Study of Adenosine Deaminase Activity As A Biochemical Marker of Cell Mediated Immunity in Tuberculous Meningitis , Tuberculous Pleural Effusion and Tuberculous Ascites

The present study conducted with the aim that ADA estimation is a simple cheaper and quicker biochemical test and could provide additional supportive evidence for the diagnosis of tuberculous meningitis, tuberculous pleural effusion, tuberculous ascites in clinically suspected cases and will therefore help in early institution of therapy to these patients. The data was collected and interpreted at department of Physiology and Department of Medicine Gandhi Medical College, Bhopal. Keyword: ADA, Tubeculous, Biochemical test, Diagnosis 1. Assistant Professor, Department of Physiology, Jhalawar Medical College Correspondence: Dr. Shashikant Agarwal, Assistant Professor, Department of Physiology, Jhalawar Medical College ; E Mail inbiosci@gmail.com showing a significant increase in its values during the mythogenic and antigenic response of lymphocyte.1,2 ADA plays a part in the differentiation of lymphid cells and the maturation of monocytes to macrophages. The congential and genetically determined deficit of this enzyme with its autosomal receive trait. It is reported to be associated with severe form of combined immuno deficiency and its responsible for an increase in toxic nucleotides that prevent the differentiation or proliferation (or both) of T lymphocytes and thus a normal immune function mediated by cells. The raising of the levels of ADA activity under antigenic stimulation shows the importance of this enzyme in the rapid proliferation of cells in order to prevent the accumulation of toxic metabolistis. Therefore, an increased ADA activity is present in several circumstances in pleural, pericardiac and peritoneal effusion of a tuberculous nature and in tuberculous cerebrospinal fluid, where ADA values are significantly higher than in normal group ad in other neurological disease. Tuberculosis occurs worldwide and is rampant in many countries. Increased level of ADA is found in various forms of tuberculosis making it a marker for the same. Hence in the present study, ADA activity was studied for easy diagnosis of tuberculous meningitis, tuberculous pleural effusion and tuberculous ascites. The present work was done in the department of Physiology and Department of Medicine Gandhi Medical College, Bhopal. The biochemical investigations were carried out in the department of Biochemistry of the institutions. J MEDICINE 2012; 13 : 32-38

showing a significant increase in its values during the mythogenic and antigenic response of lymphocyte. 1,2A plays a part in the differentiation of lymphid cells and the maturation of monocytes to macrophages.The congential and genetically determined deficit of this enzyme with its autosomal receive trait.It is reported to be associated with severe form of combined immuno deficiency and its responsible for an increase in toxic nucleotides that prevent the differentiation or proliferation (or both) of T lymphocytes and thus a normal immune function mediated by cells.The raising of the levels of ADA activity under antigenic stimulation shows the importance of this enzyme in the rapid proliferation of cells in order to prevent the accumulation of toxic metabolistis.Therefore, an increased ADA activity is present in several circumstances in pleural, pericardiac and peritoneal effusion of a tuberculous nature and in tuberculous cerebrospinal fluid, where ADA values are significantly higher than in normal group ad in other neurological disease.
Tuberculosis occurs worldwide and is rampant in many countries.Increased level of ADA is found in various forms of tuberculosis making it a marker for the same.Hence in the present study, ADA activity was studied for easy diagnosis of tuberculous meningitis, tuberculous pleural effusion and tuberculous ascites.The present work was done in the department of Physiology and Department of Medicine Gandhi Medical College, Bhopal.The biochemical investigations were carried out in the department of Biochemistry of the institutions.

Materials and methods:
The study comprised of 30 cases of tuberculous meningitis, 30 cases of tuberculous pleural effusion and 30 cases of tuberculous ascites who volunteered for the study.The study population consisted of 45 men 45 women with an average age range of 14-70 years.Clinical examination was done to assess cardiorespiratory status and general condition of study population.

Diagnostic Criteria of Tuberculous
• Assessment of nutritional status: Anthropometric measurements were done and Body mass index was calculated as per the standard procedure.• Statistical Analysis: For the data analysis the mean and standard deviation were calculated.The statistical significance was calculated using paired 't' test.Correlation coefficient 'r' was calculated to establish association between the variables.

Results and observations:
The study comprised of 30 normal healthy control aged 18-48 years and newly diagnosed 30 cases of tuberculous meningitis, 30 cases of tuberculous pleural effusion, 30 cases of tuberculous ascites who volunteered for the study.The study population consisted of 45 men and 45 women with an average age range of 14-70 years.The adenosine deaminase was measured and correlated with various parameters.
Table-I depicted, the low mean hemoglobin levels in all the three groups reflect the poor nutritional status of the patients under study.On the other hand the mean ESR values recorded were on higher side supporting the tuberculous pathology.Total leucocyte count was in the normal range in all the three groups.In the present study specimens from 90 patients were examined for ADA activity including tuberculous meningitis (30 CSF), tuberculous pleural effusion (30 pleural fluid), Tuberculous ascites (ascitic fluid 30) and 30 serum sample from control group.The probable diagnosis in all cases of TB was assigned according to clinical presentation which suggestive signs and symptoms, radiological, bacteriological and cytological criteria in all cases standard diagnostic procedure were followed.

CSF Biochemistry and Cytology :
On physical examination 93% CSF sample appeared clear only 70% were found hazy.Cobweb appearance was found in only one sample.

CSF Cytology :
There is no general agreement on nomenclature for cytologic elements in normal CSF, Sheth identified lymphocytes, piaarchnoid mesothelial (PAM) cell, monocytes, and neutrophils as normally present, Kolmel identified lymphocytes monocytes, monocytoid" cells, neutrophils and ependymal cells normally present in CSF. 3,4There has been considerable controversy on whether neutrophils are present in "normal" CSF.Some authors consider even one neutrophil as abnormal (Cole) while others suggested that up to 10 percent neutrophils (e.g. 5 out of 50cells counted) are within Normal limits (Sheth). 3,4,5According to Kolmel, neutrophils in CSF probably come from contamination by peripheral blood owing to traumatic tap.A neutrophilic reaction classically suggest, meningitis owing to pyogenic organisms.Increased numbers of lymphocytes have been reported in infections, including (1) viral maningoencephalitis (plasma cells, and some macrophages also may be present, (2) tuberculous meningitis.

CSF Biochemistry :
In the present study mean CSF protein measured was 175.29+158.88similar observations were reported by Kennedy et al. 6 Fishman suggested that increased permeability of the blood CSF barrier is a common cause for pathologic increases in CSF proteins.In meningitis there is damage to the blood CSF barrier, plus decreased removal of protein molecules at the arachnoid villi. 7e glucose content of the CSF samples collected were analyzed the mean value obtained was 42.   10,11 They concluded that ADA does not cross normal blood brain barrier.The related increased in the levels of ADA and protein in CSF in TBM may thus have their original least in part in damage to the blood brain barrier permitting ADA to enter the CSF from either the blood or adjacent cerebral tissue, alternatively raised level of CSF ADA and protein may originate in a local immune response as the result of lymphocytes proliferating in response to the relevant antigen.
In the present series it was observed that in patients having CSF glucose <40 mg/dL CSF ADA measured was maximum (36.03+19.21U/L) as compared to patients having CSF glucose >40 mg/dL (  10,13 Tuberculous pleural effusion is thought to result from a delayed hypersensitivity reaction in response to the presence of mycobacterial antigens in the pleural space.Delayed hypersensitivity reaction causes the stimulation and differentiation of lymphocytes that perform a variety of functions, including the release of certain lymphokines that activate macrophages for enhanced mycobactericidal effect.Because the chief role of ADA concerns the proliferation and differentiation of lymphocytes ADA has been looked on as a marker of cell mediated immunity which encompasses the delayed hypersensitivity reaction.
Previous reports have coated a wide range of cutoff values from 45 U/L to 80 U/L.In the present study ADA PF cutoff value was <45 U/L.This difference likely reflects the differences in study population and differing methods of ADA FF analysis therefore when interpreting the results physician should be aware of the different cutoff levels.
In the present series on the basis of pleural fluid ADA estimated study population was classified into two groups having pleural fluid ADA activity <40 U/L respectively.
It was observed that in ADA positive samples (90.78+27.89U/L) the protein content was high (4.75+1.08 g/L), glucose content was appreciably low (47.49+1.08 mg/L) and high leucocyte count (830.33+390.27)as compared to ADA negative samples.The findings correlate with the findings of Riantawan et al. 14 An attempt was made to correlate ADA level with all these three parameters.Highly significant positive correlation was found between ADA activity and protein content and leucocyte count (p <0.001).They reported high leucocyte count varying between 472-5840 per cu mm and lymphocyte percentage between 30% -100%.
On biochemical examination protein content was greater than 3 gm/dL in majority of the patients.The ascitic fluid glucose content was low (52.62+18.57mg/dL) as compared to the normal values coated.Hillbrand et al. reported ascitic fluid total protein content >2.5 g/dL in tuberculous peritonitis patients.In recent years, investigators have noted that ADA is markedly raised in tuberculous ascites. 17 patients having ADA activity <40 U/L the mean ADA was significantly lower as compare to the patients having ADA activity >40 U/L.On correlating the ADA activity with protein and lymphocyte percentage, highly significant positive correlation was obtained (p <0.001).
Martinez Vazquez et al. reported no correlation between absolute lymphocyte count and the ADA concertration. 15n the present study highly significant negative correlation (p<0.001) was observed between ascitic fluid ADA and glucose content.
The value of ADA estimation for diagnosis of tuberculous meningitis, tuberculous pleural effusion and tuberculous ascites was evaluated in terms of its sensitivity, specificity, positive predictive value (PPV), and Negative predictive value (NPV).The sensitivity and specificity of ADA depends upon the prevalence of tuberculosis in the population.The ADA measurement is used commonly in European and Asian countries where there is a higher incidence of tuberculosis.The problem with using the ADA assay in a population with a lower incidence of tuberculosis is that the positive predictive value decreases; stated another way, there is a higher likelihood that a test would give a false positive result.The downside of this could be unnecessary tuberculosis chemotherapy.
Hence, it is concluded that ADA estimation is a simple cheaper and quicker biochemical test and could provide additional supportive evidence for the diagnosis of tuberculous meningitis, tuberculous pleural effusion, tuberculous ascites in clinically suspected cases and will therefore help in early institution of therapy to these patients.

Table - I
Hematological Profile of The Study PopulationThe diagnosis of tuberculous pleural effusion was established by correlating the clinical findings with pleural fluid changes in relation to physical, biochemical and cytological examination.Table-IV showed the results of comparison of biochemical and leucocytes count based on ADA levels in tuberculous pleural effusion patients.Analysis of pleural fluid ADA level and leucocyte count in group I and group II showed statistically significant difference in the mean value (p <0.01) although mean values of the pleural fluid protein in group II were on higher side as compared to group I and glucose level was also appreciably low (<60 mg/ dL).

II
Comparison of biochemical and leucocyte count based on ada levels in tuberculous meningitis patients

Table - IV
Comparison of biochemical and leucocyte count based on ada levels in tuberculous pleural effusion patients

Table - V
, depicted the statistically significant (p < 0.001) positive correlation between the pleural fluid ADA, protein and lymphocyte percentage.A negative correlation could be established between the pleural fluid ADA and glucose values.The observations were in conformity with the diagnostic criteria laid down.

Table - V
Correlation of pleural fluid ada activity with parameters measured in tuberculous pleural effusion patients

Table - VI
Comparison Of Biochemical And Leucocyte Count Based On Ada Levels In Tuberculous Ascites PatientsTuberculosis has emerged as one of the most lethal disease men has faced.Inspite of presence of effective chemotherapy epidemiological data showed worldwide rise in incidence specially since AIDS incidence rose steeply.There is considerable urgency in establishing the correct diagnosis in patients with tuberculosis meningitis, tuberculous pleural effusion and tuberculous ascites because specific therapy is most effective when instituted early in the course of illness.
The body fluids like CSF, pleural fluid and peritoneal fluid contain sensitive biochemical markers, these includes ADA, INF-α TNF-α and IL-1.The determination of ADA activity in these body fluids has been reported to be a valuable adjunct in the diagnosis of extra pulmonary tuberculosis.Since the value of ADA activity is mainly related to cellular immunity, in immuno-compromised patients.ADA activity reported was low as compared to immunocopetent hosts.

Table - VIII
Validity of ADA Test As A Diagnostic Tool 9ADA activity is much higher in blood than in CSF Piras.et al. and Galanti studied lymphocyte ADA activity in tuberculous meningitis typhoid fever respectively.
Table No.II).Kennedy et al. reported CSF glucose levels between 30 and 45 mg/dL in 48% of the TBM cases in their study. 6A highly significant (P<0.001)negative correlation (r=0.58) could be established between CSF ADA and CSF glucose in the present study.Similar finding were reported by Malan et al. in 157 cases of TBM (r=-0.33,P <0.001). 9Decreased CSF glucose may be due to impairment active transport to increased utilization of glucose by CNS tissue.Later, Piras et al. reported the usefulness of ADA in diagnosing tuberculous pleurisy.
15citic fluid white cell count was 680.46+348.69 per cumm and consist of lymphocyte predominantly.Martinez Vazuez et al. studied 10 patients of tuberculous peritonitis and reported higher than 70% of lymphocytes in the samples analysed.15