Linezolid Exposure Is Associated with Cytopenias in Patients Treated for Multidrug-Resistant Tuberculosis.

Although linezolid is effective for multidrug-resistant TB (MDR-TB) tuberculosis treatment, it is associated with cytopenias after 4 weeks of administration. Data on toxicities with long-term use of linezolid and drug pharmacodynamics in MDR-TB treatment are limited, and concerns about toxicity present barriers to wider implementation. This was a secondary analysis of a prospective cohort study of patients treated for MDR-TB in the country of Georgia from 2015 to 2017. Intensive blood sampling 4 to 6 weeks after treatment initiation with linezolid 600 mg daily was performed for pharmacokinetic (PK) analysis, including linezolid trough concentration (Cmin) and area under the curve from 0 to 24 hours (AUC0-24). Linezolid exposure was defined using literature-reported thresholds. Cytopenias were defined using an NIH adverse event (AE) scale. Logistic regression was used to evaluate the relationship between linezolid exposure and cytopenias. Among 76 patients receiving linezolid in their baseline treatment regimen and who had PK data available, cytopenia AEs occurred in 30 (39.5%) for an incidence rate of 46 per 100 person-years. The median duration of linezolid therapy was 526 days. No patients required dose reduction or interruption due to cytopenias. Median linezolid Cmin was 0.235 mg/L (interquartile range [IQR], 0.069 to 0.529), and median AUC0-24 was 89.6 mg·h/L (IQR, 69.2 to 116.2). Cytopenias were associated with linezolid PK parameters (Cmin > 2 mg/L and AUC0-24 > 160 mg·h/L). Cytopenias occurred frequently with long-term use of linezolid 600 mg/day and were associated with PK parameters but did not result in the need for treatment interruption in the management of a cohort of patients with MDR-TB.

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.

pncA Gene Mutations Associated with Pyrazinamide Resistance in Drug-Resistant Tuberculosis, South Africa and Georgia.

Although pyrazinamide is commonly used for tuberculosis treatment, drug-susceptibility testing is not routinely available. We found polymorphisms in the pncA gene for 70% of multidrug-resistant and 96% of extensively drug-resistant Mycobacterium tuberculosis isolates from South Africa and Georgia. Assessment of pyrazinamide susceptibility may be prudent before using it in regimens for drug-resistant tuberculosis.

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.

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.

High-throughput mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping for Mycobacterium tuberculosis epidemiological studies.

The emergence of drug-resistant forms of tuberculosis (TB) represents a major public health concern. Understanding the transmission routes of the disease is a key factor for its control and for the implementation of efficient interventions. Mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) marker typing is a well-described method for lineage identification and transmission tracking. However, the conventional manual genotyping technique is cumbersome and time-consuming and entails many risks for errors, thus hindering its implementation and dissemination. We describe here a new approach using the QIAxcel system, an automated high-throughput capillary electrophoresis system that also carries out allele calling. This automated method was assessed on 1,824 amplicons from 82 TB isolates and tested with sets of markers of 15 or 24 loci. Overall allele-calling concordance between the methods from 140 to 1,317 bp was 98.9%. DNA concentrations and repeatability and reproducibility performances showed no biases in allele calling. Furthermore, turnaround time using this automated system was reduced by 81% compared to the conventional manual agarose gel method. In sum, this new automated method facilitates MIRU-VNTR genotyping and provides reliable results. Therefore, it is well suited for field genotyping. The implementation of this method will help to achieve accurate and cost-effective epidemiological studies, especially in countries with a high prevalence of TB, where the high number of strains complicates the surveillance of circulating lineages and requires efficient interventions to be carried out in an urgent manner.

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates.

BACKGROUND: Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains. RESULTS: To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal. CONCLUSION: Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.

Investigating resistance in clinical Mycobacterium tuberculosis complex isolates with genomic and phenotypic antimicrobial susceptibility testing: a multicentre observational study.

BACKGROUND: Whole-genome sequencing (WGS) of Mycobacterium tuberculosis complex has become an important tool in diagnosis and management of drug-resistant tuberculosis. However, data correlating resistance genotype with quantitative phenotypic antimicrobial susceptibility testing (AST) are scarce. METHODS: In a prospective multicentre observational study, 900 clinical M tuberculosis complex isolates were collected from adults with drug-resistant tuberculosis in five high-endemic tuberculosis settings around the world (Georgia, Moldova, Peru, South Africa, and Viet Nam) between Dec 5, 2014, and Dec 12, 2017. Minimum inhibitory concentrations (MICs) and resulting binary phenotypic AST results for up to nine antituberculosis drugs were determined and correlated with resistance-conferring mutations identified by WGS. FINDINGS: Considering WHO-endorsed critical concentrations as reference, WGS had high accuracy for prediction of resistance to isoniazid (sensitivity 98·8% [95% CI 98·5-99·0]; specificity 96·6% [95% CI 95·2-97·9]), levofloxacin (sensitivity 94·8% [93·3-97·6]; specificity 97·1% [96·7-97·6]), kanamycin (sensitivity 96·1% [95·4-96·8]; specificity 95·0% [94·4-95·7]), amikacin (sensitivity 97·2% [96·4-98·1]; specificity 98·6% [98·3-98·9]), and capreomycin (sensitivity 93·1% [90·0-96·3]; specificity 98·3% [98·0-98·7]). For rifampicin, pyrazinamide, and ethambutol, the specificity of resistance prediction was suboptimal (64·0% [61·0-67·1], 83·8% [81·0-86·5], and 40·1% [37·4-42·9], respectively). Specificity for rifampicin increased to 83·9% when borderline mutations with MICs overlapping with the critical concentration were excluded. Consequently, we highlighted mutations in M tuberculosis complex isolates that are often falsely identified as susceptible by phenotypic AST, and we identified potential novel resistance-conferring mutations. INTERPRETATION: The combined analysis of mutations and quantitative phenotypes shows the potential of WGS to produce a refined interpretation of resistance, which is needed for individualised therapy, and eventually could allow differential drug dosing. However, variability of MIC data for some M tuberculosis complex isolates carrying identical mutations also reveals limitations of our understanding of the genotype and phenotype relationships (eg, including epistasis and strain genetic background). FUNDING: Bill & Melinda Gates Foundation, German Centre for Infection Research, German Research Foundation, Excellence Cluster Precision Medicine of Inflammation (EXC 2167), and Leibniz ScienceCampus EvoLUNG.

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.

Xpert MTB/RIF Ultra for detection of Mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study.

BACKGROUND: The Xpert MTB/RIF assay is an automated molecular test that has improved the detection of tuberculosis and rifampicin resistance, but its sensitivity is inadequate in patients with paucibacillary disease or HIV. Xpert MTB/RIF Ultra (Xpert Ultra) was developed to overcome this limitation. We compared the diagnostic performance of Xpert Ultra with that of Xpert for detection of tuberculosis and rifampicin resistance. METHODS: In this prospective, multicentre, diagnostic accuracy study, we recruited adults with pulmonary tuberculosis symptoms presenting at primary health-care centres and hospitals in eight countries (South Africa, Uganda, Kenya, India, China, Georgia, Belarus, and Brazil). Participants were allocated to the case detection group if no drugs had been taken for tuberculosis in the past 6 months or to the multidrug-resistance risk group if drugs for tuberculosis had been taken in the past 6 months, but drug resistance was suspected. Demographic information, medical history, chest imaging results, and HIV test results were recorded at enrolment, and each participant gave at least three sputum specimen on 2 separate days. Xpert and Xpert Ultra diagnostic performance in the same sputum specimen was compared with culture tests and drug susceptibility testing as reference standards. The primary objectives were to estimate and compare the sensitivity of Xpert Ultra test with that of Xpert for detection of smear-negative tuberculosis and rifampicin resistance and to estimate and compare Xpert Ultra and Xpert specificities for detection of rifampicin resistance. Study participants in the case detection group were included in all analyses, whereas participants in the multidrug-resistance risk group were only included in analyses of rifampicin-resistance detection. FINDINGS: Between Feb 18, and Dec 24, 2016, we enrolled 2368 participants for sputum sampling. 248 participants were excluded from the analysis, and 1753 participants were distributed to the case detection group (n=1439) and the multidrug-resistance risk group (n=314). Sensitivities of Xpert Ultra and Xpert were 63% and 46%, respectively, for the 137 participants with smear-negative and culture-positive sputum (difference of 17%, 95% CI 10 to 24); 90% and 77%, respectively, for the 115 HIV-positive participants with culture-positive sputum (13%, 6·4 to 21); and 88% and 83%, respectively, across all 462 participants with culture-positive sputum (5·4%, 3·3 to 8·0). Specificities of Xpert Ultra and Xpert for case detection were 96% and 98% (-2·7%, -3·9 to -1·7) overall, and 93% and 98% for patients with a history of tuberculosis. Xpert Ultra and Xpert performed similarly in detecting rifampicin resistance. INTERPRETATION: For tuberculosis case detection, sensitivity of Xpert Ultra was superior to that of Xpert in patients with paucibacillary disease and in patients with HIV. However, this increase in sensitivity came at the expense of a decrease in specificity. FUNDING: Government of Netherlands, Government of Australia, Bill & Melinda Gates Foundation, Government of the UK, and the National Institute of Allergy and Infectious Diseases.

Peptide Epitalon activates chromatin at the old age.

UNLABELLED: OBJECTIVES and design. We have studied the effect of synthetic peptide Epitalon on the activity of ribosomal genes, denaturation parameters of total heterochromatin, polymorphism of structural C-heterochromatin and the variability of facultative heterochromatin in cultured lymphocytes of persons aged 76-80 years. RESULTS: The obtained data demonstrate that Epitalon induces the activation of ribosomal genes, decondensation of pericentromeric structural heterochromatin and the release of genes repressed due to the age-related condensation of euchromatic chromosome regions. CONCLUSIONS: Epitalon has shown its ability to activate chromatin by modifying heterochromatin and heterochromatinized chromosome regions in the cells of older persons.

Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain G-12-005.

Infection caused by drug-resistant Mycobacterium tuberculosis is a growing concern, especially in eastern Europe. We report an annotated draft genome sequence of M. tuberculosis strain G-12-005 obtained from a patient in Georgia.

Use of a molecular diagnostic test in AFB smear positive tuberculosis suspects greatly reduces time to detection of multidrug resistant tuberculosis.

BACKGROUND: The WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB). There are limited data on the performance and impact of these tests in field settings. METHODS: The performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST) using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program. RESULTS: Among 500 AFB smear-positive sputum specimens, 458 (91.6%) had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH) resistance directly from the sputum specimen in 159 (89.8%) of 177 specimens and MDR-TB in 109 (95.6%) of 114 specimens compared to conventional methods. There was high agreement between the MTBDRplus assay and conventional DST results in detecting MDR-TB (kappa = 0.95, p<0.01). The most prevalent INH resistance mutation was S315T (78%) in the katG codon and the most common rifampicin resistance mutation was S531L (68%) in the rpoB codon. Among 13 specimens from TB suspects with negative sputum cultures, 7 had a positive MTBDRplus assay (3 with MDR-TB). The time to detection of MDR-TB was significantly less using the MTBDRplus assay (4.2 days) compared to the use of standard phenotypic tests (67.3 days with solid media and 21.6 days with broth-based media). CONCLUSIONS: Compared to conventional methods, the MTBDRplus assay had high accuracy and significantly reduced time to detection of MDR-TB in an area with high MDR-TB prevalence. The use of rapid molecular diagnostic tests for TB and drug resistance should increase the proportion of patients promptly placed on appropriate therapy.

Combined species identification, genotyping, and drug resistance detection of Mycobacterium tuberculosis cultures by MLPA on a bead-based array.

The population structure of Mycobacterium tuberculosis is typically clonal therefore genotypic lineages can be unequivocally identified by characteristic markers such as mutations or genomic deletions. In addition, drug resistance is mainly mediated by mutations. These issues make multiplexed detection of selected mutations potentially a very powerful tool to characterise Mycobacterium tuberculosis. We used Multiplex Ligation-dependent Probe Amplification (MLPA) to screen for dispersed mutations, which can be successfully applied to Mycobacterium tuberculosis as was previously shown. Here we selected 47 discriminative and informative markers and designed MLPA probes accordingly to allow analysis with a liquid bead array and robust reader (Luminex MAGPIX technology). To validate the bead-based MLPA, we screened a panel of 88 selected strains, previously characterised by other methods with the developed multiplex assay using automated positive and negative calling. In total 3059 characteristics were screened and 3034 (99.2%) were consistent with previous molecular characterizations, of which 2056 (67.2%) were directly supported by other molecular methods, and 978 (32.0%) were consistent with but not directly supported by previous molecular characterizations. Results directly conflicting or inconsistent with previous methods, were obtained for 25 (0.8%) of the characteristics tested. Here we report the validation of the bead-based MLPA and demonstrate its potential to simultaneously identify a range of drug resistance markers, discriminate the species within the Mycobacterium tuberculosis complex, determine the genetic lineage and detect and identify the clinically most relevant non-tuberculous mycobacterial species. The detection of multiple genetic markers in clinically derived Mycobacterium tuberculosis strains with a multiplex assay could reduce the number of TB-dedicated screening methods needed for full characterization. Additionally, as a proportion of the markers screened are specific to certain Mycobacterium tuberculosis lineages each profile can be checked for internal consistency. Strain characterization can allow selection of appropriate treatment and thereby improve treatment outcome and patient management.

Bioregulator Vilon-induced reactivation of chromatin in cultured lymphocytes from old people.

The effect of the synthetic peptide bioregulator Vilon on structural and facultative heterochromatin of cultured lymphocytes from old people has been studied. The data obtained indicate that Vilon (a) induces unrolling (deheterochromatinization) of total heterochromatin; (b) activates synthetic processes caused by the reactivation of ribosomal genes as a result of deheterochromatinization of nucleolus organizer regions; (c) releases the genes repressed due to the condensation of euchromatic regions forming facultative heterochromatin; (d) does not induce decondensation of pericentromeric structural heterochromatin. Our results indicate that Vilon causes progressive activation (deheterochromatinization) of the facultative heterochromatin with increased aging.