Semiquantitative Detection by Real-Time PCR ofAspergillus fumigatusin Bronchoalveolar Lavage Fluids and Tissue Biopsy Specimens from Patients with Invasive Aspergillosis

Abstract

A real-time PCR method was developed and used to detect Aspergillus fumigatus mitochondrial DNA (mtDNA) in bronchoalveolar lavage (BAL) fluids and tissue biopsy specimens. The analytical sensitivity of the assay was one A. fumigatus conidium per reaction, and the assay was linear at least over 4 orders of magnitude above the detection limit. BAL fluids from 66 immunocompromised patients at risk of invasive pulmonary aspergillosis (IPA) and 33 immunocompetent controls and tissue biopsy specimens from 10 immunocompromised patients were analyzed. The results were related to the clinical diagnosis established according to recently published consensus criteria. A. fumigatus mtDNA positivity was encountered in 16 of 81 (20%) BAL fluid specimens from patients at risk and 1 of 33 (3%) specimens from immunocompetent controls. PCRs were positive in six of seven, two of four, and four of five of the patients with proven, probable, and possible IPA, respectively, as well as in four patients at risk but without any other evidence of IPA. With qualitative detection, the diagnostic sensitivity of PCR was 73%, specificity was 93%, and predictive values of positive (PPV) and negative (NPV) results were 73 and 95%, respectively. Using a threshold cycle of <35 as a limit for positive PCR, the specificity and PPV of PCR in the diagnosis of invasive aspergillosis were 100%, but its sensitivity was only 45% and NPV was 92%. PCR was positive in tissue biopsy specimens from all patients with invasive aspergillosis caused by A. fumigatus. Semiquantitative detection of A. fumigatus mtDNA in BAL fluid may be helpful in the diagnosis of IPA. PCR is well suited for the verification of the presence of A. fumigatus in tissue biopsy specimens.

Diagnostic Accuracy of PCR Alone Compared to Galactomannan in Bronchoalveolar Lavage Fluid for Diagnosis of Invasive Pulmonary Aspergillosis: a Systematic Review

“…A total of 19 studies published between 1993 and 2011 were included (6, 7, 15, 19-22, 24, 27-33, 38, 40, 42). All studies reported on the diagnostic accuracy of PCR in BAL fluid, and 10 studies also reported on GM in BAL fluid (15,19,24,27,28,30,31,32,33,40).…”

Invasive pulmonary aspergillosis in patients with chronic obstructive pulmonary disease

“…Although PCR on BAL fluid demonstrated poor specificity and standardisation, with 23% false-positive results [125,126], other studies demonstrated a higher sensitivity and specificity, up to 100 and 100%, respectively [127][128][129][130][131][132][133]. Furthermore, PCR may be too sensitive, so that the difference between colonisation and infection can often be difficult to establish [134].…”

Prospective Comparison of the Diagnostic Potential of Real-Time PCR, Double-Sandwich Enzyme-Linked Immunosorbent Assay for Galactomannan, and a (1→3)-β- d -Glucan Test in Weekly Screening for Invasive Aspergillosis in Patients with Hematological Disorders

“…We used our realtime PCR system (GeniQ-Asper) (17) because it is most widely used in Japan. Several authors, including Loeffler et al and Costa et al, also published excellent real-time PCR detection systems for Aspergillus DNA (9,21,26,28), and their systems might produce superior results in the diagnosis of IA, which should be addressed in future studies.…”

“…A specialized laboratory as well as an expensive assay system and reagents are also required. These problems should be addressed before PCR is widely accepted as a standard screening test for IA, although it still seems to have value in making a diagnosis when a variety of clinical samples are used (20,26,28,31).…”