Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory.

BACKGROUND: Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed. METHODS: The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis. RESULTS: Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74). CONCLUSIONS: READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.

The problem of Mycobacterium abscessus complex: multi-drug resistance, bacteriophage susceptibility and potential healthcare transmission.

OBJECTIVES: Mycobacterium abscessus complex is responsible for 2.6-13.0% of all non-tuberculous mycobacterial pulmonary infections and these are notoriously difficult to treat due to the complex regimens required, drug resistance and adverse effects. Hence, bacteriophages have been considered in clinical practice as an additional treatment option. Here, we evaluated antibiotic and phage susceptibility profiles of M. abscessus clinical isolates. Whole-genome sequencing (WGS) revealed the phylogenetic relationships, dominant circulating clones (DCCs), the likelihood of patient-to-patient transmission and the presence of prophages. METHODS: Antibiotic susceptibility testing was performed using CLSI breakpoints (n = 95), and plaque assays were used for phage susceptibility testing (subset of n = 88, 35 rough and 53 smooth morphology). WGS was completed using the Illumina platform and analysed using Snippy/snp-dists and Discovery and Extraction of Phages Tool (DEPhT). RESULTS: Amikacin and Tigecycline were the most active drugs (with 2 strains resistant to amikacin, and one strain with Tigecycline MIC of 4 µg/mL). Most strains were resistant to all other drugs tested, with Linezolid and Imipenem showing the least resistance, at 38% (36/95) and 55% (52/95), respectively. Rough colony morphotype strains were more phage-susceptible than smooth strains (77%-27/35 versus 48%-25/53 in the plaque assays, but smooth strains are not killed efficiently by those phages in liquid infection assay). We have also identified 100 resident prophages, some of which were propagated lytically. DCC1 (20%-18/90) and DCC4 (22%-20/90) were observed to be the major clones and WGS identified 6 events of possible patient-to-patient transmission. DISCUSSION: Many strains of M. abscessus complex are intrinsically resistant to available antibiotics and bacteriophages represent an alternative therapeutic option, but only for strains with rough morphology. Further studies are needed to elucidate the role of hospital-borne M. abscessus transmission.

[Determination of nicotine and cotinine in human biological materials and their significance in toxicological studies]. / Problematyka oznaczania nikotyny i kotyniny w ludzkim materiale biologicznym w aspekcie badan toksykologicznych.

The aim of this study was the preparation of reliable procedure of the determination of nicotine and cotinine both in classic (serum, urine) and alternative biological materials (hair, saliva) and evaluation of their significance for clinical and forensic toxicology. Biological material samples (blood, urine, saliva) were taken from patients after Percutaneous Trans-luminal Coronary Angioplasty (PTCA). The determination of cotinine and nicotine concentration in the biological material should be optimized depending on the aim of analysis. Liquid-liquid extraction procedure and high performance liquid chromatography HPLC/UV-DAD are reliable, specific and relatively cheap. Serum and saliva are valuable biological materials which allow to determine temporary nicotine and cotinine content on the similar level of concentrations. In the near future it will be able to replace blood with saliva sample because of an easy and non-invasive way of sampling. Evaluation of cotinine concentration in urine allows to distinguish the passive from the active tobacco smokers. Hair analysis allows to control a nicotine abstinence as well as a long-term evaluation of the history of smoking. However usage of hair is limited because of difficulty with sampling. Interpretation of results in analysis of alternative materials (hair, saliva) pose a problem because of lack of sampling standardization and lack of standardization of final analysis method.