TABLE 4

Clinical studies evaluating Aspergillus sp. PCR methods a

Study (reference no.)Date (yr) publishedStudy designPatient populationType of PCRType of specimen testedPrimer targetMethod utilized to determine accuracySensitivity (%)Specificity (%)Comments
PCR studies
    Bucheidt et al. (126)2001Retrospective67 febrile neutropenic patients and 33 immunocompetent individuals were tested with BAL fluid PCR, and 218 patients with hematologic malignancies and 60 immunocompetent individuals were tested with blood PCRNested PCRBAL fluid and whole blood18S rDNAComparison to MSG criteria100 for BAL fluid, 91.7 for whole blood92.6 for BAL fluid, 83.6 for whole blood
    Raad et al. (127)2002Prospective54 patients with cancer and pulmonary infiltrates; 4 had definite infectionTraditional PCR with detection through ethidium bromide stainingWhole bloodMitochondrial DNA and alkaline protease geneComparison to EORTC/MSG criteria100 for definite IA, 57 for probable and possible IA100
    Bucheidt et al. (128)2002Retrospective176 patients, including 141 febrile neutropenic patientsNested PCRBAL fluid18S rDNAComparison to EORTC/MSG criteria93.994.4
    Raad et al. (129)2002Prospective249 cancer patients with pulmonary infiltratesTraditional PCR with ethidium bromide staining or Southern blottingBAL fluidMitochondrial DNA and alkaline protease geneComparison to EORTC/MSG criteria80 for proven IA, 64 for probable IA93
    Las-Flörl et al. (130)2004Prospective36 patients receiving antifungals due to suspicious pulmonary infiltratesPCR-ELISA205 whole-blood specimens, 15 FNA or biopsy specimens, 21 BAL fluid or tracheal secretion specimens18S rRNA geneComparison to EORTC/MSG criteriaFor proven IA, 100 for FNA/biopsy specimens and 40 for whole blood; for probable IA, 66 for lung fluid and 44 for whole blood100 (all possible IA patients were considered truly negative)
    Buchheidt et al. (131)2004Prospective165 patients with hematologic malignancies or HSCT from 6 centersNested PCR followed by ethidium bromide staining. Positive nested PCR specimens were also tested by qPCR with fluorescent probes1,522 samples of various types18S rRNA gene for nested PCR and mitochondrial cytochrome b gene for real-time PCRComparison to EORTC/MSG criteria63.6 for nested PCR63.5 for nested PCRPossible IA cases were not included in the sensitivity and specificity determinations. Sensitivity dropped to 36.4% and specificity increased to 92.3% when only patients with at least 2 positive PCR results were considered “PCR positive.”
    Las-Flörl et al. (133)2005Retrospective16 hematologic malignancy patients with proven or probable IAPCR-ELISA108 whole-blood specimens, 9 FNA or tissue biopsy specimens, and 7 BAL fluid or tracheal secretion specimens18S rRNA geneComparison to EORTC/MSG criteriaFor proven IA, 100 for FNA/tissue samples and 66 for whole blood; for probable IA, 85 for BAL fluid/tracheal secretions and 57 for whole bloodNA due to study designSensitivity of whole-blood PCR dropped to 54 and 42% for proven and probable IA, respectively, when tested during antifungal therapy. Consecutive positive PCR results were associated with fatal outcomes.
    Halliday et al. (134)2005Prospective29 adults and 36 children with febrile neutropenia, undergoing intensive chemotherapy for hematologic malignancy or having received a hematopoietic stem cell transplantNested PCR followed by ethidium bromide staining998 whole-blood samples from 95 episodes of febrile neutropenia18S rRNA geneComparison to EORTC/MSG criteria; proven and probable cases were considered true-positive cases, cases with no evidence of IA were considered true-negative cases, and possible cases were examined differently100 for methods A and B, 70.6 for method C, 100 for method D75.4 for methods A and B, 75.4 for method C, 74.7 for method DAt least two positive PCR results were required for a case to be considered PCR positive. Positive PCR was the earliest indicator of IA, by a mean of 14 days. Antifungal therapy did not affect positive PCR results.
    Scotter and Chambers (132)2005Retrospective25 patients with hematologic malignanciesPCR-ELISABlood Comparison to EORTC/MSG criteria10085Possible IA cases were considered truly negative. GM assay of the same samples resulted in a sensitivity and specificity of 60 and 95%, respectively.
    Florent et al. (135)2006Prospective201 patients with hematologic malignanciesPCR-ELISASerumMitochondrial DNAComparison to EORTC/MSG criteriaFor proven cases, 100; for probable cases, 58.6–86.2*; for possible cases, 27.8–72.287.3–89.7 for consecutive positive results, 51.5–55.2 for single positive resultsCombined use of PCR-ELISA and galactomannan assay increased the sensitivity to 83.3%
    Hummel et al. (136)2006Retrospective6 patients with hematologic malignancies and probable, proven, or possible IANested PCR35 CSF samples18S rRNAComparison to EORTC/MSG criteriaEach patient had at least one positive CSF sampleNA
    Badiee et al. (137)2008Prospective194 patients with hematologic malignanciesPCR-ELISAWhole bloodrRNAComparison to EORTC/MSG criteria66 for proven and probable IA96
    Shahid et al. (138)2008Retrospective69 patients with bronchogenic carcinoma and 18 healthy controlsTraditional PCR with ethidium bromide stainingBAL fluid Comparison to EORTC/MSG criteria100 for proven and probable IA cases97 for non-IA cases, 100 for healthy controls
    Hummel et al. (139)2009Prospective71 pediatric and adolescent immunocompromised patientsNested PCR followed by ethidium bromide stainingVarious18S rRNAComparison to EORTC/MSG criteria80 for proven/probable IA, 32.4 for possible IA81 (drops to 71 if cases with possible IA are considered truly negative)Only 5 patients had proven/probable IA. Results were pooled for all different specimens tested. Patients with at least one positive PCR result were considered PCR positive
    Lopes Da Silva et al. (140)2010Prospective172 patients who received high-dose chemotherapyTraditional PCR followed by ethidium bromide stainingSerum and BAL fluid18S rRNAComparison to EORTC/MSG criteria75 (only proven and probable IA patients were considered truly positive)91.9The sensitivity and specificity of serum galactomannan assay were also tested (87.5% and 93%, respectively). The reported sensitivity and specificity refer to serum PCR. BAL fluid PCR was more sensitive (exact sensitivity not reported)
    Hummel et al. (141)2010Prospective91 patients within the AmBiLoad trialNested PCR followed by ethidium bromide staining454 blood samples (not specified), 3 BAL fluid samples, 1 bronchial aspirate, 1 muscle biopsy specimen18S rRNAComparison to EORTC/MSG criteria43 for proven IA, 39 for probable IANA due to study designLow sensitivity might be explained by the fact that all samples were received during antifungal treatment. Positive PCR results were associated with worse outcomes.
    Badiee et al. (142)2012Prospective62 pediatric patients at increased risk for IANested PCR followed by ethidium bromide stainingSerum Comparison to EORTC/MSG criteria8096.2Possible IA cases were excluded from the analysis.
    Reinwald et al. (143)2012Retrospective226 patients with hematologic malignanciesNested PCR followed by ethidium bromide stainingBAL fluid18S rRNAComparison to EORTC/MSG58 for proven/probable IA87 (possible IA cases were considered truly negative)Sensitivity dropped to 17% in considering only patients who were receiving at least two antifungals. Treatment with one antifungal agent during BAL sampling did not affect the PCR performance.
    Reinwald et al. (144)2012Prospective87 patients at high risk for IANested PCR followed by ethidium bromide stainingBAL fluid18S rRNAComparison to EORTC/MSG criteria5987 (possible IA cases were considered truly negative)For comparison, the sensitivity and specificity of BAL fluid GM testing on the same samples were 79% and 96%, respectively.
    Buess et al. (145)2012Prospective191 immunocompromised patients undergoing bronchoscopy for suspected pulmonary infectionNested PCR followed by ethidium bromide staining and sequencingBAL fluid18S rRNA and 5.8S rRNAComparison to EORTC/MSG criteria0 for proven IA, 50 for probable IA, 24 for possible IA70 when only no-IA patients were considered truly negativeOnly 3 patients had proven IA, and 8 had probable IA.
    Reinwald et al. (9)2013Prospective55 immunocompromised patients for whom central nervous system aspergillosis was suspectedNested PCR followed by ethidium bromide stainingCSF18S rRNAComparison to EORTC/MSG criteria100 for proven and probable IA93Possible IA cases were excluded from the analysis.
Real-time PCR studies
    Costa et al. (146)2002Retrospective20 patients with hematologic malignancies who had proven or probable IAReal-time PCR with fluorescein-labeled probesSerumMitochondrial DNAComparison to EORTC/MSG criteria70NAPlasma and white blood cell pellets were also tested by qPCR for some of the patients, yielding the same results as those obtained with the serum fraction. No frank increase in the DNA load during the course of disease was observed.
    Spiess et al. (147)2003Retrospective18 patients with hematologic malignancies with positive nested PCR results for Aspergillus and 50 healthy controlsReal-time PCR with fluorescein-labeled probesBAL fluid and whole bloodMitochondrial cytochrome b DNAComparison to EORTC/MSG criteria100 for BAL fluid, 43 for blood100Only samples that tested positive with a previously validated nested PCR test were included in the study.
    Sanguinetti et al. (148)2003Prospective44 patients undergoing bronchoscopy for suspicious pulmonary infiltratesReal-time PCR with fluorescein-labeled probeBAL fluid18S rRNAComparison to EORTC/MSG criteria90 for proven and probable IA100 (possible IA cases were considered truly negative)Galactomannan testing of the same BAL fluid samples proved to have 100% sensitivity. Nested PCR testing of the same samples also had 90% sensitivity and 100% specificity.
    Rantakokko-Jalava et al. (149)2003Retrospective66 patients at risk for IA and 33 immunocompetent controlsReal-time PCR with fluorescein-labeled probesBAL fluidMitochondrial tRNAComparison to EORTC/MSG criteria86 for proven IA, 50 for probable IA, 80 for possible IA93Due to the primer and probe design, the assay only detected A. fumigatus infection.
    Challier et al. (150)2004Retrospective41 immunocompromised patients at risk for IA and 29 controlsReal-time PCR with fluorescein-labeled probeSerum28S rRNAComparison to EORTC/MSG criteria100 for proven cases, 78.9 for probable casesAll controls had negative qPCR resultsELISA galactomannan testing of the same samples showed 75.2% sensitivity for proven and probable IA. The combination of galactomannan assay and qPCR testing yielded a 100% sensitivity for proven and probable IA.
    Kawazu et al. (52)2004Prospective96 patients at risk for IAReal-time PCR with fluorescein-labeled probesPlasma18S rRNA geneComparison to EORTC/MSG criteria5593The cutoff for positive PCR was selected to achieve a 93% specificity. Possible IA cases were considered truly positive. Galactomannan ELISA achieved a sensitivity of 100% at a cutoff value that had the same specificity.
    Musher et al. (72)2004Retrospective99 patients (49 cases of IA and 50 controls)Real-time PCR with fluorescein-labeled probeBAL fluid18S rRNAComparison to EORTC/MSG criteria67100The sensitivity and specificity of the BAL fluid galactomannan assay for the same patients were 76% and 94%, respectively, with a cutoff of 0.5. The probe for the PCR assay was designed to detect most Aspergillus species as well as Penicillium species.
    Millon et al. (151)2005Retrospective29 patients with at least one positive galactomannan testReal-time PCR with fluorescein-labeled probesSerumMitochondrial DNAComparison to EORTC/MSG criteria57.163.6Possible IA cases were disregarded. A PCR-positive result after the first GM-positive result was associated with a poor prognosis.
    White et al. (152)2006Prospective203 patients at risk for IFIReal-time nested PCR with hydrolysis (TaqMan) probesWhole blood28S rRNAComparison to EORTC/MSG criteria92.394.6Possible IA cases were disregarded. Only patients with serial positive PCR results were considered “PCR positive.”
    Cesaro et al. (153)2008Prospective62 pediatric patients at risk for IAReal-time PCR with fluorescent probesWhole blood18S rRNA geneComparison to EORTC/MSG criteria8837When two PCR-positive results were required for a case to be considered PCR positive, the sensitivity and specificity changed to 63% and 81%, respectively.
    Botterel et al. (154)2008Retrospective25 patients with at least 1 GM-positive serum sampleReal-time PCR with fluorescent probesSerumMitochondrial DNAComparison to EORTC/MSG criteria61.5 for probable and possible IA cases100Possible IA cases were considered true-positive cases and were PCR positive. Sensitivity decreases to 54.5% if only probable cases are considered.
    Suarez et al. (155)2008Prospective124 patients with hematologic malignancies undergoing chemotherapy or HSCTReal-time PCR with fluorescent probesSerum28S rRNAComparison to EORTC/MSG criteria100 when using large serum volumes for DNA extraction, 76.5 when using small serum volumes for DNA extraction96.7Two possible IA cases were considered truly positive. For comparison, GM test results for the same samples showed a sensitivity and specificity of 88.2% and 95.8%, respectively.
    Khot et al. (156)2008Retrospective81 patients with pneumoniaReal-time PCR with fluorescein-labeled probesBAL fluid18S rDNAComparison to EORTC/MSG criteria7788
    Ramirez et al. (157)2009Prospective127 patients at risk for IAReal-time PCR with fluorescein-labeled probes; species were determined by melting curve analysisWhole blood18S rRNAComparison to EORTC/MSG criteria100 for proven cases, 0 for probable cases100 if possible IA cases are disregarded.Only 1% of the 948 tested samples were PCR positive.
    Frealle et al. (158)2009Retrospective57 patients at risk for IAReal-time PCR with fluorescein-labeled probesBAL fluidMitochondrial DNAComparison to EORTC/MSG criteria50 for proven and probable IA cases100
    Cuenca-Estrella et al. (159)2009Prospective83 patients with febrile neutropeniaReal-time PCR with hydrolysis probe1,122 whole-blood samples and 1,122 serum samplesITS1Comparison to EORTC/MSG criteria91.694.4Cases with two consecutive positive PCR results were considered PCR positive. Combined with GM assay, the sensitivity increased to 100%. Possible IA cases were considered truly positive.
    Springer et al. (10)2011Prospective46 patients receiving either allogeneic SCT or myeloablative chemotherapyReal-time PCR with fluorescein-labeled probesWhole bloodMulticopy ribosomal operon region from ITS1 to 5.8S regionComparison to EORTC/MSG criteria55 for probable and possible IA (dropped to 27 when having more than one positive PCR result was considered “PCR positive”)75 (increased to 100 when having more than one positive PCR result was considered “PCR positive”)Possible IA cases were considered truly positive. Selective pathogen DNA enrichment using affinity purification unexpectedly caused a decrease in the sensitivity of the assay.
    Millon et al. (160)2011Retrospective44 patients with two sequential positive serum galactomannan results and a risk factor for IATwo different real-time PCR assays with hybridization probesSerumAssay 1, mitochondrial DNA; assay 2, 18S rRNAComparison to EORTC/MSG criteriaFor assay 1, 57.7; for assay 2, 50 (dropped to 53.8 and 46.2, respectively, when at least two positive results were needed for a PCR-positive outcome)For assay 1, 94.4; for assay 2, 66.7 (increased to 100 for both when at least two positive results were needed for a PCR-positive outcome)Due to the study design, no possible IA cases were included. The combination of the ribosomal and mitochondrial PCRs increased the sensitivity of IA diagnosis to 65.4%. Positive ribosomal PCR results were associated with a poor prognosis.
    White et al. (161)2011Retrospective31 patients (10 with proven/probable IA and 21 with no IA)Two different real-time PCR assays with fluorescently labeled probesSerumAssay 1, 28S rRNA; assay 2, 18S rRNAComparison to EORTC/MSG criteriaFor assay 1, 80; for assay 2, 70 (dropped to 50 and 60, respectively, when at least two positive results were needed for a PCR-positive outcome)For assay 1, 100; for assay 2, 90.5 (both reached 100 when at least two positive results were needed for a PCR-positive outcome)Assay 2 is a commercially available PCR assay for the diagnosis of IA.
    Bernal-Martinez et al. (162)2011Retrospective38 adult patients with a high clinical suspicion of IAReal-time PCR with fluorescently labeled probesSerum and whole bloodITS1Comparison to EORTC/MSG criteria100 for serum and 94.4 for blood for proven/probable IANAThe aim of the study was to compare the sensitivities of the same PCR on serum and blood specimens. The results show that both specimens achieve similar sensitivities. One positive PCR result was necessary to classify a patient as PCR positive.
    Luong et al. (163)2011Retrospective137 lung transplant recipientsReal-time PCR with fluorescently labeled probesBAL fluidNot specifiedComparison to EORTC/MSG criteria100 for proven/probable IA88For comparison, GM testing of the same BAL fluid samples resulted in a sensitivity and specificity of 93% and 89%, respectively, at a cutoff of 0.5
    Torelli et al. (164)2011Prospective158 patients from hematology and intensive care unitsReal-time PCR with fluorescently labeled probesBAL fluid18S rRNA geneComparison to EORTC/MSG criteria94.1 for proven and probable IA98.6
    Springer et al. (165)2013Retrospective, multicenter47 patients with proven/probable IA and 31 controlsVarious real-time PCR assaysSerum and whole bloodVariousComparison to EORTC/MSG criteria85.1 for whole blood, 78.7 for serum (dropped to 46.8 and 51.1, respectively, when two positive PCR results were needed to consider a case “PCR positive”)64.5 for blood, 83.9 for serum (increased to 93.5 and 100, respectively, when two positive PCR results were needed to consider a case “PCR positive”)Overall, no significant difference between the performances of the PCR assays on serum versus whole-blood specimens was found.
    Rogers et al. (166)2013Prospective278 patients undergoing intensive chemotherapy or HSCTTwo different real-time PCR assays (a nested and a single run assay)Whole blood28S rRNA (nested assay), ITS (single-run assay)Comparison to EORTC/MSG criteria69–87 for nested assay and 55–80 for single-run PCR assay36–63 for nested assay and 57–84 for single-run assayPossible IA cases were excluded from the analysis. Two centers were involved in the study, and the results were different between them, as evidenced by the ranges of sensitivity and specificity values.
    Li et al. (167)2013Prospective72 patients with hematologic malignancies and suffering from fever, 4 with normal temperatures, and 10 healthy volunteersReal-time PCR with hydrolysis probesWhole blood and plasma28S-ITS2 rRNA genesComparison to EORTC/MSG criteria90.9 for proven and probable IA73.4Possible IA cases were considered truly negative.
    Guinea et al. (168)2013Prospective175 patients with hematologic malignancies and at risk for IAReal-time PCR with fluorescent probesLower respiratory tract samples18S rRNAComparison to EORTC/MSG criteria93.382.9No proven IA cases were included.
  • a BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; EORTC/MSG: European Organization for Research and Treatment of Cancer/Mycoses Study Group; FNA, fine-needle aspiration; GM, galactomannan; HSCT, hematopoietic stem cell transplant; IA, invasive aspergillosis; ITS, internal transcriber spacer; qPCR, quantitative PCR; NA, not applicable.