Absolute Concentration Method Research Articles

Molecular characteristics of fluoroquinolone-resistant <i>Mycobacterium tuberculosis</i> strains from newly diagnosed tuberculosis patients in the Northwest of Russia

Introduction. Fluoroquinolones remain the key second-line anti-tuberculosis drugs. The aim of the study was the molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis strains from newly diagnosed tuberculosis patients in the Northwest of the Russian Federation. Materials and methods. The retrospective study collection included M. tuberculosis isolates isolated in 2015–2019 from previously untreated tuberculosis patients. Susceptibility to antituberculosis drugs (including the fluoroquinolone ofloxacin) was determined using the BACTEC MGIT960 or absolute concentration method. Mutations in the gyrA gene as a marker of resistance to fluoroquinolones, were detected by real-time PCR. Beijing genotype and its subtypes were detected by PCR and real-time PCR methods. Non-Beijing strains were spoligotyped. Results and discussion. Phenotypic resistance to ofloxacin was detected in 6.7% (40/599) of strains and in 17.4% (40/230) of MDR strains. 34 of 40 (85%) ofloxacin-resistant strains belonged to the Beijing genotype. 18 (45%) strains were assigned to the Russian epidemic subtype Beijing B0/W148 and 12 (30%) to Beijing Central Asian/Russian. The remaining 6 ofloxacin-resistant strains belonged to the Euro-American phylogenetic lineage. Mutations in the gyrA gene were found in 97.5% (39/40) of strains. The most common were mutations in codon 94 (69.2%, 27/39). The Asp94Gly substitution was identified in 57.5% (23/40) of ofloxacin-resistant strains and was dominant among Beijing (19/34) and non-Beijing (4/6) strains. The second most common substitution was Ala90Val (25%, 10/40). More than half of the ofloxacin-resistant strains, Beijing B0/W148 (10/18) and Central Asian/Russian (7/12), carried the Asp94Gly mutation. Conclusion. In the Northwest of Russia in 2016-2019, primary resistance of M. tuberculosis to fluoroquinolones was 6.7% in the total collection and 17.4% of MDR strains, and was mainly caused by the gyrA Asp94Gly and Ala90Val mutations. Beijing B0/W148 genotype was characterized by the largest proportion of fluoroquinolone-resistant strains.

The problem of formation of new risks of medicinal resistance of patients with tuberculosis in condition of active use of antibiotics by population under COVID-19

In pandemic conditions, situation of active and uncontrolled use by population of antimicrobial preparations treating COVID-19 occurs. So, new risks of development of medication resistance among patients with various infectious diseases, tuberculosis included, appear. The purpose of the study is to characterize prevalence of antimicrobial preparations use by population in relationship with development of medication resistance in patients with tuberculosis during COVID-19 pandemic. Material and methods. The analysis of sales of antimicrobial medicines was implemented on the basis of published official data from the joint-stock company DSM Group presenting monthly audit of the Russian pharmaceutical market. The determination of primary antibiotic resistance was carried out in 2018-2020 on 3312 patients with tuberculosis. The modified method of proportions on liquid nutrient medium in system with automated accounting of microorganisms growth, the method of absolute concentrations and the method of polymerase chain reaction with real-time detection were applied. The results of the study. It was established that the most demanding antimicrobial medications among population were ceftriaxone, azithromycin, levofloxacin, moxifloxacin, azithromycin. At the same time, the maximum increase in sales in 2020 up to 150% as compared with of 2019 was determined in medications derived from quinolone moxifloxacin, levofloxacin, which began to be used in treatment of coronavirus infection. At the same time, these medications are traditionally used in tuberculosis treatment. But in 2020, alarming trend was established that limits treatment of tuberculosis patients. The primary resistance of mycobacteria was also established in newly diagnosed tuberculosis patients, also for the same antimicrobial medications of quinolone derivatives, and increasing in proportion of patients with primary medication resistance to levofloxacin, moxifloxacin in 2020 as compared to 2018 was 189-480%. At the same time, increasing of resistance to other antibiotics made up to 60.8% on average. Conclusion. The study results imply alarming scenario of medication resistance shifts towards very virulent and highly medication-resistant genotypes. This trend can result in conditions of successful transmission of deadly medication-resistant mutants that can seriously undermine effectiveness of implemented programs of struggle with tuberculosis worldwide.

Performance Evaluation of the BACTEC MGIT 960 System for Rifampin Drug-Susceptibility Testing of Mycobacterium tuberculosis Using the Current WHO Critical Concentration.

The World Health Organization recently lowered the rifampin (RIF) critical concentration (CC) for drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) using the mycobacterial growth indicator tube (MGIT) 960 system. Here, we evaluated the diagnostic performance of the MGIT system with the revised CC for determining MTBC RIF resistance with 303 clinical MTBC isolates, including 122 isolates with rpoB mutations, of which 32 had single borderline-resistance mutations, and 181 wild-type rpoB isolates. The phenotypic RIF resistance was determined via the absolute concentration method (AC) and via MGIT using both previous (1 mg/L) and revised (0.5 mg/L) CCs for the latter method. The diagnostic accuracy of each phenotypic DST (pDST) was assessed based on rpoB genotyping as the reference standard. The overall sensitivity of the AC was 95.1% (95% confidence interval [CI], 89.6 to 98.2%), while the MGIT results with previous and revised CCs were 82.0% (95% CI 74.0 to 88.3%) and 83.6% (95% CI 75.8 to 89.7%), respectively. The 32 MTBC isolates with single borderline-resistance mutations showed a wide range of MICs, and sensitivity was not significantly increased by reducing the MGIT CC. All 181 wild-type rpoB isolates were RIF-susceptible in the AC and with MGIT using the previous CC, whereas 1 isolate was misclassified as RIF-resistant with the revised CC. Our results demonstrate that the overall diagnostic performances of the MGIT DST with the revised RIF CC and previous CC were comparable. A further large-scale study is required to demonstrate the optimal RIF CC for MGIT.

Comparison of Different Methods for Drug Susceptibility Testing of Mycobacterium tuberculosis to Rifampicin

The objective: to compare results of drug susceptibility testing to rifampicin by molecular genetic methods and phenotypic tests of Mycobacterium tuberculosis isolates obtained from clinical specimens of tuberculosis patients.Subjects and Methods. 915 samples of M. tuberculosis DNA and 426 cultures were used in this study. Genotypic tests (TB-TEST (BIOCIP-IMB, Russia), GenoType MTBDRplusV2) and phenotypic technologies (absolute concentration method, Bactec MGIT 960 system, Sensititre Myco TB kit) were used.Results. A high percentage (98.7%; CI 97.7-99.7%) of confirmation of the results of the molecular genetic test (TB-TEST) by the phenotypic test (absolute concentration method) was demonstrated. In some cases, the Bactec MGIT 960 system as well as the absolute concentration method were shown to produce false negative results of rifampicin resistance in some cases.

Clinical characteristics and drug resistance profile of 276 non-tuberculosis mycobacterium pulmonary disease patients

Objective To investigate and analyze the clinical characteristics, pathogen species composition and drug sensitivity to first-line anti-tuberculosis drugs of patients with non-tuberculosis mycobacterium (NTM) pulmonary disease from Nanyang, Henan province, and provide evidence to improve the diagnosis and treatment of NTM pulmonary disease. Methods A total of 276 patients with NTM pulmonary disease (all living in Nanyang) who visited the Sixth People's Hospital of Nanyang City from January 2015 to December 2019 were enrolled retrospectively. The baseline data, clinical symptoms, imaging manifestations and other information of the patients were collected, PCR reverse dot blot hybridization was used to identify NTM strains, and absolute concentration method was used for drug sensitivity test. SPSS18.0 was used for statistical analysis. Results From 2015 to 2019, the identification rates of NTM patients in suspected tuberculosis (TB) patients were 1.05%, 1.11%, 1.23%, 1.54% and 1.70%, respectively. Among the 276 NTM patients, men accounted for 61.59% (170/276), and the average age of the patients at onset was（51.21 ± 8.33）years. The main clinical manifestations were cough, expectoration, chest tightness, shortness of breath or anhelation. The most common imaging manifestations were pleural thickening (53.62%, 148/276), bronchiectasis (42.39%, 117/276) and cavity (40.58%, 112/276). Eight NTM strains were identified, and the top four strains were Mycobacterium intracellular (32.61%, 90/276), Mycobacterium kansaii (23.91%, 66/276), Mycobacterium abscessus (21.38%, 59/276) and Mycobacterium avium (14.49%, 40/276). The drug resistance rates of the four major NTM to isoniazid, rifampicin, ethambutol, streptomycin, kanamycin and ofloxacin were 92.55% (236/255), 83.53% (213/255), 54.51% (139/255), 89.02% (227/255), 78.82% (201/255) and 64.31% (164/255), respectively. In NTM subspecies, M. aeruginosa was more likely to cause symptoms such as fever, chest tightness, shortness of breath or anhelation and imaging findings such as bronchiectasis and cavity compared with M. avium. Conclusion The identification rate of NTM pulmonary disease in Nanyang increased year by year. The clinical characteristics were similar to those of patients with pulmonary TB. Close attention should be paid to the patients with risk factors and complications in clinical practice, and the imaging manifestations and bacteriological characteristic should be differentiated. The common NTM strains had high resistance to the first-line anti-TB drugs, so the treatment plan should be standardized.

ИЗУЧЕНИЕ РЕЗИСТЕНТНОСТИ К ПРОТИВОТУБЕРКУЛЕЗНЫМ СРЕДСТВАМ У БОЛЬНЫХ, НАХОДЯЩИХСЯ НА СТАЦИОНАРНОМ ЛЕЧЕНИИ В ОТДЕЛЕНИИ РЕЗИСТЕТНЫХ ФОРМ ТУБЕРКУЛЕЗА ЛЕГКИХ

A comparative study of resistance to anti-tuberculosis drugs in patients with newly diagnosed pulmonary tuberculosis and in patients with chronic forms of tuberculosis who were treated in an anti-tuberculosis hospital was carried out. We examined 142 patients treated in the department of resistant forms of tuberculosis. A complete bacteriological study of sputum for Mycobacterium tuberculosis was carried out with the determination of sensitivity to anti-tuberculosis drugs during the cultivation of microorganisms on a liquid nutrient medium in a system with automated registration of bacterial growth and on a dense nutrient medium of Lowenstein-Jensen using the method of absolute concentrations. Methods of PCR analysis were also used. According to the anamnesis, two groups of patients were formed: 82 patients with newly diagnosed tuberculosis and 60 patients with a chronic course of the disease or with a relapse of the disease. According to the age structure, patients under the age of 50 prevailed in both groups. At the same time, in the first group there was a significant number of patients aged 18-29 years - 20.7%. In the second group, the number of patients aged 18-29 years was significantly less - 5%. Despite the fact that all patients were assigned to the category of multidrug-resistant pathogen and were treated in the unit of resistant forms of tuberculosis, it was important to assess the level of Mycobacterium tuberculosis resistance separately for patients in each of the selected groups. The largest differences between groups were found in the assessment of second-line drug resistance. Patients of the second group showed high resistance to fluoroquinolones - 30% and amikacin - 41.6%. In patients with newly diagnosed pulmonary tuberculosis (first group), resistance to fluoroquinolones was 9.7%, and to amikacin - 17.1%. Capreomycin was the most effective in both groups. The resistance of the causative agent of tuberculosis in patients of the first group was 4.9%, in the second - 11.6%.

Neurotransmitter Release of Reprogrammed Cells Using Electrochemical Detection Methods.

The detection of neurotransmitter release from reprogrammed human cell is an important demonstration of their functionality. Electrochemistry has the distinct advantages over alternative methods that it allows for the measuring of the analyte of interest at a high temporal resolution. This is necessary for fast events, such as neurotransmitter release and reuptake, which happen in the order of milliseconds to seconds. The precise description of these kinetic events can lead to insights into the function of cells in health and disease and allows for the exploration of events that might be missed using methods that look at absolute concentration values or methods that have a slower sampling rate. In the present chapter, we describe the use of constant potential amperometry and enzyme-coated multielectrode arrays for the detection of glutamate in vitro. These biosensors have the distinct advantage of "self-referencing," a method providing high selectivity while retaining outstanding temporal resolution. Here, we provide a step-by-step user guide for a commercially available system and its application for in vitro systems such as reprogrammed cells.

Resazurin assay for rapid drug susceptibility testing of Mycobacterium tuberculosis

Introduction: Tuberculosis, a disease of antiquity continues to cause high morbidity and mortality. In Sustainable Development Goals and End Tb Strategy proposed by WHO includes Drug Susceptibility Testing as major component. Conventional methods are time consuming and molecular methods are rapid but expensive and demanding. Resazurin assay is a rapid colorimetric redox indicator method of Drug Susceptibility Testing. Materials and Methods: 67 strains of M. tuberculosis isolated from pulmonary tuberculosis patients were tested using indirect absolute concentration method by resazurin assay and compared with drug incorporated Lowenstein – Jensen media for Isoniazid and Rifamycin. Results: There was 91.5 % and 91.7% concordance between both methods for INH and Rifamycin respectively. Resazurin assay for INH was 100% sensitive and 61.54% specific. Sensitivity and specificity for RMP was 98.04% and 62.5% respectively. The duration required for INH was 7.0 ± 2.07 and for RMP 6.58 ± 1.58 days. Conclusions: Resazurin assay is sensitive, rapid, cheap, technically simple drug susceptibility method. Keywords: M. tuberculosis, resazurin assay, drug susceptibility.

Genetic polymorphism and phenotypic resistance of Mycobacterium tuberculosis to ofloxacin and moxifloxacin

The objective: to study the phenotypic sensitivity of Mycobacterium tuberculosis (MTB) to fluoroquinolones, ofloxacin and moxifloxacin and correlate it with MTB genome mutations associated with resistance to fluoroquinolones to determine the possibility to use these data when compiling chemotherapy regimens IV and V. Subjects and methods. The study included 108 multiple drug resistant Mycobacterium tuberculosis cultures obtained in 2018-2019 from specimens of tuberculosis patients from three regions of Russia. To test drug susceptibility of the isolated cultures, standard methods of the modified method of proportions on a liquid medium by Bactec MGIT 960 were used. Genetic determinants of multiple and extensive drug resistance of tuberculosis mycobacteria were detected using the test system of TB-TEST, OOO Biochip-IMB. Results. Of 66 cultures resistant to ofloxacin, 26 (39.4%) were found to be sensitive to moxifloxacin, while 40 (60.6%) were resistant. Thus, the interpolation of data of ofloxacin drug susceptibility testing to moxifloxacin is unjustified, and using the absolute concentration method as the only phenotypic drug susceptibility testing method in modern laboratories is unacceptable. It was demonstrated that the high level of resistance to moxifloxacin (0.5 μg/ml) was mainly due to the presence of the gyrAD94G, gyrAD94N, and gyrAD94H substitutions in the genome. It was suggested to revise the existing concentrations for moxifloxacin during phenotypic drug susceptibility testing.

Experience of using TB test kit for the rapid drug susceptibility testing of M. tuberculosis.

The results of the comparative testing of the susceptibility of M. tuberculosis clinical strains to isoniazid, streptomycin, rifampicin and ethambutol using the TB test kit, developed in SCRAMB, (Obolensk) and the absolute concentrations method; the TB test kit and the BACTEC MGIT 960 automated system are presented in the study. A total of 629 and 220 strains, respectively, were tested. A high degree of agreement of the results was shown: 89.1-98.6% for isoniazid, 96.2-98.0% for rifampicin, 91.5-98.2% for streptomycin and 89.1-95.9% for ethambutol. The smallest number of discrepancies in the results was obtained when comparing the TB test kit and BACTEC MGIT 960. The discrepant results analysis was performed by the proportion method, PCR sequencing, or re-testing on new lots of the TB test kit and Lowenstein-Jensen medium with anti-tuberculosis drugs, after which the sensitivity, the specificity and the efficiency of the TB test kit have exceeded 95 % for all anti-tuberculosis drugs. The turnaround time with the TB test kit (median 9.25-9.9 days, ranged from 8 to13 days) was significantly shorter than that with the absolute concentration method (median 21-23 days, ranged from 20 to 28 days) and is commensurate with the turnaround time with BACTEC MGIT 960 (average 7.2 days, ranged from 5 to 12 days). The TB test kit is easy to use, does not require expensive equipment and special staff training.

Clinical Validation of the QMAC-DST System for Testing the Drug Susceptibility of Mycobacterium tuberculosis to First- and Second-Line Drugs.

There is a high demand for novel approaches to counter the various challenges of conventional drug susceptibility testing (DST) for tuberculosis, the most prevalent infectious disease with significant global mortality. The QMAC-DST system was recently developed for rapid DST using image technology to track the growth of single cells of Mycobacterium tuberculosis (MTB). The purpose of this study was to clinically validate the QMAC-DST system compared to conventional DST. In total, 178 MTB isolates recovered from clinical specimens in Asan Medical Center in 2016 were tested by both QMAC-DST and absolute concentration methods using Lowenstein-Jensen media (LJ-DST). Among the isolates, 156 were subjected to DST using BACTEC MGIT 960 SIRE kits (BD, Sparks, MD, United States) (MGIT-DST). The susceptibility/resistance results obtained by QMAC-DST were read against 13 drugs after 7 days of incubation and compared with those of LJ-DST. Based on the gold standard LJ-DST, the agreement rates of QMAC-DST for all drugs were 97.8%, 97.9%, and 97.8% among susceptible, resistant, and total isolates, respectively, while the overall agreement of MGIT-DST tested for 156 isolates against first-line drugs was 95.5%. QMAC-DST showed the highest major error of 6.4% for rifampin, however, it could be corrected by a revised threshold of growth since false-resistant isolates showed grew only half than the true-resistant isolates. The rapid and accurate performance of QMAC-DST warrants ideal phenotypic DST for a wide range of first-line and second-line drugs.

Measuring Protein Concentration by Diffusion-Filtered Quantitative Nuclear Magnetic Resonance Spectroscopy.

The concentration of macromolecules in solution is a crucial property in many areas of research, including the development and commercialization of biological therapeutics. For proteins in particular, none of the reported methods for measuring concentration detect a molecular property that is known a priori; rather, they rely on ligand binding, degradation and derivitization, or an intrinsic property that must be determined experimentally. The purpose of this report is to describe (1) a diffusion-filtered qNMR experiment (DF-qNMR) for quantitating macromolecules in complex matrices and (2) an overall method for measuring absolute protein concentration based on this DF-qNMR experiment. This method combines protein denaturation with the diffusion filter to produce clean spectra of the protein with well-resolved resonances, regardless of the matrix complexity. The concentration is then obtained by comparing the peak area of the valine/isoleucine/leucine methyl groups to an external, certified, small-molecule quantitation standard. The method, which is referred to as VILMHA (valine isoleucine leucine methyl hydrogen analysis), was tested on three proteins of various sizes. In all cases, the measured concentration was within 1.8% of the labeled value for the undiluted standard reference material evaluated. In addition, the RSD's were less than 1.25% in all cases and less than 1% in most cases. The accuracy, precision, and ease of use make this method superior to existing absolute protein concentration methods. Furthermore, VILMHA is ideally suited to serve as the basis for converting the relative protein concentration methods into absolute methods or establishing molecular-specific parameters. Finally, DF-qNMR has the potential to quantitate other types of macromolecules (e.g., such as polymers, surfactants, etc.) in the presence of small-molecule contaminants.

Advantages of the AdvanSure MDR-TB GenoBlot assay containing disputed rpoB mutation-specific probes in a routine clinical laboratory setting.

The AdvanSure MDR-TB GenoBlot Assay detects isoniazid- and rifampin-resistant tuberculosis using mutation-specific probes, including probes to disputed rpoB mutations. The aim of this study was to evaluate the clinical usefulness of molecular drug susceptibility testing (DST) using the AdvanSure assay with weekly batch testing in routine clinical laboratory settings in a country with an intermediate tuberculosis burden. The AdvanSure assay was evaluated against an absolute concentration (AC) method and the Mycobacterial Growth Indicator Tube (MGIT) 960 System, which are phenotypic DST methods, using 496 Mycobacterium tuberculosis (MTB) isolates. We retrospectively reviewed and compared DST results and turnaround times (TATs), the time intervals from MTB culture identification to final reporting, for these methods. For rifampin, the AdvanSure assay showed 99.2% (492/496) concordance with both the AC and MGIT methods. AdvanSure also detected an rpoB mutation (D516Y) conferring low-level resistance in three isolates categorized as rifampin-susceptible by both phenotypic DST methods. For isoniazid, AdvanSure concordance rates with the AC method and MGIT DST were 96.6% (479/496) and 95.4% (473/496), respectively. The median TAT of AdvanSure in weekly batch testing was 5.8 days, shorter than the times for the phenotypic DST methods, which were 35.1 days for the AC method and 8.9 days for MGIT DST. AdvanSure shows promising clinical usefulness for rapid detection of rifampin- and/or isoniazid-resistant tuberculosis when used as a complementary method to phenotypic DST assays in weekly batch testing. Furthermore, MTB isolates with disputed mutations for rifampin resistance were detectable by the AdvanSure assay.

A rapid culture system uninfluenced by an inoculum effect increases reliability and convenience for drug susceptibility testing of Mycobacterium tuberculosis

The Disc Agarose Channel (DAC) system utilizes microfluidics and imaging technologies and is fully automated and capable of tracking single cell growth to produce Mycobacterium tuberculosis (MTB) drug susceptibility testing (DST) results within 3~7 days. In particular, this system can be easily used to perform DSTs without the fastidious preparation of the inoculum of MTB cells. Inoculum effect is one of the major problems that causes DST errors. The DAC system was not influenced by the inoculum effect and produced reliable DST results. In this system, the minimum inhibitory concentration (MIC) values of the first-line drugs were consistent regardless of inoculum sizes ranging from ~103 to ~108 CFU/mL. The consistent MIC results enabled us to determine the critical concentrations for 12 anti-tuberculosis drugs. Based on the determined critical concentrations, further DSTs were performed with 254 MTB clinical isolates without measuring an inoculum size. There were high agreement rates (96.3%) between the DAC system and the absolute concentration method using Löwenstein-Jensen medium. According to these results, the DAC system is the first DST system that is not affected by the inoculum effect. It can thus increase reliability and convenience for DST of MTB. We expect that this system will be a potential substitute for conventional DST systems.

Mutations of rpoB, katG, inhA and ahp genes in rifampicin and isoniazid-resistant Mycobacterium tuberculosis in Kyrgyz Republic

BackgroundThe aim of this study was to identify mutations of rpoB, katG, inhA and ahp-genes associated Mycobacterium tuberculosis resistance to rifampicin (RIF) and isoniazid (INH) in Kyrgyz Republic. We studied 633 smear samples from the primary pulmonary tuberculosis (TB) patients. We verified Mycobacterium tuberculosis susceptibility to RIF and INH using culture method of absolute concentrations, and commercially available test named “TB-BIOCHIP” (Biochip-IMB, Moscow, Russian Federation).ResultsFor RIF-resistance, TB-BIOCHIP’s sensitivity and specificity were 88% and 97%, 84% and 95% for INH-resistance, and 90% and 97% for multi-drug resistance (MDR). In RIF-resistant strains, TB-BIOCHIP showed mutations in codons 531 (64.8%), 526 (17.3%), 516 (8.1%), 511 (5.4%), 533 (3.2%), 522 (0.6%) and 513 (0.6%) of rpoB gene. The most prevalent was Ser531 > Leu mutation (63.7%). 91.2% of mutations entailing resistance to INH were in katG gene, 7% in inhA gene, and 1.8% in ahpC gene. Ser315→Thr (88.6%) was the most prevalent mutation leading to resistance to INH.ConclusionsIn Kyrgyz Republic, the most prevalent mutation in RIF-resistant strains was Ser531 → Leu in rpoB gene, as opposed to Ser315 → Thr in katG gene in INH-resistant Mycobacterium tuberculosis. In Kyrgyz Republic, the major reservoir of MDR Mycobacterium tuberculosis were strains with combined mutations Ser531 → Leu in rpoB gene and Ser315 → Thr in katG gene. TB-BIOCHIP has shown moderate sensitivity with the advantage of obtaining results in only two days.

In Vitro Susceptibility of Mycobacterium tuberculosis to Amikacin, Kanamycin, and Capreomycin.

Amikacin, kanamycin, and capreomycin are among the most important second-line drugs for multidrug-resistant tuberculosis. Although amikacin and kanamycin are administered at the same dose and show the same pharmacokinetics, they have different WHO breakpoints, suggesting that the two drugs have different MICs. The aim of this study was to investigate possible differences in MICs between the aminoglycosides and capreomycin. Using the direct concentration method, a range of concentrations of amikacin, kanamycin, and capreomycin (0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0, and 64.0 mg/liter) were tested against 57 clinical Mycobacterium tuberculosis strains. The 7H10 agar plates were examined for mycobacterial growth after 14 days. At 2 mg/liter, 48 strains (84%) were inhibited by amikacin and only 5 strains (9%) were inhibited by kanamycin (P < 0.05, Wilcoxon signed-rank test). The median MICs of amikacin, kanamycin, and capreomycin were 2, 4, and 8 mg/liter, respectively. No difference in amikacin, kanamycin, and capreomycin MIC distributions was observed between multidrug-resistant strains and fully susceptible strains. The results indicate that amikacin is more active than kanamycin and capreomycin against M. tuberculosis with the absolute concentration method. Determination of the impact of this difference on clinical outcomes in daily practice requires a prospective study, including pharmacokinetic and pharmacodynamic evaluations.

Direct Resazurin Microplate Assay in Drug Susceptibility Testing of Smear-Positive Sputum Samples against Mycobacterium tuberculosis.

BackgroundA new direct microplate-based colorimetric drug susceptibility test that omits the initial isolation of Mycobacterium tuberculosis from sputum specimens was evaluated.MethodsA total of 51 M. tuberculosis acid fast bacilli (AFB) smear-positive sputum specimens were inoculated directly into drug-free and serial dilutions of drug-containing Middlebrook 7H9 broth media. With this direct resazurin micro plate assay (REMA) method, resazurin dye was used as a growth indicator in microplate wells. The minimum inhibitory concentrations (MIC) of isoniazid (INH) and rifampicin (RIF) were compared with those of the ‘gold standard’ absolute concentration method (ACM). The turnaround time (TAT) of the direct REMA and the ACM were also determined.ResultsAt the selected cut-off points (INH: 0.0625 μg/mL; RIF: 0.125 μg/mL), good drug susceptibility test results were obtained for INH and RIF with an average sensitivity, specificity and accuracy of 90%, 100% and 97%, respectively, with a TAT of 15 days. The REMA method also correctly classified the resistant isolates with positive predictive values of 95% and negative predictive values of 98% for the two drugs.ConclusionsThe direct REMA was reliable in routine diagnostic laboratories for the drug susceptibility testing of M. tuberculosis and the rapid detection of multi-drug-resistant tuberculosis.

Antibacterial effect of anti-tuberculosis decoction against clinical isolated mycobacterium tuberculosis in vitro

Objective To study the antibacterial activity of anti-tuberculosis decoction against clinical isolated rifampicin drug-resistant mycobacterium tuberculosis (number: A) and rifampicin sensitive mycobacterium tuberculosis (number: B) in vitro. Methods Rifampicin was selected as a positive control medicine.Anti-tuberculosis decoction and rifampicin were respectively used to test the bacteria through absolute concentration method and the direct effect method, then recorded and analyzed the observation phenomenon. Results The MIC of anti-tuberculosis decoction for A, B was 64.00mg/mL by using the absolute concentration method, and the MIC of anti-tuberculosis decoction for A, B was 8.00mg/mL by using direct effect method. Conclusion Anti-tuberculosis decoction had a good antibacterial activity for rifampicin resistant mycobacterium tuberculosis and rifampicin sensitive mycobacterium tuberculosis in vitro. Key words: Anti-Tuberculosis decoction; Rifampicin; Mycobacterium tuberculosis; Antibacterial

LUCIFERASE REPORTER MYCOBACTERIOPHAGES FOR EVALUATING NORBORNENE-BASED ANTITUBERCULOSIS DRUG SUSCEPTIBILITY TESTING ON MYCOBACTERIUM TUBERCULOSIS

Objective: In 2015, 9.6 million people around the world became sick with tuberculosis (TB) disease and 1.5 million TB-related deaths worldwide. Recent increasing incidence of multidrug-resistant (MDR; resistance to at least rifampicin (RIF) and isoniazid [INH]) and extensively drug-resistant (MDR resistance plus resistance to a fluoroquinolone and an aminoglycoside) makes TB a serious concern. Lot of research is needed to deal with this infectious disease for a better alternative in treatment or modification of these older TB drugs. The present study aimed at evaluating antimycobacterial activity of norbornene (NOR) derived INH copolymer with poly ethylene glycol (NOR- polyethylene glycol [PEG]-INH) a novel nanocarrier along with the anti-TB drug using luciferase reporter phages (LRPs).Methods: NOR derived INH accounts for 74% of INH, 24% of NOR, and 2% of PEG. H37Rv control strain, a sensitive, and a resistant strain of Mycobacterium TB (MTB) used in this study. The in vitro activity of the drug was evaluated using absolute concentration method. The resistant strain was evaluated using LRP assay to observe the minimum inhibitory concentration of the drug.Results: Reduction in light units was observed for the resistant strain exposed to plain INH and NOR-PEG-INH separately. 24% of reduction was observed in strains exposed to plain INH whereas 37% of reduction was observed in strains exposed to NOR-PEG-INH.Conclusion: NOR-based INH had better antimycobacterial activity compared to plain INH and RIF. Antimycobacterial activity of INH and RIF increases even with very low dosage with NOR conjugate.

Use of DNA microarray chips for the rapid detection of Mycobacterium tuberculosis resistance to rifampicin and isoniazid.

The objective of the present study was to evaluate the potential development of DNA microarray chips to detect rifampicin (RFP) and isoniazid (INH) resistance in Mycobacterium tuberculosis (MTB), using samples from clinical tuberculosis (TB) patients in Soochow City, China. The sputum samples of 42 patients with TB in the Affiliated Hospital of Infectious Diseases of Soochow University (Soochow, China) were collected. The conventional Lowenstein-Jensen culture medium (Gold Standard) was used to assess drug sensitivity using the absolute concentration method. GeeDom MTB drug detection kits were also used to create a DNA microarray chip and examine the RFP-resistance associated gene mutation points rpoB-RRDR 511, 513, 516, 526, 531 and 533, and the INH-resistance associated gene mutation points katG315 and inhA-15 of the sputum samples. Compared with the results from the absolute concentration method, the susceptibility and specificity of RFP sensitivity detected by the DNA microarray chip were 92.8 and 93.8%, respectively. The susceptibility and specificity of INH sensitivity detected were 66.7 and 81%, respectively. The rpoB-RRDR 526, 531 mutations were the primary causes of MTB RFP resistance and the katG315 mutation was the primary cause of INH resistance. The detection of rpoB and katG gene mutation points by a DNA microarray chip may be used as a rapid, accurate and bulk clinical detection method for RFP and INH resistance in MTB. This is very valuable for the control of TB epidemics.