Assessment of the GenoType MTBDRsl VER 2.0 compared to the phenotypic drug susceptibility testing and whole genome sequencing for the rapid detection of resistance to fluoroquinolone and second-line injectable drugs among rifampicin-resistant Mycobacterium tuberculosis isolates.

Molecular techniques have considerable advantages for rapid detection, a reduction of infectiousness, prevention of further resistance development and surveillance of drug-resistant TB. MTBDRsl VER 2.0 was used to detect resistance to second-line anti-tuberculosis drugs on 35 rifampicin-resistant M. tuberculosis (RR-MTB) isolates compared to the minimum inhibitory concentrations (MICs) and whole genome sequencing (WGS). The MTBDRsl VER 2.0 (Hain Life Science, Nehren, Germany) and WGS (San Diego, CA, USA) were performed for tracing mutations in resistant-related genes involved in resistance to fluoroquinolone (FLQ) and second-line injectable drugs. The broth microdilution method using 7H9 Middlebrook media supplemented with OADC was used to determine the MICs. The MTBDRsl VER 2.0 correctly detected 5/6 (83.3%) of FLQ-resistant strains. The MUT1 A1401G (seven strains) and MUT2 G1484T (one strain) mutations in rrs gene were detected in eight AMK/KAN/CAP-resistant strains. Four low-level KAN-resistant strains with the G-10A/C-12T (three strains) and eis C-14T (one strain) mutations in eis gene was diagnosed using MTBDRsl VER 2.0. Five errors were found in detecting resistance to kanamycin and capreomycin compared to the phenotypic drug susceptibility testing and WGS. Failling wild-type bands without improved mutant bands did not indicate a reliable resistance. WGS could efficiently resolve the discrepancies of the results. MTBDRsl showed better performance in detecting XDR strains than pre-XDR.

Genotyping and Drug Susceptibility Patterns of M. Tuberculosis Isolated from HIV Seropositive Patients in Tehran Iran.

AIM: This study aims to investigate the prevalence and drug-resistance M. tuberculosis isolated from HIV seropositive individuals in Tehran, Iran. BACKGROUND: Human immunodeficiency virus (HIV) is one of the most important risk factors for developing active tuberculosis (TB). OBJECTIVE: The objective is to determine the rate of transmission and drug-resistant M. tuberculosis (MTB) strains isolated from HIV seropositive patients in Tehran province, Iran. METHODS: This study consecutively enrolled 217 TB/HIV coinfected patients from April 2018 to August 2019 at Emam Khomeini referral hospital and 5 other health centers in Tehran province. The isolates were genotyped using 15 loci Mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTR). Minimum inhibitory concentration (MIC) was determined for 6 drugs. In addition, mutations were assessed in rpoB, katG, inhA, and ahpC genes using Reverse Blot Hybridization Assay System. RESULTS: A 20 (9.2%) patients were culture-positive for M. tuberculosis and typed by MIRU-VNTR, 13 (65%) strains formed 5 clusters, but 6 (30%) isolates had a unique pattern. The total Hunter- Gaston discrimination index (HGDI) for all 15 loci was 0.846, and the cluster size was 2 to 4 patients. The estimated proportion of recent transmission was 45%. The mutation was identified in 1 isolate, lost inhAW1 and mutation in MT1 loci, which was resistant to isoniazid (INH). Moreover, 1 (5%) and 3 (15%) isolates were resistant to INH and ethambutol (EMB), respectively, of which 1 was resistant to INH and EMB. CONCLUSION: The transmission rate of TB in HIV patients was relatively high; however, the prevalence of drug-resistant strains and TB infection in females was insignificant in this study (p < 0.05); none of the isolates was MDR strains.

Incidence, Clinical Manifestation, Treatment Outcome, and Drug Susceptibility Pattern of Nontuberculous Mycobacteria in HIV Patients in Tehran, Iran.

BACKGROUND: Nontuberculous mycobacterial (NTM) infections have radically increased worldwide due to the increase in HIV infections. The disease activity increases with progressive immunodeficiency. METHODS: A total of 216 HIV seropositive patients suspected of having mycobacterial infection were recruited for this study. Clinical samples were collected from each patient and cultured on Lowenstein-Jensen media. Detection and species identification were simultaneously done using Reverse Blot Hybridization Assay System. Also, the minimum inhibitory concentrations (MIC) for each isolate were determined in 7H9 broth media for 10 antibiotics. RESULTS: In this study, 4 rapid and 4 slow-growing NTM species were isolated and identified. Mycobacterium fortuitum was the most common NTM species, 3/8 (37.5%), followed by Mycobacterium kansasii, 2/8 (25%). The cases were identified as pulmonary disease, 5/8 (62.5 %), disseminated infection, 2/8 (25%), and skin abscess, 1/8 (12.5%). M. chelonae and Mycobacterium avium were isolated from patients diagnosed with disseminated infection with treatment failure. The skin abscess was caused by infection with M. simiae. The results of the MIC testing were as follows: M. kansasii and M. fortuitum were susceptible to amikacin (AMK); M. avium to clarithromycin (CLA); M. fortuitum 2/3 (67%) to ciprofloxacin (CIP); 1/2 (50%) of M. kansasii isolates to CLA, and M. chelonae to rifampin (RIF), linezolid (LIN), AMK, and CIP at medium and high concentrations. CONCLUSION: AMK showed incredible in vitro activity against M. kansasii and M. fortuitum. Also, M. avium was susceptible to CLA, whereas M. simiae and M. chelonae were resistant to the tested drugs in this study.

Whole Genome Sequencing Results Associated with Minimum Inhibitory Concentrations of 14 Anti-Tuberculosis Drugs among Rifampicin-Resistant Isolates of Mycobacterium Tuberculosis from Iran.

Accurate and timely detection of drug resistance can minimize the risk of further resistance development and lead to effective treatment. The aim of this study was to determine the resistance to first/second-line anti-tuberculosis drugs in rifampicin/multidrug-resistant Mycobacterium tuberculosis (RR/MDR-MTB) isolates. Molecular epidemiology of strains was determined using whole genome sequencing (WGS)-based genotyping. A total of 35 RR/MDR-MTB isolates were subjected to drug susceptibility testing against first/second-line drugs using 7H9 Middlebrook in broth microdilution method. Illumina technology was used for paired-end WGS applying a Maxwell 16 Cell DNA Purification kit and the NextSeq platform. Data analysis and single nucleotide polymorphism calling were performed using MTBseq pipeline. The genome-based resistance to each drug among the resistant phenotypes was as follows: rifampicin (97.1%), isoniazid (96.6%), ethambutol (100%), levofloxacin (83.3%), moxifloxacin (83.3%), amikacin (100%), kanamycin (100%), capreomycin (100%), prothionamide (100%), D-cycloserine (11.1%), clofazimine (20%), bedaquiline (0.0%), and delamanid (44.4%). There was no linezolid-resistant phenotype, and a bedaquiline-resistant strain was wild type for related genes. The Beijing, Euro-American, and Delhi-CAS were the most populated lineage/sublineages. Drug resistance-associated mutations were mostly linked to minimum inhibitory concentration results. However, the role of well-known drug-resistant genes for D-cycloserine, clofazimine, bedaquiline, and delamanid was found to be more controversial.

Molecular identification of mutations conferring resistance to rifampin, isoniazid and pyrazinamide among Mycobacterium tuberculosis isolates from Iran.

Here, we aimed to determine the susceptibility of 70 Mycobacterium tuberculosis isolates obtained from different regions of the country to 8 anti-tuberculosis (anti-TB) drugs and possible underlying mechanisms causing resistance to rifampin, isoniazid, and pyrazinamide. The susceptibility of 70 isolates of M. tuberculosis to anti-TB drugs was tested using proportion method. Strains showing resistance to the first line anti-TB drugs were subjected to PCR amplification and sequencing of the rpoB, katG, ahpC, pncA genes, inhA promoter and oxyR-ahpC intergenic regions to detect resistance conferring mutations. Overall, 77.1% and 77.1% of isolates were resistant to at least one of the tested first- and second-line drugs, respectively. Within the rpoB gene the highest rate of mutation was found in codons 531(450) (56.3%), and 533(452) (12.5%). Also, codons 315 (42.4%) of katG, positions -48, -72 and -77 of oxyR-ahpC (total= 3, 9.1%) and -15 of inhA promoter region (33.3%) were the most altered positions in isoniazid resistant isolates. Only a single mutation was detected for pncA among resistant isolates. High prevalence of resistance to essential anti-TB drugs among M. tuberculosis strains isolated from retreated tuberculosis cases is alarming issue necessitating immediate action to prevent the spread of drug resistant isolates in the country.

The Chemical Composition and Anti-mycobacterial Activities of Trachyspermum copticum and Pelargonium graveolens Essential Oils.

BACKGROUND: Microbial resistance to antibiotics and their adverse effects related to these antibiotics are a matter of global public health in the 21th century. The emergence of drug-resistant strains, has gained the interest of the scientists to discover new antimicrobial agents from the essential oil of medicinal plants. METHODS: Anti-mycobacterial effects of Trachyspermum copticum and Pelargonium graveolens essential oils were determined against multi-drug resistant clinical strains of Mycobacterium tuberculosis, Mycobacterium kansasii, Mycobacterium fortuitum and standard strain of Mycobacterium tuberculosis H37Rv by a Broth micro-dilution method. Pelargonium graveolens plant named Narmada was discovered by Kulkarni R.N et al. (Patent ID, USPP12425P2) and a formulation comprising thymol obtained from Trachyspermum is useful in the treatment of drug-resistant bacterial infections (Patent ID, US6824795B2). The chemical composition of hydro-distilled essential oils was determined by GC and GC-MS. RESULTS: Minimum Inhibitory Concentration (MIC) values for T. copticum essential oil against tested isolates were ranged from 19.5 µg/mL to 78 µg/mL. The least minimum inhibitory concentration of P. graveolens extract against M. Kansasii and MDR-TB was 78 µg/ml. CONCLUSION: The results of the present research introduced T. copticum and P. graveolens essential oils as a remarkable natural anti-mycobacterial agent, but more pharmacological studies are required to evaluate their efficacy in animal models.