Evaluation of polymerase chain reaction using protein b primers for rapid diagnosis of tuberculous meningitis
Correspondence Address: Source of Support: DST (Chandigarh), Conflict of Interest: None DOI: 10.4103/0028-3886.72189
Source of Support: DST (Chandigarh), Conflict of Interest: None
Background: Rapid and specific diagnosis of tubercular meningitis (TBM) is of utmost importance. Aim: To evaluate polymerase chain reaction (PCR) using protein b primers directed against M. tuberculosis for the diagnosis of tuberculous meningitis. Materials and Methods: PCR using protein b primers was performed in ten patients with confirmed TBM (culture positive), 60 patients with clinically suspected TBM and 40 patients with no TBM (control group). Results: Protein b PCR had a sensitivity of 90% and a specificity of 100% in patients with confirmed TBM. In 60 clinically diagnosed TBM patients, protein b PCR was positive in 49 (81.7%) patients. The overall sensitivity of microscopy, culture and PCR using protein b primers was 1.4%, 14.3%, and 82.8% and specificity was 100%, 100%, and 100% respectively. Conclusion: Protein b PCR is valuable in rapid diagnosis of TBM.
Keywords: Diagnosis, PCR, tubercular meningitis
Diagnosis of tuberculous meningitis (TBM) is often presumptive, and is based on clinical features, cerebrospinal fluid (CSF) findings, imaging (computerized tomography (CT) scan and magnetic resonance imaging (MRI)) findings and response to anti-tuberculous drug therapy.  Conventional methods like microscopy and culture, although considered as gold standards, are quite inadequate. Acid fast staining requires a large number of bacilli (> 10 4 cells/ml) and culture requires 6-8 weeks, which is often negative due to small number of bacilli in CSF. , The newer tests like radiometric BACTEC system, serological tests and IFN-γ being evaluated these days are not cost effective.,
The development of a rapid, sensitive and specific test for detection of mycobacterium has been a long standing need. Nucleic acid amplification techniques such as polymerase chain reaction (PCR) have been reported to be more sensitive and specific as these techniques attempt to detect specific DNA sequence from the organism under investigation. Several Mycobacterium tuberculosis specific DNA sequences like MPB64, 65kDa antigen and IS6110 have been evaluated.  The reported sensitivity and specificity varies between 50% and 91% and 60% and 100% respectively.  The repetitive nature of IS6110 insertion sequence in M. tuberculosis makes it an attractive target for PCR amplification and several studies have evaluated IS6110 for the diagnosis of tuberculosis.  However either absence, or presence of a few copies of this sequence have already been reported in some isolates. ,,, Study from India has also reported that a large number of clinical isolates (40%) had either a single copy or no copy of insertion sequence.  Another nucleotide sequence (419 bp) of protein antigen b (Pab) gene of molecular weight 38 kda (total gene size of 1993 bp), specific for M. tuberculosis has been shown to be a useful target for detection in a few studies. ,, This protein b gene has not been properly evaluated in patients with tuberculous meningitis in this region of the world. In the present study we report our experience with protein b based PCR assay in the CSF of patients with TBM and non-TBM and the comparison with conventional techniques like microscopy and culture.
Between September 2008 and April 2009, 110 CSF samples were received in the laboratory of Medical Microbiology department, Post Graduate Institute of Medical Education and Research, Chandigarh, India for acid fast staining and culture. Patients' age ranged between 1 and 90 years. The relevant history and other details of the patients were noted from the case records. The patients were into 3 groups: Group I: TBM (n=70): (a) Confirmed TBM-culture/smear positive (n=10), and (b) suspected TBM: smear/ culture negative, clinical and laboratory features suggestive of TBM and response to anti-tuberculosis therapy  (n=60); Group II: Non-TBM infectious meningitis (n=20): (a) pyogenic meningitis (n=9), (b) viral meningitis (n=10), and (c) fungal meningitis (n=1); Group III: Non infectious neurological disorder group (n=20): Guillain Barre' syndrome (n=5), brain tumors (n=10), and multiple sclerosis (n=5). This study is a part of DST sponsored project approved by the institute ethics committee.
Processing of CSF sample
All the 110 CSF samples were subjected to three microbiological tests: Ziehl-Neelsen staining (ZN), culture on Lowenstein-Jensen medium and PCR with protein b. The CSF samples of the subjects were processed in a biosafety cabinet placed in a specially assigned room. Abour 200-300μl of specimens was aliquot and stored at -20°C. PCR was done for only those specimens which were used for smear and culture examination for M. tuberculosis. Using 200μl of centrifuge deposit for PCR, the rest of the deposit was used for acid fast microscopy by Ziehl-Neelsen method and culture was performed on 2 slopes of Lowenstein-Jensen medium. PCR was standardized and was found to have quantitative sensitivity to detect the DNA equivalent to 2-3 organisms. It tested positive with standard strain of M. tuberculosis, H 37 RV.
DNA was extracted according to the CTAB-phenol-chloroform extraction method. Briefly 0.2 ml of CSF was centrifuged at 10,000 rpm for 10 min. The supernatant was discarded and pellet was suspended in 500 μl of TE Buffer (Tris -EDTA,) 30 μl 10% SDS and 3μl proteinase k (20 mg/ml), mixed and incubated at 37°C for 1 hr. After incubation, 100 μl of 5M NaCl and 80μl of high salt CTAB (cetyle- trimethyl ammonium bromide) were added and mixed followed by incubation at 65°C for 10 min. An approximate equal volume (0.7-0.8 μl) of chloroform-isoamyle alcohol (24:1) were added, mixed thoroughly and centrifuged for 5 min at 10,000 rpm.
The aqueous viscous supernatant was carefully decanted and transferred to a new tube. An equal volume of phenol: chloroform- isoamylalcohol (25:24:1) was added followed by a 5 min spin at 10,000 rpm. The supernatant was separated and then mixed with 0.6 volume of isopropanol to get a precipitate. The precipitated nucleic acids were washed with 75% ethanol, dried and re-suspended in 100μl of TE buffer.
Polymerase chain reaction
In each independent PCR assay, test results were compared with the results for one positive and one negative control. The positive control included the DNA of H 37 Rv and negative control included the PCR grade water. Identification of M. tuberculosis was done using a specific pair of primers designed to amplify protein b antigen in the M. tuberculosis complex and the expected band size was about 419bp. The sequence of primers used Pab f and Pab r were: 5'-ACC ACC GAG CGG TTC GCC TGA-3' and 5'-GAT CTG CGG GTC GTC CCA GGT-3' respectively.
A 25 μl reaction contained 10x assay buffer (Banglore Genei, Banglore, India), 10 mM dNTPs (Banglore Genei), 10pmole of each primers, 2.5 units Taq DNA Polymerase (Banglore Genei, Banglore, India) and 5μl of extracted DNA. Amplification was carried in a thermal cycler, which involved 35 cycles including denaturation at 95°C for 4 min, annealing of primers at 63°C for 1 min. and primer extension at 72°C for one min. the amplification product were separated on 1.5% agarose gel. The samples showing the presence of 419bp band under ultraviolet transilumination were considered positive.
The sensitivity, specificity, positive predictive value and the negative predictive value were calculated using the standard formulae.
Of the 110 CSF samples studied, ten were samples form patients with confirmed TBM, 60 were from patients with clinically suspected TBM, 20 were from patients with non-TBM infections, and 20 were from patients with non-infectious neurological diseases. [Figure 1] shows the 419bp amplification product of protein b gene of M. tuberculosis by PCR.
Only one sample was positive for acid fast bacilli (AFB) by microscopy, the sample was also positive by culture and PCR. Of the ten confirmed TBM with positive CSF culture, nine were positive by PCR with protein b. In suspected TBM group of the 60 patients, PCR was positive in 49 patients [Table 1] and all 49 patients had good response to anti-tuberculous drug therapy. The response to treatment was judged by the experienced neurologists in treating TBM. In control group all 40 patiens showed negative result in all the three tests, thus giving 100 percent specificity for all the tests used.
A final diagnosis of TBM was made in 70 patients, based on results of culture, microscopy, PCR and response to anti tuberculous drug therapy [Table 2]. Of these 70 patiets, PCR was positive in 58, culture in 10, and microscopy in one. Thus the sensitivity of PCR with protein b , culture and microscopy was 82.8%, 14.3% and 1.4% respectively. However the sensitivity of PCR in the confirmed TBM and suspected TBM group was 90% and 81.7% respectively. In non TBM group PCR was negative in all cases. Hence, the specificity was 100%.
TBM is a common clinical condition which requires early and prompt treatment to prevent neurological complications.  Among the various methods studied, PCR is considered to be most sensitive and specific diagnostic method especially in cases where suspicion is high but AFB staining is negative. 
The majority of PCR based studies have used IS6110 as target. , The reason for using IS6110 is the presence of multiple copies in M. tuberculosis genome, which was thought to confer higher sensitivity.  It has however been shown that there are M.tuberculosis strains originating from India which do not contain IS6110.  It is believed that more studies are required to establish its utility in the diagnosis of TBM.  Therefore, in this study we have evaluated protein b gene PCR for diagnosis of large number of patients of TBM and non-TBM and also compared the results of PCR with conventional methods like microscopy and culture.
In our study, the protein b PCR detected the presence of M.tuberculosis DNA in 90% of patients with confirmed TBM and 81.7% patients with clinically suspected TBM. Our results are similar to some previous studies which showed 85-98% sensitivity ,,,,,,, and differs from other studies, which had a low sensitivity of 32-75%. ,,, The reason for the low sensitivity in many studies could be due to low volume of CSF available, insufficient lyses of cells, loss of DNA during purification or different target used for amplification.  In our study, there was one patient who was culture positive but PCR negative and there were 11 clinically suspected patients who were also PCR negative. All these 11 patients responded to antitubercular therapy. The various reasons for PCR negativity in the culture positive case and the clinically suspected cases which responded to anti tuberculous drug therapy could be small volume of CSF received in the microbiology laboratory, low number of bacteria in the CSF, poor lyses of bacteria in the CSF samples or the presence of some PCR inhibitors like bacterial contaminants, phenol etc in the CSF samples. Similar factors have also been reported to be responsible for PCR negativity in culture confirmed as well as clinically suspected cases of TBM in other studies. , Sometimes the tough cell wall of M. tubecculosis makes the isolation of target DNA difficult. 
For the detection of overall TBM cases, the protein b PCR was useful in terms of sensitivity (82.8%) and specificity (100%) as compared to culture results showing 14.3% sensitivity and 100% specificity [Table 2]. Protein b PCR is a good rapid method for diagnosis in clinically suspected TBM cases, particularly where AFB staining and culture are negative. Some other studies have also shown that CSF PCR is more sensitive in cases of clinically suspected TBM that responded to empirical treatment. In our study of 81% clinically suspected patients who were positive for protein b PCR, had shown good response to anti tuberculou drug therapy. Protein b PCR was positive in 90% of confirmed TBM cases. The limitation of this study is that there is no diagnostic test which can serve as a 'gold standard'. While the culture has low sensitivity, clinical assessment may be subjective. Commercially available kits in India such as MTD Gen Probe and Roche Amplicor, which are PCR based tests have shown to give low sensitivities of 33% and 60% respectively in patients with TBM. , As the sensitivity of protein b in detecting TBM was 82.8% in our study, there would be cases of TBM in endemic country like India, which would be missed by protein b and hence further studies with multiplex PCR using IS6110 and protein b should be done to look whether their combined use increases the sensitivity of the diagnosis of TBM.
We have evaluated this protein b primer for the first time in large number of TBM and non-TBM CSF samples. A number of other studies have used IS611O as target for amplification, but this IS611O may be missing in our population as mentioned above. The sensitivity and specificity in the present study was much higher than earlier studies and suggest the utility of protein b PCR as an important tool to guide a clinician in the diagnosis, where conventional methods fail.
According to our study, protein b PCR could make a considerable impact in the diagnosis of TBM, which is often missed by conventional tests producing negative results or cause an unacceptable delay in diagnosis. This is especially true in TBM cases in which early diagnosis is essential for better outcome of the disease. In conclusion, protein b PCR can play potentially important role in strengthening the diagnosis of TBM.
The study is a part of the research project funded by DST (Chandigarh).
[Table 1], [Table 2]