
Table 1. Trichosporon species identification by the VITEK 2 system and IGS1 sequencing analyses
CNM-CL = Yeast Collection Spanish National Center of Microbiology a (%) Probability values provided by the VITEK 2 database b (%) Identity with sequences available in GenBank and CBS (Centraal Bureau voor Schimmelcultures) databases
