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Since numbers of drug-resistant Mycobacterium tuberculosis strains are on the rise, the simple classification into "susceptible" and "resistant" strains based on susceptibility testing at "critical concentrations" has to be reconsidered. While future studies have to address the correlation of phenotypic resistance levels and treatment outcomes, a prerequisite for corresponding investigations is the ability to exactly determine levels of quantitative drug resistance in clinical M. tuberculosis isolates. Here we have established the conditions for quantitative drug susceptibility testing for first-and second-line agents using MGIT 960 instrumentation and EpiCenter software equipped with the TB eXiST module. In-depth comparative analysis of a range of wellcharacterized susceptible and resistant clinical isolates has allowed us to propose conditions for testing and to develop criteria for interpretation.Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Developing countries are the most vulnerable, with more than 95% of the cases (32, 33). The present trend is characterized by an alarming emergence of drug resistance (7,8,30). Much attention has focused on the burden of multidrug-resistant (MDR) TB, i.e., resistance to the firstline drugs isoniazid and rifampin (rifampicin) (32,33), and the emergence of extensively drug resistant TB (6, 26). The rise of drug-resistant TB and the increased susceptibility of the human population to TB due to coinfection with human immunodeficiency virus are driving the worldwide TB pandemic and will worsen the situation in the years ahead, with devastating effects in poor countries, whose economies suffer most from this development (19,20).In the diagnostic laboratory, testing of mycobacteria for drug susceptibility is substantially different from the general testing procedures used in bacteriology. Rather than determining MICs, a single drug concentration, termed the critical concentration, is usually used to categorize a clinical isolate as susceptible or resistant. This "critical concentration" is more an epidemiological parameter (to distinguish "wild-type" strains from "non-wild-type" strains that are able to grow in the presence of higher drug concentrations [5]) than a clinical cutoff value established to guide treatment decisions (14). With growing knowledge about the mechanisms that underlie drug resistance, it has become evident that drug resistance is multifaceted and that different mutations may lead to different levels of resistance. The acquisition of a resistance mutation leading to a decrease in drug susceptibility should not inevitably exclude an anti-TB drug from a treatment regimen, since low-level resistance does not necessarily imply clinical resistance (3). However, until now, different levels of phenotypic resistance have only rarely been taken into account in the procedures used for in vitro drug susceptibility testing (DST) of mycobacteria (4)."Critical concentration"-based DST of primary and secondary drugs has been established for the...

Universal surveillance upon patient admission is important in reducing the transmission of methicillinresistant Staphylococcus aureus (MRSA) and associated disease in hospitals. High costs for the health care system in conjunction with MRSA have promoted the development of rapid screening methods to detect MRSA carriers. This study compared two real-time PCR methods, the BD GeneOhm MRSA assay (BDGO) and the Xpert MRSA assay, with broth-enriched culture to define their performance characteristics and rapidity in an area with low MRSA prevalence. In total, 414 swabs from the nose and 389 swabs from the groin from 425 patients were tested. Of those 425 patients, 378 had swabs from both the nose and groin in parallel. Two hundred thirty-one and 194 patients were randomly assigned to the BDGO group and the Xpert MRSA group, respectively. In general, sensitivity, specificity, and negative predictive value (NPV) were high for the BDGO (100%, 98.5%, and 100%, respectively) and the Xpert MRSA (100%, 98.2%, and 100%, respectively), irrespective of whether or not nasal and inguinal specimens were considered alone or combined. In contrast, the positive predictive value (PPV) was lower: before the resolution of discrepant results, the PPVs for nasal and inguinal specimens alone and combined were 87.5%, 86.7%, and 82.4% for the BDGO and 91.7%, 66.7%, and 92.9% for the Xpert MRSA, respectively. After the resolution of discrepant results, PPVs were 93.8%, 93.3% and 94.1% for the BDGO and 91.7%, 88.9% and 92.9% for the Xpert MRSA, respectively. With the BDGO, 4 of 16 carriers were each identified by nasal or inguinal swabs alone, whereas in the Xpert MRSA group, 4 of 13 carriers were exclusively identified by nasal swabs and 2 of 13 were identified by inguinal swabs alone. Both PCR methods showed no significant difference in the number of discrepant results (odds ratio, 0.70 [P ‫؍‬ 0.789]), but specimens from wounds and other body sites (axilla, vagina, and throat) produced discrepancies more often than nasal and groin specimens (odds ratios, 4.724 [P ‫؍‬ 0.058] and 12.163 [P < 0.001], respectively). The facts that no false-negative PCR results were detected and increased PPVs were found after the resolution of discrepant results point to PCR as the actual gold standard. Since both sensitivity and NPV were exceptionally high for PCR, backup cultures may, therefore, be unnecessary in an area with low prevalence and with a preemptive isolation strategy but may still be useful for PCR-positive specimens because of the lower PPV for both methods and the possibility of susceptibility testing. The median time for analysis, including extraction, hands-on time, and actual PCR was 2 h 20 min for the Xpert MRSA versus 5 h 40 min for the BDGO. Concerning reporting time, including administration and specimen collection, the Xpert MRSA was faster than the BDGO (7 h 50 min versus 17 h).

The implementation of internal transcribed spacer (ITS) sequencing for routine identification of molds in the diagnostic mycology laboratory was analyzed in a 5-year study. All mold isolates (n ‫؍‬ 6,900) recovered in our laboratory from 2005 to 2009 were included in this study. According to a defined work flow, which in addition to troublesome phenotypic identification takes clinical relevance into account, 233 isolates were subjected to ITS sequence analysis. Sequencing resulted in successful identification for 78.6% of the analyzed isolates (57.1% at species level, 21.5% at genus level). In comparison, extended in-depth phenotypic characterization of the isolates subjected to sequencing achieved taxonomic assignment for 47.6% of these, with a mere 13.3% at species level. Optimization of DNA extraction further improved the efficacy of molecular identification. This study is the first of its kind to testify to the systematic implementation of sequence-based identification procedures in the routine workup of mold isolates in the diagnostic mycology laboratory.

cThe 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.