Prospective clinical evaluation of a LightCycler^TM‐mediated polymerase chain reaction assay, a nested‐PCR assay and a galactomannan enzyme‐linked immunosorbent assay for detection of invasive aspergillosis in neutropenic cancer patients and haematological stem cell transplant recipients

Abstract

Summary Invasive aspergillosis (IA) is a considerable clinical problem in neutropenic patients with haematological malignancies but its diagnosis remains difficult. We prospectively evaluated a LightCyclerTM polymerase chain reaction (PCR) assay, a nested‐PCR assay and a galactomannan (GM) enzyme‐linked immunosorbent assay (ELISA) to validate their significance in diagnosing IA. During 205 treatment episodes in 165 patients from six centres, a nested‐PCR assay and GM testing was performed at regular intervals. Positive nested‐PCR results were quantified by a LightCyclerTM PCR assay. Patient episodes were stratified according to the 2002 European Organization for Research and Treatment of Cancer/Mycosis Study Group consensus criteria and the PCR and serology results were correlated with the clinical diagnostic classification. Sensitivity and specificity rates for the nested‐PCR assay were up to 63·6% [95% confidence interval (CI): 30·8–89%) and 63·5% (95% CI: 53·4–72·7%) respectively, and 33·3% and 98·9% (95% CI: 7·5–70·1% and 94·2–99·9%) for GM respectively. The LightCyclerTM PCR assay yielded positive results in 21·4%, lacking discrimination by quantification across the different clinical categories. In this prospective comparison, PCR was superior to GM with respect to sensitivity rates. In patients at high risk for IA, positive results for Aspergillus by PCR of blood samples are highly suggestive for IA and contribute to the diagnosis.

Polymerase chain reaction screening for fungemia and/or invasive fungal infections in patients with hematologic malignancies

“…On the other hand, since fungi are ubiquitous in the environment, panfungal PCR methods are more likely to yield contaminated products, while the use of specific probes will reduce this possibility [13,20]. Sensitivity will probably improve if amplification is achieved by nested PCR [13,19], although some reports have showed similar sensitivity results [26] to ours. Sensitivity will also improve by using real-time PCR [26].…”

“…Although the procedure is easy to perform, the sensitivity of the assay was quite low (75%) compared to some published data [19,26]. However, in these reports, only IA was screened.…”

Randomized PCR-based therapy and risk factors for invasive fungal infection following reduced-intensity conditioning and hematopoietic SCT

“…In addition, there have been several reports of antifungal treatment being associated with conversion of PCR test results from positive to negative. 18,31,32 These findings may partly explain the many single positive PCR tests for Candida in this study, as many of these patients received prophylaxis with fluconazole. However, the single positive PCR tests for Aspergillus are difficult to explain because only 4 of these 15 patients received active treatment or prophylaxis against Aspergillus.…”

“…This finding is consistent with earlier reports in which a single positive PCR test did not correlate with proven or probable fungal disease and resolved without treatment. 31,32 One possible explanation is laboratory contamination that is hard to eliminate even though extensive precautions were used. Another possible reason for false positive Aspergillus PCR was recently reported to be contaminated nutritional supplement.…”

Clinical and radiologic predictors of invasive pulmonary aspergillosis in cancer patients

“…For example, recent studies have reported variable sensitivity (range, 30 -90%) and specificity (range, 89 -97%) with the GM assay. [12][13][14][15] In studies comparing GM with PCR, Buchheidt et al 16 reported that PCR had a sensitivity of 63% compared with 33% for GM. The specificity of PCR was 63%, compared with 98% for enzyme immunoassay.…”