Diagnosis of Invasive Fungal Infections by a Real-Time Panfungal PCR Assay in Immunocompromised Pediatric Patients

Home , Basidiobolus ranarum, Geotrichum candidum, Malassezia, Malassezia furfur

Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients

C Landlinger1, S Preuner1, L Basˇkova´1, M van Grotel2, NG Hartwig3, M Dworzak4, G Mann4, A Attarbaschi4, L Kager4, C Peters4, S Matthes-Martin4, A Lawitschka4, MM van den Heuvel-Eibrink2 and T Lion1

1Division of Molecular Microbiology, Children’s Cancer Research Institute (CCRI), Vienna, Austria; 2Department of Pediatric Oncology/Hematology, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands; 3Department of Infectious Diseases, Erasmus MC-Sophia Children’s Hospital, Rotterdam, The Netherlands and 4Department of Hematology/Oncology/Stem Cell Transplant Unit, St Anna Children’s Hospital, Vienna, Austria

Invasive fungal disease (IFD) is a life-threatening event in Timely detection of the fungal pathogens is a prerequisite for immunocompromised patients, and there is an urgent need for successful therapy and the clinical outcome in patients with reliable screening methods facilitating rapid and broad detec- IFD. In view of the changing epidemiology and the increasing tion of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection variety of fungal genera and species observed in immunosup- of more than 80 fungal pathogens, covering a large spectrum pressed patients, there is a growing demand for broad-spectrum of moulds, yeasts and Zygomycetes. To assess the clinical diagnostic tests. Many of the published technical approaches potential of the assay, more than 600 consecutive specimens based on real-time PCR methods are rapid and sensitive, from 125 pediatric patients carrying a high risk of IFD were but their detection spectrum is mostly restricted to a variety analyzed. An excellent correlation between PCR positivity and of Candida13,14 or Aspergillus species,15,16 or representatives the presence of proven, probable or possible fungal infection 17–21 according to the European Organization for Research and of both genera. Diagnostic assays permitting the detection Treatment of Cancer criteria was demonstrated, as revealed of a wide range of fungal pathogens including fungal species of by the sensitivity of the assay of 96% (95% CI: 82–99%). The both common and emerging fungal genera are therefore negative predictive value of the panfungal PCR assay presented required. To address the urgent clinical need, we have was 98% (95% CI: 90–100%), while the specificity and the developed a panfungal real-time PCR assay permitting the positive predictive value were 77% (95% CI: 66–85%) and 62% detection of at least 80 pathogenic fungal species (European (95% CI: 47–75%), respectively. The results indicate that molecular screening of patients during febrile neutropenic patent application 06817468.9). Herein, we describe the episodes by the assay presented could help prevent unneces- technical features of the assay, and present a combined sary toxicity resulting from empirical antifungal treatment prospective and retrospective study in severely immunocom- in individuals who may not be at risk of imminent fungal promised pediatric patients carrying a high risk of invasive disease. Our observations raise the possibility that rapid fungal infection, with the aim to assess the potential clinical species identification may be required to increase the positive benefit of molecular screening by a highly sensitive broad- predictive value for impending fungus-related disease. Leukemia (2010) 24, 2032–2038; doi:10.1038/leu.2010.209; spectrum fungus detection test. published online 30 September 2010 Keywords: invasive fungal infection; real-time PCR; panfungal PCR Materials and methods

Fungal strains, bacterial and viral isolates Fungal strains for PCR testing (Table 1) were obtained from Introduction different institutions, including the American Type Culture Collection (ATCC, Rockville, USA), the German Collection of Invasive fungal disease (IFD) is a leading cause of morbidity and Micro-organisms (DSM, Braunschweig, Germany) and the mortality in severely immunocompromised individuals, includ- Institute of Hygiene and Medical Microbiology of the University ing particularly patients with hematological malignancies of Vienna (IHMM, Austria). and bone-marrow transplant recipients. The vast majority of IFD events are still caused by Candida and Aspergillus species; Clinical materials however, changes in the epidemiology have occurred over the Clinical specimens from consecutive patients were obtained past decades.1–5 Recent studies from North American and after informed consent, and were prospectively collected as European centers indicate an increasing incidence of previously specified below. In total, 618 peripheral blood specimens from uncommon fungal genera, such as Cryptococcus, Trichosporon, 125 pediatric hemato-oncological patients undergoing intensive Malassezia,6,7 Fusarium,8,9 Scedosporium, Pseudallescheria,10,11 chemotherapy (n ¼ 65) or allogeneic stem cell transplantation and, very importantly, different members of the class Zygomycetes, (n ¼ 60) were analyzed during 150 episodes of febrile neutro- such as Rhizopus and Mucor.12 penia. Whenever possible, specimens were collected at first onset of fever, within 48 h thereafter, and at subsequent time Correspondence: Professor T Lion, Division of Molecular Microbiology, points in the course of the febrile episode, upon availability. On Children’s Cancer Research Institute (CCRI), Zimmermannplatz 10, average, four peripheral blood samples were investigated during Vienna A-1090, Austria. E-mail: [email protected] each episode (range 1–15). In selected instances, depending Received 15 March 2010; revised 16 July 2010; accepted 29 July on availability, additional types of specimens derived from 2010; published online 30 September 2010 primarily sterile sites of suspected infection were collected and Panfungal real-time PCR assay for diagnosis of IFD C Landlinger et al 2033 Table 1 Fungal species/isolates detected by the panfungal Table 1 (Continued ) real-time PCR assay Trichosporon asahii Reaction I Trichosporon cutaneum Experimentally tested Trichosporon inkin Acremonium strictum Saccharomyces cerevisiae Alternaria alternata Aspergillus candidus Based on sequence alignments Aspergillus clavatus Apophysomyces elegans Aspergillus flavus Basidiobolus ranarum Aspergillus fumigatus Cokeromyces recurvatus Aspergillus glaucus Mucor circinelloides Aspergillus nidulans Mucor ramosissimus Aspergillus niger Mucor rouxii Aspergillus terreus Rhizomucor variabilis Aspergillus versicolor Rhizopus azygosporus Fusarium oxysporum Rhizopus microsporus Fusarium proliferatum Rhizopus stolonifer Fusarium solani Rhodotorula mucilaginosa Fusarium verticillioides Saksenaea vasiformis Penicillium chrysogenum Trichosporon asteroides Penicillium citrinum Trichosporon moniliiforme Penicillium marneffei Trichosporon mucoides Penicillium purpurogenum Trichosporon ovoides Penicillium simplicissmum Scedosporium apiospermum Human pathogenic fungi covered by the panfungal PCR detection assay including reactions I and II. Based on sequence alignments Aspergillus ochraceus Aspergillus penicillioides Aspergillus ustus Bipolaris eleusines Cladosporium cladosporioides subjected to molecular analysis. These included cerebrospinal Cladosporium oxysporum fluid (n ¼ 11) and lung biopsies (n ¼ 2) in patients with suspected Histoplasma capsulatum central nervous system or pulmonary involvement, respectively, Scedosporium prolificans in order to correlate the data with the PCR findings in peripheral blood samples. The specimens used as training set for initial data Reaction II Experimentally tested assessment were provided by the Erasmus MC-Sophia Children’s Absidia corymbifera Hospital, Rotterdam, The Netherlands, and the specimens used Blastoschizomyces capitatus as validation set were provided by the St Anna Children’s Candida albicans Hospital of Vienna, Austria. Plasma or serum was usually Candida allociferrii collected from 3-ml samples of peripheral blood and all samples Candida colliculosa were stored at À80 1C until further processing. Peripheral blood Candida cylindracea Candida dubliniensis specimens from healthy volunteer donors were used to test for Candida famata cross-reactivity with human DNA (see below). Candida glabrata Candida guilliermondii Candida inconspicua DNA extraction Candida kefyr The isolation and purification of fungal DNA were performed Candida krusei 17 Candida lambica essentially as described earlier, with minor modifications. All Candida lipolytica steps were performed in a laminar flow hood using one-way Candida lusitaniae sterile utensils and sterile-filtered reagents. DNA isolation Candida membranaefaciens from bacteria and virus specimens for the assessment of cross- Candida norvegensis reactivity was performed using the QIAamp DNA Mini Kit Candida parapsilosis (QIAGEN GmbH, Hilden, Germany). Candida pelliculosa Candida rugosa Candida sake Candida tropicalis Real-time PCR Candida utilis The panfungal PCR detection assay included two separate Candida zeylanoides reactions, I and II, each covering different subsets of fungal Cryptococcus albidus pathogens, as outlined in Table 1. Both PCR reactions targeted a Cryptococcus laurentii Cryptococcus neoformans highly conserved region of the 28S ribosomal RNA multicopy Cunninghamella bertholletiae gene. The PCR reactions were set up in a total volume of 25 ml Geotrichum candidum containing Gene Expression Mastermix 2 Â with uracil Malassezia furfur N-glycosylase (UNG) (Applied Biosystems, Foster City, CA, Malassezia sympodialis USA), primers and probes as listed in Table 2, and molecular Mucor hiemalis biology grade water (Eppendorf, Hamburg, Germany). Locked Mucor mucedo nucleic acid nucleotides were included in the primer and probe Mucor racemosus 22 Rhizopus oryzae sequences to obtain high specificity despite short length. The amplification was performed on a TaqMan 7900 instrument

Leukemia Panfungal real-time PCR assay for diagnosis of IFD C Landlinger et al 2034 Table 2 Primers and probe used in the panfungal real-time PCR-assay

a Name Primer/probe Oligonucleotide sequence [50–30] Conc. (nM) Acc. no. Position

Reaction I Fw I TAAAGCTAAATAYYGGCCRGAGA 400 U28460 257–279 Rev I CT[T]TYCAAAGTGCTTTTCA[T]C 400 U28460 329–309 Z48339 277–255 Rev II CTCT[T]TTCAAAGTTCTTTTCA[T]C 400 U28460 331–309 Z48339 277–255 Probe I ACT[T]GT[G]CG[C]TA[T]CG 40 U28460 295–281 Reaction II Fw I GGGTGGTRARYTCCWTCTAARGCTAA 400 Z48339 186–211 Fw II GGGWGGTAAATCYCWCCTAAAGCTAA 400 Z48339 186–211 Rev I CTCT[T]TYCAAAGTKCTTTTCA[T]C 400 U284460 329–309 Z48339 277–255 Probe II A[C]TT[G]T[T]C[G][C]TA[T]CG 50 Z48339 241–227 Primer and probe sequences included in the panfungal PCR assay. Reaction I covers moulds and reaction II primarily yeasts and Zygomycetes. Nucleotides within brackets carry LNA modiﬁcations. aThe sequence accession numbers are based on A. fumigatus (U28460) and C. albicans (Z48339).

(Applied Biosystems) using the following standard protocol: 2 min p36.5 in reaction I or p37.5 in reaction II, reflecting the at 50 1C(uracilN-glycosylase-mediated degradation of potentially respective y-intercept values. In the event of single positive present contaminating amplicons containing dUTP), 10 min at values of the duplicate measurement, the CT had to be p34.5 in 95 1C, followed by 50 cycles of 15 s at 95 1C and 60 s at 60 1C.23 reaction I and p35.5 in reaction II to be regarded as PCR- positive. For the diagnosis of fungal DNAemia, a minimum of two PCR-positive peripheral blood specimens derived at Assessment of cross-reactivity with bacterial, viral and subsequent time points during close follow-up investigation human DNA were required. In the rare instances in which only one specimen Potential cross-reactivity of the panfungal detection assay with from a febrile neutropenic episode was available, a single PCR- non-fungal micro-organisms was analyzed by testing a total of positive result was regarded as indicative of DNAemia. 20 different bacterial and viral pathogens. Multiple isolates of DNA derived from different human peripheral blood specimens were also tested for cross-reactivity in this setting (not shown). Statistical analysis Results of panfungal PCR analysis in relation to the presence or absence of possible, probable or proven IFD by the European Controls Organization for Research and Treatment of Cancer (EORTC) A number of positive and negative controls were included in the 23 criteria were used for the calculation of sensitivity and panfungal real-time assay as described earlier. In view of the specificity, and the corresponding negative predictive values lack of fungus-grade reagents guaranteed to be free of (NPV) and positive predictive values (PPV) of the assay. contamination by fungal particles or DNA, highly stringent Individual values and the corresponding 95% confidence measures to control and exclude contamination were performed intervals (CIs) were calculated using the method of Wilson.24 to minimize the risk of false-positive results of the molecular screening assay. Results Specificity of the real-time PCR assays Differences in the detection efficiency of individual pathogens Design and principle of the panfungal PCR assay determined by testing defined concentrations of fungal species were The universal primers and probes for the two-reaction panfungal in the range of ±1 cycle threshold (CT) value. The only exceptions PCR assay were judiciously selected on the basis of their were the fungal species Geotrichum candidum, Malassezia furfur, homology to highly conserved sequences within the 28S gene Mucor hiemalis, Candida lipolytica and Acremonium strictum, based on the sequence alignment of more than 80 fungal species which revealed higher CT values (in the range of þ 3), apparently (Tables 1 and 2). The primers display a degenerated code due to mismatches within regions targeted by the primers. required for efficient amplification of fungal species that differ from others at single-nucleotide positions. In order to obtain optimal binding affinity, locked nucleic acid modifications of Sensitivity of the real-time PCR assays individual nucleotides were included in the hybridization To determine the detection limit of the real-time PCR assays, probes and the reverse primers (Table 2). Owing to the serial logarithmic dilutions across a 6-log range (1 fg to 100 pg) exploitation of a multi-copy target gene, the detection limit of of genomic DNA derived from A. fumigatus and C. albicans, as the panfungal PCR assay is in the range of 1 fg fungal DNA per representatives for moulds and yeasts, respectively, were reaction, which corresponds to a fraction of a single fungal analyzed. Standard curves were generated on the basis of the genome equivalent. amplification profile. Detection spectrum and specificity of the panfungal Definition of PCR positivity and DNAemia PCR assay All clinical specimens were analyzed in duplicates. A PCR result To document the detection spectrum of the two-reaction was regarded as positive when both CT values were either panfungal PCR assay, multiple strains/isolates of 61 different

Leukemia Panfungal real-time PCR assay for diagnosis of IFD C Landlinger et al 2035 fungal species (Table 1) were analyzed experimentally instances, both PCR reactions revealed positive results in repeated tests. However, based on extensive sequence indicative of mixed infections (Table 3). alignments, the panfungal real-time PCR assay presented can Five patients received empirical antifungal therapy on the be expected to cover more than 80 different fungal species basis of clinical suspicion of fungal infection, which did not (Table 1). Reaction I was designed to permit detection and meet the EORTC definition of possible IFD (generally by the quantitative assessment of a variety of moulds (nX29), whereas absence of an appropriate clinical criterion), and three of these reaction II targets a broad range of yeasts and Zygomycetes patients were also positive by the panfungal PCR assay (Table 3). (nX58). The assay therefore permits the detection of co- However, consecutive PCR-positive results were also obtained infections by different fungal genera, which may not be in 6 of 27 instances, with no clinical indication of IFD in patients uncommon in immunocompromised individuals. who were on antifungal prophylaxis only or did not receive any Cross-reactivity between reactions I and II was observed at a kind of antimycotic treatment (Table 3). Hence, analysis of the high concentration of fungal template DNA (X100 fg/reaction). training data set indicated a good correlation between repeated However, the amplification efficiencies for moulds and yeasts/ positivity of the panfungal PCR assay and the presence of Zygomycetes by the appropriate detection systems were about 3 proven, probable and possible IFD by the EORTC criteria, by logs higher, thereby permitting clear identification of the revealing a sensitivity of the panfungal screening assay of 91% dominant class of fungal pathogen present. At the concentra- (95% CI: 62–98%), and an NPV of 95% (95% CI: 78–99%). tions of fungal pathogens observed in most clinical specimens of Although it might be more appropriate to calculate the NPV of immunocompromised individuals, the cross-reactivity was not the panfungal PCR assay on the basis of proven and probable relevant. The lack of cross-reactivity of the panfungal PCR assay IFD only, we have also included patients classified as having a with non-fungal pathogens or human genomic DNA was possible IFD in the calculation in order to obtain a value assessed by extensive testing of various bacterial and viral applicable to any level of IFD according to the EORTC criteria. DNA isolates, and peripheral blood specimens of healthy A problem in the specificity of panfungal PCR-positive results volunteer donors, as specified in the Materials and methods became particularly apparent in the presence of single positive section. tests, and in follow-up samples collected at extended time intervals (43 days) during febrile neutropenia. Based on the presence of positive PCR tests, according to the definition indicated in Materials and methods, the specificity of the Analysis of clinical specimens by the panfungal PCR assay was 78% (95% CI: 59–89%), and the PPV 63% (95% CI: assay in relation to the EORTC criteria for IFD 39–82%). The analyses of clinical specimens were performed in a double- On the basis of the insights gained from analysis of the blinded manner to prevent any bias or influence on treatment training set, a larger cohort of immunocompromised pediatric decisions. First, a training set of 165 peripheral blood specimens patients was investigated, with a focus on febrile neutropenic derived from 43 pediatric patients with a high risk of IFD was episodes and the requirement of consecutive samples collected analyzed by the panfungal PCR assay in a retrospective study in at short time intervals (1–2 days). A total of 453 consecutive order to generate preliminary data on the potential clinical peripheral blood samples derived from 107 febrile neutropenic utility of the test system. The patients were generally in febrile episodes in 82 pediatric patients with hemato-oncological neutropenia at the time of sample collection. In total, 71 malignancies (n ¼ 22) or hematopoietic stem cell transplant specimens (43%) tested PCR-positive, 34 in reaction I, 15 in recipients (n ¼ 60) were analyzed. Similar to the first cohort reaction II and 22 in both reactions, with CT values clearly tested, 212 specimens (47%) tested PCR-positive, 122 in beyond the level of potential cross-reactivity. The results of reaction I (58%) and 45 in reaction II (21%). Forty-five samples molecular testing were evaluated in relation to the criteria (21%) were positive in both reactions, with differences in CT proposed by the EORTC,25 and indicated that all patients with values clearly beyond the level of potential cross-reactivity, proven or probable IFD, and all but one case with possible IFD hence indicating a mixed infection. Overall, 51 of 107 febrile tested repeatedly PCR-positive, as specified in Table 3. In three neutropenic episodes investigated revealed repeatedly positive

Table 3 Analysis of clinical specimens: training set

EORTC IFD-positive EORTC IFD-negativea

Empirical Antifungal No antifungal Proven IFD Probable IFD Possible IFD treatment prophylaxis prophylaxis/treatment

Clinical assessment Asp Can Asp Can Asp Can Febrile episodes (n ¼ 43) 1 1 4 5 5 14 13 PCR reaction I positive (n ¼ 10) 3 3 1 2 1 PCR reaction II positive (n ¼ 2) 1 1 PCR reaction I and II positive (n ¼ 7) 1 1 1 2 1 1 PCR negative (n ¼ 24) 1 2 11 10 Abbreviations: Asp, Aspergillus, Can, Candida; EORTC, European Organization for Research and Treatment of Cancer. Correlation of PCR results with clinical findings according to the EORTC criteria of IFD in the training cohort including febrile episodes in 43 patients. The cases of proven, probable and possible IFD revealed the presence of moulds (n ¼ 6; reaction I), yeasts/Zygomycetes (n ¼ 1; reaction II) or positivity in both reactions (n ¼ 3), respectively. Clinical assessment based on blood culture, HR-CT or histological analysis of targeted biopsies revealed findings indicative of Candida (Can) in one patient, and Aspergillus (Asp) in 10 patients identified as having IFD according to the EORTC criteria. aEORTC negative indicates the lack of evidence for possible, probable or proven IFD according to the consensus criteria. For these cases, the therapeutic (empirical) and prophylactic employment of antifungal agents as well as the absence of any administration of antifungals is indicated.

Leukemia Panfungal real-time PCR assay for diagnosis of IFD C Landlinger et al 2036 Table 4 Analysis of clinical specimens: validation set

EORTC IFD-positive EORTC IFD-negativea

Empirical Antifungal No antifungal Proven IFD Probable IFD Possible IFD treatment prophylaxis prophylaxis/treatment

Clinical assessment Asp Can Asp Can Asp Can Febrile episodes (n ¼ 107) 3 1 10 2 48 11 32 PCR reaction I positive (n ¼ 29) 2 1 7 14 1 4 PCR reaction II positive (n ¼ 6) 1 2 1 2 PCR reaction I and II positive (n ¼ 16) 1 3 1 9 1 1 PCR negative (n ¼ 56) 23 8 25 Abbreviations: Asp, Aspergillus, Can, Candida; EORTC, European Organization for Research and Treatment of Cancer; IFD, Invasive fungal disease. Correlation of PCR results with clinical findings according to the EORTC criteria of IFD in the validation cohort including 107 febrile episodes in 82 patients. The cases of proven, probable and possible IFD revealed the presence of moulds (n ¼ 10; reaction I), yeasts/Zygomycetes (n ¼ 1; reaction II) and positivity in both reactions (n ¼ 5), respectively. Clinical assessment based on blood culture, HR-CT or histological analysis of targeted biopsies revealed findings indicative of Can in two patients and Asp in 14 patients identified as having IFD according to the EORTC criteria. aEORTC negative indicates the lack of evidence for possible, probable or proven IFD according to the consensus criteria. For these cases, the therapeutic (empirical) and prophylactic employment of antifungal agents as well as the absence of any administration of antifungals is indicated.

test results by the panfungal real-time PCR assay (Table 4). There positive PCR results as a diagnostic criterion.29,30 However, the was no statistically significant difference between allogeneic diagnostic reliability of PCR assays in peripheral blood and stem cell transplant recipients and non-transplant patients with serum specimens has been described to be rather limited.31,32 regard to the occurrence of panfungal PCR positivity The panfungal real-time PCR assay presented was specifically in peripheral blood during the neutropenic febrile episodes adapted to highly sensitive detection of fungal pathogens in (50 vs 39%; two-sided P-value ¼ 0.29). PCR positivity was peripheral blood or serum. In the current combined prospective observed in all cases of IFD according to EORTC criteria and retrospective study, more than 600 clinical specimens from (n ¼ 16), revealing a sensitivity of 100% (95% CI: 81–100%) and 150 febrile neutropenic episodes in pediatric patients with high an NPV of 100% (95% CI: 90–100%). risk of IFD were investigated by the panfungal real-time PCR All patients with proven, probable and possible IFD (n ¼ 16) assay described. Evaluation of the molecular screening data, tested repeatedly PCR positive, as specified in Table 4. In five which had been generated in a double-blinded manner, instances, both PCR reactions revealed positive results indica- revealed an excellent correlation with the EORTC definitions tive of mixed infections with different fungal genera (Table 4). of proven, probable and possible invasive fungal infection. The In all 16 patients with IFD, positive PCR results were already sensitivity and NPV in the validation cohort of patients were in detected in the specimens collected at first onset of fever within the range of 100%, indicating that molecular screening might be a febrile neutropenic episode. instrumental in preventing unnecessary treatment. The possible In 48 instances, patients with febrile neutropenia received benefit of this notion is underlined by the observation that nearly empirical antifungal treatment despite the lack of findings half of the febrile neutropenic episodes studied were treated supporting the presence of IFD according to the EORTC criteria. empirically by antifungal agents, despite the lack of evidence for Only 25 of these patients revealed evidence of IFD by panfungal IFD according to the EORTC criteria. The absence of fungemia PCR analysis (Table 4). In patients with no clinical indication of determined by the panfungal PCR assay in about 50% of these IFD, including individuals receiving antifungal prophylaxis only episodes suggests that a high proportion of patients were and individuals without any antimycotic treatment, repeatedly probably over-treated. positive test results by the panfungal PCR assay were obtained in The limited specificity and PPV of the panfungal PCR assay 10 out of 43 (23%) febrile neutropenic episodes (Table 4). The reflect the fact that even in severely immunocompromised observations made in the validation patient cohort therefore patient populations, repeated or persistent detection of fungemia reflected a specificity of the assay at the level of 77% (95% CI: by sensitive, broad-spectrum PCR methods may not generally 62–87%) and a PPV of 62% (9% CI: 42–76%). indicate an imminent risk of severe fungal disease. This Evaluation of the molecular assay in both patient cohorts observation might be interpreted as frequent occurrence of false combined revealed a sensitivity of 96% (95%CI: 82–99%) and a positivity, most likely attributable to contamination. However, specificity of 77% (95%CI: 66–85%), corresponding to an owing to the processing of test materials in a sterile environ- NPV of 98% (95%CI: 90–100%) and a PPV of 62% (95%CI: ment, the use of internally tested reagents and the number of 47–75%). controls included in each assay, it may be an explanation for some, but very likely not all, seemingly false-positive findings. Of the PCR-positive patients who received either no antifungal Discussion treatment at all or prophylaxis only (n ¼ 16), fungal disease was diagnosed during the subsequent clinically documented febrile Over the previous years, a small number of prospective studies episode early thereafter in two patients, raising the possibility have assessed the clinical utility of different PCR assays in that subclinical fungemia was present at the time of the febrile hematopoietic stem cell transplantation recipients and/or episode investigated. In two additional patients, fungus-positive patients with hematological malignancies.18,19,26–28 The rep- cultures from urine and throat were obtained at the time of PCR orted sensitivity and the NPV were moderate to high. The positivity, indicating the presence of fungal infection or specificity was generally lower, but could be significantly colonization. However, based on the current EORTC criteria, improved by establishing the requirement of two consecutive these patients could not be classified as having IFD at any level

Leukemia Panfungal real-time PCR assay for diagnosis of IFD C Landlinger et al 2037 of probability. In five patients, treatment with semi-synthetic innovative interdisziplina¨re Krebsforschung. We are grateful to penicillins was documented during the febrile episodes Pia Reindl for collecting clinical data relating to the patient investigated. These substances were reported to yield false- specimens obtained from the St Anna Children’s Hospital, Vienna. positive results in serological fungus detection tests, and it Furthermore, we want to thank Ulrike Po¨tschger for her help with cannot be excluded that the occurrence of cross-reactivity with statistical analysis. molecular detection assays might have accounted for false positivity of the panfungal PCR assay. In seven patients, no References findings possibly explaining the PCR-positive results could be identified. The limited correlation between positive panfungal 1 Enoch DA, Ludlam HA, Brown NM. 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Molecular detection and identification of Candida and Aspergillus spp. from clinical samples using real-time Acknowledgements PCR. Clin Microbiol Infect 2006; 12: 745–753. 20 Schabereiter-Gurtner C, Selitsch B, Rotter ML, Hirschl AM, Willinger B. Development of novel real-time PCR assays This work was supported by grants from the Austrian Center for detection and differentiation of eleven medically important for Innovation and Technology (ZIT), the Austrian Science Fund Aspergillus and Candida species in clinical specimens. J Clin (FWF; Project No. P16929-B13) and Fonds der Stadt Wien fu¨r Microbiol 2007; 45: 906–914.

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