Diagnostic performance of nucleic acid tests in tuberculous pleurisy

Tuberculosis (TB), the leading cause from a single infectious agent, typically affects the lungs (pulmonary TB), but can also affect other sites (extrapulmonary TB). Extrapulmonary TB (EPTB) represented 14% of the 6.4 million incident cases notified in 2017, globally [1]. The most common form of EPTB is tuberculous pleurisy (TBP) [2]. However, the sensitivity of acid-fast bacilli (AFB) in pleural effusion (PE) smear is unacceptably low and non-tuberculos Mycobacterium (NTM) is also positive [3]. The definite diagnosis of TBP is made by detecting Mycobacterium tuberculosis (MTB) from PE or pleural tissue [4], but culturing M. tuberculosis will take 2–8 weeks to obtain the results, which can delay effective medical interventions [5]. Delayed antituberculosis treatment may result in pleural thickening or tuberculous empyema that requires surgical resolution [6, 7]. Therefore, diagnosis of TBP is sometimes referred to pleural biopsy. However, pleural biopsy is invasive and adds considerable cost to the workup. In addition, biopsy of pleural tissue for histological examination may still have false negative rate of about 20% [8]. Technological advances in nucleic acid amplification tests (NAATs) have led to breakthroughs in TB diagnosis with turnaround time under 2 h [9]. Xpert MTB/RIF (Xpert), endorsed by the Scientific and Technical Advisory Board of the WHO, integrates hemi-nested real-time Mycobacterium tuberculosis-specific DNA amplification and simultaneous detection of mutations in the rifampicin resistance-associated rpoB mutations [10]. However, the requirement of expensive specialized equipment and the high cost of the assay make it unaffordable for large-scale use in developing countries. Loop-mediated isothermal amplification (LAMP) is a DNA amplification at a constant temperature by one type of enzyme with rapid and simple features which make it a promising diagnostic method for point-of-care testing and for resources limited countries [11]. Simultaneous amplification and testing for detection of Mycobacterium tuberculosis complex (MTBC) (SAT-TB assay) is a relatively new method based on real-time fluorescence simultaneous isothermal RNA amplification. Since RNA is much more unstable than DNA, so SAT-TB assay (SAT-TB) has the advantage of lower false-positive rates and good reproducibility [12]. Previous studies of NAATs have demonstrated superior sensitivity and specificity for the diagnosis of pulmonary TB with sputum specimens [13‐18]. However, there is still limited data on the performance of NAATs on the diagnosis of TBP with pleural fluid specimens. Whether these tests are sensitive enough to rule out TBP remains unclear.

Thus, we designed the current prospective study to evaluate the diagnostic performance of Xpert, LAMP and SAT-TB with PE specimens from confirmed TBP patients in a country with high TB incidence.

In this study, we evaluated LAMP, Xpert and SAT-TB assays for the diagnosis of TBP in PE culture positive patients, compared with PE smear and found that all these methods were suboptimal for the detection of MTB in PE, whereas each of them demonstrated high specificity. Similar previous investigations of NAATs for detecting MTB in PE have also reported modest sensitivities [21‐24]. Touré et al. reported Xpert MTB/RIF with pleural liquid was positive in only 3.3% of 301 TBP patients [25]. Tyagi et al. conducted a meta-analysis, collecting 58 studies on pleural fluid-based Xpert MTB/RIF and found that the pooled sensitivity was inadequate [26]. IS1081- based LAMP was developed in a study by B. Yang et al., for the detection of MTB in PE, that was positive in 25% TBP patients (18 / 72), while no positive reaction was observed in non-TBP patients [27]. In a meta-analysis of 40 studies of NAATs for TBP, PAI et al. reported that these tests had low sensitivities (43–77%), but high specificities (95%) [28]. The reasons for the low sensitivity of NAATs in PE specimens but high sensitivity in sputum samples are not clear. The presence of inhibitory substances in PE is not a satisfactory explanation, as studies have shown that some substances of potential inhibitors of nucleic acid detection, such as RNases, were similar in sputum and non-sputum specimens [29]. The paucity of MTB in PE may play some role, but the low sensitivity is more likely to be relevant to techniques of nucleic acid extraction. Therefore, the consistent high specificities of NAATs indicated their potential role in confirming TBP as ‘rule-in’ tests and were not useful in excluding the disease. Caution should be exercised when interpreting negative NAATs results in PE.

In addition, it’s worth mentioning that ADA remains the most widely used diagnostic PE marker as a screening tool for TBP in resource-limited settings where tuberculosis is endemic, since it has the advantage of cost-effectiveness, efficiency, noninvasiveness, and ease of operation [30, 31]. In our current study, 201out of 223 TBP patients (90.13%) had an ADA level over 25 U/l, while 10 out of 42 (23.8%) non-TBP also had an ADA level over 25 U/l. Nevertheless, apart from tuberculosis, high ADA levels in lymphocytic pleural effusions have also been reported in mesothelioma, lymphoma, rheumatoid immune system diseases and other infectious disease [3, 32, 33].

One possible shortcoming of this article was the number of cases is relatively small, because the diagnostic index we used is culture positive of MTB in PE, the “gold standard” for the diagnosis of tuberculosis.

In conclusion, our research and previous work by other groups have suggested that NAATs are not the first choice in the diagnosis of TBP. If this type of test must be selected, the SAT-TB assay is recommended because of its low cost, relatively high sensitivity and high specificity compared with the other two tests. The diagnostic measure for TBP with high efficiency, low cost, rapid and convenient operation remains to be further studied.