Comparison of PCR-ELISA and Real-Time PCR for invasive aspergillosis diagnosis in patients with hematological malignancies

Abstract

This study aimed at comparing a real-time PCR assay and a PCR-ELISA assay of both serum and bronchoalveolar lavage (BAL) samples for the diagnosis of invasive aspergillosis (IA) in patients with hematological malignancies. Using a nested case-control design, 163 patients at risk were prospectively monitored and PCR assays were performed on frozen aliquots of 459 sera which were prospectively sampled twice weekly and 42 BAL specimens sampled from 43 probable and one proven IA cases and 47 matched controls. The data from three patients classified as possible IA were excluded from the nested case-control study. The sensitivity of real-time PCR and PCR-ELISA assays in serum was 73% and 86%, respectively and specificity was 100% for both. In BAL, sensitivity was 64% for real-time PCR, 71% for PCR-ELISA and 86% for Galactomannan antigen (GMA) assays with specificities of 96%, 96%, and 93%, respectively. While slightly less sensitive, the real time-PCR assay was highly specific and considerably faster and more workable than PCR-ELISA. Combining real-time PCR and GMA detection for both serum and BAL samples enhances routine laboratory IA diagnosis.

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…This study, however, did not include any antifungal treatment, which can result in a significant reduction of serum marker levels, whereas marker levels may remain elevated in BAL fluid [105]. The usefulness of BAL fluid is underlined by the finding that a combination of PCR, PCR-ELISA and GM testing in the BAL fluid of patients with hematological malignancies was shown to enhance routine laboratory diagnosis of IA [147].…”

The Effect Analysis of Different Experimental Methods for the Diagnosis of Invasive Pulmonary Aspergillosis in a Rat Model

“…Although Aspergillus sp. DNA was usually detected at a higher frequency in BAL samples [27], Hadrich et al [5] observed that RT-PCR and PCR-ELISA assays exhibited higher sensitivities in serum samples than in BAL samples, with sensitivities ranging from 64% -94%. In our rat model, the sensitivity of the qRT-PCR was 25% with whole blood samples harvested from infected, immunosuppressed rats on days 1 -7, which was similar to the 26% sensitivity using serum samples from a guinea pig IA model (days 1 -7) reported by Lengerova et al [6].…”

“…qRT-PCR analysis of serum samples of patients with hematological malignancies at risk for IA was shown to have a sensitivity of 72.7% [5]. A commercially available qRT-PCR for the detection of Aspergillus DNA (MycAssay™ Aspergillus) has shown a sensitivity of 60% -70% and a specificity of 95% for the detection of IA [22].…”

“…However, poor specificity has been noted due to several interference factors, including environmental contamination [19]. In addition, the PCR-ELISA assay is more cumbersome for clinical laboratories [5].…”

“…Traditional histopathological examination and fungal culture relying on invasive procedures are relatively insensitive and not commonly used in clinical diagnosis due to the challenges of sampling pulmonary fluids or tissues from critically ill patients. While examination of bronchialveolar lavage (BAL) fluid yields a higher detection rate than examination of blood or serum [5][6][7], obtaining BAL fluid is invasive, and many patients with IA have other severe diseases which limit BAL collection. Consensus on the most reliable assays to detect IA from minimally or noninvasive samples has not been reached, and is a major topic of current research [8,9].…”