Quinolones are one of the largest classes of antimicrobial agents used worldwide. This review considers the quinolones that are available currently and used widely in Europe (norfoxacin, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin) within their historical perspective, while trying to position them in the context of recent and possible future advances based on an understanding of: (1) their chemical structures and how these impact on activity and toxicity; (2) resistance mechanisms (mutations in target genes, efflux pumps); (3) their pharmacodynamic properties (AUC/MIC and Cmax/MIC ratios; mutant prevention concentration and mutant selection window); and (4) epidemiological considerations (risk of emergence of resistance, clonal spread). Their main indications are examined in relation to their advantages and drawbacks. Overall, it is concluded that these important agents should be used in an educated fashion, based on a careful balance between their ease of use and efficacy vs. the risk of emerging resistance and toxicity. However, there is now substantial evidence to support use of the most potent drug at the appropriate dose whenever this is required.

Nucleic acid amplification technology is examined from the critical viewpoint of a clinical microbiologist working in a routine diagnostic bacteriology laboratory. Widely recognised limitations of amplification technology include those of false-positive and false-negative results, the difficulty of obtaining quantitative results, the problem of using this technology for susceptibility testing, and the difficulty of detecting routinely the wide range of possible pathogens contained in a clinical sample. On the positive side, amplification technology brings welcome new possibilities for rapid detection of specific pathogens in a sample, including viruses, slowly growing bacteria, fastidious o r uncultivable bacteria, fungi and protozoa. Other possible applications include screening normally sterile clinical samples for non-specific bacterial contamination and the use of amplification-based DNA fingerprinting methods for identification and typing of microorganisms. Nevertheless, it is predicted that-in contrast to research and reference facilities-routine bacteriology laboratories will continue to rely on culture as the preferred 'amplification method' for most diagnostic applications.

Interpretive reading analyses the complete resistance profiles of bacteria to multiple antibiotics and infers the resistance mechanisms present; it aids therapeutic choice and enhances surveillance data. We evaluated the Advanced Expert System (AES), which interprets MICs generated by the VITEK 2. Ten European laboratories tested 42 reference strains and 76-106 of their own strains, representing important resistance genotypes. Interpretive reading by the VITEK 2 AES achieved full agreement with genotype data for 88-89% of strains, with the correct mechanism identified as one of two possibilities for a further 5-6%. Mechanisms inferred with 90% agreement with reference data included methicillin resistance in staphylococci, glycopeptide resistance in enterococci, quinolone resistance in staphylococci and Enterobacteriaceae, AAC(6')-APH(2")-mediated aminoglycoside resistance in Gram-positive cocci, erm-mediated macrolide resistance in pneumococci, extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae and Pseudomonas aeruginosa, and acquired penicillinases in Enterobacteriaceae. VanA, VanB and VanC phenotypes of enterococci were distinguished reliably, and ESBL production was accurately inferred in AmpC-inducible species as well as Escherichia coli and Klebsiella spp. Mechanisms identified, but only as possibilities among several, included IRT-type beta-lactamases and individual aminoglycoside-modifying enzymes in Enterobacteriaceae. Most disagreements with reference data concerned pneumococci found to have high-level penicillin resistance by the VITEK 2 AES but previously determined, phenotypically, to have intermediate resistance. When ESBL production was inferred in E. coli and klebsiellae, the VITEK 2 AES edited susceptible results for cephalosporins (except cefoxitin) to resistant; when an acquired penicillinase was inferred in Enterobacteriaceae, piperacillin results were edited to resistant; and when staphylococci were found methicillin resistant, resistance was reported for all beta-lactams. Further editing may be desirable (e.g. of cephalosporin results for salmonellas inferred to have ESBLs).

The performance of the m1000 system (Abbott Laboratories, Illinois) as a front-end extraction system for high-throughput "in-house" quantitative real-time PCR assays was analyzed and compared to that of manual extraction of plasma and serum samples ( hepatitis C virus [HCV] and hepatitis B virus [HBV]) and EDTAblood samples (cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). Linearity of extraction was tested on dilution series of HCV and HBV reference materials. The correlation coefficient for standard curves based on repeated extraction runs was 0.97 ؎ 0.06 for HCV and 0.97 ؎ 0.03 for HBV, indicating a linear extraction from 100 to 1.0 ؋ 10 5 HCV IU/ml and from 100 to 1.0 ؋ 10 6 HBV IU/ml. Intra-and interrun variability was below 0.23 log 10 IU/ml for 2.98 to 5.28 log 10 HCV IU/ml and 2.70 to 5.20 log 10 HBV IU/ml. Correlation between automated and manual extraction was very good. For HCV, the correlation coefficient was 0.91 and the mean difference in viral load was 0.13 log 10 HCV IU/ml. For HBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.61 log 10 HBV IU/ml. For CMV and EBV, the correlation coefficient was 0.98 and the mean difference in viral load 0.33 log 10 copies/ml. Accuracy was confirmed with a reference panel (QCMD, Glasgow, Scotland) for all four assays. No cross-contamination was observed when extracting strongly positive polyomavirus samples (8.10 log 10 copies/ml) interspersed with polyomavirus-negative samples. Automated extraction via the m1000 system offers a high reliability of extraction and resulted in a strong reduction of the required extraction hands-on time for high-throughput PCR compared to manual extraction protocols.Real-time thermocyclers have greatly decreased the amount of hands-on time in nucleic acid (NA)-based diagnostics for pathogens. The use of commercially available universal master mixes that contain all reagents except the pathogen-specific primer-probe combination has further decreased the number of pipetting steps and thus reduced labor and the possibility of errors.NA extraction has now become the most critical and laborintensive step in NA-based diagnostic assays. The overall sensitivity of the assay is determined by the NA yield, its purity, and the amount of sample equivalents that can be transferred into the amplification reaction. Conventional manual sample preparation methods are labor intensive and susceptible to contamination, handling variations, or errors (3, 9, 12).Since both the pathogen range and the number of different sample types are expanding and since multiplex downstream testing is becoming standard practice, there is a need for a generic extraction method (2, 5, 10). Ideally, the NA extraction procedure should yield pure NA from different pathogens and from a broad range of sample types.Recently, Abbott introduced m1000, an automated generic RNA and DNA extraction system using magnetic microparticle processing (17, 18). The m1000 system provides the necessary features for full automation of NA extraction of up to...