Drug susceptibility testing of Mycobacterium tuberculosis using next generation sequencing and Mykrobe software

Introduction. Mycobacterium tuberculosis is the causative agent of tuberculosis. Drug susceptibility testing is performed by phenotypic and molecular tests. Commonly used for phenotypic drug susceptibility testing is the automated BACTEC system in a liquid culture medium. Drug susceptibility by line probe molecular tests was introduced almost 15 years ago. Recently whole genome sequencing (WGS) analysis of M. tuberculosis strains demonstrated that genotyping of drug-resistance could be accurately performed. Several software tools were developed.Our study aimed to perform whole-genome sequencing on phenotypically confirmed multi-drug resistant (MDR) M. tuberculosis strains, to identify drug-resistant mutations and to compare whole-genome sequencing profiles with line probe assay and phenotypic results.Materials and methods. We performed analysis on 34 MDR M. tuberculosis Bulgarian strains. Phenotypic drug susceptibility testing was performed on the BACTEC system. For molecular testing of drug susceptibility to first- and second-line tuberculostatics, we applied line probe assay Geno Type MTBDR plus v.1.0 и Geno Type MTBDR sl v.1.0. Sequencing was performed on MiSeq. Generated FASTQ files were analyzed for known drugresistant mutations with the software platform Mykrobe v.0.8.1.Results. All three methods — phenotypic analysis using the BACTEC system, genetic analysis of strains applying the Geno Type test and Mykrobe software gave comparable sensitivity/resistance results for the studied strains. All phenotypically proven rifampicin and isoniazid-resistant strains were 100% confirmed using Mykrobe software. The C-15T mutation is a marker for isoniazid resistance in strains of the SIT41 spoligotype. We observed a 75% (21/28) agreement between BACTEC and Mykrobe for ethambutol resistance. Phenotypically, 87% (n = 27) of the strains are resistant to streptomycin, but only 59% (n = 19) are proven by Mykrobe software. Comparing phenotypic and genotypic resistance to ofloxacin, amikacin and kanamycin, we observed 100% coincidence of results.Conclusions. Whole-genome sequencing approach is relatively expensive and laborious but useful for detailed analysis such as epidemiological genotyping and molecular drug susceptibility testing.

Bacteriological and molecular characterization of Mycobacterium bovis isolates from tuberculous lesions collected among slaughtered cattle, Northwest Ethiopia

Background In Ethiopia, the distribution of bovine tuberculosis (BTB) has long been known and documented as a major problem of animal health. However, the burden of circulating M. bovis strains is poorly understood in the country. Therefore; this study aimed to identify and characterize the mycobacterial isolates responsible for BTB in Northwest Ethiopia. Methods A cross-sectional study was conducted on tuberculous lesions that had been collected from slaughtered cattle between September 2018 to June 2019. Collected lesions were cultured and tested for tuberculous bacilli. The MPT64 assay and Genotype line probe assay (LPA) were used for identification of mycobacterial isolates, and region of deletion 4 (RD4) typing and spoligotyping were used to characterize the M. bovis strains. Results Of the total 1458 examined slaughtered cattle, only 62 (4.3, 95%CI; 0.0328–0.0542) had tuberculous lesions. The highest number of gross tuberculous lesions were observed from the lymph nodes of the thoracic cavity; at the mediastinal (40.3%, 25/62) and bronchial (22.6%, 14/62) lymph nodes. Of the 62 collected tuberculous lesions; 18 (29.0%) were culture positive for mycobacterium isolates, and only five isolates were confirmed for M. tuberculosis complex (MTBc) by the MPT64 assay and LPA. All the five MTBc isolates were positive for RD4 typing of M. bovis with a PCR product size of 446 bp, and no isolate was noticed to have M. tuberculosis. The detected M. bovis strains displayed five spoligotypes; with the common SB1176 and SB0133 M. bovis strains, although the two spoligotypes had not been previously reported. Conclusion The present study shows that BTB in North Gondar, Ethiopia, is caused by M. bovis strains SB1176 and SB0033, with low frequency. Thus, the finding highlights the importance of continuous surveillance for mycobacterial strains in cattle populations.

Effectiveness of GenoType MTBDRsl in excluding TB drug resistance in a clinical trial

OBJECTIVES: To assess the performance of the GenoType MTBDRsl v1, a line-probe assay (LPA), to exclude baseline resistance to fluoroquinolones (FQs) and second-line injectables (SLIs) in the Standard Treatment Regimen of Anti-tuberculosis Drugs for Patients With MDR-TB 1 (STREAM 1) trial.METHODS: Direct sputum MTBDRsl results in the site laboratories were compared to indirect phenotypic drug susceptibility testing (pDST) results in the central laboratory, with DNA sequencing as a reference standard.RESULTS: Of 413 multidrug-resistant TB (MDR-TB) patients tested using MTBDRsl and pDST, 389 (94.2%) were FQ-susceptible and 7 (1.7%) FQ-resistant, while 17 (4.1%) had an inconclusive MTBDRsl result. For SLI, 372 (90.1%) were susceptible, 5 (1.2%) resistant and 36 (8.7%) inconclusive. There were 9 (2.3%) FQ discordant pDST/MTBDRsl results, of which 3 revealed a mutation and 5 (1.3%) SLI discordant pDST/MTBDRsl results, none of which were mutants on sequencing. Among the 17 FQ- and SLI MTBDRsl-inconclusive samples, sequencing showed 1 FQ- and zero SLI-resistant results, similar to frequencies among the conclusive MTBDRsl. The majority of inconclusive MTBDRsl results were associated with low bacillary load samples (acid-fast bacilli smear-negative or scantily positive) compared to conclusive results (P < 0.001).CONCLUSION: MTBDRsl can facilitate the rapid exclusion of FQ and SLI resistances for enrolment in clinical trials.

Recent Advances in Drug Susceptible Pulmonary Tuberculosis Diagnosis and Treatment

The most important thing for the management of drug susceptible pulmonary tuberculosis is to diagnose active pulmonary tuberculosis as soon as possible and prevent the occurrence of new patients through appropriate treatment. Therefore, it should be a priority to quickly detect tuberculosis mycobacterium and quickly exclude drug-resistant tuberculosis before treatment begins. To this end, recent guidelines recommend the general use of Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) tests, Xpert MTB/RIF tests, and rapid sensitivity tests through line probe assay (LPA). In addition, if the results of the test are positive, it is important to establish an in-hospital reporting system so that rapid reporting can be made. The treatment principle for drug susceptible pulmonary tuberculosis is 2 months of initial intensive phase (isoniazid, rifampin, ethambutol, pyrazinamide) followed by 4 months of maintenance phase (isoniazid, rifampin). Despite global efforts to shorten the duration of the treatment, the treatment of drug susceptible pulmonary tuberculosis has not changed for more than 35 years, and problems such as increased side effects and reduced drug adherence are serious obstacles to tuberculosis management. Therefore, efforts have been steadily made to shorten the treatment period through the combination of new drugs worldwide, and after many failures, they are finally paying off. A recently published Study 31/A5349 study found that 4 months short-term regimen using rifapentine (RPT) and moxifloxacin (MFX) demonstrated non-inferiority in existing standard regimen, as the result, a revision of World Health Organization guidelines is scheduled that 4 months short-term regimen using RPT and MFX may be an alternative. However, it is unlikely that RPT/MFX 4 months short-term regimen will be applied immediately in Korea because the use of RPT is currently limited in Korea due to the high frequency of side effects.

Early detection of Pre-XDR TB with line probe assay in a high TB burden country

Background: Worldwide, tuberculosis (TB) is one of the top 10 causes of death. Drug resistant tuberculosis has lately become a major public health problem that threatens progress made in Tuberculosis (TB) care and control worldwide. The aim of this study was to determine the prevalence of Pre-extensive drug resistant TB among MDR TB in North Central of Nigeria. Methods: This study was conducted from October, 2018 to August, 2019 with 150 samples. In Nigeria, guidelines for DR-TB as recommended by WHO is followed. All the samples from the patients who gave their consent were transported to a zonal reference TB laboratory (ZRL). Results: Mean age was 38.6 ± 13.4 years with peak age at 35-44. Out of these 103 samples processed with LPA, 101(98%) were rifampicin resistant and 2 were rifampicin sensitive, 99(96%) were INH resistant and 4 (4%) were INH sensitive, 5(5%) were fluoroquinolone resistant, 98(95%) were fluoroquinolone sensitive, 12 (12%) were Aminoglycoside + Capreomycin resistant, 91(83%) were Aminoglycoside + Capreomycin sensitive. Conclusion: Multidrug resistant TB and its severe forms (Pre-extensive & extensively drug resistant TB) can be detected early with rapid tool- Line Probe Assay rapid and prevented timely by early initiation on treatment. Keywords: Pre-XDR TB; line probe assay in a high TB burden country.

Comparison of Line Probe Assay (LPA) and Mycobacterium Growth Indicator Tubes (MGIT) Assay for Second-line TB Drug Susceptibility Testing

BACKGROUND: Tuberculosis (TB) infection is one of the most prominent health issues in the world, including in Indonesia. TB is evolving into multidrug-resistant tuberculosis (MDR-TB) and requiring second-line TB drugs. Mycobacterium growth indicator tube (MGIT) is the gold standard for susceptibility testing of second-line TB drugs. Alternatively, line probe assay (LPA), which detects genes resistant to second-line TB drugs, takes a shorter time to run. This study aims to compare MGIT and LPA's ability to detect TB resistance to second-line TB drugs and observe mutation patterns of genes encoding second-line TB drugs.METHODS: This was an observational analytic study, using cross-sectional method. The data were acquired from the MDR-TB clinic’s medical records at the Dr. Hasan Sadikin Hospital from September to December 2019. LPA and MGIT test were conducted at the Health Laboratory Hall of West Java Province, then tested using Kolmogorov-Smirnov and chi-square statistic.RESULTS: From 121 subjects, 113 people were not resistant to the second-line TB drugs, which was examined using both LPA and MGIT (93.4%), p=0.991. Mutations were found in gyrA and rrs gene. There was no significant difference between the proportion of subjects resistant to the second-line of TB drugs tested using LPA and MGIT.CONCLUSION: LPA is an alternative method to MGIT because it requires a shorter time and reduces the risk of exposure that will improve MDR-TB patients management.KEYWORDS: line probe assay (LPA), multidrug-resistant TB, mycobacterium growth indicator tube (MGIT), second-line TB drugs

Estimation of genotype MTBDRPLUS line probe assay in detection of rifampicin and isoniazid resistance in comparison to liquid culture (BACTEC-960) drug susceptibility testing in a tertiary care hospital from Eastern India

Introduction and Aim: India has the uppermost trouble of Multidrug resistant tuberculosis (MDR-TB) is a major challenge controlling resistance, reducing transmission and improving handling outcomes in MDR-TB patients is dependent on susceptibility testing. Isoniazid (INH) and rifampicin (Rif) are the key first-line antituberculosis drugs, and resistance to these drugs i.e., MDR-TB, is likely to result in treatment failure and poor clinical outcomes. The present study was done to compare the performance of line probe assay test (GenoType® MTBDRplus) with liquid culture (MGIT 960) system for the detection of resistance to first-line drugs. Materials and Methods: We estimate the performance of LPAs to BACTEC MGIT 960 system for susceptibility testing of bacterial resistance to first-line drugs: rifampicin (RIF), isoniazid (INH). Results: We performing Drug susceptibility testing (DST), 219/258 MTB cultures were viable after subculture the results of DST using the MGIT 960 system were compared to those obtained by line probe assay. LPA detected a total 46/258 (17.81%) samples as drug resistant, of which 35/258 (13.70%) were resistant to both rifampicin and isoniazid (MDR), 6/258 (2.28%) were rifampicin mono?resistant samples and 11/258 (4.11%) were isoniazid mono?resistant. Out of the culture?positive samples (219), LPA detected 39/219 (17.83%) as drug?resistant, of which 31/219 (14.2%) were resistant to both rifampicin and isoniazid, 5/193 (2.08%) were rifampicin mono?resistant and 8/219 (3.7%) were isoniazid mono?resistant. Conclusion: Drug resistant TB poses an enormous threat to TB control programs worldwide. Effective treatment of MDR-TB is very expensive, particularly in middle income countries such as India.

Bivalirudin Attenuates Thrombin-Induced Endothelial Hyperpermeability via S1P/S1PR2 Category: Original Articles

Aims: To explore the role of the Sphingosine 1-Phosphate (S1P)/Receptor2 (S1PR2) pathway in thrombin-induced hyperpermeability (TIP) and to test whether bivalirudin can reverse TIP via the S1P-S1PRs pathway.Methods and Results: Using western blot, we demonstrated that Human umbilical vein endothelial cells (HUVECs) that were cultured with 2 U/ml thrombin showed significantly increased S1PR2 expression while S1PR1and three kept unchanged. Such increment was attenuated by JTE-013 pretreatment and by presence of bivalirudin. Exposure of 2 U/ml of thrombin brought a higher level of S1P both intracellularly and extracellularly within the HUVECs by using ELISA detecting. Thrombin induced S1P and S1PR2 increment was restored by usage of PF543 and bivalirudin. Bivalirudin alone did not influenced the level of S1P and S1PR1,2, and S1PR3 compare to control group. As a surrogate of cytoskeleton morphology, phalloidin staining and immunofluorescence imaging were used. Blurry cell edges and intercellular vacuoles or spaces were observed along thrombin-exposed HUVECs. Presence of JTE-013 and bivalirudin attenuated such thrombin-induced permeability morphological change and presence of heparin failed to show the protective effect. Transwell chamber assay and probe assay were used to measure and compare endothelial permeability in vitro. An increased TIP was observed in HUVECs cultured with thrombin, and coculture with bivalirudin, but not heparin, alleviated this increase. JTE-013 treatment yielded to similar TIP alleviating effect. In vivo, an Evans blue assay was used to test subcutaneous and organ microvascular permeability after the treatment of saline only, thrombin + saline, thrombin + bivalirudin, thrombin + heparin or thrombin + JTE-013. Increased subcutaneous and organ tissue permeability after thrombin treatment was observed in thrombin + saline and thrombin + heparin groups while treatment of bivalirudin and JTE-013 absent this effect.Conclusion: S1P/S1PR2 mediates TIP by impairing vascular endothelial barrier function. Unlike heparin, bivalirudin effectively blocked TIP by inhibiting the thrombin-induced S1P increment and S1PR2 expression, suggesting the novel endothelial protective effect of bivalirudin under pathological procoagulant circumstance.