Real-time Quantitative PCR in the Diagnosis of Tuberculosis in Formalin-fixed Paraffin-embedded Pleural Tissue in Patients From a High HIV Endemic Area

Abstract

The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis.

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

“…The sensitivity of PCR in this study is comparable to rates reported in other studies targeting IS6110 (1,5,8). One study reported 100% sensitivity for real-time PCR, but this study tested only lung samples and demonstrated a specificity of 81% (13).…”

“…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.…”

“…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.…”

Be alert to tuberculosis-mediated glomerulonephritis: a retrospective study

“…Therefore, newer diagnostic modalities for TB-GN remain a challenge for clinicians. Some clinical studies have shown that the specificity of PCR in the diagnosis of TB was dramatically higher than that of a urine M. tuberculosis culture, with low rates of false-positive and false-negative results [24]. In our study, M. tuberculosis DNA was detected in 39 (84.8%) patients by PCR on renal biopsy specimens with a much higher positive rate than that found with either of the other two methods used.…”

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).…”