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Background Gram-negative pathogens, such as Escherichia coli, are common causes of bloodstream infections (BSIs) and increasingly demonstrate antimicrobial resistance. Molecular rapid diagnostic tests (mRDTs) offer faster pathogen identification and susceptibility results, but higher costs compared with conventional methods. We determined the cost-effectiveness of the BioFire FilmArray Blood Culture Identification (BCID) Panel, as a type of mRDT, compared with conventional methods in the identification of E. coli BSIs. Methods We constructed a decision analytic model comparing BCID with conventional methods in the identification and susceptibility testing of hospitalized patients with E. coli BSIs from the perspective of the public healthcare payer. Model inputs were obtained from published literature. Cost-effectiveness was calculated by determining the per-patient admission cost, the QALYs garnered and the incremental cost-effectiveness ratios (ICERs) where applicable. Monte Carlo probabilistic sensitivity analyses and one-way sensitivity analyses were conducted to assess the robustness of the model. All costs reflect 2019 Canadian dollars. Results The Monte Carlo probabilistic analyses resulted in cost savings ($27 070.83 versus $35 649.81) and improved QALYs (8.65 versus 7.10) in favour of BCID. At a willingness to pay up to $100 000, BCID had a 72.6%–83.8% chance of being cost-effective. One-way sensitivity analyses revealed length of stay and cost per day of hospitalization to have the most substantial impact on costs and QALYs. Conclusions BCID was found to be cost-saving when used to diagnose E. coli BSI compared with conventional testing. Cost savings were most influenced by length of stay and cost per day of hospitalization.

Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large-scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller-scale studies, they can be challenging to apply to larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performances of the instrument. Herein, we introduce an analytical strategy for addressing this problem in large-scale microbial studies. Our approach quantifies microbial boundary fluxes using two zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns that are plumbed to enable offline column equilibration. Using this strategy, we show that over 397 common metabolites can be resolved in 4.5 min per sample and that metabolites can be quantified with a median coefficient of variation of 0.127 across 1100 technical replicates. We illustrate the utility of this strategy via an analysis of 960 strains of

Liquid chromatography mass spectrometry (LC–MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC–MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools. One important distinction between research versus diagnostics-based applications of LC–MS is throughput. Clinical LC–MS must enable quantitative analyses of target molecules in hundreds or thousands of samples each day. Currently, the throughput of these clinical applications is limited by the chromatographic gradient lengths, which—when analyzing complex metabolomics samples—are difficult to conduct in under ~ 3 min per sample without introducing serious quantitative analysis problems. To address this shortcoming, we developed sequential quantification using isotope dilution (SQUID), an analytical strategy that combines serial sample injections into a continuous isocratic mobile phase to maximize throughput. SQUID uses internal isotope-labelled standards to correct for changes in LC–MS response factors over time. We show that SQUID can detect microbial polyamines in human urine specimens (lower limit of quantification; LLOQ = 106 nM) with less than 0.019 normalized root mean square error. Moreover, we show that samples can be analyzed in as little as 57 s. We propose SQUID as a new, high-throughput LC–MS tool for quantifying small sets of target biomarkers across large cohorts. Graphical Abstract

To determine if the Study To Optimize Peritoneal Infection Therapy (STOP-IT) randomized controlled trial (RCT) changed antibiotic prescribing in patients with Complicated Intra-Abdominal Infection (CIAI).CIAI is common and causes significant morbidity. In May 2015, the STOP-IT RCT showed equivalent outcomes between four-day and clinically determined antibiotic duration.This was a population-based retrospective cohort study using interrupted time series methods. The STOP-IT publication date was the exposure. Median duration of inpatient antibiotic prescription was the outcome. All adult patients admitted to four hospitals in Calgary, Canada between July 2012 and December 2018 with CIAI who survived at least four days following source control were included. Analysis was stratified by infectious source as appendix or biliary tract (group A) versus other (group B).Among 4384 included patients, clinical and demographic attributes were similar before vs after publication. In Group A, median inpatient antibiotic duration was 3 days and unchanged from the beginning to the end of the study period (adjusted median difference -0.00 days, 95% confidence interval [CI] -0.37 - 0.37 days). In Group B, antibiotic duration was shorter at the end of the study period (7.87 vs 6.73 days; -1.14 days, CI -2.37 - 0.09 days), however there was no change in trend following publication (-0.03 days, CI -0.16 - 0.09).For appendiceal or biliary sources of CIAI, antibiotic duration was commensurate with the experimental arm of STOP-IT. For other sources, antibiotic duration was long and did not change in response to trial publication. Additional implementation science is needed to improve antibiotic stewardship.

Background Pyogenic liver abscess (PLA), although uncommon in North America, is associated with significant morbidity and mortality. We sought to re-examine the epidemiology, risk factors, and outcomes of PLA in a large, diverse Canadian health zone. Methods All Calgary Health Zone (CHZ) residents aged ≥20 with PLA between 2015 and 2017 were identified. Incidence and mortality rates were calculated using census data. Risk factors for PLA were identified using a multivariate analysis. Data was compared to 1999–2003 data, also collected in the CHZ. Results There were 136 patients diagnosed with PLA between 2015 and 2017. Incidence rate during this period increased significantly relative to 1999–2003 (3.7 vs 2.3 cases/100,000 population, p Conclusions Pyogenic liver abscess is a health concern with rising incidence rate. The increasing prevalence of comorbidities in our population and factors that are associated with risk of PLA suggests this will continue to be an emerging diagnosis of concern. Increasing prevalence of antibiotic resistant organisms compounding unclear optimal treatment regimens is an issue that requires urgent study.

Background Escherichia coli is an important pathogen in humans and is the most common cause of bacterial bloodstream infections (BSIs). The objectives of our study were to determine factors associated with E. coli BSI incidence rate and third-generation cephalosporin resistance in a multinational population-based cohort. Methods We included all incident E. coli BSIs (2014–2018) from national (Finland) and regional (Australia [Canberra], Sweden [Skaraborg], and Canada [Calgary, Sherbrooke, and western interior]) surveillance. Incidence rates were directly age and sex standardized to the European Union 28-country 2018 population. Multivariable negative binomial and logistic regression models estimated factors significantly associated with E. coli BSI incidence rate and third-generation cephalosporin resistance, respectively. The explanatory variables considered for inclusion in both models were year (2014–2018), region (six areas), age ( Results We identified 31,889 E. coli BSIs from 40.7 million person-years of surveillance. Overall and third-generation cephalosporin-resistant standardized rates were 87.1 and 6.6 cases/100,000 person-years, respectively, and increased 14.0% and 40.1% over the five-year study. Overall, 7.8% (2483/31889) of E. coli BSIs were third-generation cephalosporin-resistant. Calgary, Canberra, Sherbrooke, and western interior had significantly lower E. coli BSI rates compared to Finland. The significant association between age and E. coli BSI rate varied with sex. Calgary, Canberra, and western interior had significantly greater odds of third-generation cephalosporin-resistant E. coli BSIs compared to Finland. Compared to 2014, the odds of third-generation cephalosporin-resistant E. coli BSIs were significantly increased in 2016, 2017, and 2018. The significant association between age and the odds of having a third-generation cephalosporin-resistant E. coli BSI varied with sex. Conclusions Increases in overall and third-generation cephalosporin-resistant standardized E. coli BSI rates were clinically important. Overall, E. coli BSI incidence rates were 40–104% greater than previous investigations from the same study areas. Region, sex, and age are important variables when analyzing E. coli BSI rates and third-generation cephalosporin resistance in E. coli BSIs. Considering E. coli is the most common cause of BSIs, this increasing burden and evolving third-generation cephalosporin resistance will have an important impact on human health, especially in aging populations.

Background Escherichia coli is the most common cause of bloodstream infections (BSIs) and mortality is an important aspect of burden of disease. Using a multinational population-based cohort of E. coli BSIs, our objectives were to evaluate 30-day case fatality risk and mortality rate, and determine factors associated with each. Methods During 2014–2018, we identified 30-day deaths from all incident E. coli BSIs from surveillance nationally in Finland, and regionally in Sweden (Skaraborg) and Canada (Calgary, Sherbrooke, western interior). We used a multivariable logistic regression model to estimate factors associated with 30-day case fatality risk. The explanatory variables considered for inclusion were year (2014–2018), region (five areas), age ( Results From 38.7 million person-years of surveillance, we identified 2961 30-day deaths in 30,923 incident E. coli BSIs. The overall 30-day case fatality risk was 9.6% (2961/30923). Calgary, Skaraborg, and western interior had significantly increased odds of 30-day mortality compared to Finland. Hospital-onset and 3GC-resistant E. coli BSIs had significantly increased odds of mortality compared to community-onset and 3GC-susceptible. The significant association between age and odds of mortality varied with sex, and contrasts were used to interpret this interaction relationship. The overall standardized 30-day mortality rate was 8.5 deaths/100,000 person-years. Sherbrooke had a significantly lower 30-day mortality rate compared to Finland. Patients that were either ≥70-years-old or male both experienced significantly higher mortality rates than those Conclusions In our study populations, region, age, and sex were significantly associated with both 30-day case fatality risk and mortality rate. Additionally, 3GC resistance and location of onset were significantly associated with 30-day case fatality risk. Escherichia coli BSIs caused a considerable burden of disease from 30-day mortality. When analyzing population-based mortality data, it is important to explore mortality through two lenses, mortality rate and case fatality risk.

Background Staphylococcus aureus bacteriuria (SABU) may represent multiple processes ranging from asymptomatic colonization to a marker of S. aureus bacteremia (SAB). Our objective was to describe SABU at a population-based level and determine patient characteristics associated with SAB. Methods A retrospective study was performed using electronic databases. All urine cultures positive for S. aureus between 2010 and 2013 within the Calgary Health Zone were included. Patient characteristics were compared among patients with and without SAB and risk factors identified using multiple logistic regression modeling. Results A total of 2540 urine cultures positive for S. aureus from 2054 patients were analyzed. The incidence of SABU was greatest among geriatric males with multiple comorbidities. SAB occurred in 175 (6.9%) of SABU patients. Those with SAB were more likely to be hospitalized, male, have a recent urinary procedure, have pure S. aureus culture in urine, and have laboratory findings suggesting systemic infection. Patients with isolated SABU were more likely to be ≥65 years, have dementia, and have abnormal urinalyses with pyuria and urine nitrites. In-hospital mortality in patients with SABU and SABU+SAB was 9.2% and 17.5%, respectively. Patients with SABU detected ≥48 hours before SAB had the highest risk of death. Conclusions Less than 7% of patients with SABU have or will develop SAB. Characteristics associated with SABU were identified that established higher risk for systemic infection. Investigating SABU patients with these characteristics for systemic infection is warranted because a delay in diagnosis is associated with increased mortality.

BACKGROUND A previous study in Calgary showed that travel to India was associated with high risk of community-onset infections with extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. We performed a follow-up study to determine the rate of rectal acquisition of ESBL-producing E. coli among travellers to South Asia and to identify the behaviours putting such travellers at high risk for acquiring ESBL-producing E. coli. METHODS: The study was performed at a travel clinic in Calgary. Travellers 18 years or older who were planning to visit South Asia for a period of at least 5 days were included. Three rectal swabs were obtained, and 2 questionaires were administered (before and after travel). RESULTS A total of 149 travellers participated between January 2012 and July 2014; of these, 116 (78%) provided rectal swabs upon return to Calgary and completed both pre- and post-travel questionaires. Of the 109 travellers without colonization with ESBL-producing E. coli upon enrollment, 70 (64%) acquired ESBL-producing E. coli during travel. Of the 90 participants who visited India, 66 (73%) were positive for ESBL-producing E. coli upon their return to Calgary. Most ESBL-producing E. coli specimens were identified as producing the enzyme CTX-M-15. Behaviours associated with a statistically significant risk of acquiring ESBL-producing E. coli included visiting India (odds ratio [OR] 19.9, 95% confidence interval [CI] 4.5-88.8), consuming meals with the local population (OR 6.9, 95% CI 1.2-39.6), taking any type of antibiotic during travel (OR 4.3, 95% CI 1.3-14.3) and travelling for any purpose other than business (OR 12.4, 95% CI 2.8-55.2). INTERPRETATION In this study, travel to India was associated with the highest risk of acquiring ESBL-producing E. coli relative to travel to other countries in South Asia. Nonbusiness travel, consuming foods with the local population and the use of antibiotics while travelling were associated with an increased risk of acquiring these antibiotic-resistant organisms while in India. Trial registration: ClinicalTrials.gov, no. NCT01296165.

Life-threatening infection in neonates due to group B Streptococcus (GBS) is preventable by screening of near-term pregnant women and treatment at delivery. A total of 295 vaginal-rectal swabs were collected from women attending antepartum clinics in Calgary, Alberta, Canada. GBS colonization was detected by the standard culture method (Strep B Carrot Broth subcultured to blood agar with a neomycin disk) and compared to recovery with Strep Group B Broth (Dalynn Biologicals) subcultured to StrepB Select chromogenic medium (CM; Bio-Rad Laboratories) and the Fast-Track Diagnostics GBS real-time PCR (quantitative PCR [qPCR]) assay (Phoenix Airmid Biomedical Corp.) performed with broth-enriched samples and the Abbott m2000 sp/ m2000 rt system. A total of 62/295 (21%) women were colonized with GBS; 58 (19.7%) cases were detected by standard culture, while CM and qPCR each found 61 (20.7%) cases. The qPCR and CM were similar in performance, with sensitivities, specificities, and positive and negative predictive values of 98.4 and 98.4%, 99.6 and 99.6%, 98.4 and 98.4%, and 99.6 and 99.6%, respectively, compared to routine culture. Both qPCR and CM would allow more rapid reporting of routine GBS screening results than standard culture. Although the cost per test was similar for standard culture and CM, the routine use of qPCR would cost approximately four times as much as culture-based detection. Laboratories worldwide should consider implementing one of the newer methods for primary GBS testing, depending on the cost limitations of different health care jurisdictions.

International guidelines for Pneumocystis jirovecii pneumonia (PJP) prevention recommend prophylaxis for ≥6 months following allogeneic hematopoietic cell transplantation, and longer in patients with graft-versus-host disease (GVHD) or on immunosuppressive therapy (IST). These recommendations are based on cohorts of patients who did not routinely receive anti-thymocyte globulin (ATG) for GVHD prophylaxis.We performed a retrospective chart review of 649 patients, all of whom received ATG as part of GVHD prophylaxis.The cumulative incidence of definite PJP was 3.52% at both 3 and 5 years (median follow up, 1648 days for survivors). PJP occurred in 13 non-GVHD patients between days 207 and 508, due in part to low CD4 T-cell counts (200 CD4 T cells/µL). PJP occurred in eight GVHD patients between days 389 and 792, due in part to non-adherence to PJP prophylaxis guidelines (discontinuation of PJP prophylaxis at3 months after discontinuation of IST). Breakthrough PJP infection was not observed in patients receiving prophylaxis with cotrimoxazole, dapsone or atovaquone, whereas three cases were observed with inhaled pentamidine.In conclusion, for non-GVHD patients receiving ATG-containing GVHD prophylaxis, 6 months of PJP prophylaxis is inadequate, particularly if the CD4 T-cell count is200 cells/µL or if there is a high incidence of PJP in the community. For patients with GVHD receiving ATG-containing GVHD prophylaxis, continuing PJP prophylaxis until ≥3 months post-discontinuation of IST is important. Cotrimoxazole, dapsone and atovaquone are preferred over inhaled pentamidine.

A multicentre retrospective study was performed between 2012 and 2014 in a large Canadian Health Zone in order to determine factors associated with IDC and performance of key quality of care determinants in SAB management and clinical outcomes. Factors subject to quality of care determinants were established a priori and studied for associations with IDC and 30-day all-cause mortality using multivariable analysis.Of 961 SAB episodes experienced by 892 adult patients, 605 episodes received an IDC. Patients receiving IDC were more likely to have prosthetic valves and joints and to have community-acquired and known sources of SAB, but increasing age decreased IDC occurrence. IDC was the strongest independent predictor for quality of care performance metrics, including repeat blood cultures and echocardiography. Mortality at 30 days was 20% in the cohort, and protective factors included IDC, achievement of source control, targeted therapy within 48 hours, and follow-up blood cultures but not the performance of echocardiography.There were significant gaps between the treatments and investigations that patients actually received for SAB and what is considered the optimal management of their condition. IDC is associated with improved attainment of targeted SAB quality of care determinants and reduced mortality rates. Based on our findings, we propose a policy of mandatory IDC for all cases of SAB to improve patient management and outcomes.La bactériémie àEntre 2012 et 2014, les auteurs ont réalisé une étude rétrospective multicentrique dans une grande zone de santé canadienne afin de déterminer les facteurs associés aux CeI et l’exécution des principaux déterminants de la qualité des soins dans la prise en charge et les résultats cliniques de la BSA. Au moyen d’une analyse multivariée, ils ont établiSur les 961 épisodes de BSA qu’ont vécus 892 patients adultes, 605 ont donné lieu à une CeI. Les patients dirigés vers une telle consultation étaient plus susceptibles d’avoir reçu des valvules et des articulations artificielles et de souffrir d’une BSA d’origine communautaire ou d’une autre source connue, mais ces consultations diminuaient avec le vieillissement. Les CeI étaient le meilleur prédicteur indépendant de mesures démontrant la qualité des soins, y compris des hémocultures et des échocardiographies répétées. La mortalité au bout de 30 jours s’élevait à 20 % dans la cohorte, et les facteurs protecteurs incluaient les CeI, la réalisation du contrôle des sources, un traitement ciblé dans les 48 heures et des hémocultures de suivi, mais pas des échocardiographies.Les auteurs ont constaté un écart important entre les traitements et les explorations que les patients ont subis en raison de leur BSA et ce qui est considéré comme une prise en charge optimale de leur maladie. La CeI s’associe à une meilleure réalisation des déterminants de la qualité des soins ciblés et d’un taux de mortalité réduit pour la BSA. D’après leurs constatations, les auteurs proposent que la CeI soit obligatoire en cas de BSA, afin d’améliorer la prise en charge et le pronostic des patients.

BACKGROUND: Overuse of investigations, treatments, and procedures contribute to rising health care costs and may cause patient harm. In an attempt to promote higher-value health care, the Choosing Wisely Canada campaign has encouraged professional societies to develop statements that are directly actionable by their members. Currently, there are variations in infectious diseases practice that lead some patients to receive therapies and investigations that lack benefit and are potentially harmful. METHODS: The Association of Medical Microbiology and Infectious Disease Canada (AMMI) Canada established its Choosing Wisely Canada top five list of recommendations using the framework put forward by Choosing Wisely Canada. Following an electronic survey of its members regarding low-value practices within infectious diseases, AMMI Canada convened a working group that developed a list of draft recommendations and ranked the top five recommendations by consensus. This list was shared with the AMMI Canada membership electronically and during a national open forum. Following revisions based on feedback received, the AMMI Canada Executive Council and Guidelines Committee endorsed the final list, which was disseminated online. RESULTS: The top five declarative statements on infectious diseases practices that physicians and patients should question include: do not routinely prescribe intravenous forms of highly bioavailable antimicrobial agents for patients who can reliably take and absorb oral medications; do not prescribe alternative second-line antimicrobials to patients reporting nonsevere reactions to penicillin when beta-lactams are the recommended first-line therapy; do not routinely repeat CD4 measurements in patients with HIV infection with HIV-1 RNA suppression for >2 years and CD4 counts >500/μL, unless virological failure occurs or intercurrent opportunistic infection develops; do not routinely repeat radiologic imaging in patients with osteomyelitis demonstrating clinical improvement following adequate antimicrobial therapy; and do not prescribe aminoglycosides for synergy to patients with bacteremia or native valve infective endocarditis caused by Staphylococcus aureus. CONCLUSIONS: The Choosing Wisely Canada statements in infectious diseases endorsed by AMMI Canada represent a starting point to engage AMMI Canada members in broader discussions related to resource stewardship within infectious diseases practice and to take action.

Background PLA is a significant cause of morbidity and mortality. However, its epidemiology and outcomes have not been recently evaluated in the CHZ. Understanding current trends will help guide management. Methods In this population-based study, we evaluated epidemiology, risk factors, and treatment of patients with PLA in the CHZ. CHZ residents aged ≥ 20 years diagnosed with PLA in 2015-2017 were included. Charts were reviewed for demographics and clinical outcomes. Multivariate logistic regression was used to determine factors associated with 30-day mortality. Findings were compared to a previous assessment of PLA in the CHZ from 1999-2003 (Kaplan et al., 2004). Results A total of 136 patients with PLA were identified, representing an annual incidence rate of 3.7 cases per 100,000 population. Compared to 1999-2003, incidence of PLA was increased (2.3 per 100,000; p< 0.01) but mortality was similar (1999-2003: 0.22 per 100,000 vs. 2015-2017: 0.26 per 100,000; p=0.6). The most common culprit organisms were Streptococcus anginosus group (40%), Klebsiella species (25%), Escherichia coli (18%), and obligate anaerobes (16%). Pathogen prevalence was similar to the prior cohort. Compared to 1999-2003, antibiotic resistant organisms were more frequent (8% vs 1%, p=0.04). In our cohort, liver aspirations were less frequent (p=0.02) but aspirate culture was more often positive (p< 0.01). The median duration of intravenous antibiotic therapy was longer compared to previous (2015-2017: 23 days (IQR 9-38) vs. 1999-2003: 17 days (IQR 10-29); p=0.001). Similarly, the total duration of antibiotic therapy was longer (2015-2017: 42 days (IQR 25-65) vs. 1999-2003: 31 days (IQR 18-45); p< 0.001). Thirty-day mortality from admission was 7% and did not differ amongst cohorts. Risk factors are shown in Table-1. Table-1: Risk factors for 30-day mortality in PLA Conclusion Incidence of PLA in the CHZ is rising with more antimicrobial resistance. Diagnostic liver aspirations are less frequent. Antibiotic durations are longer with no reduction in mortality. Understanding changing trends is valuable in directing future care. Encouraging liver aspirations to obtain a microbiologic diagnosis, especially with increasing resistance, is crucial. Considering shorter antibiotic durations in light of stable mortality warrants further exploration. Disclosures All Authors: No reported disclosures

A previous study in Calgary showed that travel to India was associated with high risk of community-onset infections with extended-spectrum β-lactamase (ESBL)-producingA total of 149 travellers participated between January 2012 and July 2014; of these, 116 (78%) provided rectal swabs upon return to Calgary and completed both pre- and post-travel questionaires. Of the 109 travellers without colonization with ESBL-producingIn this study, travel to India was associated with the highest risk of acquiring ESBL-producing

Diagnosis of bacterial pharyngitis is confirmed by detection of group A Streptococcus (GAS) in patient throat samples. Testing of throat samples has historically relied on culture, but new molecular methods allow much faster test turnaround time (i.e., same day versus 48 to 72 h for culture). Our laboratory uses the Hologic GAS Direct (GASD) assay for screening more than 125,000 throat samples per year. Simplexa GAS Direct is a new real-time quantitative PCR (qPCR) assay that does not require initial DNA extraction. Performance of Simplexa qPCR was compared to GASD. A total of 289 throat swabs were collected from patients attending ambulatory clinics in Calgary, Alberta, Canada. A total of 60 (20.8%) of the samples were initially GAS positive by either method: 54 by both methods, 4 by Simplex qPCR alone, and 2 by GASD alone. An in-house PCR using a unique GAS primer set was used to resolve the 6 discrepant results. Overall, GASD compared to Simplexa qPCR had a sensitivity, specificity, positive predictive value, and negative predictive value of 93.1% versus 100%, 100% versus 100%, 100% versus 100%, and 98.31% versus 100%, respectively. Implementation of Simplexa qPCR in our laboratory setting would cost more but allow the high sample volume to be reported in half the time and save 0.62 medical laboratory technician (MLT) full-time equivalent (FTE). In comparison to culture, the implementation of Simplexa qPCR would save 2.79 medical laboratory assistant (MLA) FTE plus 0.94 MLT FTE. Simplexa qPCR has improved performance and diagnostic efficiency in a high-volume laboratory compared to GASD for GAS detection in throat swabs.

Life-threatening infection in neonates due to group B Streptococcus (GBS) is preventable by screening of near-term pregnant women and treatment at delivery. A total of 295 vaginal-rectal swabs were collected from women attending antepartum clinics in Calgary, Alberta, Canada. GBS colonization was detected by the standard culture method (Strep B Carrot Broth subcultured to blood agar with a neomycin disk) and compared to recovery with Strep Group B Broth (Dalynn Biologicals) subcultured to StrepBSelect chromogenic medium (CM; Bio-Rad Laboratories) and the Fast-Track Diagnostics GBS real-time PCR (quantitative PCR [qPCR]) assay (Phoenix Airmid Biomedical Corp.) performed with broth-enriched samples and the Abbott m2000sp/m2000rt system. A total of 62/295 (21%) women were colonized with GBS; 58 (19.7%) cases were detected by standard culture, while CM and qPCR each found 61 (20.7%) cases. The qPCR and CM were similar in performance, with sensitivities, specificities, and positive and negative predictive values of 98.4 and 98.4%, 99.6 and 99.6%, 98.4 and 98.4%, and 99.6 and 99.6%, respectively, compared to routine culture. Both qPCR and CM would allow more rapid reporting of routine GBS screening results than standard culture. Although the cost per test was similar for standard culture and CM, the routine use of qPCR would cost approximately four times as much as culture-based detection. Laboratories worldwide should consider implementing one of the newer methods for primary GBS testing, depending on the cost limitations of different health care jurisdictions.

Background The recognition of infective endocarditis (IE) as a serious complication of SAB has led to a low threshold for echocardiography, extended treatment (eTx) with anti-staphylococcal agents (anti-SA), and even surgery. However, indications for eTx outside of IE are numerous. We sought to determine the frequency in which findings from a TEE changed clinical SAB management. Methods Adults with SAB within the Calgary Health Zone between 2012 and 2014 were included if both a transthoracic echocardiogram (TTE) and TEE were performed within 7 days of each other. Patients potentially benefiting from eTx courses were a priori defined as having ≥1 of; (1) metastatic phenomena, (2) complicated SAB (defined as community acquired (CA), persistent fever/bacteremia at ≥48 hours), (3) intracardiac devices, and/or (4) Duke criteria defined IE (separately evaluated using TTE or TEE). Patient demographics, treatment (including changes to anti-SA duration and surgical indications), and clinical outcomes were extracted and evaluated. Results Of 961 SAB episodes, 179 (18% MRSA) met inclusion criteria (median 3.0 days, IQR 1.9–5.1 between TTE and TEE). Within the cohort 29% (n = 51) had metastatic phenomena (intracranial; 9% [n = 16], vertebral; 8% [n = 14]; empyema; 6% [n = 11]). Complicated bacteremia was present in 89% (n = 159), while 13% (n = 23) had intravascular hardware (39% pacemakers, 65% valve prostheses). IE was diagnosed in 22% (n = 40) of SAB episodes, with TTE identifying 58% of vegetations (n = 23) and TEE identifying an additional 30% (n = 15). Of the cohort, 89% (n = 160), 37% (n = 67), 4% (n = 7) of SAB had ≥1, 2, and 3 nonechocardiographic indications for eTx, respectively. Only one patient met criteria for eTx solely based on IE diagnosed from concordant TTE and TEE. Actual duration of therapy did not differ in SAB episodes that had ≥1 a priori eTx criteria but had a negative TEE relative to those who had a positive TEE (36.7 days, IQR 23.4–48.6 vs. 43.8 days, IQR 33.3–49.5, P = 0.17). Of the 15 episodes of TEE-only evident IE, 14 were CA, 12 had prolonged BSI, and nine embolic phenomena. Only 11 patients in the cohort required cardiac surgery, none were identified exclusively by TEE. Conclusion Routine performance of TEE may not be required in all SAB as many patients have alternate indications for eTx with anti-SA regardless of TEE findings. Disclosures All authors: No reported disclosures.

The characteristics of hypermucoviscosity isolates amongKlebsiella pneumoniaecausing community-acquired bacteremia were investigated. The hypermucoviscous phenotype was present in 8.2% ofK pneumoniaeisolates, and was associated with rmpA and the K2 serotype; liver abscesses were the most common clinical presentation. The present analysis represents the first population-based surveillance study of hypermucoviscosity amongK pneumoniaecausing bacteremia.

Background. Electronic surveillance systems (ESSs) that utilize existing information in databases are more efficient than conventional infection surveillance methods. The objective was to assess an ESS for bloodstream infections (BSIs) in the Calgary Zone for its agreement with traditional medical record review.Methods. The ESS was developed by linking related data from regional laboratory and hospital administrative databases and using set definitions for excluding contaminants and duplicate isolates. Infections were classified as hospital-acquired (HA), healthcare-associated community-onset (HCA), or community-acquired (CA). A random sample of patients from the ESS was then compared with independent medical record review.Results. Among the 308 patients selected for comparative review, the ESS identified 318 episodes of BSI of which 130 (40.9%) were CA, 98 (30.8%) were HCA, and 90 (28.3%) were HA. Medical record review identified 313 episodes of which 136 (43.4%) were CA, 97 (30.9%) were HCA, and 80 (25.6%) were HA. Episodes of BSI were concordant in 304 (97%) cases. Overall, there was 85.5% agreement between ESS and medical record review for the classification of where BSIs were acquired (kappa = 0.78, 95% Confidence Interval: 0.75–0.80).Conclusion. This novel ESS identified and classified BSIs with a high degree of accuracy. This system requires additional linkages with other related databases.

A study was designed to assess the importance of sequence types among extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates causing bacteremia over an 11-year period (2000 to 2010) in a centralized Canadian region. A total of 197 patients with incident infections were identified; the majority presented with community-onset urosepsis, with a significant increase in the prevalence of ESBL-producing E. coli during the later part of the study. The majority of E. coli isolates produced either CTX-M-15 or CTX-M-14. We identified 7 different major sequence types among 91% of isolates (i.e., the ST10 clonal complex, ST38, ST131, ST315, ST393, ST405, and ST648) and provided insight into their clinical and molecular characteristics. ST38 was the most antimicrobial-susceptible sequence type and predominated during 2000 to 2004 but disappeared after 2008. ST131 was the most antimicrobial-resistant sequence type, and the influx of a single pulsotype of this sequence type was responsible for the significant increase of ESBL-producing E. coli strains since 2007. During 2010, 49/63 (78%) of the ESBL-producing E. coli isolates belonged to ST131, and this sequence type had established itself as a major drug-resistant pathogen in Calgary, Alberta, Canada, posing an important new public health threat within our region. We urgently need well-designed epidemiological and molecular studies to understand the dynamics of transmission, risk factors, and reservoirs for E. coli ST131. This will provide insight into the emergence and spread of this multiresistant sequence type.

Background We previously identified foreign travel as a risk factor for acquiring infections due to CTX‐M (active on cefotaxime first isolated in Munich) producing Escherichia coli . The objective of this study was to assess the prevalence of extended‐spectrum β‐lactamase (ESBL)‐producing E coli among stool samples submitted from travelers as compared to non‐travelers (a non‐traveler had not been outside of Canada for at least 6 months before submitting a stool specimen). Methods Once a travel case was identified, the next stool from a non‐traveler (not been outside of Canada for at least 6 months) was included and cultured on the chromID‐ESBL selection media. Molecular characterization was done using polymerase chain reaction and sequencing for bla CTX‐Ms, bla TEMs, bla SHVs, plasmid‐mediated quinolone‐resistant determinants, O25‐ST131, phylogenetic groups, pulsed‐field gel electrophoresis (PFGE), and multilocus sequencing typing. Results A total of 226 individuals were included; 195 (86%) were negative, and 31 (14%) were positive for ESBL‐producing E coli. Notably, travelers were 5.2 (95% CI 2.1–31.1) times more likely than non‐travelers to have an ESBL‐producing E coli cultured from their stool. The highest rates of ESBL positivity were associated with travel to Africa or the Indian subcontinent. Among the 31 ESBL‐producing E coli isolated, 22 produced CTX‐M‐15, 8 produced CTX‐M‐14, 1 produced CTX‐M‐8, 12 were positive for aac(6 ′ )‐Ib‐cr , and 8 belonged to clone ST131. Conclusions Our study confirms that foreign travel, especially to the Indian subcontinent and Africa, represents a major risk for rectal colonization with CTX‐M‐producing E coli and contributed to the Worldwide spread of these bacteria.

A study was designed to characterize extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli isolates causing bacteremia over an 8-year period (2000 to 2007) in a large well-defined geographical region. Molecular characterization was done by using isoelectric focusing; PCR; and sequencing of the bla CTX-M -, bla TEM -, bla OXA -, bla SHV -, and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined by pulsed-field electrophoresis with XbaI and multilocus sequence typing. A total of 67 patients with incident bloodstream infections were identified, and the majority presented with community-acquired infections involving the urinary and biliary tracts. Of the 67 ESBL-producing E. coli isolates recovered, 60 (90%) were positive for bla CTX-M genes; 32 (48%) produced CTX-M-15, 25 (37%) produced CTX-M-14, 1 (2%) produced CTX-M-24, 1 (2%) produced CTX-M-2, and 1 (2%) produced CTX-M-3, while 2 (3%) produced TEM-52 and 5 (7%) produced SHV-2. Twenty-four (36%) isolates were positive for aac(6′)-Ib-cr. The majority of isolates were resistant to ciprofloxacin (60 [90%] isolates) and gentamicin (40 [60%] isolates). The occurrence of ESBL-producing isolates was stable during the first 5 years, but there was a substantial increase from 2005 to 2007, mostly due to clone ST131, which produces CTX-M-15 and CTX-M-14, in blood cultures submitted from the community. Our results illustrated that E. coli clone ST131, which coproduces CTX-M-15, OXA-1, TEM-1, and aac(6′)-Ib-cr , has emerged as an important cause of community-onset bacteremia caused by ESBL-producing E. coli isolates; and this is the first study to identify CTX-M-14 in E. coli clone ST131.

Extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli has recently emerged as a major risk factor for community-acquired, travel-related infections in the Calgary Health Region. Molecular characterization was done on isolates associated with infections in returning travelers using isoelectric focusing, PCR, and sequencing for bla CTX-M s, bla TEM s, bla SHV s, bla OXA s, and plasmid-mediated quinolone resistance determinants. Genetic relatedness was determined with pulsed-field gel electrophoresis using XbaI and multilocus sequence typing (MLST). A total of 105 residents were identified; 6/105 (6%) presented with hospital-acquired infections, 9/105 (9%) with health care-associated community-onset infections, and 90/105 (86%) with community-acquired infections. Seventy-seven of 105 (73%) of the ESBL-producing E. coli isolates were positive for bla CTX-M genes; 55 (58%) produced CTX-M-15, 13 (14%) CTX-M-14, six (6%) CTX-M-24, one (1%) CTX-M-2, one (1%) CTX-M-3, and one (1%) CTX-M-27, while 10 (10%) produced TEM-52, three (3%) TEM-26, 11 (11%) SHV-2, and four (4%) produced SHV-12. Thirty-one (30%) of the ESBL-producing E. coli isolates were positive for aac ( 6 ′)- Ib - cr , and one (1%) was positive for qnrS . The majority of the ESBL-producing isolates ( n = 95 [90%]) were recovered from urine samples, and 83 (87%) were resistant to ciprofloxacin. The isolation of CTX-M-15 producers belonging to clone ST131 was associated with travel to the Indian subcontinent (India, Pakistan), Africa, the Middle East, and Europe, while clonally unrelated strains of CTX-M-14 and -24 were associated with travel to Asia. Our study suggested that clone ST131 coproducing CTX-M-15, OXA-1, TEM-1, and AAC(6′)-Ib-cr and clonally unrelated CTX-M-14 producers have emerged as important causes of community-acquired, travel-related infections.

A study was designed to evaluate the ability of the DiversiLab fingerprinting kit, a type of repetitive element PCR (rep-PCR), to identify Escherichia coli clone ST131 producing β-lactamase CTX-M-15. A set of 53 nonduplicate isolates of extended-spectrum β-lactamase-producing E. coli underwent rep-PCR, pulsed-field gel electrophoresis, and multilocus sequence typing. The DiversiLab system successfully identified E. coli clone ST131 producing CTX-M-15 and provides a simple standardized typing protocol for monitoring the spread of this clone.

Although Escherichia coli is the most common cause of bloodstream infection, its epidemiology has not been well defined in non-selected populations. We sought to describe the incidence of risk factors for, and outcomes associated with, E. coli bacteraemia. Population-based surveillance for E. coli bacteraemia was conducted in the Calgary Health Region (population 1.2 million) during the period 2000–2006. In total, 2368 episodes of E. coli bacteraemia were identified for an overall annual population incidence of 30.3/100 000; 15% were nosocomial, 32% were healthcare-associated community-onset and 53% were community-acquired bacteraemias. The very young and the elderly were at highest risk for E. coli bacteraemia. Sixty per cent of the episodes occurred in females (relative risk 1.5; 95% CI 1.4–1.6). Dialysis, solid organ transplantation and neoplastic disease were the most important risk factors for acquiring E. coli bacteraemia. Rates of resistance to ampicillin, trimethoprim–sulphamethoxazole, gentamicin, ciprofloxacin, cefazolin and ceftriaxone increased significantly during the period 2000–2006. The case-fatality rate was 11% and the annual population mortality rate was 2.9/100 000. Increasing age, ciprofloxacin resistance, non-urinary focus and a number of comorbid illnesses were independently associated with an increased risk of death, and community acquisition and urinary focus were associated with a lower risk of death. This study documents the major burden of illness associated with E. coli bacteraemia and identifies groups at increased risk for acquiring and dying from these infections. The emergence of ciprofloxacin resistance and its adverse effect on patient outcome is a major concern.

Objectives A study was designed to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants among clinical isolates of Enterobacteriaceae from the Calgary Health Region (CHR). Methods During January and February 2004 and January and February 2007, 564 non-repeat isolates of Escherichia coli, Klebsiella spp., Proteus mirabilis and Morganella morganii resistant to ciprofloxacin and/or tobramycin were screened for PMQR determinants using a multiplex PCR for qnrA, qnrS and qnrB and aac(6')-Ib genes; aac(6')-Ib-cr was further identified by digestion with BstF5I. Results In 2004, 6/139 (4%) of the resistant E. coli were positive for aac(6')-Ib-cr. In 2007, 53/398 (13%) were positive for aac(6')-Ib-cr, 3/398 (0.8%) were positive for qnrS, and one isolate was positive for both. All the isolates were present in urines and the majority [40/63 (63%)] were submitted from community collection sites; 8 (13%) isolates co-produced AmpC beta-lactamases and 34 (54%) co-produced CTX-M-15. aac(6')-Ib-cr was present in one Klebsiella pneumoniae and one P. mirabilis, whereas one isolate of K. pneumoniae was positive for both aac(6')-Ib-cr and qnrB. Conclusions Our results showed that isolates with aac(6')-Ib-cr, often associated with CTX-M-15, are emerging among fluoroquinolone-resistant E. coli in the CHR. Our study suggests that surveillance for PMQR determinants should be undertaken on a regular basis.

A study was designed to describe the molecular epidemiology of CTX-M-producing Escherichia coli over a 6-year period (2000 to 2005) in a large well-defined Canadian region with a centralized laboratory system. Molecular characterization was done by isoelectric focusing, PCR, and automated sequencing, while genetic relatedness was determined by pulsed-field gel electrophoresis with XbaI. Of the 552 viable extended-spectrum β-lactamase-producing E. coli isolates isolated, 354 (64%) were positive for bla CTX-M genes associated with IS Ecp1 ; 211 produced CTX-M-14, 128 produced CTX-M-15, 5 produced CTX-M-2, 4 produced CTX-M-3, 4 produced CTX-M-24, and 2 produced CTX-M-27. CTX-M-positive isolates were significantly more resistant to the fluoroquinolones than CTX-M-negative isolates, while CTX-M-15 producers were more likely to be resistant to gentamicin and tobramycin. There was a predominance of CTX-M-14 during the first 4 years of the study period, with community outbreaks associated with cluster 14A during 2000, 2001, and 2003. A substantial increase in CTX-M-15 producers occurred during the last 18 months and was due to clusters 15A and 15AR (where AR indicates related to A) in the hospital and nursing home sectors. Our results demonstrate that the persistence and dissemination of CTX-M genes among E. coli populations in larger geographic health care regions is dynamic, with the continuous emergence of clonally related CTX-M-15. This study illustrates the importance of molecular surveillance in tracking CTX-M-producing E. coli strains in the community and investigating their influx into hospitals.

AmpC β-lactamase–producing E. coli are commonly isolated from the urinary tract of older women., In the Calgary Health Region during 2000–2003, prospective, active, population-based laboratory surveillance for all cefoxitin-resistant Escherichia coli isolates was performed. Isolates were screened with an inhibitor-based disk test, and plasmid-mediated types were identified by multiplex PCR with sequencing. A total of 369 AmpC β-lactamase–producing E. coli isolates were identified; annual incidence rates were 1.7, 4.3, 11.2, and 15 per 100,000 residents for each year, respectively. AmpC β-lactamase–producing E. coli was 5× more likely to be isolated from female than male patients across all age groups except

A study was designed to describe the molecular epidemiology of carbapenem-resistant (CR) Pseudomonas aeruginosa in a large well-defined geographical region with a centralized laboratory system serving one pediatric and three large adult hospitals (acute care centers I, II, and III). Molecular characterization was done using PCR with sequencing of the integron-associated gene cassettes. Pulsed-field gel electrophoresis (PFGE) using a modified combined Stenotrophomas maltophilia and Streptococcus pneumoniae protocol with SpeI was performed on CR P. aeruginosa strains isolated in the Calgary Health Region during 2002-2006. The majority (96%) of metallo-β-lactamase (MBL)-producing isolates produced VIM-2 with gene cassettes aacC1 and aacA4 , while 4% produced IMP-7 with gene cassettes aacC4 and aacC1 . Eighty-six percent of VIM-2 producers belonged to a cluster (MBLV) that was responsible for nosocomial outbreaks during 2003 (intensive care unit) and 2004 (bone marrow transplant unit) at acute care center I. Environmental isolates from these units also belonged to MBLV. The majority of strains from cluster MBLVR (related to MBLV) were present in acute care center III. Isolates producing IMP-7 belonged to a different cluster (MBLI) and were related to strains described during the 1990s. PFGE of the MBL-negative CR strains showed that 37% belonged to a closely related cluster, NMBL, whose members were predominantly isolated from acute care center II. Our findings suggest that CR and dissemination of MBL clusters among P. aeruginosa populations in large geographic healthcare regions are dynamic processes that require continuous molecular surveillance.

Background A study was conducted in the Calgary Health Region between May 2002 and April 2004 to define the population-based epidemiological characteristics of infections caused by imipenem-resistant Pseudomonas aeruginosa and to explore the clinical outcomes due to metallo- beta -lactamase (MBL)-producing and non-MBL-producing strains. Methods Detailed clinical information was obtained by chart review, and phenotypic and molecular characterizations were performed using the MBL E-test, polymerase chain reaction with sequencing, and pulsed-field gel electrophoresis. Results A total of 228 patients with infections caused by imipenem-resistant P. aeruginosa were identified (annual incidence, 10.5 cases/100,000 population), with the highest incidence rate in those >or=75 years old. MBL-producing strains (98/228) were associated with higher rates of multidrug resistance and bacteremia. Ninety MBL-producing strains also produced VIM-2, 4 produced IMP-7, and 4 were unclassified. A cluster of VIM-2-producing strains was responsible for a nosocomial outbreak during 2003. The case-fatality rate was significantly higher for infections caused by MBL-producing strains than for those caused by non-MBL-producing strains (25% vs. 13%; relative risk, 1.98 [95% confidence interval, 1.00-3.90]; P=.05). Conclusion MBL-producing P. aeruginosa strains were associated with a higher case-fatality rate and invasive disease. Our study highlights the potential importance of molecular laboratory techniques in infection control and patient care.

Enterobacteriaceae producing extended-spectrum β-lactamases (ESBLs) typically cause nosocomial infections. Previous surveillance in the Calgary Health Region showed that Escherichia coli strains producing ESBLs were common among community patients. During the period (2000 to 2002): 23 of 157 (15%) of the strains were positive for bla CTX-M genes from the CTX-M-I group (CTX-M-1-like) and 87 of 157 (55%) of the strains were positive for bla CTX-M genes from the CTX-M-III group (CTX-M-14-like). The objective of this study was to investigate the molecular epidemiology of these strains. The β-lactamases were characterized, and the genetic relatedness of the isolates was analyzed by digesting genomic DNA with the restriction endonuclease XbaI and by performing pulse-field gel electrophoresis (PFGE). PFGE revealed two closely related restriction patterns (clusters CTXM14A and CTXM14AR) among 67 (77%) CTX-M-14 producers. These strains from CTXM14A had nearly identical susceptibility patterns and were isolated most often from urine samples obtained at community sites during 2000 and 2001. Strains from the CTX-M-1-like and CTX-M-negative groups were unrelated to clusters CTXM14, CTXM14AR, and CTXM14NR. We conclude that clonally related strains of E. coli producing CTX-M-14 β-lactamases were responsible for a predominantly community-wide outbreak. Further studies are warranted to investigate whether community-onset diseases caused by ESBL-producing E. coli are related to a point source or transmission within the community.

Context.—Bacterial cultures of bronchoscopic samples require 1 to 2 days for results to be available for use in clinical decisions. We developed a rapid diagnostic testing strategy that is highly sensitive for screening bacteria in bronchoalveolar lavage (BAL) samples, with results available within hours of collection. Objective.—To validate the ability of a bacterial adenosine triphosphate (ATP) assay and routine Gram stain microscopy to detect significant bacterial counts in BAL samples. Design.—Four hundred seventy-seven BAL samples from 319 patients suspected of having pneumonia were tested using a rapid diagnostic strategy, consisting of Gram stain and a bacterial ATP assay. Rapid results were compared with quantitative cultures with a positive cutoff of 104 CFU/mL or higher. Results.—Significant bacterial counts were identified in 107 samples (22%). The most common etiologic agents were Staphylococcus aureus (25%), Haemophilus influenzae (17%), and Streptococcus pneumoniae (12%). The rapid test results were false negative in 5 cases (S aureus in 2, both Klebsiella pneumoniae and S aureus in 1, and Stenotrophomonas maltophilia and S pneumoniae in 1 case each). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the rapid diagnostic strategy were 95.3%, 54.9%, 37.9%, 97.6%, and 63.9%, respectively. Conclusion.—A negative result with this rapid diagnostic testing strategy rules out significant bacterial counts in BAL samples with a high degree of certainty and may allow use of narrow-spectrum antimicrobial agents or withholding of empiric antimicrobial therapy in patients suspected of having ventilator-associated pneumonia.

Pyogenic liver abscess (PLA) is relatively uncommon in North America but is associated with significant morbidity and mortality. Our objective was to characterize the incidence of, risk factors for, and outcomes of PLA in a nonselected population.Population-based surveillance was conducted in the Calgary Health Region (CHR) between April 1, 1999 and March 31, 2003. All adult CHR residents with PLA were identified, and charts were reviewed.Seventy-one CHR residents developed a PLA for an annual incidence of 2.3 per 100,000 population. There was an increasing incidence of PLA with advancing age. Men were at much higher risk of acquiring a PLA as compared to women (3.3 vs 1.3 per 100,000; relative risk [RR], 2.6; 95% confidence interval [CI], 1.5-4.6; P.001), and this was observed across all age groups. A number of comorbid conditions were associated with significantly higher risk for developing a PLA including liver transplantation patients (RR, 444.8; 95% CI, 89.5-1356.0; P.0001), diabetics (RR, 11.1; 95% CI, 6.3-19; P.0001), and patients with a history of malignancy (RR, 13.3; 95% CI, 6.9-24.4; P.0001). No other solid organ transplantation patient was at increased risk. All patients required admission to hospital (median length of stay, 16 days), and 7 (10%) patients died in hospital, corresponding to a mortality rate of 0.22 per 100,000 population.This study provides important data on the burden of PLA and identifies risk groups that might potentially benefit from preventive efforts.

Major challenges in diagnostic molecular microbiology are to develop a simple assay to distinguish Staphylococcus aureus from the less virulent but clinically important coagulase-negative staphylococci (CoNS) and to simultaneously determine their antibiotic resistance profiles. Multiplex PCR assays have been developed for the detection of methicillin- and mupirocin-resistant S. aureus and CoNS but not for the simultaneous discrimination of S. aureus from CoNS. We designed a new set of Staphylococcus genus-specific primers and developed a novel quadriplex PCR assay targeting the 16S rRNA ( Staphylococcus genus specific), nuc ( S. aureus species specific), mecA (a determinant of methicillin resistance), and mupA (a determinant of mupirocin resistance) genes to identify most staphylococci, to discriminate S. aureus from CoNS and other bacteria, and to simultaneously detect methicillin and mupirocin resistance. Validation of the assay with 96 ATCC control strains and 323 previously characterized clinical isolates, including methicillin- and mupirocin-sensitive and -resistant S. aureus and CoNS isolates and other bacteria, demonstrated 100% sensitivity, specificity, and accuracy. This assay represents a simple, rapid, accurate, and reliable approach for the detection of methicillin- and mupirocin-resistant staphylococci and offers the hope of preventing their widespread dissemination through early and reliable detection.

Context.—Surface swab cultures have attracted attention as a potential alternative to biopsy histology or quantitative culture methods for microbiological burn wound monitoring. To our knowledge, the utility of adding a Gram-stained slide in this context has not been evaluated previously. Objective.—To determine the degree of correlation of Gram stain with culture for the microbiological analysis of burn wound surfaces. Design.—Prospective laboratory analysis. Setting.—Urban health region/centralized diagnostic microbiology laboratory. Patients.—Burn patients hospitalized in any Calgary Health Region burn center from November 2000 to September 2001. Intervention.—Gram stain plus culture of burn wound surface swab specimens obtained during routine dressing changes or based on clinical signs of infection. Main Outcome Measures.—Degree of correlation (complete, high, partial, none), including weighted κ statistic (κw), of Gram stain with culture based on quantitative microscopy and degree of culture growth. Results.—A total of 375 specimens from 50 burn patients were evaluated. Of these, 239 were negative by culture and Gram stain, 7 were positive by Gram stain only, 89 were positive by culture only, and 40 were positive by both methods. The degree of complete, high, partial, and no correlation of Gram stain with culture was 70.9% (266/375), 1.1% (4/375), 2.4% (9/375), and 25.6% (96/375), respectively. The degree of correlation for all 375 specimens, as expressed by the weighted κ statistic, was found to be fair (κw = 0.32). Conclusion.—The Gram stain is not suitable for the microbiological analysis of burn wound surfaces.

Context.—A rapid, real-time, duplex, fluorescent molecular beacon probe–based polymerase chain reaction (PCR) assay was recently developed for the detection of methicillin-resistant Staphylococcus aureus. Objective.—To describe the development and validation of this unique assay. Design.—Prospective laboratory analysis. Setting.—Urban health region/centralized diagnostic microbiology laboratory. Bacterial Strains.—One hundred eighty-one previously characterized clinical and American Type Culture Collection isolates, including 50 strains each of methicillin-resistant and methicillin-sensitive S aureus, plus 50 strains of coagulase-negative staphylococci and 31 nonstaphylococcal isolates to ensure assay specificity. Intervention.—Assays were performed on purified genomic DNA extracted from growing bacterial colonies. Two sets of oligonucleotide primers were used to specifically amplify the mecA and nuc genes, followed by detection of amplicons using fluorophore-labeled molecular beacon probes. Assays were performed on the Mx4000 Multiplex Quantitative PCR System (Stratagene Inc, La Jolla, Calif). Main Outcome Measures.—(1) Assay sensitivity and specificity, and (2) analytical sensitivity. Results.—The assay demonstrated 100% sensitivity and 100% specificity, and accurately characterized isolates as methicillin-resistant S aureus, methicillin-sensitive S aureus, or methicillin-resistant coagulase-negative staphylococci, with test results available in 2.5 hours. The analytical sensitivity of the assay was determined to be between 6 and 60 genomic equivalents. Conclusions.—This assay is rapid, accurate, easy to perform, and is compatible with other real-time PCR instruments, making it a suitable alternative to conventional PCR methodologies.