Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer?

  • C.L. Halliday
    Affiliations
    Clinical Mycology Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, ICMPR – Pathology West, Westmead Hospital, NSW, Australia

    Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, NSW, Australia
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  • S.E. Kidd
    Affiliations
    National Mycology Reference Centre, SA Pathology, SA, Australia
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  • T.C. Sorrell
    Affiliations
    Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, NSW, Australia

    Marie-Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, NSW, Australia
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  • S.C.-A. Chen
    Correspondence
    Address for correspondence: Centre for Infectious Diseases, Microbiology Laboratory Services, ICPMR – Pathology West, Level 3, ICPMR Building, Westmead Hospital, Darcy Road, NSW 2145, Australia.
    Affiliations
    Clinical Mycology Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, ICMPR – Pathology West, Westmead Hospital, NSW, Australia

    Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, NSW, Australia
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      Summary

      Rapid, accurate diagnostic laboratory tests are needed to improve clinical outcomes of invasive fungal disease (IFD). Traditional direct microscopy, culture and histological techniques constitute the ‘gold standard’ against which newer tests are judged. Molecular diagnostic methods, whether broad-range or fungal-specific, have great potential to enhance sensitivity and speed of IFD diagnosis, but have varying specificities. The use of PCR-based assays, DNA sequencing, and other molecular methods including those incorporating proteomic approaches such as matrix-assisted laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) have shown promising results. These are used mainly to complement conventional methods since they require standardisation before widespread implementation can be recommended. None are incorporated into diagnostic criteria for defining IFD. Commercial assays may assist standardisation. This review provides an update of molecular-based diagnostic approaches applicable to biological specimens and fungal cultures in microbiology laboratories. We focus on the most common pathogens, Candida and Aspergillus, and the mucormycetes. The position of molecular-based approaches in the detection of azole and echinocandin antifungal resistance is also discussed.

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      References

        • Arvanitis M.
        • Anagnostou T.
        • Burgwyn Fuchs B.
        Molecular and non molecular diagnostic methods for invasive fungal infections.
        Clin Micro-biol Rev. 2014; 27: 490-526
        • Gómez B.L.
        Molecular diagnosis of endemic and invasive mycoses: advances and challenges.
        Rev Iberoam Micol. 2014; 31: 35-41
        • Chen S.C.A.
        • Halliday C.L.
        • Meyer W.
        A review of nucleic acid-based diagnostic tests for systemic mycoses with an emphasis on polymerase chain reaction-based assays.
        Med Mycol. 2002; 40: 333-357
        • White P.L.
        • Perry M.D.
        • Barnes R.A.
        An update on the molecular diagnosis of invasive fungal disease.
        FEMS Microbiol Lett. 2009; 296: 1-10
        • Khot P.D.
        • Fredericks D.N.
        PCR-based diagnosis of human fungal infections.
        Expert Rev Anti Infect Ther. 2009; 7: 1201-1221
        • Hammond S.P.
        • Bialek R.
        • Milner D.A.
        • et al.
        Molecular methods to improve diagnosis and identification of mucormycosis.
        J Clin Microbiol. 2011; 49: 2151-2153
        • Clinical and Laboaratory Standards Institute (CLSI)
        Interpretative criteria for identification of bacteria and fungi by DNA sequencing; approved guideline. CLSI document MM18-A. CLSI, Wayne, PA2008
        • Schoch C.L.
        • Seifart K.A.
        • Huhndorf S.
        • et al.
        Nuclear ribosomal internal transcribed spacer (ITS) region as a universal barcode marker for Fungi.
        PNAS. 2012; 109: 6241-6246
        • Balajee S.A.
        • Borman A.M.
        • Brandt M.E.
        Sequence-based identification of Aspergillus, Fusarium and Mucorales species in the clinical mycology laboratory: where are we and where should we go from here?.
        J Clin Microbiol. 2009; 47: 877-884
        • Lau A.
        • Chen A.
        • Sorrell T.
        Development and clinical application of a panfungal assay to detect and identify fungal in tissue specimens.
        J Clin Microbiol. 2007; 45: 380-385
        • Rickerts V.
        • Mousset S.
        • Lambrecht E.
        • et al.
        Comparison of histopathological analysis, culture and polymerase chain reaction assays to detect invasive mold infections from biopsy specimens.
        Clin Infect Dis. 2007; 44: 1078-1083
        • Landlinger C.
        • Preuner S.
        • Basková L.
        • et al.
        Diagnosis of invasive fungal infections by real-time panfungal assay in immunocompromised pediatric patients.
        Leukemia. 2010; 24: 2032-2038
        • Babouee B.
        • Goldenberger D.
        • Elzi L.
        • et al.
        Prospective study of a panfungal assay followed by sequencing, for detection of fungal in normally sterile specimens in a clinical setting: a complimentary tool in the diagnosis of invasive fungal disease.
        Clin Microbiol Infect. 2013; 19: E354-E357
        • Lass-Flörl C.
        • Mutschlechner W.
        • Aigner M.
        Utility of in diagnosis of invasive fungal infections: real-life data from a multicentre study.
        J Clin Microbiol. 2013; 51: 863-868
        • Sugawara Y.
        • Nakase K.
        • Nakumura A.
        • et al.
        Clinical utility of a panfungal polymerase chain reaction assay for invasive fungal diseases in patients with haematologic disorders.
        Eur J Haematol. 2013; 90: 331-339
        • Jordanides N.E.
        • Allan E.K.
        • McLintock L.A.
        • et al.
        A prospective study of real-time panfungal PCR for the early diagnosis of invasive fungal infection in haemato-oncology patients.
        Bone Marrow Transplant. 2005; 35: 389-395
        • Badiee P.
        • Kordbacheh P.
        • Alborzi A.
        • et al.
        Study on invasive fungal infections in immunocompromised patients to present a suitable early diagnostic procedure.
        Int J Infect Dis. 2009; 13: 97-102
        • Babady N.E.
        • Miranda E.
        • Gilhuley K.A.
        Evaluation of Luminex xTAG fungal analyte-specific reagents for rapid identification of clinically relevant fungi.
        J Clin Microbiol. 2011; 49: 3777-3782
        • Mancini N.
        • Clerici D.
        • Diotti R.
        • et al.
        Molecular diagnosis of sepsis in neutropenic patients with haematological malignancies.
        J Med Microbiol. 2008; 57: 601-604
        • von Lilienfeld-Toal M.
        • Lehmann L.E.
        • Raadts A.D.
        • et al.
        Utility of a commercially available multiplex real-time assay to detect bacterial and fungal pathogens in febrile neutropenia.
        J Clin Microbiol. 2009; 47: 2405-2410
        • Lamoth F.
        • Jaton K.
        • Prod’hom G.
        • et al.
        Multiplex blood in combination with blood cultures for improvement of the microbiological documentation of infection in febrile neutropenia.
        J Clin Microbiol. 2010; 48: 3510-3516
        • Clancy C.J.
        • Nguyen M.H.
        Finding the “missing 50%’” of invasive candidiasis: how nonculture diagnostics will improve understanding of disease spectrum and transform patient care.
        Clin Infect Dis. 2013; 56: 1284-1292
        • Pfeiffer C.D.
        • Samsa G.P.
        • Schell W.A.
        • et al.
        Quantitation of Candida CFU in initial positive blood cultures.
        J Clin Microbiol. 2011; 49: 2879-2883
        • Kourkoumpetis T.K.
        • Fuchs B.B.
        • Coleman J.J.
        • et al.
        Polymerase chain reaction-based assays for the diagnosis of invasive fungal infections.
        Clin Infect Dis. 2012; 54: 1322-1331
        • Fortún J.
        • Meije Y.
        • Buitrago M.J.
        • et al.
        Clinical validation of a multiplex real-time assay for detection of invasive candidiasis in intensive care unit patients.
        J Antimicrob Chemother. 2014; 69: 3134-3141
        • Avni T.
        • Leibovici L.
        • Paul M.
        PCR diagnosis of invasive candidiasis: systematic review and meta-analysis.
        J Clin Microbiol. 2011; 49 (): 665-670
        • Chen S.
        • Slavin M.
        • Nguyen Q.
        • et al.
        Active surveillance for candidemia, Australia.
        Emerg Infect Dis. 2006; 12: 1508-1516
        • Alam F.F.
        • Mustafa A.S.
        • Khan Z.U.
        Comparative evaluation of (1, 3)-ß-D-glucan, mannan and antimannan antibodies, and Candida species-specific snPCR in patients with candidemia.
        BMC Infect Dis. 2007; 7: 103
        • Ahmad S.
        • Khan Z.
        • Mustafa A.S.
        • et al.
        Seminested for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification.
        J Clin Microbiol. 2002; 40: 2483-2489
        • McMullan R.
        • Metwally L.
        • Coyle P.V.
        • et al.
        A prospective clinical trial of a real-time polymerase chain reaction assay for the diagnosis of candidemia in nonneutropenic, critically ill adults.
        Clin Infect Dis. 2008; 46: 890-896
        • Lau A.
        • Halliday C.
        • Chen S.C.A.
        • et al.
        Comparison of whole blood, serum, and plasma for early detection of candidemia by multiplex-tandem PCR.
        J Clin Microbiol. 2010; 48: 811-816
        • Nguyen M.H.
        • Wissel M.C.
        • Shields R.K.
        • et al.
        Performance of Candida realtime polymerase chain reaction, ß-D-glucan assay, and blood cultures in the diagnosis of invasive candidiasis.
        Clin Infect Dis. 2012; 54: 1240-1248
        • Metwally L.
        • Hogg G.
        • Coyle P.V.
        • et al.
        Rapid differentiation between fluconazole-sensitive and –resistant species of Candida directly from positive blood-culture bottles by real-time PCR.
        J Med Microbiol. 2007; 56: 964-970
        • Lau A.
        • Sorrell T.C.
        • Chen S.
        • et al.
        Multiplex-tandem PCR: a novel platform for rapid detection and identification of fungal pathogens from blood culture specimens.
        J Clin Microbiol. 2008; 46: 3021-3027
        • Metwally L.
        • Fairley D.J.
        • Coyle P.V.
        • et al.
        Comparison of serum and whole-blood specimens for the detection of Candida in critically ill, nonneu-tropenic patients.
        J Med Microbiol. 2008; 57: 1269-1272
        • Neely L.A.
        • Audeh M.
        • Phung N.A.
        • et al.
        T2 magnetic resonance enables nanoparticle-mediated rapid detection of candiemia in whole blood.
        Sci Transl Med. 2013; 5: 112ra54
        • Beyda N.D.
        • Alam M.J.
        • Garey K.W.
        Comparison of the T2Dx instrument with T2Candida assay and automated blood culture in the detection of Candida species using seeded blood samples.
        Diagn Microbiol Infect Dis. 2013; 77: 324-326
        • Halliday C.L.
        • Hoile R.
        • Sorrell T.
        • et al.
        Role of prospective screening of blood for invasive aspergillosis by polymerase chain reaction in febrile neutropenic recipients of haematopoietic stem cell transplants and patients with acute leukaemia.
        Br J Haematol. 2006; 132: 478-486
        • White P.L.
        • Linton C.J.
        • Perry M.D.
        • et al.
        The evolution and evaluation of whole blood polymerase chain reaction assay for the detection of invasive aspergillosis in hematology patients in a routine clinical setting.
        Clin Infect Dis. 2006; 42: 479-486
        • Mengoli C.
        • Cruciani M.
        • Barnes R.
        • et al.
        Use of for diagnosis of invasive aspergillosis: systematic review and meta-analysis.
        Lancet Infect Dis. 2009; 9: 89-96
        • Arvanitis M.
        • Ziakas P.
        • Zacharioudakis I.M.
        • et al.
        In Diagnosis of invasive aspergillosis: a meta-analysis of diagnostic performance.
        J Clin Microbiol. 2014; 52: 3731-3742
        • De Pauw B.
        • Walsh T.J.
        • Donnelly J.P.
        • et al.
        Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.
        Clin Infect Dis. 2008; 46: 1813-1821
        • White P.L.
        • Bretagne S.
        • Klingspor L.
        • et al.
        Aspergillus PCR: one step closer to standardization.
        J Clin Microbiol. 2010; 48: 1231-1240
        • White P.L.
        • Perry M.D.
        • Loeffler J.
        • et al.
        Critical stages of extracting from Aspergillus fumigatus in whole-blood specimens.
        J Clin Microbiol. 2010; 48: 3753-3755
        • Bernal-Martinez L.
        • Gago S.
        • Buitrago M.J.
        • et al.
        Analysis of performance of a PCR-based assay to detect of Aspergillus fumigatus in whole blood and serum: a comparative study with clinical samples.
        J Clin Microbiol. 2011; 49: 3596-3599
        • Springer J.
        • Morton C.O.
        • Perry M.
        • et al.
        Multicenter comparison of serum and whole-blood specimens for detection of Aspergillus DNA in high-risk haematological patients.
        J Clin Microbio. 2013; 51: 1445-1450
        • Suarez F.
        • Lortholary O.
        • Buland S.
        • et al.
        Detection of circulating Aspergillus fumigatus by real-time assay of large volumes improves early diagnosis of invasive aspergillosis in high-risk adult patients under hematologic surveillance.
        J Clin Microbiol. 2008; 46: 3772-3777
        • White P.L.
        • Mengoli C.
        • Bretagne S.
        • et al.
        Evaluation of Aspergillus protocols for testing serum specimens.
        J Clin Microbiol. 2011; 49: 3842-3848
        • Sun W.
        • Wang K.
        • Gao W.
        • et al.
        Evaluation of on bronchoalveolar lavage fluid for diagnosis of invasive aspergillosis: a bivariate meta-analysis and systematic review.
        PLoS One. 2011; 6: e28467
        • Heng S.C.
        • Chen S.C.A.
        • Morrissey C.O.
        • et al.
        Clinical utility of Aspergillus galactomannan and PCR in broncoalveolar lavage fluid for the diagnosis of invasive pulmonary aspergillosis in patients with haematological malignancies.
        Diagn Microbiol Infect Dis. 2014; 79: 322-327
        • White P.L.
        • Perry M.D.
        • Moody A.
        • et al.
        Evaluation of analytical and preliminary clinical performance of Myconostica MycAssay Aspergillus when testing serum specimens for diagnosis of invasive aspergillosis.
        J Clin Microbiol. 2011; 49: 2169-2174
        • Torelli R.
        • Sanguinetti M.
        • Moody A.
        • et al.
        Diagnosis of invasive aspergillosis by a commercial real-time assay for Aspergillus in bronchoalveolar lavage fluid samples from high-risk patients compared to a galactomannan enzyme immunoassay.
        J Clin Microbiol. 2011; 49: 4273-4278
        • Danylo A.
        • Courtemanche C.
        • Pelletier R.
        • et al.
        Performance of MycAssay Aspergillus real-time assay compared with the galactomannan detection assay for the diagnosis of invasive aspergillosis from serum samples.
        Med Mycol. 2014; 52: 577-583
        • Millon L.
        • Larosa F.
        • Lepiller Q.
        • et al.
        Quantitative polymerase chain reaction detection of circulating in serum for early diagnosis of mycormycosis in immunocompromised patients.
        Clin Infect Dis. 2013; 56: e95-e101
        • Almyroudis N.G.
        • Sutton D.A.
        • Fothergill A.W.
        • et al.
        In vitro susceptibilities of 217 clinical isolates of zygomycetes to conventional and new antifungal agents.
        Antimicrob Agents Chemother. 2007; 51: 2587-2590
        • Lengerova M.
        • Racil Z.
        • Hrncirova K.
        • et al.
        Rapid detection and identification of mucormycetes in bronchoalveolar lavage samples from immuno-compromised patients with pulmonary infiltrates by use of high-resolution melt analysis.
        J Clin Microbiol. 2014; 52: 2824-2828
        • Bialek R.
        • Konrad F.
        • Kern J.
        • et al.
        Based identification and discrimination of agents of mucormycosis and aspergillosis in paraffin wax embedded tissue.
        J Clin Pathol. 2005; 58: 1180-1184
        • Cornerly O.A.
        • Arikan-Akdagli S.
        • Dannaoui E.
        • et al.
        ESCMID and ECMM joint clinical guidelines for the diagnosis and management of mucormycosis 2013.
        Clin Microbiol Infect. 2014; 20: 5-25
        • Kasai M.
        • Harrington S.M.
        • Francesconi A.
        • et al.
        Detection of a molecular biomarker for zygomycetes by quantitative assays of plasma, bronchoalveolar lavage, and lung tissue ina rabbit model of experimental pulmonary zygomycosis.
        J Clin Microbiol. 2008; 46: 3690-3702
        • Shendure J.
        • Ji H.
        Next-generation sequencing.
        Nat Biotechnol. 2008; 26: 1135-1145
        • Kircher M.
        • Kelson J.
        High-throughout sequencing- concepts and limitations.
        Bioessays. 2010; 32: 524-536
        • Vitale R.G.
        • de Hoog G.S.
        • Schwarz P.
        • et al.
        Antifungal susceptibility and phylogeny of opportunistic members of the order Mucorales.
        J Clin Microbiol. 2012; 50: 66-75
        • Walther G.
        • Pawłowska J.
        • Alastruey-Izquierdo A.
        • et al.
        Barcoding in Mucorales: an inventory of biodiversity.
        Persoonia. 2013; 30: 11-47
        • Ciardo D.E.
        • Schär G.
        • Böttger E.C.
        • et al.
        Internal transcribed spacer sequencing versus biochemical profiling for identification of medically important yeasts.
        J Clin Microbiol. 2006; 44: 77-84
        • Huang C.H.
        • Lee F.L.
        • Tai C.J.
        The beta-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the Saccharomyces sensu stricto complex.
        Antonie Van Leeuwenhoek. 2009; 95: 135-142
        • Aa E.
        • Townsend J.P.
        • Adams R.I.
        • et al.
        Population structure and gene evolution in Saccharomyces cerevisiae.
        FEMS Yeast Res. 2006; 6: 702-715
        • Putignani L.
        • Paglia M.G.
        • Bordi E.
        • et al.
        Identification of clinically relevant yeast species by sequence analysis of the D2 variable region of the 25-28S rRNA gene.
        Mycoses. 2008; 51: 209-227
        • Alper I.
        • Frenette M.
        • Labrie S.
        Ribosomal polymorphisms in the yeast Geotrichum candidum.
        Fungal Biol. 2011; 115: 1259-1269
        • Katsu M.
        • Kidd Ando S.A.
        • et al.
        The Internal Transcribed Spacers and 5.
        8S rRNA gene show extensive diversity among isolates of the Cryptococcus neoformans complex. FEMS Yeast Res. 2004; 4: 377-388
        • Arabatzis M.
        • Abel P.
        • Kanellopoulou M.
        • et al.
        Sequence-based identification, genotyping and EUCAST antifungal susceptibilities of Trichosporon clinical isolates from Greece.
        Clin Microbiol Infect. 2014; 20: 777-783
        • Balajee S.A.
        • Houbraken J.
        • Verweij P.E.
        • et al.
        Aspergillus species identification in the clinical setting.
        Stud Mycol. 2007; 59: 39-46
        • O’Donnell K.
        • Sutton D.A.
        • Rinaldi M.G.
        • et al.
        Internet-accessible sequence database for identifying fusaria from human and animal infections.
        J Clin Microbiol. 2010; 48: 3708-3718
        • Barker A.P.
        • Horan J.L.
        • Slechta E.S.
        • et al.
        Complexities associated with the molecular and proteomic identification of Paecilomyces species in the clinical mycology laboratory.
        Med Mycol. 2014; 52: 537-545
        • Wang L.
        • Yokoyama K.
        • Miyaji M.
        • Nishimura K.
        Mitochondrial cytochrome b gene analysis of Aspergillus fumigatus and related species.
        J Clin Microbiol. 2000; 38: 1352-1358
        • LoBuglio K.F.
        • Taylor J.W.
        Phylogeny and identification of the human pathogenic fungus Penicillium marneffei.
        J Clin Microbiol. 1995; 33: 85-89
        • Houbraken J.
        • Samson R.A.
        Phylogeny of Penicillium and the segregation of Trichocomaceae into three families.
        Stud Mycol. 2011; 70: 1-51
        • Peterson S.W.
        Multilocus sequence analysis of Penicillium and Eupenicillium species.
        Rev Iberoam Micol. 2006; 23 (): 134-138
        • Serra R.
        • Peterson S.
        CTCOR, Venâncio A. Multilocus sequence identification of Penicillium species in cork bark during plank preparation for the manufacture of stoppers.
        Res Microbiol. 2008; 159: 178-186
        • Peterson S.W.
        Aspergillus and Penicillium identification using sequences: barcode or MLST?.
        Appl Microbiol Biotechnol. 2012; 95: 339-344
        • Chen S.C.A.
        • Ellis D.
        • Sorrell T.C.
        • Meyer W.
        Molecular Detection of Human Pathogens. 1st. CRC Press, New York2011 (Chapter 44, Trichophyton)
        • Gräser Y.
        • Scott J.
        • Summerbell R.
        The new species concept in dermatophytes - a polyphasic approach.
        Mycopathologia. 2008; 166: 239-256
        • Rezaei-Matehkolaei A.
        • Mirhendi H.
        • Makimura K.
        • et al.
        Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes.
        Med Mycol. 2014; 52: 674-688
        • Lawrence D.P.
        • Gannibal P.B.
        • Peever T.L.
        • Pryor B.M.
        The sections of Alternaria: formalising species-group concepts.
        Mycologia. 2013; 105: 530-546
        • de Hoog G.S.
        • Horré R.
        Molecular taxonomy of the Alternaria and Ulocladium species from humans and their identification in the routine laboratory.
        Mycoses. 2002; 45: 259-276
        • Abliz P.
        • Fukushima K.
        • Takizawa K.
        • Nishimura K.
        Identification of pathogenic dematiaceous fungi and related taxa based on large subunit ribosomal D1/D2 domain sequence analysis.
        FEMS Immunol Med Microbiol. 2004; 40: 41-49
        • Aroca A.
        • Raposo R.
        • Lunello P.
        A biomarker for the identification of four Phaeoacremonium species using the beta-tubulin gene as the target sequence.
        Appl Microbiol Biotechnol. 2008; 80: 1131-1140
        • Lackner M.
        • de Hoog G.S.
        Parascedosporium and its relatives: phylogeny and ecological trends.
        IMA Fungus. 2011; 2: 39-48
        • Schwarz P.
        • Bretagne S.
        • Gantier J.C.
        • et al.
        Molecular identification of zygomycetes from culture and experimentally infected tissues.
        J Clin Microbiol. 2006; 44: 340-349
        • Lechevalier P.
        • Garcia-Hermoso D.
        • Carol A.
        • et al.
        Molecular diagnosis of Saksenaea vasiformis cutaneous infection after scorpion sting in an immunocompetent adolescent.
        J Clin Microbiol. 2008; 46: 3169-3172
        • Chowdhary A.
        • Kathuria S.
        • Singh P.K.
        • et al.
        Molecular characterization and in vitro antifungal susceptibility of 80 clinical isolates of Mucormycetes in Delhi.
        India Mycoses. 2014; 57: 97-107
        • Nyilasi I.
        • Papp T.
        • Csernetics A.
        • et al.
        High-affinityiron permease (FTR1) gene sequence-based molecular identification of clinicallyimportant Zygomycetes.
        Clin Microbiol Infect. 2008; 14: 393-397
        • Tintelnot K.
        • De Hoog G.S.
        • Antweiler E.
        • et al.
        Taxonomic and diagnostic markers for identification of Coccidioides immitis and Coccidioides posadasii.
        Med Mycol. 2007; 45: 385-393
        • Millar B.C.
        • Jiru X.
        • Walker M.J.
        • et al.
        False identification of Coccidioides immitis: do molecular methods always get it right?.
        J Clin Microbiol. 2003; 41: 5778-5780
        • Bialek R.
        • Kern J.
        • Herrmann T.
        • et al.
        Assays for identification of Coccidioides posadasii based on the nucleotide sequence of the antigen 2/proline-rich antigen.
        J Clin Microbiol. 2004; 42: 778-783
        • Komori T.
        • Sano A.
        • Yarita K.
        • et al.
        Phylogenetic analysis of Histoplasma capsulatum based on partial sequence of the D1/D2 region of the 28S rRNA gene.
        Nihon Ishinkin Gakkai Zasshi. 2005; 46: 291-295
        • Zhou X.
        • Rodrigues A.M.
        • Feng P.
        • Hoog G.S.
        Global ITS diversity in the Sporothrix schenckii complex.
        Fungal Divers. 2014; 66: 153-165
        • Woo P.C.
        • Leung S.Y.
        • To K.K.
        • et al.
        Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus: implications for molecular diagnosis in clinical microbiology laboratories.
        J Clin Microbiol. 2010; 48: 208-214
        • O’Donnell K.
        • Cigelnik E.
        • Nirenberg H.
        Molecular systematics and phylogeography of the Gibberella fujikuroi species complex.
        Mycologia. 1998; 90: 465-493
        • Gilgado F.
        • Cano J.
        • Gené J.
        • Guarro J.
        Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species.
        J Clin Microbiol. 2005; 43: 4930-4942
        • Borman A.M.
        • Linton C.J.
        • Oliver D.
        • et al.
        Rapid molecular identification of pathogenic yeasts by pyrosequencing analysis of 35 nucleotides of internal transcribed spacer 2.
        J Clin Microbiol. 2010; 48: 3648-3653
        • Heinrichs G.
        • de Hoog G.S.
        • Haase G.
        Barcode identifiers as a practical tool for reliable species assignment of medically important black yeast species.
        J Clin Microbiol. 2012; 50: 3023-3030
        • Nilsson R.H.
        • Ryberg M.
        • Kristiansson E.
        • et al.
        Taxonomic reliability of sequences in public sequence databases: A fungal perspective.
        PLoS One. 2006; 1: e59
        • Bridge P.D.
        • Roberts P.J.
        • Spooner B.M.
        • Panchal G.
        On the unreliability of published sequences.
        New Phytol. 2003; 160: 43-48
        • White T.J.
        • Bruns T.
        • Lee S.
        • Taylor J.
        PCR Protocols: A Guide to Methods and Applications. 1st. Academic Press, San Diego1990 (Chapter 38, Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics)
        • Vilgalys R.
        • Hester M.
        Rapid genetic identification and mapping of enzymatically amplified ribosomal from several Cryptococcus species.
        J Bacteriol. 1990; 172: 4238-4246
        • Gerrits van den Ende A.G.
        • de Hoog G.S.
        Variability and molecular diagnostics of the neurotropic species Cladophialophora bantiana.
        Stud Mycol. 1999; 43: 151-162
        • Gardes M.
        • Bruns T.D.
        ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts.
        Mol Ecol. 1993; 2: 113-118
        • Martin K.J.
        • Rygiewicz P.T.
        Fungal-specific primers developed for analysis of the ITS region of environmental extracts.
        BMC Microbiol. 2005; 5: 28
      1. Hanner, R. Data Standards for BARCODE Records in INSDC (BRIs). BARCODE Data Standards v.2.4. 28 February 2012. http://www.barcodeoflife.org/sites/default/files/DWG%20standards%20revision%202.4%20-%2029%20Feb%2012.pdf.

        • Ciardo D.E.
        • Schär G.
        • Altwegg M.
        • et al.
        Identification of moulds in the diagnostic laboratory—an algorithm implementing molecular and phenotypic methods.
        Diagn Microbiol Infect Dis. 2007; 59: 49-60
        • Meyer W.
        • Maszewska K.
        • Sorrell T.C.
        PCR fingerprinting: a convenient molecular tool to distinguish between Candida dubliniensis and Candida albicans.
        Med Mycol. 2001; 39: 185-193
        • Cadez N.
        • Raspor P.
        • de Cock A.W.
        • et al.
        Molecular identification and genetic diversity within species of the genera Hanseniaspora and Kloeckera.
        FEMS Yeast Res. 2002; 1: 279-289
        • Sakai K.
        • Trabasso P.
        • Moretti M.L.
        • et al.
        Identification of fungal pathogens by visible microarray system in combination with isothermal gene amplification.
        Mycopathologia. 2014; 178: 11-26
        • Lau A.
        • Sorrell T.C.
        • Lee O.
        • et al.
        Colony multiplex-tandem for rapid, accurate identification of fungal cultures.
        J Clin Microbiol. 2008; 46: 4058-4060
        • Denning D.W.
        • Perlin D.S.
        Azole resistance in Aspergillus: a growing public health menace.
        Future Microbiol. 2011; 6: 1229-1232
        • Vermeulen E.
        • Lagrou K.
        • Verweij P.E.
        Azole resistance in Aspergillus fumigatus: a growing public health concern.
        Curr Opin Infect Dis. 2013; 26: 493-500
        • Perlin D.S.
        Echinocandin resistance, susceptibility testing and prophylaxis: implications for patient management.
        Drugs. 2014; 74: 1573-1585
        • Pfaller M.A.
        • Messer S.A.
        • Woosley L.N.
        • et al.
        Echinocandin and triazole antifungal susceptibility profiles for clinical opportunistic yeast and mold isolates collected from 2010 to 2011: application of new CLSI clinical breakpoints and epidemiological cut-off values for characterization of geographic and temporal trends of antifungal resistance.
        J Clin Microbiol. 2013; 5: 2571-2578
        • Castanheira M.
        • Messer S.A.
        • Jones R.N.
        • et al.
        Activity of echinocandins and triazoles against a contemporary (2012) worldwide collection of yeast and moulds collected from invasive infections.
        Int J Antimicrob Agents. 2014; 44: 320-326
        • Park S.
        • Perlin D.S.
        Establishing surrogate markers for fluconazole resistance in Candida albicans.
        Microb Drug Resist. 2005; 11: 232-238
        • Sanglard D.
        • Bille J.
        Candida and Candidiasis. 1st. ASM Press, Washington DC2002 (Chapter 25, Current understanding of the mode of action and of resistance mechanisms to conventional and emerging antifungal agents for treatment of Candida infections.)
        • Xiang M.J.
        • Liu J.Y.
        • Ni P.H.
        • et al.
        ERG11 mutations associated with azole resistance in clinical isolates of Candida albicans.
        FEMS Yeast Res. 2013; 13: 386-393
        • Wang H.
        • Kong F.
        • Sorrell T.C.
        • et al.
        Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and sequencing.
        BMC Microbiol. 2009; 9: 167
        • Frade J.P.
        • Warnock D.W.
        • Arthington-Skaggs B.A.
        Rapid quantification of drug resistance gene expression in Candida albicans by reverse transcriptase LightCycler and fluorescent probe hybridization.
        J Clin Microbiol. 2004; 42: 2085-2093
        • Alexander B.D.
        • Johnson M.D.
        • Pfeiffer C.D.
        • et al.
        Increasing echinocandin resistance in Candida glabrata: clinical failure correlates with presence of FKS mutations and elevated minimum inhibitory concentrations.
        Clin Infect Dis. 2013; 56: 1724-1732
        • Lackner M.
        • Tscherner M.
        • Schaller M.
        • et al.
        Positions and numbers of FKS mutations in Candida albicans selectively influence in vitro and in vivo susceptibilities to echinocandin treatment.
        Antimicrob Agents Chemother. 2014; 58: 3626-3635
        • Jensen R.H.
        • Justesen U.S.
        • Rewes A.
        • et al.
        Echinocandin failure case due to a previously unreported FKS1 mutation in Candida krusei.
        Antimicrob Agents Chemother. 2014; 58: 3550-3552
        • Fekkar A.
        • Dannaoui E.
        • Meyer I.
        • et al.
        Emergence of echinocandinresistant Candida spp.
        in a hospital setting: a consequence of 10 years of increasing use of antifungal therapy? Eur J Clin Microbiol Infect Dis. 2014; 33: 1489-1496
        • Dudiuk C.
        • Gamarra S.
        • Leonardeli F.
        • et al.
        Set of classical PCRs for detection of mutations in Candida glabrata FKS genes linked with echinocandin resistance.
        J Clin Microbiol. 2014; 52: 2609-2614
        • Park S.
        • Kelly R.
        • Kahn J.N.
        • et al.
        Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates..
        Antimicrob Agents Chemother. 2005; 49 (): 3264-3273
        • Garcia-Effron G.
        • Kontoyiannis D.P.
        • Lewis R.E.
        • Perlin D.S.
        Caspofungin-resistant Candida tropicalis strains causing breakthrough fungemia in patients at high risk for hematologic malignancies.
        Antimicrob Agents Chemother. 2008; 52: 4181-4183
        • Staab J.F.
        • Neofytos D.
        • Rhee P.
        • et al.
        Target enzyme mutations confer differential echinocandin susceptibilities in Candida kefyr.
        Antimicrob Agents Chemother. 2014; 58: 5421-5427
        • Shields R.K.
        • Nguyen M.H.
        • Press E.G.
        • et al.
        The presence of an FKS mutation rather than MIC is an independent risk factor for failure of echinocandin therapy among patients with invasive candidiasis due to Candida glabrata.
        Antimicrob Agents Chemother. 2012; 56: 4862-4869
        • Pfaller M.A.
        • Diekema D.J.
        • Boyken L.
        • et al.
        Effectiveness of anidulafungin in eradicating Candida species in invasive candidiasis.
        Antimicrob Agents Chemother. 2005; 49: 4795-4797
        • Pham C.D.
        • Bolden C.B.
        • Kuykendall R.J.
        • Lockhart S.R.
        Development of a Luminex-based multiplex assay for detection of mutations conferring resistance to echinocandins in Candida glabrata.
        J Clin Microbiol. 2014; 52: 790-795
        • Clinical and Laboratory Standards Institute (CLSI)
        Reference method for broth dilution antifungal susceptibility testing of yeasts; fourth informational supplement. CLSI document M27-S4. CLSI, Wayne. PA2012
        • Cuenca-Estrella M.
        Antifungal drug resistance mechanisms in pathogenic fungi: from bench to bedside.
        Clin Microbiol Infect. 2014; 20: 54-59
        • Howard S.J.
        • Cerar D.
        • Anderson M.J.
        • et al.
        Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure.
        Emerg Infect Dis. 2009; 15: 1068-1076
        • Escribano P.
        • Peláez T.
        • Muñoz P.
        • et al.
        Is azole resistance in Aspergillus fumigatus a problem in Spain?.
        Antimicrob Agents Chemother. 2013; 57: 2815-2820
        • Vermeulen E.
        • Maertens J.
        • Schoemans H.
        • Lagrou K.
        Azole-resistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation emerging in Belgium, July 2012.
        Euro Surveill. 2012; 17: 20326
        • Van Der Linden J.W.
        • Warris A.
        • Verweij P.E.
        Aspergillus species intrinsically resistant to antifungal agents.
        Med Mycol. 2011; 49: S82-S90
        • Ahmad S.
        • Khan Z.
        • Hagen F.
        • Meis J.F.
        Occurrence of triazole-resistant Aspergillus fumigatus with TR34/L98H mutations in outdoor and hospital environment in Kuwait.
        Environ Res. 2014; 133: 20-26
        • Mellado E.
        • Garcia-Effron G.
        • Alcázar-Fuoli L.
        • et al.
        A new Aspergillus fumigatus resistance mechanism conferring in vitro cross-resistance to azole antifungals involves a combination of cyp51A alterations.
        Antimicrob Agents Chemother. 2007; 51: 1897-1904
        • van der Linden J.W.
        • Snelders E.
        • Arends J.P.
        • et al.
        Rapid diagnosis of azoleresistant aspergillosis by direct using tissue specimens.
        J Clin Microbiol. 2010; 48: 1478-1480
        • Denning D.W.
        • Park S.
        • Lass-Florl C.
        • et al.
        High-frequency triazole resistance found in nonculturable Aspergillus fumigatus from lungs of patients with chronic fungal disease.
        Clin Infect Dis. 2011; 52: 1123-1129
        • Spiess B.
        • Seifarth W.
        • Merker N.
        • et al.
        Development of novel assays to detect azole resistance-mediating mutations of the Aspergillus fumigatus cyp51A gene in primary clinical samples from neutropenic patients.
        Antimicrob Agents Chemother. 2012; 56: 3905-3910
        • Vermeulen E.
        • Maertens J.
        • Schoemans H.
        • Lagrou K.
        Azole-resistant Aspergillus fumigatus due to TR46/Y121F/T289A mutation emerging in Belgium, July 2012.
        Euro Surveill. 2012; 17: 20326
        • Lescar J.
        • Meyer I.
        • Akshita K.
        • et al.
        Aspergillus fumigatus harbouring the sole Y121F mutation shows decreased susceptibility to voriconazole but maintained susceptibility to itraconazole and posaconazole.
        J Antimicrob Chemother. 2014; 69: 3244-3247
        • Ahmad S.
        • Khan Z.
        • Hagen F.
        • Meis J.F.
        Simple, low-cost molecular assays for TR34/L98H mutations in the cyp51A gene for rapid detection of triazole-resistant Aspergillus fumigatus isolates.
        J Clin Microbiol. 2014; 52: 2223-2227
        • Spiess B.
        • Postina P.
        • Reinwald M.
        • et al.
        Incidence of cyp51A key mutations in Aspergillus fumigatus - a study on primary clinical samples of immunocompromised patients in the period of 1995-2013.
        PLoS One. 2014; 9: e103113
        • Gabriel S.
        • Ziaugra L.
        • Tabbaa D.
        SNP genotyping using the Sequenom MassARRAY iPLEX platform.
        Curr Protoc Hum Genet. 2009; 12 (Chapter 2: Unit 2.)
        • Kok J.
        • Chen SC-A
        • Dwyer D.E.
        • Iredell J.R.
        Current status of matrix-assisted laser desorption ionisation-time of flight mass spectrometry in the clinical microbiology laboratory.
        Pathology. 2013; 45: 4-17
        • Borman A.M.
        • Johnson E.M.
        Genomics and proteomics as compared to conventional phenotypic approaches for the identification of the agents of invasive fungal infection.
        Curr Fungal Infect Rep. 2013; 7: 235-243
        • Clark A.E.
        • Kaleta E.J.
        • Arora A.
        • Wolk D.M.
        Matrix-assisted laser desorption ionisation-time of flight mass spectrometry: a fundamental shift in the routine practice of clinical microbiology.
        Clin Microbiol Rev. 2013; 26: 547-603
        • Firacative C.
        • Trilles L.
        • Meyer W.
        MALDI-TOF enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex..
        PLoS One. 2012; 7: e37566
        • Lau A.F.
        • Drake S.K.
        • Calhoun L.B.
        • et al.
        Development of a clinically comprehensive database and a simple procedure for identification of molds from solid media by matrix-assisted laser desorption ionizationtime of flight mass spectrometry.
        J Clin Microbiol. 2013; 51: 828-834
        • Hettick J.M.
        • Green B.J.
        • Buskirk A.D.
        • et al.
        Discrimination of Aspergillus isolates at the species and strain level by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry fingerprinting.
        Anal Biochem. 2008; 380: 276-281
        • De Carolis E.
        • Vella A.
        • Florio A.R.
        • et al.
        Use of matrix-assisted laser desorption ionization-time of flight mass spectrometry for caspofungin susceptibility testing of Candida and Aspergillus species.
        J Clin Microbiol. 2012; 50: 2479-2483
        • Vella A.
        • De CArolis E.
        • Vaccaro L.
        • et al.
        Rapid antifungal susceptibility testing by matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis.
        J Clin Microbiol. 2013; 51: 2964-2969
        • Gu Z.
        • Hall T.A.
        • Frinder M.
        • et al.
        Evaluation of repetitive sequence and PCR-mass spectrometry for the identification of clinically relevant Candida species.
        Med Mycol. 2012; 50: 259-265
        • Massire C.
        • Buelow D.R.
        • Zhang S.X.
        • et al.
        Followed by electrospray ionization mass spectrometry for broad-range identification of fungal pathogens.
        J Clin Microbiol. 2013; 51: 959-966
        • Shin J.H.
        • Ranken R.
        • Sefers S.E.
        • et al.
        Detection, identification and distribution of fungi in bronchoalveolar lavage specimens by use of multilocus coupled with electrospray ionization/mass spectrometry.
        J Clin Microbiol. 2013; 51: 136-141
        • Faulds K.
        • Smith W.E.
        • Graham D.
        DNA detection by surface enhanced resonance Raman scattering.
        Analyst. 2005; 130: 1125-1131
        • White P.L.
        • Hibbitts S.J.
        • Perry M.D.
        • et al.
        Evaluation of a commercially developed semiautomated PCR-SERS assay for the diagnosis of invasive fungal disease.
        J Clin Microbiol. 2014; 52: 3536-3543