A systematic review and meta-analysis of circulating serum and plasma microRNAs in TB diagnosis.

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).

Whole Genome Sequencing Assessing Impact of Diabetes Mellitus on Tuberculosis Mutations and Type of Recurrence in India.

BACKGROUND: Evidence describing the impact of diabetes mellitus (DM) on the recurrence and mutation rate of Mycobacterium tuberculosis (Mtb) is limited. METHODS: This study was nested in 3 cohort studies of tuberculosis (TB) patients with and without DM in India. Paired Mtb isolates recovered at baseline and treatment failure/recurrence underwent whole genome sequencing. We compared acquisition of single-nucleotide polymorphisms (SNPs), TB drug resistance mutations, and type of recurrence (endogenous reactivation [<8 SNPs] or exogenous reinfection [≥8 SNPs]) by DM status. RESULTS: Of 1633 enrolled in the 3 parent cohorts, 236 (14.5%) had microbiologically confirmed TB treatment failure/recurrence; 76 Mtb isolate pairs were available for sequencing (22 in TB-DM and 54 in TB-only). The SNP acquisition rate was overall was 0.43 (95% confidence interval [CI], .25-.64) per 1 person-year (PY); 0.77 (95% CI, .40-1.35) per 1 PY, and 0.44 (95% CI, .19-.86) per 1 PY at treatment failure and recurrence, respectively. Significant difference in SNP rates by DM status was seen at recurrence (0.21 [95% CI, .04-.61]) per 1 PY for TB-only vs 1.28 (95% CI, .41-2.98) per 1 PY for TB-DM; P = .02). No significant difference in SNP rates by DM status was observed at treatment failure. Acquired TB drug resistance was seen in 4 of 18 (22%) in TB-DM vs 4 of 45 (9%) in TB-only (P = .21). Thirteen (17%) participants had exogenous reinfection; the reinfection rate at recurrence was 25% (3/12) for TB-DM vs 17% (4/24) in TB-only (P = .66). CONCLUSIONS: Considerable intrahost Mtb mutation rates were present at recurrence among patients with DM in India. One-fourth of patients with DM had exogenous reinfection at recurrence.

Autophagy Induction as a Host-Directed Therapeutic Strategy against Mycobacterium tuberculosis Infection.

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.

The recent trend in mycobacterial strain diversity among extra pulmonary lymph node tuberculosis and their association with drug resistance and the host immunological response in South India.

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.

Prevalence of SARS-CoV-2 infection in India: Findings from the national serosurvey, May-June 2020.

BACKGROUND & OBJECTIVES: Population-based seroepidemiological studies measure the extent of SARS-CoV-2 infection in a country. We report the findings of the first round of a national serosurvey, conducted to estimate the seroprevalence of SARS-CoV-2 infection among adult population of India. METHODS: From May 11 to June 4, 2020, a randomly sampled, community-based survey was conducted in 700 villages/wards, selected from the 70 districts of the 21 States of India, categorized into four strata based on the incidence of reported COVID-19 cases. Four hundred adults per district were enrolled from 10 clusters with one adult per household. Serum samples were tested for IgG antibodies using COVID Kavach ELISA kit. All positive serum samples were re-tested using Euroimmun SARS-CoV-2 ELISA. Adjusting for survey design and serial test performance, weighted seroprevalence, number of infections, infection to case ratio (ICR) and infection fatality ratio (IFR) were calculated. Logistic regression was used to determine the factors associated with IgG positivity. RESULTS: Total of 30,283 households were visited and 28,000 individuals were enrolled. Population-weighted seroprevalence after adjusting for test performance was 0.73 per cent [95% confidence interval (CI): 0.34-1.13]. Males, living in urban slums and occupation with high risk of exposure to potentially infected persons were associated with seropositivity. A cumulative 6,468,388 adult infections (95% CI: 3,829,029-11,199,423) were estimated in India by the early May. The overall ICR was between 81.6 (95% CI: 48.3-141.4) and 130.1 (95% CI: 77.0-225.2) with May 11 and May 3, 2020 as plausible reference points for reported cases. The IFR in the surveyed districts from high stratum, where death reporting was more robust, was 11.72 (95% CI: 7.21-19.19) to 15.04 (9.26-24.62) per 10,000 adults, using May 24 and June 1, 2020 as plausible reference points for reported deaths. INTERPRETATION & CONCLUSIONS: Seroprevalence of SARS-CoV-2 was low among the adult population in India around the beginning of May 2020. Further national and local serosurveys are recommended to better inform the public health strategy for containment and mitigation of the epidemic in various parts of the country.

Diverse Clinical Isolates of Mycobacterium tuberculosis Develop Macrophage-Induced Rifampin Tolerance.

The Mycobacterium tuberculosis lineage 4 strains CDC1551 and H37Rv develop tolerance to multiple antibiotics upon macrophage residence. To determine whether macrophage-induced tolerance is a general feature of clinical M. tuberculosis isolates, we assessed macrophage-induced drug tolerance in strains from lineages 1-3, representing the other predominant M. tuberculosis strains responsible for tuberculosis globally. All 3 lineages developed isoniazid tolerance. While lineage 1, 3, and 4 strains developed rifampin tolerance, lineage 2 Beijing strains did not. Their failure to develop tolerance may be explained by their harboring of a loss-of-function mutation in the Rv1258c efflux pump that is linked to macrophage-induced rifampicin tolerance.

Current trends in tuberculosis vaccine.

Despite the global efforts made to control tuberculosis (TB) and the large number of available new anti-TB drugs, TB still affects one-third of the world population. The conventional vaccine bacille Calmette-Guérin (BCG) shows varying efficacy in different populations, and there are safety issues in immunocompromised patients. Hence, there is an urgent requirement for a new and better TB vaccine candidate than BCG. There are several alternate vaccines available for TB such as DNA, subunit, adjuvant, and live-attenuated vaccines. Use of auxotrophic vaccine is an emerging technology. Newer vaccine technologies include vaccine delivery methods such as adenovirus- and cytomegalovirus (CMV)-based vector delivery, chimeric monoclonal antibody, single-chain fragment variable, RNA-lipoplexes, and nanoparticle-based technology. Based on its application, TB vaccines are classified as conventional, prophylactic, booster, therapeutic, and reinfection preventive vaccines. Currently, there are 12 vaccine candidates in clinical trials. In this review, we have briefly discussed about each of these vaccines in different phases of clinical trials. These vaccines should be analyzed further for developing a safe and more efficacious vaccine for TB.

A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis.

A clear understanding of the genetic basis of antibiotic resistance in Mycobacterium tuberculosis is required to accelerate the development of rapid drug susceptibility testing methods based on genetic sequence.Raw genotype-phenotype correlation data were extracted as part of a comprehensive systematic review to develop a standardised analytical approach for interpreting resistance associated mutations for rifampicin, isoniazid, ofloxacin/levofloxacin, moxifloxacin, amikacin, kanamycin, capreomycin, streptomycin, ethionamide/prothionamide and pyrazinamide. Mutation frequencies in resistant and susceptible isolates were calculated, together with novel statistical measures to classify mutations as high, moderate, minimal or indeterminate confidence for predicting resistance.We identified 286 confidence-graded mutations associated with resistance. Compared to phenotypic methods, sensitivity (95% CI) for rifampicin was 90.3% (89.6-90.9%), while for isoniazid it was 78.2% (77.4-79.0%) and their specificities were 96.3% (95.7-96.8%) and 94.4% (93.1-95.5%), respectively. For second-line drugs, sensitivity varied from 67.4% (64.1-70.6%) for capreomycin to 88.2% (85.1-90.9%) for moxifloxacin, with specificity ranging from 90.0% (87.1-92.5%) for moxifloxacin to 99.5% (99.0-99.8%) for amikacin.This study provides a standardised and comprehensive approach for the interpretation of mutations as predictors of M. tuberculosis drug-resistant phenotypes. These data have implications for the clinical interpretation of molecular diagnostics and next-generation sequencing as well as efficient individualised therapy for patients with drug-resistant tuberculosis.

Association of CYP27B1 gene polymorphisms with pulmonary tuberculosis and vitamin D levels.

BACKGROUND AND OBJECTIVES: Genetic factors are reported to be connected with tuberculosis (TB) infection. Studies have shown that genetic variations in genes involved in the vitamin D pathway influence the levels of vitamin D found in the bloodstream (serum). Cyp27b1 (1α-hydroxylase) is an enzyme that activates the synthesis of bioactive vitamin D3 by hydroxylation of 25(OH)D3.The in vitro studies reported rare gene variants of Cyp27b1 such as rs118204011 and rs118204012, associated with loss of Cyp27b1 function and lower serum vitamin D levels. Globally, a critical gap exists in understanding the link between these gene variants with TB and vitamin D levels. Hence, the study objective is to comprehend the association of Cyp27b1 rs118204009 (G/A), rs118204011 (C/T), and rs118204012 (A/G) with tuberculosis susceptibility/protection and to assess the influence of gene variants on vitamin D levels in both healthy controls (HCs) and those with pulmonary tuberculosis (PTB) in South India. METHODS: Genomic DNA extraction was performed by salting-out procedure and subsequently genotyped through polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method. Vitamin D level was measured by Enzyme-Linked Immunosorbent Assay (ELISA). RESULTS: In rs118204012 (A/G), a substantial association was found with PTB susceptibility in allele 'A' [Odds Ratio (OR): 1.52 (1.02-2.26); p = 0.044] and 'AA' genotype [OR: 1.69 (1.02-2.81); p = 0.040] through the dominant model. Allele 'G' [OR: 0.66 (0.44-0.98); p = 0.044) was found to be associated with protection against TB. Males were associated with increased susceptibility towards TB compared to females in the rs118204011 "CC" [OR: 3.94 (1.94-7.98); p = 0.002] and rs118204012 'AA' [OR: 4.57 (2.13-9.79); p = 0.0001] genotypes. Vitamin D insufficiency (<30 ng/ml) was more prevalent in PTB patients (66.67 %) with the rs118201012 'AA' genotype compared with healthy controls (57.14 %). This genotype was associated with disease susceptible odds ratio of 1.5. CONCLUSION: Cyp27b1 rs118204012 'AA' genotype was found to have association with vitamin D insufficiency and TB susceptibility. In terms of gender, our findings suggest that male individuals are correlated with a higher TB risk. This suggest that the gene variants may be involved in the downstream processing of serum Vitamin D levels and its association with the disease.

Elucidating the Immune Response to SARS-CoV-2: Natural Infection versus Covaxin/Covishield Vaccination in a South Indian Population.

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.

Sex-specific differences in systemic immune responses in MIS-C children.

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-1ß, 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.

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

Background: 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. Methods: We 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. Results: The 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. Conclusion: Thus, 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.

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

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.

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.

Multisystem inflammatory syndrome in children characterized by enhanced antigen-specific T-cell expression of cytokines and its reversal following recovery.

Background: Multisystem 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. Methods: We 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. Results: Children 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. Conclusion: Our study, therefore, provides evidence of enhanced activation of CD4+ and CD8+ T-cell responses in children with MIS and reversal following recovery.

CRISPR-Cas system and its use in the diagnosis of infectious diseases.

Rapid and accurate diagnostic methods for detecting pathogens are needed for effective management and treatment of infectious diseases. The conventional pathogen detection approach based on culture is considered the gold standard method, but needs several days to corroborate its results. Using nucleic acids from pathogens as detection targets has a considerable advantage in overcoming these time-consuming issues. The development of several molecular techniques has started to change the landscape of infectious disease diagnosis. However, these require expensive reagents, equipment, and sophisticated infrastructure, as well as highly trained workers. In this context, it is necessary to identify new diagnostic strategies to overcome these issues. Recently, CRISPR/Cas based diagnosis has revolutionized the area of molecular diagnostics of pathogenic diseases. In this review, we have discussed the different classes of CRISPR-Cas systems and their functions, and then focused on recent advances in CRISPR-based diagnosis technologies and the perspective of using this as a potential biosensing platform to detect infectious disease.

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.

Mycobacterium tuberculosis Lineages Associated with Mutations and Drug Resistance in Isolates from India.

Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. In this study, we performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand the genomic diversity. A retrospective collection of 498 M. tuberculosis isolates submitted to the National Institute for Research in Tuberculosis for phenotypic susceptibility testing between 2014 to 2016 were sequenced. Genotypic resistance prediction was performed using known resistance-conferring determinants. Genotypic and phenotypic results for 12 antituberculosis drugs were compared, and sequence data were explored to characterize lineages and their association with drug resistance. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction. IMPORTANCE Current knowledge on resistance-conferring determinants in Mycobacterium tuberculosis is biased toward globally dominant lineages 2 and 4. In contrast, lineages 1 and 3 are predominant in India. We performed whole-genome sequencing of 498 MDR M. tuberculosis isolates from India to determine the prevalence of drug resistance mutations and to understand genomic diversity. Four lineages were identified although lineage 1 predominated (43%). The sensitivity of prediction for isoniazid and rifampicin was 92% and 98%, respectively. We observed lineage-specific variations in the proportion of isolates with resistance-conferring mutations, with drug resistance more common in lineages 2 and 3. Disputed mutations (codons 430, 435, 445, and 452) in the rpoB gene were more common in isolates other than lineage 2. Phylogenetic analysis and pairwise SNP difference revealed high genetic relatedness of lineage 2 isolates. WGS based resistance prediction has huge potential, but knowledge of regional and national diversity is essential to achieve high accuracy for resistance prediction.

Role of matrix metalloproteinases in multi-system inflammatory syndrome and acute COVID-19 in children.

Introduction: Multisystem 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. Methods: To 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). Results: Children 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. Discussion: Our 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.

Monocyte and Macrophage miRNA: Potent Biomarker and Target for Host-Directed Therapy for Tuberculosis.

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.