Evaluation of microbial qPCR workflows using engineered Saccharomyces cerevisiae.

AIMS: We describe the development and interlaboratory study of modified Saccharomyces cerevisiae as a candidate material to evaluate a full detection workflow including DNA extraction and quantitative polymerase chain reaction (qPCR). METHODS AND RESULTS: S. cerevisiae NE095 was prepared by stable insertion of DNA sequence External RNA Control Consortium-00095 into S. cerevisiae BY4739 to convey selectivity. For the interlaboratory study, a binomial regression model was used to select three cell concentrations, high (4 × 10(7) cells ml(-1)), intermediate (4 × 10(5) cells ml(-1)) and low (4 × 10(3) cells ml(-1)), and the number of samples per concentration. Seven participants, including potential end users, had combined rates of positive qPCR detection (quantification cycle <37) of 100%, 40%, and 0% for high, intermediate, and low concentrations, respectively. CONCLUSIONS: The NE095 strain was successfully detected by all participants, with the high concentration indicating a potential target concentration for a reference material. SIGNIFICANCE AND IMPACT OF THE STUDY: The engineered yeast has potential to support measurement assurance for the analytical process of qPCR, encompassing the method, equipment, and operator, to increase confidence in results and better inform decision-making in areas of applied microbiology. This material can also support process assessment for other DNA-based detection technologies.

Recovery balance: a method for estimating losses in a Bacillus anthracis spore sampling protocol.

AIM: The aim of this study was to develop a method to calculate the performance, and isolate error contributions occurring in a microbial surface sampling protocol. METHODS AND RESULTS: The experiments were conducted using a slip/peel tester to provide consistent pressure during the wipe collection. Fluorescence microscopy was used to count spores deposited on the coupon prior to sampling. The mean recovery efficiency (RE) as well as the efficiency of each step in the process was estimated by a recovery balance (RB), similar to a mass balance. Two studies were conducted in this work. In the first one, the recovery of spores from the solution (RE(soln)) was 57.7% (SD = 8.0), while spores left on the glass surface after wiping (RE(b+c)) was 2.8% (SD = 2.4). The RE of spores adhered to the tube wall (RE(tube)) and glass surface (RE(surf)) was 1.2% (SD = 19.6) and 5.8% (SD = 7.1), respectively. From the recovery balance, it was determined that 39.9% (SD = 21.2) of spores were lost to the wipe (RE(wipe)). The applicability of the RB method was demonstrated in a second study by examining the relative impact of parameters affecting spore collection including relative humidity, wipe material, wetting agent and nonporous surfaces. CONCLUSIONS: The approach used in this study pointed out the need for a closer analysis of the complex interaction between spores and wipe material because a substantial percentage of spores were lost to the wipe. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery balance, in association with independent controls, provides an account for error contribution and potential variability on each step of the sampling protocol. The approach is not meant to be a replacement for field or laboratory validation of wipe recoveries but promote the development of new collection methodologies and support protocol optimization in laboratory settings.

Association and decontamination of Bacillus spores in a simulated drinking water system.

The objective of this work was to elucidate the disinfectant susceptibility of Bacillus anthracis Sterne (BA) and a commercial preparation of Bacillus thuringiensis (BT) spores associated with a simulated drinking water system. Biofilms composed of indigenous water system bacteria were accumulated on copper and polyvinyl chloride (PVC) pipe material surfaces in a low-flow pipe loop and uniformly mixed tank reactor (CDC biofilm reactor). Application of a distributed shear during spore contact resulted in approximately a 1.0 and 1.6 log10 increase in the number of spores associated with copper and PVC surfaces, respectively. Decontamination of spores in both free suspension and after association with biofilm-conditioned pipe materials was attempted using free chlorine and monochloramine. Associated spores required 5- to 10-fold higher disinfectant concentrations to observe the same reduction of viable spores as in suspension. High disinfectant concentrations (103 mg/L free chlorine and 49 mg/L monochloramine) yielded less than a 2-log10 reduction in viable associated spores after 60 min. Spores associated with biofilms on copper surfaces consistently yielded higher Ct values than PVC.

Macro- and nanoscale observations of adhesive behavior for several E. coli strains (O157:H7 and environmental isolates) on mineral surfaces.

Subsurface biobarriers can be conceived to attenuate the migration of pathogens by adhesion to mineral surfaces. Candidate biobarrier materials of varied surface characteristics (dolomite, alpha-alumina, silica, pyrophyllite, and Pyrax (a composite form of pyrophyllite, mica, and silica)) were tested for Escherichia coli adhesive capacity in macroscale continuous-flow columns. Atomic force microscopy (AFM) was used to determine nanoscale interaction energies. Predicted attractive interaction energies correlated well with macroscale adhesive behavior for tested E. coli strains. AFM measurements confirmed ExDLVO model predictions of attachment in the primary minima for E. coli O157:H7 and two environmental isolates E. coli (UCFL339 and UCFL-348) with MOPS conditioned Pyrax. In macroscale column experiments, pyrophyllite and Pyrax demonstrated significantly higher bacterial retention, higher deposition coefficients and lower initial cell breakthrough values for E. coli O157:H7 than did alpha-alumina, silica, or dolomite (pyrophyllite, 0.93, 3.56 h(-1), 3.2% ODo; Pyrax, 0.95, 3.73 h(-1), 2.8% ODo; alpha-alumina, 0.74, 1.60 h(-1), 33% ODo; silica, 0.63, 0.43 h(-1), 73% ODo; and dolomite, 0.33, 0.17 h(-1), 89% ODo, respectively). Bacterial hydrophilicity impacted cell retention in Pyrax columns with the relatively hydrophobic E. coli isolate UCFL-339 (0.99, 6.13 h(-1), 0.4% ODo) retained better than the more hydrophilic E. coli isolate UCFL348 (0.94, 3.70 h(-1), 3.6% ODo). The strong adhesive behavior of Pyrax was attributed to the hydrophobic (deltaGiwi = -32.4 mJ/m2) pyrophyllite component of the mineral. Vicinal water appears poised between the bacterial and the mineral surface during initial attachment. Overall, observed behavior of the various E. coli strains and the selected mineral surfaces was consistent with surface analyses, conducted at both the macro- and nanoscale.

The ringed esophagus: histological features of GERD.

OBJECTIVE: The "ringed" or "corrugated" esophagus is a cause of chronic dysphagia and recurrent food impactions in young men. It was previously believed to be a congenital condition, but recent case series have documented histological esophagitis in these patients. We have treated 19 patients with a ringed esophagus and are impressed that this represents an acquired condition with gastroesophageal reflux disease (GERD) as its etiology. Our goals are to present the largest case series to date of ringed esophagus, discuss the evidence for GERD, and suggest a strategy for its diagnosis and management. METHODS: The charts of 19 patients with a ringed esophagus were reviewed. A single pathologist interpreted all available esophageal biopsy specimens and graded them for the presence of GERD-related abnormalities. Phone interviews were conducted to assess response to therapy and confirm historical features obtained from medical records. RESULTS: The typical patient is a young man (median age 35, M:F 17:2) with long-standing dysphagia and multiple food impactions. Endoscopy revealed multiple concentric rings that persisted despite air insufflation and caused significant stenosis. Of the 11 patients with available histology, all had basal cell hyperplasia, papillomatosis, and an increased number of intraepithelial eosinophils. Other clinical features of GERD such as heartburn, endoscopic esophagitis, and hiatal hernia were often absent. Response to dilation and acid suppression was good. CONCLUSIONS: The uniform presence of histological esophagitis with intraepithelial eosinophils, basal cell hyperplasia, and papillary lengthening strongly implicates GERD in the pathogenesis of the adult ringed esophagus. In addition to a series of gradual esophageal dilations, we suggest using a proton pump inhibitor to provide acid suppression.

Sedation for colonoscopy using a single bolus is safe, effective, and efficient: a prospective, randomized, double-blind trial.

OBJECTIVE: Practice guidelines call for the careful titration of sedatives and analgesics during endoscopy, with time taken between incremental doses to assess effect. This approach is time-consuming and has never been validated in a prospective trial. The aim of this study was to compare the safety and efficacy of titration, as outlined in practice guidelines, with a single, rapid bolus of sedatives before colonoscopy. METHODS: Consecutive colonoscopy outpatients were randomized to a single, rapid bolus of meperidine and midazolam or to a titration of doses every 3 min until predefined levels of somnolence were achieved. The colonoscopist was not present during sedation and remained blinded as to which technique was used. Supplemental O2 was given for SaO2 <90% on three or more occasions. Total physician time was calculated from the first injection of sedatives to the removal of the colonoscope. Patient assessments of pain and tolerance were obtained at the time of discharge using visual analog scales of 100 mm (0 = excellent and 100 = unbearable). RESULTS: A total of 101 patients were randomized (49 bolus, 52 titration). Demographic features were similar for both groups. Titration required more physician time than did bolus (32.2 min vs 20.1 min, p < 0.001) and was associated with an increased need for supplemental O2 (44% vs 14%, p = 0.002). Mean tolerance scores were similar (titration 16.3 vs bolus 15.3, p = 0.72). CONCLUSIONS: Rapid bolus sedation for colonoscopy saves significant endoscopist time, is associated with less O2 desaturation, and provides equivalent levels of patient comfort. A revision of the guidelines for sedation and analgesia during endoscopy should be considered.

Gastroenterologist-administered propofol for therapeutic upper endoscopy with graphic assessment of respiratory activity: a case series.

BACKGROUND: Traditional methods of sedation and analgesia for advanced endoscopic procedures can be inadequate and frequently prolong recovery room observation. Propofol is a rapidly acting agent that produces an excellent hypnotic state, but its use is typically limited to anesthesiologist-assisted cases because of the inadequacy of current monitoring standards to reliably detect early stages of respiratory depression. METHODS: Ten patients undergoing advanced upper endoscopic procedures (endoscopic retrograde cholangiopancreatography, endoscopic ultrasound, esophageal stent placement) received a propofol infusion under the control of a second qualified gastroenterologist with advanced cardiac life support skills. Graphic assessment of respiratory activity was made by using a sidestream carbon dioxide detecting cannula. Patient satisfaction was measured with a 100 mm visual analog scale. Recovery scores were measured by standardized scoring of discharge criteria. RESULTS: Monitoring with graphic assessment of respiratory activity detected early phases of respiratory depression, resulting in a timely decrease in the propofol infusion without significant hypoxemia, hypercapnia, hypotension, or arrhythmias. Satisfaction scores were extremely high (median score 92 of 100) and 9 of 10 patients met discharge criteria at 15 minutes after discontinuation of the propofol infusion. CONCLUSIONS: With the use of monitoring by graphic assessment of respiratory activity, propofol infusion by a second qualified gastroenterologist for prolonged upper endoscopic procedures is safe and results in high levels of patient satisfaction with rapid recovery times.