An improved algorithm for rapid diagnosis of pleural tuberculosis from pleural effusion by combined testing with GeneXpert MTB/RIF and an anti-LAM antibody-based assay

Tuberculosis (TB), caused by Mycobacterium tuberculosis complex (MTBC or MTB), is a serious global public health concern [1]. Despite great progress made in recent decades toward reducing TB disease burden, 10.1 million incident cases and 1.6 million deaths are currently observed each year worldwide [1]. In addition to damaging the lungs as the most commonly affected tissue, tuberculosis can involve any other organ or tissue to cause so-called extrapulmonary tuberculosis (EPTB) [2]. Globally, reported proportions of extrapulmonary cases range from 15 to 25% across countries [2, 3], with a worsening burden of EPTB disease resulting from patient co-infection with human immunodeficiency virus (HIV) [2]. Unfortunately, this TB/HIV co-infection scenario has been relatively neglected by TB control programs, mainly due to its low overall contribution to TB transmission within the community [4]. Moreover, although pulmonary TB cases are often easily recognizable due to typical radiological features and positive bacteriological evidence, EPTB is frequently more difficult to diagnose due to non-specific clinical and radiological features of EPTB that are often subject to variable interpretation [5].

Tuberculous pleurisy, one of the most common manifestations of EPTB, is the most common cause of pleural effusion in patients in many countries [6, 7]. As for other forms of EPTB, pleural TB poses a great diagnostic challenge, since conventional acid-fast bacilli (AFB) and culture methods have poor sensitivity as tools for diagnosing this disease [8]. A promising approach, PCR, has been applied to the detection of mycobacterial DNA in pleural fluid, with molecular diagnostic sensitivity ranging from 29 to 75% depending on target sequence amplified and DNA extraction procedure [9‐11]. Recently, the use of GeneXpert, a fully automatic molecular diagnostic system, provides rapid and accurate detection of EPTB. However, this assay can be difficult to perform in resource-limited countries, due to high cartridge costs and infrastructural requirements [12]. Additionally, given the poor sensitivity of GeneXpert in pleural TB, WHO has not recommended the use of GeneXpert for the diagnosis of pleural TB [13‐15]. Therefore, the lack of a reliable test for detecting MTB in pleural effusion specimens undoubtedly leads to misdiagnosis or missed diagnosis of pleural TB. These challenges thus highlight the urgent need for development of more effective diagnostics for timely diagnosis of pleural TB [12].

Pleural TB results from entry of MTB antigens into the pleural space after the rupture of a subpleural focus. This entry of antigens leads to generation of a host antibody response and accumulation of pleural effusion that result from a hypersensitivity reaction [16]. Therefore, immunological tests based on detection of antibodies present in pleural effusion specimens offer promise for improving pleural TB diagnosis [17]. Performance of immunologically based testing had been previously evaluated as a tool for diagnosing pleural TB from blood samples [17], with sensitivity estimates ranging from 26 to 59% and specificity estimates ranging from 81 to 100% [2, 18, 19]. Despite the fact that the WHO has advised against the use of commercial serological tests for diagnosing active TB [20], a serological test for diagnosis of pleural TB may be of value, since current tools are not diagnostically effective for this disease. Such a test would be based on detection of anti-MTB antibodies produced by the underlying inflammatory reaction that leads to pleural TB. This concept is based on limited data from previous studies, which had demonstrated that lipoarabinomannan (LAM), an integral component of the MTB cell wall, can stimulate production of anti-LAM antibodies by the human host [21]. We therefore conducted a retrospective study to assess the performance of an immunological method (the LAM assay) to detect anti-LAM antibodies in TB pleural effusion specimens. We then compared LAM assay sensitivity and specificity to corresponding results obtained using conventional culture and molecular testing methods.

The diagnosis of pleural TB is still an unsolved problem worldwide due to unreliable laboratory detection test results [12]. Numerous studies have documented that the GeneXpert assay is a useful confirmatory (rule in) diagnostic test for EPTB from various types of clinical specimens, such as cerebrospinal fluid and tissue samples [25‐27]. However, it is not endorsed for use as an initial test for diagnosing patients suspected of having pleural TB, due to its unsatisfactory performance for testing of pleural effusion specimens [28, 29]. In this study, detection of anti-LAM antibody in pleural effusion samples showed high specificity and moderate sensitivity for diagnosis of pleural TB, as compared with GeneXpert and MGIT culture.

Here, the LAM assay could identify approximately 40% of pleural TB cases when combined with MGIT culture or GeneXpert, a success rate significantly higher than obtained by each test alone. However, the combination of MGIT culture and GeneXpert offered limited benefit for diagnosing pleural TB from pleural effusion samples, since positive results of these etiological diagnostic methods are based on the presence of tubercle bacilli in specimens. Moreover, despite different detection limits between culture and GeneXpert methods, a high proportion of detection overlap between the two methods would undoubtedly weaken justification for their combined use for pleural TB diagnosis. On the contrary, detection of anti-LAM antibodies yielded better positivity despite the fact there were few tubercle bacilli but there were adequate LAM molecules in circulation in bodily fluids of TB patients and that could elicit significant production of anti-LAM antibodies and thus gave reasonably good sensitivity.

Notably, numerous studies have demonstrated that EPTB has been reported more frequently in females than in males [3, 30]. Conversely, in this report we found that men were more likely to have pleural TB. However, a nationwide epidemiological study from the Netherlands could reconcile these conflicting results, since in that study EPTB was relatively more prevalent among females, while pleural TB was more common in males (8.6% in males versus 6.7% in females, P < 0.01) [5]. Although the exact causes for gender biases among TB-related diseases remain unknown, we hypothesize that immunity, hormones, and socio-economic factors may be involved [31], warranting further research. Meanwhile, our analysis showed that pleural TB was more frequently observed among patients younger than 44 years of age. One possible explanation may be due to the fact that pleural TB is likely a manifestation of paucibacillary mycobacterial infection that leads to an immunologically-based hypersensitivity response [16]. Therefore, a decline in immunity with increasing age may decrease the risk of pleural TB occurrence in aging TB patients [32]. In line with this hypothesis, further analysis here revealed that younger pleural TB patients were more likely to generate an anti-LAM antibody response than were elderly patients.

In addition to advancing age, diabetes is another major comorbidity associated with low rates of pleural TB diagnosis that may contribute to false-negative LAM assay results. It is now well accepted that both humoral and cellular immunity are involved in the pathogenesis of diabetes [33], since patients with diabetes appear to exhibit reduced humoral immunity, with more rapid decay of antibody responses than observed in non-diabetic patients [33]. Therefore, poorer humoral immune function of patients in this population may have led to a lower pleural TB detection rate using the anti-LAM antibody detection method. However, it should be noted that the small number of diabetic patients in our study population may have undermined the reliability of these results. Nevertheless, the low detection rate obtained using the LAM assay highlights the need for new specific biomarkers that are more suitable for detecting pleural TB from pleural effusion samples in patients with diabetes.

Compared with other diagnostic tests for pleural TB, the LAM assay is simple and easy to perform and generates results within 1 h after sample collection. Moreover, the cost of this assay is less than 1.00 USD per test, which justifies its use as a cost-effective test for detecting pleural TB. Furthermore, since special equipment and skilled operators are not always provided to TB laboratories in resource-limited settings, another advantage of this assay is that it can be operated and interpreted without any complicated procedures or sophisticated instrumentation. Therefore, the LAM assay is an affordable and promising diagnostic test for use in diagnosis of pleural TB, especially in resource-limited settings.

This study had several obvious limitations. First, this was a retrospective study rather than one based on continuous recruitment of individuals with suspected pleural TB, which may limit the overall significance of our study conclusions. Second, the positive rate of MGIT culture for detection of MTB in pleural effusion specimens obtained here was lower than rates obtained in previous studies, a result possibly due to the small sample volume used for mycobacterial culture. As a consequence, the relatively low sensitivity may have negated the potentially beneficial role of mycobacterial culture for analysis of various diagnostic combinations. Third, we realize that the performance of the LAM assay is far from acceptable for applications needed for high-priority target product profiles [34]. The combination use of multiple biomarkers, such as 38kD, 16kD, Ag85A and MPT64 [35], may improve the sensitivity of this assay for analysis of pleural TB using pleural effusion specimens. Fourth, the performance of the LAM assay was only evaluated in pleural effusion samples rather than serum samples in this study. Despite these limitations, our study provides important insights into the use of an anti-LAM antibody-based assay for diagnosis of pleural TB.

The results of this study demonstrate that the LAM assay, which detects patient anti-LAM antibodies, shows promising performance for achieving pleural TB diagnosis from pleural effusion samples. Moreover, the combined use of the LAM assay with MGIT culture or GeneXpert could improve pleural TB diagnostic sensitivity relative to each test alone. In addition, old age and diabetes comorbidities are main factors associated with false-negative LAM assay results.