Evaluation of Polymerase Chain Reaction and Cobas TaqMan Real Time PCR in the Diagnosis of Tuberculosis: Indian Prospective

Background & Objectives: Globally, tuberculosis (TB) still remains a major public health problem. India is a high TB burden country contributing to 26 per cent of global TB burden. Pulmonary tuberculosis (PTB) cases are more common (~ 90% of cases) while extra pulmonary tuberculosis (EPTB) constitutes around 10 to 20% of all tuberculosis cases in India. The diagnosis of the EPTB cases is difficult because of few bacilli and consequently is associated with low sensitivity of Zhiel-Neelson (ZN) smear and culture on LJ media. The present study evaluates the utility of PCR for the detection of M. tuberculosis in paucibacillary extra pulmonary and pulmonary tuberculosis samples. Methods: A total of 561 samples (553 EPTB & 8 PTB cases) were collected from the extra pulmonary and pulmonary tuberculosis patients which were processed for ZN smear, culture on LJ media and conventional PCR using two gene targets (IS6110 and MPB64). Results: The PCR positivity of IS6110 and MPB64 gene targets was found to be 91.3% (N=63/69) and 89.9% (N= 62/69) in majority of smear negative & culture positive (as a gold standard) extra pulmonary cases, respectively. However the PCR positivity was observed 100% in smear positive, culture positive Line probe assay tested MDR PTB cases (true positive controls; N=34). Further the PCR specificity was determined >95% (true negative healthy controls; N=26). The positivity of M. tuberculosis by IS6110 & MPB 64 gene targets was found to be range of 88% to 100% in various clinical paucibacillary extra pulmonary samples i.e. pleural fluid, ascitic fluid, lymph node, pus, CSF and others. Our data on 64 samples (non respiratory, n=63 & respiratory samples, n=1) revealed 40.6% positivity by Cobas TaqMan Real Time PCR (utilizing 16S rRNA probe; Roche, USA). Interpretation & Conclusion: Our data revealed that utility of both PCR and Real Time PCR in rapid diagnosis of M. tuberculosis in paucibacillary extra pulmonary tuberculosis samples in Indian scenario.


Introduction
Tuberculosis (TB) remains a major global health problem, despite the availability of highly efficacious treatment for decades. India has the world's largest burden of tuberculosis (TB), accounting for one-fifth (21%) of the global TB incidence. The global annual incidence estimate is 8.7 million, most of them children (especially in endemic areas), and it leads to approximately 1.4 million deaths annually [1].
Pulmonary (PTB) are more common (in about 90% of cases) [2] while extra pulmonary tuberculosis (EPTB) constitutes around 10 to 20% of all tuberculosis cases in India (which affects mainly the lymph nodes, meningitis, kidney, spine, and growing ends of the bones), with a 25 to 50% case mortality rate within months. The most common form of extra pulmonary tuberculosis (EPTB) is tuberculous pleural effusion & lymphadenopathy. However, the disease in extra pulmonary tuberculosis cases most often remains undiagnosed and, even worse, untreated. Conventional methods like smear and culture on Lowenstein-Jenson (LJ) Media are of limited use in the diagnosis of extra pulmonary tuberculosis cases which is associated with low sensitivity because of few bacillary load, smear microscopy has both the problems of sensitivity and specificity [3 -5]. Several investigators reported 0.4-37% smear positivity & 12-45% culture positivity in variable proportional in different biological samples [6][7][8][9][10].
For the rapid and accurate diagnosis of infectious diseases, polymerase chain reaction (PCR) technique & TaqMan Real time PCR have attracted considerable interest because of high degree of sensitivity and specificity over the conventional methods, particularly with the hope of shortening the time required to detect and identify Mycobacterium tuberculosis in respiratory and non-respiratory samples. These molecular tools and methods can be used for the confirmation of identity of isolates, direct detection of gene sequences from the clinical specimen and also molecular detection of drug resistance [4,[11][12][13].
The role of PCR in the diagnosis of extra pulmonary tuberculosis cases has been evaluated extensively as an alternative diagnostic tool and has yielded variable results, with sensitivities ranging between 42 and 100% and specificities ranging between 85 and 100% using various PCR targets such as IS6110, MPB64 (MPt64), TRC4, GCRS, etc. However, due to variability in the sensitivity rates in different studies, the role of PCR remains controversial [3,9,11,[14][15][16].
Highly conserved insertion sequences, IS6110, is most commonly used in the detection of M. tuberculosis. The range of IS6110 copies among isolates varied from 0-19 in the M. tuberculosis genome [16,17]. However, the sensitivity and specificity of IS6110 sequence in the diagnosis of tuberculosis remains uncertain, and needs to be include other various PCR targets reliable screening test for tuberculosis in clinical specimen such as devR, TRC4, GCRS, MPB64 (MPt64), etc. [5,11,14,15]. MPB64 is secretary protein, it has been implicated in the virulence and pathogenesis of M.tuberculosis [18]. These assays targeting various gene segments, have abbreviated the turn around time for definitive mycobacteriological detection in the laboratory to 1-2 days, besides being more sensitive than conventional methods. A prompt diagnosis is indispensable for initiating appropriate treatment. The Cobas Amplicor M.TUBERCULOSIS assay for direct detection of M.tuberculosis complex (M.TUBERCULOSISC) in pulmonary tuberculosis samples have been used in many studies [12,19,20,21]. The Amplicor assay was approved by the FDA for testing on smear-positive respiratory samples. The Cobas Amplicor M.TUBERCULOSIS assay is based on amplification of a segment of the 16S rRNA gene, followed by colorimetric detection of the PCR product by probe hybridization. The present study evaluates the utility of PCR (IS6110 and MPB64 gene targets) & TaqMan Real Time-PCR in the detection of M.tuberculosis in majority of smear negative paucibacillary extra pulmonary samples in Indian scenario.

Study Subject
From 2011 to 2013 year, we conducted this study in National Institute of Tuberculosis and Respiratory Diseases, New Delhi, India. Different categories of samples (sputum for pulmonary tuberculosis and body fluids for extra-pulmonary cases) were collected from all study population. Current study was approved by research & ethics committee of National Institute of Tuberculosis and Respiratory Diseases in 2011.  Figure 1). Majority of the samples were pleural fluid. A detailed clinical history, sex, and age were also collected from the requisition form that accompanied with samples.

Collection of the Clinical samples
On the other hand healthy controls (N=26) TB negative and TB positive (smear positive, culture positive Line probe assay tested in MDR PTB; N=34) were also recruited as negative and positive controls H37Rv, M.tuberculsois, respectively ( Figure 2 and Figure 3).
Inclusion criteria: The inclusion criteria of the study was recruitment of tuberculosis samples which included pleural fluid, ascitic fluids, CSF, lymph nodes aspirates, pus, endometrial fluid, peritoneal fluid, sputum, bronchial wash and others samples) with required sufficient volume to process the paucibacillary samples.
Data of clinically presentations, radiological and other laboratory examinations for diagnosis of extra-pulmonary tuberculosis (included smears and culture and other cyto-pathological reports wherever available etc) were also collected from OPD and ward for these patients.

Processing of the samples
All 561 samples were decontaminated by standard protocol i.e. N-acetyl-L-cysteine (NALC)-sodium hydroxide (NaOH) procedure which included 2% NaOH, 2.9% trisodium citrate, 0.5% NALC [22]. The processed samples were used for ZN smear, culture on LJ medium (bacteriological identification as a gold standard) and for DNA extraction from all body fluids as well as sputum by QIAamp DNA Mini Kit (Qiagen, Germany) and by Amplicor Respiratory samples Preparation Kit according to manufacturer's instructions and protocols (Roche, USA). Equal volume (100µl) of decontaminated samples was used in DNA extraction for conventional PCR and Real Time PCR method. Eluted DNA was stored at -20 o C. To avoid contamination during DNA extraction and amplification, strict precautions were taken, including separate areas for DNA extraction, reagent preparation, amplification and product detection and regular meticulous cleaning of surface with 10% hypochlorite were also applied to maintain the standard molecular laboratory procedures.

DNA Amplification for Mycobacterium tuberculosis Detection
Amplification of bacterial DNA was performed by using In-House PCR (IS6110 and MPB64 gene targets) in extracted DNA of all clinical samples as per the below mentioned protocols.

In-house PCR
Total 25µl of PCR reaction volume was containing 1x PCR buffer, 0.2 mM dNTPs, 50ng of each primer (IS6110:      Immunology and Infectious Diseases 1(1): 1-9, 2013 5 100 µl neutralization reagent (RN) was added to each specimen and control tube. Samples were vortexes for 5 seconds at half speed and stored at 2-8 o C before addition into working master mix.

Results
The present study determined the significance of conventional and Real Time PCR for the early detection of M.tuberculosis in clinical samples in smear negative, culture positive paucibacillary tuberculosis cases. Out of 561 clinical suspected tuberculosis samples, 72 were found to be culture positive (data represented in Table-1) subjected to smear microscopy after ZN staining, culture on LJ media (as gold standard) to analyzed the mycobacterium growth and further conventional PCR (IS6110 and MPB64) and Real Time TaqMan PCR (16SrRNA).

Discussion
The major challenge in the diagnosis of extra pulmonary tuberculosis is detection of M.tuberculosis. Conventional methods including smear and culture have poor sensitivity due to the paucibacillary load in the samples. Without an affirmative answers, clinicians could not start treatment due to the delayed diagnosis. Therefore, a high index of suspicion is necessary to make an early diagnosis, and quite often, more than one procedure is necessary for the confirmation of the diagnosis. With the development of novel & rapid molecular techniques, this delay in the accurate diagnosis of the disease is minimized but till date it could not replace traditional techniques in contrast to diagnostic modalities for other pathogens, like Chlamydia or Mycoplasma [5,7,9,10,12,24,25]. In order to identify tuberculosis in patients of suspected extra pulmonary tuberculosis for the detection of M. tuberculosis in clinical samples, two well established gene targets for conventional PCR (IS6110 & MPB64) were used. Current study showed the different PCR positive rates in various categories of body fluids. The PCR positivity of M. tuberculosis by IS6110 & MPB 64 gene targets was found to be 88% to 100% in various clinical samples paucibacillary smear negative extra pulmonary samples i.e. pleural fluid (88%), ascitic fluid (100%), lymph node (100%), pus (89%), CSF (100%) and others (100%). Our data revealed the significance importance of M.tuberculosis PCR by utilizing two gene targets in the diagnosis of paucibacillary smear negative cases in Indian scenario.
Extra pulmonary TB is a significant health problem (10-20%) in both developing and developed countries. The reported prevalence of extra pulmonary tuberculosis in India varies between 8.3% and 13.1% in different districts according to cohort analysis [3].
Several studies reported low detection rate by smear in extra pulmonary tuberculosis cases are less than 10% in pleural fluid [6]; 0.4% [26] in endometrial samples, 13% in lymph node, skin and other body fluids [19], 5.4% in peritoneal fluids, CSF, cervical lymph node biopsies, tissue biopsy and pericardial fluid [10] , zero percentage in endometrial biopsy, gastric aspirate and pus [9] whereas culture positivity was 4% in peritoneal fluids, CSF, cervical lymph node biopsies, tissue biopsy and pericardial fluid [ [19], 12 to 70 % in pleural fluid [6] and 4.3% in endometrial biopsy, gastric aspirate and pus [9]. In contrast to above reported studies, the current study showed a significant utility of PCR and Real time PCR in smear negative, paucibacillary respiratory & non respiratory TB samples. Various sensitivity of PCR using IS6110 has been reported in extra pulmonary samples i.e. 74.1% in tissue [27], 83% in pleural fluid, pleural tissue, and lymph node [15], 69.1 % and 87.5% in lymph node [5,12], 75% in clinical samples [7], 40% in EPTB [9]. Chakarvorty et al [15] reported that IS6110 PCR efficiency was 90.9% for pleural tissue and 85.5% for pleural fluid and 68.4% lymph node samples. But in our findings by IS6110 & MPB64, positivity was increased in pleural fluid (89%) and lymph node (100%) smear negative & culture positive samples. These results further emphasized the significance of molecular diagnostic method for earlier diagnosis.
It has been reported that the sensitivity of PCR can be increased by using one more sets of primers in EPTB cases i.e. 77% [5]. Similarly in our study, positivity of the PCR to detect M.tuberculosis was also increased (i.e. combined results of conventional and real time PCR in 64 tuberculosis samples) 79.7% (51/64). M.tuberculosis detection rate by single PCR was low i.e. 68.7% for IS6110, 62.5% for MPB64 PCR and 40.6% by Real time PCR. The low positivity rate through Amplicor Real time Roche assay in smear negative extra-pulmonary tuberculosis samples in our study revealed its more significance in smear-positive respiratory samples compared to smear negative paucibacillary extrapulmonary cases. It has been reported earlier that the Amplicor Real time assay has high sensitivity rate to detect M.tuberculosis in smear-positive respiratory samples [20].
Boukaline et al. 19 reported the testing of EPTB cases by Real Time PCR method for M.tuberculosis diagnosis and analyzed the 53.8% sensitivity. Rimek et al.12 reported 87.5% & 45.5% sensitivity, 100.0% & 91.3% specificity of In-house PCR and the COBAS M.tuberculosis assays, respectively. This pilot study suggested the significance of the molecular tools in different body fluids of the suspected EPTB cases, compared to conventional method like smear (3.1%) and culture (10.9%).

Conclusion
This study reveals rapid molecular test such as PCR with more than one gene targets & Real Time PCR methods have significant role for the early diagnosis (by decreasing time duration) of extra pulmonary tuberculosis cases, which is not diagnosed by conventional test (smear & culture). Further study needs to include more gene targets and to develop multiplex PCR assays for rapid diagnosis of M.tuberculosis in developing countries like India.