Spoligotyping of Mycobacterium tuberculosis - Comparing in vitro and in silico approaches.

Spoligotyping is one of the molecular typing methods widely used for exploring the genetic variety of Mycobacterium tuberculosis. The aim of this study was to compare the spoligoprofiles of M. tuberculosis clinical isolates, obtained using in vitro and in silico approaches. The study included 230 M. tuberculosis isolates, recovered from Poland and Lithuania between 2018 and 2021. Spoligotyping in vitro was performed with a commercially available kit. Whole genome sequencing (WGS) was done with Illumina NovaSeq 6000 sequencer. Spoligotype International Types (SITs) were assigned according to the SITVIT2 database or using three different in silico tools, and based on WGS data, namely SpoTyping, SpolPred, and lorikeet. Upon in vitro spoligotyping, the isolates produced 65 different spoligotypes. Spoligotypes inferred from the WGS data were congruent with in vitro generated patterns in 81.7% (188/230) for lorikeet and 81.3% (187/230) for SpolPred and SpoTyping. Spacers 18 and 31 produced the highest ratio of discrepant results between in vitro and in silico approaches, with their signals discordantly assigned for 15 (6.5%) and 9 (3.9%) isolates, respectively. All three in silico approaches used were similarly efficient for M. tuberculosis spoligotype prediction. However, only SpoTyping could predict spoligotypes without a need for manual curation. Thus, we consider it as the most accurate tool. Its use is further advocated by the shortest time of analysis. A relatively high (ca. 20%) discordance between in vitro and in silico spoligotyping results was observed. While we discourage comparing conventional spoligotyping with in silico equivalents, we advise the use of the latter, as it improves the accuracy of spoligopatterns, and thus depicts the relatedness between the isolates more reliably.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-Resistant Tuberculosis (MDR-TB): a 5-Year Prospective In Vitro Surveillance Study of Bedaquiline and Other Second-Line Drug Susceptibility Testing in MDR-TB Isolates.

Bedaquiline Drug Resistance Emergence Assessment in Multidrug-resistant tuberculosis (MDR-TB) (DREAM) was a 5-year (2015 to 2019) phenotypic drug resistance surveillance study across 11 countries. DREAM assessed the susceptibility of 5,036 MDR-TB isolates of bedaquiline treatment-naive patients to bedaquiline and other antituberculosis drugs by the 7H9 broth microdilution (BMD) and 7H10/7H11 agar dilution (AD) MIC methods. Bedaquiline AD MIC quality control (QC) range for the H37Rv reference strain was unchanged, but the BMD MIC QC range (0.015 to 0.12 µg/ml) was adjusted compared with ranges from a multilaboratory, multicountry reproducibility study conforming to Clinical and Laboratory Standards Institute Tier-2 criteria. Epidemiological cutoff values of 0.12 µg/ml by BMD and 0.25 µg/ml by AD were consistent with previous bedaquiline breakpoints. An area of technical uncertainty or intermediate category was set at 0.25 µg/ml and 0.5 µg/ml for BMD and AD, respectively. When applied to the 5,036 MDR-TB isolates, bedaquiline-susceptible, -intermediate, and -resistant rates were 97.9%, 1.5%, and 0.6%, respectively, for BMD and 98.8%, 0.8%, and 0.4% for AD. Resistance rates were the following: 35.1% ofloxacin, 34.2% levofloxacin, 33.3% moxifloxacin, 1.5% linezolid, and 2% clofazimine. Phenotypic cross-resistance between bedaquiline and clofazimine was 0.4% in MDR-TB and 1% in pre-extensively drug-resistant (pre-XDR-TB)/XDR-TB populations. Coresistance to bedaquiline and linezolid and clofazimine and linezolid were 0.1% and 0.3%, respectively, in MDR-TB and 0.2% and 0.4%, respectively, in pre-XDR-TB/XDR-TB populations. Resistance rates to bedaquiline appear to be low in the bedaquiline-treatment-naive population. No treatment-limiting patterns for cross-resistance and coresistance have been identified with key TB drugs to date.

Investigation of Adipose Tissue Fatty Acid Composition in Men with Uronephrolithiasis and Metabolic Syndrome.

BACKGROUND Fatty acids (FA) and their metabolites are closely related to some mechanisms involved in the development of uronephrolithiasis. The aim of this study was to evaluate the relationship between FA composition and type of kidney stones. MATERIAL AND METHODS Abdominal adipose tissue fatty acid methyl esters of 71 men with nephrolithiasis were identified by GC/MS, and the type of kidney stones was identified using FTIR infrared spectroscopy. Patients were divided into groups according to diagnosis of metabolic syndrome (MS) and type of kidney stone. The composition of FA was compared within different groups of patients with different types of kidney stones and between the patients and healthy individuals (control group) (n=100). RESULTS Individuals with nephrolithiasis had a significantly higher level of monounsaturated fatty acids (MUFA) and a lower level of polyunsaturated fatty acids (PUFA) versus healthy individuals. Patients with MS had a higher level of 18: 1ω9 and a lower level of 16: 1ω7 than patients without MS. Individuals with nephrolithiasis, but without MS, had a higher level of saturated fatty acids (SFA) compared to controls. The level of PUFA was higher in the control group (p<0.0001) compared to individuals with uronephrolithiasis, with or without MS. PUFA, ω - 6 PUFA, and 18: 2ω6 were higher in patients with calcium-based kidney stones without MS versus patients with uric acid kidney stones with MS. CONCLUSIONS The levels of MUFA were significantly higher and the levels of PUFA were significantly lower in patients with uronephrolithiasis compared to controls.