IJCRR - Vol 11 Issue 11, JUNE
Date of Publication: 10-Jun-2019
Download XML Download PDF
Role of Cartridge Based Nucleic Acid Amplification Test of Cerebrospinal Fluid in the Diagnosis of Tubercular Meningitis
Author: Lavanya S.R., Mahesh Dave, Archana Gokhroo
Abstract:Introduction: Tubercular meningitis is the commonest form of neurotuberculosis. Diagnosis is challenging because of paucibacillary nature, lack of specific sign and symptoms. CBNAAT is a real time Polymerase Chain Reaction (PCR) test for the diagnosis of tubercular meningitis. So we used this rapid test to assess its role in diagnosis of Tubercular Meningitis(TBM).
Material and Methods: This study was done over a period of one year in a tertiary centre. Patients with symptoms suggestive of tubercular meningitis were our study population. Their detailed clinical history, followed by a thorough general physical & systemic examination were done and documented in a predesigned proforma. Chest x-ray as well as neuroimaging were done in patients whose condition permitted. 3ml CSF fluid was drawn by lumbar puncture, 2 ml was sent for routine and bacteriological examination test and 1 ml for CBNAAT.
Results: 100 patients were included, with a male to female ratio of 1.7:1. Mean age of the affected population was 37.53 years. 47% had radiological finding suggestive of tuberculosis.18 out of 100 were HIV reactive. MRI brain in 64 patients showed meningeal enhancement as the most common finding (60.93%). In Cerebrospinal Fluid (CSF) analysis mean CSF protein was 136.5 mg/dl, mean CSF glucose was 56.4 mg/dl and CSF cell count was 66.7 cells/microliter. Mean Adenosine Deaminase (ADA) was 11.22 U/L.CSF CBNAAT was positive in 9 patients out of which 8 were sensitive to rifampicin and one resistant to it.
Conclusion: Even though CSF cytology gives good estimate of suspected TBM patient the test is not confirmative for bacilli demonstration. CBNAAT being a rapid accurate test would play a major role in the diagnosis, treatment, as well as for estimating rifampicin resistance of one of the common medical emergency in India by clear guidance from WHO.
Keywords: CBNAAT, Tubercular Meningitis, Cerebrospinal Fluid, Rifampicin resistance, WHO
DOI URL: http://dx.doi.org/10.31782/IJCRR.2019.11113
Tuberculosis remains one of the deadliest communicable diseases. Worldwide, TB is one of the top 10 causes of death and the leading cause from a single infectious agent (above HIV/AIDS)1. India is the highest TB burden country in the world, accounting for about 23.3% of the global prevalence and estimated incidence being 2.84 million cases2. About 2.2% of new cases and 15% of previously treated cases have MDR-TB in India.
Tuberculosis was first recognized as a clinical entity in the early 19thcentury by schonlein, who used the term tuberculosis in 1830, which was derived from the English term “tubercle” or “lesion of consumption”3. According to World Health Organization (WHO), TB is a worldwide pandemic. While pulmonary tuberculosis is the most common presentation, extra-pulmonary tuberculosis (EPTB) is also an important clinical problem4,5.
CNS Tuberculosis includes three clinical categories, tubercular meningitis, tuberculoma and spinal arachnoiditis6. Tubercular meningitis is the commonest form of neurotuberculosis in Indian subcontinent accounting for 70%-80% of cases7. The burden of CNS TB is directly proportional to the prevalence of TB infection. Tubercular meningitis is the most devastating form of extra-pulmonary TB with 30% mortality and disabling neurological sequelae in >25% survivors7. Important risk factors includes HIV, overcrowding of urban population, poor nutritional status, appearance of drug resistant strains of tuberculosis7
The diagnosis of EPTB is challenging because of its paucibacillary nature, lack of specific signs symptoms and often negative acid fast bacilli smear of biological specimens8.
Patients who demonstrate a subacute clinical course with headache, vomiting, pyrexia and anorexia should be suspected of having TBM. Diagnosis is based on the clinical symptoms and cerebrospinal fluid changes (increased protein, low glucose and mononuclear pleocytosis)9.
Definitive diagnosis of tuberculosis involves demonstration of MTB by microbiological, cytological or histo-pathological methods
Gene Xpert or CBNAAT (Cartridge Based Nucleic Acid Amplification Test) is a real time PCR test approved by WHO Policy in 2010, initially used in diagnosing MDR-TB and HIV associated TB. RNTCP policy update in 2013 expanded its uses, including for the diagnosis of TB in children, on selected specimens for the diagnosis of EPTB and for all individuals suspected of having pulmonary TB.10 Based on systematic review, WHO recommends Xpert over conventional tests for diagnosis of EPTB which permits rapid TB diagnosis through detection of the DNA of mycobacterium TB and simultaneous identification of a majority of the mutations that confirm Rifampicin resistance which is highly predictive of MDR TB.
In the present study we prospectively determined the utility of this test in detection of MTB in CSF samples obtained from the patients suspected to have tubercular meningitis.
Aim and Objective.
To determine utility of Gene Xpert (CBNAAT) test in detection of MTB in CSF obtained from the patients who are clinically diagnosed case of tubercular meningitis.
Materials and Methods.
The present study was conducted for a period of one year at R.N.T. Medical College and attached group of hospitals, Udaipur. After obtaining approval from the institutional ethical committee and written informed consent from patient, 100 patients with symptoms and signs suggestive of TBM were included. Their detailed clinical history, previous history of tuberculosis, history of contact with pulmonary tuberculosis, past history of medical illness were taken. After general physical and central nervous system examination, Chest x-ray as well as neuroimaging (CT / MRI brain) were done if patients condition permitted. Sputum samples from study population, who had cough for any duration, was sent for AFB examination. 3ml CSF fluid was drawn by lumbar puncture, 2 ml was sent for routine biochemical and bacteriological examination and 1 ml for CBNAAT. Bacterial, viral and fungal meningitis were ruled out by clinico-radiological basis & biochemical and bacteriological examination of CSF. All the information was recorded in predesigned proforma formed in Microsoft excel for final analysis.
1.All patients with suspicion of TBM admitted in MBG (Maharana Bhupal Government) hospital in medicine and neurology ward.
2.Age group >18yrs.
1.Age less than 18yrs
2.Patients with other causes of altered sensorium such as dyselectrolytemia, cerebrovascular accident, bacterial /viral meningitis/meningoencephalitis etc
3.Not willing to give consent.
In this study of 100 patients 63 patients (63%) were male and 37 patients (37%) were females. Male to female ratio was 1.7:1. Majority of them were in adolescent/early adult age group. Mean value for age was 41.3 years. Most common co-morbidity associated with study population was past history of pulmonary tuberculosis and diabetes mellitus.18 patients were HIV positive. Fever was the most common symptom followed by altered sensorium headache and vomiting. Chest x-ray suggestive of pulmonary tuberculosis was 39 (39%) out of which only 4 patients were positive for sputum AFB.8(8%) patients had x-ray chest suggestive of old healed lesion and 53(53%) patients had a normal chest x-ray. CSF analysis showed mean CSF protein value of 136.5 mg/dl, CSF cell count value of 58.8 cells/microliter (90% lymphocytes) CSF glucose value of 56.4mg/dl and CSF ADA level had a mean value of 11.3 U/L. MTB was detected in 9 CSF samples out of 100 sent for CBNAAT. Out of 9 samples 8 were Rifampicin sensitive and 1 showed resistance. Among 8 positive and rifampicin sensitive result one patient was HIV positive. Neuroimaging (MRI) was done on 64 patients out of which 55(85.97%) was abnormal and normal in 9 patients. Most common finding was meningeal enhancement (60.93%) followed by hydrocephalus(11.76%). 39 patients recovered completely, 20 patients recovered with residual morbidity and 16 patients had mortality. We lost the track of 25 patients as they did not show up in further follow ups.
In this study of 100 patients final outcome was variable, 39% cases were completely recovered from the disease and 20% patients had some residual morbidity, while 16% mortality was observed. 25% cases outcome could not be documented as we lost the follow up.
In this study 63 patients (63%) were male and 37 patients (37%) were females. Male to female ratio was 1.7:1 and mean age of population was 37.53 years, Most of the patients were in the age group of 8 to 30 yrs (49%). In a study by Rakesh Bhatia et al11. 20 were males and 14 were females and male to female ratio was 1.4:1. Patients of productive age groups were frequently involved by this clinical entity. Co-morbidities in our study group included 18 patients of diabetes mellitus, 14 of hypertension, 24 with past history of tuberculosis/contact and 2 of bronchial asthma. In Inês Sanches et al study, HIV, DM and cancer were frequent co-morbidities associated with extra-pulmonary tuberculosis and seen in 15.8% (20), 6.3% (8) and 4.8% (6) patients respectively12. DM is a well known risk factor for Tuberculosis. depressed cellular immunity, dysfunction of alveolar macrophages, low levels of interferon gamma, pulmonary microangiopathy, and micronutrient deficiency have been implicated in the occurrence of tuberculosis in Diabetic patients.
18 (18%) patients out of 100 were HIV positive in our study population. In a Study by Nathan C Bahr et al out of 257 patients with meningitis 105 (40%) patients were HIV positive13. HIV is a major risk factor for tuberculosis. Patients with HIV and active tuberculosis have an increased risk of extrapulmonary tuberculosis, and this risk will also increase with declining CD4+ count.
In symptomatology of our study population, all patients (100%) had fever followed by altered sensorium in 58 (58%) patients. Headache and vomiting had a prevalence of 41 (41%) and 46 (46%) respectively among patients, followed by seizure (30%), cough (17%), focal neurological deficit (9%,one with facial nerve involvement) and loss of consciousness(5%). In study by Modi M et al14 out of 209 patients 195 patients had fever followed by headache (199), vomiting(169), loss of appetite(139), altered sensorium (101), loss of weight (94), focal deficit (67) and seizure (49).
In our study chest x-ray suggestive of pulmonary tuberculosis was 39, out of which only 4 patients were positive for sputum AFB. 8 patients had x-ray chest suggestive of old healed lesion and 53(53%) patients had a normal chest x-ray. Chest x-ray lesions included apical infiltrates (unilateral >bilateral), miliary infiltrate and old healed calcified lesion and pleural thickening. In a study conducted by Solomons R S et al out of 84 children 37 (44%) TBM patients had CXR findings suggestive of TB, 9 (11%) with disseminated (miliary) TB.
In a study conducted by Sidra Aurangzeb et al15 out of 100 TBM patients radiographic findings of pulmonary TB was found only in 30(30%) patients .The predominant patterns on CXR were apical infiltration (26.6%), miliary mottling (20%) and hilar enlargement (16.6%).The relationship between pulmonary and cranial miliary lesions is controversial and there is a paucity of work done on adults in this regard.
CSF analysis conducted in our study population had mean CSF protein value of 136.5 mg/dl and CSF cell count value of 58.8 cells/microliter ( 90% lymphocytes) and CSF glucose value of 56.4mg/d. In the study conducted by Modi M et al14 out of 203 patients 179 (88%) patients had lymphocytic predominant (>90%) pleocytosis,101 (44%) patients had glucose <30mg/dl. In a study by Moghtaderi et al16 mean CSF protein was 80 mg/dl, mean CSF glucose was 34 mg/dl and lymphocyte predominance (80%) mean value being 18 cells/microliter. In the study conducted by Kumar K et al17, 156 patients had lymphocyte predominance (>50%). High protein (>45mg/dL) was seen in 173 patients. Characteristic CSF findings of TBM include the following: (i)lymphocytic-predominant pleocytosis. Total white cell counts are usually between 100 and 500 cells/μL. Very early in the disease, lower counts and neutrophil predominance may be present, (ii)elevated protein levels, typically between 100 and 500 mg/dL, (iii)low glucose, usually less than 45 mg/dL or CSF: plasma ratio <0.518
CSF ADA level in our study population had a mean value of 11.3 U/L (range 10-20 U/Litre).In the study conducted by Lely solari et al19. The validity of cerebrospinal fluid parameters for the diagnosis of tuberculous meningitis , ADA level >6 U/l had a sensitivity of 60% and was 94% specific. ADA is released by T cells during cell mediated immune response (CMI) to the tubercle bacilli. Raised levels of ADA in CSF are not specific to meningeal inflammatory disease but it can be a test for confirming its etiology with good predictive value. CSF ADA level 10 μ/L is sensitive and can suggest the diagnosis of TBM, especially if the clinical suspicion is high20,21.
In this study MTB was detected in 9 CSF samples out of 100 sent for Gene Xpert technique and 1 with error. Out of 9 samples 8 were Rifampicin sensitive and 1 showed resistance. Among 8 positive and rifampicin sensitive result, one patient was HIV positive.
In study by Nguyen Thi Quynh Nhu et al22 X-pert MTB/ RIF was positive in 108 (59.3%) patients with sensitivity of 59.3% and specificity 99.5%. 4 cases of RIF resistance(4/108) was identified by Xpert. Patel and colleagues23 report the diagnostic performance of the Gene Xpert system's Xpert MTB/RIF assay for the diagnosis of TBM assay's overall sensitivity was 62%, and specificity was 95%.
In the study conducted by Sharma Kusum et al24 multiplex PCR was positive in 84.78% cases. The overall sensitivity and specificity was 86.63% and 100 % respectively. In CSF, the pooled sensitivity from metaanalysis of Xpert MTB/RIF compared against culture as a reference standard was 79.5% (95% CI, 62.0-90.2%) (16 studies, 709 specimens)25. Various studies conducted worldwide has varied sensitivity and specificity depending on various factors such as volume, centrifugation. Despite improved diagnostic accuracy using centrifuged CSF for Xpert compared with un-centrifuged CSF, the ideal CSF volume to collect is unknown. Xpert has an analytical sensitivity detection threshold of approximately ~100 CFU/mL M. tuberculosis organisms.
In our study neuroimaging (MRI) was done on 64 patients out of which 55(85.97%) showed abnormalities, 39 (60.93%) had meningeal enhancement, 8 patients (11.76%) had hydrocephalus, 5 patients (7.81%) had vasculitic infarct, 2 patients(3.12%) had meningoencephalitis and 1 patient (1.56%) had dural sinus thrombosis.MRI brain was normal in 9 patients.
In study conducted by Modi M et al14. Exudates were present in 82.3% patients Hydrocephalus was present in 52.1% and infarcts were seen in 23.9% of patients. Tuberculomas were present in 45.9% of patients. R. Abdelmalek et al16 in their retrospective study reviewed 29 patients out of which 26 patients showed abnormalities in their MRI and concluded that Cerebrospinal MRI performed when TBM is suspected aids in its diagnosis and is also a useful means of monitoring the course of the disease under treatment. Common findings on imaging are abnormal meningeal enhancement in the basal cisterns, hydrocephalus, and vascular complications.
Out of 100 patients in our study population 39 patients (39%) recovered completely, 20 patients (29%) recovered with residual morbidity and 16 patients(16%) had mortality. We lost the track of 25 patients as they did not show up in further follow ups. Prognosis of TBM largely depends on neurologic status at the time of presentation, and time-to-treatment initiation. While the course of TBM is generally not as rapid or fulminant as meningitis due to pyogenic bacteria, empiric treatment should be initiated as soon as the diagnosis is suspected as any delay in treatment can worsen outcome18 Mortality risk is highest in those with comorbidities, severe neurologic involvement on admission, rapid progression of disease, and advanced or very young age.
The main limitation of our study was a small study population and a significant loss of case follow up. The other being overburden of number of samples (both pulmonary and extrapulmonary) as the test was facilitated by a single machine for a tertiary centre. Due to which there was delay in processing of the sample and hence a high false negative result. The third limitation being our inability to repeat CSF samples for comparing different factors such as volume and centrifugation which could have further decreased false negative result of CSF samples study population.
In order to reach a quick diagnosis using CSF specimens, CBNAAT should be preferentially used as rapid diagnosis and treatment is a strong prognostic indicator for reduced death and neurologic deficit. Eventhough CSF cytology gives good estimate of suspected TBM patient the test is not confirmative for bacilli demonstration. Hence CBNAAT has to be endorsed in every centres as the test gives rapid result and also detects rifampicin resistance which is the major concern for every clinician. To increase the value of this test which has gained popularities in detection of MTB and its resistance in sputum samples, a good amount as well as centrifuged CSF sample has to be considered. Clear guidance should be given by WHO regarding CBNAAT testing of CSF samples in suspected TBM patients so that this rapid test would play a major role in diagnosis and treatment of one of the most common medical emergency in India.
CBNAAT-Cartridge Based Nucleic Acid Test
WHO-World Health Organisation
HIV-Human Immunodeficiency Virus
MRI-Magnetic Resonance Imaging
AIDS-Acquired Immune Deficiency Syndrome
MDR-TB-Multi Drug Resistant Tuberculosis
PLHA-People living With HIV/AIDS
EPTB-Extra Pulmonary Tuberculosis
MTB/RIF-Mycobacterium Tuberculosis/Rifampicin resistance
RNTCP-Revised National Tuberculosis Control Programme
CFU-Colony Forming Unit
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
1.Tuberculosis key facts-World Health Organisation, cited at https://www.who.int
2.Gobal Tuberculosis report 2018.Geneva :World Health Organisation 2018
3.Kligman, Stanton, Stgeme, Schor, Paediatric Infectious Disease. Nelson textbook of pediatrics. Volume 2, Ch 215, pp 1445-1453.
4.Fanning A. Tuberculosis: Extrapulmonary disease. Canadian Med Asso J. 1999; 160: 1597-603.
5.Iscman MD. Tuberculosis in relation to human immunodeficiency virus and acquired immunodeficiency syndrome. In: Iseman MD, editor. A clinician’s guide to tuberculosis. Philadelphia: Lippincott Williams and Wilkins; 2000 p. 199-252
6. John M Leonard et al, Central Nervous System Tuberculosis. Aminoff’s neurology and General medicine, cited at www.sciencedirect.com, 5th edition, 2014.
7. Virendra Kr. Goyal, Approach to Neurotuberculosis, cited at www.apiindia.org, Ch 24 Medicine updates 2016.
8.Sunil Kumar Komanapalli, Uma prasad, Bhagyalakshmi Atla, Vasundhara nammi et al. Role Of CBNAAT in diagnosing extrapulmonary tuberculosis in correlation with FNA in a tertiary care centre, International Journal of Research in Medical sciences, Vol 6
9. Suzuki Y, Nihon Rhinso et al .Tuberculosis infection in the nervous system. www.ncbi.nlm.nih.gov/m/pubmed/21838041/, 2011 Aug ;69(8):1422-6.
10.Xpert MTB/RIF implementation manual Technical and operational ‘how-to’: practical considerations, 2014, cited at www.who.int
11. Rakesh Bhatia, Dayal R, Jindal S, Agrawal D, Goyal A ; Gene Xpert for Diagnosis of Tubercular Meningitis. Indian J Pediatr.2016 Nov;83(11):1353-1355 [pubmed]
12. InêsSanches a, Aurora Carvalho, Raquel Duarte Who are the patients with extra-pulmonary tuberculosis? Rev Port Pneumol. 2015; 21(2):90--93. https://journalpulmonology.org
13. Nathan C Bahr, Lillian Tugume, David R Boulware; Improved Diagnostic Sensitivity for TB Meningitis with Xpert MTB/RIF of centrifuged CSF : A Prospective study. Int J Tuberc Lung Dis.2015 Oct;19(10):1209-1215.
14. M Modi, Sharma, Prabhakar, Goyal MK, Takkar A et al. Clinical and radiological predictors of outcome in tubercular meningitis: A prospective study of 209 patients. Clinical Neurology and Neurosurgery 161 (2017) 29–34.[pubmed]
15. Sidra Aurangzeb et al. Chest Radiographic Findings in Neurotuberculosis without Pulmonary Signs and Symptoms. Cited at www.jcpsp.com Journal of College of Physicians and Surgeons Pakistan, 2008; vol.18(1);27-30.
16. Ali Moghtaderi, Niazi A, Alavi-Naini R, Yaghoobi S, Naroule B et al. Comparative analysis of CSF ADA in Tuberculous and Nontuberculous meningitis. Clinical Neurology and Neurosurgery.112(2010) 459-462.[pubmed]
17. Kumar K et al. Diagnosis and treatment of tuberculosis :latest developments and future priorties. ARH. Cited at www.arh.amegroups.com/article/view/3820/4592, 22 Aug 2017.
18. Grace E Marx et al. Tuberculous Meningitis: Diagnosis and Treatment Overview. Tuberculosis Research and Treatment Volume 2011, cited at https://www.hinawi.com/journals/trt/2011/798764, Article ID 798764, 9 pages.
19. Lely Solari et al. The Validity of CSF parameters for the diagnosis of tuberculous meningitis. Int J of Infect Dis. Vol 17:issue 12,Dec 2013; e1111-e1115. https://doi.org/10.1016/j.ijid.2013.06.003
20. Bharat Kumar Gupta et al. Adenosine Deaminase Levels in CSF of Tuberculous Meningitis Patients. J Clin Med Res. 2010 Oct; 2(5): 220–224. doi:10.4021/jocmr429w
21. Gopal Chandra Gosh et al.CSF ADA Determination in Early Diagnosis of Tuberculous Meningitis in HIV-Infected Patients. Scientifica Volume 2016. doi:10.1155/2016/5820823.
22. Nguyen ThiQuynhNhu, Dorothee Heemskerk, Do Dang Anh Thu. Evaluation of GeneXpert MTB/RIF for Diagnosis of Tuberculous Meningitis. Journal of Clinical Microbiology p. 226–233 January 2014 .doi:10.1128/JCM.01834-13.
23. Vinod B Patel, Grant Theron, Laura Lenders, Brian Matinyena, Cathy Connolly, Ravesh Singh. Diagnostic accuracy of quantitative PCR (Xpert MTB/RIF) for Tuberculous Meningitis in a high burden setting: A Prospective Study. PLoS Med.2013 Oct. doi:10.1371/journal.pmed.1001536
24. Sharma Kusum et al. Multiplex PCR for rapid diagnosis of tuberculous meningitis. J Neurol (2011) 258:1781–1787. J Glob Infect Dis.
25. Xpert MTB/RIF implementation manual Technical and operational ‘how-to’: practical considerations. www.who.int, SBN: 978 92 4 150670 0 © World Health Organization 2014.