Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents.

Revisiting susceptibility testing in MDR-TB by a standardized quantitative phenotypic assessment in a European multicentre study.

OBJECTIVES: Treatment outcome of MDR-TB is critically dependent on the proper use of second-line drugs as per the result of in vitro drug susceptibility testing (DST). We aimed to establish a standardized DST procedure based on quantitative determination of drug resistance and compared the results with those of genotypes associated with drug resistance. METHODS: The protocol, based on MGIT 960 and the TB eXiST software, was evaluated in nine European reference laboratories. Resistance detection at a screening drug concentration was followed by determination of resistance levels and estimation of the resistance proportion. Mutations in 14 gene regions were investigated using established techniques. RESULTS: A total of 139 Mycobacterium tuberculosis isolates from patients with MDR-TB and resistance beyond MDR-TB were tested for 13 antituberculous drugs: isoniazid, rifampicin, rifabutin, ethambutol, pyrazinamide, streptomycin, para-aminosalicylic acid, ethionamide, amikacin, capreomycin, ofloxacin, moxifloxacin and linezolid. Concordance between phenotypic and genotypic resistance was >80%, except for ethambutol. Time to results was short (median 10 days). High-level resistance, which precludes the therapeutic use of an antituberculous drug, was observed in 49% of the isolates. The finding of a low or intermediate resistance level in 16% and 35% of the isolates, respectively, may help in designing an efficient personalized regimen for the treatment of MDR-TB patients. CONCLUSIONS: The automated DST procedure permits accurate and rapid quantitative resistance profiling of first- and second-line antituberculous drugs. Prospective validation is warranted to determine the impact on patient care.

Transmission of multidrug-resistant tuberculosis in a low-incidence setting, Switzerland, 2006 to 2012.

The goal of the present study was to examine the transmission dynamics of multidrug-resistant tuberculosis (MDR-TB) in Switzerland. Between 2006 and 2012, a total of 49 MDR-TB cases were reported to the Swiss Federal Office of Public Health, 46 of which were of foreign origin. All 49 initial strains were evaluated by molecular epidemiologic methods at the Swiss National Reference Centre for Mycobacteria. In 43 strains, unique DNA fingerprint patterns were identified. Twelve strains were grouped into six clusters. Data from contact tracing suggest likely in-country transmission in four clusters, mostly among close contacts. In the remaining two clusters, no contact tracing data were available, but the identified genotypes were known to be prevalent in the countries of origin of the patients, suggesting the possibility that the infection was acquired there. While most MDR-TB cases are imported to Switzerland, at least four of the 49 MDR-TB cases were due to transmission within the country. The imported cases, however, did not lead to secondary cases outside the circles of close contacts. The results also indicate that prevention of MDR-TB transmission among immigrants may require closer monitoring.

Evaluation of the AID TB resistance line probe assay for rapid detection of genetic alterations associated with drug resistance in Mycobacterium tuberculosis strains.

The rapid accurate detection of drug resistance mutations in Mycobacterium tuberculosis is essential for optimizing the treatment of tuberculosis and limiting the emergence and spread of drug-resistant strains. The TB Resistance line probe assay from Autoimmun Diagnostika GmbH (AID) (Strassburg, Germany) was designed to detect the most prevalent mutations that confer resistance to isoniazid, rifampin, streptomycin, amikacin, capreomycin, fluoroquinolones, and ethambutol. This assay detected resistance mutations in clinical M. tuberculosis isolates from areas with low and high levels of endemicity (Switzerland, n=104; South Africa, n=52) and in selected Mycobacterium bovis BCG 1721 mutant strains (n=5) with 100% accuracy. Subsequently, the line probe assay was shown to be capable of rapid genetic assessment of drug resistance in MGIT broth cultures, the results of which were in 100% agreement with those of DNA sequencing and phenotypic drug susceptibility testing. Finally, the line probe assay was assessed for direct screening of smear-positive clinical specimens. Screening of 98 clinical specimens demonstrated that the test gave interpretable results for >95% of them. Antibiotic resistance mutations detected in the clinical samples were confirmed by DNA sequencing. We conclude that the AID TB Resistance line probe assay is an accurate tool for the rapid detection of resistance mutations in cultured isolates and in smear-positive clinical specimens.

The critical influence of the intermediate category on interpretation errors in revised EUCAST and CLSI antimicrobial susceptibility testing guidelines.

Erroneous assignments of clinical isolates to the interpretative categories susceptible, intermediate and resistant can deprive a patient of successful antimicrobial therapy. The rate of major errors (ME) and very major errors (vME) is dependent on: (i) the precision/standard deviation (σ) of the antibiotic susceptibility testing (AST) method, (ii) the diameter distributions, (iii) clinical breakpoints, and (iv) the width of the intermediate zone. The European Committee on AST (EUCAST) has abandoned or decreased the intermediate zone for several drug/species combinations. This study focused on the effects of discontinuing the intermediate category on the rate of interpretation errors. In total, 10,341 non-duplicate clinical isolates were included in the study. For susceptibility testing the disc diffusion method was used. Error probabilities were calculated separately for diameter values flanking the interpretative category borders. Error probabilities were then applied to the actual numbers of clinical isolates investigated and expected rates of ME and vME were calculated. Applying EUCAST AST guidelines, significant rates of ME/vME were demonstrated for all drug/species combinations without an intermediate range. Virtually all ME/vME expected were eliminated in CLSI guidelines that retained an intermediate zone. If wild-type and resistant isolates are not clearly separated in susceptibility distributions, the retaining of an intermediate zone will decrease the number of ME and vME. An intermediate zone of 2-3 mm avoids almost all ME/vME for most species/drug combinations depending on diameter distributions. Laboratories should know their epidemiology settings to be able to detect problems of individual species/drug/clinical breakpoint combinations and take measures to improve precision of diameter measurements.

Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

Evaluation of a diagnostic flow chart for detection and confirmation of extended spectrum ß-lactamases (ESBL) in Enterobacteriaceae.

This study aimed to develop a modular, diagnostic algorithm for extended spectrum ß-lactamase (ESBL) detection in Enterobacteriaceae. Clinical Enterobacteriaceae strains (n = 2518) were screened for ESBL production using Clinical and Laboratory Standards Institute (CLSI) breakpoints for third-generation cephalosporins and by synergy image detection (clavulanic acid/extended-spectrum cephalosporins). Isolates screening positive for ESBL (n = 242, 108 by critical CLSI diameters alone, five by double disk synergy test (DDST) alone, and 129 by both critical diameters and DDST) and 138 ESBL screening negative isolates (control group) were investigated by molecular methods considered to be the reference standard (multiplex CTX-M type PCR, TEM and SHV type sequence characterization). One hundred and twenty-four out of 242 Enterobacteriaceae isolates screening positive for ESBL were confirmed to be ESBL positive by the reference standard, the majority of them in E. coli, K. pneumoniae and E. cloacae (94, 17 and nine isolates, respectively). Prevalence of ESBL production ranged from <1% for P. mirabilis to 4.7%, 5.1% and 6.6%, for K. pneumoniae, E. cloacae and E. coli, respectively. Combining CLSI ceftriaxone and cefpodoxime critical ESBL diameters was found to be the most sensitive phenotypic screening method (sensitivity 99.2%). Combining critical diameters of cefpodoxime and ceftriaxone with DDST for cefpodoxime resulted in a sensitivity of 100%. For phenotypic confirmation, combining the CLSI recommended combined disk test (CDT) for ceftazidime and cefotaxime amended with a cefepime CDT was highly sensitive (100%) and specific (97.5%). With respect to the studied population, the diagnostic ESBL algorithm developed would have resulted in sensitivity and specificity of 100%. The corresponding flow chart is simple, easy to use, inexpensive and applicable in the routine diagnostic laboratory.

The ins and outs of Mycobacterium tuberculosis drug susceptibility testing.

Drug susceptibility testing of Mycobacterium tuberculosis in the diagnostic laboratory classifies clinical isolates as either drug-'resistant' or drug-'susceptible', on the basis of their ability to grow in the presence of a 'critical concentration' of the test compound. From knowledge of the mechanisms that underlie drug resistance, it has become evident that drug resistance in M. tuberculosis is quite heterogeneous and involves low-level, moderate-level and high-level drug resistance phenotypes. Different mutations are associated with different levels of phenotypic resistance, and the acquisition of a genetic alteration leading to a decrease in drug susceptibility does not inevitably exclude the affected compound from treatment regimens. As a result, the simple categorization of clinical M. tuberculosis isolates as 'resistant' on the basis of susceptibility testing at 'critical concentrations' may need to be revised and supplemented by quantitative measures of resistance testing to reflect the biological complexity of drug resistance, with the view of optimally exploiting the compounds available for treatment.

Detection of AmpC beta-lactamase in Escherichia coli: comparison of three phenotypic confirmation assays and genetic analysis.

Two mechanisms account for AmpC activity in Escherichia coli, namely, mutations in the ampC promoter and attenuator regions resulting in ampC overexpression and acquisition of plasmid-carried ampC genes. In this study, we analyzed 51 clinical E. coli isolates with reduced susceptibility to amoxicillin-clavulanic acid, piperacillin-tazobactam, or extended-spectrum cephalosporins for the presence of AmpC production. Three phenotypic AmpC confirmation assays (cefoxitin-cloxacillin disk diffusion test, cefoxitin-EDTA disk diffusion test, and AmpC Etest) were compared for the detection of AmpC activity. All 51 isolates were characterized genetically by mutational analysis of the chromosomal ampC promoter/attenuator region and by PCR detection of plasmid-carried ampC genes. Altogether, 21/51 (41%) E. coli isolates were considered true AmpC producers. AmpC activity due to chromosomal ampC promoter/attenuator mutations was found in 12/21 strains, and plasmid-carried ampC genes were detected in 8/21 isolates. One strain contained both ampC promoter mutations and a plasmid-carried ampC gene. All three phenotypic tests were able to detect the majority (>90%) of AmpC-positive strains correctly. Cefoxitin resistance was found to be a discriminative parameter, detecting 20/21 AmpC-producing strains. Susceptibility to extended-spectrum cephalosporins, e.g., ceftriaxone, ceftazidime, and cefotaxime, was found in 9 of the 21 AmpC-positive strains. Considering the elevated zone diameter breakpoints of the 2010 CLSI guidelines, 2/21 AmpC-positive strains were categorized as susceptible to extended-spectrum cephalosporins.

[Selected interventional methods for the treatment of chronic pain : part 2: regional anesthetic techniques close to the spinal cord and neuromodulative methods]. / Ausgewählte interventionelle Verfahren zur Behandlung chronischer Schmerzen : Teil 2: rückenmarknahe und neuromodulative Verfahren.

Approximately 5-8 million people in Germany suffer from chronic pain and some patients can profit from specific interventional techniques. In detail these are regional anesthetic techniques close to the spinal cord, neuromodulation, blocks of the sympathetic chain and peripheral nerve blocks. Part 2 of the article presents regional anesthetic techniques close to the spinal cord and neuromodulative methods. Regional anesthetic techniques close to the spinal cord are of high importance for the treatment of chronic low back pain although the efficiency is highly disputed due to the lack of evidence. Neuromodulation includes amongst others intrathecal pharmacotherapy and spinal cord stimulation, which are used for highly selected patients and can lead to very good results for these patients.

[Selected interventional methods for the treatment of chronic pain: Part 1: peripheral nerve block and sympathetic block]. / Ausgewählte interventionelle Verfahren zur Behandlung chronischer Schmerzen : Teil 1: periphere Nervenblockade und Sympathikusblockade.

Approximately 5-8 million people in Germany suffer from chronic pain. Some patients can obtain relief from specific interventional techniques. In detail these are blocks of the sympathetic chain and peripheral nerve blocks, regional anesthetic techniques close to the spinal cord and neuromodulation. Part 1 of this article presents peripheral nerve blocks using the example of intercostal blocks and blocks of the sympathetic chain. Peripheral nerve blocks are important for postoperative pain treatment. Only a few methods are used for chronic pain and this applies primarily to the intercostal block which is used for the treatment of pain occurring after thoracotomy, intercostal neuralgia and pain associated with infiltration of cancer. Blocks of the vegetative nervous system are accomplished on the ganglions of the head and the sympathetic chain and are therefore most commonly applied to treat headache, neuropathic and sympathetic pain in the area of abdomen and the extremities.

Detection and identification of Mycobacterium spp. in clinical specimens by combining the Roche Cobas Amplicor Mycobacterium tuberculosis assay with Mycobacterium genus detection and nucleic acid sequencing.

We have recently developed a PCR assay for detection of Mycobacterium spp. at the genus level based on the Cobas Amplicor platform. The sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. The specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. In a follow-up study, we have systematically evaluated the Mycobacterium genus assay in parallel with the Cobas Amplicor Mycobacterium tuberculosis assay on 2,169 clinical specimens, including respiratory and nonrespiratory specimens. Based on the genus assay, nontuberculous mycobacteria were readily detected and identified to the species level by PCR-mediated sequencing. In addition, our data point to a limited specificity of the Cobas Amplicor M. tuberculosis assay.

Development and evaluation of a molecular assay for detection of nontuberculous mycobacteria by use of the cobas amplicor platform.

We have developed and evaluated a semiautomated assay for detection of nontuberculous mycobacteria (NTM) from clinical samples based on the Cobas Amplicor Mycobacterium tuberculosis test (Roche Diagnostics, Switzerland). A capture probe, specific for mycobacteria at the genus level, was linked to magnetic beads and used for the detection of amplification products obtained by the Cobas Amplicor M. tuberculosis assay. We demonstrate that the analytical sensitivity of the genus assay is similar to that of Cobas Amplicor M. tuberculosis detection. Four hundred sixteen clinical specimens were evaluated for the presence of NTM DNA. Sensitivities for smear-positive and smear-negative specimens were found to be 100% and 47.9%, respectively. Specificity was 97.7%, the positive predictive value 84.6%, and the negative predictive value 93.1%. The genus assay is easy to perform, produces reliable results, and was found to be a valuable diagnostic tool for rapid diagnosis of infections with NTM. The genus assay has the potential to detect NTM not routinely recovered by culture and to discover new mycobacterial species.

Evaluation of the colorimetric VITEK 2 card for identification of gram-negative nonfermentative rods: comparison to 16S rRNA gene sequencing.

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than Achromobacter xylosoxidans, Acinetobacter sp., Burkholderia cepacia complex, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.

A genetic model to investigate drug-target interactions at the ribosomal decoding site.

Recent advances in X-ray crystallography have greatly contributed to the understanding of the structural interactions between aminoglycosides and the ribosomal decoding site. Efforts to genetically probe the functional relevance of proposed drug-nucleotide contacts have in part been hampered by the presence of multiple rRNA operons in most bacteria. A derivative of the Gram-positive Mycobacterium smegmatis was rendered single rRNA operon allelic by means of gene inactivation techniques. In this system, genetic manipulation of the single chromosomal rRNA operon results in cells carrying homogeneous populations of mutant ribosomes. An exhaustive mutagenesis study of the ribosomal A site has been performed to define the importance of individual drug-nucleotide contacts. Mutational alterations in the M. smegmatis decoding site are discussed here, comparing the results with those obtained in other organisms. Implications for the selectivity of antimicrobial agents and for the fitness cost of resistance mutations are addressed.

16S rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB card for identification of nonfermenting Gram-negative bacteria in the clinical laboratory.

Over a period of 26 months, we have evaluated in a prospective fashion the use of 16S rRNA gene sequencing as a means of identifying clinically relevant isolates of nonfermenting gram-negative bacilli (non-Pseudomonas aeruginosa) in the microbiology laboratory. The study was designed to compare phenotypic with molecular identification. Results of molecular analyses were compared with two commercially available identification systems (API 20 NE, VITEK 2 fluorescent card; bioMérieux, Marcy l'Etoile, France). By 16S rRNA gene sequence analyses, 92% of the isolates were assigned to species level and 8% to genus level. Using API 20 NE, 54% of the isolates were assigned to species and 7% to genus level, and 39% of the isolates could not be discriminated at any taxonomic level. The respective numbers for VITEK 2 were 53%, 1%, and 46%, respectively. Fifteen percent and 43% of the isolates corresponded to species not included in the API 20 NE and VITEK 2 databases, respectively. We conclude that 16S rRNA gene sequencing is an effective means for the identification of clinically relevant nonfermenting gram-negative bacilli. Based on our experience, we propose an algorithm for proper identification of nonfermenting gram-negative bacilli in the diagnostic laboratory.

Internal transcribed spacer sequencing versus biochemical profiling for identification of medically important yeasts.

In this study, we established an in-house database of yeast internal transcribed spacer (ITS) sequences. This database includes medically important as well as colonizing yeasts that frequently occur in the diagnostic laboratory. In a prospective study, we compared molecular identification with phenotypic identification by using the ID32C system (bioMérieux) for yeast strains that could not be identified by a combination of CHROMagar Candida and morphology on rice agar. In total, 113 yeast strains were included in the study. By sequence analysis, 98% of all strains were identified correctly to the species level. With the ID32C, 87% of all strains were identified correctly to the species or genus level, 7% of the isolates could not be identified, and 6% of the isolates were misidentified, most of them as Candida rugosa or Candida utilis. For a diagnostic algorithm, we suggest a three-step procedure which integrates morphological criteria, biochemical investigation, and sequence analysis of the ITS region.

Mutagenesis of 16S rRNA C1409-G1491 base-pair differentiates between 6'OH and 6'NH3+ aminoglycosides.

Using a single rRNA allelic Gram-positive model system, we systematically mutagenized 16S rRNA positions 1409 and 1491 to probe the functional relevance of structural interactions between aminoglycoside antibiotics and the A-site rRNA that were suggested by X-ray crystallography. At the structural level, the interaction of the 2-deoxystreptamine aminoglycosides with the rRNA base-pair C1409-G1491 has been suggested to involve the following features: (i) ring I of the disubstituted 2-deoxystreptamines stacks upon G1491 and H-bonds to the Watson-Crick edge of A1408; (ii) ring III of the 4,5-disubstituted aminoglycosides shows hydrogen bonding to G1491. However, we found that mutants with altered 16S rRNA bases 1409 and 1491 discriminated poorly between 4,5-disubstituted and 4,6-disubstituted 2-deoxystreptamines, but differentially affected aminoglycosides with a hydroxyl group versus an ammonium group at position 6' of ring I, e.g. G1491U conferred high-level drug resistance to paromomycin and geneticin, but not to neomycin, tobramycin or gentamicin.