Zeenath Begum¹, P V Satya Narayana^2*

 

 

Abstract               Background: Tuberculosis is one of the oldest human diseases with the highest mortality rate among infectious diseases and is caused by bacteria Mycobacterium tuberculosis complex and usually affects the lungs, although other organs are involved in up to one-third of cases. Adenosine deaminase (ADA) is now being recognized as a marker of cell mediated immunity particularly as a marker of T lymphocyte activation. Adenosine deaminase levels (ADA) have also been considered by several researchers to differentiate tubercular disease from non-tubercular. Purpose: 1) To evaluate the usefulness of enzyme Adenosine deaminase in diagnosis of Tuberculous Meningitis. Methodology: A case control study was conducted and a total of 100 patients were included in the study in which 50 suspected and diagnosed cases of tubercular meningitis patients and 50 healthy subjects taken as controls. Estimation of ADA in both serum and CSF was done by GUISTI AND GALANTI METHOD Results: In the present study, the Mean±SD of serum ADA in TBM cases was 37.54±10.81 and in controls was 11.56±3.240. This implied that tuberculous 56 meningitis patients had high levels of serum ADA levels than controls and the P value was <0.0001. Simultaneous estimation of serum and CSF-ADA have an added advantage to diagnose TB meningitis and differentiate between TBM and pyogenic meiningitis. Conclusions: To conclude, ADA is an enzyme that increases in TB because of the stimulation of T- cell lymphocytes by microbial antigens, SERUM and CSF ADA can be used for early diagnostic marker of Tubercular Meningitis.

Key Words: Tuberculous meningitis, Adenosine deaminase, Cerebrospinal fluid.

 

 

 

INTRODUCTION

Tuberculosis (TB) is one of the top 10 causes of death worldwide. In 2016, 10.4 million people fell ill with TB, and 1.7 million died from the disease (including 0.4 million among people with HIV). In 2016, an estimated 1 million children became ill with TB and 2,50,000 children died of TB (including children with HIV associated TB). TB is a leading killer of HIV-positive people in 2016, 40% of HIV deaths were due to TB. Over 95% of TB deaths occur in low- and middle-income countries. Seven countries account for 64% of the total, with India leading the count, followed by Indonesia, China, Philippines, Pakistan, Nigeria, and South Africa Multidrug-resistant TB (MDR-TB) remains a public health crisis and a health security threat. WHO estimates that there were 6, 00,000 new cases, of which 4,90,000 had MDR-TB¹. Among various forms of extra pulmonary TB, tuberculous meningitis (TBM) is the most severe form and remains a major global health problem with a high mortality rate². Early recognition of TB meningitis is of paramount importance. Clinical diagnosis of TBM is difficult as the clinical features are nonspecific and vary widely, and is often diagnosed when brain damage has already occurred^[3,4]Available methods of diagnosis of TBM were evaluated and all of them were shown to have low sensitivity and specificity. Direct evidence of acid fast bacilli (AFB) is available only in small percentage of cases⁵. So it is necessary to introduce simple, reliable and cost effective method for rapid diagnosis and differentiation of various types of meningitis. Adenosine deaminase (ADA) is an enzyme in the purine salvage pathway. ADA levels are ten times higher in T-lymphocytes than in erythrocytes. ADA is released by T cells during cell mediated immune response (CMI) to the tubercle bacilli. ADA is now being recognized as a marker of cell mediated immunity particularly as a marker of T lymphocyte activation. Adenosine deaminase levels (ADA) have also been considered by several researchers to differentiate tubercular disease from non-tubercular^6,7 The ADA2 isoenzyme is the major contributor to increased ADA activity in CSF of patients with tubercular meningitis, probably reflecting the monocyte-macrophage origin of the ADA. The 4 source of raised ADA in CSF of TBM patients may be damaged blood brain barrier permitting ADA to enter into CSF from the blood or adjacent cerebral tissue and/or as a result of lymphocyte-macrophage proliferation indicating local immune response. So the present study was carried to evaluate the usefulness of enzymes such as Adenosine deaminase in serum and CSF for the diagnosis of Tuberculous Meningitis as it is difficult to demonstrate on culture⁸

 

MATERIALS AND METHODS

A case control study was conducted during December 2015 to May 2017. A total of 100 patients were recruited for the study which included 50 suspected and diagnosed cases of tubercular meningitis patients and 50 healthy subjects taken as controls. Blood and CSF samples were collected

Estimation of ADA: GUISTI AND GALANTI METHOD (52)

Principle: ADA hydrolyses adenosine to ammonia and inosine. Ammonia further reacts with phenol and hypochlorite in an alkaline medium to form blue iodophenol complex with sodium nitroprusside as a catalyst. Intensity of blue is proportional to ADA. Adenosine + H2O ADA Inosine + Ammonia 27 Ammonia + Phenol + Hypochlorite Alkaline medium Blue indophenols The intensity of blue colour formed is proportional to ADA concentration and measured Spectrophotometer at 630nm.

RESULTS

The present study, a total of 100 patients were recruited for the study which included 50 suspected and diagnosed tubercular meningitis patients and 50 healthy individuals as controls. In Serum ADA and CSF ADA were evaluated and the results were expressed in IU/l for ADA. The data was analyzed using Graph Pad Prism software version 6.0. Descriptive

 

Table 1: Study parameters in all groups

 

Table 2: Unpaired t-test between 2 group

P value was significant in all the parameters (SERUM ADA,CSF ADA ) Pearson’s correlation was done to assess the correlation of parameters within the groups.

 

Table 3: Pearsons correlation between different parameters in cases

Pearson’s correlation of cases: Serum ADA was positively correlated with CSF ADA which was not statistically significant.

Table 4: Pearson’s correlation between different parameters in controls

 

Pearson’s correlationin controls: Serum ADA was negatively correlated with CSF ADA

Roc curve analysis: In order to assess the maximum sensitivity, specificity and the best cut off values of ADA are calculated using ROC analysis. Best cut off values are established by selecting a point closer to the left hand curve that provides greatest sum of sensitivity and specificity. Area under curve provides unbiased estimates of sensitivity and specificity. It is a comprehensive representation of pure accuracy discriminating ability over the entire range of the test.

 

Table 5:

          Figure 1                                                      Figure 2

Figure 3                                                                                Figure 4

Figure 1: Graphical representation of Mean ± SD of SERUM ADA; Figure 2: Graphical representation of Mean ± SD of CSF ADA; Figure 3: ROC curve of Serim ada in cases vs controls; Figure 4: ROC curve of csf ada in cases vs controls

DISCUSSION

In the present study, the mean±SD of serum ADA in cases was 37.54±10.81 and in controls was 11.56±3.240. This implied that tuberculous meningitis patients had high levels of serum ADA levels than controls and the P-value was <0.0001. These findings were in concordance with the following studies of ChoBH, Km BC, Yoon GJ et al⁹, Jyotsna Shrivastava et al¹⁰, Donald et al¹¹, On the contrary, Chaturvedi et al⁷³ reported that the specificity and sensitivity for CSF-ADA were 86 and 87%, respectively on using the CSF-ADA value of 17.06 IU/L as cut-off value while specificity and sensitivity for serum ADA were 83 and 96% respectively on using a cut-off value 31.23 IU/L. They have suggested that simultaneous estimation of serum and CSF-ADA have an added advantage to diagnose TBM and differentiate between TBM and pyogenic meiningitis. These different results can be due to the different study method, age and the disease stage during which ADA was determined. Measurement of CSF-ADA is a simple, cost-effective and reliable biochemical test to differentiate TBM from non-tuberculous meningitis. On the other hand, simultaneous estimation of serum and CSF ADA measurement has no added advantage over CSF-ADA alone. Thus, CSF-ADA estimation can be a useful tool to diagnose TBM cases especially in resource-limited countries like India where the incidence of tuberculosis is high and availability of other diagnostic tests for tuberculous meningitis is limited. Dr J.P.Bhartiya et al¹² studied ADA activity in Serum and CSF of 80 patients with tuberculous meningitis. Serum ADA findings was mean 71.96, SD16.3, sensitivity 87% and specificity 85%. CSF ADA mean 13.95, SD 3.91 sensitivity 91 and specificity 90. The author found that aimed to find out the association between CSF and serum ADA levels in tubercular meningitis and found a significant positive correlation between CSF and serum ADA levels in patients as compared to the controls. ADA has a major role in proliferation and differentiation of T lymphocytes. It also acts in maturation of monocytes and transforming them to macrophages. ADA is a significant indicator of active cellular immunity. Cell-mediated immunity plays an important role in tuberculosis infection. AD Aactivity in Serum has been studied in pulmonary and serosal tuberculosis and it has been proposed to be a useful surrogate marker for TB because it can be detected in body fluids such as pleural, pericardial and peritoneal fluid. The levels of ADA increase in TB because of the stimulation of T cells by mycobacterial antigens in TB patients^. P. R. Donald et al¹³ studied simultaneous determination of cerebrospinal fluid and plasma adenosine deaminase activity diagnostic aid in tuberculous meningitis, According to the author CSF/plasma ADA ratio is of value in differentiating TBM from other forms of bacterial meningitis. In the present study, following findings are observed in cases of CSF ADA was Mean 23.6, SD 4.84, Cutoff valve 11, sensitivity 100 and specificity 100. We have observed a statistically significant difference in the CSF - ADA levels of TBM and controls (P <. 0001). These findings were in concordance with the following studiesof B K Gupta et al¹⁴ This is as similar to the finding seen In the study conducted by Ali Moghtader et al¹⁵ observed mean 23.05, SD 13.1,Cutoff value 10.5,sensitivity 81 and specificity 86 in CSF of tb meningitis patients, AparnaYerramilli et al^[16],DrJ.P.Bhartiya et al^[12],Raviraj et al¹⁷, R. Baheti et al¹⁸, M. Mehta et al¹⁹. The mean CSF-ADA activity in TBM group was significantly higher than in non-TBM group, and it is statistically significant with a p-value of <0.001. CSF values of ADA have long been used as the investigation of choice in the diagnosis of tubercular meningitis, which is why many studies have been conducted to improve the accuracy of the test. Many studies have focused on the mean value of the CSF ADA levels in diagnosed cases and tried to compare them with suspected cases. Researches have also tested the sensitivity and specificity of the test by increasing the Cutoff value. The findings in the present study are similar to the studies conducted by BK Gupta.et.al and Ali Moghtader.et.al with near similar cutoff values obtained. We achieved a sensitivity and specificity of 100% respectively which was 94.33% and 90.47% in BK Gupta.et.al and 81% and 86% in Ali Moghtader.et.al studies. The difference in the values can be attributed to less number of samples in the aforementioned studies and their series in suspected cases, whereas the present study was carried out in diagnosed cases with more number of cases.

 

CONCLUSION

Adenosine deaminase activity in Serum and CSF is a rapid, relatively inexpensive and easy procedure and can be of great value in the early differentiation of tuberculous meningitis, bacterial meningitis and aseptic meningitis and also in early diagnosis of tuberculous meningitis, helping in earlier institution of appropriate treatment and thereby decreasing mortality and complications. Though demonstration of AFB in CSF, CSF culture, CSF cytochemistry, and CT scan are the various means to diagnose TBM, the CSF - ADA estimation is a cost-effective and reliable means to establish a diagnosis of TBM.

 

ACKNOWLEDGEMENT

The authors are thankful to faculty, technicians, non-teaching staff of Department of Biochemistry, Kakatiya Medical College, Warangal, Telangana, India, for providing facilities to carry out the research work. We are also thankful to Medicine, Pulmonology and SPM departments for their support to carry out this work.

 

REFERENCES

1. Jahantigh, F.F. and Ameri, H. (2017) Evaluation of TB Patients Characteristics Based on Predictive Data Mining Approaches. Journal of Tuberculosis Research, 5, 13-22
2. Kashyap RS, Agarwal NP, Chandak NH, Taori GM, Biswas SK, Purohit HJ, et al. The application of the Mancini technique as a diagnostic test in the CSF of tuberculous meningitis patients. Med SciMonit 2002;8:MT95-8.
3. Thwaites GE, Hein TT. Tuberculous meningitis: Many questions, too few answers. Lancet Neurol2005;4:160-70.
4. Udani PM, Parekh UC, Dastur DK. Neurological and related syndromes in CNS tuberculous meningitis: Clinical features and pathogenesis. J NeurolSci1971;14:341-57.
5. Whiteman M, Espinoza L, Post MJ, Bell MD, Falcone S. Central nervous system tuberculosis in HIV-infected patients: Clinical and radiographic findings. AJNR Am J Neuroradiol1995;16:1319-27.
6. Sharma SK, Mohan A. Adenosine deaminase in the diagnosis of Tuberculosis pleural effusion. Indian J Chest Dis Allied Sci 1996;38(2):69-71
7. Kashyap RS, Kainthla RP, Mudaliar AV, Purohit HJ, Taori GM, Daginawala HF. Cerebrospinal fluid adenosine deaminase activity: a complimentary tool in the early diagnosis of tuberculous meningitis. Cerebrospinal FluidRes2006;3:5.
8. Gupta B K, Bharat Vinay, BandyopadhyayDebapriya, Role of Adenosine deaminase estimation in differentiation of tuberculous and nontuberculous exudative pleural effusions, J Clin Med Res. Apr 2010;2(2):79-84.
9. ChoBH, Km BC, Yoon GJ ClinNeurolNeurosurg. 2013 Sep;115(9):1831-6. Do10.1016/j.clineuro.2013.05.017. Epub 2013 Jun 12.
10. JyotsnaShrivastava J, Agrawal A, Patel D. Simultaneous estimation of serum and cerebrospinal fluid adenosine deaminase level to differentiate tuberculous and non-tuberculous meningitis. Int J Med Res Rev 2014;2(5):414- 419.doi:10.17511/ijmrr.2014.i05.03.
11. Donald PR, Malan C, vander Walt A, Schoeman JF. The simultaneous determination of cerebrospinal fluid and plasma adenosine deaminase activity as a diagnostic aid in tuberculous meningitis. S Afr Med J. 1986 Apr 12;69(8):505
12. Dr. Jai Prakash Bhartiya, Dr.Rakhee Yadav Indian Journal of Basic and Applied Medical Research; September 2015: Vol.-4, Issue- 4, P. 208-214
13. Donald PR, Golden M, Taljaard JJ. Adenosine deaminaselevels in cerebrospinal fluid in the diagnosis of tuberculous meningitis. JTrop Med Hyg 1984;87(1):33-40.
14. B K Gupta J Clin Med Res. 2010 Oct; 2(5): 220–224 84
15. Ali Moghtader, Niazi A, Alavi-Naini R, Yaghoobi S, Narouie B. Comparative analysis of cerebrospinal fluid adenosine deaminase in tubercular and nontubercular meningitis. ClinNeurolNeurosurg2010;112:459–462
16. Yerramilli A, Mangapati P, Prabhakar S, Sirimulla H, Vanam S, Voora Y. A study on the clinical outcomes and management of meningitis at a tertiary care centre. Neurol India 2017;65:1006-12.
17. Raviraj, renoy a henry, g. ganapathirao; Determination and Validation of a Lower Cut Off Value of Cerebrospinal Fluid Adenosine Deaminase (CSF-ADA) Activity in Diagnosis of Tuberculous Meningitis Journal of Clinical and Diagnostic Research. 2017 Apr, Vol-11(4): OC22-OC24
18. Rajesh Baheti, Purnima Laddha,RSGehlot CSF - Adenosine Deaminase (ADA) Activity in Various Types of Meningitis Journal, Indian Academy of Clinical Medicine Vol. 2, No. 4 October-December 2001
19. Dr Margeyi Mehta, Dr JigishShah, Dr A T Leuva; CSF-ADA As Diagnostic Tool For Tuberculous Meningitis Patients ;NJIRM 2012; Vol. 3(3). July –August eISSN: 0975-9840 pISSN: 2230 – 9969.