EVALUATION OF POLYMERASE CHAIN REACTION FOR RAPID DIAGNOSIS OF TUBERCULOUS MENINGITIS

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1 Original Article Ind. J Tub., 2002,49, 133 EVALUATION OF POLYMERASE CHAIN REACTION FOR RAPID DIAGNOSIS OF TUBERCULOUS MENINGITIS Joy Sarojini Michael 1, M.K. Lalitha 1,Thomas Cherian 2, Kurien Thomas 3, Dilip Mathai 3, O.C. Abraham 3 and K.N. Brahmadathan 1 (Received on ; Accepted on ) Summary : Background: Rapid and specific diagnosis of tuberculous meningitis is of utmost importance. Aim: To evaluate polymerase chain reaction (PCR) using primers directed against the IS6110 gene of M.tuberculosis in the diagnosis of tuberculous meningitis (TBM). Methods: PCR was performed on CSF samples from 34 patients suspected to have tuberculous meningitis (TBM) and 68 likely to be having meningitis due to other causes (controls). The first group was further divided into definite TBM and probable TBM using culture as the gold standard. Results : In 17 culture positive patients (definite TBM), PCR was positive in 13 while in 17 patients who were diagnosed clinically, PCR picked up 11 cases all of whom were culture negative. There were 2 PCR positives among the control group : one patient was HIV positive having cryptococcal meningitis, with disseminated tuberculosis in whom true positivity could not be ruled out. For final diagnosis of tuberculous meningitis, the overall sensitivity of microscopy, culture and PCR were 3%, 50%, and &71% and specificity 100%, 100% and 97% respectively. Conclusion : PCR is valuable in the rapid diagnosis of tuberculous meningitis. Key words : Tuberculous meningitis; PCR; Tuberculosis diagnosis INTRODUCTION Tuberculosis continues to be an important cause of morbidity and mortality throughout the world 1, especially in a developing country like India. Tuberculous meningitis (TBM), the most dangerous form of extra-pulmonary tuberculosis, occurs in 7-12% of tuberculous patients in our country and has high fatality despite availability of effective chemotherapy. Delay in diagnosis is directly related to neurologic sequelae in 20-25% of patients who do not receive early treatment. Demonstration of tubercle bacilli in cerebrospinal fluid (CSF) is necessary for definitive diagnosis. Bacteriologic methods are inadequate for the early diagnosis of TBM because there are far too few organisms in the cerebrospinal fluid for consistent demonstration by direct smear, and culture identification takes 6-8 weeks. Diagnosis in the critical early stage of the disease is, therefore, often presumptive and a rapid diagnostic method like polymerase chain reaction (PCR) is urgently needed. In Christian Medical College & Hospital, Vellore, tuberculous meningitis is an important causation of meningitis despite diagnosis being still dependent on clinical and laboratory parameters. However, PCR results can be obtained within 24 hours of the receipt of the sample and its sensitivity and specificity far exceeds that of microscopy. Several studies have reported PCR sensitivity ranging between 50 and 91% and specificity ranging between 95 and 100%, using culture as the gold standard 2-5. Therefore, PCR method using IS 6110 antigen was evaluated at our centre for rapid and specific diagnosis of tuberculous meningitis. 1. Department of Clinical Microbiology 2. Department of Child Health 3. Department of Medicine Christian Medical College & Hospital, Vellore Correspondence: Dr. M.K. Lalitha, Professor, Department of Microbiology, Christian Medical College & Hospital, Vellore

2 134 J.S MICHAEL ET AL MATERIAL AND METHODS Subjects The subjects comprised patients and controls who were admitted between January 1999 and January 2000 with provisional diagnosis of meningitis. The patients were those who got the presumptive diagnosis of tuberculous meningitis. They were further divided into three categories depending on the clinical and laboratory findings. Category I : Definite cases of TBM: based on fever, persistent headache, neck stiffness, vomiting, alteration of sensorium or a focal deficit, mental changes and confusion, lethargy and stupor, for more than seven days. Supportive evidence was obtained by examination of CSF showing the presence of > 10 WBC/ml and >80% lymphocytes, protein concentration of >40 mg/dl and sugar concentration less than 60% of corresponding blood glucose. The suggestive radiological and CT findings included basal exudate, hydrocephalus, infarcts and gyral enlargement. Also taken into consideration were clinical response to anti-tuberculosis therapy with resolution of signs and symptoms of tuberculous meningitis and failure to respond to general antibiotics. Smear and / or culture positive results for M.tuberculosis were confirmatory. Category II :Possible cases of TBM, who had abnormal neurological signs and symptoms of meningitis and supportive evidence of one of the criteria listed under definite case of TBM but had negative smear/culture for M.tuberculosis. Category III (controls): Patients who had signs and symptoms of meningitis and whose CSF samples were smear and culture negative for M.tuberculosis but smear/culture/antigen positive for other bacteria, viruses or fungi or those with CNS disorders other than meningitis. Samples The CSF samples of the subjects were processed in a biosafety cabinet placed in a specially assigned room. Culture positivity was taken as the gold standard for evaluating the polymerase chain reaction (PCR) methodology used. At least l of the specimen was aliquoted into provials whenever possible and stored at -20ºC to avoid frequent freezing and thawing. PCR was done only on those specimens which were used for smear and culture examinations, for M.tuberculosis as well as for other organisms. Using 200 l of the centrifuged deposit for PCR, the rest of the deposit was used for smear and inoculation on two slopes of Lowenstein- Jensen medium. Where the specimen was < 2ml, it was not centrifuged but directly taken for PCR, smear and culture. The CSF samples underwent all the three microbiological tests namely microscopy by auramine staining, culture on Lowenstein Jensen medium and PCR. All the CSF samples belonging to category I Definite TBM were retrospective samples except one which was positive by PCR and then became culture positive after 6 weeks. The CSF samples belonging to category II Possible TBM, and category III Non TBM controls were all prospective samples. PCR was standardised and was found to have a quantitative sensitivity to detect the DNA equivalent to 10 organisms. It tested positive with standard strain of M.tuberculosis, H 37 RV, whereas it yielded a negative result with other causative agents like Streptococcus pneumoniae, Haemophilus influenzae and Cryptococcus neoformans. DNA extraction DNA was extracted from 200l of CSF using the QIAamp blood kit using manufacturer s instructions (QIAGEN Gmg H, Hilden Germany). Same quantity of distilled water was processed in parallel in each run as a control for the extraction step. In each step there was one specimen from the Definitive TBM group, 2 from the Probable TBM group, 2 from the Non-TBM group and a water blank as negative control. The strain of H 37 RV at concentration of 10 4 organisms/ml was used as positive control.

3 PCR IN TUBERCULOUS MENINGITIS 135 Polymerase chain reaction (PCR) PCR was performed by a single step method to detect the presence of multi copy IS6110 (Insertion sequence 6110) element specific for bacteria belonging to the M.tuberculosis complex. In all, 10l of the extract from the previous step was added to 40l of the mix to give a final volume of 50l. For reaction, final concentration included 5l of 10x buffer (1.5mM MgCl2, 10mM Tris-HCl, 50mM KCL and Triton X-100), 1l of dntps of 10 pmols each, 1l of the primers ISL-l (5 CGC CGC GAG CTG CGC GAT GGC GA 3 ) ISL 2 (5 GGT GCT GGT GGT CCG AAG CGG CG 3 ) of 20 pmols each, 0.75l of Taq polymerase and 31.25l of millipore water. Taq polymerase used was Dynazyme a thermostable DNA polymerase enzyme purified from a strain of Thermus brockianus (F500), which is a Finnzymes proprietary strain 2 U/l. Amplification was done in a thermal cycler (MJ Research). The reaction cycles consisted of 5 minutes at 95º followed by 40 cycles of 1 minute at 95ºC and 1 minute at 72ºC and final holding step of 7 minutes at 72ºC. Water blanks were also used in the amplification step to monitor carry over of amplicons. The microfuge tubes containing the amplified products were taken out after the appropriate cycling time. The amplified products were stored at 4ºC till their use for detection. Amplified DNA was detected by electrophoresis of 20l of the amplified product on 2.5% agarose gel with 0.1% ethidium bromide. The electrophoresis was done in a submarine gel electrophoresis equipment. The amplified product at 263bp region was detected with the help of ethidium bromide in agarose gel electrophoresis. GEL DOC (Bio-Rad) was used for documenting the gel picture. Standardization of PCR Assays were performed by spiking both normal saline and culture/smear negative (sterile) CSF samples with the other organisms causing meningitis such as Streptococcus pneumoniae, Haemophilus influenzae, and Cryptococcus neoformans. The additions were not picked up by this assay, demonstrating specificity of the test. H 37 RV, a standard strain of M.tuberculosis was also spiked in both normal saline and sterile CSF. A concentration of 10 4 organisms/ml was used as positive control in every run. Serial doubling dilutions of the H 37 RV in normal saline from 10 6 to 1 organisms/m were prepared and 200l of the sample from each dilution was taken and the above mentioned steps were performed. By the above-mentioned protocol, PCR was able to detect upto 10 organisms/ml demonstrating sensitivity of the test. Four different work stations were employed, one each for the above mentioned procedures to minimize the chances of contamination through extraneous DNA and amplicon carry over. RESULTS In all, 102 subjects were studied, of whom 17 belonged to Definite TBM, 17 to Possible TBM and the remaining 68 to Non-TBM groups (18 of bacterial meningitis, 5 viral meningitis, 20 cryptococcal meningitis and 25 other CNS disorders). Microscopy contributed an AFB positive result only in one sample, which was also positive by culture and PCR, of the 17 cases in the Definite TBM group. All these patients had culture positive tuberculous meningitis of which 13 were positive by PCR. Of the four samples which were PCR negative, one patient had acanthamoeba meningitis which did not respond to first and second line antibiotics even though the CSF specimen grew M.tuberculosis. In the Possible TBM group of 17 patients, in whom microscopy and culture results were negative, PCR was positive in 11 patients (Table 1), out of whom 8 responded well to anti-tuberculosis treatment, one died and two were discharged against medical advice. In the Controls group, PCR was positive in two patients, one of whom had an initial diagnosis of cryptococcal meningitis but was later found to be HIV infected and having both disseminated tuberculosis as well as cryptococcal infection while the other patient had pyogenic meningitis caused by Streptococcus pneumoniae.

4 136 J.S. MICHAEL ET AL Table 1: Clinical and laboratory parameters of patients with CSF specimen positive by PCR but negative by smear and culture Sl. Total WBC Lymphocytes Protein Sugar ATT No. count % mg% mg% Outcome Improved Improved DAMA Improved Improved Improved Improved Improved DAMA Died Improved DAMA - Discharged against medical advice ATT : Anti-tuberculosis treatment A final diagnosis of tuberculous meningitis was made for 34 patients, based on results of culture, PCR and microscopy/response to anti-tuberculosis treatment (Table 2). PCR was positive in 24 of the 34 cases, culture in 17 cases and microscopy only in one case. The sensitivity of PCR, culture and microscopy was, thus, 71%, 50% and 3% respectively. However, the sensitivity of PCR in the Definite TBM and the Possible TBM groups was 77% and 65% respectively. In the non-tbm group, PCR was positive in 2 cases. Hence, the specificity was 97%. Table 2 : PCR, culture and microscopy results in TBM and Non-TBM groups and sensitivity and specificity of each test DISCUSSION Among the different forms of tuberculosis, tuberculous meningitis (TBM) is a medical emergency that requires urgent treatment because early intervention reduces the risk of mortality. Rapid detection of Mycobacterium tuberculosis is of vital importance for the proper diagnosis and management of tuberculous meningitis. Among the many rapid methods studied, polymerase chain reaction is considered to be the most specific diagnostic method. In India, only a few studies have been reported and this study has the largest number of CSF samples studied. In addition, PCR was conducted on all the patients having symptoms and signs compatible with TBM without selection and PCR was done without the knowledge of the status of the patient or the results of any other tests. Test Result Final Diagnosis TBM Non- Sensiti- Specifi- TBM vity(%) city(%) PCR Positive Negative Culture Positive Negative Microscopy Positive Negative Until now, microscopy using the Ziehl- Neelsen or auramine staining procedure has been the main means of rapidly confirming muyobacterial etiology in the acutely ill patient. Only one of our suspected tuberculous meningitis cases had a CSF sample with positive auramine result. The detection limit of microscopy is 10 4 mycobacteria per milliliter whereas the majority of patients with TBM have fewer mycobacteria in their CSF samples and are thus missed as tuberculous meningitis cases. Centrifugation of the sample prior to microscopy is advisable. Even then, only 5% to 20% of patients have Ziehl Neelsen positive results 2,6-7.

5 PCR IN TUBERCULOUS MENINGITIS BY 137 Among the 34 cases considered either definitely tuberculous or possibly TBM, on the basis of clinical and laboratory findings, PCR proved to be a more sensitive method, detecting 24 out of 34 cases (71%), whereas only 17 were culture positive (50%). Additional cases were diagnosed as tuberculous meningitis on the basis of response to therapy.some studies report a relatively higher sensitivity of PCR for the diagnosis of TBM, ranging from 75-90%, but some authors had tested very small number of patients 8 or had used a selected patient group 9 or had a considerable number of false positives 4. An explanation for the lower sensitivity of PCR in our study could be the sometimes small volume of CSF (mean volume l) available for testing (after using for smear and culture) so that the sample could not be concentrated. The volume of sample is of great significance in PCR, especially in tuberculous meningitis, due to frequent low number of bacteria in the CSF. Culture of CSF also requires larger volume and when both culture and PCR have to be done, the minimum volume of CSF should be 2 ml. Another reason for low sensitivity of PCR may be presence of PCR inhibitors in the CSF as well as poor lysis of mycobacteria 10. False negatives have occurred in two studies, in which reported CSF PCR sensitivities 8,11 were 32% and 85%. For detection of M.tuberculosis, PCR is not affected by the presence of other infecting organisms, as may occur in the immuno-compromised patients. One patient in the Non-TBM group with HIV infection had disseminated tuberculosis and cryptococcal meningitis (diagnosed in the laboratory by smear and India ink preparation of the CSF) for which he was receiving treatment. PCR was positive in this patient and anti-tuberculosis drugs were started along with Amphotericin B to which he responded well. We found another CSF positive by PCR in a patient with pyogenic meningitis caused by Streptococcus pneumoniae. This positive result could be due to cross contamination during initial handling. It is unlikely that amplicon carry over occurred, as we had taken all the necessary steps to avoid it. Our study supports the thesis that PCR deserves a place in the laboratory diagnosis of tuberculosis but careful adherence to the test protocol is mandatory. Results are available with a speed comparable to microscopy; sensitivity is higher than both microscopy and culture and the direct identification of the organism, as belonging to the M.tuberculosis complex is possible if the samples are concentrated. Culture should be used concurrently for drug sensitivity testing, for restricted fragment length polymorphism (RFLP) studies and as a back up for PCR. Overreliance on PCR should be avoided, as premature cessation of treatment will have serious consequence in patients with TBM, in whom PCR is negative. Hence, a combination of clinical criteria and PCR is needed in the diagnosis of TBM. REFERENCES 1. Ellner,J.J.,A.R.Hinman,S.W.Dooley,.A.Fischl,K.A.Septowitz, M.J.Goldberberger, T.M.Shinnick, M.D.Iselman, and W.R.Jacobs, Jr.. Tuberculosis symposium :Emerging problems and promise. J.Infect.Dis.1993;168: Kox L.F.F. Ing S.K, Kolk A.H.J. Early diagnosis of tuberculous meningitis by polymerase chain reaction, Neurology. 1995;45 (12): Shankar P, Manjunath N, Mohan K.K.et al,. Rapid diagnosis of tuberculous meningitis by polymerase chain reaction.lancet. 1991;337:5 4. Miorner H, Sjorbring U, Nayak P, Chandramukhi A. Diagnosis of tuberculous meningitis: a comparative analysis of 3 immunoassays, a immuno-complex assay and polymerase chain reaction. Tubercle & Lung Dis.1995;76: Ahuja G.K, Mohan K.K, Prasad K, Behari M. Diagnostic criteria for tuberculous meningitis and their validation. Tubercle & Lung Dis. 1994; 775: Liu PYF, Shi ZY, Lau YJ, Hu BS. Rapid diagnosis of tuberculous meningitis : a simplified nested amplification protocol. Neurology. 1994; 44; Folgueira L, Delgado R, Palenque Em Noriega AR. Polymerase chain reaction in the diagnosis of tuberculous meningitis in AIDS patients. 1994; Neurology 44: Seth P, Ahuja G.K., Vijayabhanu N, Behari M, Bhowmik S, Broor S, Dar L,Chakraborty M. Evaluation of polymerase chain reaction for rapid diagnosis of clinically suspected tuberculous meningitis. Tubercle & Lung Dis.1996; 77: Scarpellini,P., S.Racca, P.Cinque, F.Delfanti, N.Gianotti, M.R.Terreni, L.Vago and A.Lazzarin. Nested polymerase chain reaction for the diagnosis and monitoring of response in AIDS patients with tuberculous meningitis. AIDS.1995;9: Gasconyme, D.M., Hay.P.M. False negative polymerase chain reaction on cerebrospinal fluid samples in tuberculous meningitis. J Neurol Neurosurg Psychiatry 1999; 67: Nguyen LN, Kox LFF, Pham LD, et al. The potential contribution of the polymerase chain reaction to the diagnosis of tuberculous meningitis. Arch Neurol 1996; 53:771

6 138 Smoking Withdrawal Syndrome Inhaled nicotine bonds with some receptors in the body which provide perceived pleasurable feelings. When the effect of nicotine dissipates, the receptors send signals to the brain which are perceived as a displeasurable feeling - the withdrawal syndrome-compelling the smoker to light up. The act of smoking does not, however, happen in a vacuum. It is almost always attended with particular settings, situations or emotions peculiar to each individual s desires. These unfulfilled desires make the withdrawal symptoms all the more painful. It is mainly the psychological associations which urge a smoker to resume smoking or when the nicotine level in the body is low enough to trigger the receptors. Smokers associate cigarettes and smoking with many aspects of their daily life. And whenever these associations recur, there is a craving to smoke. After stopping smoking, the physical effects of the withdrawal persist for between 7 to 30 days. However, the craving for a cigarette continues for months, despite the withering away of receptors after having been deprived of nicotine. The craving persists because of the associated settings and human emotions of the smoker. YOU HAVE TO QUIT Published by the Tuberculosis Association of India in the interest of public health