Impact of a Laboratory-Developed Phenotypic Rapid Susceptibility Test Directly From Positive Blood Cultures on Time to Narrowest Effective Therapy in Patients With Gram-Negative Bacteremia: A Prospective Randomized Trial.

Background: Antimicrobial susceptibility testing (AST) is often needed prior to antimicrobial optimization for patients with gram-negative bloodstream infections (GN-BSIs). Rapid AST (rAST) in combination with antimicrobial stewardship (AS) may decrease time to administration of narrower antibiotics. Methods: This was a prospective, nonblinded, randomized trial evaluating the impact of a phenotypic rAST method vs conventional AST (cAST) in hospitalized patients with GN-BSI and source control. The primary outcome was time to narrowest effective therapy. Results: Two hundred seventy-four patients were randomized and 205 underwent analysis (97 cAST, 108 rAST). Median (interquartile range [IQR]) time to susceptibility results was 23 hours shorter in the rAST group (cAST: 62 [59-67] hours vs rAST: 39 [IQR, 35-46] hours; P < .001). Median (IQR) time to narrowest effective therapy was similar between groups (cAST: 73 [44-138] hours vs rAST: 64 [42-92] hours; P = .10). Median (IQR) time to narrowest effective therapy was significantly shorter in a prespecified subgroup of patients not initially on narrowest therapy and during AS working hours (cAST: 93 [56-154] hours vs rAST: 62 [43-164] hours; P = .004). Significant decreases were observed in median (IQR) time to oral therapy (cAST: 126 [76-209] hours vs rAST: 91 [66-154] hours; P = .02) and median (IQR) length of hospital stay (cAST: 7 [4-13] days vs rAST: 5 [4-8] days; P = .04). Conclusions: In patients with GN-BSI, rAST did not significantly decrease time to narrowest effective therapy but did decrease time to oral antibiotics and length of hospital stay. Rapid AST using existing microbiology platforms has potential to optimize patient outcomes.

Multicenter Clinical Validation of the Molecular BD Max Enteric Viral Panel for Detection of Enteric Pathogens.

The conventional methodology for gastrointestinal pathogen detection remains time-consuming, expensive, and of limited sensitivity. The objective of this study was to evaluate the performance of the BD Max enteric viral panel (Max EVP) assay for identification of viral pathogens in stool specimens from individuals with symptoms of acute gastroenteritis, enteritis, or colitis. Prospective and archival stool specimens from adult and pediatric patients with diarrhea were collected in Cary-Blair medium or unpreserved containers. The results for specimens tested by the Max EVP (on the BD Max platform) were compared to those obtained by the reference method (alternate PCR assays, followed by bidirectional sequencing). Positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. A total of 2,239 specimens were collected, with 2,148 being included for analysis. In this population, 39.6% of specimens were from outpatients, 42.1% were from patients <21 years old, and 49.7% were from females. Prevalence rates for prospective specimens were 7.3%, 4.5%, 3.5%, 2.4%, and 1.2% for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. PPA was 92.8%, 84.9%, 93.0%, 100%, and 95.6%, for norovirus, sapovirus, astrovirus, rotavirus, and adenovirus, respectively. NPA was ≥99.4% for all targets. In conjunction with the clinical presentation, laboratory findings, and epidemiological information, the Max EVP assay is effective for the differential diagnosis of enteric disease caused by norovirus, sapovirus, astrovirus, rotavirus, and adenovirus. This assay can be used individually for patients at high risk for a viral enteropathogen (e.g., in outbreak settings) or as an adjunct to other enteric bacterial panels.

Carbapenem-Resistant Pseudomonas aeruginosa at US Emerging Infections Program Sites, 2015.

Pseudomonas aeruginosa is intrinsically resistant to many antimicrobial drugs, making carbapenems crucial in clinical management. During July-October 2015 in the United States, we piloted laboratory-based surveillance for carbapenem-resistant P. aeruginosa (CRPA) at sentinel facilities in Georgia, New Mexico, Oregon, and Tennessee, and population-based surveillance in Monroe County, NY. An incident case was the first P. aeruginosa isolate resistant to antipseudomonal carbapenems from a patient in a 30-day period from any source except the nares, rectum or perirectal area, or feces. We found 294 incident cases among 274 patients. Cases were most commonly identified from respiratory sites (120/294; 40.8%) and urine (111/294; 37.8%); most (223/280; 79.6%) occurred in patients with healthcare facility inpatient stays in the prior year. Genes encoding carbapenemases were identified in 3 (2.3%) of 129 isolates tested. The burden of CRPA was high at facilities under surveillance, but carbapenemase-producing CRPA were rare.

A randomized trial of single versus double high-level disinfection of duodenoscopes and linear echoendoscopes using standard automated reprocessing.

BACKGROUND AND AIMS: In a pilot study, we demonstrated that current guidelines for duodenoscope and linear echoendoscope (DLE) reprocessing using a single cycle of high-level disinfection (HLD) in an automated reprocessor may be inadequate. In August 2015, the U.S. Food and Drug Administration offered double HLD as a possible response to address this concern. As a result, Providence Health and Services adopted double HLD as standard procedure for DLEs, but no rigorous clinical studies supported this practice. We undertook a quality improvement study to compare single HLD versus double HLD at 4 of our 34 hospitals. METHODS: HLD of DLE was randomized, separately in each facility, to either single HLD or double HLD on weekdays, with standard double HLD on weekends or holidays. There was 99.7% compliance with the randomization scheme. Daily qualitative surveillance cultures of dried, post-HLD DLEs were collected for 6 months (1 swab sample from the elevator mechanism and 1 combined brush sample from the suction and working channels for each encounter), and each sample was incubated for 48 hours. Positivity rates of any microbial growth and growth of high-concern pathogens (potentially pathogenic enteric flora) were compared between the 2 study arms. RESULTS: Altogether, 5850 surveillance culture specimens were obtained during 2925 encounters from the 45 DLEs in clinical use in the participating hospitals. Of these, 3052 (52.2%) were from endoscopes cleaned by double HLD. Double HLD demonstrated no benefit over single HLD because similar positivity rates were observed (all P > .05). The elevator mechanism was more frequently colonized than the biopsy channel (5.2% vs 2.9%, P < .001). Among the 224 encounters with positive growth, 140 (62.5%) recovered microbes from only the elevator mechanism specimens, 73 (32.6%) recovered microbes from only the channel specimens, and 11 (4.9%) recovered microbes from both types of specimens. Double HLD failed to improve contamination rates for either sample site at any of the 4 endoscopy facilities, although there were significant overall differences in contamination rates among the facilities (P < .001), as observed in our previous study. Only 8 high-concern pathogens were recovered from 5 DLEs, all from the elevator mechanism. Persistent growth was observed on 2 duodenoscopes. One grew Enterococcus spp (not vancomycin-resistant enterococci) on 3 occasions, and Escherichia coli was present on 2 of these occasions, 1 of which was a multidrug-resistant organism. The other grew different enteric flora on 2 specimens. CONCLUSIONS: Our prospectively randomized study, involving 4 separate endoscopy facilities and standard automated endoscope reprocessing, showed that double HLD did not reduce culture positivity rates compared with single HLD in facilities with an already low positive culture rate. Alternative risk mitigation strategies will be assessed in an ongoing effort to reduce endoscope contamination.

Multisite Evaluation of the BD Max Extended Enteric Bacterial Panel for Detection of Yersinia enterocolitica, Enterotoxigenic Escherichia coli, Vibrio, and Plesiomonas shigelloides from Stool Specimens.

The purpose of this study was to perform a multisite evaluation to establish the performance characteristics of the BD Max extended enteric bacterial panel (xEBP) assay directly from unpreserved or Cary-Blair-preserved stool specimens for the detection of Yersinia enterocolitica, enterotoxigenic Escherichia coli (ETEC), Vibrio, and Plesiomonas shigelloides The study included prospective, retrospective, and prepared contrived specimens from 6 clinical sites. BD Max xEBP results were compared to the reference method, which included standard culture techniques coupled with alternate PCR and sequencing, except for ETEC, for which the reference method was two alternate PCRs and sequencing. Alternate PCR was also used to confirm the historical results for the retrospective specimens and for discrepant result analysis. A total of 2,410 unformed, deidentified stool specimens were collected. The prevalence in the prospective samples as defined by the reference method was 1.2% ETEC, 0.1% Vibrio, 0% Y. enterocolitica, and 0% P. shigelloides Compared to the reference method, the positive percent agreement (PPA) (95% confidence interval [CI]), negative percent agreement (NPA) (95% CI), and kappa coefficient (95% CI) for the BD Max xEBP assay for all specimens combined were as follows: ETEC, 97.6% (87.4 to 99.6), 99.8% (99.5 to 99.9), and 0.93 (0.87 to 0.99); Vibrio, 100% (96.4 to 100), 99.7% (99.4 to 99.8), and 0.96 (0.93 to 0.99); Y. enterocolitica, 99.0% (94.8 to 99.8), 99.9% (99.8 to 99.9), and 0.99 (0.98 to 1); P. shigelloides, 100% (96.4 to 100), 99.8% (99.5 to 99.9), and 0.98 (0.95 to 1), respectively. In this multicenter study, the BD Max xEBP showed a high correlation (kappa, 0.97; 95% CI, 0.95 to 0.98) with the conventional methods for the detection of ETEC, Vibrio, Y. enterocolitica, and P. shigelloides in stool specimens from patients suspected of acute gastroenteritis, enteritis, or colitis.

Surveillance of guideline practices for duodenoscope and linear echoendoscope reprocessing in a large healthcare system.

BACKGROUND AND AIMS: To assess the adequacy of currently recommended duodenoscope and linear echoendoscope (DLE) automatic endoscope reprocessing (AER) and high-level disinfection (HLD), we collected daily post-reprocessing surveillance cultures of 106 DLEs in 21 Providence and Affiliate Hospitals. METHODS: Daily qualitative surveillance of dried, post-HLD DLEs was conducted for a minimum of 30 days at each facility. Positivity rates for any microbial growth and growth of high-concern pathogens were reported. Potential effects of DLE manufacturer, age, and AER processor on culture-positivity rate were assessed. RESULTS: Microbial growth was recovered from 201 of 4032 specimens (5%) or 189 of 2238 encounters (8.4%), including 23 specimens (.6%) or 21 encounters (.9%) for a high-concern pathogen. Wide variations in culture-positivity rate were observed across facilities. No striking difference in culture-positivity rate was seen among 8 DLE models, 3 DLE manufacturers, DLE age, manual or bedside cleanser, or automatic flushing system use. However, there was suggestive evidence that Custom Ultrasonics AER (Warminster, Pa, USA) had a lower culture-positivity rate than Medivators AER (Cantel Medical Corp., Little Falls, NJ, USA) for high-concern pathogen growth (0/1079 vs 21/2735 specimens or 0/547 vs 20/1582 encounters). Two endoscopes grew intestinal flora on several occasions despite multiple HLD. No multidrug-resistant organism was detected. CONCLUSIONS: In this multicenter DLE surveillance study, microbial growth was recovered in 5.0% of specimens (8.4% of encounters), with most being environmental microbes. Enteric bacterial flora was recovered in .6% of specimens (.9% of encounters), despite compliance with 2014 U.S. guidelines and manufacturers' recommendations for cleaning and HLD process. The observed better performance of Custom Ultrasonics AER deserves further investigation.

Genotypic resistance testing creates new treatment challenges: two cases of oxacillin-susceptible methicillin-resistant Staphylococcus aureus.

Oxacillin-susceptible, mecA-positive Staphylococcus aureus isolates create a treatment challenge for the clinician. In this article, we describe two cases of bacteremia from isolates that carried the mecA gene but were susceptible to oxacillin (oxacillin-susceptible methicillin-resistant S. aureus [OS-MRSA]). DNA microarray analysis was used to characterize these isolates as a mecA-positive, clonal complex 5, pediatric strain and a mecA-positive, clonal complex 8, USA300 strain.

Clinical impact of a real-time PCR assay for rapid identification of staphylococcal bacteremia.

The purpose of this study was to evaluate the impact of real-time PCR reporting both on timely identification of clustered Gram-positive cocci (GPC) in blood cultures and on appropriate antibiotic treatment. This retrospective, interventional cohort study evaluated inpatients with blood cultures positive for GPC in the pre-PCR (15 January 2009 to 14 January 2010) and post-PCR (15 January 2010 to 14 January 2011) periods. Post-PCR implementation, laboratory services completed batched PCR; results other than methicillin-resistant Staphylococcus aureus (MRSA) were reported in the electronic medical record without additional interventions. The assay's sensitivity and specificity, time to identification of staphylococcal bacteremia, and clinically relevant outcomes, including time to optimal antibiotic therapy, were evaluated. Demographic information was also collected and analyzed. Sixty-eight and 58 patients with Staphylococcus aureus bacteremia from the pre- and post-PCR periods, respectively, met inclusion criteria. Similar numbers of consecutive patients with coagulase-negative staphylococci were analyzed for comparison. The time to identification was significantly reduced post-PCR implementation (mean, 13.2 h; 95% confidence interval [95% CI], 10.5 to 15.9 h; P < 0.0001). However, the time to optimal antibiotic therapy was not significantly reduced. We conclude that implementation of a PCR assay demonstrated the potential to improve appropriate antibiotic use based on clinically meaningful and statistically significant reductions in the time to microbiologic identification. However, in order to realize this potential benefit, processes must be optimized and additional interventions initiated to facilitate providers' use of the PCR result.

Diagnosing periprosthetic infection: false-positive intraoperative Gram stains.

BACKGROUND: Intraoperative Gram stains have a reported low sensitivity but high specificity when used to help diagnose periprosthetic infections. In early 2008, we recognized an unexpectedly high frequency of apparent false-positive Gram stains from revision arthroplasties. QUESTIONS/PURPOSES: The purpose of this report is to describe the cause of these false-positive test results. METHODS: We calculated the sensitivity and specificity of all intraoperative Gram stains submitted from revision arthroplasty cases during a 3-month interval using microbiologic cultures of the same samples as the gold standard. Methods of specimen harvesting, handling, transport, distribution, specimen processing including tissue grinding/macerating, Gram staining, and interpretation were studied. After a test modification, results of specimens were prospectively collected for a second 3-month interval, and the sensitivity and specificity of intraoperative Gram stains were calculated. RESULTS: The retrospective review of 269 Gram stains submitted from revision arthroplasties indicated historic sensitivity and specificity values of 23% and 92%, respectively. Systematic analysis of all steps of the procedure identified Gram-stained but nonviable bacteria in commercial broth reagents used as diluents for maceration of periprosthetic membranes before Gram staining and culture. Polymerase chain reaction and sequencing showed mixed bacterial DNA. Evaluation of 390 specimens after initiating standardized Millipore filtering of diluent fluid revealed a reduced number of positive Gram stains, yielding 9% sensitivity and 99% specificity. CONCLUSIONS: Clusters of false-positive Gram stains have been reported in other clinical conditions. They are apparently rare related to diagnosing periprosthetic infections but have severe consequences if used to guide treatment. Even occasional false-positive Gram stains should prompt review of laboratory methods. Our observations implicate dead bacteria in microbiologic reagents as potential sources of false-positive Gram stains.

Distinction between intact and antibiotic-inactivated bacteria by real-time PCR after treatment with propidium monoazide.

One limitation to the use of the polymerase chain reaction (PCR) to identify orthopedic infections has been apparent false-positive results, possibly due to the detection of dead bacteria. We recently showed that the use of DNA-binding agent propidium monoazide (PMA) could distinguish viable from heat-inactivated bacteria, and, in this study, we investigated whether the same technique can be applied to bacteria killed by two antibiotics with distinctly different mechanisms of action, a test of greater clinical relevance than thermal inactivation. Staphylococcus aureus and S. epidermidis were inactivated by vancomycin and gentamicin and treated with PMA or left untreated before DNA extraction. The threshold cycle difference of antibiotic-treated bacteria with and without PMA pretreatment was investigated with PCR primers for the 16S rDNA and tuf genes. Our results indicated that PMA effectively inhibited detection by PCR of bacteria, which had been inactivated by either vancomycin or gentamicin. The effect was statistically significant at 24 h after treatment (C(t) difference consistently >3; p < 0.05) and after 10 days of treatment (C(t) difference >4; p < 0.01), when compared to viable cells (C(t) difference 1-2). Vancomycin had a stronger effect on the C(t) value than gentamicin, reflecting the different mechanism of action of each antibiotic. Techniques of this type may help reduce clinically false-positive PCR results caused by the detection of dead bacteria, and may be especially useful in patients who have received antibiotics, such as patients undergoing the second stage of a two-stage revision for infected arthroplasty.

Limiting false-positive polymerase chain reaction results: detection of DNA and mRNA to differentiate viable from dead bacteria.

We compared 2 methods for determining Escherichia coli viability in vitro. A 16S rDNA polymerase chain reaction (PCR) assay detected bacteria irrespective of viability. A groEL mRNA reverse transcriptase PCR was positive for 72 h but later became negative. Detecting mRNA holds promise but is tedious, and groEL may not be the best target.

Improving clinical significance of PCR: use of propidium monoazide to distinguish viable from dead Staphylococcus aureus and Staphylococcus epidermidis.

Molecular techniques, such as the polymerase chain reaction (PCR) have high sensitivity when used to diagnose infection, but may detect DNA, RNA, and proteins from dead, as well as viable, bacteria. Propidium monoazide (PMA) is a DNA binding agent, that has the ability to penetrate only dead cells with compromised membranes and has been used in conjunction with real-time PCR to distinguish intact from dead bacterial cells. In this study, intact, heat-inactivated (dead), and intact/dead admixed Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were treated with PMA or left untreated before DNA extraction. We quantified levels of 16S rDNA and tuf gene by real-time quantitative PCR (qPCR), to test the ability of PMA to distinguish intact from dead bacteria. Our results indicated that PMA inhibited detection of dead bacteria, and the qPCR results reflected the number of intact bacteria without being impacted by the presence of the dead bacteria. This approach of combining qPCR with and without PMA treatment has promise to limit false-positive PCR results when used to diagnose infections, but needs to be further validated in clinical samples.

Characterization of blaKPC-containing Klebsiella pneumoniae isolates detected in different institutions in the Eastern USA.

BACKGROUND: The emergence of bla(KPC)-containing Klebsiella pneumoniae (KPC-Kp) isolates is attracting significant attention. Outbreaks in the Eastern USA have created serious treatment and infection control problems. A comparative multi-institutional analysis of these strains has not yet been performed. METHODS: We analysed 42 KPC-Kp recovered during 2006-07 from five institutions located in the Eastern USA. Antimicrobial susceptibility tests, analytical isoelectric focusing (aIEF), PCR and sequencing of bla genes, PFGE and rep-PCR were performed. Results By in vitro testing, KPC-Kp isolates were highly resistant to all non-carbapenem beta-lactams (MIC(90)s >or= 128 mg/L). Among carbapenems, MIC(50/90)s were 4/64 mg/L for imipenem and meropenem, 4/32 mg/L for doripenem and 8/128 for ertapenem. Combinations of clavulanate or tazobactam with a carbapenem or cefepime did not significantly lower the MIC values. Genetic analysis revealed that the isolates possessed the following bla genes: bla(KPC-2) (59.5%), bla(KPC-3) (40.5%), bla(TEM-1) (90.5%), bla(SHV-11) (95.2%) and bla(SHV-12) (50.0%). aIEF of crude beta-lactamase extracts from these strains supported our findings, showing beta-lactamases at pIs of 5.4, 7.6 and 8.2. The mean number of beta-lactamases was 3.5 (range 3-5). PFGE demonstrated that 32 (76.2%) isolates were clonally related (type A). Type A KPC-Kp isolates (20 bla(KPC-2) and 12 bla(KPC-3)) were detected in each of the five institutions. rep-PCR showed patterns consistent with PFGE. CONCLUSIONS: We demonstrated the complex beta-lactamase background of KPC-Kp isolates that are emerging in multiple centres in the Eastern USA. The prevalence of a single dominant clone suggests that interstate transmission has occurred.

Improved detection of biofilm-formative bacteria by vortexing and sonication: a pilot study.

Bacteria such as staphylococci commonly encountered in orthopaedic infections form biofilms and adhere to bone implants and cements. Various methods to disrupt the biofilm and enhance bacterial detection have been reported. We will describe the effectiveness of vortexing and sonication to improve the detection of biofilm-formative bacteria from polymethylmethacrylate by conventional quantitative bacterial culture and real-time quantitative PCR. We used a single biofilm-formative Staphylococcus aureus strain and 20 polymethylmethacrylate coupons as an in vitro biofilm model; four coupons were used for each of two control groups or three experimental sonication times (1, 5, and 30 minutes). Vortexing the cement without sonication increased the yield of adherent bacteria to a considerable extent. The combination of vortexing and sonication further enhanced the yield regardless of the duration of sonication. Quantitative conventional cultures correlated with quantitative PCR assay. The combination of vortexing and sonication to disrupt the bacterial biofilm followed by quantitative PCR and/or culture seems to be a sensitive method for detecting bacteria adherent to bone cement.

Efficiency of the ortho VITROS assay for detection of hepatitis C virus-specific antibodies increased by elimination of supplemental testing of samples with very low sample-to-cutoff ratios.

The clinical significance of specimens with low sample-to-cutoff (S/Co) ratios in the Ortho VITROS chemiluminescence assay (CIA) for detection of antibodies to hepatitis C virus (HCV) was evaluated. In one study of 482 CIA-reactive samples, none of the 83 samples with S/Co ratios of < 5 was HCV RNA positive. In a subsequent study, 332 samples with S/Co ratios of between 1 and 20 were tested with the recombinant immunoblot assay (RIBA). None of the 163 samples with S/Co ratios of < 5 was RIBA positive, 83% were RIBA negative, and 28 samples (18%) were RIBA indeterminate. HCV RNA and/or clinical evidence of hepatitis was not found in the 27 indeterminate cases examined. These results show that over 99% of samples with very low S/Co ratios (< or = 5) have no evidence of HCV infection. Therefore, we suggest that the HCV antibody testing algorithm for the VITROS assay might be modified to eliminate supplemental testing of samples with very low S/Co ratios.