Biomarkers of Disease Activity, Cure, and Relapse in Tuberculosis

Tuberculosis (TB) diagnosis among sputum-scarce patients is time consuming. Thus, a nonsputum diagnostic alternative is urgently needed. The Mycobacterium tuberculosis–specific transrenal (Tr) DNA from urine is a potential target for TB diagnostics. In this study, a new urine-based Tr-DNA molecular assay was evaluated for diagnosis of pulmonary tuberculosis among 428 adults suspected of having pulmonary TB (164 HIV positive, 263 HIV negative) from Cape Town, South Africa. Tr-DNA was isolated from 4 mL of EDTA urine, and a rapid, double-stranded, primer-based PCR method was performed targeting the Mycobacterium tuberculosis–specific direct repeat region. Each Tr-DNA eluate was tested in triplicate using an automated molecular analyzer with controls included in each test. With liquid culture used as the gold standard, the Tr-DNA assay showed sensitivity of 42.9% (n = 75/175; 95% CI, 35.4%–50.5%) and specificity of 88.6% (n = 210/237; 95% CI, 83.9%–92.4%). Among HIV-infected patients with TB, sensitivity and specificity were 45.2% and 89.0%, respectively. The combination of smear microscopy and Tr-DNA increased the sensitivity to 83.8% (smear microscopy alone, 75.1%), with 96.6% specificity. This study indicates that Tr-DNA has a moderate specificity with low sensitivity for diagnosis of pulmonary TB. Despite low sensitivity, this diagnostic test may have potential in combination with smear microscopy to support TB diagnosis in HIV-endemic regions, where sputum-scarce patients are common.

Transcriptomic host biomarkers could assist in developing effective diagnostics, vaccines and therapeutics for tuberculosis (TB). However, different biomarkers may be discriminatory in different populations depending on the host and bacillary genetics and HIV infection, and need to be addressed.

The expression levels of 45 genes that are known to be involved in or affected by TB pathogenesis were analyzed using dual color Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (dcRT-MLPA) assay in whole blood of 106 HIV positive individuals including active TB patients (TB⁺HIV⁺, n = 29), and non TB patients that are tuberculin skin test positive (TST+) (TST⁺HIV⁺, n = 26), or TST negative (TST⁻HIV⁺, n = 51).

Between the two clinical groups (TB⁺HIV⁺ vs. TST⁻HIV⁺) 8 genes were differently expressed (CCL19, CD14, CD8A, FPR1, IL7R, CCL22, TNFRSF1A, and FCGR1A); between TB⁺HIV⁺ vs. TST⁺HIV⁺, 6 genes (CD14, IL7R, TIMP2, CCL22, TNFRSF1A, and FCGR1A) were differently expressed. Since no difference in gene expression was revealed between TST⁺HIV⁺ vs. TST⁻HIV⁺, we clustered both the TST⁺HIV⁺ and TST⁻HIV⁺ individuals as one group (TST^+/−HIV⁺) and compared gene expression with TB⁺HIV⁺ patients. Thus, the results revealed that the levels of five genes (CD8A, TIMP2, CCL22, FCGR1A and TNFRSF1A) were the most accurate single gene markers for differentiation between TB⁺HIV⁺ and TST^+/−HIV⁺, with AUCs of 0.71, 0.71, 0.79, 0.83 and 0.73, respectively. However, the combination of two genes (CCL22 + FCGR1A) and FCGR1A alone were the most accurate marker for differentiation between the two groups (TB⁺HIV⁺ and TST^+/−HIV⁺) with AUC of 0.85 and 0.83, respectively.

We showed that five genes (CD8A, TIMP2, CCL22, FCGR1A and TNFRSF1A), specifically FCGR1A and CCL22 have the potential to discriminate active TB from non-active TB in HIV patients in Ethiopia and could be used to improve diagnostic tools for active TB in HIV patients, and to understand the pathogenesis of TB/HIV coinfection.

Identification of Mtb specific induced cytokine/chemokine host biomarkers could assist in developing novel diagnostic, prognostic and therapeutic tools for TB.

Levels of IFN-γ, IL-2, IL-17, IL-10, IP-10 and MIP-1α were measured in supernatants of whole blood stimulated with Mtb specific fusion protein ESAT-6/CFP-10 using xMAP technology. The study groups were HIV positive TB patients (HIV⁺TB⁺), HIV negative TB patients (HIV⁻TB⁺), HIV positive tuberculin skin test positive (TST+) (HIV⁺TST⁺), HIV negative TST+ (HIV⁻TST⁺), and HIV⁻TST⁻ individuals.

Compared to HIV⁻TST⁻, latent TB infection led to increased levels of IP-10, IFN-γ and IL-17, while levels of IL-2 and IP-10 were increased with active TB. Levels of IFN-γ, IL-17, MIP-1α, and IL-10 were increased in HIV⁻TST⁺ individuals compared to HIV⁻TB⁺ patients. HIV coinfection decreased the level of IFN-γ, IL-17, IP-10 and IL-2. After six months (M6) of anti-TB treatment (ATT) in HIV⁻TB⁺ patients, IFN-γ, IL-10, and MIP-1α levels normalized. After M6 and M18 of ATT plus HAART in HIV⁺TB⁺ patients, levels of MIP-1α and IL-10 normalized, while this was not the case for IFN-γ, IL-2, IL-17, and IP-10 levels. In HIV⁺TST⁺ patients on HAART, levels of IFN-γ, IL-17, IL-10 and MIP-1α normalized, while no change in the levels of IL-2 and IP-10 were observed.

In conclusion, the simultaneous measurement of IFN-γ, IL-17 and IP-10 may assist in diagnosing LTBI; IL-2 and IP-10 may assist in diagnosing active TB; while IFN-γ, IL-17, MIP-1α, and IL-10 levels could help to discriminate LTBI and active TB. In addition, IL-10 and MIP-1α levels could help to monitor responses to TB treatment and HAART.

Mycobacterium tuberculosis remains one of the most pernicious of human pathogens. Current vaccines are ineffective, and drugs, although efficacious, require prolonged treatment with constant medical oversight. Overcoming these problems requires a greater appreciation of M. tuberculosis in the context of its host. Upon infection of either macrophages in culture or animal models, the bacterium realigns its metabolism in response to the new environments it encounters. Understanding these environments, and the stresses that they place on M. tuberculosis, should provide insights invaluable for the development of new chemo- and immunotherapeutic strategies.