Evaluation of Staphylococcus aureus eradication therapy in orthopaedic surgery.

PURPOSE: Despite WHO recommendations, there is currently no national screening and eradication policy for the detection of methicillin-sensitive Staphylococcus aureus (MSSA) in the UK prior to elective orthopaedic surgery. This study aimed to evaluate the effectiveness of current standard methicillin-resistant S. aureus (MRSA) eradication therapies in the context of S. aureus (both MRSA and MSSA) decolonization in an elective orthopaedic population. METHODOLOGY: A total of 100 patients awaiting joint replacement surgery who were positive for S. aureus on PCR nasal screening underwent the current standard MRSA pre-operative decolonization regimen for 5 days. Prior to commencement of the eradication therapy, swabs of the anterior nares, throat and perineum were taken for culture. Further culture swabs were taken at 48-96 h following treatment, at hospital admission for surgery and at hospital discharge. Following the completion of treatment, patients were asked to provide feedback on their experience using Likert rating scales. The primary outcome of this study was S. aureus clearance 48-96 h following eradication treatment.Results/Key Findings. Clearance of S. aureus 48-96 h following treatment was 94 % anterior nares, 66 % throat and 88 % groin. Mean completion with nasal mupirocin was 98 %. There was no statistically significant recolonization effect between the end of the eradication treatment period and the day of surgery (P>0.05) at a median time of 10 days. CONCLUSION: Current MRSA decolonisation regimens are well tolerated and effective for MSSA decolonization for the anterior nares and groin. The decolonization effect is preserved for at least 10 days following treatment.

Underestimation of Staphylococcus aureus (MRSA and MSSA) carriage associated with standard culturing techniques: One third of carriers missed.

OBJECTIVES: Nasal carriers of Staphylococcus (S.) aureus (MRSA and MSSA) have an increased risk for healthcare-associated infections. There are currently limited national screening policies for the detection of S. aureus despite the World Health Organization's recommendations. This study aimed to evaluate the diagnostic performance of molecular and culture techniques in S. aureus screening, determine the cause of any discrepancy between the diagnostic techniques, and model the potential effect of different diagnostic techniques on S. aureus detection in orthopaedic patients. METHODS: Paired nasal swabs for polymerase chain reaction (PCR) assay and culture of S. aureus were collected from a study population of 273 orthopaedic outpatients due to undergo joint arthroplasty surgery. RESULTS: The prevalence of MSSA nasal colonization was found to be between 22.4% to 35.6%. The current standard direct culturing methods for detecting S. aureus significantly underestimated the prevalence (p = 0.005), failing to identify its presence in approximately one-third of patients undergoing joint arthroplasty surgery. CONCLUSION: Modelling these results to national surveillance data, it was estimated that approximately 5000 to 8000 S. aureus surgical site infections could be prevented, and approximately $140 million to $950 million (approximately £110 million to £760 million) saved in treatment costs annually in the United States and United Kingdom combined, by using alternative diagnostic methods to direct culture in preoperative S. aureus screening and eradication programmes.Cite this article: S. T. J. Tsang, M. P. McHugh, D. Guerendiain, P. J. Gwynne, J. Boyd, A. H. R. W. Simpson, T. S. Walsh, I. F. Laurenson, K. E. Templeton. Underestimation of Staphylococcus aureus (MRSA and MSSA) carriage associated with standard culturing techniques: One third of carriers missed. Bone Joint Res 2018;7:79-84. DOI: 10.1302/2046-3758.71.BJR-2017-0175.R1.

An outbreak of mumps with genetic strain variation in a highly vaccinated student population in Scotland.

An outbreak of mumps within a student population in Scotland was investigated to assess the effect of previous vaccination on infection and clinical presentation, and any genotypic variation. Of the 341 cases, 79% were aged 18-24. Vaccination status was available for 278 cases of whom 84% had received at least one dose of mumps containing vaccine and 62% had received two. The complication rate was 5·3% (mainly orchitis), and 1·2% were admitted to hospital. Genetic sequencing of mumps virus isolated from cases across Scotland classified 97% of the samples as genotype G. Two distinct clusters of genotype G were identified, one circulating before the outbreak and the other thereafter, suggesting the virus that caused this outbreak was genetically different from the previously circulating virus. Whilst the poor vaccine effectiveness we found may be due to waning immunity over time, a contributing factor may be that the current mumps vaccine is less effective against some genotypes. Although the general benefits of the measles-mumps-rubella (MMR) vaccine should continue to be promoted, there may be value in reassessing the UK vaccination schedule and the current mumps component of the MMR vaccine.

Comparison of ePlex Respiratory Pathogen Panel with Laboratory-Developed Real-Time PCR Assays for Detection of Respiratory Pathogens.

Infections of the respiratory tract can be caused by a diversity of pathogens, both viral and bacterial. Rapid microbiological diagnosis ensures appropriate antimicrobial therapy as well as effective implementation of isolation precautions. The ePlex respiratory pathogen panel (RP panel) is a novel molecular biology-based assay, developed by GenMark Diagnostics, Inc. (Carlsbad, CA), to be performed within a single cartridge for the diagnosis of 25 respiratory pathogens (viral and bacterial). The objective of this study was to compare the performance of the RP panel with those of laboratory-developed real-time PCR assays, using a variety of previously collected clinical respiratory specimens. A total of 343 clinical specimens as well as 29 external quality assessment (EQA) specimens and 2 different Middle East respiratory syndrome coronavirus isolates have been assessed in this study. The RP panel showed an agreement of 97.4% with the real-time PCR assay regarding 464 pathogens found in the clinical specimens. All pathogens present in clinical samples and EQA samples with a threshold cycle (CT ) value of <30 were detected correctly using the RP panel. The RP panel detected 17 additional pathogens, 7 of which could be confirmed by discrepant testing. In conclusion, this study shows excellent performance of the RP panel in comparison to real-time PCR assays for the detection of respiratory pathogens. The ePlex system provided a large amount of useful diagnostic data within a short time frame, with minimal hands-on time, and can therefore potentially be used for rapid diagnostic sample-to-answer testing, in either a laboratory or a decentralized setting.

Multicenter Evaluation of the Cepheid Xpert Hepatitis C Virus Viral Load Assay.

Viral load monitoring for hepatitis C virus (HCV) is necessary to diagnose infection and monitor response to therapy, but the tests involved are currently confined to specialist institutions. There is a need for a fast, accurate assay with limited operator input to enhance the access to viral load monitoring. We evaluated the quantification of HCV RNA in serum and plasma by the Cepheid Xpert HCV Viral Load assay in comparison to the Abbott RealTime HCV assay. Serum and plasma samples were gathered from HCV-infected individuals at four international sites. These were tested with the Xpert HCV Viral Load assay, and results were compared to quantification by the Abbott RealTime HCV assay. An external quality assessment panel of eight samples was also tested. In total, 614 samples were analyzed in the study, and the qualitative results agreed on the two platforms for 588 (95.8%) samples. Further analysis of 396 samples quantified by both tests showed strong correlation (correlation coefficient r = 0.99) across the quantifiable range, with Bland-Altman plot data showing a mean difference (±1.96 standard deviation) of 0.03 ± 0.44 log10 IU/ml. In the external quality assessment panel, the Xpert HCV Viral Load assay results (quantified in log10 IU per milliliter) were within 1 standard deviation of the target value for all but one sample, which was also similarly misquantified by the Abbott RealTime HCV assay. The Xpert HCV Viral Load assay performs well compared to a market-leading HCV viral load test and should be considered for instances where rapid near-to-patient testing is required.

Fatal disseminated varicella zoster infection following zoster vaccination in an immunocompromised patient.

A 79-year-old man with chronic lymphocytic leukaemia presented with fever and a widespread vesicular rash on 19 November 2014. The patient had not been under immunosuppressive regime for 6 months. He had received a shingles vaccine on 14th October and developed flu-like symptoms after 2 weeks. Intravenous antimicrobial therapy including aciclovir was started. He remained stable with no evidence of systemic involvement. On day 5, he developed respiratory and renal failure that required transfer to intensive care unit. Vesicle fluid, bronchoalveolar lavage and plasma were positive for varicella zoster virus by PCR. Slight clinical improvement allowed extubation on day 16. He subsequently deteriorated and died on day 25. Multiorgan failure was considered the immediate cause of death whereas disseminated varicella zoster infection was stated in the medical certificate as the other condition leading to this outcome. Varicella zoster Oka vaccine strain was detected in vesicle fluid, using PCR.

Low-pathogenicity Mycoplasma spp. alter human monocyte and macrophage function and are highly prevalent among patients with ventilator-acquired pneumonia.

BACKGROUND: Ventilator-acquired pneumonia (VAP) remains a significant problem within intensive care units (ICUs). There is a growing recognition of the impact of critical-illness-induced immunoparesis on the pathogenesis of VAP, but the mechanisms remain incompletely understood. We hypothesised that, because of limitations in their routine detection, Mycoplasmataceae are more prevalent among patients with VAP than previously recognised, and that these organisms potentially impair immune cell function. METHODS AND SETTING: 159 patients were recruited from 12 UK ICUs. All patients had suspected VAP and underwent bronchoscopy and bronchoalveolar lavage (BAL). VAP was defined as growth of organisms at >10(4) colony forming units per ml of BAL fluid on conventional culture. Samples were tested for Mycoplasmataceae (Mycoplasma and Ureaplasma spp.) by PCR, and positive samples underwent sequencing for speciation. 36 healthy donors underwent BAL for comparison. Additionally, healthy donor monocytes and macrophages were exposed to Mycoplasma salivarium and their ability to respond to lipopolysaccharide and undertake phagocytosis was assessed. RESULTS: Mycoplasmataceae were found in 49% (95% CI 33% to 65%) of patients with VAP, compared with 14% (95% CI 9% to 25%) of patients without VAP. Patients with sterile BAL fluid had a similar prevalence to healthy donor BAL fluid (10% (95% CI 4% to 20%) vs 8% (95% CI 2% to 22%)). The most common organism identified was M. salivarium. Blood monocytes from healthy volunteers incubated with M. salivarium displayed an impaired TNF-α response to lipopolysaccharide (p=0.0003), as did monocyte-derived macrophages (MDMs) (p=0.024). MDM exposed to M. salivarium demonstrated impaired phagocytosis (p=0.005). DISCUSSION AND CONCLUSIONS: This study demonstrates a high prevalence of Mycoplasmataceae among patients with VAP, with a markedly lower prevalence among patients with suspected VAP in whom subsequent cultures refuted the diagnosis. The most common organism found, M. salivarium, is able to alter the functions of key immune cells. Mycoplasmataceae may contribute to VAP pathogenesis.

Lessons learned from a prolonged and costly norovirus outbreak at a Scottish medicine of the elderly hospital: case study.

BACKGROUND: Norovirus outbreaks are a major burden for healthcare facilities globally. AIM: Lessons learned to inform an action plan to improve facilities as well as responses to norovirus within the medicine of the elderly (MoE) hospital as well as other NHS (National Health Service) Lothian facilities. METHODS: This study investigated the impact of a prolonged outbreak at an MoE hospital in one of the 14 Scottish health boards between February and March 2013. FINDINGS: In all, 143 patients (14.80 cases per 1000 inpatient bed-days) and 30 healthcare staff (3.10 cases per 1000 inpatient bed-days) were affected clinically and 63 patients were confirmed virologically. Restricting new admissions to affected units resulted in 1192 lost bed-days. The cost due to lost bed-days in addition to staff absence and management of the outbreak was estimated at £341,534 for this incident alone. At certain points during the outbreak, the whole facility was closed with resulting major impact on the health board's acute care hospitals. CONCLUSION: Due to the outbreak, new measures were implemented for the first time within NHS Lothian that included floor-by-floor (instead of individual) ward closures, enhanced cleaning with chlorine-based products throughout the hospital, reduction in bed capacity with enhanced bed-spacing and interruption to direct admissions from the Board's general practice surgeries, and temporary suspension of visitors to affected areas. Together with regular communication to staff, patients, relatives, and the public throughout the outbreak and good engagement of staff groups in management of the incident, the outbreak was gradually brought under control.

Development of two real-time multiplex PCR assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections.

The frequent lack of a positive and timely microbiological diagnosis in patients with lower respiratory tract infection (LRTI) is an important obstacle to antimicrobial stewardship. Patients are typically prescribed broad-spectrum empirical antibiotics while microbiology results are awaited, but, because these are often slow, negative, or inconclusive, de-escalation to narrow-spectrum agents rarely occurs in clinical practice. The aim of this study was to develop and evaluate two multiplex real-time PCR assays for the sensitive detection and accurate quantification of Streptococcus pneumoniae, Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. We found that all eight bacterial targets could be reliably quantified from sputum specimens down to a concentration of 100 CFUs/reaction (8333 CFUs/mL). Furthermore, all 249 positive control isolates were correctly detected with our assay, demonstrating effectiveness on both reference strains and local clinical isolates. The specificity was 98% on a panel of nearly 100 negative control isolates. Bacterial load was quantified accurately when three bacterial targets were present in mixtures of varying concentrations, mimicking likely clinical scenarios in LRTI. Concordance with culture was 100% for culture-positive sputum specimens, and 90% for bronchoalveolar lavage fluid specimens, and additional culture-negative bacterial infections were detected and quantified. In conclusion, a quantitative molecular test for eight key bacterial causes of LRTI has the potential to provide a more sensitive decision-making tool, closer to the time-point of patient admission than current standard methods. This should facilitate de-escalation from broad-spectrum to narrow-spectrum antibiotics, substantially improving patient management and supporting efforts to curtail inappropriate antibiotic use.

An international, prospective, multicenter evaluation of the combination of AdvanDx Staphylococcus QuickFISH BC with mecA XpressFISH for detection of methicillin-resistant Staphylococcus aureus isolates from positive blood cultures.

Sepsis caused by Staphylococcus aureus is a major health problem worldwide. Better outcomes are achieved when rapid diagnosis and determination of methicillin susceptibility enable early optimization of antimicrobial therapy. Eight large clinical laboratories, seven from the United States and one from Scotland, evaluated the combination of the Staphylococcus QuickFISH BC and the new mecA XpressFISH assay (both AdvanDx, Woburn, MA, USA) for the detection of methicillin-resistant S. aureus in positive blood cultures. Blood cultures flagged as positive by automated blood culture instruments and demonstrating only Gram-positive cocci in clusters on Gram stain were tested by QuickFISH, a 20-min assay. If only S. aureus was detected, mecA XpressFISH testing followed. The recovered S. aureus isolates were tested by cefoxitin disk diffusion as the reference method. The QuickFISH assay results were concordant with the routine phenotypic testing methods of the testing laboratories in 1,211/1,221 (99.1%) samples and detected 488/491 S. aureus organisms (sensitivity, 99.4%; specificity, 99.6%). Approximately 60% of the samples (730) contained coagulase-negative staphylococci or nonstaphylococci as assessed by the QuickFISH assay and were not tested further. The 458 compliant samples positive exclusively for S. aureus by the QuickFISH assay were tested by the mecA XpressFISH assay, which detected 209 of 211 methicillin-resistant S. aureus organisms (sensitivity, 99.1%; specificity, 99.6%). The mecA XpressFISH assay also showed high reproducibility, with 534/540 tests performed by 6 operators over 5 days achieving reproducible results (98.9% agreement). The combination of the Staphylococcus QuickFISH BC and mecA XpressFISH assays is sensitive, specific, and reproducible for the detection of methicillin-resistant S. aureus and yields complete results in 2 h after the blood culture turns positive.

Development of a PCR-free electrochemical point of care test for clinical detection of methicillin resistant Staphylococcus aureus (MRSA).

An MRSA assay requiring neither labeling nor amplification of target DNA has been developed. Sequence specific binding of fragments of bacterial genomic DNA is detected at femtomolar concentrations using electrochemical impedance spectroscopy (EIS). This has been achieved using systematic optimisation of probe chemistry (PNA self-assembled monolayer film on gold electrode), electrode film structure (the size and nature of the chemical spacer) and DNA fragmentation, as these are found to play an important role in assay performance. These sensitivity improvements allow the elimination of the PCR step and DNA labeling and facilitate the development of a simple and rapid point of care test for MRSA. Assay performance is then evaluated and specific direct detection of the MRSA diagnostic mecA gene from genomic DNA, extracted directly from bacteria without further treatment is demonstrated for bacteria spiked into saline (10(6) cells per mL) on gold macrodisc electrodes and into human wound fluid (10(4) cells per mL) on screen printed gold electrodes. The latter detection level is particularly relevant to clinical requirements and point of care testing where the general threshold for considering a wound to be infected is 10(5) cells per mL. By eliminating the PCR step typically employed in nucleic acid assays, using screen printed electrodes and achieving sequence specific discrimination under ambient conditions, the test is extremely simple to design and engineer. In combination with a time to result of a few minutes this means the assay is well placed for use in point of care testing.

Increased reports of Mycoplasma pneumoniae from laboratories in Scotland in 2010 and 2011 - impact of the epidemic in infants.

In common with reports from other European countries, we describe a substantial increase in the number of laboratory reports of Mycoplasma pneumoniae in Scotland in 2010 and 2011. The highest number of reports came from those aged one year and younger. However, reports from young children were more likely to come from PCR testing than serological testing.

Molecular epidemiology of norovirus in Edinburgh healthcare facilities, Scotland 2007-2011.

Norovirus (NoV) is a leading cause of outbreaks of gastroenteritis worldwide, and a major burden for healthcare facilities. This study investigated the NoV genotypes responsible for outbreaks in Edinburgh healthcare facilities between June 2008 and July 2011, and studied their temporal distribution to enable a better understanding of the epidemiology of the outbreaks. A total of 287 samples positive for NoV genogroup II (GII) RNA by reverse transcription-polymerase chain reaction (RT-PCR) during routine diagnostic testing were investigated. Nested RT-PCR (nRT-PCR) and sequencing was used to genotype the NoV strains. Overall, a total of 69 NoV strains belonging to six different genoclusters (GII.1, GII.2, GII.3, GII.4, GII.6, GII.13) were detected. The predominant genotype was GII.4 that included four variants, GII.4 2006a, GII.4 2006b, GII.4 2007 and GII.4 2010. Importantly, increases in NoV activity coincided with the emergence of new GII.4 strains, highlighting the need for an active surveillance system to allow the rapid identification of new strains.

Population exposure to a novel influenza A virus over three waves of infection.

BACKGROUND: The influenza A(H1N1)2009 virus has been spreading throughout the world since April 2009. Since then, several studies have been undertaken to measure the frequency of antibodies that react against the virus. Microneutralisation assays have regularly been used for these analyses, and titres of ≥40 have conventionally been taken to represent significant levels of antibodies (this significance is derived from it being four times the minimum level of antibodies that the assay can detect rather an established correlate of protection). However a microneutralisation titre that correlates with protection against influenza A(H1N1)2009 has not been established. OBJECTIVES: Analysing influenza A(H1N1)2009 antibody seroprevalence in Scotland at multiple timepoints, and in different age groups and geographical locations, and comprehensively describing the spread of the virus in Scotland (taken alongside previously published data). This study presents for the first time the effects of a novel influenza virus on a naïve population that has been followed from the initial outbreak to a time when the majority of the population have reactive antibodies. STUDY DESIGN: A microneutralisation titre ≥10 represents the minimum level of antibodies detectable by the assay. Blood samples (taken in April 2009 and April 2010 in Edinburgh (n=400 each year), and in February 2011 in Aberdeen, Edinburgh, Glasgow, and Inverness (n=1600)) were tested for the presence of influenza A(H1N1)2009 antibodies at this titre. This represents an effective indicator of the proportion of a population who have been exposed to the virus. RESULTS: Following the 2010/2011 influenza season, there is evidence of exposure to influenza A(H1N1)2009 in approximately four fifths of the Scottish population. CONCLUSIONS: This study provides impetus to the call for further research in establishing robust correlates of susceptibility to influenza infection and the development of clinical illness, provides useful information for future outbreaks, and is relevant to public health policy in planning for future influenza seasons.

Disease burden of the most commonly detected respiratory viruses in hospitalized patients calculated using the disability adjusted life year (DALY) model.

BACKGROUND: The most common acute infections occur in the respiratory tract. Recent discoveries of several novel viruses have markedly increased the repertoire of agents understood to cause presentations of acute respiratory disease. OBJECTIVES: Further understanding is needed of the relative importance of newly discovered pathogens in the clinical setting to provide clinicians with an indication of appropriate diagnostic and therapeutic targets. To address this, quantification of the disease burden of respiratory viruses in hospitalized patients was undertaken. STUDY DESIGN: Disease burden caused by respiratory viruses in hospitalized patients was quantified using the World Health Organization endorsed DALY model. Diagnostic testing results from samples collected over three years for adenovirus (AdV), influenzas A and B, parainfluenza viruses 1, 2 and 3 (PIV-1, -2 and -3), respiratory syncytial virus (HRSV), and previously published retrospective screening for human metapneumovirus, rhinoviruses, and four respiratory coronaviruses were applied to the DALY model. Disability weights were calculated per 1000 hospitalized patients in age banded groups. RESULTS: Strikingly different disease burden profiles were observed in children and adults. Adenoviruses were among the leading cause of respiratory presentations in children but not adults. HRSV and influenza A were consistently one of the greatest causes of disease regardless of sampled population. Rhinoviruses and PIV-3 were significant pathogens in all groups except those aged 16-64 years. In immunocompromised patients rhinoviruses were the leading viral cause of disease. CONCLUSIONS: These analyses provide a framework which can be used to identify where finite resources should be directed in respiratory therapeutics and vaccine development.

Epidemiology and costs associated with norovirus outbreaks in NHS Lothian, Scotland 2007-2009.

Healthcare-associated gastroenteritis outbreaks are becoming more common and are recognized challenges in hospital and community settings. In Edinburgh [NHS (National Health Service) Lothian], all the hospitals and the community were actively monitored for outbreaks of gastroenteritis from September 2007 to June 2009. In total, 1732 patients and 599 healthcare staff were affected in 192 unit outbreaks. In the acute sector, 1368 patients (0.99 cases/1000 inpatient bed-days) and 406 healthcare staff (0.29 cases/1000 inpatient bed-days) were affected in 155 unit outbreaks (0.23 unit outbreaks/day). Noroviruses were detected in 142 outbreaks (74%); 50 were not laboratory confirmed but were presumed to be noroviruses on epidemiological grounds. The closure of affected units to new admissions resulted in the loss of 3678 bed-days. By extrapolation, lost bed-days and staff absence due to gastroenteritis outbreaks cost NHS Lothian £1.2 million for the two norovirus seasons. Outbreaks in which the affected unit was closed within the first three days of recognizing the index case were contained in a mean of six days, and outbreaks in units that were closed later persisted for a mean of seven days; this difference was not statistically significant. Rapid implementation of control measures was effective in the control of outbreaks.

Influenza A(H1N1)2009 antibody seroprevalence in Scotland following the 2010/11 influenza season.

Following the 2010/11 influenza season, we determined the age- and location-specific seroprevalence of antibodies against the influenza A(H1N1)2009 virus in Scotland. Samples were analysed by microneutralisation assay. Age/seropositivity profiles varied significantly between cities. The increases in seroprevalence relative to the previous influenza season (2009/10) were similar across age groups and geographic locations. However, the increased seropositivity in older adults appeared to be driven by exposure to vaccination, indicating significantly lower levels of infection than in younger age groups.

Rapid testing for respiratory syncytial virus in a paediatric emergency department: benefits for infection control and bed management.

Respiratory syncytial virus (RSV) is responsible for annual winter outbreaks of respiratory tract infection among children in temperate climates, placing severe pressure on hospital beds. Cohorting of affected infants has been demonstrated to be an effective strategy in reducing nosocomial transmission of RSV, and may keep cubicles free for other patients who require them. Testing of symptomatic children for RSV is standard practice, but unfortunately traditional laboratory testing is not rapid enough to aid decision-making processes. Rapid point-of-care testing (POCT) in the emergency department has been suggested as an alternative. We performed a prospective study to quantify the amount of cubicle time saved by using POCT results to allow a targeted cohorting strategy. Over the four-month study period, the POCT allowed 183 children to be admitted directly to a designated cohort area, thus saving 568.5 cubicle-days for other patients. This is equivalent to five cubicles being left free for each day of the study period. This is the first time the benefits of using POCT have been quantified in this way. POCT for RSV is a safe, cost-effective and efficient way to improve bed management.

Real-time evaluation of an optimized real-time PCR assay versus Brilliance chromogenic MRSA agar for the detection of meticillin-resistant Staphylococcus aureus from clinical specimens.

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

Comparison of two multilocus variable-number tandem-repeat methods and pulsed-field gel electrophoresis for differentiating highly clonal methicillin-resistant Staphylococcus aureus isolates.

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson's index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.