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Pyrazinamide (PZA) is a key first-line antituberculosis drug that plays an important role in eradicating persister Mycobacterium tuberculosis (TB) bacilli and shortening the duration of tuberculosis treatment. However, PZA-resistance is on the rise, particularly among persons with multidrug-resistant (MDR) tuberculosis. This nationwide study was conducted to explore the prevalence of mutations conferring PZA resistance, catalogue mutation diversity, investigate the associations of PZA resistance with specific lineages, examine co-resistance to 13 first- and second-line drugs, and evaluate the diagnostic accuracy of sequencing pncA and panD genes for predicting PZA resistance. Whole genome sequencing was performed on 2,207 M. tuberculosis isolates from 25 States and 4 Union Territories of India. The majority of phenotypically PZA-resistant isolates (77%) harbored 171 distinct mutations in pncA; however, a small number of mutations in panD, rpsA and clpC1 were also observed. A set of novel mutations associated PZA resistance was uncovered, along with an additional 143 PZA resistance-conferring mutations in pncA based on application of WHO-endorsed grading rules. PZA resistance was predominately observed in Lineage 2 and eight lineage-specific resistance markers were identified. Mutations distributed across pncA correlate to 94% of PZA resistance and were the predominant drivers of phenotypic resistance; evidence generated herein substantiates sequencing the entire gene and promoter for comprehensive genotypic-based prediction of PZA resistance. This work provides key insights into the scope of PZA-resistance in India, a high drug-resistant TB burden country, and can support the effectiveness of TB prevention and control efforts.

Abstract Background Tuberculosis (TB) ranks as the second leading cause of death globally among all infectious diseases. This problem is likely due to the lack of biomarkers to differentiate the heterogeneous spectrum of infection. Therefore, the first step in solving this problem is to identify biomarkers to distinguish the different disease states of an individual and treat them accordingly. Circulating microRNA (miRNA) biomarkers are promising candidates for various diseases. In fact, we are yet to conceptualize how miRNA expression influences and predicts TB disease outcomes. Thus, this systematic review and meta-analysis aimed to assess the diagnostic efficacy of circulating miRNAs in Latent TB (LTB) and Active Pulmonary TB (PTB). Methods Literature published between 2012 and 2021 was retrieved from PubMed, Web of Science, Cochrane, Scopus, Embase, and Google Scholar. Articles were screened based on inclusion and exclusion criteria, and their quality was assessed using the QUADAS-2 tool. Funnel plots and forest plots were generated to assess the likelihood of study bias and heterogeneity, respectively. Results After the screening process, seven articles were selected for qualitative analysis. The study groups, which consisted of Healthy Control (HC) vs. TB and LTB vs. TB, exhibited an overall sensitivity of 81.9% (95% CI: 74.2, 87.7) and specificity of 68.3% (95% CI: 57.8, 77.2), respectively. However, our meta-analysis results highlighted two potentially valuable miRNA candidates, miR-197 and miR-144, for discriminating TB from HC. The miRNA signature model (miR197-3p, miR-let-7e-5p, and miR-223-3p) has also been shown to diagnose DR-TB with a sensitivity of 100%, but with a compromised specificity of only 75%. Conclusion miRNA biomarkers show a promising future for TB diagnostics. Further multicentre studies without biases are required to identify clinically valid biomarkers for different states of the TB disease spectrum. Systematic review registration PROSPERO (CRD42022302729).

Abstract Multisystem Inflammatory Syndrome in Children (MIS-C) is a rare manifestation of Severe Acute Respiratory Syndrome-CoronaVirus-2 (SARS-CoV-2) infection that can result in increased morbidity and mortality. Mounting evidence describes sex disparities in the clinical outcomes of coronavirus disease 2019 (COVID-19). However, there is a lack of information on sex-specific differences in immune responses in MIS-C. This study is an observational and cross-sectional study and we wanted to examine immune parameters such as cytokines, chemokines, acute phase proteins (APPs), growth factors, microbial translocation markers (MTMs), complement components and matrix metalloproteinases (MMPs) in MIS-C children, based on sex. Male children were associated with heightened levels of pro-inflammatory cytokines—IFNγ, IL-2, TNFα, IL-1α, IL-1β, IL-6, IL-12, G-CSF and GM-CSF, chemokines-CCL2, CCL11, CXCL1, CXCL8 and CXCL10, acute phase proteins-α-2M, CRP, growth factors VEGF and TGFα, microbial translocation markers- iFABP, LBP, EndoCAb, complement components—C1q, MBL and C3 and matrix metalloproteinases MMP-8 and MMP-9 compared to female children with MIS-C. These results indicate that the heightened immune response in males is a characteristic feature of MIS-C. These findings might explain the differential disease pathogenesis in males compared to females with MIS-C and facilitate a deeper understanding of this disease.

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.

A natural infection or a vaccination can initially prime the immune system to form immunological memory. The immunity engendered by vaccination against COVID-19 versus natural infection with SARS-CoV-2 has not been well studied in the Indian population. In this study, we compared the immunity conferred by COVID-19 vaccines to naturally acquired immunity to SARS-CoV-2 in a South Indian population. We examined binding and neutralizing antibody (NAb) levels against the ancestral and variant lineages and assessed the ex vivo cellular parameters of memory T cells, memory B cells, and monocytes and finally measured the circulating cytokine response. COVID-19 vaccination stimulates heightened levels of IgG antibodies against the original strain of SARS-CoV-2, as well as increased binding to the spike protein and neutralizing antibody levels. This enhanced response extends to variant lineages such as B.1.617.2 (Delta, India), B.1.1.529 (Omicron, India), B.1.351 (Beta, South Africa), and B.1.1.7 (Alpha, UK). COVID-19 vaccination differs from SARS-CoV-2 infection by having increased frequencies of classical memory B cells, activated memory B and plasma cells, CD4/CD8 T cells of effector memory, effector cells, stem cell-like memory T cells, and classical and intermediate monocytes and diminished frequencies of CD4/CD8 T cells of central memory and non-classical monocytes in vaccinated individuals in comparison to those with natural infection. Thus, COVID-19 vaccination is characterized by enhanced humoral responses and robust activation of innate and memory T cell responses in comparison to natural infection in a South Indian population.

BackgroundMultisystem inflammatory syndrome (MIS) in children is considered to be a post-infectious complication of COVID-19. T-cell responses in children with this condition have not been well-studied.MethodsWe aimed to study the immune responses in children with MIS in comparison to children with acute COVID-19 and children with other infections. Whole blood was stimulated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)–specific antigens and flow cytometry was performed to examine CD4+ and CD8+ T-cell responses.ResultsChildren with MIS had higher frequencies of CD4+ and CD8+ T cells expressing cytokines at baseline and upon SARS-CoV-2 antigen–specific stimulation in comparison to children with COVID-19 and/or other infections. Children with COVID-19 also exhibited higher frequencies of CD4+ and CD8+ T cells expressing cytokines at baseline and upon SARS-CoV-2 antigen–specific stimulation in comparison to children with other infections. At 6–9 months following treatment and recovery, this enhanced response against SARS-CoV-2 antigens was down modulated in children with MIS.ConclusionOur study, therefore, provides evidence of enhanced activation of CD4+ and CD8+ T-cell responses in children with MIS and reversal following recovery.

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.

BackgroundMonocyte 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.MethodsWe sorted total monocytes including three subsets (HLA-DR+CD14+, HLA-DR+CD14+CD16+, and HLA-DR+CD16+cells) from peripheral blood mononuclear cells (PBMCs) of healthy and TB-infected individuals through flow cytometry and subjected them to NanoString-based miRNA profiling.ResultsThe outcome was the differential expression of 107 miRNAs particularly the downregulation of miRNAs in the active TB groups (both drug-resistant and drug-sensitive). The miRNA profile revealed differential expression signatures: i) decline of miR-548m in DR-TB alone, ii) decline of miR-486-3p in active TB but significant elevation only in LTB iii) elevation of miR-132-3p only in active TB (DR-TB and DS-TB) and iv) elevation of miR-150-5p in DR-TB alone. The directionality of functions mediated by monocyte miRNAs from Gene Set Enrichment Analysis (GSEA) facilitated two phenomenal findings: i) a bidirectional response between active disease (activation profile in DR-TB and DS-TB compared to LTB and HC) and latent infection (suppression profile in LTB vs HC) and ii) hyper immune activation in the DR-TB group compared to DS-TB.ConclusionThus, monocyte miRNA signatures provide pathological clues for altered monocyte function, drug resistance, and disease severity. Further studies on monocyte miRNAs may shed light on the immune regulatory mechanism for tuberculosis.

IntroductionMultisystem Inflammatory Syndrome in children (MIS-C) is a serious inflammatory sequela of SARS-CoV2 infection. The pathogenesis of MIS-C is vague and matrix metalloproteinases (MMPs) may have an important role. Matrix metalloproteinases (MMPs) are known drivers of lung pathology in many diseases.MethodsTo elucidate the role of MMPs in pathogenesis of pediatric COVID-19, we examined their plasma levels in MIS-C and acute COVID-19 children and compared them to convalescent COVID-19 and children with other common tropical diseases (with overlapping clinical manifestations).ResultsChildren with MIS-C had elevated levels of MMPs (P < 0.005 statistically significant) in comparison to acute COVID-19, other tropical diseases (Dengue fever, typhoid fever, and scrub typhus fever) and convalescent COVID-19 children. PCA and ROC analysis (sensitivity 84–100% and specificity 80–100%) showed that MMP-8, 12, 13 could help distinguish MIS-C from acute COVID-19 and other tropical diseases with high sensitivity and specificity. Among MIS-C children, elevated levels of MMPs were seen in children requiring intensive care unit admission as compared to children not needing intensive care. Similar findings were noted when children with severe/moderate COVID-19 were compared to children with mild COVID-19. Finally, MMP levels exhibited significant correlation with laboratory parameters, including lymphocyte counts, CRP, D-dimer, Ferritin and Sodium levels.DiscussionOur findings suggest that MMPs play a pivotal role in the pathogenesis of MIS-C and COVID-19 in children and may help distinguish MIS-C from other conditions with overlapping clinical presentation.

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.

Abstract Background Tuberculosis (TB) though primarily affects the lungs it may also affect the other parts of the body and referred as extra pulmonary (EPTB). This study is focused on understanding the genetic diversity and molecular epidemiology of Mycobacterium tuberculosis (M.tb) among tuberculous lymphadenitis (TBL), a form of EPTB patients identified in Chennai, Tamil Nadu. Methods The genetic diversity was identified by performing spoligotyping on the M.tb clinical isolates that were recovered from lymph node samples. A total of 71 M.tb isolates were recovered from extra pulmonary lymph node samples and subjected to Drug susceptibility testing and spoligotyping was carried out. In addition, immunological characterization from blood of same individuals from whom M.tb was isolated was carried out between the two major lineages groups East African Indian 3 (EAI3) and non-EAI3 strains by ELISA. The results of spoligotyping patterns were compared with the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). Results We found 41 spoligotype patterns and their associated lineages. Out of 41 spoligotype pattern, only 22 patterns are available in the spoldB4 database with Spoligotype international Type (SIT) number and remaining patterns were orphan strains without SIT number. The most predominant spoligotype lineage that was found in lymph node sample in this region of India was EAI (36), followed by central Asian strain (CAS) (6), T1 (5), Beijing (3), Latin American & Mediterranean (LAM) (2), U (1), X2 (1) and orphan (22). In addition to EAI, CAS and Beijing, our study identified the presence of orphan and unique spoligotyping patterns in Chennai region. We observed six drug resistant isolates. Out of six drug resistant isolates, four were resistant to isoniazid drug and associated with EAI family. Moreover, we observed increased levels of type 2 and type 17 cytokine profiles between EAI3 and non-EAI family, infected individuals. Conclusions The study confirms that EAI lineage to be the most predominant lineages in EPTB patients with lymphadenitis and were found to have increased type 1 and type 17 proinflammatory cytokine profiles.

Introduction: Differentiation between relapse and reinfection in cases with tuberculosis (TB) recurrence has important implications for public health, especially in patients with human immunodeficiency virus (HIV) co-infection. We compared Mycobacterial Interspersed Repeat Unit (MIRU) typing and spoligotyping with whole genome sequencing (WGS) to differentiate between relapse and reinfection in patients (HIV-positive and HIV-negative) with TB recurrence. We also assessed the value of WGS to track acquired drug resistance in those with relapse after successful treatment. Method: Forty-one paired M. tuberculosis isolates collected from 20 HIV-positive and 21 HIV-negative patients were subjected to WGS in addition to spoligotyping and MIRU typing. Phylogenetic and Single Nucleotide Substitution (SNP) clustering analyses were performed to determine whether recurrences were due to relapse or re-infection. Results: Comparison of M. tuberculosis genomes indicated that 95% of TB recurrences in the HIV-negative cohort were due to relapse, while the majority of TB recurrences (75%) in the HIV-positive cohort was due to reinfection (P = 0.0001). New drug resistance mutations were acquired in 5/24 cases (20.8%) that experienced relapse. Conclusions: WGS provided increased resolution, but differentiation between relapse and reinfection was broadly consistent with MIRU and spoligotyping. The high contribution of reinfection among HIV infected patients experiencing TB recurrence warrants further study to explore risk factors for TB exposure.

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013–2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB.

The end TB strategy reinforces the essentiality of readily accessible biomarkers for early tuberculosis diagnosis. Exploration of microRNA (miRNA) and pathway analysis opens an avenue for the discovery of possible therapeutic targets. miRNA is a small, non-coding oligonucleotide characterized by the mechanism of gene regulation, transcription, and immunomodulation. Studies on miRNA define their importance as an immune marker for active disease progression and as an immunomodulator for innate mechanisms, such as apoptosis and autophagy. Monocyte research is highly advancing toward TB pathogenesis and biomarker efficiency because of its innate and adaptive response connectivity. The combination of monocytes/macrophages and their relative miRNA expression furnish newer insight on the unresolved mechanism for Mycobacterium survival, exploitation of host defense, latent infection, and disease resistance. This review deals with miRNA from monocytes, their relative expression in different disease stages of TB, multiple gene regulating mechanisms in shaping immunity against tuberculosis, and their functionality as biomarker and host-mediated therapeutics. Future collaborative efforts involving multidisciplinary approach in various ethnic population with multiple factors (age, gender, mycobacterial strain, disease stage, other chronic lung infections, and inflammatory disease criteria) on these short miRNAs from body fluids and cells could predict the valuable miRNA biosignature network as a potent tool for biomarkers and host-directed therapy.

Geographically, most tuberculosis (TB) cases in 2018 were reported from India. This TB burden is compounded by MDR-TB and XDR-TB. The strategies for the management and control of TB in the community depend on an understanding of the mode of spread of the different strains of TB isolates in the community. To determine the distribution and trends of M. tb strains over the time period in the community due to treatment, we carried out the present study on changes over two decades. Design/Methods. A total of 1218 M. tb isolates (year: 2001–2018) from Tiruvallur, India, were genotyped by spoligotyping after DNA extraction and subjected to anti-TB drug susceptibility testing for the first-line anti-TB drugs. Results. On analysis with the SpolDB4 database, majority (2001–2003: 53.32% and 2015–2018: 46.3%) of the isolates belonged to East African Indian (EAI) lineage, and the orphans designated in comparison to SpolDB4 stood 33% among 2001–2003 strain collection and 46.3% among 2015–2018 strain collection. 10.2% (2001–2003) and 9.26% (2015 to 2018) of isolates were monoresistant to isoniazid (H). MDR strains were less common among EAI strains (3.2%) compared to non-EAI strains (10.32%). Conclusions. EAI is the most predominant lineage in Tiruvallur, despite the presence of highly transmissible lineages like Beijing for the last two decades. The prevalence of MDR-TB is below the national average of 2-3% among the new TB cases in the last two decades. The reason can be attributed to the well-established nature of the locally circulating strains in this region which are not associated with drug resistance.

Tuberculosis (TB), a bacterialinfectious disease caused by Mycobacterium tuberculosis (M.tb), which causes significant mortality in humans worldwide. Current treatment regimen involve the administration of multiple antibiotics over the course of several months that contributes to patient non-compliance leading to relapse and the development of drug-resistant M.tb (MDR and XDR) strains. Together, these facts highlight the need for the development of shorter TB treatment regimens. Host-directed therapy (HDT) is a new and emerging concept that aims to augment host immune response using drugs/compounds with or without adjunct antibiotics against M.tb infection. Autophagy is a natural catabolic mechanism of the cell that involves delivering the cytosolic constituents to the lysosomes for degradation and recycling the components; thereby maintaining the cellular and energy homoeostasis of a cell. However, over the past decade, an improved understanding of the role of autophagy in immunity has led to autophagy activation by using drugs or agents. This autophagy manipulation may represent a promising host-directed therapeutic strategy for human TB. However, current clinical knowledge on implementing autophagy activation by drugs or agents, as a stand-alone HDT or as an adjunct with antibiotics to treat human TB is insufficient. In recent years, many reports on high-throughput drug screening and measurement of autophagic flux by fluorescence, high-content microscopy, flow cytometry, microplate reader and immunoblotting have been published for the discovery of drugs that modulate autophagy. In this review, we discuss the commonly used chemical screening approaches in mammalian cells for the discovery of autophagy activating drugs against M.tbinfection. We also summarize the various autophagy-activating agents, both pre-clinical candidates and compounds approved for advanced clinical investigation during mycobacterial infection. Finally, we discuss the opportunities and challenges in using autophagy activation as HDT strategy to improve TB outcome and shorten treatment regimen.

Monocytes are critical defense components that play an important role in the primary innate immune response. The heterogeneous nature of monocytes and their ability to differentiate into either monocyte-derived macrophages or monocyte-derived dendritic cells allows them to serve as a bridge between the innate and adaptive immune responses. Current studies of monocytes based on immunofluorescence, single-cell RNA sequencing and whole mass spectrometry finger printing reveals different classification systems for monocyte subsets. In humans, three circulating monocyte subsets are classified based on relative expression levels of CD14 and CD16 surface proteins, namely classical, intermediate and non-classical subsets. Transcriptomic analyses of these subsets help to define their distinct functional properties. Tuberculosis (TB) is a disease instigated by the deadly pathogen Mycobacterium tuberculosis. Current research on monocytes in TB has indicated that there are alterations in the frequency of intermediate and non-classical subsets suggesting their impact in bacterial persistence. In this review, we will focus on these monocyte subsets, including their classification, frequency distribution, cytokine profiles, role as a biomarker and will comment on future directions for understanding the salient phenotypic and functional properties relevant to TB pathogenesis.