Multiplex Real-Time PCR Assay for Rapid Detection of Methicillin-Resistant Staphylococci Directly from Positive Blood Cultures

Methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent cause of bloodstream infections (BSIs) and is recognized as a major nosocomial pathogen. This study aimed to evaluate a newly designed multiplex real-time PCR assay capable of the simultaneous detection of mecA, S. aureus, and coagulase-negative staphylococci (CoNS) in blood culture specimens. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays (M&D, Republic of Korea) use the TaqMan probes 16S rRNA for Staphylococcus spp., the nuc gene for S. aureus, and the mecA gene for methicillin resistance. The detection limit of the multiplex real-time PCR assay was 10³ CFU/ml per PCR for each gene target. The multiplex real-time PCR assay was evaluated using 118 clinical isolates from various specimen types and a total of 350 positive blood cultures from a continuous monitoring blood culture system. The results obtained with the multiplex real-time PCR assay for the three targets were in agreement with those of conventional identification and susceptibility testing methods except for one organism. Of 350 positive bottle cultures, the sensitivities of the multiplex real-time PCR kit were 100% (166/166 cultures), 97.2% (35/36 cultures), and 99.2% (117/118 cultures) for the 16S rRNA, nuc, and mecA genes, respectively, and the specificities for all three targets were 100%. The Real-MRSA and Real-MRCoNS multiplex real-time PCR assays are very useful for the rapid accurate diagnosis of staphylococcal BSIs. In addition, the Real-MRSA and Real-MRCoNS multiplex real-time PCR assays could have an important impact on the choice of appropriate antimicrobial therapy, based on detection of the mecA gene.     

Characterisation of Coagulase-Negative Staphylococci Isolated from Blood Infections: Incidence, Susceptibility to Glycopeptides, and Molecular Epidemiology

The purpose of this study was to determine incidence of coagulase-negative staphylococci (CNS) bacteraemia and to characterise the epidemiology of isolates with reduced susceptibility to glycopeptides. CNS isolates from bloodstream infections were collected and characterised by determination of the species, analysis of antibiotic susceptibility, and restriction fragment length polymorphism using pulsed-field gel electrophoresis. The medical records of patients with positive cultures and the trends in glycopeptide use were reviewed to determine the effect of previous antibiotic treatment on the susceptibility profile of these organisms. The incidence of bacteraemia caused by CNS was 0.26 per 100 patients or 0.36 per 1,000 days of hospitalisation. According to genomic fingerprinting typing, 41 (67.2%) cases of bacteraemia were caused by a unique strain of CNS and 20 were caused by several strains. Nineteen of the 61 cases of bacteraemia studied were caused by an isolate with decreased susceptibility to teicoplanin. Genomic DNA analysis of the 90 CNS isolates recovered from the 61 cases of bacteraemia generated 50 unique profiles (1 isolate per major PFGE pattern) and 13 multiple profiles (several isolates per major PFGE pattern). Neither decreased susceptibility of an isolate to teicoplanin nor hospital acquisition was associated with a multiple profile. There was a significant correlation between the incidence of bacteraemia caused by CNS with decreased susceptibility to teicoplanin and glycopeptide use at the unit level but not in individual patients. Cross-transmission did not play an important role in the dissemination of CNS with decreased susceptibility to teicoplanin, thus strains probably become resistant as a result of antibiotic pressure. Prudent use of glycopeptides is necessary to minimise the spread of resistance to these agents. Electronic Publication     

Clinical Importance of Identifying Coagulase-Negative Staphylococci Isolated from Blood Cultures: Evaluation of MicroScan Rapid and Dried Overnight Gram-Positive Panels versus a Conventional Reference Method

We evaluated the clinical usefulness of species identification of blood isolates of coagulase-negative staphylococci as a predictor of the clinical significance of the isolates. In addition, we compared results of species identification obtained with MicroScan Rapid Gram-Positive Identification panels and Dried Overnight (Conventional) Gram-Positive Identification panels with those obtained by a tube reference method. Two hundred eighty-five blood isolates were tested, including 92 judged to represent true bacteremia and 193 judged to represent contamination. The most common species detected were Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus. These three species accounted for nearly 98% of the clinically significant isolates and 89% of the contaminants. The isolation of other species almost always represented contamination. However, identification of the three most common species did not help distinguish pathogens from contaminants. Both the Rapid and the Dried Overnight Gram-Positive panels identified S. epidermidis strains accurately, but the panels performed less well for the other species. Analysis revealed that S. hominis was frequently misidentified due to the presence of a previously unknown subspecies. Based on the initial results, revised investigational Dried Overnight Gram-Positive Identification panels (CPID-2) were prepared and tested. The CPID-2 panels identified 85 to 95% of S. epidermidis strains, 76 to 86% of S. hominis strains, and 88 to 92% of S. haemolyticus strains with high probability (>85%) and, overall, represented a significant improvement over the other panels for identification of these staphylococcal species.     

Evaluation of Sensititre Plates for Identification of Clinically Relevant Coagulase-Negative Staphylococci

Coagulase-negative staphylococcus isolates were identified using Sensititre GPID plates and API strips (n = 156). For selected isolates, partial sequencing of the 16S rRNA, sodA, and tuf genes was performed. The Sensititre plates correctly identified 68.9% of isolates, with a concordance of 86% for Staphylococcus haemolyticus and 73% for Staphylococcus epidermidis.Infections caused by coagulase-negative staphylococci (CoNS) are increasing (2-4, 6, 8, 12, 14). None of the commercially available options for the identification of staphylococcal species is 100% accurate for all CoNS (1, 5, 7, 9). Sensititre identification GPID plates (TREK Diagnostic Systems Inc., Cleveland, OH) identify the most common staphylococci and include in its taxa Staphylococcus aureus, Staphylococcus capitis subsp. capitis, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis subsp. hominis, Staphylococcus lugdunensis, Staphylococcus saprophyticus subsp. saprophyticus, and Staphylococcus warneri. Our objective was to evaluate the Sensititre system for the identification of CoNS species for routine diagnostic use.Clinically relevant CoNS isolates (n = 156) were collected from May 2006 to May 2009 from the Hospital Civil de Guadalajara and the Hospital Universitario in Monterrey. Only one isolate per patient was included. Pure bacterial isolates were stored in Brucella broth containing 15% glycerol at −70°C.All isolates were identified using both Sensititre GPID plates and API Staph strips following manufacturer''s instructions. Sensititre plates were read with a Sensititre AutoReader, and API STAPH V4.1 identification software was used for the API strips. For both phenotypic methods, when the result was inconclusive, manual tests recommended by the manufacturer were performed to resolve the identity of the test isolate. If no satisfactory identification was obtained, the identification was repeated.For all isolates with discordant results between the two phenotypic methods and for randomly selected clinical isolates with the same result from both phenotypic methods, partial sequencing of the 16S rRNA gene was performed. For these isolates, 3 to 5 colonies were suspended in sterile water, and the suspension was boiled for 5 min and then centrifuged; the supernatant was used as the DNA template for PCR assays. PCR was performed as described previously (10), and PCR products were purified using the commercial Wizard PCR Preps DNA purification system (Promega, Madison, WI). Sequencing of the ∼1,200-bp products was performed in both directions at the Instituto de Biotecnología, Universidad Nacional Autónoma de México. When no definitive identification could be made based on 16S rRNA analysis, partial sequencing of the sodA and tuf genes was performed as described previously (5, 11).DNA sequence fragments were compared to NCBI GenBank sequence entries using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST). Homology values above 97% were considered reliable (13). If more than one species fulfilled this criterion, definitive species identification was archived based on the species with the higher percentage homology.The quality control strains S. haemolyticus ATCC 29970, S. capitis ATCC 35661, Staphylococcus xylosus ATCC 700404, S. lugdunensis ATCC 49576, S. hominis ATCC 27844, S. saprophyticus ATCC 15305, S. epidermidis ATCC 14990, Staphylococcus sciuri ATCC 29060, and Staphylococcus intermedius ATCC 29663 were tested with both the API strips and the Sensititre plates.The quality control strains were correctly identified by both phenotypic methods for S. saprophyticus, S. epidermidis, S. haemolyticus, S. capitis, and S. hominis. The API strips correctly identified S. sciuri, S. xylosus, and S. lugdunensis, but the Sensititre system did not identify any of these species. S. intermedius was not identified by either of the two methods.Ninety-six (62%) isolates showed agreement in the phenotypic test results (Table ​(Table1),1), and identification was confirmed by partial sequencing of the 16S rRNA gene in 32/36 (89%) isolates.

TABLE 1.

Identification results for CoNS isolates

Comparison of the Staph-Ident System with a Conventional Method for Species Identification of Urine and Blood Isolates of Coagulase-Negative Staphylococci

The Staph-Ident system (Analytab Products) for species identification of coagulase-negative staphylococci was compared with the conventional method of Kloos and Schleifer (21). A total of 101 clinical isolates from urine cultures and 95 clinical isolates from blood cultures were studied: overall agreement between the two methods was 86%. We concluded that the Staph-Ident system is a practical test for most clinical microbiology laboratories and that results obtained from this rapid test are comparable to those obtained from the more cumbersome conventional method. Additional investigations are needed to determine the clinical relevance of such species identification.     

Detection of Methicillin-Resistant Coagulase-Negative Staphylococci by the Vitek 2 System

The accurate performance of the Vitek 2 GP67 card for detecting methicillin-resistant coagulase-negative staphylococci (CoNS) is not known. We prospectively determined the ability of the Vitek 2 GP67 card to accurately detect methicillin-resistant CoNS, with mecA PCR results used as the gold standard for a 4-month period in 2012. Included in the study were 240 consecutively collected nonduplicate CoNS isolates. Cefoxitin susceptibility by disk diffusion testing was determined for all isolates. We found that the three tested systems, Vitek 2 oxacillin and cefoxitin testing and cefoxitin disk susceptibility testing, lacked specificity and, in some cases, sensitivity for detecting methicillin resistance. The Vitek 2 oxacillin and cefoxitin tests had very major error rates of 4% and 8%, respectively, and major error rates of 38% and 26%, respectively. Disk cefoxitin testing gave the best performance, with very major and major error rates of 2% and 24%, respectively. The test performances were species dependent, with the greatest errors found for Staphylococcus saprophyticus. While the 2014 CLSI guidelines recommend reporting isolates that test resistant by the oxacillin MIC or cefoxitin disk test as oxacillin resistant, following such guidelines produces erroneous results, depending on the test method and bacterial species tested. Vitek 2 cefoxitin testing is not an adequate substitute for cefoxitin disk testing. For critical-source isolates, mecA PCR, rather than Vitek 2 or cefoxitin disk testing, is required for optimal antimicrobial therapy.     

Evaluation of Screening and Commercial Methods for Detection of Methicillin Resistance in Coagulase-Negative Staphylococci

The National Committee for Clinical Laboratory Standards recommends 48 h of incubation by the oxacillin salt agar screen (OSAS) method for the detection of methicillin-resistant coagulase-negative staphylococci (CoNS). An earlier identification of methicillin resistance is desirable. The time to detection of the mecA gene by PCR was compared with the times to detection by OSAS, by the oxacillin disk diffusion (ODD) method, and with MicroScan Gram Positive Combo type 6 panels (MicroScan Inc. Sacramento, Calif.) and Vitek GPS-SA cards (bioMérieux Vitek Inc., Hazelwood, Mo.). The combination of the Vitek card and the ODD method detected 92 of 99 methicillin-resistant strains of CoNS at 24 h; however, 6 mecA-positive strains were phenotypically methicillin susceptible. We conclude that most methicillin-resistant CoNS can be detected and the results can be reported after overnight incubation by a combination of methods.     

Changing Epidemiology of Coagulase-Negative Staphylococci in Normal Flora of Skin

Coagulase negative staphylococci (CoNS) have recently emerged as important pathogens causing nosocomial blood stream infections. To evaluate the prevalence of CoNS in cutaneous normal flora, skin swabs were collected from voluntary blood donors and processed for culture and identification using matrix assisted laser desorption ionisation-time of flight (MALDI-TOF). CoNS were isolated from 96% of blood donors, most commonly Staphylococcus hominis (86%), followed by Staphylococcus epidermidis (22%) and Staphylococcus haemolyticus (9%). There has been a shift in the prevalent species of CoNS in the community in India, from the earlier known S. epidermidis towards resistant species such as S. hominis and S. haemolyticus. Routine and rapid speciation of CoNS in clinical isolates with MALDI-TOF should be used effectively to manage these resistant species.     

头孢西丁和拉氧头孢低密度菌落法检测耐甲氧西林凝固酶阴性葡萄球菌

目的评价头孢西丁和拉氧头孢低密度菌落法检测耐甲氧西林凝固酶阴性葡萄球菌（MRCNS）的可靠性和临床实用性。方法临床收集的93株凝固酶阴性葡萄球菌（CNS）,按法国生物学会（CASFM）推荐的头孢西丁和拉氧头孢30μg低密度菌落法检测MRCNS,并与mecA基因PCR扩增法进行比较。结果以mecA基因PCR扩增法为金标准,其中MRCNS56株（阳性率60.2%）,低密度菌落法头孢西丁的敏感性和特异性分别为94.6%和100%,拉氧头孢的敏感性和特异性分别为100%和97.3%。结论头孢西丁和拉氧头孢低密度菌落法是鉴定异质性MRCNS的良好方法。     

Stewardship Approach for Optimizing Antimicrobial Therapy through Use of a Rapid Microarray Assay on Blood Cultures Positive for Enterococcus Species

Enterococci are a major cause of bloodstream infections in hospitalized patients and have limited antimicrobial treatment options due to their many resistance mechanisms. Molecular technologies have significantly shortened the time to enterococcal isolate identification compared with conventional methods. We evaluated the impact of rapid organism identification and resistance detection with the Verigene Gram-positive blood culture microarray assay on clinical and economic outcomes for patients with enterococcal bacteremia. A single-center preintervention/postintervention quasiexperimental study compared inpatients with enterococcal bacteremia from 1 February 2012 to 9 September 2012 (preintervention period) and 10 September 2012 to 28 February 2013 (postintervention period). An infectious disease and/or critical care pharmacist was contacted with the microarray assay results, and effective antibiotics were recommended. The clinical and economic outcomes for 74 patients were assessed. The mean time to appropriate antimicrobial therapy was 23.4 h longer in the preintervention group than in the postintervention group (P = 0.0054). A nonsignificant decrease in the mean time to appropriate antimicrobial therapy was seen for patients infected with vancomycin-susceptible Enterococcus isolates (P = 0.1145). For patients with vancomycin-resistant Enterococcus bacteremia, the mean time to appropriate antimicrobial therapy was 31.1 h longer in the preintervention group than in the postintervention group (P < 0.0001). In the postintervention group, the hospital length of stay was significantly 21.7 days shorter (P = 0.0484) and mean hospital costs were $60,729 lower (P = 0.02) than in the preintervention group. The rates of attributed deaths in the two groups were not statistically different. Microarray technology, supported by pharmacy and microbiology departments, can decrease the time to appropriate antimicrobial therapy, the hospital length of stay, and health care costs.     

Impact of Antimicrobial Stewardship Intervention on Coagulase-Negative Staphylococcus Blood Cultures in Conjunction with Rapid Diagnostic Testing

Rapid diagnostic testing with matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) decreases the time to organism identification by 24 to 36 h compared to the amount of time required by conventional methods. However, there are limited data evaluating the impact of MALDI-TOF with real-time antimicrobial stewardship team (AST) review and intervention on antimicrobial prescribing and outcomes for patients with bacteremia and blood cultures contaminated with coagulase-negative Staphylococcus (CoNS). A quasiexperimental study was conducted to analyze the impact of rapid diagnostic testing with MALDI-TOF plus AST review and intervention for adult hospitalized patients with blood cultures positive for CoNS. Antibiotic prescribing patterns and clinical outcomes were compared before and after implementation of MALDI-TOF with AST intervention for patients with CoNS bacteremia and CoNS contamination. A total of 324 patients with a positive CoNS blood culture were included; 246 were deemed to have contaminated cultures (117 in the preintervention group and 129 in AST the intervention group), and 78 patients had bacteremia (46 in the preintervention group and 32 in the AST intervention group). No differences in demographics were seen between the groups, and similar rates of contamination occurred between the preintervention and AST intervention groups (64.3% versus 72.6%, P = 0.173). Patients with bacteremia were initiated on optimal therapy sooner in the AST intervention group (58.7 versus 34.4 h, P = 0.030), which was associated with a similarly decreased mortality (21.7% versus 3.1%, P = 0.023). Patients with CoNS-contaminated cultures had similar rates of mortality, lengths of hospitalization, recurrent bloodstream infections, and 30-day hospital readmissions, but the AST intervention group had a decreased duration of unnecessary antibiotic therapy (1.31 versus 3.89 days, P = 0.032) and a decreased number of vancomycin trough assays performed (0.88 versus 1.95, P < 0.001). In patients with CoNS bacteremia, rapid pathogen identification integrated with real-time stewardship interventions improved timely organism identification and initiation of antibiotic therapy. Patients in the AST group with blood cultures contaminated with CoNS had decreased inappropriate antimicrobial prescribing and decreased unnecessary serum vancomycin trough assays.     

Evaluation of FilmArray and Verigene Systems for Rapid Identification of Positive Blood Cultures

The Verigene tests for Gram-positive and Gram-negative organisms in blood culture and the FilmArray blood culture identification panel were assessed for their ability to identify pathogens from positive blood cultures. Both platforms correctly identified bacteria in 92% of monomicrobial cultures analyzed, with times to identification that were significantly shorter than those for identification from subcultures.     

Impact of a Rapid Microarray-Based Assay for Identification of Positive Blood Cultures for Treatment Optimization for Patients with Streptococcal and Enterococcal Bacteremia

Implementation of the Verigene Gram-positive blood culture test led to reductions in time to acceptable antibiotic overall (1.9 versus 13.2 h, respectively; P = 0.04) and time to appropriate antibiotic for patients with vancomycin-resistant Enterococcus (4.2 versus 43.7 h; P = 0.006) and viridans group Streptococcus (0.2 versus 7.1 h; P = 0.02).     

Adjunctive mecA PCR for Routine Detection of Methicillin Susceptibility in Clinical Isolates of Coagulase-Negative Staphylococci

Concerns over the reliability of routine sensitivity testing in coagulase-negative staphylococci often lead to the use of potentially less-effective antibiotics as few laboratories have access to routine tests for the mecA resistance gene. Although previous studies have shown a reasonable correlation between oxacillin disc and automated sensitivity testing, changing epidemiology and methodology dictate periodic reappraisal of these methods. In the present study, we evaluated two real-time PCR assays against novel targets in the mecA gene as an adjunct to routine susceptibility testing using the Vitek II AST-P620 card. All samples were further examined for the presence of the mecC gene. Of 118 strains of coagulase-negative staphylococci tested, 81 were oxacillin resistant and 37 oxacillin susceptible by the Vitek II assay compared with 103 positive and 15 negative by mecA PCR. In-house PCR results correlated well with a previously published reference PCR, though little correlation was found between mecA PCR or Vitek II and PBP 2a latex agglutination. Incubation conditions may have affected the accuracy of the latter test. None of the strains tested were mecC PCR positive. The inclusion of dual-target PCRs in the testing algorithm was inexpensive and offered the safest strategy for determining beta-lactam susceptibility in coagulase-negative staphylococci in our laboratory.     

Evaluation of a Latex Agglutination Test (MRSA-Screen) for Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci

The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84 mecA-positive strains and 116 mecA-negative strains) consisting of 108 Staphylococcus epidermidis, 37 S. saprophyticus, 15 S. haemolyticus, 11 S. hominis, 10 S. capitis, 10 S. warneri, and 3 S. lugdunensis species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 microg/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the mecA gene was used as the "gold standard." The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.     

Evaluation of Three Rapid Diagnostic Methods for Direct Identification of Microorganisms in Positive Blood Cultures

The identification of organisms from positive blood cultures generally takes several days. However, recently developed rapid diagnostic methods offer the potential for organism identification within only a few hours of blood culture positivity. In this study, we evaluated the performance of three commercial methods to rapidly identify organisms directly from positive blood cultures: QuickFISH (AdvanDx, Wolburn, MA), Verigene Gram-Positive Blood Culture (BC-GP; Nanosphere, Northbrook, IL), and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) with Sepsityper processing (Bruker Daltonics, Billerica, MA). A total of 159 blood cultures (VersaTREK Trek Diagnostic Systems, Cleveland, OH) positive for Gram-positive and Gram-negative bacteria as well as yeast were analyzed with QuickFISH and MALDI-TOF MS. In all, 102 blood cultures were analyzed using the BC-GP assay. For monomicrobial cultures, we observed 98.0% concordance with routine methods for both QuickFISH (143/146) and the BC-GP assay (93/95). MALDI-TOF MS demonstrated 80.1% (117/146) and 87.7% (128/146) concordance with routine methods to the genus and species levels, respectively. None of the methods tested were capable of consistently identifying polymicrobial cultures in their entirety or reliably differentiating Streptococcus pneumoniae from viridans streptococci. Nevertheless, the methods evaluated in this study are convenient and accurate for the most commonly encountered pathogens and have the potential to dramatically reduce turnaround time for the provision of results to the treating physician.     

Improving the Yield of Blood Cultures for Patients with Early Lyme Disease

This study was designed to improve the recovery of Borrelia burgdorferi from blood. With the techniques used, B. burgdorferi could be recovered from the blood of approximately 25% of patients with early Lyme disease associated with erythema migrans. Serum was a better source of culture material than whole blood. The volume of blood cultured correlated directly with yield, particularly for patients with a single erythema migrans lesion.     

Rapid and Accurate Species-Level Identification of Coagulase-Negative Staphylococci by Using the sodA Gene as a Target

Simple PCR and sequencing assays that utilize a single pair of degenerate primers were used to characterize a 429-bp-long DNA fragment internal (sodA(int)) to the sodA gene encoding the manganese-dependent superoxide dismutase in 40 coagulase-negative staphylococcal (CNS) type strains. The topology of the phylogenetic tree obtained was in general agreement with that which was inferred from an analysis of their 16S rRNA or hsp60 gene sequences. Sequence analysis revealed that the staphylococcal sodA genes exhibit a higher divergence than does the corresponding 16S ribosomal DNA. These results confirm that the sodA gene constitutes a highly discriminative target sequence for differentiating closely related bacterial species. Clinical isolates that could not be identified at the species level by phenotypical tests were identified by use of this database. These results demonstrate the usefulness of this method for rapid and accurate species identification of CNS isolates, although it does not allow discrimination of subspecies. The sodA sequence polymorphisms observed with staphylococcal species offer good opportunities for the development of assays based on DNA chip technologies.