Sixty-four of 85 (75.3%) smear-negative respiratory (n ‫؍‬ 78) and nonrespiratory (n ‫؍‬ 7) samples with positive cultures of Mycobacterium tuberculosis complex (MTC) were detected by the GeneXpert system using the Xpert MTB/RIF assay (GX). In addition, GX found rpoB mutations in all six of the rifampin-resistant strains detected. The test was negative in 20 culture-negative and 20 nontuberculous culture-positive samples (100% specificity). GX offers high potential for the diagnosis of tuberculosis due to its capacity for direct detection of MTC, its rapidity, and its simplicity.Tuberculosis (TB) remains one of the biggest health problems in developing and industrialized countries and is associated with high rates of morbidity and mortality (19). The emergence and spread of Mycobacterium tuberculosis strains resistant to multiple drugs represent a serious threat to TB control worldwide (18). Early diagnosis of active TB and detection of multidrug-resistant (MDR) strains are essential to interrupt transmission. Traditionally, acid-fast bacillus (AFB) smear microscopy has been the initial method for diagnosis, due to its speed, simplicity, and low cost. However, its low sensitivity (45 to 80% of positive cultures), as well as the fact that a significant percentage (17%) of bacillus transmission is due to smear-negative pulmonary cases, limits the usefulness of this technique (1). On the other hand, due to the intrinsic slow growth of these microorganisms, mycobacterial culture (the gold standard method for TB diagnosis) takes several weeks to provide microbiological confirmation. Therefore, other techniques are needed to reduce turnaround time to diagnosis (13,16).In recent years, direct-detection methodologies, most of them based on nucleic acid amplification, have appeared as potentially useful tools for the rapid diagnosis of TB (7)(8)(9)(11)(12)(13)(14). Guidelines for their use have been defined and recently updated (6).A real-time automated integrated system, the GeneXpert system using the Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) (GX), has recently been developed and evaluated (2, 3, 10). GX integrates DNA extraction, genomic amplification (heminested PCR), semiquantitative detection of M. tuberculosis complex (MTC), and rifampin (RIF) resistance determination in a single cartridge, thus reducing labor time and crosscontamination risk.The aim of the present study was to evaluate the effectiveness of GX for direct detection of MTC and RIF resistance in smear-negative clinical respiratory and nonrespiratory samples.One hundred twenty-five smear-negative clinical samples from 122 patients, collected over a 10-year period, were retrospectively studied; 85 samples had MTC-positive cultures, 20 samples (all respiratory) had isolates of nontuberculous mycobacteria (5 rapidly growing mycobacteria, 4 Mycobacterium avium-intracellulare complex isolates, 3 Mycobacterium kansasii isolates, and 8 others), and 20 samples had negative mycobacterial cultures (Table 1). Nonsterile clinical samples were pretreated accordi...

In the absence of availability of molecular biology techniques, the cefoxitin disc was the best predictor of methicillin resistance in S. aureus from among the techniques tested.

During the course of a molecular epidemiology study of mechanisms of antibiotic resistance in the area served by our hospital (516,000 inhabitants), we isolated the gene encoding CTX-M-14 ␤-lactamase. Thirty clinical strains (27 Escherichia coli and 3 Klebsiella pneumoniae isolates) with a phenotype of extended-spectrum ␤-lactamase were collected from January to October 2001 and studied for the presence of the CTX-M-14 ␤-lactamase gene. By isoelectric point determination, PCR, and nucleotide sequencing, we detected the presence of this gene in 17 E. coli strains belonging to 15 different genotypes (REP-PCR) causing infections in 17 different patients. Epidemiological studies based on medical records did not suggest any relationship between the patients infected with these E. coli strains and, interestingly, 7 of 30 patients harboring strains with extended-spectrum ␤-lactamases never had contact with the hospital environment before the clinical E. coli isolation. Conjugation experiments revealed that this gene was plasmid mediated in the 17 E. coli strains, and plasmid restriction fragment length polymorphisms showed 9 different patterns in the 17 E. coli strains. By PCR, the sequence of the tnpA transposase gene of the insert sequence ISEcp-1 was detected in all the plasmids harboring the CTX-M-14 gene. These results strongly suggest that plasmid dissemination between different E. coli strains in addition to a mobile element (transposon) around the ␤-lactamase gene may be involved in the spreading of the CTX-M-14 gene. This study reinforces the hypothesis that the epidemiology of the prevalence of the ␤-lactamase genes is changing and should alert the medical community to the increase in the emergence of the CTX-M ␤-lactamases worldwide.

A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (bla CTX-M-32 ) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis