Evaluation of Polymerase Chain Reaction for Detection of Mycobacterium tuberculosis in Pleural Fluid

doi:10.1378/chest.119.6.1737

Chest

Volume 119, Issue 6, June 2001, Pages 1737-1741

https://doi.org/10.1378/chest.119.6.1737 Get rights and content

Objectives

Tuberculosis, a reemergent killer, is threatening to assume serious proportions all over the world, particularly in view of the AIDS pandemic. The detection of mycobacterial DNA by polymerase chain reaction (PCR) in clinical samples is a promising approach for the rapid diagnosis of tuberculous infections. The aims of this study were to evaluate PCR for detection of Mycobacterium tuberculosis in pleural fluids and to correlate the results with adenosine deaminase activity (ADA) estimation and acid-fast bacilli (AFB) screening.

Methods

The sensitivity and specificity of PCR in detection of mycobacterial DNA in 20 samples of tuberculous pleural effusion were evaluated using 40 samples of nontubercular pleural effusion as controls. The results were correlated with the ADA in all 60 pleural fluids. In addition, AFB detection by Ziehl-Neelsen staining on cytospin smears of all pleural fluids was also compared.

Results

Of the 20 samples of tuberculous pleural effusion, mycobacterium could be detected by AFB staining in 4 samples. Fourteen samples were PCR positive. None of the samples from the control group were AFB or PCR positive. The sensitivity of PCR, therefore, was 70.0% with specificity of 100% (positive predictive value, 100%; negative predictive value, 86.95%). The sensitivity of AFB screening was at best 20%. The mean of ADA values in tubercular pleural effusions was 63.21 U/L (SD, 33.01), and the mean in the control samples was 51.1 U/L (SD, 29.71). Taking a cut-off value of 50 U/L, both the sensitivity and specificity of ADA estimation in diagnosing tuberculosis were only 55%.

Conclusion

PCR represents a rapid and sensitive method for the detection of mycobacterial DNA in tuberculous pleural effusions. AFB screening has low sensitivity, and ADA estimation has both low sensitivity and specificity. Therefore, when the clinical suspicion is high and smear result is negative, but the signs and symptoms of M tuberculosis are apparent, PCR is the method of choice for identifying the infection.

Section snippets

Patients and Pleural Fluid

The population under study comprised 60 patients with pleural effusion. The study period was from July 1997 to December 1998. Any patient coming to the outpatient department within the study period with pleural effusion and with no other specific clinical history for disease, such as lung cancer, and who had not received any antituberculous treatment (ATT) was included in the study. Pleural fluid was aspirated from these patients and transported to the laboratory for investigation.

Study Design

The study was

Results

Sixty samples of pleural effusion were obtained for this study from the outpatient unit of the Department of Pulmonary Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India. Twenty samples were tubercular pleural effusion, and 40 samples were nontubercular pleural effusion. The latter were obtained as a disease control group. The ages of the patients with tubercular pleurisy (study group) ranged from 6 to 70 years and included 9 female (45%) and 11 male patients

Discussion

Traditional methods of diagnosing tuberculosis have been isolation of bacilli in culture or recognition of AFB in clinical specimens. AFB staining lacks sensitivity. So far, the “gold standard” has been culture. But with a dividing time of 48 h, it takes up to 10 weeks using the culture method. Besides, microscopy-positive specimens fail to yield mycobacterium on culture, perhaps due to harsh chemical treatment used in decontamination or the presence of nonviable mycobacterium in partially

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Pleural tuberculosis (pTB) is diagnosed by using a composite reference standard (CRS) since microbiological methods are grossly inadequate and an accurate diagnostic test remains an unmet need. The present study aimed to evaluate the utility of Mycobacterium tuberculosis (Mtb) antigen and DNA-based tests for pTB diagnosis. Patients were classified as ‘Definite TB’, ‘Probable TB’ and ‘Non-TB’ disease according to the CRS. We assessed the performance of in-house antigen detection assays, namely antibody-based Enzyme-Linked ImmunoSorbent Assay (ELISA) and aptamer-based Aptamer-Linked Immobilized Sorbent Assay (ALISA), targeting Mtb HspX protein and DNA-based tests namely, Xpert MTB/RIF and in-house devR-qPCR. ROC curves were generated for the combined group of ‘Definite TB’ and ‘Probable TB’ vs. ‘Non-TB’ disease group and cut-off values were derived to provide specificity of ≥98%. The sensitivity of ALISA was ∼93% vs. ∼24% of ELISA (p-value ≤0.0001). devR-qPCR exhibited a sensitivity of 50% vs. ∼22% of Xpert (p-value ≤0.01). This novel aptamer-based ALISA test surpasses the sensitivity criterion and matches the specificity requirement spelt out in the ‘Target product profile’ for extrapulmonary tuberculosis samples by Unitaid (Sensitivity ≥80%, Specificity 98%). The superior performance of the aptamer-based ALISA test indicates its translational potential to bridge the existing gap in pTB diagnosis.
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There are several kinds of methods to diagnose tuberculous pleural effusion. Several reports showed that nucleic acid amplification (PCR) was used to detect M. tuberculosis in pleural effusion specimens, with highly variable sensitivities (11%–81%) [9–13]. Thus, the clinical utility of PCR testing seemed highly limited for specimens such as pleural effusion, in which the bacillary load was very low [14].
Tuberculous pleural effusion is the second most common form of extrapulmonary tuberculosis, which is very difficult to rapidly distinguish from malignant pleural effusion in the clinical setting. A time-resolved fluoroimmunoassay (TRF) of CFP-10, a low molecular weight protein secreted by pathogenic Mycobacterium tuberculosis, was developed to differentiate tuberculous pleural effusion from malignant one. The measuring range was 0.3–187.5 ng/ml with the dose–response coefficient of 0.9998 and detection limit of 0.036 ng/ml. The intra-assay and inter-assay coefficients of variation were 3.6–9.2% and 10.0–12.4%, respectively. The concentration of CFP-10 in malignant pleural effusion was less than 0.8 ng/ml. The negative predictive value was 93.1% in malignant pleural effusion (n = 247) while the positive predictive value was 83.0% in tuberculous pleural effusion (n = 235). Moreover, there was a statistically significant difference in the CFP-10 concentration of pleural effusion between the groups before and after clinical therapy of tuberculosis (P < 0.001, n = 81). In addition, the stability of the diagnostic reagents lasted at least 1 year at 4 °C. Therefore, the TRF of CFP-10 may be used for the rapid diagnosis of tuberculous pleural effusion and further monitoring the clinical therapeutic efficacy of tuberculosis.
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Chest

Objectives

Methods

Results

Conclusion

Section snippets

Patients and Pleural Fluid

Study Design

Results

Discussion

References (22)

The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosisDNA probes

Tubercle

Diagnosis of tuberculosis by DNA amplification in clinical practice evaluation

Lancet

Adenosine deaminase

Value of direct amplified test for diagnosis of tuberculosis

Lancet

Tuberculous pleurisy

Chest

Enumeration of tubercle bacilli in sputum of patients with pulmonary tuberculosis

Antimicrob Agents Chemother

New approaches to the rapid diagnosis of tuberculous meningitis

J Infect Dis

Diagnosis of pulmonary tuberculosis by detection of tuberculostearic acid in sputum by using gas chromatography-mass spectrometry with selected ion monitoring

J Infect Dis

Repetitive DNA sequences as probes for Mycobacterium tuberculosis

J Clin Microbiol

A cloned DNA fragment for identification of Mycobacterium tuberculosis

Rev Infect Dis

Use of polymerase chain reaction for rapid diagnosis of tuberculosis

J Clin Microbiol

Cited by (95)

A novel aptamer-based test for the rapid and accurate diagnosis of pleural tuberculosis

Development of a time-resolved fluoroimmunoassay of CFP-10 for rapid diagnosis of tuberculous pleural effusion

Comparison of LCD array and IS6110-PCR with conventional techniques for detection of Mycobacterium bovis isolated from Egyptian cattle and Buffaloes

Acute Pneumonia

A Postpartum Immigrant with Pulmonary, Pleural, and Intestinal Tuberculosis

Flow cytometry, molecular analysis, and other special techniques (in Serous Fluid Cytopathology)

Chest

Clinical InvestigationsTuberculosisEvaluation of Polymerase Chain Reaction for Detection of Mycobacterium tuberculosis in Pleural Fluid

Objectives

Methods

Results

Conclusion

Section snippets

Patients and Pleural Fluid

Study Design

Results

Discussion

Tubercle

Lancet

Lancet

Chest

Enumeration of tubercle bacilli in sputum of patients with pulmonary tuberculosis

Antimicrob Agents Chemother

New approaches to the rapid diagnosis of tuberculous meningitis

J Infect Dis

Diagnosis of pulmonary tuberculosis by detection of tuberculostearic acid in sputum by using gas chromatography-mass spectrometry with selected ion monitoring

J Infect Dis

Repetitive DNA sequences as probes for Mycobacterium tuberculosis

J Clin Microbiol

A cloned DNA fragment for identification of Mycobacterium tuberculosis

Rev Infect Dis

Use of polymerase chain reaction for rapid diagnosis of tuberculosis

J Clin Microbiol

Clinical Investigations
Tuberculosis
Evaluation of Polymerase Chain Reaction for Detection of Mycobacterium tuberculosis in Pleural Fluid