Study of Serum Adenosine Deaminase (ADA) Activity in Pulmonary Tuberculosis Patients of Indian Subpopulation

Introduction

Tuberculosis (TB) is a contagious bacterial disease caused by the tubercle bacilli, Mycobacterium tuberculosis. It is one of the oldest diseases known to affect humans and a major cause of death worldwide.¹ Approximately nine million active TB cases occur every year. Moreover, about 1.5 to 2 million people die of TB worldwide each year.² Approximately 0.5 million patients of tuberculosis die every year in India.³

The presence of acid-fast bacilli on sputum smear examination of patient provides a distinct diagnosis of pulmonary tuberculosis (PTB). A dilemma arises when sputum smear results repeatedly feature negative for acid fast bacilli. Further, chest skiagram provides only a probable diagnosis and culture for tubercle bacilli is a complicated and time-consuming process. To overcome this complexity, biochemical test Adenosine deaminase (ADA) estimation in serum may help in confirming the diagnosis of PTB.⁴

ADA is considered as an enzyme which catalyzes the deamination reaction from adenosine to inosine. It is also an essential enzyme of the purine catabolic pathway, which is produced by cells throughout the body. There are two isoforms of ADA: ADA-1 and ADA- 2. ADA-1 is found in many tissues including red blood cells. ADA-2 is found in macrophages and monocytesonly.¹ ADA is a major marker of active cellular immunity. It increases in biological fluids if infected by micro-organisms in macrophages causing infectious disease.⁵ Due to the stimulation of T cells by mycobacterial antigens, the level of ADA increases in TB patients.¹ ADA may be released and detected in biological fluid (serum) of patients with active pulmonary tuberculosis as the tubercle bacilli infect lung macrophages.⁵ It would be advantageous to use serum levels since access to effusion liquids in pulmonary and extra-pulmonary TB is not always possible everywhere⁷ .

There are various ultrasensitive molecular tests available to diagnose tuberculosis, such as Interferon Gamma Release Assays (IGRA), Tuberculosis-Polymerase Chain Reaction (TB-PCR) and Cartridge based Nucleic Acid Amplification Test (CBNAAT). However, these tests are very costly and not affordable for a common man, and even the facilities to conduct these tests may not be available in remote areas. Determination of serum ADA levels is advantageous as the test is rapid, simple, and safe to perform, even in small laboratories. A study has reported serum ADA levels as a consistent chemical biomarker when suspicion of tuberculosis arose in certain endemic areas.³ The present study was aimed to evaluate the clinical utility of serum ADA levels as a biomarker in the diagnosis of PTB.

Materials and Methods

Selection of participants and specimen collection

A total of sixty subjects (n=60), with 30 cases and 30 controls were included in the study. The patients who visited the medicine department of a referral hospital in Bangalore, for the diagnosis of PTB with positive sputum AFB were taken as cases and the healthy individuals who visited the diagnostic center at Vijayanagar, Bangalore with normal blood reports and negative sputum AFB smear were chosen as controls. Written informed consent was obtained from the participants. Demographic details, body mass index, dietary habits, smoking, alcohol, drug intake, and family history were collected prior to the collection of blood samples.

Subjects with age ≥18 years, AFB positive for tubercle bacilli and radiographic findings suggestive of PTB were included in the study. Individuals who have had contact with PTB patients and individuals with respiratory symptoms with AFB negative for tubercle bacilli were excluded from the study. The study was approved by the Institutional review board.

Specimen handling and analysis

The blood specimens received in the laboratory were centrifuged at 4000 rpm for 15 min to separate the cellular components and the cell free serum was processed for the analysis of routine biochemical parameters sought by the treating clinicians and the sample portions of ADA analysis were stored at -20˚C until used for the further analysis. Aliquots were thawed appropriately on the day of analysis of samples.

Analytical method

With the method described by Giusti and Galanti (1984) which was based on the Bertholet reaction, serum ADA activity was determined at 37^o C. In brief, the colored indophenol complex formed from ammonia liberated from adenosine was quantified spectrophotometrically. One unit of ADA is defined as the amount of enzyme required to release 1 µmoL of ammonia/min from adenosine at standard assay conditions. ADA activity was expressed as international unit (IU).⁶

Statistical analysis

SPSS was used for data entry and management. The obtained data were expressed as mean±SD. The two tailed- t test was employed to assess the differences between the groups comparing with reference interval from available literature. Pearson correlation coefficient was used for data analysis. p value <0.05 was considered statistically significant at 95% Confidence interval.

Results

The study participants (n=60) were divided into two groups - Test and Control. Each group consisted of 30 subjects; test group was comprised of 10 females and 20 males while the control group included 12 male and 18 female participants (Figure 1 and Figure 2).

Biological reference intervals (BRI) of ADA

The manufacturer recommends the clinical laboratories to establish their own BRI for the population they serve. According to the reagent manufacturer, the BRI for serum ADA was 12±11 U/L.

Serum ADA levels

ADA levels in the control and test population were 3.2±2.2 U/L and 12.6±5.1 U/L, respectively. In our study, we compared the ADA results of test population against ADA results of the control group subjects. Significant high levels of serum ADA activity was observed in pulmonary tuberculosis patients (***p <0.001) (Figure 3). The ADA results of the test population did not show significant increase compared to BRI (p >0.05) (Figure 4). Thus, it is evident that the BRI recommended by the manufacturer may not be suitable for the population in the geographical location where the study was conducted. 

Discussion

Tuberculosis is an ancient disease that is still existing, affecting all the age groups and causing life threats worldwide. It is an unsolved question from millions of years and challenging to all the governments. Despite taking all the necessary precautions by the Government of India, through Revised National Tuberculosis Control Program (RNTCP), the outcome does not appear to be satisfactory. Inadequate co-operation from the patients, lack of awareness and inadequate or discontinuation of treatment by the patients could be a few contributing reasons for the same.

There are many tests to diagnose PTB which include direct methods such as smear test by Ziehl-Neelsen stain and Auramine stain. These tests are simple and are considered as gold standard to diagnose the disease, but poor sampling and/or low bacterial load can result in false negative results. There are ultrasensitive molecular tests available to diagnose TB, such as Interferon Gamma Release Assays (IGRA), Tuberculosis- Polymerase Chain Reaction (TB-PCR) and Cartridge based Nucleic Acid Amplification Test (CBNAAT). But these tests are expensive and not affordable for a common man and the test facility may not be available in remote areas. Serum ADA test is simple, rapid, and safe to perform, even in small laboratories. There is a pressing need to evaluate the diagnostic sensitivity and clinical utility of such biomarkers in order to diagnose tuberculosis.⁹ Our study was an attempt in that direction.

ADA is concerned with the metabolism of purine and catalysis of the hydrolytic deamination of adenosine to inosine and deoxyadenosine to deoxyinosine.² This enzyme plays a vital role in regulation of the effects of these compounds on immunological, neurological and vascular processes.² The ADA has a major role in proliferation and differentiation of T lymphocytes.^5,10 In comparison to peripheral blood, the percentage of T lymphocyte subpopulation in tuberculous pleural fluid is higher. However, this does not correspond to the ADA levels. This suggests that the enzyme activity may correlate more to the maturation stage of T cells rather than its quantity⁸ . Increased serum ADA levels can be seen in diseases associated with cellular system stimulation such as tuberculosis, cancer, and pneumonia. Therefore, serum ADA levels were significantly increased in pulmonary tuberculosis patients compared to controls.

In the present study, the serum ADA levels in TB patients did not correlate with the BRI provided by the manufacturer. Nevertheless, the BRI may not be suitable for Indian sub population. As per the manufacturer’s recommendations, the present study made an attempt to determine the same.

Serum ADA levels found in control population 3.2±2.2 U/L could serve as new reference interval for the geographical population. However, further assessment and validation with large sample population is required to confirm the findings.

Certain limitations in our study could have biased our results. Sample size included in this study was low to conclude ADA as a biomarker for pulmonary tuberculosis. Other diseases like liver disease, typhoid fever, rheumatoid arthritis, leukemia, sarcoidosis, brucellosis, and infectious mononucleosis are likely to be correlated with high serum levels of ADA.² These diseases have been excluded in the clinical presentation of PTB. Therefore, further controlled studies are recommended.

Conclusion

Serum ADA levels showed a significant increase in pulmonary tuberculosis patients compared to healthy individuals. Serum ADA levels can be used as diagnostic and prognostic biomarker in patients with PTB. It is important for the clinical laboratories to establish their own BRI for serum ADA.

Conflict of Interest

All the authors have contributed equally. The authors of this study have no financial interest nor received any financial support for this research work.

Acknowledgement

This research was supported by Padmashree Institute of Medical Laboratory Technology, Padmashree Institute of Clinical Research and Padmashree Diagnostics. We thank all the authors who provided us insights and their expertise which greatly assisted the research