BackgroundNewer molecular diagnostics have brought paradigm shift in early diagnosis of tuberculosis [TB]. WHO recommended use of GeneXpert MTB/RIF [Xpert] for Extra-pulmonary [EP] TB; critics have since questioned its efficiency.MethodsThe present study was designed to assess the performance of GeneXpert in 761 extra-pulmonary and 384 pulmonary specimens from patients clinically suspected of TB and compare with Phenotypic, Genotypic and Composite reference standards [CRS].ResultsComparison of GeneXpert results to CRS, demonstrated sensitivity of 100% and 90.68%, specificity of 100% and 99.62% for pulmonary and extra-pulmonary samples. On comparison with culture, sensitivity for Rifampicin [Rif] resistance detection was 87.5% and 81.82% respectively, while specificity was 100% for both pulmonary and extra-pulmonary TB. On comparison to sequencing of rpoB gene [Rif resistance determining region, RRDR], sensitivity was respectively 93.33% and 90% while specificity was 100% in both pulmonary and extra-pulmonary TB. GeneXpert assay missed 533CCG mutation in one sputum and dual mutation [517 & 519] in one pus sample, detected by sequencing. Sequencing picked dual mutation [529, 530] in a sputum sample sensitive to Rif, demonstrating, not all RRDR mutations lead to resistance.ConclusionsCurrent study reports observations in a patient care setting in a high burden region, from a large collection of pulmonary and extra-pulmonary samples and puts to rest questions regarding sensitivity, specificity, detection of infrequent mutations and mutations responsible for low-level Rif resistance by GeneXpert. Improvements in the assay could offer further improvement in sensitivity of detection in different patient samples; nevertheless it may be difficult to improve sensitivity of Rif resistance detection if only one gene is targeted. Assay specificity was high both for TB detection and Rif resistance detection. Despite a few misses, the assay offers major boost to early diagnosis of TB and MDR-TB, in difficult to diagnose pauci-bacillary TB.

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of drug resistance necessitate the search for therapeutic vaccine strategies for tuberculosis (TB). Such strategies ought to elicit not only IFN-γ, but polyfunctional response including TNF-α, which is essential for protective granuloma formation. Here, we investigated the impact of PD-1 inhibition in facilitating protective polyfunctional T cells (PFTs), bacillary clearance, and disease resolution. We have observed PD-1 inhibition preferentially rescued the suppressed PFTs in active tuberculosis patients. In addition, polyfunctional cytokine milieu favored apoptosis of infected MDMs over necrosis with markedly reduced bacillary growth ( CFU) in our in vitro monocyte-derived macrophages (MDMs) infection model. Furthermore, the animal study revealed a significant decline in the bacterial burden in the lungs and spleen of infected mice after in vivo administration of α-PD-1 along with antitubercular treatment. Our findings suggest that rescuing polyfunctional immune response by PD-1 inhibition works synergistically with antituberculosis chemotherapy to confer improved control over bacillary growth and dissemination. In summary, our data strongly indicate the therapeutic potential of α-PD-1 as adjunct immunotherapy that can rejuvenate suppressed host immunity and enhance the efficacy of candidate therapeutic vaccine(s). Keywords: polyfunctional T cells r PD-1 r Treg cells r tuberculosis r immunotherapyAdditional supporting information may be found online in the Supporting Information section at the end of the article. Abbreviations: MDM: monocyte-derived macrophage · M.tb: Mycobacterium tuberculosis · PFT: polyfunctional T cell · TB: tuberculosis

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Host T cell response plays a critical role in containment of M. tuberculosis (Mtb) infection by granuloma formation. The prevalence of multidrug-resistance tuberculosis (MDR-TB) among immunocompromised hosts (HIV co-infection & posttransplant patients) indicate the influence of host T cell response on MDR TB. Previously, we have shown critical role of regulatory T (Treg) cells and PD-1 pathway causing suppressed state of T cell response against Mycobacterium tuberculosis (Mtb) among TB patient.In this study, we attempted to understand the status of host immune response among the MDR and Drug Sensitive (DS) TB patients. We also checked the contribution of PD-1 pathway on poly-functional T cells (PFTs), critical for protective immunity in TB. METHODS:For immune response profile and in vitro experiments, polychromatic flowcytometry based immunological assays were performed. PD1 blocking experiments were performed in mice infected with Mtb and invitro model where monocyte derived macrophages (MDM) were infected with virulent Mtb DS strains. Furthermore, the impact of cytokine on the efflux pump of bacteria was evaluated using virulent Mtb DS strains infected MDM in presence of pro and anti-inflammatory cytokines. RESULTS:We observed marked reduction of polyfunctional T cells (PFTs) in TB patients. PFTs could be significantly rescued by blocking PD-1 pathway, which resulted in effective clearance of Mtb in MDM in vitro model. Blocking PD-1 pathway in mice infected with Mtb, demonstrated decrease in Tregs and restoration of PFTs with enhanced reduction of bacillary load in the lung & spleen relative to chemotherapy alone. Among MDR patients, we observed increase in the frequency of Tregs and decrease in frequency of Mtb specific T cell cytokine producers (IFN-g or TNF-a or IFN-gþ TNF-aþ) compared to DS TB patients, suggesting a tight correlation of Treg mediated suppression with MDR status. Furthermore, we observed higher expression of efflux pump in DR strains which could be substantially modulated by pro-inflammatory (IFN-g, TNF-a) and anti-inflammatory cytokines (IL10 and TGF b) in in vitro MDM model. Suppressive cytokines (IL10 and TGF b) increased the efflux pump of Mtb. This is suggestive of possible causal relation of host immune status with the drug efflux pump expression in Mtb. CONCLUSIONS:Our results demonstrate elicitation of weaker effector T cell response in DR TB compared to DS TB patients. Additionally, our findings suggest critical role of PD-1 in suppressing the protective immune response in TB. Rescuing PFTs by blocking PD-1 pathway may offer a novel strategy for adjunct immunotherapy in TB. This may be more relevant in MDR TB as PFT response may modulate the drug pump so as to increase the efficacy of chemotherapy.CLINICAL IMPLICATIONS: Rescuing appropriate immune response may improve the efficacy of anti-tubercular therapy in TB, especially for MDR TB with possible reduction of duration of drug therapy and relapse.

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