Pathology (April 2015) 47(3), pp. 257–269

MOLECULAR DIAGNOSTICS IN MICROBIOLOGY

Molecular diagnostic methods for invasive fungal disease: the horizon draws nearer? C. L. HALLIDAY1,2, S. E. KIDD3, T. C. SORRELL2,4

AND

S. C-A. CHEN1,2

1Clinical Mycology Reference Laboratory, Centre for Infectious Diseases and Microbiology Laboratory Services, ICMPR – Pathology West, Westmead Hospital, NSW, 2Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, NSW, 3National Mycology Reference Centre, SA Pathology, SA, and 4Marie-Bashir Institute

for Infectious Diseases and Biosecurity, University of Sydney, NSW, Australia

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 molecularbased approaches in the detection of azole and echinocandin antifungal resistance is also discussed. Key words: DNA sequencing, fungal infections, molecular diagnosis, PCR. Received 11 November 2014, revised 7 January, accepted 22 January 2015

INTRODUCTION Invasive fungal disease (IFD) causes significant morbidity and mortality in hospitalised patients despite advances in antifungal therapies. Early diagnosis, which necessarily includes species identification, is essential for improving patient outcomes, but standard histological and culture methods are slow and insensitive.1,2 To overcome these limitations, rapid culture-independent molecular (and serological) tests, which are also noninvasive, are increasingly used. This article summarises recent advances in molecular methods, in the context of the clinical mycology laboratory, for the detection and identification of fungal pathogens in (i) clinical specimens, and (ii) fungal cultures with emphasis on invasive candidiasis (IC), invasive aspergillosis (IA) and mucormycosis. Diagnosis of Pneumocystis jirovecii is not discussed. We also review the molecular approaches in Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0000000000000234

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detection of resistance to antifungal agents since these, and their applicability as potential alternatives to phenotypic methods of resistance detection, have become increasingly important. More recent applications of matrix-associated laser desorption ionisation-time of flight mass spectroscopy (MALDI-TOF MS) that complement molecular-based diagnostics are also briefly discussed.

GENERAL CONSIDERATIONS Parameters influencing selection of a molecular test and its utility include: (i) the local epidemiology of fungal pathogens; (ii) body site(s) of infection; (iii) appropriate specimen selection; (iv) method used to isolate and concentrate fungal DNA from different clinical specimens; (v) selection of the fungal target gene; and (vi) the amplification and detection method.2,3 Regardless of the efficiency of a polymerase chain reaction (PCR) assay and the platform employed, its overall performance will be limited by the effectiveness of DNA extraction, which in turn is influenced by the specimen tested.4 Here we discuss only the laboratory-related issues of importance. PCR and other molecular tests are used to either (i) screen for a particular IFD, i.e. to pre-emptively diagnose this IFD in highrisk patient groups, or (ii) enable a definite diagnosis where an IFD is clinically evident. The selection of specimen to be tested, frequency of testing and result interpretation necessarily depends on the indication, as above, for testing. In both settings, PCR is often used to complement culture methods but may also represent the primary diagnostic approach. Specimens Determining the most appropriate specimen for PCR testing is usually straightforward but will depend on whether the assay is to be utilised for screening high-risk patients for IFD, or as a diagnostic test per se. Blood specimens (whole blood, plasma, serum) are easy to obtain and are the most widely used for screening for infection. The choice of blood fraction determines whether free circulating DNA is targeted during extraction, as for serum/plasma, or if cell-associated DNA is targeted as for whole blood.4 Where DNA is predominantly cell-associated, large volumes (>3 mL) of whole blood should be centrifuged to obtain sufficiently concentrated DNA.5 In the setting of pulmonary pathology, testing respiratory tract specimens other than lung tissue is often performed. Since these specimens are non-sterile, both pathogens and commensal fungi will be detected resulting in higher ‘clinical’ false positives.5 Bronchoalveolar lavage (BAL)

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as well as the more common pathogens, Aspergillus and Candida species. Ideally, the PCR target will be: (i) of a suitable product size (
500 bp) to allow ease of sequencing; (ii) long enough to provide sufficient species-specific discriminatory information; and (iii) available in a public sequence database.2 Identification of the PCR product is usually achieved by DNA sequencing or by the inclusion of genus- or speciesspecific probes and melting curve analysis.10–15 Sequencing is time consuming but comprehensive, whilst assays based on probe design are targeted at specific pathogens and therefore fail to identify other fungi. Lau et al.10 developed a panfungal PCR assay targeting the ITS1 region of the rDNA gene cluster followed by sequencing to detect and identify fungal DNA in fresh and paraffin embedded tissue specimens from patients with culture- or histologically-proven IFDs. The assay identified a diverse range of fungi and was successfully applied to other specimen types including vitreous fluid and cerebrospinal fluid (CSF). In our hands, the panfungal PCR approach is complementary to culture and, as reported by others, most valuable for identifying fungi in culture negative, histologically-proven infection, where species identification helps guide antifungal therapy.10,11,13,14 However, in our experience, the clinical utility of applying broad-range PCR to non-sterile samples, including BAL fluid, is poor due to amplification of commensal fungi, e.g., Candida species. We recently reviewed panfungal PCR results from 136 BAL fluid/washing specimens; 48% (n ¼ 65) tested positive by PCR, however all but two of the organisms identified were not considered to be clinically significant [Candida species (n ¼ 35), non-Candida yeast (n ¼ 7), saprophytic moulds (n ¼ 6), mixed fungi (n ¼ 15)] (Halliday et al., unpublished). Panfungal PCR assays have also been used to detect fungi in the blood of high-risk patients,12,15–17 although the majority of the studies targeted patients with suspected IA and IC. Sugawara et al.15 used a 18S rDNA-targeted panfungal PCR assay to prospectively screen blood for IFD (n ¼ 64 at risk episodes). They reported 44.4% of fungi detected were neither Aspergillus or Candida species, and in those cases PCR provided valuable information for selecting suitable therapies. The greatest drawback of panfungal PCR assays is exogenous contamination of specimens and/or PCR master mixes by environmental fungal spores. To minimise contamination, it is essential that laboratory staff follow strict precautions

fluid is an example of a common specimen that is tested but it is not suitable for high-frequency screening.4 Conversely, the detection of fungal DNA from normally sterile tissue and other samples is diagnostic of IFD. This is particularly helpful in the diagnosis of mucormycosis where the aetiological agent may not be cultured.6 Fresh tissue is preferred to paraffin embedded specimens.3 Gene target Ideally the fungal gene target to be amplified should be: (i) present in multiple copies to maximise PCR sensitivity, and (ii) sufficiently conserved to allow amplification of target fungi, but with adequate sequence variation to define a particular genus or species. The majority of fungal PCR-based assays target one or more regions of the multi-copy (50–100 copies in the haploid genome) ribosomal DNA (rDNA) gene cluster comprising the 18S, 5.8S and 28S genes and the intervening internal transcribed spacer (ITS) regions, ITS1 and ITS2 (Fig. 1).7 These regions contain both highly conserved and variable regions, allowing the design of universal primers within the conserved regions to amplify DNA from a large number of fungal species. At the same time, genus or species specific primers/probes can be designed based on the variable gene regions.3 Since the ITS region is the most variable, it is the most likely to enable species identification. As such, it has been proposed as the primary fungal barcode marker by the Consortium for the Barcode of Life8 (see later). Other useful multicopy targets include mitochondrial cytochrome (mtCytB), alkaline proteinase, and cytochrome P450 lanosterol C-14ademethylase genes.3 In some instances, single copy genes, e.g., house-keeping candidates such as RNA polymerase I (RBP1), RNA polymerase II (RBP2),8 b-tubulin (BT2), or translation elongation factor (EF-1a) may also be suitable targets.9

DETECTION OF FUNGI IN CLINICAL SPECIMENS There are two main PCR-based approaches to detect fungi directly in clinical specimens: broad-range or panfungal assays, and more directed genus- or species-specific tests. Panfungal approach The use of panfungal PCR assays mirrors the growing need to detect a broad range of ‘unknown’ fungi in clinical specimens,

1 rDNA repeat unit

ITS1

18S rDNA

5.8S rDNA

ITS2

IGS1

28S rDNA

5S rDNA

IGS2

Major rDNA transcript (1 copy) Conserved regions targeted by primers Fig. 1 Schematic diagram of the fungal rDNA gene cluster (adapted from CLSI7). The 18S, 5.8S and 28S rDNA genes are separated by the internal transcribed spacers 1 (ITS1) and 2 (ITS2). The 28S and 5S rDNA genes are separated by the intergenic spacer 1 (IGS1). The intergenic spacer 2 (IGS2) separates the repeat units from each other.

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MOLECULAR DIAGNOSTIC METHODS FOR INVASIVE FUNGAL DISEASE

throughout the whole procedure, including the use of negative controls during both DNA extraction and PCR amplification, and maintain a unidirectional workflow.10 Commercial PCR systems The Luminex xTAG system (Luminex, USA) offers 23 analytespecific reagents (ASRs) for the detection of common fungi (including 7 species complexes of Candida, 4 species complexes of Aspergillus, Cryptococcus neoformans, Scedosporium apiospermum, Scedosporium prolificans, Fusarium species and several mucormycetes) either from culture or in clinical specimens. Here molecular reagents are standardised and laboratories may create their own panel of target pathogens based on clinical need. A small retrospective study to identify moulds directly in respiratory samples (n ¼ 43) reported a sensitivity of 58% (11/19) and specificity of 92% (25/27) for the detection of Aspergillus fumigatus.18 Another commercial system, the LightCycler SeptiFast (Roche Diagnostics, Germany) is a multiplex real-time PCR assay capable of detecting bacteria and fungi from blood. Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata and A. fumigatus are detected by dual fluorescence resonance energy transfer (FRET) probes targeting species-specific ITS regions. The assay has performed well, compared with blood culture, for detection of A. fumigatus and Candida species in febrile neutropenic patients.19–21 However, in practice, its clinical utility is limited by detection only of those species targeted by the assay and by high running costs. Studies are required to determine if the information provided by LightCycler SeptiFast assay leads to improved clinical outcomes. Other broad range commercial assays include the PLEX-ID system, which couples PCR amplification with electrospray ionisation-mass spectrometry (ESI-MS). Its utility is discussed under ‘Combined proteomic-genomic approaches’. Diagnosis of invasive candidiasis (IC) High mortality rates of up to 40% have been reported for IC, due in part to failure to initiate antifungal therapy early enough and inadequate sensitivity of blood and sterile-site cultures.22 The very high sensitivity afforded by PCR-based approaches, detecting 90%) were noted by Kourkoumpetis et al.24 Positive predictive values (PPVs) vary, although most studies report PPVs of approximately 50%. Negative predictive values (NPVs) were higher (88–100%), suggesting that PCR-based assays are more useful in excluding, rather than establishing,

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the diagnosis of IC,24,25 and may provide justification for stopping antifungal therapy.22 Whether PCR assays to detect IC are superior to traditional culture-based tests is under ongoing discussion. A recent metaanalysis reported a pooled sensitivity and specificity of 95% and 92%, respectively, for the diagnosis of candidaemia. In patients with suspected or probable IC, the positivity rate of PCR was 85% compared with 38% for blood cultures. The specificity for the same patients was >90% and decreased among patients colonised with Candida species.26 Higher sensitivities were observed with whole-blood samples (compared with serum), and the use of rDNA or cytochrome P450 gene targets, with an in vitro detection limit of
10 CFU/mL. An added advantage of PCR is the ability to detect more than one species in a single blood sample. In the Australian Candidaemia Study (2001–2004) polycandidal infections were present in 2.2% of cases.27 Two studies utilising a speciesspecific semi-nested PCR assay reported 18.5% (5/27)28 and 41% (5/12)29 of patients were infected with more than one Candida species. Of note, studies using real-time PCR and in situ hybridisation with commercial probes have shown the best results (i.e., high sensitivity, specificity, PPV and NPVs) for the diagnosis of IC using blood specimens.30–32 McMullan et al.30 prospectively evaluated the performance of three real-time PCR assays to detect Candida species in 157 critically ill patients (assay 1 targeted C. albicans, C. tropicalis, C. parapsilosis and C. dubliniensis; assay 2 targeted C. glabrata; and assay 3 targeted C. krusei). The overall sensitivity, specificity, PPV and NPV of the assays was 87%, 100%, 100% and 99.6%, respectively.30 More recently, Nguyen et al.32 reported that a Candida realtime PCR was more sensitive than the Fungitell 1,3-b-D-glucan (BDG) assay (Associates of Cape Cod, USA) for diagnosing IC (80% versus 56%) and deep-seated (blood culture negative) candidiasis (89% versus 53%), with comparable specificity (70% versus 73%). Both PCR and BDG were more sensitive than blood cultures among patients with deep-seated candidiasis (88% and 62% versus 17%). In patients with IC, the sensitivity of blood cultures combined with PCR or the BDG assay was 98% and 79%, respectively. Thus, these two non-culture-based assays, when combined with blood cultures, increase the sensitivity of culture methods. These results were supported by another study25 comparing a multiplex real-time PCR assay with blood culture and BDG. The PCR-based assay had a sensitivity of 90.9% versus 45.4% for blood culture. Despite their low sensitivity, blood cultures remain essential due to their ease of use and provision of an isolate for susceptibility testing. In addition, maintaining culture-based methods also provide an important method of detecting strains, which may not be detected due to the development of target sequence variation. An alternative to the detection of Candida in blood, is to use PCR to identify the Candida species directly from blood culture bottles.33,34 Conventional species identification requires a pure culture and takes 24–72 h. One study used a multiplex real-time PCR assay to differentiate between four fluconazolesensitive and two fluconazole-resistant Candida species. Results were 100% concordant (n ¼ 33) with conventional identification, and available in 90–95% concordance in comparison to conventional methods.152–154 In practice, the advantage of fungal identification by MALDI-TOF MS is the reduction in time to identification and corroboration of direct morphological examination, e.g., for filamentous fungi. Essential to the success of MALDI-TOF MS is the adoption of conditions to standardise (i) fungal growth, and (ii) protein extraction to achieve reproducible profiles for specific fungi. This is particularly important with filamentous fungi. One pragmatic approach is the method developed by Lau et al.156 which allows fungal proteins to be extracted directly from clinical isolates grown on solid agar. The second critical element in fungal identification is to query unknown spectra against databases that contain the appropriate ‘reference’ spectra; at the time of writing, the construction of robust spectral databases using well-characterised isolates of interest to supplement those provided by the manufacturer remains necessary for accurate identification. Lau et al.156 produced a comprehensive database for use with the BioTyper software which allowed identification of 152 fungal species including many cryptic and difficult-to-identify members of the Aspergillus, Scedosporium and Fusarium complexes which often require molecular identification. Construction of specific databases also lends itself as an alternative approach to traditional genotyping approaches to delineate strain differences.155,157 Proteomic analysis by MALDI-TOF MS can also be applied to directly identify fungi in clinical specimens, of which blood cultures have been the most often tested, reducing turnaround times even further.153 This has good accuracy for common Candida species and high (100%) specificity. However detection of mixed fungaemia is problematic. Pre-treatment of blood samples to lyse erythrocytes is required.152–154 A MALDI method to assess antifungal susceptibility of yeast and Aspergillus isolates has also been described. Changes in the protein composition of fungal cells after exposure to a known quantity of antifungal drug are compared with the spectra obtained in the absence of the drug. Spectral changes in Candida after exposure to a certain quantity of caspofungin correlate well with minimum inhibitory concentrations (MICs) measured by standard CLSI methods and only take 3 h (versus 24 h).158,159 PCR electrospray ionisation mass spectrometry (PCR-ESI) An extension of the MALDI-TOF MS approach is the combination of PCR amplification with electrospray mass spectrometry (PCR-ESI MS), which is capable of identifying pathogens either from cultures or directly from clinical specimens. Assay primers can be designed to target a broad range of fungi. Few studies have described its use for fungal identification. Gu et al.160 evaluated the detection of a limited number of Candida species on the PLEX ID instrument (Abbott Molecular, USA) where 51 of 61 isolates were correctly identified to species level. Recently, the approach performed well in identifying 81.4% of 264 mould, and 98.4% of 130 yeast

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isolates to either the genus or species level, and in identifying fungi in BAL fluid.161,162 Data from these few studies are not sufficiently robust to determine the clinical relevance of PCRESI MS. Reliable reference spectra are required.

Surface enhanced resonance Raman spectroscopy (SERRS) A novel spectroscopic approach in identifying fungal cultures is SERRS.163 SERRS uses sensors that detect light scattered through the production of PCR-amplified DNA, in conjunction with a specific dye placed against a roughened surface such as gold or silver. By employing probe-based technology and spectroscopic detection, analyte-specific spectral profiles can be generated and the assay is suited to the simultaneous detection of multiple targets. A new platform, the RenDx (Renishaw Diagnostics, UK), was developed for detection of nucleic acid targets by Raman spectroscopy, including multiplex detection of up to 10 targets per reaction. White et al.164 further developed the RenDx Fungiplex assay (Renishaw Diagnostics) to detect Aspergillus and Candida species in clinical samples including blood, by using a generic Aspergillus probe and probes to differentiate between diploid Candida species, as well as to identify C. glabrata and C. krusei.164 There was a 95% limit of detection of Candida and Aspergillus of 200 copies/reaction with overall reproducibility of 92.1%. Clinical evaluation showed a sensitivity of 82.8% (80% for Candida; 85.7% for Aspergillus) and a specificity of 87.5%. Prospective studies in defined patient populations are needed to establish its diagnostic potential.

CONCLUSIONS Significant recent advances in molecular diagnostics for clinical mycology include: the development towards a standardised Aspergillus PCR to allow comparability of results, and hence incorporation into clinical trials and possibly into definitions for IFD; commercial kits to detect Aspergillus in clinical specimens; the first FDA approved molecular test to detect Candida species in positive blood cultures; and the widespread use of MALDI-TOF MS to identify fungal cultures in combination with molecular methods. Many of these methods are moving towards implementation into clinical mycology laboratories, e.g., panfugal PCR, for identification of fungi in tissue biopsy specimens. However, currently none have been incorporated into diagnostic criteria for IFD when performed on blood or BAL fluid because of inconsistent approaches, difficulties with DNA extraction and/or insufficient data with regards to clinical utility. Clinicians need to determine how best to integrate these tools into their practice, and to combine them with traditional diagnostics. Molecular methods to detect azole and echinocandin resistance in Candida species and A. fumigatus have matured, with the implementation of FKS gene mutation detection a realistic goal. Conflicts of interest and sources of funding: The authors state that there are no conflicts of interest to disclose. Address for correspondence: A/Prof Sharon Chen, Centre for Infectious Diseases, Microbiology Laboratory Services , ICPMR – Pathology West, Level 3, ICPMR Building, Westmead Hospital, Darcy Road, NSW 2145, Australia. Email: [email protected]

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