Quantitative Multiplexed Detection of Common Pulmonary Fungal Pathogens by Labeled Primer Polymerase Chain Reaction Zhengming Gu, PhD; Daelynn R. Buelow, PhD; Ruta Petraitiene, PhD; Vidmantas Petraitis, PhD; Thomas J. Walsh, MD, PhD; Randall T. Hayden, MD

 Context.—Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients. Objective.—To design and evaluate a multiplexed assay aimed at quantitative detection and differentiation of the 5 molds that are most commonly responsible for pulmonary infections. Design.—Using labeled primer polymerase chain reaction chemistry, an assay was designed to target the 5.8S and 28S ribosomal RNA genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the order Mucorales (Rhizopus oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). This assay was split into 2 multiplexed reactions and was evaluated using both samples seeded with purified nucleic acid from 42 well-characterized clinical fungal isolates and 105 archived samples (47 blood [45%], 42 bronchoalveolar lavage fluid [40%], and 16 tissue [15%]) collected from rabbit models of invasive pulmonary fungal infections.

Results.—Assay detection sensitivity was less than 25 copies of the target sequence per reaction for Aspergillus spp, 5 copies for Fusarium spp and Scedosporium spp, and 10 copies for the Mucorales. The assay showed quantitative linearity from 5 3 101 to 5 3 105 copies of target sequence per reaction. Sensitivities and specificities for bronchoalveolar lavage fluid, tissue, and blood samples were 0.86 and 0.99, 0.60 and 1.00, and 0.46 and 1.00, respectively. Conclusions.—Labeled primer polymerase chain reaction permits rapid, quantitative detection and differentiation of common agents of invasive fungal infection. The assay described herein shows promise for clinical implementation that may have a significant effect on the rapid diagnosis and treatment of patients’ severe infections caused by these pulmonary fungal pathogens. (Arch Pathol Lab Med. 2014;138:1474–1480; doi: 10.5858/arpa.2013-0592-OA)

I

However, the broad range of pathogens causing invasive fungal infections limits the utility of molecular methods that target only a single pathogen or small group of organisms. Reports in recent years have indicated an increasing incidence of mucormycosis, as well as infections caused by other hyaline molds and by dematiaceous fungi.4–8 The use of an Aspergillus-specific polymerase chain reaction (PCR) test, for example, would miss other causes of mold infections. Moreover, evidence supports the need for accurate identification of those pathogens because antifungal drug susceptibility and treatment efficacy varies significantly among known pathogens.5,9,10 Therefore, early and reliable knowledge, to at least the genus level of identification, for a broad range of potential pathogenic, filamentous fungi that are capable of causing pulmonary mycoses may directly inform initial decisions of antifungal therapy with benefits measured in patient outcome and resource use. In an effort to address that need, we describe the development and evaluation of a multiplexed, real-time PCR assay that is capable of detection and quantification of several of the most commonly encountered pathogenic, filamentous fungi. The assay employs labeled primer chemistry. This method uses 2 modified nucleotides, iso-

nvasive fungal infections represent a significant cause of morbidity and mortality, particularly among immunocompromised patients. Although the past decade has seen increased use of newer mold-active agents for prophylaxis and treatment, development of molecular diagnostic methods for medically important fungi has been slow. Efforts at development of molecular diagnostic methods for pulmonary mycoses have most commonly been limited to the detection of invasive aspergillosis or, less commonly, to other individual genera or to the Mucorales.1–3 Accepted for publication January 8, 2014. From the Department of Pathology, St. Jude Children’s Research Hospital, Memphis, Tennessee (Drs Gu, Buelow, and Hayden); and the Transplantation-Oncology Infectious Diseases Program, Weill Cornell Medical Center, New York, New York (Drs Petraitiene, Petraitis, and Walsh). Dr Hayden has a research agreement with Luminex Corporation (Austin, Texas). The other authors have no relevant financial interest in the products or companies described in this article. Presented in part as a poster at the 51st Annual Interscience Conference on Antimicrobial Agents and Chemotherapy; September 17–20, 2001; Chicago, Illinois. Reprints: Randall T. Hayden, MD, Department of Pathology, St. Jude Children’s Research Hospital, 262 Danny Thomas Pl., Memphis, TN 38105 (e-mail: [email protected]). 1474 Arch Pathol Lab Med—Vol 138, November 2014

Labeled Primer PCR for Fungal Detection—Gu et al

Table 1.

Fungal Strains and Isolates Used in the Study

Fungal Strains

ATCC No.

Reference strainsa Absidia corymbifera Absidia glauca Absidia griseola Acremonium strictum Alternaria alternate Apophysomyces elegans Aspergillus aculeatus Aspergillus brasiliensis Aspergillus fischeri Aspergillus flavus Aspergillus fumigatus Aspergillus fumigatus Aspergillus microcysticus Aspergillus nidulans Aspergillus niger Aspergillus ochraceus Aspergillus oryzae Aspergillus versicolor Aspergillus wentii Clavispora lusitaniae Cryptococcus luteolus Cryptococcus neoformans Curvularia lunata Exophiala dermatitidis Exserohilum parlierensis Fusarium solani Issatchenkia orientalis Mucor hiemalis Mucor racemosus Paecilomyces lilacinus Paecilomyces variotii Penicillium chrysogenum Penicillium citrinum Penicillium marneffei Penicillium primulinum Rhizomucor miehei Rhizomucor pusillus Scedosporium apiospermum Scopulariopsis brevicaulis Trichosporon inkin

22743 7852a 22618 10141 6662 200277 42745 9642 1020 24133 14110 16424 16826 38163 10249 1008 42149 16853 1023 MYA-2636 10671 66031 74066 MYA-884 MYA-2456 36031 6258 16636 18361 10114 10865 10106 9846 18224 10438 16457 16458 9258 1102 18020

Clinical isolatesb Aspergillus fumigatus Aspergillus flavus Aspergillus terreus Aspergillus niger Fusarium solani Fusarium oxysporum Scedosporium apiospermum Scedosporium prolificans Rhizopus oryzae Rhizopus microspores Mucor spp Absidia corymbifera Rhizomucor Cunninghamella bertholletiae

No. of isolates 3 3 3 3 3 2 3 3 3 3 3 3 4 3

a

Reference strains used to determine specificity of test and their American Type Culture Collection (ATCC) numbers are given. b Clinical isolates used to determine limit of detection, linearity, and limit of quantification.

deoxyguanosine triphosphate and iso-deoxycytidine triphosphate, together with fluorescence resonance energy– based detection to enable real-time quantitative PCR detection of molecular targets without the need for separate oligonucleotide probes.11–14 The latter aspect of this technology simplifies assay design, enabling the use of more limited stretches of conserved sequence in detecting targets Arch Pathol Lab Med—Vol 138, November 2014

or organism groups with sequence heterogeneity. Initial analytic evaluation in artificially seeded samples was followed by the use of samples from well-characterized animal models of invasive pulmonary fungal infection, which permitted assay refinement and confidence in test performance characteristics before the use of human samples, which will be used in future studies. MATERIALS AND METHODS Fungal Strains and Seeded Samples Forty fungal reference strains (American Type Culture Collection) and 42 well-characterized clinical fungal isolates (Table 1) were used for analytic evaluation. Seeded samples were used to determine the lower limit of detection and the limit of quantification for each targeted organism group. The seeded samples were prepared by performing 10-fold, serial dilutions of purified DNA from the appropriate fungal reference strains, which were quantified spectrophotometrically. The DNA was diluted in TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) to create concentrations from 1 3 101 to 1 3 106 genome copies/lL, followed by spiking normal human whole blood and serum to a final fungal DNA concentration of 1 to 1 3 105 genome copies/lL. An aliquot of 5 lL of DNA solution was used in the PCR test. Extraction of Fungal Reference Strains.—Fungal strains were grown on Sabouraud glucose agar (Fisher Scientific, Suwanee, Georgia) at 298C for 2 to 7 days. The DNA was extracted and purified using MO BIO UltraClean Microbial DNA Isolation Kit (MO BIO Laboratories Inc, Carlsbad, California), with slight modification to the standard protocol. Briefly, conidia and sporangiospores were collected by rubbing sterile polyester–tipped applicators over the mycelium, which were then transferred to a collection tube, washed in 1 mL PCR-grade water, and centrifuged for 1 minute at 10 000g to collect the spores. The conidia and sporangiospores were resuspended in Microbead solution (Mo BIO), transferred to Microbead tubes containing MD1 solution, vortexed for 10 minutes, and then boiled for 10 minutes. Upon addition of solution MD2, the mixture was kept at 48C overnight. The DNA was collected in a spin filter and eluted in a final volume of 35 lL of PCR-grade water. The DNA was quantitated using a NanoDrop 2000c spectrophotometer (Thermo Scientific, Waltham, Massachusetts) and stored at 208C before use. An 5-lL aliquot of DNA solution, containing 0.005 or 0.5 ng fungal DNA, was used in the PCR test. Extraction of Clinical Fungal Isolates.—Clinical fungal isolates were pretreated and extracted using the MagNA Pure LC system (Roche Applied Science, Indianapolis, Indiana). Briefly, samples were centrifuged for 10 minutes at 16 000g, and supernatants were discarded. An 150-lL aliquot of spheroplast buffer (1.0 M sorbitol [S-1876, Sigma, St Louis, Missouri], 50.0 mM sodium phosphate monobasic [S-0751, Sigma], 0.1% 2-mercaptoethanol [M-3148, Sigma], 10 mg/mL lyticase [L-2524, Sigma]), 10-lL lysing enzymes (Novozyme 20 mg/mL [L-1412, Sigma]), and lysing matrix were added to each specimen. Samples were briefly vortexed and incubated at 308C for 5 minutes at 1200 gpm in an Eppendorf thermomixer (Eppendorf, Westbury, New York). Mixing was terminated and sample incubation continued for 25 minutes. Samples were processed using a Fast Prep instrument (QBIOgene/ MP Biomedicals, Irvine, California) with glass milk binding matrix (Bio 101, Inc, La Jolla, California) at speed 5 for 30 seconds and placed on ice for 5 minutes; this process was performed 3 times. Samples were equilibrated to room temperature and centrifuged for 1 minute at 1000g. The samples were then processed with a MagNA Pure LC instrument using a MagNA Pure LC DNA isolation kit III (Roche), as recommended by the manufacturer. Samples were eluted in 100 lL of kit elution buffer. The DNA concentration in the eluate was determined spectrophotometrically. Aliquots of 5 lL DNA solution, containing 0.25 ng DNA, were used in the PCR test. Extraction of Fungal DNA Seeded Samples.—Normal human whole blood and serum were seeded with serial, 10-fold dilutions Labeled Primer PCR for Fungal Detection—Gu et al 1475

of purified fungal DNA and were extracted to create a concentration of fungal DNA of 1 to 1000 copies of target/lL. Fungal DNAseeded human whole blood (200 lL) was extracted with the QIAsymphony DNA Mini Kitt (QIAGEN, Valencia, California), whereas fungal DNA-seeded human serum (200 lL) was extracted with the QIAsymphony Virus/Bacteria Mini Kit. The DNA was eluted to 60 lL of kit elution buffer and stored at 208C before use. An aliquot of 5 lL of DNA solution was used in the PCR test.

Animals, Organisms, and Inoculation Female New Zealand white rabbits (Covance Research Products Inc, Denver, Pennsylvania), weighing 2.8 to 3.6 kg each, were individually housed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. They were monitored according to the guidelines of the National Research Council for the humane care and use of laboratory animals and under approval by the Animal Care and Use Committee of the National Cancer Institute.15 Vascular access for each rabbit, as previously described,16 was established under general anesthesia by the surgical placement of a Silastic tunneled central venous catheter that permitted nontraumatic, repeated blood sampling and administration of parenteral agents. Well-characterized clinical isolates of Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus, Pseudallescheria boydii, Rhizopus oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, and Mucor circinelloides were used for endotracheal inoculation.17,18 All animals received endotracheal inoculum of 1.0 to 1.25 3 108 conidia in a volume of 250 to 350 lL under general anesthesia. Fusarium solani isolates were inoculated intravenously (1.0 3 108 conidia). Neutropenic Rabbit Models of Invasive Pulmonary Mycoses.—Neutropenia in rabbits was established, as previously described,17 with intravenous administration of cytarabine (AraC) (Cytosar-U; Pharmacia-Upjohn, Kalamazoo, Michigan) 1 day before inoculation with an initial course of Ara-C at 525 mg/m2 for 5 consecutive days (days 1–5) and methylprednisolone (Pfizer, New York, New York) at a dose of 5 mg/kg/day administered on days 1– 2. A maintenance dose of Ara-C of 484 mg/m2 was administered on days 8 and 9 and on days 13 and 14 of the experiments. Ceftazidime (75 mg/kg twice daily [GlaxoSmithKline, Research Triangle Park, North Carolina]), gentamicin (5 mg/kg/day every other day [formerly Abraxis, now Celgene Corporation, Summit, New Jersey]), and vancomycin (15 mg/kg/day [Hospira, Lake Forest, Illinois]) were administered intravenously from day 4 until study completion to prevent opportunistic bacterial infections. To prevent antibiotic-associated diarrhea from Clostridium spiroforme, all experimental animals received continuous vancomycin 50 at mg/ L in drinking water. Neutrophil counts were monitored twice weekly by measuring total leukocyte counts with a Beckman Coulter Z1 Dual (Beckman Coulter, Inc, Brea, California) and by peripheral blood tests and differential cell counts, respectively. Bronchoalveolar Lavage.—Bronchoalveolar lavage (BAL) was performed on each postmortem lung preparation by the instillation and subsequent withdrawal of 10 mL of sterile normal saline twice into the clamped trachea with a sterile 12-mL syringe. The BAL fluid was then centrifuged for 10 minutes at 400g. The supernatant was discarded, leaving the pellet, which was then resuspended in 2 mL of sterile normal saline. A 0.1-mL sample of that fluid and 0.1 mL of a dilution (101) of that fluid were cultured on 5% synthetic genetic array plates. The remainder of the BAL fluid was stored at 808C until analyzed. Animal Samples.—The 105 animal specimens from the aforementioned rabbit models of pulmonary fungal infection were collected for analysis, with 47 blood (44%; 20 serum and 27 plasma, including one negative control), 42 BAL fluids (40%; of which 6 were negative controls), and 16 formalin-fixed, paraffin-embedded tissue samples (15%; including one negative control). Blood Specimens.—The DNA was extracted from 200 lL of blood sample into 60 lL of elution buffer using the QIAsymphony Virus/Bacteria Mini Kit on the QIAsymphony instrument according to the manufacturer’s instruction. The DNA was stored at 208C prior use. An aliquot of 5 lL was amplified. 1476 Arch Pathol Lab Med—Vol 138, November 2014

BAL Specimens.—The DNA was extracted from 1000 lL of the BAL fluid sample using the QIAamp Pathogen Mini Kit (QIAGEN) following manufacturer’s instructions. The DNA was eluted into 50 lL of elution buffer and stored at 208C prior use. An aliquot of 5 lL of DNA solution was used in the PCR test. Formalin-Fixed, Paraffin-Embedded Tissue Sections.—The DNA was extracted from 2 tissue sections (6–10 lm thick; approximately 6 3 6 mm) using QIAamp kit, according to the manufacturer’s instructions. The DNA was eluted in a final volume of 50 lL of kit elution buffer and stored at 208C before use. An aliquot of 5 lL of eluate was used in the PCR test. Consecutive paraffin sections from each case were processed and stained with hematoxylin-eosin and with Grocott methenamine silver. Slides were then examined microscopically by one of the authors (R.T.H.), blinded to PCR results. Sections were graded from 0 to 5þ, based on a subjective evaluation of the fungal burden, where 0 represented no morphologically identifiable fungal elements, and 5 represented most of the slide involved by large numbers of fungal hyphae.

Internal Control Internal-control DNA consisted of a plasmid constructed by inserting a 357 base pair DNA fragment of Phocid herpesvirus 1 gB gene into vector pUC57 (designed in-house at St. Jude Children’s Research Hospital (Memphis, Tennessee) and manufactured at GenScript USA Inc, Piscataway, New Jersey). Control DNA was added to all samples before extraction and served to assess both DNA extraction and PCR inhibition. Control DNA was detected in processed samples with a dedicated primer set developed by St. Jude Children’s Research Hospital (Table 2).

Fungal Amplicon Standards Five pairs of PCR primers were designed to flank conserved regions of Aspergillus spp, Fusarium spp, Scedosporium spp, and Mucorales (2 primer pairs), respectively. Concentration of purified amplicon DNA was measured spectrophotometrically. Analytic linearity of detection for each targeted genus was assessed using 5 lL of each 10-fold, serial dilution of the amplicon, ranging from 1 to 1 3 105 copies/lL of PCR-grade water.

Labeled Primer Quantitative Real-Time PCR Labeled primer real-time PCR uses a customized PCR primer that contains a 5 0 -terminal fluorescent reporter adjacent to an isodeoxycytosine residue. Quencher iso-deoxyguanosine triphosphate is preferentially incorporated opposite of isodeoxycytosine in the primer, resulting in quenching of the fluorescent dye on the isodeoxycytosine and a reduction in fluorescence in PCR; that allows detection and quantification without the use of separate oligonucleotide probes. Accumulation of product is accompanied by a decrease in fluorescence, and melt-curve analysis after amplification may be used to assess reaction specificity. A reagent kit (Plexor qPCR System, Promega Corporation, Madison, Wisconsin) was used as the PCR master mixture in the study. The MultiCode-RTx analysis software v1.6.2 (EraGen Biosciences, Madison, Wisconsin) was used to analyze the data. Primer pairs (Table 2) were designed to target highly specific regions in the 5.8S and 28S ribosomal RNA (rRNA) genes of Aspergillus spp, Fusarium spp, Scedosporium spp, and members of the Mucorales order (Rhizopus oryzae, Rhizopus microsporus, Cunninghamella bertholletiae, Mucor circinelloides, Lichtheimia corymbifera, and Rhizomucor pusillus). These primers were grouped into 2 multiplexed reactions. The first PCR reaction detected the Aspergillus spp, Fusarium spp, and Scedosporium spp, whereas the second PCR reaction detected the Mucorales: 12.5-lL of 23 PCR master mix (Plexor qPCR system) and a 7.5-lL primer mix was prepared as a 20-lL reaction mix, followed by the addition of 5 lL of DNA sample. The primer mix in the first PCR contained 0.5 lL of each primer of Aspergillus-forward-labeled (15lM) and Aspergillus-reverse (15lM), 0.25 lL of each Fusarium-forward (10lM) and Fusarium-reverse-labeled (10lM), and 0.5 lL of each Scedosporium-forward-labeled (10lM) and Scedosporium-reverse Labeled Primer PCR for Fungal Detection—Gu et al

Table 2. Polymerase Chain Reaction Primer Sequences, Molecular Target, and Corresponding Fungal Targets Primer

Sequence

Asp-F-L Asp-R Fus-F Fus-R-L Sce-F-L Sce-R Muc1-F-L Muc1-R Muc2-F-L Muc2-R IC-F-L IC-R

TTTGAAAGCTGGCTCCTTCGG CACTTAGACGGGGGCTGCA TGAGAGCCCCGTCTGGTT TGGCCGGTATTTAGCTTTAGAAGAC AACAGCACGTGAAATTGTTGAAA GAGCGACGGCTGATTCGA CCCTAGTAACGGCGAGTGAAGA CTCACGGTACTTGTTCGCTATCG ACTTTAAGCAATGGATCACTTGGTT CAAAGGCTGCAGATCGCATTA AGATTGAATCTGATGATACAGCAACAT GCGGTTCCAAACGTACCAA

Molecular and Fungal Targets 28S rRNA gene Aspergillus 28S rRNA gene Fusarium 28S rRNA gene Pseudallescheria/Scedosporium 28S rRNA gene Absidia, Mucor/Rhizopus, Cunninghamella 5.8S rRNA gene Rhizomucor Internal control

Abbreviations: Asp-F-L, Aspergillus-forward-labeled; Asp-R, Aspergillus-reverse; Fus-F, Fusarium-forward; Fus-R-L, Fusarium-reverse-labeled; IC-F-L, internal control-forward-labeled; IC-R, internal control-reverse; Muc-F-L, Mucorales-forward-labeled; Muc-R, Mucorales-reverse; rRNA, ribosomal RNA; Sce-F-L, Scedosporium-forward-labeled; Sce-R, Scedosporium-reverse.

(10lM). The primer mix in the second PCR reaction contained 0.5 lL of each Mucorales-forward-labeled 1 (10lM) and Mucoralesreverse 1 (10lM) and 0.5 lL of each Mucorales-forward-labeled 2 (10lM) and Mucorales-reverse 2 (10lM). Also included was 0.5 lL of each internal control–forward-labeled primer (10lM) and internal control–reverse primer (10lM) in both PCR reactions, which detect an extractable control DNA used for quality control in the assay. Each sample was amplified in duplicate. Thermal cycling on a 7500 real-time PCR system (Applied Biosystems, Life Technologies, Foster City, California) included 4 stages: 1 cycle at 958C for 2 minutes; 5 cycles at 958C for 5 seconds, at 658C for 10 seconds, and at 728C for 35 seconds; 45 cycles at 958C for 5 seconds, then at 638C for 10 seconds and at 728C for 35 seconds; and a dissociation stage from 638C to 958C with ramp rate set at auto. Fluorescent data were collected during stage 3 at 728C for 35 seconds. A true target was defined by an amplicon melting temperature of 82.38C (61.58C) for Aspergillus spp, 80.5 (61.58C) for Fusarium spp, 82.7 (61.58C) for Scedosporium spp, and 81.8 (638C) for members of the Mucorales, which was determined by nucleotide composition of the amplicon and experimentally confirmed, respectively.

Statistics To measure the diagnostic accuracy of the PCR results for a given fungal pathogen in its corresponding animal model of pulmonary mycosis, results were stratified by specimen type, and specificities and sensitivities were calculated overall and for each specimen type; the score confidence interval was used to calculate the confidence interval (CI) for each parameter.19 Data were tabulated in a results matrix with rows summarizing the presence of the organism and the columns summarizing the test results. Quantitative linearity was based on maintaining precision sufficient to detect a 10-fold difference in copy number with a power of 90%. The corresponding standard deviation (SD) was calculated by the following equation: SD ¼

log10 ð10Þ pffiffiffi 3ð1:96 þ 1:282Þ 2

where 1.96 is the standard normal deviate corresponding to a type 1 error rate of 0.05, and 1.282 is the standard normal deviate corresponding to the power of 0.90. The numerator, log10(10), reflects a 10-fold difference between 2 concentrations.20

RESULTS Limits of Detection, Quantification, and Linearity Seeded samples of fungal DNA in normal human whole blood and serum were used to determine the limit of detection for each targeted organism group. Based on a minimum 95% detection rate in at least 20 replicates during Arch Pathol Lab Med—Vol 138, November 2014

three runs, the limit of detection was demonstrated as less than 25 target copies per reaction for Aspergillus, 5 copies for Fusarium and Scedosporium, and 10 copies for Mucorales, corresponding to 1.5 3 103, 3 3 102, and 6 3 102 copies/mL, respectively. Suspension in whole blood and serum demonstrated no effect on limit of detection when compared with aqueous buffer. In silico analysis using FASTA (EMBL-EBI, Hinxton, Cambridgeshire, England) nucleotide sequence similarity search (GenBank, National Center for Biotechnology Information, Bethesda, Maryland) and test of human genomic DNA under studied experimental conditions showed no evidence of potential of crossreactivity with human genomic DNA. Quantitative linearity was achieved from 5 3 101 to 5 3 105 genome copies (Figures 1, A through C, and 2, A through C) per reaction with a calculated PCR efficiency of greater than 90% (efficiency, 10(1/slope)  1)21 for all targets, corresponding to 3 3 103 to 3 3 107 genome copies/mL, which were defined as the limits of quantification based on a 90% power to detect a 10-fold difference in genome copies between 2 samples (SD, 0.22). Sensitivity and Specificity by Fungal Reference Strains and Clinic Isolates Forty fungal reference strains were tested (Table 1). All species of Fusarium, Scedosporium, and the Mucorales, as well as 11 of 13 Aspergillus species (85%; Aspergillus versicolor and Aspergillus wentii excepted) used in the study were accurately detected and differentiated. There was no evidence of cross-reactivity of fungal species, outside of those targeted by the assay, nor was there evidence of crossreactivity among targeted groups when challenged with 0.005 ng fungal DNA. When total fungal DNA was increased to 0.5 ng, very low levels of cross-reactivity were seen for Paecilomyces lilacinus (Fusarium probes) and for Trichosporon inkin (Mucorales probes). Forty-two well-characterized clinical fungal strains (Table 1) were also tested. The assay correctly detected all Aspergillus isolates (12 of 12, 100%; 3 of 3 A. fumigatus, 3 of 3 A. flavus, 3 of 3 A. niger, 3 of 3 A. terreus), F. oxysporum/F. solani (2 of 3; 67%), Scedosporium apiospermum/Scedosporium prolifcans (3 of 3; 100%), and all Mucorales (16 of 16, 100%; 3 of 3 A. corymbifera, 3 of 3 C. bertholletiae, 3 of 3 Mucor spp, 4 of 4 Rhizomucor spp, and 3 of 3 Rizopus micosporus/R. oryzae). No evidence of cross-reactivity was seen among clinical fungal strains. Labeled Primer PCR for Fungal Detection—Gu et al 1477

Figure 1. Labeled primer polymerase chain reaction standard curve. A representative amplification plot (A) and melt curves (B), as well as a standard curve plot (C) are shown for detection of Aspergillus spp. A 10-fold, serial dilution of Aspergillus amplicon standards, ranging from 5 3 101 to 5 3 105 copies, was used to generate the plots. Abbreviations: dRFU/dTemp, negative first-derivative analyses of postreaction thermodynamic melt curve versus temperature in Celsius; eff, efficiency; NTC, no template control; PCR, polymerase chain reaction; RFU, relative fluorescence units.

Figure 2. Labeled primer polymerase chain reaction standard curve. A representative amplification plot (A) and melt curves (B), as well as a standard curve plot (C), are shown for detection of Mucorales. A 10fold, serial dilution of Cunninghamella amplicon standards, ranging from 5 3 101 to 5 3 105 copies, was used to generate the plots. Abbreviations: dRFU/dTemp, negative first-derivative analyses of postreaction thermodynamic melt curve versus temperature in Celsius; eff, efficiency; NTC, no template control; PCR, polymerase chain reaction; RFU, relative fluorescence units.

Samples from Animal Models The 105 animal specimens included 47 blood specimens (45%; 46 positive [98%] and one negative [2%] control), 42 BAL fluid specimens (40%; 36 positive [86%] and 6 negative [14%] controls), and 16 formalin-fixed, paraffin-embedded tissue samples (15%; 15 positive [94%] and 1 negative [6%] control) were tested. Twenty-one of 46 positive blood samples (46%) were detected (Aspergillus, 19 of 32, 59%; Mucorales, 2 of 14, 14%) for a sensitivity of 45.7% (CI, 1478 Arch Pathol Lab Med—Vol 138, November 2014

32.2%–59.8%) and a specificity of 100% (CI, 97.9%–100%) (Figure 3, A). A detection sensitivity of 86.1% (CI, 71.3%–93.9%) was achieved from 36 BAL samples (Aspergillus, 15 of 17, 88%; Mucorales, 16 of 19, 84% positive) with a specificity of 99.2% (CI, 95.8%–99.9%) (Figure 3, B). One Mucoralespositive BAL fluid sample gave a weak false-positive signal for Aspergillus (28 copies/reaction). Labeled Primer PCR for Fungal Detection—Gu et al

Figure 3. Polymerase chain reaction results for detection of fungal DNA from animal specimens. The sensitivities and specificities of the detection are summarized and show for 47 blood samples (A), 42 bronchoalveolar lavage fluid samples (B), 16 tissue samples (C), and all specimen types combined (D).

The DNA from 15 formalin-fixed, paraffin-embedded tissue samples from infected animals was tested. Nine of 15 samples (sensitivity, 60%) tested positive (Aspergillus spp, 5 of 6, 83%; Fusarium spp, 2 of 5, 40%; Scedosporium spp, 2 of 4; 50%) (CI, 35.7%–80.2%) and a specificity of 100% (CI, 92.7%–100%) (Figure 3, C). Results correlated well with fungal burden based on histopathology. Four of 6 falsely negative samples (67%) showed no fungal elements on microscopic examination (score, 0), whereas the remaining 2 (33%) showed minimal involvement by fungal hyphae (one each, 1þ and 2þ). All negative control samples (one each, blood and tissue, and 6 BAL fluid samples) tested negative in all replicates. Overall, 105 animal specimens produced a sensitivity and specificity of 62.9% (CI, 53%–71.8%) and 99.7% (CI, 98.3%–99.9%) (Figure 3, D). Internal controls were detected in all samples, without evidence of inhibition. COMMENT Labeled primer PCR chemistry was shown here to successfully detect several important groups of fungal pathogens in a multiplexed format. Analytic performance demonstrated a high degree of sensitivity, corresponding to single-genome detection or less for each targeted organism. The method was shown to be highly specific, without interference by human genomic DNA and with minimal cross-reactivity either between the genera of interest or with numerous other fungi commonly seen as either environmental contaminants or human pathogens. Sensitivity and specificity was maintained whether aqueous buffer, whole blood, or serum was used as a matrix for seeded suspensions, and no evidence of inhibition was noted. Sensitivity was confirmed using a wide variety of strains from each genus or target group, corresponding to most Arch Pathol Lab Med—Vol 138, November 2014

clinically relevant genospecies. Performance using specimens collected from rabbit models of fungal infection varied depending on the sample type, with all showing a high degree of specificity, evidenced both by the absence of cross-reactivity among targets and primers and by the low to absent false-positive results. Variation in sensitivity by sample type is to be expected, and relative performance was similar to that reported by others using single target assays. Most such published works have focused on the detection of Aspergillus species, with a few studies related to the Mucorales, and with most data derived from series with few samples. Sensitivities are typically highest in BAL fluid samples, with lower rates of detection in tissue or blood specimens.3,22–25 Although higher detection rates in tissue have been reported by at least one group,26 that publication did not provide information on the fungal burden of the samples tested. In the present study, 100% of samples with 3þ to 5þ visible fungal hyphae on histologic section and Grocott methenamine silver staining were positive by PCR. Other factors that may affect sensitivity in various sample types include the number of samples tested per patient, EORTC (European Organisation for Research and Treatment of Cancer) case classification, and the volume of blood or the number of histologic sections used for nucleic acid extraction.26,27 Overall, both sensitivity in seeded samples and in specimens collected from animal models in the present study are comparable to other reports using single-target assays. The use of specimens from well-characterized and clinically applicable animal models in this study allows for a robust number of specimens from a wide range of invasive fungal infections that would otherwise be difficult to obtain from human disease. Because bronchoalveolar lavage may be difficult to perform in patients with thrombocytopenia, Labeled Primer PCR for Fungal Detection—Gu et al 1479

such specimens are difficult to acquire in a clinical setting. All infections in the animal models were histologically verified and quantified to provide the strongest controls for blood and BAL fluid. Such histologic verification is difficult to obtain in patients, and quantitative cultures are seldom performed. Thus, the animal model systems provide a highly reliable platform for understanding the performance of analytic molecular diagnostic assays to prevent the consumption of precious human samples and to allow for optimization of the assays before analysis of clinical specimens. Although the value in using animal models cannot be underestimated, so, too, is it necessary to evaluate this method using human samples before making conclusions about its ultimate utility. As has repeatedly been seen in studies of fungal diagnostics, other limitations here relate to the number of samples available for testing and the ability to adequately represent the wide degree of strain variation seen in targeted organisms. An additional challenge is to ensure specificity, given the incredibly broad scope of environmental fungi and other potential sources of falsepositive results. Nonetheless, we feel that the data presented here represent a reasonable sampling, allowing conclusions with respect to likely performance in clinical practice. Although both accuracy and utility in clinical care will need to be evaluated in future studies, this test shows promise as a rapid and sensitive means for diagnosis of invasive pulmonary fungal infection. The ability to detect and differentiate between 4 of the most common causes of invasive fungal infection with a high degree of sensitivity and specificity offers a potentially powerful tool for the care of these often critically ill patients. Early detection is thought to lead to better outcome in these cases. When this ability is paired with differentiation among the detected organisms, it should allow more informed decision making in the selection among empiric treatment options. Similarly, quantitative data can help assess therapeutic responsiveness and may assist in decisions to alter or end therapy. Although determination of the ultimate utility of this method awaits further testing using human samples and prospective evaluation in the context of ongoing clinical care, it may bring us one step closer to rapid, specific diagnosis in these challenging cases. This work was supported in part by the American Lebanese Syrian Associated Charities (ALSAC), by the Anderson Charitable Foundation, by the intramural research program of the National Cancer Institute, and by the Henry Schueler 41&9 Foundation. Dr Walsh is a Henry Schueler Foundation Scholar in Mucormycosis. We thank Markus Morgenstern, BS, and Alicia Rodriguez, MS, CCRP, for their technical and statistical support of this project. References 1. Hata DJ, Buckwalter SP, Pritt BS, Roberts GD, Wengenack NL. Real-time PCR method for detection of Zygomycetes. J Clin Microbiol. 2008;46(7):2353– 2358. 2. Schabereiter-Gurtner C, Selitsch B, Rotter ML, Hirschl AM, Willinger B. Development of novel real-time PCR assays for detection and differentiation of eleven medically important Aspergillus and Candida species in clinical specimens. J Clin Microbiol. 2007;45(3):906–914.

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3. Walsh TJ, Wissel MC, Grantham KJ, et al. Molecular detection and speciesspecific identification of medically important Aspergillus species by real-time PCR in experimental invasive pulmonary aspergillosis. J Clin Microbiol. 2011; 49(12):4150–4157. 4. Cortez KJ, Roilides E, Quiroz-Telles F, et al. Infections caused by Scedosporium spp. Clin Microbiol Rev. 2008;21(1):157–197. 5. Kriengkauykiat J, Ito JI, Dadwal SS. Epidemiology and treatment approaches in management of invasive fungal infections. Clin Epidemiol. 2011; 3:175–191. doi:10.2147/CLEP.S12502. 6. Low CY, Rotstein C. Emerging fungal infections in immunocompromised patients. F1000 Med Rep. 2011;3:14. doi:10.3410/M3-14. 7. Revankar SG, Sutton DA. Melanized fungi in human disease. Clin Microbiol Rev. 2010;23(4):884–928. 8. Ribes JA, Vanover-Sams CL, Baker DJ. Zygomycetes in human disease. Clin Microbiol Rev. 2000;13(2):236–301. 9. Nucci M. Use of antifungal drugs in hematology. Rev Bras Hematol Hemoter. 2012;34(5):383–391. 10. Van Thiel DH, George M, Moore CM. Fungal infections: their diagnosis and treatment in transplant recipients. Int J Hepatol. 2012;2012:106923. doi:10. 1155/2012/106923. 11. Chen X, Zehnbauer B, Gnirke A, Kwok PY. Fluorescence energy transfer detection as a homogeneous DNA diagnostic method. Proc Natl Acad Sci U S A. 1997;94(20):10756–10761. 12. Johnson SC, Marshall DJ, Harms G, et al. Multiplexed genetic analysis using an expanded genetic alphabet. Clin Chem. 2004;50(11):2019–2027. 13. Moser MJ, Prudent JR. Enzymatic repair of an expanded genetic information system. Nucleic Acids Res. 2003;31(17):5048–5053. 14. Sherrill CB, Marshall DJ, Moser MJ, et al. Nucleic acid analysis using an expanded genetic alphabet to quench fluorescence. J Am Chem Soc. 2004; 126(14):4550–4556. 15. Committee on the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council. Guide for the Care and Use of Laboratory Animals. Washington, DC: National Academy Press; 1996. 16. Walsh TJ, Bacher J, Pizzo PA. Chronic silastic central venous catheterization for induction, maintenance and support of persistent granulocytopenia in rabbits. Lab Anim Sci. 1988;38(4):467–471. 17. Francis P, Lee JW, Hoffman A, et al. Efficacy of unilamellar liposomal amphotericin B in treatment of pulmonary aspergillosis in persistently granulocytopenic rabbits: the potential role of bronchoalveolar d-mannitol and serum galactomannan as markers of infection. J Infect Dis. 1994;169(2):356–368. 18. Petraitis V, Petraitiene R, Antachopoulos C, et al. Increased virulence of Cunninghamella bertholletiae in experimental pulmonary mucormycosis: correlation with circulating molecular biomarkers, sporangiospore germination and hyphal metabolism. Med Mycol. 2013;51(1):72–82. 19. Agresti A, Coull BA. Approximate is better than ‘‘exact’’ for interval estimation of binomial proportions. Am Stat. 1998;52(2):119–126. 20. Madej RM, Caliendo AM, Day SP, et al. Limits of quantitation In: Madej RM, Caliendo AM, Day SP, et al, eds. Quantitative Molecular Methods for Infectious Diseases; Approved Guideline. 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute; 2010 NCCLS document MM6-A;23(28):7.5.3. 21. Higuchi R, Fockler C, Dollinger G, Watson R. Kinetic PCR analysis: realtime monitoring of DNA amplification reactions. Biotechnology (N Y). 1993; 11(9):1026–1030. 22. Gish RG. Standards of treatment in chronic hepatitis C. Semin Liver Dis. 1999;19(suppl 1):S35–S47. 23. Torelli R, Sanguinetti M, Moody A, et al. Diagnosis of invasive aspergillosis by a commercial real-time PCR assay for Aspergillus DNA in bronchoalveolar lavage fluid samples from high-risk patients compared to a galactomannan enzyme immunoassay. J Clin Microbiol. 2011;49(12):4273–4278. 24. White PL, Bretagne S, Klingspor L, et al. Aspergillus PCR: one step closer to standardization. J Clin Microbiol. 2010;48(4):1231–1240. 25. White PL, Perry MD, Moody A, Follett SA, Morgan G, Barnes RA. 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(6):2169–2174. 26. Munoz-Cadavid C, Rudd S, Zaki SR, et al. Improving molecular detection ˜ of fungal DNA in formalin-fixed paraffin-embedded tissues: comparison of five tissue DNA extraction methods using panfungal PCR. J Clin Microbiol. 2010; 48(6):2147–2153. 27. Khot PD, Fredricks DN. PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther. 2009;7(10):1201–1221.

Labeled Primer PCR for Fungal Detection—Gu et al

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Quantitative multiplexed detection of common pulmonary fungal pathogens by labeled primer polymerase chain reaction.

Invasive fungal infections are an important cause of morbidity and mortality among immunocompromised patients...
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