Detection of pyrazinamide heteroresistance in Mycobacterium tuberculosis

AbstractAimPyrazinamide (PZA) is a first-line key agent in the effective treatment of tuberculosis (TB), including PZA susceptible multidrug-resistant tuberculosis (MDR-TB). Occasionally, TB patients might have mixed infections with both drug-sensitive and -resistant strains. This is termed heteroresistance. If 10% of the bacterial population is resistant to PZA, there is an increased risk for poor treatment outcome. The aim of this study is to evaluate the ability of the three established drug susceptibility testing (DST) techniques – BACTEC MGIT 960, Wayne’s pyrazinamidase test and Sanger sequencing of the pncA gene – to detect 10% PZA heteroresistance.MethodsMixed cultures of the fully drug susceptible Mycobacterium tuberculosis H37Rv reference strain and two laboratory-generated isogenic H37Rv mutants (with C475G and T254C pncA mutations, respectively) were made in proportions of 100%, 10%, 5% and 1% of the PZA-resistant (PZA-R) strain. Corresponding mixed cultures were also made using one drug-susceptible and one PZA-resistant MDR clinical isolate with the T62G pncA mutation, both belonging to one specific MIRU cluster. Additional mixes of 50%, 75%, 90% and 99% of the PZA-R strains were prepared for the Wayne’s test. Tests were for all methods performed in duplicates at two separate occasions.ResultsUsing the MGIT system, the in vitro-generated PZA-R strains were generally detected at a 5% proportion while the clinical PZA-R isolate only was detected at the critical 10% proportion, except for one test occasion. Sanger sequencing was unable to detect 10% PZA heteroresistance. Wayne’s test also failed to detect the critical level of 10% PZA resistance; instead it displayed misguiding results determining highly resistant samples as susceptible.ConclusionHeteroresistance is caused by present drug-resistance development and/or dual infections with one resistant and one susceptible strain. Mixed infections with resistant strains may occur in up to 20% of all TB cases in high burden areas, according to limited data. This study showed that only the phenotypic BACTEC MGIT system was capable in determining the critical proportion of 10% PZA resistance, whereas neither the Sanger nor the Wayne’s test were successful in this respect. This indicates a need for diagnostic tools with increased sensitivity to determine heteroresistance in M. tuberculosis

EU-mosaik

Who influences our notion of the European Union? This paper examines the Swedish discourse of the European Union. The study shows how the Swedish parties in the Riksdag (Parliament) build up the picture of the European Union, including and excluding aspects and issues after their interests. The discursive theory of Laclau and Mouffe is used in order to conceptualize and analyze the discursive constitution of the Swedish view on the European Union. The theory is of great importance for the analysis, stating that the social reality is constructed by us humans, by our statements and acts. Using a theory-based qualitative analysis of the texts published on the parties’ web pages, I show which issues can be seen as accepted by all, and which issues they are struggling about. The main areas of disagreement and consensus are pointed out. The study also discusses the central role of Sweden and the Swedish identity in the discourse. The findings show that the discursive struggle is fought between the EU-sceptical and the EU-friendly parties

Att bita den hand som föder en? EU-kritiska parlamentarikers arbete i Europaparlamentet

The study seeks knowledge about the Swedish euro-scepticism and about the work of the Swedish euro-sceptical Members of the European Parliament (MEPs). How do they work in an environment where the norm is to have a positive attitude towards a future federal state of Europe? How do they regard their own situation and possibilities to make a change? The study includes a debate on today's Swedish euro-scepticism as background. My finding about the MEPs? work is that their way of acting in the European Parliament does not distinguish from other MEPs? in large. The euro-sceptical MEPs seem to see small opportunities to change the EU essentially, but I also show that they do not use all possible means. They have adopted themselves to the Parliament's organisation standard and they seem to do their daily work like anyone else, with the only visible difference that they work more. I show that though these MEPs are in a difficult situation, they do not have a Swedish euro-sceptical co-operation. This picture leads me to the conclusion that euro-sceptical MEPs do not make a big difference in the European Parliament

Kontroll av demokrativärden i kommunal förvaltning

Demokrati är ett omhuldat begrepp som är svårt att definiera och har otydliga avgränsningar. Detta medför svårigheter vid kontrollen av hur demokratisk den svenska förvaltningen är. Avsaknaden av ett verktyg för att undersöka verksamheten ur ett demokratiskt perspektiv gör att demokratigranskningen har svårt att få samma förutsättningar som en ekonomirevision. Denna uppsats har som syfte att granska om en kontroll av demokratiska värden sker och hur den ser ut. Uppsatsen bygger på en fördjupad granskning av revisionen i Malmö stad. Studien har grundat sig på analys av tidigare forskning och textanalys av befintliga dokument som styr den kommunala revisionen samt intervjuer med i ämnet insatta personer. Vi fann att det förekommer en demokratisk kontroll i Malmö stad, men att den saknar en tydlig demokratidefinition som kunde vara ett verktyg för att ytterligare utveckla hur en sådan kontroll ska se ut. Denna otydlighet återfinns i de regleringar som styr den kommunala revisionen, varför vi efterlyser en levande demokratidiskussion

Dynamics of Antibiotic Resistant Mycobacterium tuberculosis during Long-Term Infection and Antibiotic Treatment

For an infecting bacterium the human body provides several potential ecological niches with both internally (e.g. host immunity) and externally (e.g. antibiotic use) imposed growth restrictions that are expected to drive adaptive evolution in the bacterium, including the development of antibiotic resistance. To determine the extent and pattern of heterogeneity generated in a bacterial population during long-term antibiotic treatment, we examined in a monoclonal Mycobacterium tuberculosis infection antibiotic resistant mutants isolated from one patient during a 9-years period. There was a progressive accumulation of resistance mutations in the infecting clone. Furthermore, apparent clonal sweeps as well as co-existence of different resistant mutants were observed during this time, demonstrating that during treatment there is a high degree of dynamics in the bacterial population. These findings have important implications for diagnostics and treatment of drug resistant tuberculosis infections

Evaluation of loop mediated isothermal amplification for diagnosis of Mycobacterium tuberculosis complex in clinical samples

Tuberculosis (TB) remains an important global public health problem. The lack of rapid and accurate diagnostic testing is an important impediment to global tuberculosis control. Loop mediated isothermal amplification (LAMP) is a rapid method for nucleic acid amplification. In this study, we assessed the performance of an in-house LAMP assay for the detection of tuberculosis. Six oligonucleotide primers specific for Mycobacterium tuberculosis complex were designed corresponding to IS6110 gene sequence. Optimization of LAMP reaction was performed. A total of 133 clinical sputum samples and 80 bacterial cultures were studied by LAMP method. Sensitivity of this assay for detection of genomic DNA was 5 fg. This assay successfully detected M. tuberculosis complex not only in the bacterial cultures but also in the clinical sputum samples from patients with TB. The sensitivity of LAMP in culturepositive samples was 100% (60/60) and the specificity in culture-negative samples was 95.9% (70/73, 95% confidence interval 91.3 to 98.7%). Thus, LAMP is a rapid, highly sensitive and specific DNA amplification technique for early diagnosis of TB.Key words: Loop mediated isothermal amplification (LAMP), polymerase chain reaction (PCR), IS6110 gene, Mycobacterium tuberculosis, diagnosis

Little difference between minimum inhibitory concentrations of Mycobacterium tuberculosis wild-type organisms determined with BACTEC MGIT 960 and Middlebrook 7H10

AbstractThe MIC wild-type (WT) distribution for Mycobacterium tuberculosis in BACTEC 960 MGIT is not defined, which may result in poor reproducibility for drug susceptibility testing (DST), as several DST methods with different breakpoints are in use. In a comparison between MGIT and Middlebrook 7H10 medium of seven first- and second-line drugs, including 133 MIC determinations of 15 WT isolates, we found an agreement of 91.7% within ± one MIC dilution step. The results confirm the agreement in MIC testing between 7H10 and MGIT and indicate that breakpoints could be harmonized in order to avoid misclassification

Multicentre testing of the EUCAST broth microdilution reference method for MIC determination on mycobacterium tuberculosis

Objectives: the first objective of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) subcommittee for antimycobacterial susceptibility testing (AMST), launched in 2016, was to set a reference method for determining the MICs of antituberculous agents, since many protocols are used worldwide and a consensus one is needed for the determination of microbiological breakpoints. Methods: during 2017 and 2018, MIC determination protocols were evaluated prospectively in a multicentre study within the four AMST laboratories. MIC results were obtained for isoniazid, levofloxacin and amikacin on the reference strain Mycobacterium tuberculosis H37Rv ATCC 27294. Broth microdilution (BMD) in Middlebrook 7H9 and solid medium dilution (SMD) in Middlebrook 7H10 were performed using two inoculum concentrations. MICs were interpreted with regard to visual and 99% inhibition after 7, 14 or 21 days of incubation for BMD and 21 days for SMD. Results: following the EUCAST reference protocol, intra- and inter-assay agreements were within ±1 MIC dilution for >95% of the observations for the three drugs in both methods. MIC values, presented as MIC mode (range) for BMD and SMD respectively, were: 0.03 (0.015-0.06) mg/L and 0.12 (0.06-0.25) mg/L for isoniazid, 0.25 mg/L (0.25-0.5) and 0.5 mg/L (0.12-0.5) for levofloxacin, and 0.5 mg/L (0.5-1.0) and 0.5 mg/L (0.5-1.0) for amikacin. Conclusions: both SMD and BMD were reproducible and eligible as a reference method for MIC determination of the Mycobacterium tuberculosis complex (MTBC). BMD was finally selected as the EUCAST reference method. From now on it will be used to set epidemiological cut-off values and clinical breakpoints of new and old antituberculous agents

Antimicrobial susceptibility testing of mycobacterium tuberculosis complex isolates - the EUCAST broth microdilution reference method for MIC determination

Scope:Several methods are used worldwide for antibiotic susceptibility testing (AST) for theMycobac-terium tuberculosiscomplex (MTBC). The variability in the results obtained with these methods hamperssetting epidemiological cut-off (ECOFF) values and clinical breakpoints according to EUCAST guidelines.Methods for susceptibility testing and determination of the minimal inhibitory concentrations (MICs)need to be standardized for MTBC isolates for old and new agents. Our objective was to establish astandardized reference method for MIC determination for MTBC.Methods:The EUCAST antimycobacterial susceptibility testing subcommittee (AMST) compared pro-tocols of MIC determination with regard to medium, inoculum preparation, antituberculous agentpreparation, incubation, reading of the results and interpretation.Recommendations:The EUCAST reference method of MIC determination for MTBC is the broth micro-dilution method in Middlebrook 7H9-10% OADC medium. Thefinal inoculum is a 105CFU/mL suspension,obtained from a 10 2dilution of a 0.5 McFarland suspension prepared after vortexing bacterial colonieswith glass beads before suspending them in sterile water. The culture is maintained in a U-shaped 96-well polystyrene microtitre sterile plate with a lid incubated at 36 ±1 C. Reading is done using aninverted mirror as soon as the 1:100 diluted control (i.e. 103CFU/mL suspension) shows visual growth.The MIC, expressed in mg/L, is the lowest concentration that inhibits visual growth.MycobacteriumtuberculosisH37Rv ATCC 27294 is used as the reference strain and its targeted MIC values are within therange 0.03e0.12 for isoniazid, 0.12e0.5 for levofloxacin and 0.25e1 mg/L for amikacin.Conclusions:The EUCAST reference method for MTBC was endorsed by EUCAST after public consultationand will from now on be used to define EUCAST ECOFFs and clinical breakpoints. This reference methodis not primarily intended to be used under routine conditions and the AST methods will need to b