Characterization and Comparison of Galactomannan Enzyme Immunoassay and Quantitative Real-Time PCR Assay for Detection of Aspergillus fumigatus in Bronchoalveolar Lavage Fluid from Experimental Invasive Pulmonary Aspergillosis

Abstract

Bronchoalveolar lavage (BAL) is widely used for evaluation of patients with suspected invasive pulmonary aspergillosis (IPA). However, the diagnostic yield of BAL for detection of IPA by culture and direct examination is limited. Earlier diagnosis may be facilitated by assays that can detect Aspergillus galactomannan antigen or DNA in BAL fluid. We therefore characterized and compared the diagnostic yields of a galactomannan enzyme immunoassay (GM EIA), quantitative real-time PCR (qPCR), and quantitative cultures in experiments using BAL fluid from neutropenic rabbits with experimentally induced IPA defined as microbiologically and histologically evident invasion. The qPCR assay targeted the rRNA gene complex of Aspergillus fumigatus. The GM EIA and qPCR assay were characterized by receiver operator curve analysis. With an optimal cutoff of 0.75, the GM EIA had a sensitivity and specificity of 100% in untreated controls. A decline in sensitivity (92%) was observed when antifungal therapy (AFT) was administered. The optimal cutoff for qPCR was a crossover of 36 cycles, with sensitivity and specificity of 80% and 100%, respectively. The sensitivity of qPCR also decreased with AFT to 50%. Quantitative culture of BAL had a sensitivity of 46% and a specificity of 100%. The sensitivity of quantitative culture decreased with AFT to 16%. The GM EIA and qPCR assay had greater sensitivity than culture in detection of A. fumigatus in BAL fluid in experimentally induced IPA (P ؎ 0.04). Use of the GM EIA and qPCR assay in conjunction with culture-based diagnostic methods applied to BAL fluid could facilitate accurate diagnosis and more-timely initiation of specific therapy.Invasive pulmonary aspergillosis (IPA) is a major cause of morbidity and mortality in immunocompromised patients (7-9, 13, 14, 18, 27). Mortality rates of this severe infection range from 30% to 90% in neutropenic patients (9,10,15,20). Accurate diagnosis of IPA routinely relies upon bronchoalveolar lavage (BAL) as a standard of care in assessing immunocompromised patients (1, 12). However, current culture-based methods for evaluation of BAL fluid may have low sensitivity (3,19,32,33). Other methods, such as tissue biopsy, are often not optimal due to the fragile condition of the patient with respect to sustaining an invasive procedure. Early diagnosis of IPA remains difficult. Improved prognosis for IPA requires early diagnosis (40). The development of nonculture and noninvasive methods may facilitate an early diagnosis of IPA (5,22,25).The sandwich enzyme immunoassay (EIA) based on the detection of the Aspergillus antigen galactomannan (GM) (3,4,21,38) and real-time PCR methods (25, 34) for the detection of Aspergillus-specific DNA are encouraging alternatives to biopsy and culture. However, the sensitivity and optimal interpretive cutoff values of the GM EIA in studies using BAL fluid are not well defined (4,34,38). Although quantitative real-time PCR (qPCR) assays have been developed for the detection of IPA (16,26,34,35), their diagnostic sensitivity...

A prospective randomized controlled trial comparing PCR-based and empirical treatment with liposomal amphotericin B in patients after allo-SCT

“…in bronchoalveolar fluid was confirmed by another group. 28 The high sensitivity of a PCR assay in the detection of Aspergillus spp. was shown in patients with cerebral aspergillosis.…”

Automated and Manual Methods of DNA Extraction for Aspergillus fumigatus and Rhizopus oryzae Analyzed by Quantitative Real-Time PCR

“…Four microliters of RNase A (100 mg/ml) was added to each sample, vortexed vigorously, and incubated for 10 min at 65°C in an Eppendorf thermomixer at 1,200 rpm. The samples were further processed according to the DNeasy Plant minikit (Qiagen, Valencia, CA) protocol with the following modification: after 200 l preheated (65°C) AE buffer was applied to the column, the entire apparatus (column and collection tube) was heated at 65°C in the Eppendorf thermomixer for 5 min (10,26).…”

High-Throughput Identification and Quantification of Candida Species Using High Resolution Derivative Melt Analysis of Panfungal Amplicons

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