Rapid screening and identification of methicillin-resistant Staphylococcus aureus from clinical samples by selective-broth and real-time PCR assay

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) by using selective broth and real-time PCR (broth-PCR) was developed and evaluated. The samples (n = 304) were cultured in the broth overnight, followed by nuc gene detection by real-time PCR. nuc-negative samples were further checked for the presence of nuc amplification inhibitors by a PCR internal inhibitor assay. nuc-positive samples and nuc-negative samples with PCR inhibitors were cultured onto plates and processed further. The diagnostic values for this MRSA screening method were 93.3% sensitivity, 89.6% specificity, 31.8% positive predictive value, and 99.6% negative predictive value. The application of the broth-PCR method will be able to report most of the negative samples (258 of 289 [89.3%]) on the next morning and can save as much as 84.9% (258 of 304) of the labor and cost spent on processing the nuc-negative specimens on plates. In the study, all the samples were processed in parallel by the broth enrichment method and the plating method for comparison. To identify MRSA, the isolated oxacillin-resistant S. aureus strains were tested by a duplex real-time PCR targeting the mecA gene and the nuc gene. A collection of MRSA, methicillin-susceptible Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, and methicillin-susceptible Staphylococcus epidermidis strains and a panel of standard strains of 11 bacterial species other than S. aureus were also tested by this method, which was proved to be a valuable tool for MRSA identification in a routine microbiological laboratory.     

Evaluation of four selective agars and two enrichment broths in screening for methicillin-resistant Staphylococcus aureus

To evaluate methicillin-resistant Staphylococcus aureus detection, we tested in vitro four selective agars and two enrichment broths apart and in combination. Tryptone soya broth with salt, aztreonam, and cefoxitin appeared to be the most sensitive medium. This broth was superior to a phenol red mannitol broth with aztreonam and ceftizoxime.     

Rapid screening for carriage of methicillin-resistant Staphylococcus aureus by PCR and associated costs

PCR tests for the rapid and valid detection of methicillin-resistant Staphylococcus aureus (MRSA) are now available. We evaluated the costs associated with contact screening for MRSA carriage in a tertiary-care hospital with low MRSA endemicity. Between 1 October 2005 and 28 February 2006, 232 patients were screened during 258 screening episodes (644 samples) for MRSA carriage by GenoType MRSA Direct (Hain Lifescience GmbH, Nehren, Germany). Conventional culture confirmed all PCR results. According to in-house algorithms, 34 of 258 screening episodes (14.7%) would have qualified for preemptive contact isolation, but such isolation was not done upon negative PCR results. MRSA carriage was detected in 4 (1.5%) of 258 screening episodes (i.e., in four patients), of which none qualified for preemptive contact isolation. The use of PCR for all 258 screening episodes added costs (in Swiss francs [CHF]) of CHF 104,328.00 and saved CHF 38,528.00 (for preemptive isolation). The restriction of PCR screening to the 34 episodes that qualified for preemptive contact isolation and screening all others by culture would have lowered costs for PCR to only CHF 11,988.00, a savings of CHF 38,528.00. Therefore, PCR tests are valuable for the rapid detection of MRSA carriers, but high costs require the careful evaluation of their use. In patient populations with low MRSA endemicity, the broad use of PCR probably is not cost-effective.     

Rapid PCR-based identification of methicillin-resistant Staphylococcus aureus from screening swabs

A PCR identification of methicillin-resistant Staphylococcus aureus (MRSA), obviating the need for subculture on agar media, was investigated. The combination of MRSA detection by mecA femB PCR with prior enrichment in selective broth was tested for 439 swabs. PCR identified 36 MRSA-positive samples, in concordance with conventional methods.     

Rapid detection of vanA and vanB genes directly from clinical specimens and enrichment broths by real-time multiplex PCR assay

A real-time PCR assay previously developed for use on the Roche LightCycler platform was investigated as an alternative to culture for the direct detection of vancomycin-resistant enterococci (VRE) in clinical specimens. PCR primers and fluorescence resonance energy transfer hybridization probes specific for the vanA and vanB genes were combined in a multiplex real-time PCR assay performed directly with fecal material obtained by rectal swabbing and with enrichment broth samples. DNA was prepared from the rectal swabs and enrichment broths with a commercially available DNA preparation column designed specifically for use with fecal specimens. One hundred eighty duplicate rectal swabs were obtained from 42 patients who were previously found to be positive for VRE and who were being monitored for carriage of VRE. Direct and enrichment broth cultures were performed with one swab, while PCR was performed with the other swab as well as any corresponding presumptive positive enrichment broth. In total, 100 specimens from 30 patients remained positive for VRE by at least one method. The multiplex real-time PCR was positive for 88 enrichment broths of rectal swabs from 27 patients but for only 45 rectal swabs from 15 patients. Direct culture was positive for VRE for only 43 specimens from 11 patients, while enrichment broth culture was positive for VRE for 75 specimens from 22 patients. Inhibition studies for the multiplex real-time PCR assay, performed by spiking the DNA extracts from 50 negative rectal swabs and the corresponding enrichment broths with between 1 and 10 CFU of a VanB Enterococcus faecium strain, detected inhibition rates of 55.1 and 10%, respectively. PCR performed directly with enrichment broths was found to be significantly more sensitive than enrichment broth culture (P < 0.025). Negative samples were identified significantly earlier by PCR than by culture alone.     

Rapid identification of methicillin-resistant Staphylococcus aureus and simultaneous species confirmation using real-time fluorescence PCR

A duplex LightCycler PCR assay targeting the mecA gene and a Staphylococcus aureus-specific marker was used to test 165 S. aureus strains and 80 strains of other bacterial species. Within an assay time of 60 min plus 10 min for sample preparation, S. aureus as well as the presence or absence of the mecA gene was correctly identified.     

Multiplex real-time PCR assay for detection of methicillin-resistant Staphylococcus aureus and associated toxin genes

We describe a real-time PCR assay for the detection of methicillin-resistant Staphylococcus aureus and genes encoding toxic shock syndrome toxin 1 and Panton-Valentine leukocidin. Rapid screening and detection of toxins is a useful tool for surveillance studies and outbreak investigations involving large numbers of isolates.     

Detection of methicillin-resistant Staphylococcus aureus directly from nasal swab specimens by a real-time PCR assay

Screening for colonization with methicillin-resistant Staphylococcus aureus (MRSA) is a key aspect of infection control to limit the nosocomial spread of this organism. Current methods for the detection of MRSA in clinical microbiology laboratories, including molecularly based techniques, require a culture step and the isolation of pure colonies that result in a minimum of 20 to 24 h until a result is known. We describe a qualitative in vitro diagnostic test for the rapid detection of MRSA directly from nasal swab specimens (IDI-MRSA; Infectio Diagnostic, Inc., Sainte-Foy, Quebec, Canada), based upon a real-time PCR and direct detection of MRSA via amplicon hybridization with a fluorogenic target-specific molecular beacon probe. Samples from 288 patients were analyzed for the presence of MRSA with the IDI-MRSA assay, compared to detection by either direct plating or enrichment broth selective culture methods. The diagnostic values for this MRSA screening method were 91.7% sensitivity, 93.5% specificity, 82.5% positive predictive value, and 97.1% negative predictive value when compared to culture-based methods. The time from the start of processing of specimen to result was approximately 1.5 h. In our hands, the IDI-MRSA assay is a sensitive and specific test for detection of nasal colonization with MRSA and providing for same-day results, allowing more efficient and effective use of infection control resources to control MRSA in health care facilities.     

Multilocus sequence typing of methicillin-resistant Staphylococcus aureus from an area of low endemicity by real-time PCR

A protocol for multilocus sequence typing (MLST) of methicillin-resistant Staphylococcus aureus (MRSA) was adapted to real-time LightCycler System PCR for efficient and rapid amplification of seven housekeeping genes in the same PCR run and real-time detection of the products. The method was evaluated on a representative and well-characterized collection of clinical MRSA isolates (n = 57) obtained from an area of low endemicity. Twenty sequence types (STs) and nine clonal complexes were identified. Combining STs and the staphylococcal cassette chromosome mec (SCCmec) type identified 27 different genotypes, and type IV SCCmec was present in 11 different STs. The presence of the Panton Valentine leukocidin (PVL) genes was found in isolates of four different STs. Eleven different STs were found among the community-acquired as well as among the hospital-acquired MRSA. The genetic heterogeneity was also denoted by pulsed-field gel electrophoresis analysis that showed 24 different pulsotypes among the 57 MRSA isolates. The presence of more than one different type of SCCmec in the same ST indicates that the MRSA clones have arisen at several occasions in the same genetic background by independent acquisition of SCCmec into methicillin-sensitive strains. This circumstance shows the importance of combining MLST data with SCCmec-typing results when investigating the origins of MRSA.     

Use of broth enrichment and real-time PCR to exclude the presence of methicillin-resistant Staphylococcus aureus in clinical samples: a sensitive screening approach

A rapid and sensitive method for excluding the presence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples was developed and evaluated. The method utilised an MRSA-selective enrichment broth for 16 h, followed by PCR quantification of the nuc gene. Samples below a quantitative PCR threshold were reported as MRSA-negative. Broths from PCR-positive samples were subcultured for MRSA isolation. Clinical samples (n = 334) in a constructed high prevalence population were analysed in parallel with a selective plating method. The new broth-PCR assay increased the number of positive samples by 35% (49 vs. 66), and 94% of negative samples were reported within 24 h. To reduce costs and workload, 665 clinical samples were grown separately in enrichment broth and then pooled in the PCR step. The broth-PCR assay increased the number of MRSA positive samples from 11 to 15 compared with selective plating. Most (89%) of the culture-negative samples were also PCR-negative and could be reported within 24 h. The growth of 25 European EMRSA strains was tested in the selective enrichment broth. On average, the MRSA strains showed a 300 000-fold increase in CFU, compared with 30-fold for the eight methicillin-sensitive Staphylococcus aureus strains tested.     

Rapid detection of methicillin-resistant staphylococci by real-time PCR directly from positive blood culture bottles

For the rapid detection of methicillin-resistant staphylococci directly from blood cultures containing gram-positive cocci in clusters, we implemented a real-time (LightCycler) polymerase chain reaction (PCR) specific for the Staphylococcus aureus nuc gene encoding nuclease and the mecA gene encoding methicillin resistance. For the 475 positive blood cultures tested, the assay turned out to have 100% sensitivity and 100% specificity for the identification of methicillin-susceptible (n = 108) and methicillin-resistant (n = 34) S. aureus. When coagulase-negative staphylococci (CoNS) were included, the overall sensitivity for the detection of methicillin resistance was 93% and the specificity was 99%. Real-time PCR for nuc and mecA from blood culture bottles with staphylococci yields therefore a rapid (2–3 h) identification of S. aureus and CoNS including methicillin resistance.     

Rapid detection of Staphylococcus aureus bacteremia and methicillin resistance by real-time PCR in whole blood samples

We prospectively evaluated a real-time polymerase chain reaction (PCR) approach for the rapid diagnosis of Staphylococcus aureus bacteremia and presence of the mecA gene in 902 blood samples from 468 infectious episodes of 384 patients. Eight of 12 blood culture (BC)-confirmed samples were positive by the S. aureus-specific PCR. In addition, the mecA gene PCR correctly detected all cases of BC-confirmed methicillin-resistant Staphylococcus aureus (MRSA) infection. A positive PCR result was also obtained in ten of 462 BC-negative infectious episodes, including three patients with culture-confirmed S. aureus infection at other body sites. Juliane Winter and Susanne Gebert contributed equally to this work.     

Rapid detection of methicillin-resistant Staphylococcus aureus by Crystal MRSA ID System.

A commercially available method for the rapid detection of methicillin-resistant Staphylococcus aureus (BBL Crystal MRSA ID System) was evaluated and compared with conventional methods. All 52 isolates of methicillin-susceptible and 142 isolates of intrinsic methicillin-resistant S. aureus were correctly identified in 4 h by the test method, whereas correct identification took 11 to 24 h by conventional methods. The test is simple, rapid, and easy to perform and the results are easy to interpret.     

New real-time PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus directly from specimens containing a mixture of staphylococci

Molecular methods for the rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) are generally based on the detection of an S. aureus-specific gene target and the mecA gene. However, such methods cannot be applied for the direct detection of MRSA from nonsterile specimens such as nasal samples without the previous isolation, capture, or enrichment of MRSA because these samples often contain both coagulase-negative staphylococci (CoNS) and S. aureus, either of which can carry mecA. In this study, we describe a real-time multiplex PCR assay which allows the detection of MRSA directly from clinical specimens containing a mixture of staphylococci in <1 h. Five primers specific to the different staphylococcal cassette chromosome mec (SCCmec) right extremity sequences, including three new sequences, were used in combination with a primer and three molecular beacon probes specific to the S. aureus chromosomal orfX gene sequences located to the right of the SCCmec integration site. Of the 1,657 MRSA isolates tested, 1,636 (98.7%) were detected with the PCR assay, whereas 26 of 569 (4.6%) methicillin-susceptible S. aureus (MSSA) strains were misidentified as MRSA. None of the 62 nonstaphylococcal bacterial species or the 212 methicillin-resistant or 74 methicillin-susceptible CoNS strains (MRCoNS and MSCoNS, respectively) were detected by the assay. The amplification of MRSA was not inhibited in the presence of high copy numbers of MSSA, MRCoNS, or MSCoNS. The analytical sensitivity of the PCR assay, as evaluated with MRSA-negative nasal specimens containing a mixture of MSSA, MRCoNS, and MSCoNS spiked with MRSA, was approximately 25 CFU per nasal sample. This real-time PCR assay represents a rapid and powerful method which can be used for the detection of MRSA directly from specimens containing a mixture of staphylococci.     

Evaluation of the Xpert methicillin-resistant Staphylococcus aureus (MRSA) assay using the GeneXpert real-time PCR platform for rapid detection of MRSA from screening specimens

The need for rapid methods to accurately detect methicillin-resistant Staphylococcus aureus (MRSA) is widely acknowledged, and a number of molecular assays are commercially available. This study evaluated the Xpert MRSA assay, which is run on the GeneXpert real-time PCR platform (Cepheid) for use in a clinical laboratory. The following parameters were investigated: (i) the limits of detection (LoDs) for four MRSA strains; (ii) the ability to detect isolates of MRSA from a collection representative of MRSA in Ireland since 1974 (n = 114) and the ability to detect control strains with staphylococcal cassette chromosome mec types IVa (IV.1.1.1), IVb (IV.2.1.1), IVc (IV.3.1.1), IVd (IV.4.1.1), V (V.1.1.1), V_T, and VI; and (iii) performance in a clinical trial with swabs from nose, throat, and groin/perineum sites from 204 patients, where results were compared with those obtained by direct and enrichment cultures. The average LoD of the four test strains was 610 CFU/ml (equivalent to 58 CFU/swab). All 114 MRSA isolates and 7 control strains tested were detected. Sensitivity, specificity, and positive and negative predictive values for clinical specimens from all sites investigated were 90%, 97%, 86%, and 98%, respectively, but throat specimens yielded poor sensitivity (75%). Sensitivity, specificity, and positive and negative predictive values for nasal specimens were 95%, 98%, 90%, and 99%, respectively. Overall, the assay was rapid and easy to perform, but performance might be enhanced by the inclusion of an equivocal interpretive category based on analysis of all available amplification data.     

Development of a real-time PCR assay for rapid identification of methicillin-resistant Staphylococcus aureus from clinical samples

A major drawback of mecA PCR to detect methicillin-resistant Staphylococcus aureus (MRSA) directly from patient materials is the high frequency of methicillin-resistant coagulase-negative staphylococci. Therefore, a reliable detection method for MRSA from clinical samples using real-time PCR was developed. The PCR assay targeting the integration site (orfX) of the staphylococcal cassette chromosome mec (SCCmec) was evaluated in MRSA SCCmec reference strains (n = 9), MRSA ST strains (n = 16) and clinical isolates of MRSA (n = 124), MSSA (n = 53), methicillin-resistant coagulase-negative staphylococci (n = 47), and methicillin-susceptible, coagulase-negative staphylococci (n = 32). The diagnostic values of the assay were 98% sensitivity and 100% specificity. Furthermore, the PCR detection method was evaluated with 60 swabs from different body sites which were incubated overnight in brain-heart infusion. The PCR gave positive results for 27 of 29 swabs which were found to contain MRSA by conventional methods. The diagnostic values of the PCR assay for these samples were 93% sensitivity and 100% specificity. To determine the in vitro sensitivity of the assay, swabs were inoculated with serially diluted bacterial suspensions. After overnight enrichment the detection limit of the PCR was less than 10 CFU/swab. This new real-time PCR assay proved to be a fast, sensitive and specific tool for MRSA detection in a routine microbiological laboratory.