The Clinical Utility of Polymerase Chain Reaction and Adenosine Deaminase (ADA), for the Diagnosis of Pleural Tuberculosis: Indian Scenario

Background and Objectives: In spite of higher incidence and prevalence of tuberculosis, the diagnosis of extra-pulmonary tuberculosis (EPTB) in various clinical specimens (such as pleural fluid, ascitic fluids, CSF, lymph node aspirate etc), remains true challenge. Current tools for the diagnosis of tuberculosis in various body fluids are suboptimal. Clinicians underestimate these diseases, and use of insensitive conventional analytical method has contributed to the difficulties in managing patient with extra pulmonary tuberculosis. It is important to develop rapid, sensitive and specific test for early diagnosis of extra pulmonary tuberculosis because of the lack of sensitivity & specificity of the conventional methods as AFB smear by ZN technique and culture on LJ media. Pleural tuberculosis (TB) diagnosis often requires invasive procedures such as pleural biopsy. The study was undertaken to evaluate the combined utility of polymerase chain reaction (PCR) for different gene targets (IS6110, MPB64 and protein antigen b; Pab ) especially in pleural fluid specimens with adenosine deaminase (ADA) levels in the diagnosis of pleurisy. Methods: Total 430 clinical specimens (412 extra-pulmonary and 18 pulmonary tuberculosis) were recruited from the outdoor and indoor Department of National Institute of Tuberculosis and Respiratory Diseases, New Delhi during the 2011-2013 periods. All specimens were further processed for AFB smear, culture on LJ media, ADA level and conventional PCR (IS6110 & MPB 64 and Pab gene targets). Results: The PCR positivity IS6110, MPB64 P 181/412) gene target was found to be significantly low as compared to the IS6110 (65.3%; 269/412; X2=37.058; pc=0.000; Odds ratio 2.401; 95% CI=1.795-3.213) & MPB 64 (63.6%; 262/412; X2=31.245; pc=0.000; Odds ratio 2.229; 95% CI=1.669-2.978) gene targets in extra pulmonary tuberculosis cases. Further we have analyzed the combined utility of PCR with ADA levels among the body fluids (165 pleural fluid, 15 ascitic fluid, 1 lymph node and 1 cerebrospinal fluid; CSF). Our results indicated that the PCR alone can detect total 72.5% (132/182) TB cases, whereas ADA alone can detect 61.5% (112/182; considering cutoff value >40IU/L or confirmed cases of TB on clinic-radiological findings), M.tuberculosis in body fluids. Further data was compared in between single, two and three gene targets considering cut off value ADA >40IU/L levels in body fluids. Our observation showed that the positivity of tuberculosis cases were significantly higher through three gene targets (N=48/83; 57.8%; Mean of ADA >40IU/L =127.3) as compared to single gene target (N=10/83; 12.1%; Mean of ADA >40IU/L =68.2; X2=36.27; pc=0.000; Odds ratio 10.011; 95% CI=4.272-24.008) utilizing conventional PCR technology. No significant difference has been observed in other body fluids. The combined evaluation of both techniques (PCR and ADA) raised 14-15 % additional diagnosis of tuberculosis in body fluids (158/ 182; 86.8%; in pleural fluid= 147/165; 89.1%). Interpretation and Conclusion: Our results suggested that Protein antigen b (Pab) gene target showed less sensitivity as compared to IS6110 & MPB64. This study demonstrated the combined utility of both techniques (multigene target PCR with ADA level), enhanced the sensitivity of diagnosis of tuberculosis in body fluids. The study also confirmed the high diagnostic utility of PCR and ADA methods in diagnosis of tuberculosis in various paucibacillary body fluids in Indian scenario.