Comparison of Single-Copy and Multicopy Real-Time PCR Targets for Detection of Mycobacterium tuberculosis in Paraffin-Embedded Tissue

Abstract

Real-time PCR can rapidly identify Mycobacterium tuberculosis in paraffin-embedded tissue in the absence of microbiological culture. In a comparison of single-copy and multicopy PCR targets in 70 tissue samples, the sensitivities were 26% and 54%, respectively, with 100% specificity. Sensitivity was 75% for newer samples and was not decreased for acid-fast bacillus (AFB) stain-negative specimens.With a third of the world's population infected by Mycobacterium tuberculosis complex, accurate and timely diagnosis of tuberculosis is critical for management of this global epidemic (12). Although tuberculosis is often diagnosed by sputum smear and microbiological culture, the disease can also present as extrapulmonary mass lesions, which are frequently biopsied without clinical suspicion for infection (3). Under histopathologic examination, biopsy sections typically demonstrate necrotizing granulomas with or without acid-fast bacilli, neither of which is specific for tuberculosis (1, 5). Without an alternative method for diagnosis, a repeat biopsy is often necessary to procure tissue for mycobacterial culture.More recently, real-time PCR has been utilized for detection of M. tuberculosis in culture and sputum (4, 6, 9, 10). Real-time PCR is faster than conventional PCR and does not require postamplification specimen handling (10). Several studies have also evaluated the usefulness of real-time PCR for M. tuberculosis in formalin-fixed, paraffin-embedded tissue, with sensitivities ranging from 67% to 100% (1,3,5,8,13). However, most of these studies did not have complete microbiological culture results to use as their reference standard and instead relied on histologic findings and/or clinical data to identify presumptive tuberculosis cases. Furthermore, none evaluated multiple tissue sites or other factors that may have impacted the sensitivity of real-time PCR in fixed tissue. An older study using conventional PCR suggested that IS6110, a multicopy PCR target, is more sensitive than a single-copy PCR target; however, this study also did not use culture as the reference standard, and the assays were compared across different laboratories using different instruments and reagents (11). Thus, the purpose of this study was to evaluate the sensitivity and specificity of real-time PCR for detection of culture-positive M. tuberculosis in formalin-fixed paraffin-embedded tissue and to identify factors that enhance or impede the sensitivity of this assay.A retrospective study was performed on 70 formalin-fixed paraffin-embedded tissue biopsy specimens collected at a major academic hospital over a 10-year period. Biopsies of 35 consecutive specimens with M. tuberculosis complex, 23 specimens with nontuberculous mycobacteria, and 12 noninfectious specimens were included. Biopsy sites included lung (n ϭ 30), lymph nodes (n ϭ 17), bone (n ϭ 5), skin (n ϭ 5), and other sites (n ϭ 13), such as soft tissue, with sample types evenly distributed over the study time period. Microbiological culture using reference methods (10) from tissu...

Hepatic Granulomas: A Review With Emphasis on Infectious Causes

“…However, performing polymerase chain reaction on acid-fast bacillus-negative tissue sections often will not yield a positive result. 13 It is also important to recognize that current culture methods and molecular tests cannot distinguish bacillus Calmette-Guerin from M tuberculosis.…”

Performance Characteristics of Nested Polymerase Chain Reaction vs Real-Time Polymerase Chain Reaction Methods for Detecting Mycobacterium tuberculosis Complex in Paraffin-Embedded Human Tissues

“…[16][17][18][19][20] However, only a few studies have evaluated the usefulness of RT-PCR for the detection of MTB using FFPE specimens compared with culture results as a reference. 7,21 In this study, we aimed to compare the diagnostic performances of N-PCR and four RT-PCR methods, including three commercial methods for detecting MTB. The sensitivities and specificities were analyzed using mycobacterial culture results as a reference.…”

Clinical Laboratory Diagnostics for Mycobacterium tuberculosis

“…Although paraffin-embedded tissues are not optimal samples for PCR, often, they are the only specimens available to rule out tuberculosis. The sensitivity and specificity of PCR assays, conventional and real-time, on paraffin-embedded tissues varies from 64-100% and 73-100%, respectively (Beqaj et al, 2007;Baba et al, 2008;Nopvichai et al, 2009;Luo et al, 2010).…”