Use of a multiplex molecular beacon platform for rapid detection of methicillin and vancomycin resistance in Staphylococcus aureus

Drug resistance, particularly vancomycin and methicillin resistance, in Staphylococcus aureus continues to emerge as a significant public health threat in both the hospital and community settings. In addition to the limited treatment options, S. aureus strains acquire and express numerous virulence factors that continue to increase its ability to cause a wide spectrum of human disease. As a result, empirical treatment decisions are confounded and there is a heightened need for a diagnostic test (or assay) to rapidly identify antibiotic resistance and specific virulence determinants and indicate the appropriate treatment. To that end we developed a platform using multiplex molecular beacon probes with real-time PCR for the rapid detection of drug resistance-determining genes and virulence factors in S. aureus. In this study, we demonstrate the specificity and sensitivity of our platform for detection of the genes conferring methicillin (mecA) and vancomycin (vanA) resistance as well as a gene encoding the virulence factor Panton-Valentine leucocidin (lukF) in S. aureus isolates.     

Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus

The probe-based Velogene Rapid MRSA Identification Assay (ID Biomedical Corp., Vancouver, British Columbia, Canada) and the latex agglutination MRSA-Screen (Denka Seiken Co., Tokyo, Japan) were evaluated for their ability to identify methicillin-resistant Staphylococcus aureus (MRSA) and to distinguish strains of MRSA from borderline oxacillin-resistant S. aureus (BORSA; mecA-negative, oxacillin MICs of 2 to 8 microgram/ml). The Velogene is a 90-min assay using a chimeric probe to detect the mecA gene. MRSA-Screen is a 15-min latex agglutination test with penicillin-binding protein 2a antibody-sensitized latex particles. We compared these assays with the BBL Crystal MRSA ID System (Becton Dickinson, Cockeysville, Md.) and with PCR for mecA gene detection. A total of 397 clinical isolates of S. aureus were tested, consisting of 164 methicillin-susceptible strains, 197 MRSA strains, and 37 BORSA strains. All assays performed well for the identification of MRSA with sensitivities and specificities for Velogene, MRSA-Screen, and BBL Crystal MRSA ID of 98.5 and 100%, 98.5 and 100%, and 98.5 and 98%, respectively. Three MRSA strains were not correctly identified by each of the Velogene and MRSA-Screen assays, but repeat testing with a larger inoculum resolved the discrepancies. The BBL Crystal MRSA ID test misclassified four BORSA strains as MRSA. Both the Velogene and the MRSA-Screen assays are easy to perform, can accurately differentiate BORSA isolates from MRSA isolates, and provide a rapid alternative for the detection of methicillin resistance in S. aureus in clinical laboratories, especially when mecA PCR gene detection is unavailable.     

Clinical impact of a real-time PCR assay for rapid identification of Staphylococcus aureus and determination of methicillin resistance from positive blood cultures

The full identification and susceptibility profile of staphylococci from positive blood cultures (BCs) generally takes 24–48 h using phenotypic methods. The aim of this prospective study was to evaluate the clinical impact of a real-time PCR strategy for rapid identification of staphylococci and determination of methicillin resistance directly from positive BCs. During a 12-month period, 250 episodes of positive BCs with organism morphology resembling staphylococci were enrolled. Two strategies were compared: conventional (n = 128) using standard phenotypic methods or rapid (n = 122) using a real-time PCR assay that is able to detect specific genes of Staphylococcus aureus (nuc and sa442) and the encoding gene for methicillin resistance (mecA). Overall, 97 episodes (39%) were clinical-significant bloodstream infections. The prevalence of methicillin resistance of S. aureus was 24%. A favorable outcome (defined as clinical cure with resolution of signs and no evidence of recurrence or relapse at 12 weeks follow-up) was observed in similar proportions of episodes with (58%) or without (60%) PCR testing (p 0.8). In multivariate analyses, age and infection due to methicillin-susceptible S. aureus (adjusted OR 0.96, 95% CI 0.93–0.99; and adjusted OR 3.11, 95% CI 1.12–8.65, respectively) were the unique factors independently associated with a favorable outcome. Among the 153 episodes of contaminated BCs, similar proportions received unjustified antibiotic therapy (PCR strategy: 17%, conventional testing: 10%; p 0.33). In a setting with a moderate level of methicillin-resistant S. aureus and relatively high contamination of BCs, real-time PCR testing was not beneficial compared to conventional methods.     

Immuno-flow cytometry for the rapid identification of Staphylococcus aureus and the detection of methicillin resistance

Methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) can be reliably differentiated by flow cytometry when labeled with nucleic acid dyes. The purpose of this study was to determine if this differentiation can be achieved while labeling with a S. aureus-specific anti-staphylococcal protein A antibody instead of nucleic acid dyes. A total of 103 S. aureus isolates were incubated for 4 h at 37°C in Mueller Hinton broth with and without oxacillin, then stained with anti-staphylococcal protein A antibody, and analyzed by flow cytometry using the Micro PRO? instrument. Dot plots (side scatter vs. fluorescence intensity) of isolates exposed to oxacillin were examined to define two gates encompassing the majority of MSSA and MRSA signal events, respectively. The ratio of signal event counts in the two gates was called the gate signal count ratio (GSCR), and its performance was evaluated using receiver operating characteristic (ROC) curves. The GSCR could differentiate MRSA from MSSA with 98% sensitivity and 100% specificity using a cut-off of 0.6868 when the two gates were defined as follows: gate 1, fluorescence intensity 2-10, side scatter 5-70; gate 2, fluorescence intensity 7-700, side scatter 70-500. MRSA and MSSA can be accurately detected and differentiated by flow cytometry after 4 h of oxacillin exposure when labeled with anti-staphylococcal protein A antibody.     

Evaluation of a single-locus real-time polymerase chain reaction as a screening test for specific detection of methicillin-resistant Staphylococcus aureus in ICU patients

The aim of the present study was to determine the diagnostic value of a single-locus real-time polymerase chain reaction (PCR) recently proposed for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) from clinical samples (IDI-MRSA; Infectio Diagnostic, Sainte-Foy, Québec, Canada). This test, which was developed on the basis of studies of the sequence analysis of the mecA gene carried by staphylococcal cassette chromosome mec (SCCmec), was used to screen nasal swabs of 320 intensive care unit (ICU) patients at admission. The results were compared with those of conventional culture of swabs from several body sites. When compared with culture of swabs from the nose, throat, and wounds, the diagnostic values of the real-time PCR test from nasal swabs were as follows: 92.3% sensitivity, 98.6% specificity, 75.0% positive predictive value, and 99.6% negative predictive value. Fifteen (4.7%) samples could not be evaluated because the PCR reaction was inhibited, even after the samples were frozen and thawed for retesting. Culture of nasal swabs showed that 78 of the patients were colonized with methicillin-susceptible S. aureus. Unexpectedly, 4 (5.1%) of these samples gave false-positive results in the IDI-MRSA. These isolates were all single clones, as shown by pulsed-field gel electrophoresis and spa typing. Reliable results were obtained with the IDI-MRSA assay, even in a patient population with a low prevalence (approximately 4%) of MRSA and even when compared with swabs of different body sites. Nevertheless, further work is needed to reduce the inhibition rate of the PCR and to explain why false-positive results were obtained with methicillin-susceptible S. aureus.This study was presented in part at the 6th Symposium Krankenhausinfektionen, Ulm, Germany, March 2005.     

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.     

Validation of a high-throughput real-time polymerase chain reaction assay for the detection of capripoxviral DNA

Capripoxviruses, which are endemic in much of Africa and Asia, are the aetiological agents of economically devastating poxviral diseases in cattle, sheep and goats. The aim of this study was to validate a high-throughput real-time PCR assay for routine diagnostic use in a capripoxvirus reference laboratory. The performance of two previously published real-time PCR methods were compared using commercially available reagents including the amplification kits recommended in the original publication. Furthermore, both manual and robotic extraction methods used to prepare template nucleic acid were evaluated using samples collected from experimentally infected animals. The optimised assay had an analytical sensitivity of at least 63 target DNA copies per reaction, displayed a greater diagnostic sensitivity compared to conventional gel-based PCR, detected capripoxviruses isolated from outbreaks around the world and did not amplify DNA from related viruses in the genera Orthopoxvirus or Parapoxvirus. The high-throughput robotic DNA extraction procedure did not adversely affect the sensitivity of the assay compared to manual preparation of PCR templates. This laboratory-based assay provides a rapid and robust method to detect capripoxviruses following suspicion of disease in endemic or disease-free countries.     

Thermostabilization of indigenous multiplex polymerase chain reaction reagents for detection of enterotoxigenic Staphylococcus aureus

Background/Purpose

Among DNA-based techniques, polymerase chain reaction (PCR) is the most widely accepted molecular tool for the detection of pathogens. However, the technique involves several reagents and multiple pipetting steps that often lead to error-prone results. Additionally, the reagents entail a cold-chain facility to maintain their stability during storage and transportation. The main aim of the present study was to simplify the utility of a pre-optimized multiplex PCR format that was developed to detect toxigenic strains of Staphylococcus aureus by providing stable, pre-mixed, and ready-to-use master mix in a lyophilized formulation.

Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus

Infectious laryngotracheitis is an acute viral respiratory disease of chickens with a worldwide distribution. Sensitive detection of the causative herpesvirus is particularly important because it can persist in the host at a very low copy number and be transmitted to other birds. Quantification of viral genome copy number is also useful for clinical investigations and experimental studies. In the study presented here, a quantitative polymerase chain reaction (qPCR) assay was developed using SYBR Green chemistry and the viral gene UL15a to detect and quantify infectious laryngotracheitis virus (ILTV) in ILTV-inoculated chicken embryos or naturally infected birds. The specificity of the assay was confirmed using a panel of viral and bacterial pathogens of poultry. The sensitivity of the assay was compared with two conventional PCR assays, virus titration and an antigen-detecting enzyme-linked immunosorbent assay. The qPCR developed in this study was highly sensitive and specific, and has potential for quantification of ILTV in tissues from naturally and experimentally infected birds and embryos.     

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.     

Development of a real-time reverse transcription polymerase chain reaction assay for detection of marine caliciviruses (genus Vesivirus)

Marine caliciviruses form a distinct lineage within the genus Vesivirus (family Caliciviridae). This group includes vesicular exanthema of swine virus (VESV) and San Miguel sea lion virus (SMSV) and other related viruses which have been proposed to be marine in origin isolated from a variety of terrestrial and marine animals. Rapid and reliable detection of marine caliciviruses is important as these viruses appear to be widespread and can cause vesicular disease in a wide variety of susceptible hosts including pigs and experimentally infected cattle where clinical signs cannot be easily distinguished from foot-and-mouth disease (FMD), swine vesicular disease (SVD) and vesicular stomatitis (VS). A real-time RT-PCR assay targeting conserved nucleotide sequences in the RNA-dependent RNA polymerase (3D) region of the genome successfully detected cell culture-grown virus preparations of more than thirty marine calicivirus serotypes. Only the atypical SMSV serotypes 8 and 12 failed to be detected, which provided further indication of genetic divergence between these and the other calicivirus serotypes said to be marine in origin. The real-time RT-PCR assay also specifically amplified RNA from samples collected following experimental inoculation of pigs with VESV. No cross-reactivity was demonstrated when the assay was tested with RNA prepared from representative viruses of FMD, SVD and VS. The real-time RT-PCR assay described is a sensitive and specific tool for detection and differential diagnosis of these viruses from other vesicular-disease causing viruses.     

Prospective validation of quantitative fluorescent polymerase chain reaction for rapid detection of common aneuploidies

PurposeTo prospectively validate a quantitative fluorescent polymerase chain reaction (PCR) assay as a method of rapid prenatal aneuploidy detection for chromosomes 13, 18, 21, X, and Y.MethodsA commercial quantitative fluorescent PCR kit was validated on 200 known, blinded, prenatal DNA specimens. The kit was then validated prospectively on 1069 amniotic fluid specimens, and the results were compared with the karyotype results and the results of interphase fluorescence in situ hybridization testing, when performed in the course of standard care. Turnaround time was monitored in a subset of the prospective specimens.ResultsThe analytical sensitivity and specificity of testing in the validation specimens were 98.9% and 100%, respectively. There were no false positives and a single false negative, a mosaic sex chromosome aneuploidy interpreted as normal. In the prospective study, the analytical sensitivity and specificity were 98% and 100%, respectively. No false positives and a single false negative, again a sex chromosome mosaic, were detected. Overall, 72.5% of all chromosomal anomalies and 87.7% of clinically significant chromosome anomalies were detected by quantitative fluorescent PCR. The average and median turnaround times were 30.5 and 25.1 hours, respectively.ConclusionsQuantitative fluorescent PCR is a robust and accurate method of rapid prenatal aneuploidy detection. Genet Med 2011:13(2):140–147.     

Development of a rapid and sensitive polymerase chain reaction assay for detection of bovine herpesvirus type 1 in bovine semen.

We developed a polymerase chain reaction (PCR) assay to detect bovine herpesvirus type 1 (BHV-1) in bovine semen. Since bovine semen contains components that inhibit PCR amplification, a protocol was developed to purify BHV-1 DNA from bovine semen. To identify failures of PCR amplification, we used an internal control template that was coamplified by the same PCR primers. When separated fractions of BHV-1-contaminated semen were analyzed by the PCR, we found that more than 90% of the BHV-1 DNA was present in a pooled fraction consisting of seminal fluid, nonsperm cells, and virus adsorbed to spermatozoa. By using this fraction, three to five molecules of BHV-1 DNA in 50 microliters of bovine semen could be detected. A pilot study to compare this PCR assay with the routinely used virus isolation method showed that this PCR assay is 2- to 100-fold more sensitive. In addition, the results of the PCR assay are available in 1 day, whereas the virus isolation method takes 7 days. Therefore, the PCR assay may be a good alternative to the virus isolation method.     

Clinical validation of the molecular BD GeneOhm StaphSR assay for direct detection of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus in positive blood cultures

The rapid detection of Staphylococcus aureus bacteremia and a swift determination of methicillin susceptibility has serious clinical implications affecting patient mortality. This study evaluated the StaphSR assay (BD GeneOhm, San Diego, CA), a real-time PCR assay, for the identification and differentiation of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) from 300 positive blood cultures. The BD GeneOhm StaphSR assay was performed and interpreted according to the manufacturer's recommendations. Positive blood cultures (containing predominantly gram-positive cocci in clusters) were subcultured on 5% sheep blood agar plates. After 18 to 24 h of incubation, isolates morphologically consistent with S. aureus were presumptively identified by latex agglutination (Staphaurex Plus; Remel, Lenexa, KS). Susceptibility testing was initially performed with the Phoenix automated microbiology system (BD Diagnostics, Sparks, MD). Additional susceptibility testing of samples with discrepant results was done using BBL oxacillin screen agar (BD Diagnostics, Sparks, MD), oxacillin and cefoxitin Etests (AB Biodisk, Solna, Sweden) on Mueller-Hinton agar, an immunoassay for penicillin binding protein 2' (Denka Seiken Co., Tokyo, Japan), and mecA PCR. The sensitivity, specificity, and positive and negative predictive values of the BD GeneOhm StaphSR assay for MSSA detection were 98.9, 96.7, 93.6, and 99.5%, respectively. For the detection of MRSA, the BD GeneOhm StaphSR assay was 100% sensitive and 98.4% specific; positive and negative predictive values for MRSA detection were 92.6 and 100%, respectively. Inhibition was seen with only one sample, and the issue was resolved upon retesting. The BD GeneOhm StaphSR assay appears to be a valuable diagnostic tool for quickly differentiating bacteremia caused by MSSA and MRSA from that caused by other gram-positive cocci.     

Double triplex real-time PCR assay for simultaneous detection of Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus haemolyticus and determination of their methicillin resistance directly from positive blood culture bottles

We developed and validated here a double triplex real-time PCR assay to simultaneously detect and identify Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus haemolyticus and their methicillin resistance in a single reaction directly from Gram-positive cocci-in-clusters (GPCs)-positive blood culture bottles. From August 15, 2009 through February 15, 2010, 238 GPC-positive samples were collected and identified by conventional methods as 11 methicillin-resistant S. aureus (MRSA), 28 methicillin-susceptible S. aureus (MSSA), 176 MR coagulase-negative staphylococci (MRCoNS), 21 MSCoNS and two Enterococcus faecalis. The double triplex real-time PCR assay was targeted and detected tuf, nuc and mecA genes in the first tube and atlE, gap and mvaA genes in the second tube which could be run simultaneously. The detection limit of the assay was found at 10³ CFU/ml for the atleE gene, 10⁴ CFU/ml for the mva gene and 10⁵ CFU/ml for gap, nuc, mecA and tuf genes based on seeding experiments. All Staphylococcus species except two S. epidermidis were correctly identified by the assay. The double triplex real-time PCR assay quickly and accurately detects S. aureus, S. epidermidis, S. hominis and S. haemolyticus and their methicillin resistance in a single reaction directly from positive blood culture bottles within 83 min.     

Detection of methicillin-resistant Staphylococcus aureus by polymerase chain reaction]

The low-affinity penicillin-binding protein (PBP 2') is associated with methicillin-resistance of Staphylococcus aureus and its structural gene (mecA) not present in methicillin-susceptible S. aureus could be detected by the polymerase chain reaction (PCR) method, in which a 533 bp region of mecA was amplified and detected by agarose gel electrophoresis. Survey for the mecA gene in 210 clinical isolates of S. aureus revealed that, while there was a gross correlation between the presence of the mecA gene and the resistance level to beta-lactams, three strains of mecA (+) tested showed beta-lactam susceptibility similar to those of mecA (-) strains. These three strains did not produce a detectable amount of PBP 2' constitutively nor inducibly, which was the cause of their high susceptibility to beta-lactams. One of them yielded a typical methicillin-resistant variant at a low frequency with a concomitant recovery of PBP 2' production when the bacterial cells of high density were spread onto an agar plate containing 10 micrograms/ml of oxacillin. These findings suggested that typical methicillin resistant S. aureus occurred during chemotherapy with beta-lactam antibiotics even when resistant strains could not be detected by the susceptibility test and thus all mecA (+) strains including those with high susceptibility should be precisely detected.