Evaluation of the QMAC-dRAST System Version 2.5 for Rapid Antimicrobial Susceptibility Testing of Gram-Negative Bacteria From Positive Blood Culture Broth and Subcultured Colony Isolates.

BACKGROUND: Rapid antimicrobial susceptibility testing (AST) for bloodstream infections (BSIs) facilitates the optimization of antimicrobial therapy, preventing antimicrobial resistance and improving patient outcomes. QMAC-dRAST (QuantaMatrix Inc., Korea) is a rapid AST platform based on microfluidic chip technology that performs AST directly using positive blood culture broth (PBCB). This study evaluated the performance of QMAC-dRAST for Gram-negative bacteria using PBCB and subcultured colony isolates, comparing it with that of VITEK 2 (bioMérieux, France) using broth microdilution (BMD) as the reference method. METHODS: We included 141 Gram-negative blood culture isolates from patients with BSI and 12 carbapenemase-producing clinical isolates of Enterobacterales spiked into blood culture bottles. QMAC-dRAST performance was evaluated using PBCB and colony isolates, whereas VITEK 2 and BMD were tested only on colony isolates. RESULTS: For PBCB, QMAC-dRAST achieved 92.1% categorical agreement (CA), 95.3% essential agreement (EA), with 1.8% very major errors (VMEs), 3.5% major errors (MEs), and 5.2% minor errors (mEs). With colony isolates, it exhibited 92.5% CA and 95.1% EA, with 2.0% VMEs, 3.2% MEs, and 4.8% mEs. VITEK 2 showed 94.1% CA and 96.0% EA, with 4.3% VMEs, 0.4% MEs, and 4.3% mEs. QMAC-dRAST yielded elevated error rates for specific antimicrobial agents, with high VMEs for carbapenems and aminoglycosides. The median time to result for QMAC-dRAST was 5.9 h for PBCB samples and 6.1 h for subcultured colony isolates. CONCLUSIONS: The QMAC-dRAST system demonstrated considerable strengths and comparable performance to the VITEK 2 system; however, challenges were discerned with specific antimicrobial agents, underlining a necessity for improvement.

Performance Evaluation of the BACTEC MGIT 960 System for Rifampin Drug-Susceptibility Testing of Mycobacterium tuberculosis Using the Current WHO Critical Concentration.

The World Health Organization recently lowered the rifampin (RIF) critical concentration (CC) for drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) using the mycobacterial growth indicator tube (MGIT) 960 system. Here, we evaluated the diagnostic performance of the MGIT system with the revised CC for determining MTBC RIF resistance with 303 clinical MTBC isolates, including 122 isolates with rpoB mutations, of which 32 had single borderline-resistance mutations, and 181 wild-type rpoB isolates. The phenotypic RIF resistance was determined via the absolute concentration method (AC) and via MGIT using both previous (1 mg/L) and revised (0.5 mg/L) CCs for the latter method. The diagnostic accuracy of each phenotypic DST (pDST) was assessed based on rpoB genotyping as the reference standard. The overall sensitivity of the AC was 95.1% (95% confidence interval [CI], 89.6 to 98.2%), while the MGIT results with previous and revised CCs were 82.0% (95% CI 74.0 to 88.3%) and 83.6% (95% CI 75.8 to 89.7%), respectively. The 32 MTBC isolates with single borderline-resistance mutations showed a wide range of MICs, and sensitivity was not significantly increased by reducing the MGIT CC. All 181 wild-type rpoB isolates were RIF-susceptible in the AC and with MGIT using the previous CC, whereas 1 isolate was misclassified as RIF-resistant with the revised CC. Our results demonstrate that the overall diagnostic performances of the MGIT DST with the revised RIF CC and previous CC were comparable. A further large-scale study is required to demonstrate the optimal RIF CC for MGIT.

Performance Evaluation of MolecuTech REBA Myco-ID Using HybREAD480 for Identification of Nontuberculous Mycobacteria.

BACKGROUND: Rapid and accurate identification of nontuberculous mycobacteria (NTM) species is essential for the diagnosis and treatment of NTM disease. MolecuTech REBA Myco-ID (YD Diagnostics, Yongin, Korea) is a line probe assay for identification of NTM species and can be performed using HybREAD480, an instrument for automating the post-PCR steps. In this study, we assessed the performance of MolecuTech REBA Myco-ID using HybREAD480. METHODS: Seventy-four reference strains, including 65 Mycobacterium strains and nine non-Mycobacterium strains within the order Mycobacteriales, were used to determine the analytical specificity of MolecuTech REBA Myco-ID. The clinical performance of this assay was evaluated with 192 clinical Mycobacterium strains, and the assay results were compared to those of multigene sequencing-based typing. RESULTS: The accuracy of MolecuTech REBA Myco-ID for the 74 reference strains and 192 clinical strains was 77.0% (57/74; 95% confidence interval [CI], 65.8 - 86.0%) and 94.3% (181/192; 95% CI, 90.0 - 97.1%), respectively. Although some rarely isolated NTM species are misidentified, the most commonly isolated NTM species, including M. avium complex, M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. fortuitum com-plex, were all correctly identified. Of note, all M. lentiflavum strains tested (reference strain, n = 1; clinical strain, n = 10) were misidentified as M. gordonae. CONCLUSIONS: MolecuTech REBA Myco-ID using HybREAD480 was accurate for identifying commonly isolated NTM species and for discriminating between M. abscessus subsp. abscessus and M. abscessus subsp. massiliense. However, the main limitations of this assay, including misidentification of some rarely isolated NTM species and cross-reactivity between M. lentiflavum and M. gordonae, should be considered.

GenoType NTM-DR Performance Evaluation for Identification of Mycobacterium avium Complex and Mycobacterium abscessus and Determination of Clarithromycin and Amikacin Resistance.

We evaluated the GenoType NTM-DR (NTM-DR) line probe assay for identifying Mycobacterium avium complex (MAC) species and Mycobacterium abscessus subspecies and for determining clarithromycin and amikacin resistance. Thirty-eight reference strains and 145 clinical isolates (58 MAC and 87 M. abscessus isolates), including 54 clarithromycin- and/or amikacin-resistant strains, were involved. The performance of the NTM-DR assay in rapid identification was evaluated by comparison with results of multigene sequence-based typing, whereas performance in rapid detection of clarithromycin and amikacin resistance was evaluated by comparison with sequencing of the erm(41), rrl, and rrs genes and drug susceptibility testing (DST). The accuracies of MAC and M. abscessus (sub)species identification were 92.1% (35/38) and 100% (145/145) for the 38 reference strains and 145 clinical isolates, respectively. Three MAC strains other than M. intracellulare were found to cross-react with the M. intracellulare probe in the assay. Regarding clarithromycin resistance, NTM-DR detected rrl mutations in 52 isolates and yielded 99.3% (144/145) and 98.6% (143/145) concordant results with sequencing and DST, respectively. NTM-DR sensitivity and specificity in the detection of clarithromycin resistance were 96.3% (52/54) and 100% (91/91), respectively. The NTM-DR yielded accurate erm(41) genotype results for all 87 M. abscessus isolates. Regarding amikacin resistance, NTM-DR detected rrs mutations in five isolates and yielded 99.3% (144/145) and 97.9% (142/145) concordant results with sequencing and DST, respectively. Our results indicate that the NTM-DR assay is a straightforward and accurate approach for discriminating MAC and M. abscessus (sub)species and for detecting clarithromycin and amikacin resistance mutations and that it is a useful tool in the clinical setting.

Performance evaluation of the QMAC-dRAST for staphylococci and enterococci isolated from blood culture: a comparative study of performance with the VITEK-2 system.

Objectives: To evaluate the performance of a rapid antimicrobial susceptibility testing (AST) platform based on microfluidic chip technology, the QMAC-dRAST, which enables AST from colony isolates or positive blood culture broth (PBCB), and to compare the performance of the QMAC-dRAST for staphylococci and enterococci with that of the VITEK-2 system based on reference broth microdilution (BMD). Methods: A total of 110 staphylococcal and enterococcal isolates from blood cultures were included. AST was performed directly using the QMAC-dRAST with PBCB. Thereafter, colony isolates derived from subculture of PBCB were used for the QMAC-dRAST, the VITEK-2 system and BMD. Results: The overall agreement between the QMAC-dRAST with PBCB and BMD was 91.5%. There were 1.2% very major errors (VMEs), 4.3% major errors (MEs) and 5.4% minor errors (mEs). The QMAC-dRAST with colony isolates yielded 94.6% agreement and error rates of 1.0% VMEs, 1.8% MEs and 4.0% mEs. The VITEK-2 system showed 96.2% agreement and error rates of 2.3% VMEs, 0.5% MEs and 2.6% mEs. The incubation time in the QMAC-dRAST was significantly shorter than in the VITEK-2 system (median of 6 versus 10 h; P < 0.0001). Conclusions: The QMAC-dRAST system provided rapid results and represents an alternative to conventional AST methods. The QMAC-dRAST with colony isolates produced more reliable results for staphylococci and enterococci than the QMAC-dRAST with PBCB. The QMAC-dRAST system also performed comparably to BMD and the VITEK-2 system.

Communication: Evaluation of the Humasis COVID-19 Antigen Rapid Diagnostic Test for the Diagnosis of SARS-CoV-2 Infection.

OBJECTIVE: We assessed the performance of the Humasis COVID-19 AgHS Test (Humasis, Korea), a novel antigen rapid diagnostic test (Ag-RDT) based on lateral flow immunoassay. METHODS: 85 SARS-CoV-2-positive and 155 SARS-CoV-2-negative nasopharyngeal swab specimens confirmed by rRT-PCR were tested using the Humasis and PBCheck Ag-RDTs. The analytical specificity of the Humasis Ag-RDT was evaluated using 27 strains of human respiratory pathogens. RESULTS: The overall sensitivity and specificity were 72.9% and 99.4% for the Humasis Ag-RDT and 64.7% and 100% for the PBCheck Ag-RDT, respectively. The sensitivity for specimens with Ct≤25 was 100% for both Ag-RDTs. The Humasis Ag-RDT showed no cross-reactivity with other respiratory pathogens. CONCLUSION: Our data suggests that the Humasis Ag-RDT can be a useful diagnostic tool for the detection of SARS-CoV-2 infection.

Evaluation of the AdvanSure One-Stop COVID-19 Plus Kit for SARS-CoV-2 Detection Using a Streamlined RNA Extraction Method.

Real-time reverse transcription (rRT)-PCR, which is the reference standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, generally involves a time-consuming and costly RNA extraction step prior to amplification. We evaluated the performance of the AdvanSure One-Stop COVID-19 Plus Kit (LG Chem, Seoul, Korea), a novel rRT-PCR assay that can detect SARS-CoV-2 within 90 minutes using a streamlined RNA extraction method. In total, 509 nasopharyngeal swab (NPS) specimens (SARS-CoV-2 positive: N=205; SARS-CoV-2 negative: N=304) previously tested using the PowerChek SARS-CoV-2 Real-time PCR Kit (Kogene Biotech, Seoul, Korea) were tested using the AdvanSure assay. The limit of detection (LOD) of the AdvanSure assay was determined using serially diluted inactivated SARS-CoV-2. The positive and negative percent agreements between the AdvanSure and PowerChek assays were 99.5% (204/205) and 99.3% (302/304), respectively. The LODs of the AdvanSure assay for SARS-CoV-2 nucleocapsid and spike/RNA-dependent RNA polymerase genes were 672 and 846 copies/mL, respectively. The results show that the performance of the AdvanSure assay is comparable to that of the PowerChek assay used for routine SARS-CoV-2 testing, suggesting that the AdvanSure assay is a useful diagnostic tool for rapid and accurate detection of SARS-CoV-2 infection.

Comparison between the STANDARD™ F S. Pneumoniae Ag FIA and BinaxNOW S. Pneumoniae Antigen Card for Detection of Streptococcus Pneumoniae Urinary Antigen.

We compared the performance of STANDARD F S. pneumoniae Ag FIA with that of BinaxNOW S. pneumoniae Antigen Card using 206 urine samples. The performance of STANDARD F was highly comparable to that of BinaxNOW. STANDARD F assay could be a valuable tool for diagnosis of invasive pneumococcal disease.

Performance Evaluation of the PowerChek SARS-CoV-2, Influenza A & B Multiplex Real-Time PCR Kit in Comparison with the BioFire Respiratory Panel.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza viruses may pose enormous challenges to our healthcare system. We evaluated the performance of the PowerChek SARS-CoV-2, Influenza A & B Multiplex Real-time PCR Kit (PowerChek; Kogene Biotech, Seoul, Korea) in comparison with the BioFire Respiratory Panels 2 and 2.1 (RP2 and RP2.1; bioMérieux, Marcy l'Étoile, France), using 147 nasopharyngeal swabs. The limit of detection (LOD) of the PowerChek assay was determined using SARS-CoV-2, influenza A, and B RNA standards. The LOD values of the PowerChek assay for SARS-CoV-2 and influenza A and B were 1.12, 1.24, and 0.61 copies/µL, respectively. The positive and negative percent agreements of the PowerChek assay compared with RP2 and RP2.1 were 97.5% (39/40) and 100% (107/107) for SARS-CoV-2; 100% (39/39) and 100% (108/108) for influenza A; and 100% (35/35) and 100% (112/112) for influenza B, respectively. The performance of the PowerChek assay was comparable to that of RP2 and RP2.1 for detecting SARS-CoV-2 and influenza A and B, suggesting its use in diagnosing SARS-CoV-2 and influenza infections.

Comparison of the PowerChek SARS-CoV-2, Influenza A&B, RSV Multiplex Real-time PCR Kit and BioFire Respiratory Panel 2.1 for simultaneous detection of SARS-CoV-2, influenza A and B, and respiratory syncytial virus.

The potential co-circulation of SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) could pose an unprecedented challenge to healthcare systems worldwide. Here, we compared the performance of the PowerChek SARS-CoV-2, Influenza A&B, RSV Multiplex Real-time PCR Kit (PowerChek) for simultaneous detection of SARS-CoV-2, influenza A and B, and respiratory syncytial virus with that of BioFire Respiratory Panel 2.1 (RP2.1) using 175 nasopharyngeal swab (NPS) specimens. Positive percent agreement and negative percent agreement of the PowerChek assay compared to RP2.1 were as follows: 100 % (40/40) and 100 % (135/135) for SARS-CoV-2; 100 % (39/39) and 100 % (136/136) for influenza A; 100 % (35/35) and 100 % (140/140) for influenza B; and 93.1 % (27/29) and 100 % (146/146) for RSV, respectively. The limit of detection (LOD) was accessed using RNA standards for each virus, and the LOD values of the PowerChek assay for SARS-CoV-2, influenza A and B, and RSV were 0.36, 1.24, 0.09, and 0.63 copies/µL, respectively. Our results demonstrate that the PowerChek assay is sensitive and accurate for detection of SARS-CoV-2, influenza A and B, and RSV, suggesting that this assay can be a valuable diagnostic tool when SARS-CoV-2, influenza, and RSV are co-circulating.

Evaluation of the ASTA MicroIDSys matrix-assisted laser desorption ionization time-of-flight mass spectrometry system for identification of mycobacteria directly from positive MGIT liquid cultures.

OBJECTIVES: We evaluated the performance of the MicroIDSys Elite system, a newly developed matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry system for identification of mycobacteria directly from positive MGIT liquid cultures. METHODS: Analytical specificity was evaluated with 63 reference strains grown in mycobacteria growth indicator tube media. Prospective performance evaluation was conducted with primary liquid cultures of sputum samples for identification of mycobacteria, and results were compared to multigenerational sequencing as the reference method. Liquid media subcultures were also analyzed. RESULTS: The accuracy for the 63 reference strains was 98.4% (62/63). A total of 167 paired mycobacterial primary cultures and subcultures in liquid media, comprised of seven Mycobacterium tuberculosis isolates, 109 slowly growing nontuberculous mycobacterial isolates, and 51 rapidly growing nontuberculous mycobacterial isolates, was identified by the MicroIDSys Elite system. Using primary liquid cultures, the MicroIDSys Elite system correctly identified 143 (85.6%) isolates; 21 (12.6%) resulted in "no identification"; and three (1.8%) isolates were misidentified. Using liquid media subcultures with this system, 159 (95.2%) isolates were correctly identified; seven (4.2%) resulted in "no identification"; and one (0.6%) isolate was misidentified. CONCLUSION: The MicroIDSys Elite system is a useful routine diagnostic tool for identification of mycobacterial species from liquid culture.

Linezolid Resistance in Methicillin-Resistant Staphylococcus aureus in Korea: High Rate of False Resistance to Linezolid by the VITEK 2 System.

As various linezolid resistance mechanisms have been identified in methicillin-resistant Staphylococcus aureus (MRSA), we investigated the molecular characteristics of MRSA with elevated linezolid minimum inhibitory concentrations (MICs), using the VITEK 2 system (bioMérieux, Marcy-l'Étoile, France). Twenty-seven MRSA isolates from 14 patients exhibiting linezolid MICs ≥8 µg/mL were examined by broth microdilution (BMD) test as well as by sequencing for mutations in the 23S rRNA gene or ribosomal proteins (L3, L4, and L22) and the presence of the optrA, cfr, and cfr(B) genes. Of the 27 isolates, four (14.8%) from one patient were confirmed as linezolid resistant by BMD and harbored a 23S rRNA T2500A mutation. The remaining 23 were confirmed as linezolid susceptible, indicating that the linezolid-resistant results were major errors generated by VITEK 2. The most commonly detected mutation (19/27, 70.4%), L3 Gly152Asp, was detected in only linezolid-susceptible isolates. No isolates contained optrA, cfr, or cfr(B) or any L4 or L22 protein alterations. Our results show that the 23S rRNA T2500A mutation was mainly associated with linezolid resistance, while the L3 Gly152Asp mutation was not related to linezolid resistance. A confirmatory test is recommended for VITEK 2 linezolid-resistant results owing to the high probability of false resistant results.

Diagnostic Performance of the GENEDIA MTB/NTM Detection Kit for Detecting Mycobacterium tuberculosis and Nontuberculous Mycobacteria With Sputum Specimens.

The GENEDIA MTB/NTM Detection Kit (GENEDIA MTB/NTM; Green Cross Medical Science Corp., Chungbuk, Korea) is a multiplex real-time PCR assay used for differential identification of Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). While the importance of differential identification of MTB/NTM is recognized, there is limited data on the performance of GENEDIA MTB/NTM assay to date. A total of 687 consecutive sputum specimens were cultured and analyzed with the GENEDIA MTB/NTM and GENEDIA MTB assays. Nineteen specimens (2.8%) were MTBC-positive, and 69 (10.0%) were NTM-positive based on mycobacterial culture. All specimens showed concordant results for MTBC using both assays, with a kappa value of 1.00, overall sensitivity of 63.2% (12/19), and specificity of 100% (668/668). The overall NTM sensitivity and specificity were 23.2% (16/69) and 99.7% (616/618) for GENEDIA MTB/NTM. The association between NTM-positivity using GENEDIA MTB/NTM and the diagnosis of NTM pulmonary disease was not statistically significant. In conclusion, the two real-time PCR assays showed similar diagnostic performance for MTBC detection. However, the sensitivity for NTM detection was lower than that for MTBC detection.

Evaluation of the BioFire FilmArray Pneumonia Panel for rapid detection of respiratory bacterial pathogens and antibiotic resistance genes in sputum and endotracheal aspirate specimens.

OBJECTIVES: The performance of the investigational-use-only version of the BioFire FilmArray Pneumonia Panel (FA-Pneumo), a high-order nested multiplex PCR, was evaluated for the detection of typical respiratory bacterial pathogens and antibiotic resistance genes in sputa and endotracheal aspirate (ETA) specimens. METHODS: Thirty-one sputa and 69 ETA specimens were analyzed. The diagnostic performance of FA-Pneumo was assessed using routine microbiological methods as the reference standard. RESULTS: Overall sensitivity and specificity for organism detection using FA-Pneumo were 98.5% and 76.5%, respectively. The sensitivity for each pathogen was 100%, except for Klebsiella aerogenes, and the range of specificity was 83.3-99.0%. FA-Pneumo detected antimicrobial resistance genes in 17 out of 18 specimens (94.4%) that were resistant by antimicrobial susceptibility testing. FA-Pneumo additionally detected 25 resistance genes in 22 specimens, and sequencing for the presence of resistance genes confirmed the majority of these results (20/25, 80%). Semi-quantitative analysis of bacterial nucleic acid amounts by FA-Pneumo revealed that 88.2% of the identified bacteria (67/76) with ≥106 copies/ml also gave culture-positive results with significant amounts of bacteria. CONCLUSIONS: FA-Pneumo is a rapid test with high sensitivity for the detection of bacteria and antimicrobial resistance genes in sputum and ETA specimens and could aid in determining antibiotic therapy.

Comparative Evaluation Between the RealStar Pneumocystis jirovecii PCR Kit and the AmpliSens Pneumocystis jirovecii (carinii)-FRT PCR Kit for Detecting P. jirovecii in Non-HIV Immunocompromised Patients.

BACKGROUND: Real-time PCR is more sensitive than microscopic examination for detecting Pneumocystis jirovecii. We compared the performance of two assays for detecting P. jirovecii DNA: the RealStar Pneumocystis jirovecii PCR Kit 1.0 CE (Altona Diagnostics, Hamburg, Germany) and the AmpliSens Pneumocystis jirovecii (carinii)-FRT PCR kit (InterLabService Ltd., Moscow, Russia). METHODS: We used 159 samples from the lower respiratory tract (112 bronchoalveolar lavage [BAL] fluid, 37 sputum, and 10 endotracheal aspirate [ETA] samples) of non-HIV immunocompromised patients. Nested PCR and sequencing were used to resolve discordant results. The performance of the two assays was evaluated according to clinical categories (clinical Pneumocystis pneumonia [PCP], possible PCP, or unlikely PCP) based on clinical and radiological observations. RESULTS: The positive and negative percent agreement values were 100% (95% confidence interval [CI], 85.4-100%) and 96.6% (95% CI, 90.9-98.9%), respectively, and kappa was 0.92 (95% CI, 0.84-0.99). P. jirovecii DNA load was significantly higher in the clinical PCP group than in the other groups (P<0.05). When stratified by sample type, the positive rate for BAL fluids from the clinical PCP group was 100% using either assay, whereas the positive rate for sputum/ETA samples was only 20%. CONCLUSIONS: The two assays showed similar diagnostic performance and detected low P. jirovecii burden in BAL fluids. Both assays may be useful as routine methods for detecting P. jirovecii DNA in a clinical laboratory setting, though their results should be interpreted considering sample type.

Resistance mechanisms of linezolid-nonsusceptible enterococci in Korea: low rate of 23S rRNA mutations in Enterococcus faecium.

PURPOSE: To investigate linezolid-resistance mechanisms in linezolid-nonsusceptible enterococci (LNSE) isolated from a tertiary hospital in Korea. METHODOLOGY: Enterococcal isolates exhibiting linezolid MICs ≥4 mg l-1 that were isolated between December 2011 and May 2016 were investigated by PCR and sequencing for mutations in 23S rRNA or ribosomal proteins (L3, L4 and L22) and for the presence of cfr, cfr(B) and optrA genes.Results/Key findings. Among 135 LNSE (87 Enterococcus faecium and 48 Enterococcus faecalis isolates), 39.1 % (34/87) of E. faecium and 18.8 % (9/48) of E. faecalis isolates were linezolid-resistant. The optrA carriage was the dominant mechanism in E. faecalis: 13 isolates, including 10 E. faecalis [70 % (7/10) linezolid-resistant and 30 % (3/10) linezolid-intermediate] and three E. faecium [33.3 % (1/3) linezolid-resistant and 66.7 % (2/3) linezolid-intermediate], contained the optrA gene. G2576T mutations in the 23S rRNA gene were detected only in E. faecium [14 isolates; 71.4 % (10/14) linezolid-resistant and 28.6 % (4/14) linezolid-intermediate]. One linezolid-intermediate E. faecium harboured a L22 protein alteration (Ser77Thr). No isolates contained cfr or cfr(B) genes and any L3 or L4 protein alterations. No genetic mechanism of resistance was identified for 67.6 % (23/34) of linezolid-resistant E. faecium. CONCLUSION: A low rate of 23S rRNA mutations and the absence of known linezolid-resistance mechanisms in the majority of E. faecium isolates suggest regional differences in the mechanisms of linezolid resistance and the possibility of additional mechanisms.