Comparison of the Vitek Gram-Positive Susceptibility 106 card and the MRSA-screen latex agglutination test for determining oxacillin resistance in clinical bloodstream isolates of Staphylococcus aureus.

The Vitek automated susceptibility testing system with a modified Gram-Positive Susceptibility (GPS) 106 Card (bioMerieux Vitek, Inc., Hazelwood, Mo.) and a rapid slide latex agglutination test (MRSA-Screen; Denka Seiken Co., Ltd., Tokyo, Japan) were evaluated for their ability to detect oxacillin resistance in Staphylococcus aureus. The oxacillin-salt agar screen (OS) test, the reference broth microdilution method, and the detection of the mecA gene by PCR were compared with the commercial products. A total of 200 contemporary (1999) bloodstream infection isolates were collected from the SENTRY Antimicrobial Surveillance Program, representing diverse geographic areas throughout the world. Among the 99 mecA-positive isolates, 3 isolates were found negative by the MRSA-Screen. Another two isolates did not grow on OS plates and had MICs of 0.5 and 2 microg/ml with the Vitek GPS card. All 101 mecA-negative isolates were also found negative by the MRSA-Screen and were categorized as susceptible by the GPS card. Overall, the MRSA-Screen, GPS card, and OS test had sensitivities of 96.9, 98.0, and 98.0% and specificities of 100.0, 100.0, and 98.0%, respectively. MRSA-Screen was a rapid (

Occurrence of single-point gyrA mutations among ciprofloxacin-susceptible Escherichia coli isolates causing urinary tract infections in Latin America.

To detect if isolates susceptible to quinolones already carry mutations in the gyrA and parC genes, we selected 12 ciprofloxacin-susceptible Escherichia coli strains collected from patients with urinary tract infections in Latin America in 1998, as part of ongoing SENTRY Antimicrobial Surveillance Program. The isolates studied exhibited minimal inhibitory concentrations (MICs) for ciprofloxacin between < or = 0.015 microg/mL and 0.5 microg/mL. The molecular characterization of quinolone resistance was determinated by amplification of the gyrA and parC by PCR followed by sequencing of the respective amplicons. We observed that E. coli isolates exhibiting MIC, < or = 0.06 microg/mL for ciprofloxacin did not show mutations in either topoisomerase. On the other hand, all isolates with MIC between 0.12 microg/mL and 0.5 microg/mL demonstrated single mutation in the gyrA gene. The most frequent mutation occurred at position 83, where the amino acid serine was replaced by leucine. No mutations in the parC gene were observed. To preserve the potency and prevent the development of resistance, we suggest that quinolone usage should be rational, especially in the treatment of urinary tract infections, and in the prophylaxis of immunosupressed patient populations.

Activity and spectrum of 22 antimicrobial agents tested against urinary tract infection pathogens in hospitalized patients in Latin America: report from the second year of the SENTRY antimicrobial surveillance program (1998).

The potency and spectrum of various antimicrobial agents tested against 434 bacterial isolates causing urinary tract infection (UTI) in hospitalized patients in Latin America were evaluated. The genotypes of the extended-spectrum beta-lactamase-producing and selected multi-resistant isolates were also evaluated by molecular typing techniques. Escherichia coli (60.4%) was the most common aetiological agent causing UTI, followed by Klebsiella spp. (11.2%) and Pseudomonas aeruginosa (8.3%). In contrast, Enterococcus spp. isolates caused only 2.3% of UTIs. Fewer than 50% of E. coli isolates were susceptible to broad-spectrum penicillins. The resistance rates to ciprofloxacin and the new quinolones were also high among these isolates. The molecular characterization of ciprofloxacin-resistant E. coli showed that most of them have a double mutation in the gyrA gene associated with a single mutation in the parC gene. The Klebsiella pneumoniae isolates studied demonstrated high resistance rates to beta-lactam drugs, including broad-spectrum cephalosporins. The carbapenems were the compounds with the highest susceptibility rate among these isolates (100.0% susceptible) followed by cefepime (91.7% susceptible). Meropenem, imipenem and cefepime were also the most active drugs against Enterobacter spp. Among P. aeruginosa isolates, meropenem (MIC(50), 2 mg/L) was the most active compound, followed by imipenem (MIC(50), 4 mg/L), cefepime (MIC(50), 8 mg/L) and ceftazidime (MIC(50), 16 mg/L). The results presented in this report confirm that bacterial resistance continues to be a great problem in Latin American medical institutions.

Use of molecular and reference susceptibility testing methods in a multicenter evaluation of MicroScan dried overnight gram-positive MIC panels for detection of vancomycin and high-level aminoglycoside resistances in enterococci.

Modified MicroScan gram-positive MIC no. 8 panels (PM-8) were analyzed for their improved ability to detect vancomycin resistance (VR) and high-level aminoglycoside resistance (HLAR) in enterococci. A validation study design that utilized selected challenge strains, recent clinical isolates, and reproducibility experiments in a multicenter format was selected. Three independent medical centers compared the commercial panels to reference broth microdilution panels (RBM) and Synergy Quad Agar (QA). Resistance was verified by demonstration of VR and HLAR genes by PCR tests. The study was conducted in three phases. (i) In the challenge phase (CP), two well-characterized sets of enterococci were obtained from the Centers for Disease Control and Prevention; one set contained 50 isolates for VR testing and one contained 48 isolates for HLAR testing. In addition, a set of 47 well-characterized isolates representing diverse geographic areas, obtained from earlier national surveillance studies, was tested at the University of Iowa College of Medicine (UICM). (ii) In the efficacy phase (EP), each laboratory tested 50 recent, unique clinical isolates by all methods. (iii) In the reproducibility Phase (RP), each laboratory tested the same 10 strains by all methods in triplicate on three separate days. All isolates from the EP were sent to the UICM for molecular characterization of vanA, -B, -C1, -C2-3, and HLAR genes. In the CP, the ranking of test methods by error rates (in parentheses; very major and major errors combined, versus PCR results) were as follows: for high-level streptomycin resistance (HLSR), QA (12.0%) > PM-8 (5.2%) > RBM (1.6%); for high-level gentamicin resistance (HLGR), RBM (3.7%) > PM-8 (3.1%) > QA (2.6%); and for VR, RBM = QA (3.0%) > PM-8 (1.2%). In the EP, agreement between all methods and the reference PCR result was 98.0% for HLSR, 99.3% for HLGR, and 98. 6% for VR. In the RP, the percentages of results +/- 1 log2 dilution of the all-participant mode were as follows: for VR, 100% (PM-8), 98.9% (QA), and 90.0% (RBM); for HLSR, 99.6% (RBM), 98.5% (PM-8), and 82.2% (QA); and for HLGR, 99.6% (RBM), 99.3% (PM-8), and 98.1% (QA). The ability of the PM-8 to detect VR and HLAR in enterococci was comparable to those for reference susceptibility and molecular PCR methods and was considered acceptable for routine clinical laboratory use.

Staphylococcus aureus and coagulase-negative staphylococci from blood stream infections: frequency of occurrence, antimicrobial susceptibility, and molecular (mecA) characterization of oxacillin resistance in the SCOPE program.

Staphylococci are major causes of nosocomial blood stream infection. The recently completed SCOPE Surveillance Program found that coagulase-negative staphylococci (CoNS) and Staphylococcus aureus were the first and second most common etiologic agents, respectively, causing nosocomial blood stream infection in the USA. The frequency of oxacillin resistance was 68% among 1553 strains of CoNS and 26% among 787 strains of S. aureus in this study. Extended susceptibility profiles were generated for a subset of 150 S. aureus and 300 CoNS against 16 antimicrobial agents. Oxacillin-susceptible strains of both CoNS and S. aureus were uniformly susceptible to beta-lactam agents with the exception of ampicillin and penicillin. Oxacillin-susceptible S. aureus were also highly susceptible to the fluoroquinolones, aminoglycosides, and trimethoprim/sulfamethoxazole. The oxacillin-susceptible CoNS were less susceptible to these agents, and only glycopeptides were reliably active against oxacillin-resistant strains. PCR detection of the mecA gene was used to scrutinize current NCCLS interpretive breakpoint MICs for determining susceptibility or resistance to oxacillin. We found complete concordance between the presence or absence of mecA and the NCCLS oxacillin interpretive breakpoint categories for S. aureus. In contrast, the NCCLS breakpoints for oxacillin significantly underestimate the degree of true oxacillin resistance among CoNS. Using the presence of mecA as the reference standard, we detected 15.7% false susceptibility to oxacillin using a MIC susceptible breakpoint concentration of < or = 2 micrograms/mL. Lowering the oxacillin MIC breakpoint to < or = 0.25 microgram/mL for CoNS would greatly improve the accuracy of the MIC test performance. We found that both the current oxacillin disk test and the 30-microgram ceftizoxime disk test functioned quite well in predicting those strains of CoNS that contain mecA. These studies have demonstrated both a high level of antimicrobial resistance among nosocomial blood stream isolates of staphylococci as well as significant problems with the current NCCLS breakpoints for oxacillin when testing CoNS.

In vitro activity of cefepime and other broad-spectrum beta-lactams tested against 129 mec A-negative Staphylococcus spp. isolates: a multicenter sample.

The in vitro activity of cefepime was compared to that of penicillin, piperacillin/tazobactam, ceftazidime, ceftriaxone, imipenem, vancomycin, and teicoplanin by using the broth microdilution method against 129 isolates of Staphylococcus [50 S. aureus and 79 coagulase-negative staphylococci (CoNS], selected for their lack of the mec A gene as determined by the polymerase chain reaction. These isolates were obtained from a recent (1995-1996) surveillance of nearly 5000 nosocomial blood stream isolates from more than 40 geographically diverse U.S. medical centers. These results were compared to CoNS results from the same collection selected for their phenotypic susceptibility to oxacillin (OS; MIC < or = 2 micrograms/ml) regardless of their med A genotype. Cefepime, as well as piperacillin/ tazobactam, ceftriaxone, and imipenem, showed 100% susceptibility against OS and mec A-negative staphylococci. Ceftazidime showed relative resistance (30.2% resistant) against CoNS classified as OS based on phenotypic characteristics (MIC < or = 2 micrograms/mL) as compared to strains of mec A-negative CoNS (5.1% resistant). Accurate phenotypic detection of mec A-positive staphylococci by simple standardized in vitro susceptibility tests becomes very important to guide empirical use of beta-lactams for therapy. Furthermore, previously published MIC90 and range data for broad-spectrum beta-lactams versus OS have been falsely elevated by the presence of mec A-positive strains. The greater use of these potent beta-lactams against true mec A-negative staphylococci should enhance clinical outcomes and reduce the need for vancomycin.

Antimicrobial activity of 12 broad-spectrum agents tested against 270 nosocomial blood stream infection isolates caused by non-enteric gram-negative bacilli: occurrence of resistance, molecular epidemiology, and screening for metallo-enzymes.

A total of 270 recent nosocomial blood stream isolates of non-Enterobacteriaceae Gram-negative bacilli representing nearly 50 U.S. medical centers were characterized. The numbers of isolates of individual organisms were: Pseudomonas aeruginosa (n = 204), Acinetobacter spp. (n = 48), and Stenotrophomonas maltophilia (n = 18). MICs were determined using the broth microdilution susceptibility method with 12 antimicrobial agents: piperacillin, piperacillin/tazobactam, ceftriaxone, ceftazidime, cefepime, imipenem, ciprofloxacin, ofloxacin, amikacin, gentamicin, tobramycin, and trimethoprim/sulfamethoxazole. Based on current National Committee for Clinical Laboratory Standards breakpoints, rates of resistance to cefepime, ceftazidime, and imipenem were as follows: P. aeruginosa, 3, 9, and 5%; Acinetobacter spp., 2, 37, and 0%; and S. maltophilia, 88.7, 35.3, and 100%, respectively. Trimethoprim/sulfamethoxazole was the most active agent against S. maltophilia (100% susceptible). Twenty-eight isolates of P. aeruginosa that expressed high levels of resistance to ceftazidime (MIC, > 256 micrograms/mL) and imipenem (MIC, > 32 micrograms/mL) were examined for potential metallo-beta-lactamase production by polymerase chain reaction and were found to be negative. Molecular typing of P. aeruginosa isolates revealed many patient-unique strains, but also noted clustering of infections due to isolates of the same DNA type, suggesting possible nosocomial transmission in 9 of 14 medical centers. Given the resistance profile and pathogenic potential of these non-enteric Gram-negative bacilli, considerable effort should be exerted to develop and enforce infection control and antimicrobial utilization practices that will limit the spread of these organisms in the hospital environment.

Nosocomial enterococcal blood stream infections in the SCOPE Program: antimicrobial resistance, species occurrence, molecular testing results, and laboratory testing accuracy. SCOPE Hospital Study Group.

Characteristics of nosocomial enterococcal blood stream infection (NEBSI) isolates obtained from patients at 41 U.S. hospitals participating in the SCOPE Program were studied. Isolates from 480 episodes of NEBSI were characterized according to species and antimicrobial susceptibility profile. Selected isolates were also identified to species and vancomycin resistance genotype using polymerase chain reaction based methods. Polymerase chain reaction genotyping and ribotyping were used as genetic markers for molecular epidemiologic typing. Enterococci were the third most common cause of nosocomial blood stream infection in this study, accounting for 11.7% of all isolates reported. Enterococcus faecalis was the most common species (59.6%), followed by E. faecium (19.4%). Species identification errors involving E. faecium, E. durans, E. avium, and E. raffinosus were observed. Vancomycin resistance was observed in 36.4% of all participating medical centers and varied from 11.1% of medical centers in the Northwest to 60.9% of medical centers in the Southwest. Vancomycin-resistant enterococci accounted for 20.6% of NEBSI in the Northeast, 11.4% in the Southeast, 11.1% in the Southwest, and 9.5% in the Northwest regions. VanA genotypes predominated in the Northeast and Southwest, whereas vanA and vanB genotypes were equally prevalent in the Northwest and Southeast. Molecular typing studies identified strains that were unique to individual hospitals as well as strains that were prevalent in several different hospitals. NEBSI with vancomycin-resistant enterococci continues to escalate among hospitalized patients in all geographic areas of the USA.

Vancomycin-resistant Enterococcus raffinosus: molecular epidemiology, species identification error, and frequency of occurrence in a national resistance surveillance program.

Enterococcal blood stream infections are the third most common among all nosocomial blood stream infections in the United States and the occurrence of glycopeptide (vancomycin, teicoplanin) resistance in these isolates has markedly increased. Control of hospital-acquired infections with vancomycin-resistant enterococci requires high quality antimicrobial susceptibility test methods and species identification procedures as a supplement to epidemiologic investigation and appropriate infection control procedures. In this report, bacteremias caused by Enterococcus avium (BioMerieux Vitek, Hazelwood, MO, USA) were observed to be Enterococcus raffinosus infections (six of eight cases; 1.1% of all cases) when reference biochemical identification methods were applied. The vancomycin-susceptible E. raffinosis (two strains) and E. avium (two strains) had unique phenotypic and genotypic molecular profiles. In contrast, four vancomycin-resistant E. raffinosus strains (van A by polymerase chain reaction) from a single institution had the same phenotypic and molecular (PCR, PFGE, ribotyping) pattern, indicating clonal dissemination among four patients over a 66-day period. Clinical laboratories should be aware of the high probability that van A genes may be transferred from Enterococcus faecium or Enterococcus faecalis to other more rarely encountered Enterococcus species. Also contemporary, widely used commercial identification systems may fail to accurately identify those rare species. Errors appear to be most prevalent for E. avium, Enterococcus durans, and E. raffinosus based on the experience of the SCOPE Program.

Phenotypic detection of mec A-positive staphylococcal blood stream isolates: high accuracy of simple disk diffusion tests.

Detection of oxacillin-resistance in staphylococci by phenotypic methods remains problematic. Although standardized susceptibility test methods are adequate for Staphylococcus aureus, many are less satisfactory for the coagulase-negative staphylococci (CNS). We have studied 108 consecutive blood culture isolates of staphylococci. The mec A gene was detected by PCR in one S. aureus and 55 CNS isolates. Susceptibility testing was performed as follows: oxacillin (1-microgram), ceftizoxime (30-microgram), and cephalothin (30-microgram) by disk diffusion; oxacillin, ceftizoxime, cephalothin, methicillin, ampicillin, ampicillin/ sulbactam, penicillin, cefazolin, imipenem, and meropenem by the broth microdilution method. In addition, isolates were tested by the oxacillin agar screen plate method. The single oxacillin-resistant S. aureus strain was detected by all oxacillin susceptibility test methods and by the ceftizoxime disk and MIC methods. Two oxacillin-susceptible S. aureus were intermediate (minor error) by ceftizoxime broth microdilution (MIC, 16 micrograms/mL). The most sensitive, simple phenotypic methods for detection of oxacillin-resistant CNS (mec A positive) were as follows: oxacillin disk diffusion at 98%, oxacillin screen plate at 91%, oxacillin broth microdilution at 87%, ceftizoxime disk diffusion at 100%, ceftizoxime broth microdilution at 87%, and methicillin broth microdilution at 83%. These results indicate that oxacillin and ceftizoxime disk diffusion tests are the most accurate phenotypic methods in routine clinical use for detection of oxacillin-resistant CNS. Oxacillin broth microdilution MIC testing (2% NaCl supplement) would perform more satisfactorily (100% sensitivity) with an adjusted interpretive breakpoint at < or = 0.5 microgram/mL, in contrast to the lower accuracy of the "so-called" reference agar screen test.

Automation of polymerase chain reaction tests. Reduction of human errors leading to contamination.

We compared the performance over 21 months of manually performed polymerase chain reaction (PCR)-based DNA analysis experiments with 25 months of automated PCR performed by a Zymark robotic system. Automation of the PCR technique resulted in a sixfold reduction in the number of experiments reporting carryover contamination and decreased the overall rate of contamination among total reactions 68-fold. Whereas contamination occurrences among manual experiments were evenly dispersed over the study interval and correlated with the lack of experience of laboratory personnel, the contamination that occurred with the robotic system was confined to the first 10 months of operation. In manual experiments, many of the 81 no-target false positives were sufficiently strong to result in the invalidation of 151 samples and positive controls. The seven no-target control false positives in the automated system were weak bands that were easily subtracted as background. Because none of the negative samples had DNA bands, no sample on the automated system has ever been invalidated as a result of contamination. Automation of PCR tests appears to offer great promise in reducing contamination to acceptable levels (e.g., < or = 0.1%).

Oncogene amplification screening by labeled primer multiplex polymerase chain reaction.

This paper describes an improved procedure for rapid detection of amplified genes in fresh or formalin-fixed, paraffin-embedded tissues. Utilizing a multiplex differential polymerase chain reaction with radioactively labeled primers and electrophoresis of the products through thin gels, it is possible to screen for oncogene amplification more rapidly and reproducibly than has been previously demonstrated. This procedure takes advantage of thin vertical gels with external cooling, which allows sharp band resolution. Four separate gels can be electrophoresed at the same time in a single gel box. Because each gel slab contains 10 or more lanes, 40 or more samples can be assayed for gene amplification simultaneously. The entire procedure can be carried out from formalin-fixed, paraffin-embedded tissue to finish in 8 h when combined with a sonication technique for DNA extraction.

Use of thiazole orange homodimer as an alternative to ethidium bromide for DNA detection in agarose gels.

Detection of polymerase chain reaction-amplified DNA fragments is commonly accomplished by visualizing the products in electrophoretic agarose beds with the use of ethidium bromide under ultraviolet light. However, ethidium bromide is mutagenic, and special handling and disposal precautions must be used. We report the use of a nonmutagenic dye, thiazole orange dimer (TOTO), which can be substituted for ethidium bromide. The excitation maximum for TOTO under ultraviolet light is 488 nm, and the absorption maximum is 510 nm, necessitating photographic filters different from those used for ethidium bromide for optimal results. Of particular importance in TOTO's use is the quantity used for each gel lane, since excess TOTO will cause unacceptable product mobility retardation. TOTO is only slightly more expensive than ethidium bromide. Overall, this stain provides very good visualization of polymerase chain reaction--amplified DNA bands in agarose gels. We believe the use of this safer reagent will become more widespread with increased regulation of laboratory activities.

H-ras-1 gene mutations in basal cell carcinoma: automated direct sequencing of clinical specimens.

Ras oncogenes are activated by point mutations occurring in codons 12, 13 or 61 and almost any base pair mutation occurring within the first two positions of any of these codons results in activation of the gene. Although ras point mutations have been reported to occur in several skin neoplasms including squamous carcinoma, keratoacanthoma and melanoma, their frequency of occurrence in basal cell carcinoma is not known. We examined basal cell carcinomas from 13 patients for activating mutations in the H-ras-1 gene by automated direct sequencing of polymerase chain reaction assay amplified targets. We amplified sequences in exon 1 that flanked codons 12 and 13 and amplified sequences in exon 2 that flanked codon 61. The PCR products were centrifuged and directly sequenced using antisense primers in an automated sequencer using fluorescent dideoxyterminators. One tumor was found to show an activating G to A transversion in codon 13 which code for aspartic acid instead of glycine. Although H-ras mutations may be found in some skin tumors, they are not frequent in basal cell carcinomas. From this study we believe that direct sequencing of clinical material is of value and has advantages over other techniques. Additional studies need to be undertaken to understand the true clinical significance of ras mutations in basal cell carcinomas when they occur.

Multiplex polymerase chain reaction.

The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.

DNA extraction by sonication: a comparison of fresh, frozen, and paraffin-embedded tissues extracted for use in polymerase chain reaction assays.

DNA extraction from fixed tissues can be the most laborious and complex step in amplifying DNA by the polymerase chain reaction (PCR). We have previously reported a rapid and efficient method for extracting DNA by the use of sonication and glass beads. We have extended our experiences with this technique using fresh, frozen, and formalin-fixed paraffin-embedded tissues with and without the use of glass beads and report their results. Multiple tissue types were obtained at autopsy or as part of a surgical specimen. DNA was extracted from identical tissue when the sample was fresh, frozen, or formalin-fixed paraffin-embedded. Our results indicate that in most instances the sonication technique, which takes only 30 min from start to finish, can rapidly extract fresh, frozen, or formalin-fixed paraffin-embedded tissue and is superior to other rapid extraction techniques in terms of quality and quantity of DNA. It is much more rapid than those techniques that use long digestion periods. This technique will be of great value to those investigators extracting DNA for polymerase chain reaction assays.