Suitability of saliva for Tuberculosis diagnosis: comparing with serum

Globally, tuberculosis (TB) ranks alongside HIV as the leading cause of mortality and morbidity [1]. The diagnosis of the disease remains a major problem, due to several shortcomings in the currently available diagnostic tests. Smear microscopy, the most widely available TB diagnostic test has poor sensitivity [2]. Sputum culture, the gold standard has a long turnaround time (up to 42 days) [3], whereas the recently developed GeneXpert MTB/RIF test (Cepheid Inc., Sunnyvale, USA), although rapid, is expensive, amongst other limitations which hamper its use in resource-poor countries [4]. All these tests also depend on the quality of sputum provided by the patient, to yield reliable results. This is highly problematic in children who cannot cough, and in individuals with extra pulmonary TB.

Current research has given birth to immunodiagnostic tests including the interferon gamma release assays (IGRA) such as the Quantiferon TB Gold and the T-SPOT assays. These tests require a blood draw to evaluate the body’s T cell response towards Mycobacterium tuberculosis (Mtb), as a means to diagnose infection with Mtb infection. These assays are based on the principle that individuals who have previously been exposed to Mtb habour pre-activated T cells in circulation, which rapidly respond with the secretion of IFN-γ after re-challenge in vitro, with Mtb specific antigens. IGRAs have been shown to be useful in the diagnosis of Mtb infection especially in comparison with the tuberculin skin test. However, they are not able to discriminate with latent Mtb infection and active TB disease. This therefore means that the assays are not useful in settings with high prevalence of latent TB. Another limitation of these assays is the fact that they are overnight assays, requiring a second visit to the hospital. Assays based on ex-vivo samples such as serum, saliva or whole blood, may be more beneficial as they may lead to a more rapid diagnosis, and tests based on such assays may be easily converted to point-of-care tests.

When compared to serum, saliva has advantages which include; low protein content, easy non invasive collection and ease of storage [5‐7]. Saliva has previously been used for molecular DNA testing in diagnosis of systemic diseases like hepatitis [8] HIV, renal diseases, cardiovascular diseases, autoimmune diseases, cancer, diabetes and other infectious diseases [9]. Recently, more studies have ventured into the search for biomarkers of TB in saliva [10, 11]. Compared to blood, saliva has advantages as a specimen for TB diagnosis which include none-invasiveness, no need for skilled personnel for collection, none clotting ability and ease to handle [12]. A study by Phalane et al. [13], compared serum with saliva and it was shown that some host inflammatory biomarkers are expressed in much higher concentrations in saliva than are in blood. Further studies also showed that some of the host markers detected in saliva showed potential as diagnostic biomarkers for TB disease [10, 11]. However all these previous TB studies have only been done on samples collected from a single study site. It is known from previous immunological studies [14] that immune responses tend to differ in patients recruited from different African countries, thereby highlighting the need for potential immunological based biomarkers to be investigated in different geographical regions. In the present study, we evaluated the expression of host biomarkers in serum in comparison to saliva, and further investigated whether any of these biomarkers had potential in differentiating active TB disease from latent or no TB infection, in individuals with presumed TB disease, recruited from Mulago hospital study site in Uganda. Replication of the findings from previous South African studies [10, 11, 13] in the present study would make the case for further investigation of the candidate markers identified so far and other recently identified markers in future larger studies and ultimately, the possible development of fiend-friendly TB diagnostic tests based on such salivary signatures. Furthermore, as saliva is a mucosal/airway linked sample and is relatively closer to the site of TB disease than peripheral blood, saliva may be a more informative sample for biomarker discovery purposes.

The search for biomarkers of TB disease and infection requires the investigation and research on non-sputum samples for TB diagnosis [17]. Early in biomarker research, IFN-γ took the lead as a relevant TB biomarker. It has however shown not to be a reliable biomarker if not used in conjunction with other markers. With the search for other biomarkers in TB research on the lead, there’s a need for a non-sputum biological sample and biomarker [18]. This study evaluated whether saliva could serve as an alternative specimen to serum for TB diagnosis. This owes to its non invasive nature of collection which can be applicable in children and the very ill groups [19]. Recently, studies evaluating the relevance of salivary host markers have shown promising markers that could be incorporated in a point-of-care test following validation [10, 11] and [13]. Overall, the levels of markers were higher in serum for all participants except for GM-CSF and VEGF. This confirms the findings by Phalane et al. [13]. This study found increased levels of VEGF and IL-6 in serum samples of active PTB patients compared to those with other respiratory diseases. These were also identified in previous studies [10, 11] and [13]. Increased VEGF in serum of patients with active TB makes it a useful prognostic indicator in granulomatous diseases, such as TB as identified by [20]. This suggests its role in pathogenesis of pulmonary TB in the development of the chronic inflammatory reaction.

In line with a study by Phalane et al. [13], increased saliva levels of GM-CSF in this study are relevant to enhance T cell responses, regulate phagocytosis and innate immune responses in alveolar macrophages [21]. High GM-CSF levels have also been identified in saliva of patients with oral squamous cell carcinoma [22].

Serum levels of MIP-1α, IL-6, TNF-α, IFN-γ and G-CSF were noticeably higher than were in saliva. IL-6 and TNF-α are pro-inflammatory cytokines which have a role in granuloma formation [23]. MIP-1α and β are important chemo attractants for lymphocytes to the site of TB infection leading to suppression of Mycobacterium growth [24]. Findings by other investigators [25, 26] also identified MIP-1α and β to be produced in high amounts in TB disease. IFN-γ on the other hand is important in promoting antigen presentation and recruiting T helper and cytotoxic T cells involved in killing the bacilli. In combination with TNF-α, they activate macrophages in order to kill intracellular pathogens and induce production of reactive nitrogen intermediates [27]. Serum levels of markers are insufficient to base on for Mtb complex diagnosis as we can’t pin their origin entirely to one immune condition such as PTB [28]. For this reason, salivary markers are being evaluated for their use in PTB disease diagnosis.

When salivary marker levels were evaluated for their ability to differentiate between active TB disease from No TB disease, there was no significant difference between the individual marker levels. Comparing with serum, the concentrations of IL-6 and VEGF were significantly different between the active TB disease group and the ORD group. This may suggest that salivary biomarkers may not be very useful individually in the diagnosis of TB disease. This may however, only apply to the markers investigated in the current study as previous reports identified salivary biomarkers which showed potential in the diagnosis of TB disease (Phalane et al., [13] Jacobs et al., [10] Jacobs et al. [11]). When biomarkers were used in combinations however, a biosignatures containing salivary biomarkers and combinations between saliva and serum markers showed potential. This correlates with findings by [29] and [30] which reported high VEGF levels in active TB. Our findings also suggest that Interleukin-6 as noted by previous studies by Jacobs et al., [10] and Phalane et al., [13] may be relevant for TB diagnosis. IL-6 appeared in predictive combinations and is known to stimulate the secretion of IFN-γ, a crucial cytokine in the activation of macrophages infected with M.tb [31]. This correlates with findings by [24] who identified increased serum levels of IL-6 in active TB patients. IL-2 on the other hand was below detectable levels for both serum and saliva. It is a relevant T cell growth factor, macrophage activator [23] and contributes to T cell memory. Previous studies found IL-2 production to be reduced among active PTB patients [32]. This may explain the low IL-2 levels shown given that all samples used were baseline from TB suspects.

More investigations are required including studies done on biomarkers other than the ones investigated in the current study, given the potential that a diagnostic tool based on saliva may contribute to the management of TB disease in all patient types especially those with difficulty in providing sputum samples such as children. Such future studies should focus on the identification of biosignatures, rather than individual biomarkers given that these inflammatory biomarkers may not be highly specific for TB, especially when used alone. Validated salivary biosignatures could then be incorporated into point-of-care tests for the diagnosis of TB disease (Sutherland et al. [33]).

In conclusion, saliva may be an important alternative diagnostic sample for PTB diagnosis and biomarker discovery. Our findings suggest that biosignatures comprising of combinations between different host markers detected in saliva may be useful for the diagnosis of PTB disease. We confirmed that there are highly significant differences in the concentrations of some biomarkers expressed in saliva in comparison to serum levels and therefore biomarkers that do not show potential in serum samples may not necessarily yield the same results in saliva. Therefore saliva warrants further exploration as a sample for biomarker TB biomarker discovery given that it is a very easily accessible sample and can be collected from all patient types, and immunological assays based on saliva may be easily translated into simple, culture-free point-of-care tests. Furthermore, saliva may be a more informative sample type than serum as it is a mucosal/airway linked sample and therefore is closer to the site of disease than peripheral blood samples. Our findings require further investigation in future larger prospective studies.