RESUMO
In early June 2018, an increase in non-travel-related cases of Legionella non-pneumophila Legionnaires' disease (LD) was observed in Sweden and a national outbreak investigation was started. Outbreak cases were defined as notified confirmed or probable cases of L. non-pneumophila LD, with symptom onset after 1 April 2018. From April to August 2018, 41 cases were reported, 30 of whom were identified as L. longbeachae. We conducted a case-control study with 27 cases and 182 matched controls. Results from the case-control study indicated that gardening and handling commercial bagged soil, especially dusty dry soil, were associated with disease. L. longbeachae was isolated in soils from cases' homes or gardens, but joint analysis of soil and human specimens did not identify any genetic clonality. Substantial polyclonality was noted between and within soil samples, which made finding a genetic match between soil and human specimens unlikely. Therefore, whole genome sequencing may be of limited use to confirm a specific soil as a vehicle of transmission for L. longbeachae. Handling soil for residential gardening was associated with disease and the isolation of L. longbeachae in different soils provided further evidence for Legionella non-pneumophila infection from soil.
Assuntos
Legionella longbeachae , Legionella pneumophila , Doença dos Legionários , Estudos de Casos e Controles , Surtos de Doenças , Jardinagem , Humanos , Legionella pneumophila/genética , Doença dos Legionários/diagnóstico , Doença dos Legionários/epidemiologia , Solo , Suécia/epidemiologiaRESUMO
Highly infectious diseases can spread rapidly across borders through travel or trade, and international coordination is essential to a prompt and efficient response by public health laboratories. Therefore, developing strategies to identify priorities for a rational allocation of resources for research and surveillance has been the focus of a large body of research in recent years. This paper describes the activities and the strategy used by a European-wide consortium funded by the European Commission, named EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level), for the selection of high-threat pathogens with cross-border potential that will become the focus of its preparedness activities. The approach used is based on an objective scoring system, a close collaboration with other networks dealing with highly infection diseases, and a diagnostic gaps analysis. The result is a tool that is simple, objective and adaptable, which will be used periodically to re-evaluate activities and priorities, representing a step forward towards a better response to infectious disease emergencies.
Assuntos
Controle de Doenças Transmissíveis/organização & administração , Atenção à Saúde/organização & administração , Surtos de Doenças/prevenção & controle , Laboratórios/organização & administração , Viroses/prevenção & controle , Vírus/patogenicidade , Europa (Continente) , HumanosRESUMO
OBJECTIVES: Fluoroquinolones (FQs) are important in the treatment of MDR-TB and in the definition of XDR-TB. Our objective was to investigate how discrepancies in the phenotypic and genotypic methods for antimicrobial susceptibility testing could affect the interpretation of antimicrobial susceptibility test results. METHODS: We analysed MICs of ofloxacin and levofloxacin in Middlebrook 7H10 broth (7H10) as well as sequencing of the quinolone resistance-determining region of the gyrA gene and the MTBDRsl assay in 75 resistant isolates, including MDR and XDR strains of Mycobacterium tuberculosis. RESULTS: Among 75 resistant isolates, 27 had mutations associated with FQ resistance. Among isolates with resistance mutations in gyrA, 26% (seven of 27) were susceptible to levofloxacin and ofloxacin by phenotypic testing at 1 mg/L and 2 mg/L. The most common mutation was in codon 94 and these isolates had significantly increased MICs of levofloxacin (2-8 mg/L) compared with isolates with mutations in codon 90 (0.25-2 mg/L, P <â0.05). The sensitivity and specificity for the MTBDRsl assay compared with gyrA sequencing were 96% and 98%, respectively. CONCLUSION: Current critical concentrations may classify up to 26% of isolates with gyrA mutations as susceptible to FQs due to a close relationship between susceptible and resistant populations. These results should be considered while improving clinical breakpoints for M. tuberculosis and may have an impact on the definition of XDR-TB.
Assuntos
Antituberculosos/farmacologia , DNA Girase/genética , Fluoroquinolonas/farmacologia , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA , Terminologia como AssuntoRESUMO
The ability of pyrosequencing to detect a resistant minority population of a heteroresistant Mycobacterium tuberculosis strain was investigated by performing a titration study. A mutant signal was noted only when the proportion of mutant DNA in the DNA target was 35 to 50%, showing that the sensitivity is significantly lower than that of phenotypic drug susceptibility test methods.
Assuntos
Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Análise de Sequência de DNA/métodos , Tuberculose/microbiologia , Genética Populacional , Humanos , Testes de Sensibilidade Microbiana/métodos , Sensibilidade e EspecificidadeRESUMO
Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Análise de Sequência de DNA/métodos , Proteínas de Bactérias/genética , Humanos , Testes de Sensibilidade Microbiana/métodos , Mutação , Regiões Promotoras Genéticas , Sensibilidade e EspecificidadeRESUMO
The rise in multidrug-resistant tuberculosis makes it increasingly important that antimicrobial susceptibility testing of Mycobacterium tuberculosis produce clinically meaningful and technically reproducible results. Unfortunately, this is not always the case because mycobacteriology specialists have not followed generally accepted modern principles for the establishment of susceptibility breakpoints for bacterial and fungal pathogens. These principles specifically call for a definition of the minimum inhibitory concentrations (MICs) applicable to organisms without resistance mechanisms (also known as wild-type MIC distributions), to be used in combination with data on clinical outcomes, pharmacokinetics and pharmacodynamics. In a series of papers the authors have defined tentative wild-type MIC distributions for M. tuberculosis and hope that other researchers will follow their example and provide confirmatory data. They suggest that some breakpoints are in need of revision because they either (i) bisect the wild-type distribution, which leads to poor reproducibility in antimicrobial susceptibility testing, or (ii) are substantially higher than the MICs of wild-type organisms without supporting clinical evidence, which may result in some strains being falsely reported as susceptible. The authors recommend, in short, that susceptibility breakpoints for antituberculosis agents be systematically reviewed and revised, if necessary, using the same modern tools now accepted for all other bacteria and fungi by the scientific community and by the European Medicines Agency and the European Centre for Disease Prevention and Control. For several agents this would greatly improve the accuracy and reproducibility of antimicrobial susceptibility testing of M. tuberculosis.
Assuntos
Antituberculosos/uso terapêutico , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/tratamento farmacológico , Humanos , Tuberculose Pulmonar/diagnósticoRESUMO
OBJECTIVES: The aim of this study was to clarify the conflicting data regarding cross-resistance and drug-resistance mechanisms for the cyclic peptide capreomycin and the aminoglycosides amikacin and kanamycin by comparing genotypes and phenotypes of clinical isolates and in vitro selected mutants of Mycobacterium tuberculosis. METHODS: The genes rrs and tlyA and the promoter region of eis of 152 M. tuberculosis clinical isolates (including 55 capreomycin resistant) and 44 in vitro selected capreomycin-, amikacin- and kanamycin-resistant mutants were sequenced. In addition, MICs of capreomycin, amikacin and kanamycin on Middlebrook 7H10 were determined. RESULTS: The results clearly show major differences in genotypes and cross-resistance patterns to amikacin and kanamycin between the capreomycin-resistant clinical isolates and in vitro selected mutants. tlyA mutations were found almost exclusively among the in vitro selected capreomycin-resistant mutants, while only four were found among the clinical isolates, of which two were capreomycin susceptible. In contrast, 53 of the 55 capreomycin-resistant clinical isolates had a mutation at position 1401 in rrs and were resistant to capreomycin, amikacin and kanamycin. Low-level resistance to kanamycin was correlated to mutations in the promoter region of eis. CONCLUSIONS: Our findings are consistent with the belief that a mutation at position 1401 in rrs leads to resistance to capreomycin, amikacin and kanamycin. The data also show that tlyA is not a sensitive genetic marker for capreomycin resistance in clinical isolates of M. tuberculosis, as mutations in this gene are infrequent and not all mutations in tlyA lead to capreomycin resistance.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Capreomicina/farmacologia , Farmacorresistência Bacteriana , Marcadores Genéticos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Acetiltransferases , Amicacina/farmacologia , Antígenos de Bactérias/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Humanos , Canamicina/farmacologia , Testes de Sensibilidade Microbiana , Mutação Puntual , Regiões Promotoras Genéticas , RNA Ribossômico 16S/genética , Sensibilidade e Especificidade , Análise de Sequência de DNARESUMO
OBJECTIVES: To perform a multicentre study to evaluate the performance of the colorimetric redox indicator (CRI) assay and to establish the MICs and critical concentrations of rifampicin, isoniazid, ofloxacin, kanamycin and capreomycin. METHODS: The study was carried out in two phases. Phase I determined the MIC of each drug. Phase II established critical concentrations for the five drugs tested by the CRI assay compared with the conventional proportion method. RESULTS: Phase I: a strain was considered resistant by the CRI assay if the MIC was ≥0.5 mg/L for rifampicin, ≥0.25 mg/L for isoniazid, ≥4.0 mg/L for ofloxacin and ≥5.0 mg/L for kanamycin and capreomycin. Sensitivity was 99.1% for isoniazid and 100% for the other drugs and specificity was 97.9% for capreomycin and 100% for the other drugs. Phase II: the critical concentration was 0.5 mg/L for rifampicin, 0.25 mg/L for isoniazid, 2.0 mg/L for ofloxacin and 2.5 mg/L for kanamycin and capreomycin giving an overall accuracy of 98.4%, 96.6%, 96.7%, 98.3% and 90%, respectively. CONCLUSIONS: Results demonstrate that the CRI assay is an accurate method for the rapid detection of XDR Mycobacterium tuberculosis. The CRI assay is faster than the conventional drug susceptibility testing method using solid medium, has the same turnaround time as the BACTEC MGIT 960 system, but is less expensive, and could be an adequate method for low-income countries.
Assuntos
Antituberculosos/farmacologia , Colorimetria/métodos , Farmacorresistência Bacteriana Múltipla , Tuberculose Extensivamente Resistente a Medicamentos/diagnóstico , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Indicadores e Reagentes/metabolismo , Testes de Sensibilidade Microbiana/métodos , Mycobacterium tuberculosis/isolamento & purificação , OxirreduçãoRESUMO
A reverse line blot DNA hybridization format for rapid detection of multidrug-resistant tuberculosis was developed. Simultaneous detection of rifampin and isoniazid resistance in clinical isolates of Mycobacterium tuberculosis was based on the same amplification/reverse hybridization principle of the widely used spoligotyping. The test involved probing nine DNA regions that are targets of common drug resistance-associated mutations in the genes rpoB, katG, and inhA. Addition of quaternary amine tetramethyl ammonium chloride to the hybridization buffer promoted multiple hybrid formations at a single annealing temperature irrespective of the different GC contents of probes. The assay was standardized using 20 well-documented strains from the Institute of Tropical Medicine (Belgium) and evaluated blindly in a central laboratory with 100 DNA samples that were obtained from cultured clinical isolates and shipped dried from three other countries. Compared with drug susceptibility testing, both sensitivity and specificity for rifampin resistance detection were 93.0% while for isoniazid the values were 87.7% and 97.7%, respectively. Compared with sequencing and GenoType MTBDRplus methods, sensitivity and specificity reached 96.4% and 95.5% for rifampin and 92.7% and 100% for isoniazid. Altogether, 40/45 (89%) multidrug-resistant isolates were correctly identified. Advantages of this in-house development include versatility, capacity to run up to 41 samples by triplicate in a single run, and reuse of the membrane at least 10 times. These features substantially reduce cost per reaction and make the assay an attractive tool for use in reference laboratories of countries that have a high burden of multidrug-resistant tuberculosis but that cannot afford expensive commercial tests because of limited resources.
Assuntos
Antituberculosos/farmacologia , Farmacorresistência Bacteriana Múltipla , Isoniazida/farmacologia , Mycobacterium tuberculosis/genética , Oligonucleotídeos , Rifampina/farmacologia , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Proteínas de Bactérias/genética , Técnicas Bacteriológicas/métodos , Técnicas Bacteriológicas/normas , Catalase/genética , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA , Humanos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/isolamento & purificação , Hibridização de Ácido Nucleico/métodos , Oxirredutases/genética , Sensibilidade e Especificidade , Tuberculose Resistente a Múltiplos Medicamentos/microbiologiaRESUMO
BACKGROUND: A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). METHODS: We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. RESULTS: Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). CONCLUSIONS: Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.
Assuntos
Farmacorresistência Bacteriana , Genes MDR , Mutação , Mycobacterium tuberculosis/genética , Filogenia , Antituberculosos/farmacologia , Clofazimina/farmacologia , Diarilquinolinas/farmacologia , Concentração Inibidora 50 , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/efeitos dos fármacosRESUMO
This study evaluated cross-resistance of Mycobacterium tuberculosis strains to ofloxacin, moxifloxacin, and gatifloxacin and investigated the presence of mutations in gyrA and gyrB. Fluoroquinolone susceptibilities were determined for 41 M. tuberculosis strains by the proportion method on 7H11, and MICs were determined by the resazurin microtiter assay. Forty strains shared the same resistance results for the three fluoroquinolones. However, one strain, with an Asn-533 --> Thr mutation in gyrB, was susceptible to ofloxacin but resistant to moxifloxacin and gatifloxacin.
Assuntos
Antibacterianos/farmacologia , DNA Girase/genética , Fluoroquinolonas/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Compostos Aza/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Gatifloxacina , Moxifloxacina , Mutação , Ofloxacino/farmacologia , Quinolinas/farmacologiaRESUMO
OBJECTIVES: The aim of this study was to establish wild-type MIC distributions of first-line drugs for Mycobacterium tuberculosis, as well as to explore the usefulness of such distributions when setting clinical breakpoints. METHODS: We determined the MICs of rifampicin, isoniazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460. RESULTS: The agreement with BACTEC460 was very high for isoniazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n = 6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4-8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%-88% among 26 laboratories, n = 4) for which the MICs were 4-8 mg/L. CONCLUSIONS: Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data.
Assuntos
Antituberculosos/farmacologia , Etambutol/farmacologia , Isoniazida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Rifampina/farmacologia , Tuberculose/microbiologia , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/isolamento & purificaçãoRESUMO
Thirty-four pyrazinamide-resistant and 37 pyrazinamide-susceptible Mycobacterium tuberculosis complex strains were analyzed for pncA gene mutations. None of the sensitive strains had any mutations, apart from silent mutations, whereas all but one resistant strain showed pncA mutations. By using sequencing as a means of early resistance detection, the inconsistency of phenotypic pyrazinamide assays can be circumvented.
Assuntos
Amidoidrolases/genética , Proteínas de Bactérias/genética , Mutação , Mycobacterium tuberculosis/efeitos dos fármacos , Pirazinamida/farmacologia , Antituberculosos/farmacologia , Análise Mutacional de DNA , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/genéticaRESUMO
The Swedish Forum for Biopreparedness Diagnostics (FBD) is a network that fosters collaboration among the 4 agencies with responsibility for the laboratory diagnostics of high-consequence pathogens, covering animal health and feed safety, food safety, public health and biodefense, and security. The aim of the network is to strengthen capabilities and capacities for diagnostics at the national biosafety level-3 (BSL-3) laboratories to improve Sweden's biopreparedness, in line with recommendations from the EU and WHO. Since forming in 2007, the FBD network has contributed to the harmonization of diagnostic methods, equipment, quality assurance protocols, and biosafety practices among the national BSL-3 laboratories. Lessons learned from the network include: (1) conducting joint projects with activities such as method development and validation, ring trials, exercises, and audits has helped to build trust and improve communication among participating agencies; (2) rotating the presidency of the network steering committee has fostered trust and commitment from all agencies involved; and (3) planning for the implementation of project outcomes is important to maintain gained competencies in the agencies over time. Contacts have now been established with national agencies of the other Nordic countries, with an aim to expanding the collaboration, broadening the network, finding synergies in new areas, strengthening the ability to share resources, and consolidating long-term financing in the context of harmonized European biopreparedness.
Assuntos
Doenças dos Animais/diagnóstico , Bioterrorismo , Qualidade de Produtos para o Consumidor , Laboratórios/normas , Segurança/normas , Doenças dos Animais/prevenção & controle , Doenças dos Animais/transmissão , Animais , Comportamento Cooperativo , Europa (Continente) , Recursos em Saúde , Humanos , Garantia da Qualidade dos Cuidados de Saúde , Países Escandinavos e Nórdicos , Suécia , Estados Unidos , Organização Mundial da SaúdeRESUMO
Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Mycobacterium tuberculosis/genética , Tuberculose Resistente a Múltiplos Medicamentos/genética , Antituberculosos/uso terapêutico , Proteínas de Bactérias/genética , Catalase/genética , Genômica/métodos , Humanos , Isoniazida/uso terapêutico , Mutação/genética , Mycobacterium tuberculosis/efeitos dos fármacos , Polimorfismo Genético/genética , Rifampina/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
Tuberculosis (TB) remains a global health problem. The increasing prevalence of drug-resistant Mycobacterium tuberculosis, the causative agent of TB, demands new measures to combat the situation. Rapid and accurate diagnosis of the pathogen and its drug susceptibility pattern is essential for timely initiation of optimal treatment, and, ultimately, control of the disease. We have developed a molecular method for detection of first- and second-line drug resistance in M. tuberculosis by Pyrosequencing(®). The method consists of seven Pyrosequencing assays for the detection of mutations in the genes or promoter regions, which are most commonly responsible for resistance to the drugs rifampicin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and fluoroquinolones. The method was validated on clinical isolates and it was shown that the sensitivity and specificity of the method were comparable to those of Sanger sequencing. In the protocol in this chapter we describe the steps necessary for setting up and performing Pyrosequencing for M. tuberculosis. The first part of the protocol describes the assay development and the second part of the protocol describes utilization of the method.
Assuntos
Farmacorresistência Bacteriana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Análise de Sequência de DNA/métodos , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/genética , Reação em Cadeia da PolimeraseRESUMO
Resistant Mycobacterium tuberculosis has become a serious threat to public health. In order to inhibit spreading and give effective treatment, it is of great importance, as early as possible, to detect drug-resistant bacteria. To evaluate the usefulness of the Line Probe Assay (INNO-LiPA(TM) Rif.TB) for rapid detection of rifampicin resistance, we used 52 clinical isolates of M. tuberculosis from the national strain collection at the Swedish Institute for Infectious Disease Control and the drug susceptible reference strain H37Rv. By using BACTEC 460 methodology, 27 of these strains were determined as resistant to rifampicin and 26 as sensitive to rifampicin. Mutations known to give resistance to rifampicin were detected by LiPA in all 27 rifampicin-resistant strains. Among the 26 susceptible strains, 24 had the wild-type pattern in LiPA, while in two, mutations were seen. The LiPA correctly identified the M. tuberculosis complex in all samples. The high accuracy and simplicity of LiPA makes it a very promising method for the early identification of rifampicin resistance in M. tuberculosis.
Assuntos
Antibióticos Antituberculose , Farmacorresistência Bacteriana/genética , Mycobacterium tuberculosis/genética , Kit de Reagentes para Diagnóstico , Rifampina , Humanos , Hibridização In Situ , Testes de Sensibilidade Microbiana , MutaçãoRESUMO
The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4mg/L, 8mg/L and 2mg/L, respectively) compared to extracellularly (MIC90: 0.5mg/L for AMI and EMB; 0.25mg/L for LEV). The reverse was the case for INH (IC: 0.064mg/L vs EC: 0.25mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs.
Assuntos
Antituberculosos/farmacologia , Luciferases/análise , Macrófagos/microbiologia , Testes de Sensibilidade Microbiana/métodos , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular , Contagem de Colônia Microbiana , Genes Reporter , Humanos , Medições Luminescentes , Macrófagos/fisiologia , Mycobacterium tuberculosis/fisiologiaRESUMO
SETTING: This study was performed in the city of Minsk in Belarus, where a very severe problem with MDR-TB was demonstrated in a recent drug resistant survey. OBJECTIVE: The aim of this study was to use molecular typing of MDR and pan-susceptible clinical isolates of Mycobacterium tuberculosis to increase the understanding of the transmission patterns and possible differences between the strains causing susceptible and drug-resistant tuberculosis. STUDY POPULATION AND METHODS: Consecutive isolates from pulmonary TB patients in Minsk were collected at the Belarusian National Reference Laboratory. Isolates found to be either pan-susceptible or MDR were included in the study, which totally comprised 81 MDR and 82 pan-susceptible clinical isolates. All isolates were characterized by spoligotyping. The major clusters were characterized using sequencing of the pncA gene. RESULTS: Three out of four MDR cases were caused by one out of two drug-resistant clones of M. tuberculosis belonging to the Beijing and T1 genotypes, respectively. A single T1 clone, SIT No. 266, found exclusively in the MDR cohort, was shown to cause no less than 30% of all MDR-TB cases. DISCUSSION: The findings indicate that the major cause of MDR-TB in Minsk is an ongoing transmission of certain already resistant M. tuberculosis strains. CONCLUSION: The significant transmission of MDR-TB in Minsk underlines the urgent need for strengthened infection control measures to limit the transmission in order to better control MDR-TB.