Serum Adenosine Deaminase : A Novel Biomarker Tool for the Diagnosis of Tuberculosis

Tuberculosis is a major health issues worldwide. Every year more than 9 million new cases are reported worldwide with death rate of around 2 million each year. Rapid and accurate diagnosis of tuberculosis in population is most supportive in proper medication and early recovery. The golden standard method for diagnosis of tuberculosis in patient is formerly culture method which is time consuming and cumbersome. Various other techniques used in the diagnosis of tuberculosis have poor sensitivity and specificity however serum adenosine deaminase (ADA) has emerged as a potent biochemical marker that can be used as tool for the diagnosis of tuberculosis for rapid, easy and better result. Increased level of ADA in blood generally indicates the presence of tuberculosis. In the present study, the serum ADA level was examined in 75 healthy control and 75 tuberculosis patients with positive sputum smear for acid fast bacilli (AFB), having clinical symptoms were diagnosed for pulmonary tuberculosis and radiological impression for extra pulmonary tuberculosis. The selection of cases and control were based on the inclusion and exclusion criteria. Samples were collected at the outpatient department of National Tuberculosis Centre, Nepal after asking simple questionnaires. Modified Guisti and Galanti method was adopted for estimation of serum ADA from pooled and processed blood samples. There was significant difference (p<0.001) in mean rank of level of serum ADA among the TB cases to controls. The P-TB had the highest mean rank (116.52) followed by EP-TB (99.48) and control had significantly less mean rank (40.16). At the cut-off point of 25 U/L; sensitivity, specificity, positive predictive value and negative predictive value were 90.7%, 100%, 90.66% and 100% respectively. It can be concluded from data that there were insignificant difference in mean rank among sex wise distribution with p=0.037 and sputum grading wise distribution with p=0.142.

Tuberculosis is a chronic granulomatous infectious disease commonly known with TB which is caused by the bacillus M. tuberculosis.It typically affects the lungs usually called pulmonary TB but can affect other sites as well including various organs, also called as extra-pulmonary TB.People who are infected with pulmonary TB expel bacteriain the air by coughing and the disease is spread to healthy person.TB remains a major global health problem in the world.It causes ill-health among millions of people each year and ranks as the second leading cause of death from an infectious disease worldwide after HIV/ AIDS.
Adenosine deaminase (ADA, adenosine aminohydrolase, EC 3.5.4.4), an enzyme involved in purine metabolism, catalyses the hydrolytic cleavage of adenosine and 2'deoxyadenosine, irreversible converting them into inosine and 2'deoxyinosine respectively.ADA activity increases during cellular activation for energy demand to detoxify the toxic metabolites.It plays an important role in lymphocyte and monocyte maturation and activity.ADA is essential for proliferation and differentiation of lymphoid cells, especially T cells, and helps in the maturation of monocytes to macrophages.ADA seems to be an index for cellular immunity.Increased serum level of ADA has been reported in several diseases characterized by an enhanced cell mediated immune (CMI) response, such as typhoid fever, bacterial pneumonia, infectious mononucleosis and tuberculosis.
Recent studies showed extensive delay in TB diagnosis [1][2] .There are different diagnostic methods but they have some drawbacks.To culture TB bacterium is the golden standard for TB diagnosis usually takes 8 weeks.The polymerase chain reaction (PCR) test for TB diagnosis is expensive and it requires skilled personnel and lot of sophisticated equipment.Therefore, in recent years, there has been a great demand for finding new microbiological, genetic, immunological and biomedical diagnostic methods to diagnose TB quickly and accurately.ADA is an enzyme which contributes in purine metabolism.Measuring of adenosine deaminase (ADA) activity is a biomedical method used in diagnosis of TB [3][4][5][6] .The previous studies have proved its value in TB diagnosis, even for assessing TB effusions in which activity of this enzyme increases in the infected patients 7 .In some studies, the level of ADA in body fluids and serum was used for diagnosis of TB and it was monitored during TB treatment 8 .Initial studies used effusion fluids and a very limited number of studies used patients' serum 9 .Since, it is not always possible to access effusion liquids everywhere in pulmonary and extra pulmonary TB; therefore, it would be helpful to take advantage of serum levels.The goal of this study was to assess the diagnostic value and cut off point of serum ADA levels in TB patients.This test may be ordered as one of several tests to help rule in or rule out TB.The objectives of the present studies were to determine the level of serum ADA in tuberculosis cases of different population, cut
The Mean age of study groups were 34.20 yrs. of Pulmonary TB cases followed by 30.12 yrs. of Extra Pulmonary TB and 26.13 yrs. of healthy control.
Table 2  The Mean Rank Serum ADA level was found higher in male of 41.82 U/L than in female of 30.81 U/L with p value of 0.037.The variation of Mean Rank Serum ADA level among male and female was not found statistically significant.
There was significant difference in the mean rank of the level of serum ADA among the TB patients to control group.The P-TB had higher mean rank (116.52)followed by EP-TB (99.48) and Normal Control had significantly less mean rank (40.16) with p value 0.001.We got mean ± SD of 47.99 ± 2.22 among TB cases with minimum serum ADA level 11.06 U/L and maximum 127.34 U/L whereas among control group mean ± SD was 14.98±4.32with minimum serum ADA level 4.67 U/ L and maximum 24.07 U/L.
The serum ADA mean rank in P-TB were higher among 3+ graded sputum positive patients (29.97) followed by 2+ (29.14) and 1+ (21.60) with p = 0.142.The increase in serum ADA levels among the study group may be due to stimulation of cell mediated immunity.The variation in immune response by the study group might be the reason of data being statistical insignificant.

CONCLUSION
In the rapid progression of science and technology in the field of medicine has overcome many drawbacks of conventional and traditional ways of disease diagnosis, prognosis and monitoring.It used to be time consuming and cumbersome in diagnosis of infectious diseases mainly tuberculosis.Although the study was conducted in narrow limitations, it has provided one small effort to support the easy diagnosis of tuberculosis.Serum ADA can be easily performed as the routine test in diagnosis of tuberculosis which is relatively easy, cheap, specific and sensitive biomarker in establishment of disease diagnosis.The Serum ADA estimation can be used as routine lab investigation for the diagnosis of TB even in basic lab set up.The test procedure does not require any special lab instruments and can be performed on basic lab instruments.
The study was carried out among 75 TB cases and 75 healthy controls.Of 75 healthy control 47% (35) were female and 53% (40) were male of age group ranging from 18 -47 years.The mean level of serum ADA was found to be 14.98 U/L with SD 4.32.We got maximum value of serum ADA 24.07 U/L and minimum of 4.67 U/L among healthy control.The levels of serum ADA among healthy group were within normal limit as per reference range given by reagent company leaflet.The study group of tuberculosis cases was diagnosed by the medical team of NTC along with sputum examination, clinical symptoms, radiological impressions and serological tests like Mantoux test.The TB case group included 35% (26) female and 65% (49) male of age ranging from 15 -59 years.The cases were divided into 2 category; PT (50) and EP-TB (25).The mean serum ADA level in cases was found to be 47.99 U/L with SD 2.22.The maximum level of serum ADA level was observed 127.34 U/L whereas the minimum level was 11.06 U/L.
The pulmonary TB cases include 26% (13) female and 74% (37) males of age ranging from 15 -59 years.The mean serum ADA level was found to be 53.03U/L with SD 2.36.The maximum level of serum ADA was observed 127.34 U/L and minimum of 11.06 U/L.The cases of Pulmonary TB where further categorized depending upon sputum grading: 1+ (26), 2+ (7) and 3+ (17).The ET-TB cases include 52% (13) female and 48% (12) male of age ranging from 17 -50 years.The mean serum ADA level was found to be 37.89 U/L with SD 1.47.The maximum level of serum ADA was observed 70.10 U/L and minimum of 15.52 U/L.There was significant difference in mean rank of the level of serum ADA among the TB patients to healthy control.PTB has higher mean rank (116.52)followed by EPTB (99.48) and control (40.16) with p= 0.001.There was no significant difference in mean rank of the level of serum ADA among sex wise distribution with p=0.037.There was also no significant difference in mean rank of the level of serum ADA among grading wise distribution with p=0.142.The mean rank serum ADA levels of 1+ sputum AFB grading and 3+ sputum AFB grading were also found to statistically insignificant with p= 0.08.Since the highest sensitivity and specificity was shown at cut-off point of 25 U/L, the serum ADA level can be set at 25 U/L as borderline value according to our study.The present study suggest to use serum ADA estimation as the biochemical marker in the diagnosis of TBhighlighting it as simple, rapid, cheaper and accurate diagnostic test.
off level for Serum ADA in diagnosis of TB and sensitivity and specificity determination of serum ADA in the diagnosis of tuberculosis MATERIALS AND METHODS A cross-sectional case control study was conducted among the patients visiting National Tuberculosis Centre (NTC), Bhaktapur, Nepal.NTC is the major centre for the study of tuberculosis in Nepal where every day more than 50 patients with tuberculosis visit for treatment.Since our study was concerned with tuberculosis, we could easily get the cases of tuberculosis of our concern.The patients attending NTC are from almost all parts of Nepal and we could include the cases from various parts of Nepal and of different ages and sex.We performed sample collection in the Out Patient Department (OPD) of NTC from cases with verbal consent.The separated serum samples were stored at -4 to 0 o C and were processed in the biochemistry laboratory of Dhulikhel Hospital, Nepal.The ethical consent was taken from the authority of National Tuberculosis Centre (NTC) for sample collection and from Department of Biochemistry, Dhulikhel Hospital for sample processing.Quantitative method: Determination of serum ADA from the cases and control was based on the principle of Guisti & Galanti (1974) methods of enzymatic analyses.ADA-MTB diagnostic kit from Microxpress a division of Tulip diagnostics Number of cases

Fig. 1 .
Fig. 1.Total number of cases and demonstrates that the Mean Rank of Serum ADA level were shown highest 116.52 U/L in Pulmonary TB cases followed by 99.48 U/L in Extra Pulmonary TB and lowest of 40.16 U/L in healthy control cases.The mean rank level of Serum ADA was shown to be statistically significant with p value of 0.001.The Mean Rank Serum ADA level in TB cases were shown higher 110.84 U/L than in Healthy control cases of 40.16 U/L with p value of 0.001.The variation in Mean Rank was shown to be statistically significant.

Table 1 .
Age wise distribution of study population

Table 2 .
Mean Rank distribution of Serum ADA level among Study Groups

Table 4 .
Sex wise distribution of serum ADA U/L mean rank

Table 5 .
Table showing sensitivity, specificity, PPV and NPV at different cut-off levels