Identification and Characterization of Genetic Determinants of Isoniazid and Rifampicin Resistance in Mycobacterium tuberculosis in Southern India

Abstract: Drug-resistant tuberculosis (TB), one of the leading causes of death worldwide, arises mainly from spontaneous mutations in the genome of Mycobacterium tuberculosis. There is an urgent need to understand the mechanisms by which the mutations confer resistance in order to identify new drug targets and to design new drugs. Previous studies have reported numerous mutations that confer resistance to anti-TB drugs, but there has been little systematic analysis to understand their genetic background and the potential impacts on the drug target stability and/or interactions. Here, we report the analysis of whole-genome sequence data for 98 clinical M. tuberculosis isolates from a city in southern India. The collection was screened for phenotypic resistance and sequenced to mine the genetic mutations conferring resistance to isoniazid and rifampicin. The most frequent mutation among isoniazid and rifampicin isolates was S315T in katG and S450L in rpoB respectively. The impacts of mutations on protein stability, protein-protein interactions and protein-ligand interactions were analysed using both statistical and machine-learning approaches. Drug-resistant mutations were predicted not only to target active sites in an orthosteric manner, but also to act through allosteric mechanisms arising from distant sites, sometimes at the protein-protein interface

Downregulation of monocyte miRNAs: implications for immune dysfunction and disease severity in drug-resistant tuberculosis

Monocyte miRNAs govern both protective and pathological responses during tuberculosis (TB) through their differential expression and emerged as potent targets for biomarker discovery and host-directed therapeutics. Thus, this study examined the miRNA profile of sorted monocytes across the TB disease spectrum [drug-resistant TB (DR-TB), drug-sensitive TB (DS-TB), and latent TB] and in healthy individuals (HC) to understand the underlying pathophysiology and their regulatory mechanism

Plasma chemokines CXCL10 and CXCL9 as potential diagnostic markers of drug-sensitive and drug-resistant tuberculosis

Tuberculosis (TB) diagnosis still remains to be a challenge with the currently used immune based diagnostic methods particularly Interferon Gamma Release Assay due to the sensitivity issues and their inability in differentiating stages of TB infection. Immune markers are valuable sources for understanding disease biology and are easily accessible. Chemokines, the stimulant, and the shaper of host immune responses are the vital hub for disease mediated dysregulation and their varied levels in TB disease are considered as an important marker to define the disease status. Hence, we wanted to examine the levels of chemokines among the individuals with drug-resistant, drug-sensitive, and latent TB compared to healthy individuals. Our results demonstrated that the differential levels of chemokines between the study groups and revealed that CXCL10 and CXCL9 as potential markers of drug-resistant and drug-sensitive TB with better stage discriminating abilities

Differential Frequencies of Intermediate Monocyte Subsets Among Individuals Infected With Drug-Sensitive or Drug-Resistant Mycobacterium tuberculosis

The rampant increase in drug-resistant tuberculosis (TB) remains a major challenge not only for treatment management but also for diagnosis, as well as drug design and development. Drug-resistant mycobacteria affect the quality of life owing to the delayed diagnosis and require prolonged treatment with multiple and toxic drugs. The phenotypic modulations defining the immune status of an individual during tuberculosis are well established. The present study aims to explore the phenotypic changes of monocytes & dendritic cells (DC) as well as their subsets across the TB disease spectrum, from latency to drug-sensitive TB (DS-TB) and drug-resistant TB (DR-TB) using traditional immunophenotypic analysis and by uniform manifold approximation and projection (UMAP) analysis. Our results demonstrate changes in frequencies of monocytes (classical, CD14(++)CD16(-), intermediate, CD14(++)CD16(+) and non-classical, CD14(+/-)CD16(++)) and dendritic cells (DC) (HLA-DR(+)CD11c(+) myeloid DCs, cross-presenting HLA-DR(+)CD14(-)CD141(+) myeloid DCs and HLA-DR(+)CD14(-)CD16(-)CD11c(-)CD123(+) plasmacytoid DCs) together with elevated Monocyte to Lymphocyte ratios (MLR)/Neutrophil to Lymphocyte ratios (NLR) and alteration of cytokine levels between DS-TB and DR-TB groups. UMAP analysis revealed significant differential expression of CD14(+), CD16(+), CD86(+) and CD64(+) on monocytes and CD123(+) on DCs by the DR-TB group. Thus, our study reveals differential monocyte and DC subset frequencies among the various TB disease groups towards modulating the immune responses and will be helpful to understand the pathogenicity driven by Mycobacterium tuberculosis

Systemic Inflammation and the Increased Risk of Inflamm-Aging and Age-Associated Diseases in People Living With HIV on Long Term Suppressive Antiretroviral Therapy.

The ART program in low- and middle-income countries (LMIC) like India, follows a public health approach with a standardized regimen for all people living with HIV (PLHIV). Based on the evidence from high-income countries (HIC), the risk of an enhanced, and accentuated onset of premature-aging or age-related diseases has been observed in PLHIV. However, very limited data is available on residual inflammation and immune activation in the populations who are on first-generation anti-HIV drugs like zidovudine and lamivudine that have more toxic side effects. Therefore, the aim of the present study was to evaluate the levels of systemic inflammation and understand the risk of age-associated diseases in PLHIV on long-term suppressive ART using a large number of biomarkers of inflammation and immune activation. Blood samples were obtained from therapy naïve PLHIV (Pre-ART, = 43), PLHIV on ART for >5 years (ART, = 53), and HIV-negative healthy controls (HIVNC, = 41). Samples were analyzed for 92 markers of inflammation, sCD14, sCD163, and telomere length. Several statistical tests were performed to compare the groups under study. Multivariate linear regression was used to investigate the associations. Despite a median duration of 8 years of successful ART, sCD14 ( < 0.001) and sCD163 ( = 0.04) levels continued to be significantly elevated in ART group as compared to HIVNC. Eleven inflammatory markers, including 4E-BP1, ADA, CCL23, CD5, CD8A, CST5, MMP1, NT3, SLAMF1, TRAIL, and TRANCE, were found to be significantly different ( < 0.05) between the groups. Many of these markers are associated with age-related co-morbidities including cardiovascular disease, neurocognitive decline and some of these markers are being reported for the first time in the context of HIV-induced inflammation. Linear regression analysis showed a significant negative association between HIV-1-positivity and telomere length ( < 0.0001). In ART-group CXCL1 ( = 0.048) and TGF-α ( = 0.026) showed a significant association with the increased telomere length and IL-10RA was significantly associated with decreased telomere length ( = 0.042). This observation warrants further mechanistic studies to generate evidence to highlight the need for enhanced treatment monitoring and special interventions in HIV-infected individuals

Efficacy of T Regulatory Cells, Th17 Cells and the Associated Markers in Monitoring Tuberculosis Treatment Response

Treatment monitoring is an essential aspect for tuberculosis (TB) disease management. Sputum smear microscopy is the only available tool for monitoring, but it suffers from demerits. Therefore, we sought to evaluate markers and cellular subsets of T regulatory (Treg) cells and T helper (Th) 17 cells in pulmonary TB patients (PTB) for TB treatment monitoring. Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro (with purified protein derivative (PPD)) overnight which was followed by a polychromatic flow cytometry approach to study Treg and Th17 markers and cellular subsets in PTB (n = 12) undergoing antituberculous treatment (ATT). The baseline levels of these markers and cellular subsets were evaluated in normal healthy subjects (NHS). We observed a significant decrease in the expression of CD25 (p&lt;0.01) marker and percentage of T-cell subsets like CD4+CD25+ (p&lt;0.001) and CD4+CD25+CD39+ (p&lt;0.05) at the end of intensive phase (IP) as well as in the continuation phase (CP) of ATT. A decrease in CD25 marker expression and percentage of CD4+CD25+ T cell subset showed a positive correlation to sputum conversion both in high and low sputum positive PTB. In eight PTB with cavitary lesions, only CD4+CD25+FoxP3 Treg subset manifested a significant decrease at the end of CP. Thus, results of this study show that CD25 marker and CD4+CD25+ T cells can serve as better markers for monitoring TB treatment efficacy. The Treg subset CD4+CD25+FoxP3 may be useful for prediction of favorable response in PTB with extensive lung lesions. However, these findings have to be evaluated in a larger patient cohort

Cytokine upsurge among drug-resistant tuberculosis endorse the signatures of hyper inflammation and disease severity

Abstract Tuberculosis (TB) elimination is possible with the discovery of accurate biomarkers that define the stages of infection. Drug-resistant TB impair the current treatment strategies and worsen the unfavourable outcomes. The knowledge on host immune responses between drug-sensitive and drug-resistant infection is inadequate to understand the pathophysiological differences and disease severity. The secreted proteins, cytokines display versatile behaviour upon infection with Mycobacterium tuberculosis (MTB) and their imbalances often tend to assist disease pathology than protection. Therefore, studying these soluble proteins across TB infection spectrum (drug-resistant TB, drug-sensitive TB, and latent TB) may unveil the disease mediated responses and unique stage specific cytokine signatures. Thus, we sought to determine the plasma cytokine levels from healthy, latently infected, drug-sensitive, and drug-resistant TB individuals. Our study revealed top 8 cytokines (IL-17, IL-1α, IL-2, IL-10, IL-5, IFN-γ, TNF-α and IL-6) and their biomarker abilities to discriminate different stages of infection

Plasma chemokines CXCL10 and CXCL9 as potential diagnostic markers of drug-sensitive and drug-resistant tuberculosis

Abstract Tuberculosis (TB) diagnosis still remains to be a challenge with the currently used immune based diagnostic methods particularly Interferon Gamma Release Assay due to the sensitivity issues and their inability in differentiating stages of TB infection. Immune markers are valuable sources for understanding disease biology and are easily accessible. Chemokines, the stimulant, and the shaper of host immune responses are the vital hub for disease mediated dysregulation and their varied levels in TB disease are considered as an important marker to define the disease status. Hence, we wanted to examine the levels of chemokines among the individuals with drug-resistant, drug-sensitive, and latent TB compared to healthy individuals. Our results demonstrated that the differential levels of chemokines between the study groups and revealed that CXCL10 and CXCL9 as potential markers of drug-resistant and drug-sensitive TB with better stage discriminating abilities