Nested PCR in lung tissue for diagnosis of pulmonary tuberculosis

Abstract

Diagnosis of pulmonary tuberculosis (TB) is difficult in cases with an unusual presentation and often requires a lung biopsy. The goal of this study was to determine the clinical usefulness of nested PCR on lung tissue for the diagnosis of pulmonary TB.Clinical and laboratory data were reviewed from patients who underwent diagnostic lung biopsies, followed by nested TB PCR on formalin-fixed paraffin-embedded lung tissue specimens. The diagnostic yield and clinical impact of nested PCR were investigated.Of the 223 patients studied, 142 were diagnosed with TB. Microbiologically confirmed TB was identified in 71 patients. Compared to culture results, the sensitivity, specificity, positive predictive value and negative predictive value of nested PCR were 85%, 99%, 98% and 88%, respectively. Nested PCR was more sensitive than acid-fast bacilli smear of respiratory specimens and histopathological findings. The PCR results provided an early diagnosis and initiation of treatment for TB. However, negative PCR results did not lead to discontinuation of unnecessary TB treatment in patients on medication.In conclusion, nested PCR on lung tissue specimens is a useful diagnostic test for pulmonary TB in patients with an unusual presentation.

Comparison of real time PCR with phenotypic methods in bronchoalveolar lavage in diagnosis of sputum smear negative pulmonary tuberculosis patients

“…High sensitivity and specificity of real time PCR in diagnosing pulmonary tuberculosis has been reported by several studies although the sensitivity of PCR is lower in smear negative cases in comparison to the smear positive cases. [7][8][9] Seven of our cases )6.8%( which were AFB smear and culture MTB negative but were positive on PCR for MTB were treated with antituberculosis drugs with good clinico-radiological response thereby suggesting that PCR is also useful in some culture MTB negative patients to diagnose pulmonary tuberculosis ) Table 4(. PCR for MTB was falsely positive in only one patient with history of antituberculosis treatment in the last one year and this may have occurred due to cross contamination or due to the presence of dead bacilli in the patient.…”

Performance of the Roche LightCycler Real-Time PCR Assay for Diagnosing Extrapulmonary Tuberculosis

“…The recent WHOendorsed Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA), has shown a sensitivity for nonrespiratory tissue specimens ranging from 53% to 100% and a specificity ranging from 97.3% to 100% (2,10,13,31). However, with various degrees of sensitivity being reported for different assays (8), the clinical utility of PCR-based tests for diagnosing EPTB in tissue samples is still uncertain (17). We investigated another molecular assay for application in diagnosing EPTB, the LightCycler mycobacterium detection assay (LCTB) (Roche Diagnostics, GmbH, Germany), which has the added advantage of simultaneously diagnosing three species, Mycobacterium tuberculosis, M. avium, and M. kansasii.…”

“…Possibly, the poorer performance of the LCTB with other tissue types may be due to poor quality of tissues, uneven distribution of bacteria, difficulty in extracting M. tuberculosis DNA or loss of DNA during extraction, or the presence of PCR-inhibitory substances (6,29). Similar studies have employed phenol-chloroform with ethanol precipitation (7,22,23) or manual commercial kit extraction (6,17), which are labor intensive and time-consuming. Since an automated extraction method was used, a tissue homogenization step was performed preextraction to liquefy the solid tissue specimens and release any bacteria present.…”

“…Conventional diagnosis of EPTB hinges on identification of acid-fast bacilli, histopathological examination of tissues, or culture (6,17). These methods are time-consuming, require expertise, can be nonspecific, and are frequently delayed.…”

“…The shift is toward newer molecular diagnostic tools for detecting either RNA or DNA to improve sensitivity and speed up the diagnostic process (30). A number of studies have reported on performance of PCR-based assays for diagnosis of EPTB in formalinfixed paraffin-embedded tissues (3,9,14,18,26), bone marrow, and lymph node (22,23) and lung (17) tissue. The recent WHOendorsed Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA), has shown a sensitivity for nonrespiratory tissue specimens ranging from 53% to 100% and a specificity ranging from 97.3% to 100% (2,10,13,31).…”