Tuberculosis (TB), one of the important re-emerging infectious diseases caused by Mycobacterium tuberculosis and is characterized pathologically by the formation of granulomas. [1] One third of the world’s population is latently infected with this causative agent and annual new cases of TB worldwide counts approximately 9 million. [2] It is a global health concern for both developing and developed countries because of its infectious nature. Despite being preventable and curable TB now ranks as a leading infectious disease killer globally alongside Human Immunodeficiency Virus (HIV). [3] According to WHO Global TB Report 2016, Bangladesh is one of the world’s 30 high TB burden countries with annual occurrence of 362,000 new TB cases. About 73,000 people die annually due to Tuberculosis (World Health Organization, 2017). [4] It can present as pulmonary tuberculosis (PTB) or extrapulmonary tuberculosis (EPTB). Among them pulmonary TB is the most common form of tuberculosis. [5] The main symptoms of PTB are chronic cough, low grade fever–evening rise of temperature, haemoptysis, chest pain, dyspnoea, loss of weight, unresolved pneumonia. [6] In clinical practice, rapid TB diagnosis can be difficult, and early pulmonary TB detection continues to be challenging for clinicians. Prompt diagnosis of active pulmonary TB is a priority for TB control, both for treating the individual and for public health intervention to reduce further spread in the community. Chest X-ray is useful but is not specific for diagnosing pulmonary TB. Moreover, TB can present with symptoms and atypical radiologic findings that are indistinguishable from those of community acquired pneumonia. [7] there are others different diagnostic methods but they have some drawbacks. To prepare mycobacterium culture, which is the golden standard for TB diagnosis, it may take 8 weeks. The polymerase chain reaction (PCR) test for TB diagnosis is expensive and it requires skilled personnel and lot of equipments. Finding acid-fast bacilli is the quick screening method for pulmonary TB diagnosis; nevertheless, its sensitivity is low. [8] Despite best of the efforts, a clinician may have to face difficulties in smear negative patients, and sometimes, it becomes almost impossible to diagnose this entity. The presence of co-morbidities like diabetes mellitus, HIV and other immune compromised conditions further complicate the picture as they lead to atypical clinical and radiological presentations. [9,10] This delay in diagnosis and subsequent treatment leads to increased disease transmission and chances of drug resistance. Hence, in the recent years, there has been a great demand for finding a rapid diagnostic method for the same. [11] Adenosine deaminase (ADA) is one such biomarker which is now a days being studied as a diagnostic tool in tuberculosis due to its simplicity, low cost, and quickly available results. [12] Measuring adenosine deaminase (ADA) activity is a biomedical method. ADA is an enzyme which contributes in purine metabolism. This enzyme catalyzes the hydrolytic deamination of adenosine to inosine, and deoxy adenosine to deoxy inosine. This enzyme plays an important physiological role in the regulation of the effects of these metabolites on immunological, neurological and vascular processes. ADA is essential for proliferation and differentiation of lymphoid cells, especially T cells, and helps in the maturation of monocytes to macrophages. It seems that ADA is an index for cellular immunity and previous studies have proved its value in TB diagnosis, even for assessing TB effusions. [13] Activity of this enzyme increases in TB patients. In some studies, the level of ADA in sputum and serum was used for diagnosis of TB, and it was monitored during TB treatment. However, some previous studies used effusion fluids and a very limited number of studies used patients’ serum. It is not always possible to access effusion liquids in pulmonary and extra-pulmonary TB; therefore, it would be helpful to take advantage of serum levels. [14] Therefore, this study was planned to investigate the diagnostic value of serum ADA in smear negative pulmonary tuberculosis. The results of this study would help to provide a clear picture of ADA for diagnosis of TB, hence future plans for TB could be better executed.

This cross-sectional analytical study was conducted at the Department of Medicine, Dhaka Medical College & Hospital, Dhaka between March, 2019 to September, 2021. A total of 140 newly diagnosed pulmonary cases admitted within the study period fulfilling the inclusion and exclusion criteria were included in this study by convenient purposive sampling. The study subjects were divided into two groups according to selection criteria: Group I (Smear negative Pulmonary TB, n=62), and Group II (non-TB pulmonary cases viz. pneumonia, COPD, bronchiectasis, lung malignancy, n=78). Patients with sputum smear positive pulmonary TB, history of previous pulmonary/extrapulmonary TB or ongoing anti-TB treatment, having secondary immunodeficiency states: HIV, organ transplantation, and treatment with long term corticosteroids, any malignancy in the body other than lung malignancy, presence of hepatic or renal impairment, pregnant and lactating women, having concomitant lymphoproliferative disease were excluded from the study. In those patients having a productive cough, sputum for AFB was sent to DOT corner in DMCH which was done by Light Emitting Diode (LED) fluorescence microscopy (FM). Sputum for acid fast bacilli was performed in all patients on two sputum specimens as follows - one on spot specimen which was collected on spot when a patient was sent to the DOTS. Another was the early morning specimen where patients were given a sputum container to collect the second specimen on the following morning. Early morning sputum was also collected for Xpert MTB/RIF examination which was done in college building of DMCH. Sputum was also sent for gram stain, culture in Microbiology Department of DMCH. Chest-Xray P/A view was done in Radiology Department of DMCH .Other necessary investigations - CBC with ESR, MT, Spirometry, CT scan of chest and CT guided FNAC/Biopsy where appropriate were carried out. After the final enrollment, blood samples for serum ADA were sent to the BSMMU Microbiology Department written in routine biochemistry form. Serum ADA was analyzed using sensitive colorimetric method described by Guisti and Galanti with BIOSIC kit. All the final data were collected in the semi-structured and pretested case record form. During data collection, highest standard ethical measures were ensured and maintained throughout study. These data were analyzed statistically by the standard procedure to arrive at definite conclusion in regards to the research question.

Out of 140 patients, 62 patients had smear negative pulmonary TB (SNPTB), and rest 78 patients were suffering from non-TB pulmonary diseases, of whom, 35 patients had pneumonia, 25 had COPD, 10 had lung carcinoma and rest 8 patients had bronchiectasis. SNPTB patients were assigned as Group I and non- TB pulmonary cases were assigned as Group II.

Figure 1: Distribution of study population according to diagnosis (n=140).

than Group I (SNPTB cases) with mean age 52 ± 8.56 vs 43.02 ± 8.49 years respectively; p<0.001* (determined by student’s t-test). Maximum patients from Group II were 51-60 years of age (42.30%), while maximum patients from Group I were 41-50 years of age (48.40%).

Figure2: Distribution of study population according to age group (n=140).

Male subjects were predominant in both Group I (SNPTB) and Group II (non-TB pulmonary cases) which were 71 % and 60.3%, respectively.

Figure-3: Distribution of study population according to gender (n=140).

Table I shows that majority patients in Group I were garment workers (38.7%) which was found statistically significant (p<0.05), while majority patients in Group II were service holders (24.4%).

Table 1: Distribution of study population according to occupation (n=140).

Group I= Smear negative pulmonary TB, Group II= Non-TB pulmonary cases

Majority of patients in Group I and Group II were from middle income status - 51.60% and 50% respectively, though there were no significant differences between the two groups regarding socio- economic status; p value=0.928, determined by Chi-squared Test x²).

Figure 4: Distribution of study population according to socio-economic status (n=140).

Table II shows that SNPTB patients had significantly higher frequency of cough, fever and weight loss compared to non-TB pulmonary cases (p value <0.05). In contrast, chest pain and shortness of breath were more frequent in Group II than in Group I (p<0.05).

Table 2: Distribution of study population according to clinical features (n=140).

Group I= Smear negative pulmonary TB, Group II= Non-TB pulmonary diseases.

Table 3: Distribution of study population according to ESR level (n=140).

Table V shows that Sputum for gram stain was positive in 14.5% SNPTB patients and 23.1% non-TB pulmonary disease patients without any statistical significance (p>0.05).

Table 4: Distribution of study population according to sputum examination (n=140).

Group I= Smear negative pulmonary TB, Group II= Non-TB pulmonary diseases # Chi-squared Test x²) was performed.

• Significant. Table VII shows that serum ADA was significantly higher among SNPTB patients compared to non-TB pulmonary patients (48.16±12.13 vs 18.64±7.85 IU/L, p<0.001).

Table 5: Comparison between the two groups according to serum ADA values (n=140).

Group I= Smear negative pulmonary TB, Group II= Non-TB pulmonary diseases # Student t-test was performed.

• Significant ROC analysis of serum ADA in diagnosis of patients with SNPTB found AUC of 0.9850 (95% CI 0.969- 1.00) which was statistically significant (p<0.001). A cut-off value measured ≥33 IU/L showed 93.55% sensitivity and 94.87% specificity (Fig:-5 and Table VIII).

Fig.-5: ROC analysis of serum ADA in diagnosis of patients with SNPTB (n=140).

Table 6: Result of ROC curve.

AUC: Area under the curve; CI: Confidence Interval

• Significant Table IX shows that, among 62 SNPTB cases, a cut-off value of serum ADA of ≥33 IU/L could detect truly 58 cases of SNPTB.

Table 7: shows that, among 62 SNPTB cases, a cut-off value of serum ADA of ≥33 IU/L could detect truly 58 cases of SNPTB.

A cut-off value of serum ADA≥ 33 IU/L showed sensitivity, specificity, PPV, NPV, PLR, NLR and accuracy 93.55%, 94.87%, 93.55%, 94.87%, 17.98%, 0.07% and 94.29%, respectively (fig.-6)

Positive likelihood ratio: An individual having serum ADA value ≥ 33.IU/L is 17.98 times more likely to have SNPTB compared to individual having serum ADA ≤ 33 IU/L. (PLR > 1 indicates a test has diagnostic value ).

Diagnostic accuracy: Serum ADA ≥ 33 IU/L can detect 94 individuals correctly with SNPTB among 100 individuals.

Fig: 6: Diagnostic accuracy of Serum ADA to distinguish smear negative pulmonary TB from non- TB pulmonary diseases (n=140).

In this study, majority respondents among Group I were aged between 41-50 years (48.4%) followed by (35.5%) in 31-40 years age group with the mean age of 43.02±8.49 years. Among patients in Group II, majority were in the age group of 51-60 years (42.3%) followed by (35.9%) in greater than 60 years with mean age of 52±8.56 years. In line with my study findings, previous studies also found almost similar age distribution with male preponderance among pulmonary tuberculosis patients [15-17]). [13,15,16,17] Majority of the participants were male in this study. This might be because of more exposure of economically productive males to the external environment. In a previous study, the differences in sociodemographic characteristics in two periods, from the very beginning of the 21st century and 10 years after, were examined. In both observed periods, male people suffered from tuberculosis more frequently. [18] In the low socioeconomic background of our country, females are given less attention and access for them to the health care facilities is limited. Majority of the patients were garment workers among Group 1 (38.70%) and service holders in Group II (24.40%). Most of them belonged to middle income family in our study, about 51.60% and 50% respectively in Group I and Group II. This might be due to more exposure to external environment, working in overcrowded places, inadequate nutrition, alteration in immune function, poor ventilation and poor hygiene habits. Cough was the predominant clinical feature in 100% patients of Group I, followed by 79% sputum production, 74.2% fever and 51% had weight loss. Sajith et al.[19] found in their study that cough with expectoration were prevalent in 96.5% of TB patients followed by weight loss (80.7%), fever (73.7%) and loss of appetite (54.4%). [19] Maximum patients in Group I (74.2%) had ESR in 1st hour of 50-100 mm, while maximum non-TB pulmonary cases had ESR <50 mm in 1st hour which was statistically significant (p < 0.001). Mandal and Chavan [20] found in their study that, ESR was elevated in 87% and normal in 26% of pulmonary TB patients.20 About 69.4% patients in Group I had Mantoux test positive with 100% negative in non-TB pulmonary cases in this study. Karumuri, et al. [21] also found in their study that about 75% pulmonary TB patients had positive Mantoux test. [21] Sputum for gram stain was positive in 14.5% SNPTB patients and 23.1% in non-TB pulmonary cases without any statistical significance (p > 0.05). Sputum for Xpert MTB/RIF was 100% positive in Group I. Chest x-ray showed that maximum SNPTB patients had patchy opacity (42%), followed in decreasing order upper lobe consolidation (24.2%), cavitary lesion (24.2%), consolidation in different lobes (21%), pleural effusion (16.1%), collapse (11.3%) and lymphadenopathy (4.8%), whereas Group II patients had consolidation in different lobes (44.9%) followed by hyperlucent lung (32.1%), pleural effusion (24.4%), multiple ring shadows (10.3%) and mass lesion (10.3%). In this study, serum ADA was significantly higher among Group I patients compared to Group II (48.16 ± 12.13 vs 18.64 ± 7.85 IU/L, p<0.001). Similarly,) also found in their study that mean serum ADA among smear negative TB cases was (42.26 ± 21.22 U/L) and healthy control was (18.88 ± 6.67 U/L) with statistical significance (p<0.0001). [22] Alaarag et al.  found mean ADA of (42.26 ± 21.22 U/L) and (23.31 ± 8.22 U/L) in smear negative pulmonary TB and non-TB pulmonary cases respectively in their study with statistical significance (p<0.001). [23] in their study found higher mean ADA of (41.6 ± 6.4 U/L) in smear negative TB cases compared to healthy controls (15.5 ± 0.5 U/L) with statistical significance (p<0.001). [21] reported mean ADA of (39.478 ±32.22 U/L) in sputum negative TB cases and (11.819± 8.0235 U/L) in control groups with statistical significance (p<0.00).1 In a study by the serum ADA level in smear negative pulmonary TB subjects was (35.12 ± 12.1 U/L) which was statistically significant (p<0.001) as compared to that of in healthy subjects (14.603 ± 4.69 U/L). [17] Also reported that serum ADA level in smear negative pulmonary TB subjects was found to be highly significant (38.48 ±10.56 vs 15.30 ± 0.23 U/L, p<0.001) as compared to that of in healthy subjects. [24] In the present study, ROC analysis of serum ADA in diagnosis of patients with smear negative pulmonary TB cases found AUC of 0.9850 (95% CI 0.969-1.00) which was statistically significant (p<0.001). A cut-off value of serum ADA ≥ 33 IU/L showed sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy of 93.55%, 94.87%, 93.55%, 94.87% and 94.29% respectively to correctly diagnose smear negative pulmonary TB cases. Also found 33 U/L as cut-off value for serum ADA in diagnosing pulmonary tuberculosis in their study with sensitivity 98.06% and specificity 95.35%. [21] Similarly, evaluated the usefulness of ADA with a cut-off value of 33.3 U/L in serum to diagnose pulmonary tuberculosis patients efficiently with sensitivity, specificity, and positive predictive value, negative predictive value of 96.69%, 96.69 %, 96.69% and 96.69% respectively. [25] Besides, exact similar cut-off point (serum ADA ≥ 33 U/L) was also reported by) in diagnosing TB patients from patients with non-tubercular diseases with specificity 100% and sensitivity 98%.[26] found the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of serum ADA to be 95%, 86.7%, 90.5% and 92.9% respectively, at 30 U/L cut-off point. [23] Pandey et al. (2016) found a cut-off value of serum ADA for TB diagnosis of 30 U/L, with sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of 81.3%, 100%, 81.33% and 100% respectively.[13] showed that at a serum ADA level of 30 U/L as a cut-off value, serum ADA had sensitivity, specificity, positive predictive value (PPV), negative predictive value of 83.10%, 91.25%, 94% and 69.52% respectively. [22] However, according to the cut- off point which has been used in different researches, sensitivity, specificity, PPV, and NPV have been reported differently, and therefore the outcomes of different studies must be interpreted cautiously. Since there is no program in order to standardize the ADA results, determining a cut- off point for ADA must be dependent on the type of method and defined separately for each area. Studies have shown that in areas in which tuberculosis is endemic, the test sensitivity is of high importance.[27,28] In my research project, the sensitivity for truly diagnosing smear negative pulmonary TB cases from non-TB pulmonary cases was also high (93.55%). Besides, the negative predictive value of this test was also high (94.87%) and this gives it a place as a widely usable screening test to exclude smear negative pulmonary TB. Therefore, determination of serum ADA should be done routinely, particularly if the diagnosis of tuberculosis is in doubt, and also to differentiate smear negative pulmonary tuberculosis from non- tubercular pulmonary diseases.

In this study, serum ADA was significantly higher in smear negative pulmonary TB (SNPTB) patients than in non-TB pulmonary cases, with a remarkable diagnostic accuracy. These results correspond with the findings of previous studies with slight variations. Hence, the present study suggests to use serum ADA estimation as the biochemical marker in the diagnosis of SNPTB highlighting it as simple, rapid, cheaper and accurate diagnostic test. However, further study with larger sample size is recommended, which could give us more information about role of serum ADA in diagnosing SNPTB, thus helping the clinicians to manage those patients in a better way.

Although the results of this study support the hypothesis, there are some facts to be considered which might have affected the result of the current study. It was a single center study. The number of study population was relatively small.

Data Availability: The datasets analyzed during the current study are not publicly available due to the continuation of analyses but are available from the corresponding author on reasonable request.

Conflict of Interest: The authors stated that there is no conflict of interest in this study

Funding: This research received no external funding.

Ethical consideration: The study was approved by the Ethical Review Committee of Dhaka Medical College & Hospital, Dhaka, Bangladesh. Informed consent was obtained from each participant or caregivers of the patients.

Author Contributions: All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Acknowledgments: The authors were grateful to the staffs of the Department of Medicine of Dhaka Medical College & Hospital, Dhaka, Bangladesh.

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