Retrospective Comparison of Nucleic Acid Sequence–Based Amplification, Real-Time PCR, and Galactomannan Test for Diagnosis of Invasive Aspergillosis
2014; Elsevier BV; Volume: 16; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2014.05.001
ISSN1943-7811
AutoresLipeng Wang, Yunyan He, Yun Xia, Xiaoyan Su, Huijuan Wang, Shu‐Mei Liang,
Tópico(s)Plant Pathogens and Fungal Diseases
ResumoInvasive aspergillosis is a life-threatening infection in immunocompromised patients, and treating these infections at an early stage is often crucial for a favorable outcome. Early diagnosis, however, remains challenging. We performed a retrospective comparison of three methods: real-time quantitative PCR (qPCR), nucleic acid sequence-based amplification (NASBA), and galactomannan enzyme-linked immunosorbent assay (GM-ELISA); these detect circulating Aspergillus DNA, RNA, and galactomannan, respectively. Blood samples from 80 patients at high risk for invasive aspergillosis were tested by each assay. The sensitivity of NASBA, qPCR, and GM-ELISA was 76.47% (95% CI, 58.4–88.6%), 67.65% (95% CI, 49.4–82.0%), and 52.94% (95% CI, 35.4–69.8%), respectively, and the specificity was 80.43% (95% CI, 65.6–90.1%), 89.13% (95% CI, 75.6–95.9%), and 80.43% (95% CI, 65.6–90.1%), respectively. We also evaluated the efficiency of the three tests in various combinations. Perfect specificity (100%; 95% CI, 90.4–100%) and perfect positive predictive value (100%; 95% CI, 77.1–100%) were achieved by combining NASBA and qPCR testing in series. Testing with both NASBA and qPCR in parallel was the most sensitive and had the highest Youden index. Our data support the great potential of NASBA and qPCR, singly or in combination, for diagnosis of invasive aspergillosis in high-risk populations. Invasive aspergillosis is a life-threatening infection in immunocompromised patients, and treating these infections at an early stage is often crucial for a favorable outcome. Early diagnosis, however, remains challenging. We performed a retrospective comparison of three methods: real-time quantitative PCR (qPCR), nucleic acid sequence-based amplification (NASBA), and galactomannan enzyme-linked immunosorbent assay (GM-ELISA); these detect circulating Aspergillus DNA, RNA, and galactomannan, respectively. Blood samples from 80 patients at high risk for invasive aspergillosis were tested by each assay. The sensitivity of NASBA, qPCR, and GM-ELISA was 76.47% (95% CI, 58.4–88.6%), 67.65% (95% CI, 49.4–82.0%), and 52.94% (95% CI, 35.4–69.8%), respectively, and the specificity was 80.43% (95% CI, 65.6–90.1%), 89.13% (95% CI, 75.6–95.9%), and 80.43% (95% CI, 65.6–90.1%), respectively. We also evaluated the efficiency of the three tests in various combinations. Perfect specificity (100%; 95% CI, 90.4–100%) and perfect positive predictive value (100%; 95% CI, 77.1–100%) were achieved by combining NASBA and qPCR testing in series. Testing with both NASBA and qPCR in parallel was the most sensitive and had the highest Youden index. Our data support the great potential of NASBA and qPCR, singly or in combination, for diagnosis of invasive aspergillosis in high-risk populations. CME Accreditation Statement: This activity ("JMD 2014 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. CME Accreditation Statement: This activity ("JMD 2014 CME Program in Molecular Diagnostics") has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians. The ASCP designates this journal-based CME activity ("JMD 2014 CME Program in Molecular Diagnostics") for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity. CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose. Invasive aspergillosis (IA), an opportunistic fungal infection, is increasingly recognized as a major cause of morbidity and mortality in immunocompromised patients, including those receiving aggressive chemotherapy or immunosuppressive drugs.1Chandrasekar P. Diagnostic challenges and recent advances in the early management of invasive fungal infections.Eur J Haematol. 2010; 84: 281-290Crossref PubMed Scopus (35) Google Scholar, 2Sanz Alonso M.A. Jarque Ramos I. Salavert Lletí M. Pemán J. Epidemiology of invasive fungal infections due to Aspergillus spp. and Zygomycetes.Clin Microbiol Infect. 2006; 12 Suppl s7: 2-6Abstract Full Text Full Text PDF Scopus (38) Google Scholar, 3Leventakos K. Lewis R.E. Kontoyiannis D.P. Fungal infections in leukemia patients: how do we prevent and treat them.Clin Infect Dis. 2010; 50: 405-415Crossref PubMed Scopus (120) Google Scholar At present, despite great advances in imaging and antigen-based serological detection, IA remains difficult to diagnose. Because early diagnosis is important for improved outcomes, much effort has been devoted to developing diagnostic assays targeting fungal biomarkers, with the potential for new paradigms in prevention and early treatment of IA. One of the most attractive fungal biomarkers is galactomannan (GM), a polysaccharide component of fungal cell wall, which can be released into serum and bronchoalveolar lavage (BAL) fluid during fungal infection. Measurement of GM can now be achieved by using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Platelia Aspergillus; Bio-Rad Laboratories, Hercules, CA). This assay was cleared by the US Food and Drug Administration for marketing in 2009 (http://www.accessdata.fda.gov/cdrh_docs/pdf9/k093678.pdf, last accessed May 14, 2014). Despite its substantial contribution to improving diagnosis of IA, this assay lacks species specificity and cannot differentiate among Aspergillus spp. Additionally, false-positive results may occur because of cross-reactivity with certain antibiotics or parenteral nutrition preparations.4Ascioglu S. Rex J.H. de Pauw B. Bennett J.E. Bille J. Crokaert F. Denning D.W. Donnelly J.P. Edwards J.E. Erjavec Z. Fiere D. Lortholary O. Maertens J. Meis J.F. Patterson T.F. Ritter J. Selleslag D. Shah P.M. Stevens D.A. Walsh T.J. Invasive Fungal Infections Cooperative Group of the European Organization for Research and Treatment of Cancer; Mycoses Study Group of the National Institute of Allergy and Infectious DiseasesDefining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus.Clin Infect Dis. 2002; 34: 7-14Crossref PubMed Scopus (2153) Google Scholar, 5Maertens J. Theunissen K. Lagrou K. Galactomannan testing.in: Pasqualotto A.C. Aspergillosis: from diagnosis to prevention. Springer Publishers, London2010: 105-124Google Scholar PCR can be used to detect pathogen genes in clinical samples, allowing early diagnosis of IA. Although a variety of Aspergillus PCR assays have been reported, including conventional PCR and real-time quantitative PCR (qPCR),6White P.L. Bretagne S. Klingspor L. Melchers W.J. McCulloch E. Schulz B. Finnstrom N. Mengoli C. Barnes R.A. Donnelly J.P. Loeffler J. European Aspergillus PCR InitiativeAspergillus PCR: one step closer to standardization.J Clin Microbiol. 2010; 48: 1231-1240Crossref PubMed Scopus (222) Google Scholar, 7White P.L. Mengoli C. Bretagne S. Cuenca-Estrella M. Finnstrom N. Klingspor L. Melchers W.J. McCulloch E. Barnes R.A. Donnelly J.P. Loeffler J. European Aspergillus PCR Initiative (EAPCRI)Evaluation of Aspergillus PCR protocols for testing serum specimens.J Clin Microbiol. 2011; 49: 3842-3848Crossref PubMed Scopus (115) Google Scholar, 8White P.L. Linton C.J. Perry M.D. Johnson E.M. Barnes R.A. The evolution and evaluation of a whole blood polymerase chain reaction assay for the detection of invasive aspergillosis in hematology patients in a routine clinical setting.Clin Infect Dis. 2006; 42: 479-486Crossref PubMed Scopus (170) Google Scholar these PCR applications lack standardization and clinical validation,9Mengoli C. Cruciani M. Barnes R.A. Loeffler J. Donnelly J.P. Use of PCR for diagnosis of invasive aspergillosis: systematic review and meta-analysis.Lancet Infect Dis. 2009; 9 ([Erratum appeared in Lancet Infect Dis 2009, 9:280]): 89-96Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar and thus have not yet been approved for clinical use according to the revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the NIH National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.10De Pauw B. Walsh T.J. Donnelly J.P. Stevens D.A. Edwards J.E. Calandra T. Pappas P.G. Maertens J. Lortholary O. Kauffman C.A. Denning D.W. Patterson T.F. Maschmeyer G. Bille J. Dismukes W.E. Herbrecht R. Hope W.W. Kibbler C.C. Kullberg B.J. Marr K.A. Muñoz P. Odds F.C. Perfect J.R. Restrepo A. Ruhnke M. Segal B.H. Sobel J.D. Sorrell T.C. Viscoli C. Wingard J.R. Zaoutis T. Bennett J.E. European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group; National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus GroupRevised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.Clin Infect Dis. 2008; 46: 1813-1821Crossref PubMed Scopus (4035) Google Scholar Nucleic acid sequence-based amplification (NASBA) is an RNA-directed isothermal transcription-based amplification process that specifically amplifies RNA even in the presence of genomic DNA. The amplification efficiency of NASBA is more robust than that of PCR, yielding more than 1012 amplicons in as little as 30 minutes.11Zhao Y. Park S. Kreiswirth B.N. Ginocchio C.C. Veyret R. Laayoun A. Troesch A. Perlin D.S. Rapid real-time nucleic acid sequence-based amplification–molecular beacon platform to detect fungal and bacterial bloodstream infections.J Clin Microbiol. 2009; 47: 2067-2078Crossref PubMed Scopus (67) Google Scholar, 12Compton J. Nucleic acid sequence-based amplification.Nature. 1991; 350: 91-92Crossref PubMed Scopus (1017) Google Scholar The advantages of NASBA over PCR in simplicity, speed, and sensitivity have stimulated interest in evaluating its application to detection of Aspergillus RNA in clinical samples. Results from a few studies of small scope support NASBA as an innovative way for early diagnosis of IA,13Loeffler J. Hebart H. Cox P. Flues N. Schumacher U. Einsele H. Nucleic acid sequence-based amplification of Aspergillus RNA in blood samples.J Clin Microbiol. 2001; 39: 1626-1629Crossref PubMed Scopus (86) Google Scholar, 14Zhao Y. Perlin D.S. Quantitative detection of Aspergillus spp. by real-time nucleic acid sequence-based amplification.Methods Mol Biol. 2013; 968: 83-92Crossref PubMed Scopus (12) Google Scholar but clinical validation of this test by retrospective studies has been lacking. In the present study, we enrolled 80 patients who were at high risk for IA, measured their circulating Aspergillus GM, DNA, and RNA in blood samples by GM-ELISA, qPCR, and NASBA, respectively, and retrospectively compared the diagnostic performance of these three methods by using receiver operating characteristic (ROC) curve and statistical analyses. This retrospective study was conducted at the Department of Laboratory Medicine, First Affiliated Hospital of Chongqing Medical University, from November 2012 to June 2013. A total of 80 inpatients at high risk for IA were enrolled. The definition of high risk, adapted from Galimberti et al15Galimberti R. Torre A.C. Baztán M.C. Rodriguez-Chiappetta F. Emerging systemic fungal infections.Clin Dermatol. 2012; 30: 633-650Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar and Dimopoulos et al,16Dimopoulos G. Frantzeskaki F. Poulakou G. Armaganidis A. Invasive aspergillosis in the intensive care unit.Ann N Y Acad Sci. 2012; 1272: 31-39Crossref PubMed Scopus (48) Google Scholar included patients who had a high (1,3)-β-d-glucan level (defined as >60 pg/mL), immunocompromised status, and at least one of the following six conditions: i) recipient of allogeneic stem cell transplant; ii) hematological disease; iii) severe immunodeficiency; iv) prolonged use of corticosteroids; v) fever or chest infiltrates unresponsive to routine antibiotics; and vi) implicit invasive fungal disease by radiological examination. Clinical details are provided in Supplemental Table S1. All patients were tested at enrollment for their (1,3)-β-d-glucan level using an MB-80 microbiology kinetic rapid reader (Jinshanchuan, Beijing, China); 19 patients were tested again within 1 week. GM level was not included in the criteria for high-risk IA. None of the patients received any antifungal therapy until after the first blood samples had been collected. Only the blood samples collected at enrollment were used in the comparative analysis of the NASBA, qPCR, and GM-ELISA assays. Plasma was separated from each fresh blood sample by centrifugation at 2500 × g, aliquoted, and stored at −80°C. All samples were tested in batches within 1 week after collection. The blood collection tubes were all from the same lot (Axygen; Corning Life Sciences, Tewksbury, MA) and were free of Aspergillus DNA contamination, as indicated by testing of randomly selected tubes from each package. The laboratory in which this study was conducted practices strict spatial separation of all steps involved in DNA and RNA amplification, and disposable plasticware and biosafety cabinets with regular UV irradiation are used to reduce risk of contamination. These studies were approved by the Chongqing Medical University Institutional Review Board. The GM level in blood samples was measured by ELISA using Platelia kits (Bio-Rad Laboratories, Hercules, CA) according to the manufacturer's recommendations. The optical density of each specimen and control was measured with a Sunrise microplate reader (Tecan, Männedorf, Switzerland) at 450 nm and 620 nm. Enzyme immunoassay data were expressed as the galactomannan index (GMI), defined as the double optical density value of the sample divided by the optical density value of the cutoff control provided in the test kit (GMI = ODcutoff control/2ODsample). The optimal GMI cutoff value was 0.5. DNA was extracted from plasma using a QIAamp blood mini kit (Qiagen, Hilden, Germany; Valencia, CA) according to the manufacturer's instructions. In brief, 200 μL plasma (restored to room temperature from −80°C) was mixed with 20 μL protease solution in a microcentrifuge tube. Then 200 μL buffer AL was added to the tube and mixed by pulse vortexing for 15 seconds. After incubation at 56°C for 10 minutes, 200 μL absolute ethanol was added, and the whole mixture was transferred to a QIAamp mini spin column. The column was centrifuged at 6000 × g for 1 minute, and the filtrate was discarded. After sequential washes with 500 μL buffer AW1, 500 μL buffer AW2, and 200 μL buffer AE, DNA was eluted with 40 μL buffer AE and stored at −20°C. Purified DNA was amplified by an Aspergillus genus-specific qPCR assay using SYBR Green chemistry and previously reported primers targeting the 28S rRNA gene (5′-GCACGTGAAATTGTTGAAAGG-3′ and 5′-CAGGCTGGCCGCATTG-3′).17Williamson E.C. Leeming J.P. Palmer H.M. Steward C.G. Warnock D. Marks D.I. Millar M.R. Diagnosis of invasive aspergillosis in bone marrow transplant recipients by polymerase chain reaction.Br J Haematol. 2000; 108: 132-139Crossref PubMed Scopus (103) Google Scholar PCR reactions were performed in an ABI 7500 sequence detection system (Applied Biosystems; Life Technologies, Carlsbad, CA) using 96-well plates with a total reaction volume of 20 μL per well containing 10 μL 2× SYBR Premix ExTaq II (Takara Bio, Otsu, Japan), 0.8 μL each primer, 0.4 μL 50× ROX reference dye II, 2 μL DNA, and 6 μL double-distilled water (ddH2O). Thermal cycling conditions were 95°C for 30 seconds, followed by 50 cycles of 95°C for 5 seconds and 60°C for 34 seconds. Blank control (ddH2O), negative control (DNA extracted from patients without IA), and positive control (DNA extracted from Aspergillus fumigatus in pure culture) were also included. Each sample was run in duplicate, and mean cycle numbers were used for analysis. The lower limit of detection was empirically determined to be 10 colony-forming units of Aspergillus conidia per reaction. Total RNA was extracted from plasma using a blood/liquid sample total RNA rapid extraction kit (BioTeke, Beijing, China) according to the manufacturer's instructions. In brief, 200 μL plasma (restored to room temperature from −80°C) was mixed with 600 μL lysis buffer RLS in a 1.5-mL microcentrifuge tube and agitated for 2 minutes, followed by incubation for 10 minutes at room temperature. After addition of 150 μL chloroform, the tube was vortexed vigorously for 15 seconds, followed by incubation for 3 minutes at room temperature. After centrifugation at 12,000 × g for 10 minutes at 4°C, the aqueous phase was transferred to a Spin-column AC and mixed with 500 μL of 70% ethanol. The column was washed sequentially with buffer RE and buffer RW. Finally, RNA was eluted with 100 μL RNase-free water and stored at −80°C. A highly conserved 18S rRNA region specific for the Aspergillus genus was chosen as the detection target; it was amplified using a pair of previously reported primers (P1, 5′-AATTCTAATACGACTCACTATAGGGGAGCAAAGGCCTGCTTTGAACA-3′; P2, 5′-GCCGCGGTAATTCCAGCTCCAATA-3′).13Loeffler J. Hebart H. Cox P. Flues N. Schumacher U. Einsele H. Nucleic acid sequence-based amplification of Aspergillus RNA in blood samples.J Clin Microbiol. 2001; 39: 1626-1629Crossref PubMed Scopus (86) Google Scholar, 18Perlin D.S. Zhao Y. Molecular diagnostic platforms for detecting Aspergillus.Med Mycol. 2009; 47 Suppl 1: S223-S232Crossref PubMed Scopus (24) Google Scholar Aspergillus RNA sample (5 μL) was mixed with 10 μL of amplification buffer containing 40 mmol/L Tris-HCl (pH 8.5), 70 mmol/L KCl, 5 mmol/L dithiothreitol, 12 mmol/L MgCl2, 0.375 mol/L sorbitol, 12 U ribonuclease inhibitor (Promega, Fitchburg, WI), 0.4 μmol/L each primer, and 2 mmol/L each NTP (Takara Bio) in 10% dimethyl sulfoxide. The mixture was incubated at 65°C for 5 minutes, then cooled to 41°C for 5 minutes to allow primer annealing, followed by addition of 5 μL enzyme mixture containing 2.0 μg bovine serum albumin, 0.1 U RNase H, 40 U T7 RNA polymerase (Promega), and 8 U avian myeloblastosis virus reverse transcriptase (AMV-RT) (Takara Bio).19Gill P. Ramezani R. Amiri M.V. Ghaemi A. Hashempour T. Eshraghi N. Ghalami M. Tehrani H.A. Enzyme-linked immunosorbent assay of nucleic acid sequence-based amplification for molecular detection of M. tuberculosis.Biochem Biophys Res Commun. 2006; 347: 1151-1157Crossref PubMed Scopus (34) Google Scholar The reaction mixture was incubated at 41°C for 90 minutes, and the products were analyzed by 1% agarose gel electrophoresis. Blank control (ddH2O), negative control (RNA extracted from patients without IA), and positive control (RNA extracted from Aspergillus fumigatus in pure culture) were included in each run. Performance parameters for each assay were evaluated by construction of 2 × 2 tables. For calculation of the sensitivity of the three methods, positive results from patients with proven or probable IA according to the EORTC/MSG criteria were considered to be true positives. For calculation of specificity, negative results from patients without IA evidence based on the EORTC/MSG criteria were considered to be true negatives. The ROC curves for qPCR cycle threshold (CT) values were computed using IBM SPSS Statistics software package version 20 (IBM, Armonk, NY). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio, and negative likelihood ratio were determined for each assay. Performance parameters of diagnostic assays were analyzed using the R statistical package version 3.0.0 with DT ComPair software. Comparison between two assays was performed by paired diagnosis test design, McNemar test, with a P value of <0.05 being considered significant. The Youden index (J = Sensitivity + Specificity − 1) was calculated to evaluate the synthetic ability of each assay. The κ statistic was determined and used to evaluate the consistency of the three methods,20White P.L. Parr C. Thornton C. Barnes R.A. Evaluation of real-time PCR, galactomannan enzyme-linked immunosorbent assay (ELISA), and a novel lateral-flow device for diagnosis of invasive aspergillosis.J Clin Microbiol. 2013; 51: 1510-1516Crossref PubMed Scopus (116) Google Scholar as follows: excellent agreement between tests, κ ≥ 0.80; substantial agreement, 0.60 < κ ≤ 0.80; moderate agreement, 0.40 < κ ≤ 0.60; and poor agreement, κ ≤ 0.40. A total of 80 adult patients at high risk for IA were enrolled. These patients were categorized as proven IA (n = 8), probable IA (n = 26), or non-IA (n = 46) according to the 2008 revised EORTC/MSG definitions10De Pauw B. Walsh T.J. Donnelly J.P. Stevens D.A. Edwards J.E. Calandra T. Pappas P.G. Maertens J. Lortholary O. Kauffman C.A. Denning D.W. Patterson T.F. Maschmeyer G. Bille J. Dismukes W.E. Herbrecht R. Hope W.W. Kibbler C.C. Kullberg B.J. Marr K.A. Muñoz P. Odds F.C. Perfect J.R. Restrepo A. Ruhnke M. Segal B.H. Sobel J.D. Sorrell T.C. Viscoli C. Wingard J.R. Zaoutis T. Bennett J.E. European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group; National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus GroupRevised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.Clin Infect Dis. 2008; 46: 1813-1821Crossref PubMed Scopus (4035) Google Scholar (Supplemental Table S1). All proven IA cases had evidence of Aspergillus invasion detected by histology or in vitro culture of specimens obtained from normally sterile but clinically abnormal sites, whereas all probable IA cases had radiological and/or mycological evidence of Aspergillus infection by direct examination or culture of specimens from sites that may be colonized. The 34 proven or probable IA cases came from various departments, centers, and units: 7 from intensive care, 3 each from hematology, neurology, and nephrology; 2 from geriatrics; and 1 each from pneumology, stem cell transplantation, gastroenterology, and dermatology. The sex distribution (24:10 male/female for proven or probable IA and 31:15 male/female for non-IA) and mean age (64.94 ± 13.34 years for proven or probable IA and 59.37 ± 17.52 years for non-IA) did not differ significantly between groups. No significant differences were observed in (1,3)-β-d-glucan levels between proven or probable IA and non-IA cases. The qPCR CT values were assessed by ROC analysis (Figure 1). The optimal threshold of the CT value was 39.2 (area under the curve = 0.765); the cutoff value of GMI recommended by the manufacturer and in the EORTC/MSG criteria is 0.5. A sample was considered positive if CT ≤ 39.2 or GMI ≥ 0.5; otherwise, it was considered negative. The NASBA results were determined based on presence (positive) or absence (negative) of the expected RNA band in agarose gels. All samples from proven or probable IA patients with negative qPCR or NASBA results were retested at 1:10 dilution. None of the retested samples became positive, suggesting a lack of inhibition. A total of 52 samples were identified as positive by at least one of the three tests (NASBA, qPCR, and GM-ELISA). Of these, 26 were positive in advance of clinical diagnosis as proven or probable IA and 7 were positive after clinical diagnosis as proven or probable IA; the remaining 19 were positive either before or after clinical diagnosis as non-IA (ie, false positive) (Supplemental Table S2). The results of NASBA, qPCR, and GM-ELISA assays are listed in Table 1. For each assay, the diagnostic parameters sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values, and Youden index were calculated (Table 2). Comparison of the three assays revealed that NASBA had the highest sensitivity, at 76.47% (95% CI, 58.4–88.6%), and qPCR had the highest specificity, at 89.13% (95% CI, 75.6–95.9%). Hypothesis testing of sensitivity and specificity revealed that NASBA sensitivity was significantly greater than that of GM-ELISA; there were no other significant differences among the three tests (Table 2). In addition, the NASBA assay was the best performing assay for negative likelihood ratio, negative predictive value, and Youden index, and qPCR was the best for positive likelihood ratio, positive predictive value, and κ value.Table 1Results of NASBA, qPCR, and GM-ELISA for Diagnosis of IAEORTC/MSG criteriaNASBAqPCRGM-ELISATotal+−+−+−+2682311181634−93754193746Total35452852275380EORTC/MSG, European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the NIH National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.10De Pauw B. Walsh T.J. Donnelly J.P. Stevens D.A. Edwards J.E. Calandra T. Pappas P.G. Maertens J. Lortholary O. Kauffman C.A. Denning D.W. Patterson T.F. Maschmeyer G. Bille J. Dismukes W.E. Herbrecht R. Hope W.W. Kibbler C.C. Kullberg B.J. Marr K.A. Muñoz P. Odds F.C. Perfect J.R. Restrepo A. Ruhnke M. Segal B.H. Sobel J.D. Sorrell T.C. Viscoli C. Wingard J.R. Zaoutis T. Bennett J.E. European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group; National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus GroupRevised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.Clin Infect Dis. 2008; 46: 1813-1821Crossref PubMed Scopus (4035) Google Scholar Open table in a new tab Table 2Performance of NASBA, qPCR, and GM-ELISA in 34 Cases of Proven or Probable IAParameterNASBAqPCRGM-ELISASensitivity [% (95% CI)]76.47 (58.4–88.6)67.65 (49.4–82.0)52.94 (35.4–69.8)∗P < 0.05 versus NASBA.Specificity [% (95% CI)]80.43 (65.6–90.1)89.13 (75.6–95.9)80.43 (65.6–90.1)Positive likelihood ratio3.9086.2242.705Negative likelihood ratio0.29260.36300.5851PPV [% (95% CI)]74.29 (56.4–86.9)82.14 (62.4–93.2)66.67 (46.0–82.8)NPV [% (95% CI)]82.22 (67.4–91.5)78.85 (64.9–88.5)69.81 (55.5–81.3)Youden index0.56900.56780.3337κ statistic0.5670.5810.343Combined analysis for both proven IA (n = 8) and probable IA (n = 26). Best performance values are highlighted in bold.∗ P < 0.05 versus NASBA. Open table in a new tab EORTC/MSG, European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the NIH National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.10De Pauw B. Walsh T.J. Donnelly J.P. Stevens D.A. Edwards J.E. Calandra T. Pappas P.G. Maertens J. Lortholary O. Kauffman C.A. Denning D.W. Patterson T.F. Maschmeyer G. Bille J. Dismukes W.E. Herbrecht R. Hope W.W. Kibbler C.C. Kullberg B.J. Marr K.A. Muñoz P. Odds F.C. Perfect J.R. Restrepo A. Ruhnke M. Segal B.H. Sobel J.D. Sorrell T.C. Viscoli C. Wingard J.R. Zaoutis T. Bennett J.E. European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group; National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus GroupRevised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.Clin Infect Dis. 2008; 46: 1813-1821Crossref PubMed Scopus (4035) Google Scholar Combined analysis for both proven IA (n = 8) and probable IA (n = 26). Best performance values are highlighted in bold. To determine the optimal diagnostic strategy for IA, we compared the performance of various combinations of the three tests (Table 3). Parallel testing, in which any one positive yields a positive result (in contrast to serial testing, in which any one negative yields a negative result), may increase sensitivity but with accordingly decreased specificity. Perfect specificity (100%; 95% CI, 90.4–100%) and perfect positive predictive value (100%; 95% CI, 77.1–100%) were achieved by combining NASBA and qPCR in serial testing. A combination of NASBA and qPCR in parallel testing appeared to
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