Significance of adenosine deaminase in diagnosing tuberculous pleurisy

Tuberculous pleurisy (TP) is one of the most common extra-pulmonary tuberculosis forms. Tuberculous pleurisy occurs when Mycobacterium tuberculosis antigen is released from a ruptured caseous focus into the pleural space causing hyperinflammatory response with a rapid influx of lymphocytes.

Acid-fast bacilli (AFB) staining, cultures and pathohistological biopsy finding are positive in most patients only in less than 10% of samples. Culture results take about 6-8 weeks which delays the diagnosis. A problem also occurs in the differentiation of effusions with lymphocytic predominance. Adenosine deaminase (ADA) is a biochemical marker with high sensitivity and specificity and is considered a gold standard within biomarkers when it comes to diagnosing TP. Using an algorithm for the values of ADA above or below 40 U/L we can distinguish this type of effusion from other types.

ADA in pleural punctate is a fast, efficient, and economical way for clarifying the etiology of a pleural effusion such as tuberculous pleurisy and treatment response during the follow up period.

INTRODUCTION

When it comes to infectious diseases, tuberculosis (TB) was the leading cause of death in 2019 [1],[2]. Tuberculosis remains a major problem in the modern world – although its eradication was imminent, morbidity and mortality rates have not decreased as predicted. Disease patterns have changed, so TB may or may not be accompanied by pulmonary symptoms, and there is a higher incidence of extra-pulmonary TB as well [1]. An extra-pulmonary form of tuberculosis is found in about 15-25% of all patients with tuberculosis. Lymphadenitis and tuberculous pleurisy (TP) are the two most common extra-pulmonary forms [1],[2]. Extra-pulmonary forms of infection also affect osteoarticular areas, the central nervous system, eyes, the gastrointestinal tract, and generally any organ [3].

TUBERCULOUS PLEURISY

Tuberculous pleurisy occurs when Mycobacterium tuberculosis antigen is released from a ruptured caseous focus into the pleural space [4]. This antigen causes a hyperinflammatory response with a rapid influx of neutrophils, followed by an influx of macrophages and lymphocytes. Therefore, this effusion is of lymphocyte-predominant exudative pleural type [4]. There may be a problem with malignant pleural effusion which is also lymphocyte-predominant effusion. Other characteristics of this type of effusion can be like in other pleurisy types. The TB fluid is usually clear but may be opalescent as in case of bacterial pleurisy in para-pneumonic effusion with pneumonia [5]. C-reactive protein, amylases or natriuretic brain peptide in pleural effusion sample can be used to differentiate between the effusion types [6],[7]. With antibiotic therapy (fluoroquinolones), effusion can be partial, or it resolves completely as in case of bacterial para-pneumonic disorder delaying the right diagnosis until the effusion reappears (Figure 1).

Figure 1. Tuberculous effusion in chest X-ray radiography

Lack of timely diagnosis and treatment can develop severe complications in a small percent of cases, such as empyema, bronchopleural fistulas, fibrothorax, or bronchial stenosis [1],[8]. Effusion is almost always an exudate, with lymphocytic predominance in about 90% of cases, but malignant effusion can have these characteristics as well [6],[7]. We need a biomarker, which can differentiate between the malignant form of tuberculous pleural effusion and other pleural effusions.

INVASIVE AND MICROBIOLOGICAL DIAGNOSIS OF TUBERCULOUS PLEURISY

Diagnosis of TB is challenging in patients with tuberculous pleurisy without a coexisting parenchymal lesion as they are sputum negative. In many of these cases, sputum smear through Ziehl-Neelsen staining can be negative. Therefore, a better approach to diagnosing is thoracentesis. Diagnosis of TP should meet the following criteria: acid-fast bacilli (AFB) staining or Löwenstein-Jensen cultures, pleural biopsy culture, and histology (granuloma-like changes in pleural biopsy samples and the exclusion of pleurisy due to other causes) [9]. The use of liquid culture media with inoculation of the pleural fluid can provide higher yields and faster results in comparison with conventional methods [9]. Histological analysis and mycobacterial culture from the pleural tissue obtained by biopsy have been the gold standard in diagnostics. Blind closed pleural biopsy is the most sensitive diagnostic test for tuberculous pleurisy [10]. Diacon et al. conducted a direct comparative study and obtained interesting results: the sensitivity of histology, culture and the combination of histology and culture was 66%, 48%, and 79%, respectively, for closed-needle biopsy and 100%, 76%, and 100%, respectively, for thoracoscopy. It helps visualize the entire pleural surface and allows target biopsy, adhesionolysis, and drainage of TP [10].

All these analyses have limitations. The yield of M. tuberculosis is shown to be very low in tuberculous pleural effusions [11]. AFB staining and cultures from most patients are positive in only 10% of cases [12]. Culture results take about 6-8 weeks which delays the diagnosis and the right treatment. The problem is that ess than a quarter of all results are positive, so fluid culture is a better solution [13]. Limitation of thoracentesis is a small sample for diagnosis, so the sample cannot represent the entire amount of pleural fluid and its characteristics. Pleural biopsy approach is invasive and usually negative because it is a blind closed biopsy [14]. A more invasive approach is pleuroscopy in total sedation or VATS (video assisted thoracoscopic surgery), which has many contraindications (such as comorbidities or age) and complications. Sensitivity and specificity are much better, over 90% and 100% [13]. A negative smear for acid-fast bacilli, a lack of granulomas in histopathology, and a failure to culture Mycobacterium tuberculosis do not exclude the diagnosis itself.

Because of all the above mentioned, it is difficult to diagnose TP, and it takes a lot of time to establish the right diagnosis and start the treatment. The aim of this paper is to clarify the diagnosis of TP using a highly sensitive and specific biomarker, which is practically a non-invasive approach with excellent results.

ADENOSINE DEAMINASE AS A BIOMARKER OF TUBERCULOUS PLEURISY

The biochemical marker for diagnosing TP is adenosine deaminase (ADA). Aggarwal et al. updated the sensitivity (0.92) and specificity (0.90) of ADA concluding it was a gold standard within biomarkers for detecting pleural TB among adults, which has been supported by more than 170 publications [15]. A systematic review and meta-analysis showed that this biomarker was convenient for detecting TP in pediatric population [16]. The level of ADA in the pleural effusion can be lower in elderly patients, the critically ill or those with multi-organ dysfunction [15].

ADA is an enzyme synthesized by many cells: mononuclear cells, lymphocytes, neutrophils. There are two different types of ADA biomarker: ADA1 that is ubiquitous and can be found in many cells, and ADA2 that is produced by monocytes/macrophages and is responsible for tuberculous pleuritis [5]. Mycobacterial antigens in pleural fluid stimulate Th1 lymphocytes. ADA is a T lymphocyte enzyme that catalyzes adenosine into inosine and due to this the amount of this enzyme is increased in a lymphocyte-rich exudate [14],[16]. According to numerous studies, the most accepted cutoff value for pleural ADA is 40 U/L [9]. About 30% of para-pneumonic effusions and 70% of empyema cases have ADA levels above 40 U/L. We can distinguish between these two effusions with neutrophil or lymphocyte predominant cytology. High ADA levels have also been reported in patients with lymphomas, but this effusion has extremely high ADA values (>250 U/L) [6]. Malignant mesotheliomas usually have low ADA levels [17]. Malignant mesothelioma can have lymphocyte predomination like other malignant effusions, but other biomarkers can be used to distinguish between those two types of effusion, as well as a histological biopsy profile, such as Carletinin or Cytokeratin5 [16].

According to literature and meta-analyses, ADA2 as isoenzymes may help distinguish TP from other types of pleural effusion [9],[18] with greater sensitivity and specificity in the diagnosis of TP. In high TB prevalence regions, pleural ADA values above 20 U/L showed excellent sensitivity and specificity, while in low TB prevalence regions, values between 40 U/L and 70 U/L may be associated with numerous false positives results. For that reason, some authors suggest ADA cut-off point of 70 U/L [19]. If we use established cut off values of the fluid ADA and if it is above 70 U/L with clinical presentation, the diagnosis of tuberculous pleurisy is established, and anti-tuberculous therapy can be started. If pleural fluid ADA is between 40 U/L and 70 U/L, further diagnostic procedures such as a needle biopsy or thoracoscopy should be performed. If the patient’s pleural fluid ADA level is below 40 U/L, the diagnosis of tuberculosis is unlikely (Figure 2) [9]. Due to this, the level of pleural ADA is used as a part of various ratio or scoring systems. One of the most commonly used ratios is the serum LDH/pleural ADA (cancer ratio), where values above 20 suggest malignant pleural effusion [20]. In high TB prevalence regions and in patients with the presence of lymphocyte-predominant exudate with clinical suspicion of TB and ADA >40 IU/L there is a positive predictive value of 98%. ADA is the most frequently used test for diagnosing tuberculosis in areas with moderate altitude prevalence of the disease, while in areas with low prevalence ADA can be used as an exclusion test [18],[21]. The disadvantage of this biomarker is that it does not provide information on cultivation and the type of mycobacteriosis and drug resistance [21].

Figure 2. The algorithm for diagnosing tuberculous pleurisy

The role of ADA is also important in treatment response during the follow up period. In a prospective study from India, the authors showed that the level of serum ADA could be useful for monitoring the anti-tuberculosis effect. The results showed a significant difference between ADA levels before and after the tuberculosis treatment (p < 0.001) [22].

Based on all the above-mentioned reasons, we use ADA in our daily practice in the pulmonology department. It is a fast and simple way to confirm TP when other diagnostic procedures have negative results. It is important that in the 21^st century we do not waste precious time waiting for cultures and repetition of pleural thoracentesis and pleural biopsy instead of providing treatment. The goal is the least invasive method and the minimal time for establishing the adequate diagnosis of TP and starting the treatment [23]. Our experience shows a high rate of successful response to the application of anti-tuberculosis therapy after the diagnosis of TP based on ADA results.

CONCLUSION

ADA is a sensitive and specific biochemical marker; it is available and cheap, and it should be used whenever possible. It is a fast, efficient, and economical way for clarifying the etiology of the pleural effusion as tuberculous pleurisy. Implementation of this biomarker in the routine practice shortens the path to the adequate TP diagnosis and the treatment of these patients. Further research and studies must go in the direction of diagnostics of such highly specific biomarkers for rapid detection of various diseases.