TABLE 4.
Clinical studies evaluating Aspergillus sp. PCR methodsa
| Study (reference no.) | Date (yr) published | Study design | Patient population | Type of PCR | Type of specimen tested | Primer target | Method utilized to determine accuracy | Sensitivity (%) | Specificity (%) | Comments |
|---|---|---|---|---|---|---|---|---|---|---|
| PCR studies | ||||||||||
| Bucheidt et al. (126) | 2001 | Retrospective | 67 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 PCR | Nested PCR | BAL fluid and whole blood | 18S rDNA | Comparison to MSG criteria | 100 for BAL fluid, 91.7 for whole blood | 92.6 for BAL fluid, 83.6 for whole blood | |
| Raad et al. (127) | 2002 | Prospective | 54 patients with cancer and pulmonary infiltrates; 4 had definite infection | Traditional PCR with detection through ethidium bromide staining | Whole blood | Mitochondrial DNA and alkaline protease gene | Comparison to EORTC/MSG criteria | 100 for definite IA, 57 for probable and possible IA | 100 | |
| Bucheidt et al. (128) | 2002 | Retrospective | 176 patients, including 141 febrile neutropenic patients | Nested PCR | BAL fluid | 18S rDNA | Comparison to EORTC/MSG criteria | 93.9 | 94.4 | |
| Raad et al. (129) | 2002 | Prospective | 249 cancer patients with pulmonary infiltrates | Traditional PCR with ethidium bromide staining or Southern blotting | BAL fluid | Mitochondrial DNA and alkaline protease gene | Comparison to EORTC/MSG criteria | 80 for proven IA, 64 for probable IA | 93 | |
| Las-Flörl et al. (130) | 2004 | Prospective | 36 patients receiving antifungals due to suspicious pulmonary infiltrates | PCR-ELISA | 205 whole-blood specimens, 15 FNA or biopsy specimens, 21 BAL fluid or tracheal secretion specimens | 18S rRNA gene | Comparison to EORTC/MSG criteria | For proven IA, 100 for FNA/biopsy specimens and 40 for whole blood; for probable IA, 66 for lung fluid and 44 for whole blood | 100 (all possible IA patients were considered truly negative) | |
| Buchheidt et al. (131) | 2004 | Prospective | 165 patients with hematologic malignancies or HSCT from 6 centers | Nested PCR followed by ethidium bromide staining. Positive nested PCR specimens were also tested by qPCR with fluorescent probes | 1,522 samples of various types | 18S rRNA gene for nested PCR and mitochondrial cytochrome b gene for real-time PCR | Comparison to EORTC/MSG criteria | 63.6 for nested PCR | 63.5 for nested PCR | Possible 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) | 2005 | Retrospective | 16 hematologic malignancy patients with proven or probable IA | PCR-ELISA | 108 whole-blood specimens, 9 FNA or tissue biopsy specimens, and 7 BAL fluid or tracheal secretion specimens | 18S rRNA gene | Comparison to EORTC/MSG criteria | For 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 blood | NA due to study design | Sensitivity 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) | 2005 | Prospective | 29 adults and 36 children with febrile neutropenia, undergoing intensive chemotherapy for hematologic malignancy or having received a hematopoietic stem cell transplant | Nested PCR followed by ethidium bromide staining | 998 whole-blood samples from 95 episodes of febrile neutropenia | 18S rRNA gene | Comparison 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 differently | 100 for methods A and B, 70.6 for method C, 100 for method D | 75.4 for methods A and B, 75.4 for method C, 74.7 for method D | At 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) | 2005 | Retrospective | 25 patients with hematologic malignancies | PCR-ELISA | Blood | Comparison to EORTC/MSG criteria | 100 | 85 | Possible 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) | 2006 | Prospective | 201 patients with hematologic malignancies | PCR-ELISA | Serum | Mitochondrial DNA | Comparison to EORTC/MSG criteria | For proven cases, 100; for probable cases, 58.6–86.2*; for possible cases, 27.8–72.2 | 87.3–89.7 for consecutive positive results, 51.5–55.2 for single positive results | Combined use of PCR-ELISA and galactomannan assay increased the sensitivity to 83.3% |
| Hummel et al. (136) | 2006 | Retrospective | 6 patients with hematologic malignancies and probable, proven, or possible IA | Nested PCR | 35 CSF samples | 18S rRNA | Comparison to EORTC/MSG criteria | Each patient had at least one positive CSF sample | NA | |
| Badiee et al. (137) | 2008 | Prospective | 194 patients with hematologic malignancies | PCR-ELISA | Whole blood | rRNA | Comparison to EORTC/MSG criteria | 66 for proven and probable IA | 96 | |
| Shahid et al. (138) | 2008 | Retrospective | 69 patients with bronchogenic carcinoma and 18 healthy controls | Traditional PCR with ethidium bromide staining | BAL fluid | Comparison to EORTC/MSG criteria | 100 for proven and probable IA cases | 97 for non-IA cases, 100 for healthy controls | ||
| Hummel et al. (139) | 2009 | Prospective | 71 pediatric and adolescent immunocompromised patients | Nested PCR followed by ethidium bromide staining | Various | 18S rRNA | Comparison to EORTC/MSG criteria | 80 for proven/probable IA, 32.4 for possible IA | 81 (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) | 2010 | Prospective | 172 patients who received high-dose chemotherapy | Traditional PCR followed by ethidium bromide staining | Serum and BAL fluid | 18S rRNA | Comparison to EORTC/MSG criteria | 75 (only proven and probable IA patients were considered truly positive) | 91.9 | The 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) | 2010 | Prospective | 91 patients within the AmBiLoad trial | Nested PCR followed by ethidium bromide staining | 454 blood samples (not specified), 3 BAL fluid samples, 1 bronchial aspirate, 1 muscle biopsy specimen | 18S rRNA | Comparison to EORTC/MSG criteria | 43 for proven IA, 39 for probable IA | NA due to study design | Low 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) | 2012 | Prospective | 62 pediatric patients at increased risk for IA | Nested PCR followed by ethidium bromide staining | Serum | Comparison to EORTC/MSG criteria | 80 | 96.2 | Possible IA cases were excluded from the analysis. | |
| Reinwald et al. (143) | 2012 | Retrospective | 226 patients with hematologic malignancies | Nested PCR followed by ethidium bromide staining | BAL fluid | 18S rRNA | Comparison to EORTC/MSG | 58 for proven/probable IA | 87 (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) | 2012 | Prospective | 87 patients at high risk for IA | Nested PCR followed by ethidium bromide staining | BAL fluid | 18S rRNA | Comparison to EORTC/MSG criteria | 59 | 87 (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) | 2012 | Prospective | 191 immunocompromised patients undergoing bronchoscopy for suspected pulmonary infection | Nested PCR followed by ethidium bromide staining and sequencing | BAL fluid | 18S rRNA and 5.8S rRNA | Comparison to EORTC/MSG criteria | 0 for proven IA, 50 for probable IA, 24 for possible IA | 70 when only no-IA patients were considered truly negative | Only 3 patients had proven IA, and 8 had probable IA. |
| Reinwald et al. (9) | 2013 | Prospective | 55 immunocompromised patients for whom central nervous system aspergillosis was suspected | Nested PCR followed by ethidium bromide staining | CSF | 18S rRNA | Comparison to EORTC/MSG criteria | 100 for proven and probable IA | 93 | Possible IA cases were excluded from the analysis. |
| Real-time PCR studies | ||||||||||
| Costa et al. (146) | 2002 | Retrospective | 20 patients with hematologic malignancies who had proven or probable IA | Real-time PCR with fluorescein-labeled probes | Serum | Mitochondrial DNA | Comparison to EORTC/MSG criteria | 70 | NA | Plasma 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) | 2003 | Retrospective | 18 patients with hematologic malignancies with positive nested PCR results for Aspergillus and 50 healthy controls | Real-time PCR with fluorescein-labeled probes | BAL fluid and whole blood | Mitochondrial cytochrome b DNA | Comparison to EORTC/MSG criteria | 100 for BAL fluid, 43 for blood | 100 | Only samples that tested positive with a previously validated nested PCR test were included in the study. |
| Sanguinetti et al. (148) | 2003 | Prospective | 44 patients undergoing bronchoscopy for suspicious pulmonary infiltrates | Real-time PCR with fluorescein-labeled probe | BAL fluid | 18S rRNA | Comparison to EORTC/MSG criteria | 90 for proven and probable IA | 100 (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) | 2003 | Retrospective | 66 patients at risk for IA and 33 immunocompetent controls | Real-time PCR with fluorescein-labeled probes | BAL fluid | Mitochondrial tRNA | Comparison to EORTC/MSG criteria | 86 for proven IA, 50 for probable IA, 80 for possible IA | 93 | Due to the primer and probe design, the assay only detected A. fumigatus infection. |
| Challier et al. (150) | 2004 | Retrospective | 41 immunocompromised patients at risk for IA and 29 controls | Real-time PCR with fluorescein-labeled probe | Serum | 28S rRNA | Comparison to EORTC/MSG criteria | 100 for proven cases, 78.9 for probable cases | All controls had negative qPCR results | ELISA 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) | 2004 | Prospective | 96 patients at risk for IA | Real-time PCR with fluorescein-labeled probes | Plasma | 18S rRNA gene | Comparison to EORTC/MSG criteria | 55 | 93 | The 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) | 2004 | Retrospective | 99 patients (49 cases of IA and 50 controls) | Real-time PCR with fluorescein-labeled probe | BAL fluid | 18S rRNA | Comparison to EORTC/MSG criteria | 67 | 100 | The 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) | 2005 | Retrospective | 29 patients with at least one positive galactomannan test | Real-time PCR with fluorescein-labeled probes | Serum | Mitochondrial DNA | Comparison to EORTC/MSG criteria | 57.1 | 63.6 | Possible IA cases were disregarded. A PCR-positive result after the first GM-positive result was associated with a poor prognosis. |
| White et al. (152) | 2006 | Prospective | 203 patients at risk for IFI | Real-time nested PCR with hydrolysis (TaqMan) probes | Whole blood | 28S rRNA | Comparison to EORTC/MSG criteria | 92.3 | 94.6 | Possible IA cases were disregarded. Only patients with serial positive PCR results were considered “PCR positive.” |
| Cesaro et al. (153) | 2008 | Prospective | 62 pediatric patients at risk for IA | Real-time PCR with fluorescent probes | Whole blood | 18S rRNA gene | Comparison to EORTC/MSG criteria | 88 | 37 | When 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) | 2008 | Retrospective | 25 patients with at least 1 GM-positive serum sample | Real-time PCR with fluorescent probes | Serum | Mitochondrial DNA | Comparison to EORTC/MSG criteria | 61.5 for probable and possible IA cases | 100 | Possible 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) | 2008 | Prospective | 124 patients with hematologic malignancies undergoing chemotherapy or HSCT | Real-time PCR with fluorescent probes | Serum | 28S rRNA | Comparison to EORTC/MSG criteria | 100 when using large serum volumes for DNA extraction, 76.5 when using small serum volumes for DNA extraction | 96.7 | Two 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) | 2008 | Retrospective | 81 patients with pneumonia | Real-time PCR with fluorescein-labeled probes | BAL fluid | 18S rDNA | Comparison to EORTC/MSG criteria | 77 | 88 | |
| Ramirez et al. (157) | 2009 | Prospective | 127 patients at risk for IA | Real-time PCR with fluorescein-labeled probes; species were determined by melting curve analysis | Whole blood | 18S rRNA | Comparison to EORTC/MSG criteria | 100 for proven cases, 0 for probable cases | 100 if possible IA cases are disregarded. | Only 1% of the 948 tested samples were PCR positive. |
| Frealle et al. (158) | 2009 | Retrospective | 57 patients at risk for IA | Real-time PCR with fluorescein-labeled probes | BAL fluid | Mitochondrial DNA | Comparison to EORTC/MSG criteria | 50 for proven and probable IA cases | 100 | |
| Cuenca-Estrella et al. (159) | 2009 | Prospective | 83 patients with febrile neutropenia | Real-time PCR with hydrolysis probe | 1,122 whole-blood samples and 1,122 serum samples | ITS1 | Comparison to EORTC/MSG criteria | 91.6 | 94.4 | Cases 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) | 2011 | Prospective | 46 patients receiving either allogeneic SCT or myeloablative chemotherapy | Real-time PCR with fluorescein-labeled probes | Whole blood | Multicopy ribosomal operon region from ITS1 to 5.8S region | Comparison to EORTC/MSG criteria | 55 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) | 2011 | Retrospective | 44 patients with two sequential positive serum galactomannan results and a risk factor for IA | Two different real-time PCR assays with hybridization probes | Serum | Assay 1, mitochondrial DNA; assay 2, 18S rRNA | Comparison to EORTC/MSG criteria | For 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) | 2011 | Retrospective | 31 patients (10 with proven/probable IA and 21 with no IA) | Two different real-time PCR assays with fluorescently labeled probes | Serum | Assay 1, 28S rRNA; assay 2, 18S rRNA | Comparison to EORTC/MSG criteria | For 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) | 2011 | Retrospective | 38 adult patients with a high clinical suspicion of IA | Real-time PCR with fluorescently labeled probes | Serum and whole blood | ITS1 | Comparison to EORTC/MSG criteria | 100 for serum and 94.4 for blood for proven/probable IA | NA | The 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) | 2011 | Retrospective | 137 lung transplant recipients | Real-time PCR with fluorescently labeled probes | BAL fluid | Not specified | Comparison to EORTC/MSG criteria | 100 for proven/probable IA | 88 | For 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) | 2011 | Prospective | 158 patients from hematology and intensive care units | Real-time PCR with fluorescently labeled probes | BAL fluid | 18S rRNA gene | Comparison to EORTC/MSG criteria | 94.1 for proven and probable IA | 98.6 | |
| Springer et al. (165) | 2013 | Retrospective, multicenter | 47 patients with proven/probable IA and 31 controls | Various real-time PCR assays | Serum and whole blood | Various | Comparison to EORTC/MSG criteria | 85.1 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) | 2013 | Prospective | 278 patients undergoing intensive chemotherapy or HSCT | Two different real-time PCR assays (a nested and a single run assay) | Whole blood | 28S rRNA (nested assay), ITS (single-run assay) | Comparison to EORTC/MSG criteria | 69–87 for nested assay and 55–80 for single-run PCR assay | 36–63 for nested assay and 57–84 for single-run assay | Possible 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) | 2013 | Prospective | 72 patients with hematologic malignancies and suffering from fever, 4 with normal temperatures, and 10 healthy volunteers | Real-time PCR with hydrolysis probes | Whole blood and plasma | 28S-ITS2 rRNA genes | Comparison to EORTC/MSG criteria | 90.9 for proven and probable IA | 73.4 | Possible IA cases were considered truly negative. |
| Guinea et al. (168) | 2013 | Prospective | 175 patients with hematologic malignancies and at risk for IA | Real-time PCR with fluorescent probes | Lower respiratory tract samples | 18S rRNA | Comparison to EORTC/MSG criteria | 93.3 | 82.9 | No 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.