Drug-Resistant Tuberculosis, Georgia, Kazakhstan, Kyrgyzstan, Moldova, and Ukraine, 2017-2022.

In 2021, the World Health Organization recommended new extensively drug-resistant (XDR) and pre-XDR tuberculosis (TB) definitions. In a recent cohort of TB patients in Eastern Europe, we show that XDR TB as currently defined is associated with exceptionally poor treatment outcomes, considerably worse than for the former definition (31% vs. 54% treatment success).

Benchmarking the empirical accuracy of short-read sequencing across the M. tuberculosis genome.

MOTIVATION: Short-read whole-genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences and sequencing bias reduces the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. To benchmark short-read variant calling, we used 36 diverse clinical Mycobacterium tuberculosis (Mtb) isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically studied the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias and GC content. RESULTS: Reference-based Illumina variant calling demonstrated a maximum recall of 89.0% and minimum precision of 98.5% across parameters evaluated. The approach that maximized variant recall while still maintaining high precision (<99%) was tuning the mapping quality filtering threshold, i.e. confidence of the read mapping (recall = 85.8%, precision = 99.1%, MQ ≥ 40). Additional masking of repetitive sequence content is an alternative conservative approach to variant calling that increases precision at cost to recall (recall = 70.2%, precision = 99.6%, MQ ≥ 40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52/168 PE/PPE genes (34.5%). From these results, we present a refined list of low confidence regions across the Mtb genome, which we found to frequently overlap with regions with structural variation, low sequence uniqueness and low sequencing coverage. Our benchmarking results have broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems and more generally for WGS applications in other organisms. AVAILABILITY AND IMPLEMENTATION: All relevant code is available at https://github.com/farhat-lab/mtb-illumina-wgs-evaluation. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A case report of persistent drug-sensitive pulmonary tuberculosis after treatment completion.

BACKGROUND: Mycobacterium tuberculosis (Mtb) has been found to persist within cavities in patients who have completed their anti-tuberculosis therapy. The clinical implications of Mtb persistence after therapy include recurrence of disease and destructive changes within the lungs. Data on residual changes in patients who completed anti-tuberculosis therapy are scarce. This case highlights the radiological and pathological changes that persist after anti-tuberculosis therapy completion and the importance of achieving sterilization of cavities in order to prevent these changes. CASE PRESENTATION: This is a case report of a 33 year old female with drug-sensitive pulmonary tuberculosis who despite successfully completing standard 6-month treatment had persistent changes in her lungs on radiological imaging. The patient underwent multiple adjunctive surgeries to resect cavitary lesions, which were culture positive for Mtb. After surgical treatment, the patient's chest radiographies improved, symptoms subsided, and she was given a definition of cure. CONCLUSIONS: Medical therapy alone, in the presence of severe cavitary lung lesions may not be able to achieve sterilizing cure in all cases. Cavities can not only cause reactivation but also drive inflammatory changes and subsequent lung damage leading to airflow obstruction, bronchiectasis, and fibrosis. Surgical removal of these foci of bacilli can be an effective adjunctive treatment necessary for a sterilizing cure and improved long term lung health.

Moxifloxacin target site concentrations in patients with pulmonary TB utilizing microdialysis: a clinical pharmacokinetic study.

Background: Moxifloxacin is a second-line anti-TB drug that is useful in the treatment of drug-resistant TB. However, little is known about its target site pharmacokinetics. Lower drug concentrations at the infection site (i.e. in severe lung lesions including cavitary lesions) may lead to development and amplification of drug resistance. Improved knowledge regarding tissue penetration of anti-TB drugs will help guide drug development and optimize drug dosing. Methods: Patients with culture-confirmed drug-resistant pulmonary TB scheduled to undergo adjunctive surgical lung resection were enrolled in Tbilisi, Georgia. Five serum samples per patient were collected at different timepoints including at the time of surgical resection (approximately at Tmax). Microdialysis was performed in the ex vivo tissue immediately after resection. Non-compartmental analysis was performed and a tissue/serum concentration ratio was calculated. Results: Among the seven patients enrolled, the median moxifloxacin dose given was 7.7 mg/kg, the median age was 25.2 years, 57% were male and the median creatinine clearance was 95.4 mL/min. Most patients (71%) had suboptimal steady-state serum Cmax (total drug) concentrations. The median free moxifloxacin serum concentration at time of surgical resection was 1.23 µg/mL (range = 0.12-1.80) and the median free lung tissue concentration was 3.37 µg/mL (range = 0.81-5.76). The median free-tissue/free-serum concentration ratio was 3.20 (range = 0.66-28.08). Conclusions: Moxifloxacin showed excellent penetration into diseased lung tissue (including cavitary lesions) among patients with pulmonary TB. Moxifloxacin lung tissue concentrations were higher than those seen in serum. Our findings highlight the importance of moxifloxacin in the treatment of MDR-TB and potentially any patient with pulmonary TB and severe lung lesions.

Lung Tissue Concentrations of Pyrazinamide among Patients with Drug-Resistant Pulmonary Tuberculosis.

Improved knowledge regarding the tissue penetration of antituberculosis drugs may help optimize drug management. Patients with drug-resistant pulmonary tuberculosis undergoing adjunctive surgery were enrolled. Serial serum samples were collected, and microdialysis was performed using ex vivo lung tissue to measure pyrazinamide concentrations. Among 10 patients, the median pyrazinamide dose was 24.7 mg/kg of body weight. Imaging revealed predominant lung lesions as cavitary (n = 6 patients), mass-like (n = 3 patients), or consolidative (n = 1 patient). On histopathology examination, all tissue samples had necrosis; eight had a pH of ≤5.5. Tissue samples from two patients were positive for Mycobacterium tuberculosis by culture (pH 5.5 and 7.2). All 10 patients had maximal serum pyrazinamide concentrations within the recommended range of 20 to 60 µg/ml. The median lung tissue free pyrazinamide concentration was 20.96 µg/ml. The median tissue-to-serum pyrazinamide concentration ratio was 0.77 (range, 0.54 to 0.93). There was a significant inverse correlation between tissue pyrazinamide concentrations and the amounts of necrosis (R = -0.66, P = 0.04) and acid-fast bacilli (R = -0.75, P = 0.01) identified by histopathology. We found good penetration of pyrazinamide into lung tissue among patients with pulmonary tuberculosis with a variety of radiological lesion types. Our tissue pH results revealed that most lesions had a pH conducive to pyrazinamide activity. The tissue penetration of pyrazinamide highlights its importance in both drug-susceptible and drug-resistant antituberculosis treatment regimens.

The TB Portals: an Open-Access, Web-Based Platform for Global Drug-Resistant-Tuberculosis Data Sharing and Analysis.

The TB Portals program is an international consortium of physicians, radiologists, and microbiologists from countries with a heavy burden of drug-resistant tuberculosis working with data scientists and information technology professionals. Together, we have built the TB Portals, a repository of socioeconomic/geographic, clinical, laboratory, radiological, and genomic data from patient cases of drug-resistant tuberculosis backed by shareable, physical samples. Currently, there are 1,299 total cases from five country sites (Azerbaijan, Belarus, Moldova, Georgia, and Romania), 976 (75.1%) of which are multidrug or extensively drug resistant and 38.2%, 51.9%, and 36.3% of which contain X-ray, computed tomography (CT) scan, and genomic data, respectively. The top Mycobacterium tuberculosis lineages represented among collected samples are Beijing, T1, and H3, and single nucleotide polymorphisms (SNPs) that confer resistance to isoniazid, rifampin, ofloxacin, and moxifloxacin occur the most frequently. These data and samples have promoted drug discovery efforts and research into genomics and quantitative image analysis to improve diagnostics while also serving as a valuable resource for researchers and clinical providers. The TB Portals database and associated projects are continually growing, and we invite new partners and collaborations to our initiative. The TB Portals data and their associated analytical and statistical tools are freely available at https://tbportals.niaid.nih.gov/.

Cavitary penetration of levofloxacin among patients with multidrug-resistant tuberculosis.

A better understanding of second-line drug (SLD) pharmacokinetics, including cavitary penetration, may help optimize SLD dosing. Patients with pulmonary multidrug-resistant tuberculosis (MDR-TB) undergoing adjunctive surgery were enrolled in Tbilisi, Georgia. Serum was obtained at 0, 1, 4, and 8 h and at the time of cavitary removal to measure levofloxacin concentrations. After surgery, microdialysis was performed using the ex vivo cavity, and levofloxacin concentrations in the collected dialysate fluid were measured. Noncompartmental analysis was performed, and a cavitary-to-serum levofloxacin concentration ratio was calculated. Twelve patients received levofloxacin for a median of 373 days before surgery (median dose, 11.8 mg/kg). The median levofloxacin concentration in serum (Cmax) was 6.5 µg/ml, and it was <2 µg/ml in 3 (25%) patients. Among 11 patients with complete data, the median cavitary concentration of levofloxacin was 4.36 µg/ml (range, 0.46 to 8.82). The median cavitary/serum levofloxacin ratio was 1.33 (range, 0.63 to 2.36), and 7 patients (64%) had a ratio of >1. There was a significant correlation between serum and cavitary concentrations (r = 0.71; P = 0.01). Levofloxacin had excellent penetration into chronic cavitary TB lesions, and there was a good correlation between serum and cavitary concentrations. Optimizing serum concentrations will help ensure optimal cavitary concentrations of levofloxacin, which may enhance treatment outcomes.

Genetic diversity within diagnostic sputum samples is mirrored in the culture of Mycobacterium tuberculosis across different settings.

Culturing and genomic sequencing of Mycobacterium tuberculosis (MTB) from tuberculosis (TB) cases is the basis for many research and clinical applications. The alternative, culture-free sequencing from diagnostic samples, is promising but poses challenges to obtain and analyse the MTB genome. Paradoxically, culture is assumed to impose a diversity bottleneck, which, if true, would entail unexplored consequences. To unravel this paradox we generate high-quality genomes of sputum-culture pairs from two different settings after developing a workflow for sequencing from sputum and a tailored bioinformatics analysis. Careful downstream comparisons reveal sources of sputum-culture incongruences due to false positive/negative variation associated with factors like low input MTB DNA or variable genomic depths. After accounting for these factors, contrary to the bottleneck dogma, we identify a 97% variant agreement within sputum-culture pairs, with a high correlation also in the variants' frequency (0.98). The combined analysis from five different settings and more than 100 available samples shows that our results can be extrapolated to different TB epidemic scenarios, demonstrating that for the cases tested culture accurately mirrors clinical samples.

SMA-TB: study protocol for the phase 2b randomized double-blind, placebo-controlled trial to estimate the potential efficacy and safety of two repurposed drugs, acetylsalicylic acid and ibuprofen, for use as adjunct therapy added to, and compared with, the standard WHO recommended TB regimen.

BACKGROUND: The duration and regimen of tuberculosis (TB) treatment is currently based predominantly on whether the M. tuberculosis (Mtb) strain is drug-sensitive (DS) or multidrug-resistant (MDR) with doses adjusted by patients' weight only. The systematic stratification of patients for personalized treatment does not exist for TB. As each TB case is different, individualized treatment regimens should be applied to obtain better outcomes. In this scenario, novel therapeutic approaches are urgently needed to (1) improve outcomes and (2) shorten treatment duration, and host-directed therapies (HDT) might be the best solution. Within HDT, repurposed drugs represent a shortcut in drug development and can be implemented at the short term. As hyperinflammation is associated with worse outcomes, HDT with an anti-inflammatory effect might improve outcomes by reducing tissue damage and thus the risk of permanent sequelae. METHODS: SMA-TB is a multicentre randomized, phase IIB, placebo-controlled, three-arm, double-blinded clinical trial (CT) that has been designed in the context of the EC-funded SMA-TB Project ( www.smatb.eu ) in which we propose to use 2 common non-steroidal anti-inflammatory drugs (NSAID), acetylsalicylic acid (ASA) and ibuprofen (Ibu), as an HDT for use as adjunct therapy added to, and compared with, the standard of care (SoC) World Health Organization (WHO)-recommended TB regimen in TB patients. A total of 354 South African and Georgian adults diagnosed with confirmed pulmonary TB will be randomized into SoC TB treatment + placebo, SoC + acetylsalicylic acid or SoC + ibuprofen. DISCUSSION: SMA-TB will provide proof of concept of the HDT as a co-adjuvant treatment and identify the suitability of the intervention for different population groups (different epidemiological settings and drug susceptibility) in the reduction of tissue damage and risk of bad outcomes for TB patients. This regimen potentially will be more effective and targeted: organ saving, reducing tissue damage and thereby decreasing the length of treatment and sequelae, increasing cure rates and pathogen clearance and decreasing transmission rates. It will result in better clinical practice, care management and increased well-being of TB patients. TRIAL REGISTRATION: Clinicaltrials.gov NCT04575519. Registered on October 5, 2020.

Additional drug resistance in Mycobacterium tuberculosis isolates from resected cavities among patients with multidrug-resistant or extensively drug-resistant pulmonary tuberculosis.

The pathogenesis of increasing drug resistance among patients with multidrug-resistant or extensively drug-resistant tuberculosis undergoing treatment is poorly understood. Increasing drug resistance found among Mycobacterium tuberculosis recovered from cavitary isolates compared with paired sputum isolates suggests pulmonary cavities may play a role in the development of worsening tuberculosis drug resistance.

Adjunctive surgery versus medical treatment among patients with cavitary multidrug-resistant tuberculosis.

OBJECTIVES: Surgical resection is recommended as adjunctive treatment for multidrug-resistant (MDR) tuberculosis (TB) in certain scenarios; however, data are limited. We sought to evaluate the impact of surgery by comparing TB outcomes among patients with cavitary disease who received medical versus combined medical and surgical treatment. METHODS: A cohort of all patients with cavitary MDR or extensively drug-resistant (XDR) TB treated in Tbilisi, Georgia, between 2008 and 2012. Patients meeting indications for surgery underwent adjunctive resection in addition to medical treatment. We compared TB outcomes (proportions achieving cure/complete) among patients who received adjunctive surgery to those who received medical treatment alone using an adjusted robust Poisson regression. RESULTS: Among 408 patients, 299 received medical treatment alone and 109 combined medical and surgical treatment. Patients in the non-surgical group were older and had higher rates of tobacco and alcohol use and bilateral disease compared to the surgical group. Patients in the surgical group had higher rates of XDR disease (28% vs 15%). Favourable outcomes were higher among the surgical versus non-surgical group cohort (76% vs 41%). After adjusting for multiple factors, the association between adjunctive resection and favourable outcome remained (adjusted risk ratio 1.6, 95% confidence interval 1.3-2.0); the relationship was also observed in secondary models that excluded patients with bilateral disease (contraindication for surgery) and patients receiving <6 months of treatment. Major postoperative complications occurred among 8 patients (7%) with no postoperative mortality. CONCLUSIONS: Adjunctive surgery is safe and may improve the effectiveness of treatment among select patients with cavitary MDR- and XDR-TB.

Genomic analyses of Mycobacterium tuberculosis from human lung resections reveal a high frequency of polyclonal infections.

Polyclonal infections occur when at least two unrelated strains of the same pathogen are detected in an individual. This has been linked to worse clinical outcomes in tuberculosis, as undetected strains with different antibiotic resistance profiles can lead to treatment failure. Here, we examine the amount of polyclonal infections in sputum and surgical resections from patients with tuberculosis in the country of Georgia. For this purpose, we sequence and analyse the genomes of Mycobacterium tuberculosis isolated from the samples, acquired through an observational clinical study (NCT02715271). Access to the lung enhanced the detection of multiple strains (40% of surgery cases) as opposed to just using a sputum sample (0-5% in the general population). We show that polyclonal infections often involve genetically distant strains and can be associated with reversion of the patient's drug susceptibility profile over time. In addition, we find different patterns of genetic diversity within lesions and across patients, including mutational signatures known to be associated with oxidative damage; this suggests that reactive oxygen species may be acting as a selective pressure in the granuloma environment. Our results support the idea that the magnitude of polyclonal infections in high-burden tuberculosis settings is underestimated when only testing sputum samples.

Impact of adjuvant therapeutic surgery on the health-related quality of life of pulmonary tuberculosis patients.

This study aimed to determine the health-related quality of life (HRQoL) of patients with pulmonary tuberculosis (TB) and to assess its change after a therapeutic surgical procedure. In this scenario, the purpose was to elucidate and quantify the effect of various demographic, epidemiological, clinical, surgical and psychosocial details on this variable. A prospective cohort of 40 patients undergoing therapeutic surgery for pulmonary TB (Study of Human Tuberculosis Lesions (SH-TBL) cohort) was recruited in Tbilisi, Georgia, between 2016 and 2018. HRQoL was assessed by administering the St George's Respiratory Questionnaire (SGRQ) and a novel psychosocial questionnaire, the BCN-Q, both at baseline and at 6 months post-surgery. A statistically and clinically significant improvement in the SGRQ total score was observed at follow-up, although it did not reach the values found for the healthy population. The differences between time points were statistically significant for the following groups: women, age <40 years, body mass index ≥20 kg·m-2, nonsmokers, drug-susceptible and drug-resistant participants, both new and relapsed patients, early culture negativisation, cases with a single lesion, either lesions <35 mm or ≥35 mm, and lesion, lobe and lung resections. The analysis of BCN-Q together with the SGRQ showed that several of its items, such as marital status, living conditions, nutrition, employment, external support, certain attitudes towards the healthcare system, emotional burden and sleep troubles, can impact HRQoL. These results highlight the benefit of adjuvant therapeutic surgery for pulmonary TB in selected patients in terms of HRQoL and suggest that a comprehensive approach including demographic, epidemiological, clinical and psychosocial variables may more accurately predict TB evolution and prognosis.

Type I IFN exacerbates disease in tuberculosis-susceptible mice by inducing neutrophil-mediated lung inflammation and NETosis.

Tuberculosis (TB) is a leading cause of mortality due to infectious disease, but the factors determining disease progression are unclear. Transcriptional signatures associated with type I IFN signalling and neutrophilic inflammation were shown to correlate with disease severity in mouse models of TB. Here we show that similar transcriptional signatures correlate with increased bacterial loads and exacerbate pathology during Mycobacterium tuberculosis infection upon GM-CSF blockade. Loss of GM-CSF signalling or genetic susceptibility to TB (C3HeB/FeJ mice) result in type I IFN-induced neutrophil extracellular trap (NET) formation that promotes bacterial growth and promotes disease severity. Consistently, NETs are present in necrotic lung lesions of TB patients responding poorly to antibiotic therapy, supporting the role of NETs in a late stage of TB pathogenesis. Our findings reveal an important cytokine-based innate immune effector network with a central role in determining the outcome of M. tuberculosis infection.

Deficiency in the glycosyltransferase Gcnt1 increases susceptibility to tuberculosis through a mechanism involving neutrophils.

Modulation of immunity and disease by glycans is increasingly recognized. However, how host glycosylation shapes and is shaped by tuberculosis remains poorly understood. We show that deficiency in the glucosaminyl (N-acetyl) transferase 1 (Gcnt1), a key enzyme for core-2 O-glycans biosynthesis, drives susceptibility to Mycobacterium tuberculosis infection. The increased susceptibility of Gcnt1 deficient mice was characterized by extensive lung immune pathology, mechanistically related to neutrophils. Uninfected Gcnt1 deficient mice presented bone marrow, blood and lung neutrophilia, which further increased with infection. Blood neutrophilia required Gcnt1 deficiency in the hematopoietic compartment, relating with enhanced granulopoiesis, but normal cellular egress from the bone marrow. Interestingly, for the blood neutrophilia to translate into susceptibility to M. tuberculosis infection, Gnct1 deficiency in the stroma was also necessary. Complete Gcnt1 deficiency associated with increased lung expression of the neutrophil chemoattractant CXCL2. Lastly, we demonstrate that the transcript levels of various glycosyltransferase-encoding genes were altered in whole blood of active tuberculosis patients and that sialyl Lewis x, a glycan widely present in human neutrophils, was detected in the lung of tuberculosis patients. Our findings reveal a previously unappreciated link between Gcnt1, neutrophilia and susceptibility to M. tuberculosis infection, uncovering new players balancing the immune response in tuberculosis.

Pulmonary function and respiratory health after successful treatment of drug-resistant tuberculosis.

BACKGROUND: Post-treatment morbidity among subjects with drug-resistant tuberculosis (DR-TB) is unclear. METHODS: This was a cross-sectional study of patients from Tbilisi, Georgia with cavitary DR-TB and an outcome of cure. Participants had a chest X-ray (CXR), St. George Respiratory Quality (SGRQ) survey, and pulmonary function tests (PFTs) performed. Correlations between SGRQ and PFT results and factors associated with pulmonary impairment were examined. RESULTS: Among 58 subjects (median age 31 years), 40% used tobacco, 59% had prior TB, and 47% underwent adjunctive surgical resection. The median follow-up time was 41 months. Follow-up CXR revealed fibrosis in 30 subjects (52%) and bronchiectasis in seven (12%). The median forced expiratory volume (FEV1)/forced vital capacity (FVC) ratio was 0.72, with 24 subjects (41%) having a ratio of ≤0.70. Significant correlations existed between PFT measures and overall and component SGRQ scores. In linear regression, age, prior TB, and CXR fibrosis or bronchiectasis were significantly associated with decreased pulmonary function. Adjunctive surgery was significantly associated with a higher percent predicted FEV1 and FVC. CONCLUSIONS: A high proportion of DR-TB subjects had residual pulmonary impairment, particularly with recurrent TB and severe radiological disease. The association of surgical resection with improved lung function deserves further study. PFTs and SGRQ may both be useful to evaluate lung health.

Retrospective study of clinical and lesion characteristics of patients undergoing surgical treatment for Pulmonary Tuberculosis in Georgia.

OBJECTIVES: Our aim was to retrospectively compare clinical data and characteristics of removed lesions of the cohort of patients undergoing therapeutical surgery for their tuberculosis. DESIGN AND METHODS: Demographic and epidemiological details, clinical data, data on the surgery performed, macroscopic characteristics of the TB lesions removed, and outcome were recorded retrospectively from the 137 patients who underwent therapeutical surgery for their TB in Tbilisi, Georgia during 2014 and 2015. RESULTS: Men represented 70% of the included patients, presented more comorbidities and underwent operation earlier in terms of days between diagnostic and surgery. Women underwent operation at younger ages, and in MDR/XDR-TB cases, showed higher percentages of sputum conversion at >2 months and of fresh necrosis in the surgical specimens, suggesting a worse evolution. Half of cases were MDR/XDR-TB cases. In spite of being considered microbiologically cured according to WHO, a non despricable percentage of cases showed viable bacilli in the surgical specimen. Even if no causality could be statistically demonstrated, differences could be encountered according to gender and drug susceptibility of the responsible strains. CONCLUSIONS: According to our results, host factors such as gender, type of necrosis found in the lesions, size of lesions and presence of viable bacilli in the surgical specimen, should be included in future studies on therapeutical surgery of TB. As most of studies are done in MDR/XDR-TB, more data on DS-TB operated cases are needed. Our results also highlight that, in spite of achieving the microbiologically cured status, sterilization might not occur, and thus new biomarkers and new methods to evaluate the healing process of TB patients are urgently needed and radiological assays should be taken into account.

Development of the food supplement Nyaditum resae as a new tool to reduce the risk of tuberculosis development.

Nyaditum resae (NR) is a galenic preparation of heat-killed Mycobacterium manresensis (hkMn). This is a new species that belongs to the Mycobacterium fortuitum complex, and it is present in drinking water-thus, regulatorily speaking, it is considered a food supplement. Preclinical studies in the murine model of active tuberculosis (TB) in the C3HeB/FeJ strain have demonstrated that daily administration of NR containing 103-106hkMn for 14days was able to stop the progression toward active TB [1]. The mechanism of action was linked to the induction of low dose tolerance and was related to the increase of Tuberculin Purified Protein Derivative (PPD) memory-specific Tregs (CD4+CD25+CD39+ cells) after ex vivo incubation of splenocytes for 7days. This increase of Tregs was related to the increase of interleukin (IL)-10 in the spleen and in the reduction of IL-17 in the lungs, where there was also a reduction in bacillary load and the pathology caused by a reduction of neutrophiles' infiltration [2]. Two randomized, double-blind placebo-controlled clinical trials (CTs) have been conducted in humans. The NYADATREG study (Clinicaltrials.gov identifier NCT02076139; 2013-2014) was aimed to evaluate the safety and the immunogenicity of two concentrations of NR (containing 104hkMn and 105hkMn) versus placebo (all administered orally everyday for 14days) in tuberculin-positive and tuberculin-negative volunteers (total n=51). The results demonstrated an excellent safety record, with no differences between groups in terms of adverse effects. A significant increase in PPD-specific memory regulatory T cells was also detected in both NR groups [3]. The NYADAPETRICS study (Clinicaltrials.gov identifier NCT02581579) is evaluating the safety and immunogenicity of NR 105hkMn (capsule format, orally) in the pediatric population. Currently, an efficacy study (randomized, double-blinded, placebo-controlled CT) is being conducted in Georgia. This NYADAGEORG trial includes close contacts of active TB cases with positive sputum not tributaries of chemoprophylaxis (<5-year-old children and HIV-positive individuals), which will receive NR (containing 105hkMn) or placebo (orally, every day for 14days). A total of 3300 participants will be recruited in four medical centers around Tbilissi. The participants are monitored by telephone for up to 2years to evaluate the incidence of active TB. The hypothesis is that the NR group will exhibit a 40% reduction in expected TB incidence. Thus, the anticipated TB incidence will be 3% in the NR group versus 5% in the placebo group. The CT is projected to end by 2021 (Clinicaltrials.gov identifier NCT02897180). The administration of the food supplement NR appears to be a new, easy, safe, and reliable method for reducing the risk of developing active TB, and new CTs must be encouraged to discern the particular efficacy power according to different population characteristics.