Adenosine Deaminase Activity (ADA) and Lysozyme Activity (LYSA) were measured in the CSF of tuberculous meningitis (TBM) cases : 26 bacteriologically positive TBM (Group 1), 61 bacteriologically negative TBM (Group 2), 10 non-tuberculous meningitis (Group 3) and 17 control subjects (Group 4). The mean ADA levels in different groups in that order were found to be 11.6, 4.5, 4.4 and 0.8 U/l respectively. The mean LYSA levels in the same groups were 6.3, 2.1, 2.2 and 0.5 mcg/ml respectively. In bacteriologically positive TBM, the mean ADA and LYSA levels were significantly higher than the other three groups (p < 0.0001). An ADA level of 4U/l and LYSA tests were 96%, 82% and 85%, 95% respectively. When both the criteria were considered, the sensitivity and specificity were 91% and 93% respectively. Combination of both test definitions could give additional support to the diagnosis in 49% of 61 clinically suspected but bacteriologically negative TBM cases. Correlation of ADA and LYSA levels in CSF was found to be statistically significant (r = 0.59; p < 0.01)

Duraipandian, M

Prabhakar, R

Selvakumar, N

Thillothammal , n

Vanaja, Kumar

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Original Article  Ind J. Tub., 1991, 38, 217CEREBROSPINAL FLUID ADENOSINE DEAMINASE AND LYSOZYMELEVELS IN THE DIAGNOSIS OF TUBERCULOUS MENINGITISN. Selvakumar, Vanajakumar, M. Duraipandian, N. Thillothammal and R. Prabhakar(Original received on 2151991; Accepted 2.7.1991)IntroductionConfirmation of diagnosis of TBM is oftendifficult. Bacteriological confirmation of thedisease is by demonstration of acid fast bacilli inC S F  b y  m i c r o s c o p y  a n d  i s o l a t i o n  o fM.tuberculosis in culture. Since culture takes along time and only less than 5% of TBM casesare positive by smear examination, there is anurgent need for simple, specific and rapid testsfor the diagnosis of TBM.Several  indirect tests,  such as lactatedehydrogenase level1, lactate concentration2,bromide partition test3 and many direct tests suchas detection of mycobacterial antigen4,5 a n dpresence of tuberculostearic acid6 were suggestedfor the early diagnosis of TBM. None of thesetests have been accepted as routine procedure inclinical microbiology laboratories7. T.M. Daniel8has reviewed the value of various direct andindirect tests for the rapid diagnosis of TBM.ADA catalyses the deamination of adenosineinto inosine and ammonia. It is secreted byT-lymphocytes andmacrophages duringinfections. So, there has been interest in ADA asa marker of chronic inflammatory conditions suchas tuberculous pleural effusions9, tuberculousperitonitis10 and TBM7,11 .Lysozyme, secreted by the monocytic cellseries, was reported by us12 as useful for thedifferential diagnosis of meningitis. Hence, theobjectives of the present study were to assess theusefulness of ADA test and to compare it withLYSA test in the diagnosis of TBM in children.Material and MethodsPatients : The patients included in this studywere children aged below 12 years, divided into 4groups:Group 1. (Confirmed TBM) : The CSF from allthe 26 patients in this group was culture positivefor M.tubercu1osisGroup 2. (Clinical TBM) : The 61 patients in thisgroup were clinically diagnosed as suffering fromTBM but their CSF was negative by smear andculture. The diagnosis in them was based onclinical features, history of contact, Mantoux test,biochemical findings of CSF and presence ofFrom Tuberculosis Research Centre (ICMR), Chetput, Madras-600 031 and *Institute of Child Health, Egmore,Madras-600 031Correspondence : Director, Tuberculosis Research Centre, Chetput, Madras-600 031.218 N.SELVAKUMAR ET ALevidence for tuberculosis elsewhere in the body.Group 3. (Non-TBM) : The 10 patients in thisgroup were clinically diagnosed as suffering frompyogenic meningitis. S.pyogenes was isolated fromone and H.influenzae from the other patient andthe CSF specimens from the remaining 8 patientswere reported as culture sterile.Group 4. (Control) : The CSF from patientswith febrile fits(8), epilepsy (1), neuromusculardisorder (1), seizure disorder (1), cerebrovascularaccident (3) and conditions such as hydrocele (2),anal fissure (l), where spinal anaesthesia wasgiven constituted the control group. All the 17CSF samples were biochemically normal andculture negative for AFB and non-AFB.ADA assay : The ADA assay was performed asdescribed by Giusti et al13 . The CSF specimenswere kept in the deep freezer (-20° C) for 7 to 10days before the assay was done. The assay was setup on a routine basis and the details of thespecimens were not known. On each day of theassay a pooled CSF sample was included as acontrol. The ADA content was expressed in U/Iof CSF. Coefficient of variation was found to be20% for a pooled CSF sample with a mean ADAlevel of 10.12 U/I in 9 consecutive assays.Lysozyme assay : The lysozyme content wasestimated by the turbidity measurement asdescribed by Jain at al14 . A pooled CSF samplewas included to monitor the variation in theassay. The lysozyme content was expressed inmcg/ml of CSF. The coefficient of variation forthe pooled CSF sample with a mean LYSA levelof 1.43 mcg/ml was 15% in 7 consecutive assays.AFB culture : All the CSF specimens weresubjected to smear examination by fluorescencemicroscopy and processed for culture usingmultiple media as practised in this Centre15.Non-AFB culture : The CSF specimens fromthe control and Non-TBM groups were examinedfor Non-AFB by the conventional procedures.Statistical method : Univariate statisticalanalysis was carried out for the groups,separately. The comparisons of the means ofvarious groups of meningitis were made usingKruskal-Wallis non-parametric one-way ANOVAtest. The association between ADA and LYSAlevels was determined using Pearson correlationcoefficient and their significance using t-test.Unless otherwise stated, a statistical significanceindicates P value less than 0.05.ResultsThe frequency distribution, mean, standarddeviation and range of ADA values in differentgroups are given in Table 1. The mean level ofADA in confirmed TBM was 11.6 U/l and wasignificantly higher than the other three groups(P <0.000l). In the confirmed TBM group, 25(96%) of 26 had ADA values more than 4 U/l.All the 17 in the control group and 7 of 10 inNon-TBM group had values less than 4 U/l. So,an ADA level of 4 U/l and above was considered      diagnostic of TBM. With this criterion, thesensitivity and specificity of the test were 96%and 82% respectively.The frequency distribution, mean, standarddeviation and range of LYSA levels in differentgroups are given in Table 2. The mean level ofLYSA in confirmed TBM was 6.3 mcg/ml andwas significantly higher than the other threegroups (P < 0.0001). In confirmed TBM, 23(88%) out of 26 had LYSA values higher than 2mcg/ml. All the 17 in the control group and 9 outof 10 in the Non-TBM group had values less than2 mcg/ml. So, a LYSA value of 2 mcg/ml andabove was considered diagnostic of TBM. Withthis criterion, the sensitivity and specificity of thetest were found to be 85% and 95% respectively.The results of ADA and LYSA tests inbacteriologically positive and negative TBM  groups are presented in Table 3. Inbacteriologically positive TBM, 25 (96%) out of     26 were positive by ADA test whereas 23 (88%)_were positive by LYSA test. By applying either ofthe two tests a probable diagnosis of TBM couldbe made in 49% of 61 cases withoutbacteriological confirmation.The correlation coefficient between the ADAand LYSA levels in the confirmed TBM wasfound to be statistically significant (r = 0.59;P < 0.01)DiscussionIn the present study, ADA levels ranged from2.5 to 25.5 U/l in confirmed TBM. In pediatricpopulations, Malan et al16 and Coovadia et al7reported a range of 4.7 to 32.7 U/l and 7.1 to 32.5U/l respectively. A range of 9.1 to 22.7 U/l wasreported by Ribera et al” in adults. These resultsDIAGNOSIS OF TUBERCULOUS MENINGITIS 219Table 1. Distribution of ADA levels.GroupsADA Confirmed Clinical Non-TBM Control(U/l) TBM TBM0- 0 25 3 172- 1 10 44- 6 158- 10 8 312- 9 3Total 26 61 10 17Mean 11.6 4.5 4.4 0.8SD 6.2 4.3 4.0 0.5Range 2.5-25.4 0.1-20 1.0-10.9 0.0-1.6Table 2. Distribution of LYSA levelsLYSA Confirmed Clinical Non-TBM Control(mcg/ml) TBM TBM0- 3 38 5 15l- 1 4 4 22- 2 64- 11 98- 9 4 1Total 26 61 10 17Mean 6.3 2.1 2.2 0.5\SD 3.4 2.8 3.9 0.6Range 0.0-10.8 0.0-10.5 0.3-13.1 0.0-1.6TaBLE 3. Results of ADA and LYSA tests in bacteriologi-tally positive and negative TBMBact. neg.TBM Bact. pos.TBMTest status Number  % Number %Both positive 15 24.5 22 85ADA positiveLYSA negative 11 18.0 3 11LYSA positiveADA negative 4 6.5 - -Both negative 31 51.0 1 4Total 61 100 26 100show that ADA secretion by T-lymphocytes inresponse to mycobacterial infection is similar indifferent ethnic groups.In this investigation, an ADA value of 4 U/land above was considered consistent withtuberculous pathology. Mann et all7 used thesame criterion for the differential diagnosis ofmeningitis. Malan et alI6 reported a value of 6U/l as the cut off point for classification;Coovadia et al7 had a cut-off point of 10 U/l fordifferentiation while in adults Ribera et al11considered a value of 9 U/l as the significantlevel. The variations in the diagnostic cut-offlevels in these reports could be due to the type ofcontrols investigated.In this study, a LYSA level of 2 mcg/ml wasconsidered as diagnostic while Matti Klockars etalI8 used 1.5 mcg/ml as the cut off point.In this study, 3 of 10 Non-TBM patients hadADA values more than 4 U/l . similarly, Malan etall6 reported that 13 (31%) of 42 patients withbacterial meningitis had values higher than 9 U/l.Coovadia et al7 observed that 14 of 16 Non-TBMpatients had ADA levels more than 10 U/I. It is,therefore, evident that the CSF from patientswith pyogenic meningitis had also elevated ADAlevels.  This aspect should be taken intoconsideration while interpreting ADA testresults. Ribera et al11 h d reported that only 2(1%) out of 213 cases of Non-TBM had ADAvalues higher than 9 U/l without any explanation.It is, therefore,imperative that pyogenicmeningitis is ruled out by an initial screening,before elevated levels of ADA and LYSA levelsare considered for the diagnosis of TBM.Considering either of the two test definitions,49% of 61 clinically suspected andbacteriologically negative cases of TBM in thecurrent study had additional supportive evidencein favour of their diagnosis.The agreement between ADA and LYSA testsin the classification of different groups ofmeningitis was found to be 97%. The differencebetween the two tests is not statisticallysignificant (Sign test; P > 0.2). These resultssuggest that either LYSA or ADA could beapplied as an additional test for the diagnosis ofTBM.Considering the practical aspects of the twotests, LYSA test requires only culture suspensionof Micrococcus lysodeikticus which can be grownand maintained on nutrient agar. On the otherhand, ADA test needs reagents which are220 N.SELVAKUMAR ET ALexpensive and have a short shelf life i.e.. around30 days. Also,  the assay involves themeasurement of ammonia and utmost careshould be taken to avoid contamination with tapwater. Since LYSA assay is very simple andinexpensive, compared to ADA, it can beperformed in laboratories with limited financialsupport. Thus, it may be observed that LYSAtest is simple to perform and as sensitive andspecific as the ADA test in the diagnosis ofTBM.In conclusion , ADA level of 4 U/l and aboveand LYSA level of 2 mcg/l and above in CSFcould provide additional supportive evidencefor the diagnosis of TBM in clinicallysuspected and bacteriologically negative cases.AcknowledgmentsWe thank Mrs Josephine Lima Raj and MrG. Santhanakrishnan for their technicalassistance, Dr P.K. Jayanthi for collection ofCSF and Mr P. Venkatesan for statisticaladvice.References1. Wrobleweki F., Decker B and Wroblewski R.The clinical implications of spinal fluid lacticdehydrogenase activity. New England Journalof Medicine; 1958,258 635.2. Lauwers S. Lactic acid concentration incerebrospinal fluid and differential diagnosisof meningitis. Lancet; 1978, II, 163.3. Wiggelinkhuizen J. and Mann M. Theradioactive bromide partition test in thediagnosis of tuberculous meningitis inchildren. Journal of Pediatrics; 1980, 97, 843.4. Kadival G.V., Mazarelo T.B. M.S. andChaparas S.D. Sensitivity and specificity ofELISA in the detection of antigen intuberculous meningitis cerebrospinal fluid.J.Clin. Microbial; 1986,23, 90.5. Chandramuki A., Allen P.R.J., Keen M. andIvanyi, J. Detection of mycobacterial antigenand antibodies in the CSF of patients withtuberculous meningitis. J.of Med. Microbial;1985, 20, 239.6. Mardh P.A., Larson L., Holby N., EngbackH.C. and Odham G. Tuberculostearic aqid as7.8.9.10.11.12.13.14.15.16.17.18.a diagnostic marker in tuberculous meningitis.Lancet; 1983, I, 367.Coovadia Y.M., Dawood A., Ellis M.E., CoovadiaH.M. and Daniel T.M. Evaluation of adenosinedeaminase activity and antibody toM.Tuberculosis antigen 5 in cerebrospinal fluidand the radioactive bromide partition test for theearly diagnosis of tuberculous meningitis. Arch.Dis. Child; 1986, 61, 428Thomas M. Daniel. New approaches to the rapiddiagnosis of tuberculous meningitis. J. ofInfectious Diseases; 1987,155,599.Chopra R.K., Veena Singh, Harbans Lal A.S.,Rakesh K. Chawla and Baldev Raj. Adenosinedeaminase and T-lymphocyte levels in patientswith pleural effusions. Ind. J. Tub ; 1988, 35, 22.Michael D.Voigt, Issac Kalvaria, Charies Trey,Peter Berman, Carl Lombard and Ralph E.Kirsch. Diagnostic value of ascites adenosinedeaminase in tuberculous peritonitis. The Lancet;I, 1989, 751.Ribera E., Martinex-Vazquez J.M., Ocans I.,Segura R.M. Pascual C. Activity of adenosinedeaminase in cerebrospinal fluid for the diagnosisand follow-up of tuberculous meningitis in adults.J. of Infect. Dis; 1987, 155, 603.Selvakumar N., Acharyulu G.S. and Prabhakar R.Cerebrospinal fluid lysozyme in the differentialdiagnosis of tuberculous meningitis. Ind. J. Med.Res; 1985, 82, 479.Giusti G. Adenosine deaminase. Meth. Enzy.Anal., 1974, 2, 1092.Jain V.K., Zafar Jamil and Sukla D.P. Serumlysozyme levels in healthy Indian subjects atLucknow. Ind. J. Med. Res; 1979, 69, 344.Mitchison D.A. and Aber V.R. Culture ofspecimens other than sputum for mycobacteria. J.Clin. Pathol; 1974, 27, 883.Malan C. Donold P.R., Marillyn Golden andTaljaard J.J.F. Adenosine deaminase levels incerebrospinal fluid in the diagnosis of tuberculousmeningitis. Journal of Tropical Medicine andHygiene; 1984, 87, 33.Mann M.D., MacFarlane CM., Verburg C.J. andWiggelinkhuizen J. The bromide partition test andCSF adenosine deaminase activity in the diagnosisof tuberculous meningitis in children. S. Afr. J.,1982, 62, 431.Matti Klockars, Sakari Reitano, Teddy Weber andYrjo Kerttula. Cerebrospinal fluid lysozyme inbacterial and viral meningitis. Acta. Med. Scan;1978, 203, 71.

Cerebrospinal fluid adenosine deaminase and lysozyme levels in the diagnosis of tuberculous meningitis

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Cerebrospinal fluid adenosine deaminase and lysozyme levels in the diagnosis of tuberculous meningitis

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