Search

Background: High bacterial burden stalls wound healing and can quickly progress to infection and sepsis in complex, older-adult patients in long-term care (LTC) or skilled nursing facilities (SNFs)., Objective: To investigate the outcomes of point-of-care fluorescence (FL) imaging (MolecuLight i:X) of bacterial loads, which are frequently asymptomatic, to inform customized wound treatment plans for patients in LTC/SNFs., Methods: In this retrospective pre/postinterventional cohort study, the authors compared the healing and infection-associated outcomes of 167 pressure injuries from 100 Medicare beneficiaries before and after implementation of FL imaging., Results: Most patient demographics and wound characteristics did not differ significantly between the standard-of-care (SOC; n = 71 wounds) and FL (n = 96 wounds) cohorts. Significantly more wounds (+71.0%) healed by 12 weeks in the FL cohort (38.5%) versus the SoC cohort (22.5%). Wounds in the FL cohort also healed 27.7% faster (-4.8 weeks), on average, and were 1.4 times more likely to heal per Kaplan-Meier survival analysis (hazard ratio = 1.40; 95% CI, 0.90-2.12). Infection-related complications decreased by 75.3% in the FL cohort, and a significant shift from largely systemic to topical antibiotic prescribing was evidenced., Conclusions: Fluorescence-imaging-guided management of wounds significantly improved healing and infection outcomes in highly complex and multimorbid patients in LTC/SNFs. Proactive bacterial infection management via local treatments was enabled by earlier, objective detection. These reported outcome improvements are comparable to randomized controlled trials and cohort studies from less compromised, selectively controlled outpatient populations. Fluorescence imaging supports proactive monitoring and management of planktonic and biofilm-encased bacteria, improving patient care in a complex, real-world setting., (Copyright © 2024 the Author(s). Published by Wolters Kluwer Health, Inc.)

Objective: High bacterial load contributes to chronicity of wounds and is diagnosed based on assessment of clinical signs and symptoms (CSS) of infection, but these characteristics are poor predictors of bacterial burden. Point-of-care fluorescence imaging (FL) MolecuLight i:X can improve identification of wounds with high bacterial burden (>10 4 colony-forming unit [CFU]/g). FL detects bacteria, whether planktonic or in biofilm, but does not distinguish between the two. In this study, diagnostic accuracy of FL was compared to CSS during routine wound assessment. Postassessment, clinicians were surveyed to assess impact of FL on treatment plan. Approach: A prospective multicenter controlled study was conducted by 20 study clinicians from 14 outpatient advanced wound care centers across the United States. Wounds underwent assessment for CSS followed by FL. Biopsies were collected to confirm total bacterial load. Three hundred fifty patients completed the study (138 diabetic foot ulcers, 106 venous leg ulcers, 60 surgical sites, 22 pressure ulcers, and 24 others). Results: Around 287/350 wounds (82%) had bacterial loads >10 4 CFU/g, and CSS missed detection of 85% of these wounds. FL significantly increased detection of bacteria (>10 4 CFU/g) by fourfold, and this was consistent across wound types ( p  < 0.001). Specificity of CSS+FL remained comparably high to CSS ( p  = 1.0). FL information modified treatment plans (69% of wounds), influenced wound bed preparation (85%), and improved overall patient care (90%) as reported by study clinicians. Innovation: This novel noncontact, handheld FL device provides immediate, objective information on presence, location, and load of bacteria at point of care. Conclusion: Use of FL facilitates adherence to clinical guidelines recommending prompt detection and removal of bacterial burden to reduce wound infection and facilitate healing.

We evaluated data from 10 laboratories that analyzed water samples from 82 recreational water sites across the state of Michigan between 2016 and 2018. Water sample replicates were analyzed by experienced U.S. Environmental Protection Agency (EPA) analysts and Michigan laboratories personnel, many of whom were newly trained, using EPA Draft Method C-a rapid quantitative polymerase chain reaction (qPCR) technique that provides same day Escherichia coli (E. coli) concentration results. Beach management decisions (i.e. remain open or issue an advisory or closure) based on E. coli concentration estimates obtained by Michigan labs and by the EPA were compared; the beach management decision agreed in 94% of the samples analyzed. We used the Wilcoxon one-sample signed rank test and nonparametric quantile regression to assess (1) the degree of agreement between E. coli concentrations quantified by Michigan labs versus the EPA and (2) Michigan lab E. coli measurement precision, relative to EPA results, in different years and water body types. The median quantile regression curve for Michigan labs versus EPA approximated the 1:1 line of perfect agreement more closely as years progressed. Similarly, Michigan lab E. coli estimates precision also demonstrated yearly improvements. No meaningful difference was observed in the degree of association between Michigan lab and EPA E. coli concentration estimates for inland lake and Great Lakes samples (median regression curve average slopes 0.93 and 0.95, respectively). Overall, our study shows that properly trained laboratory personnel can perform Draft Method C to a degree comparable with experienced EPA analysts. This allows health departments that oversee recreational water quality monitoring to be confident in qPCR results generated by the local laboratories responsible for analyzing the water samples., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microbial water quality monitoring for the presence of faecal indicator bacteria (FIB) is a mandatory activity in many countries and is key in public health protection. Despite technological advances and a need for methodological improvements, chromogenic and fluorogenic enzymatic techniques remain the mainstays of water quality monitoring for both public health agencies and regulated utilities. We demonstrated that bioelectroanalytical approaches to FIB enumeration are possible and can be achieved using commercially available enzyme-specific resorufin glycosides, although these are expensive, not widely available or designed for purpose. Following this, we designed two naphthoquinone glycosides which performed better, achieving Escherichia coli detection in the range 5.0 × 10 2 to 5.0 × 10 5 CFU ml -1 22-54% quicker than commercially available resorufin glycosides. The molecular design of the naphthoquinone glycosides requires fewer synthetic steps allowing them to be produced for as little as US$50 per kg. Tests with environmental samples demonstrated the low tendency for abiotic interference and that, despite specificity being maintained between β-glucuronidase and β-galactosidase, accurate enumeration of E. coli in environmental samples necessitates development of a selective medium. In comparison to a commercially available detection method, which has U.S. Environmental Protection Agency (EPA) approval, our approach performed better at high organism concentrations, detecting 500 organisms in 9 h compared with 13.5 h for the commercial method. Bioelectroanalytical detection is comparable to current approved methods and with further development could result in improved detection times. A recent trend for low-cost open-source hardware means that automated, potentiostatically controlled E. coli detection systems could be constructed for less than US$100 per channel., (© 2016 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Epidemiologic studies of the carcinogenic stomach bacterium Helicobacter pylori have been limited by the lack of noninvasive detection and genotyping methods. We developed a new stool-based method for detection, quantification, and partial genotyping of H. pylori using droplet digital PCR (ddPCR), which allows for increased sensitivity and absolute quantification by PCR partitioning., Materials and Methods: Stool-based ddPCR assays for H. pylori 16S gene detection and cagA virulence gene typing were tested using a collection of 50 matched stool and serum samples from Costa Rican volunteers and 29 H. pylori stool antigen-tested stool samples collected at a US hospital., Results: The stool-based H. pylori 16S ddPCR assay had a sensitivity of 84% and 100% and a specificity of 100% and 71% compared to serology and stool antigen tests, respectively. The stool-based cagA genotyping assay detected cagA in 22 (88%) of 25 stools from CagA antibody-positive individuals and four (16%) of 25 stools from CagA antibody-negative individuals from Costa Rica. All 26 of these samples had a Western-type cagA allele. Presence of serum CagA antibodies was correlated with a significantly higher load of H. pylori in the stool., Conclusions: The stool-based ddPCR assays are a sensitive, noninvasive method for detection, quantification, and partial genotyping of H. pylori. The quantitative nature of ddPCR-based H. pylori detection revealed significant variation in bacterial load among individuals that correlates with presence of the cagA virulence gene. These stool-based ddPCR assays will facilitate future population-based epidemiologic studies of this important human pathogen., (© 2015 John Wiley & Sons Ltd.)

Shiga toxin-producing Escherichia coli are a growing concern in the area of food safety, and the United States Department of Agriculture Food Safety and Inspection Service has identified the serotypes O26, O45, O103, O111, O121, O145, and O157 as adulterants in certain types of raw beef. The most relevant to human disease are the enterohemorrhagic E. coli (EHEC) strains that possess intimin (eae), Shiga toxin 1 and/or 2 (stx1-2), and in most cases the conserved pO157 or pO157 like virulence plasmid. Contamination of raw beef with EHEC is likely to occur via the transfer of cattle feces on hides to the carcass. To detect EHEC directly from cattle feces, we evaluated the utility of a multiplex real time PCR assay that targets the EHEC associated gene target ecf1 in combination with eae and stx1-2. Our assay had an increased sensitivity and provided a reliable limit of detection (LOD) of 1.25×10(3)colony-forming unitspermL (CFUs/mL) in an EHEC spiked fecal background. In addition, we evaluated the use of a duplex qPCR assay using ecf1 for the enumeration of total EHEC directly from cattle feces. The reliable limit of quantification (LOQ) was determined to be 1.25×10(3)CFUs/mL. Our assay requires minimal sample processing and provides LOD and LOQ of EHEC directly from cattle feces that are the lowest reported. The application of this assay towards the identification of cattle shedding EHEC at a level above 1.25×10(3)CFUs/mL could be a first line of defense in identifying cattle shedding these pathogens., (Published by Elsevier B.V.)

The U.S. EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 recreational water quality criteria (RWQC). The CCE quantification unit stems from the calibration model used to estimate enterococci densities in recreational beach waters in the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. Recent studies have demonstrated that CCE estimates from the method can vary when using different cultured Enterococcus cell preparations in calibrator samples. These differences have been attributed to differences in the quantities of targeted gene copies (target sequences) that are recovered per nominal calibrator cell by DNA extraction. Standardization of results from the calibration model will require the estimation of target sequence recoveries from the calibrator and water samples. In addition, comparisons of water sample results with the RWQC values will require a knowledge of target sequence recoveries from the NEEAR study calibrator samples. In this study recoveries of target sequences and the mean target sequence/cell ratio for the NEEAR study calibrator samples were retrospectively estimated with a corroborated standard curve. A modification of the calibration model was then used to estimate enterococci target sequence quantities in water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence quantities by the mean NEEAR study target sequence/cell ratio. This target sequence-based quantification approach resulted in a high degree of agreement in beach action decisions (determinations of whether bacterial fecal indicator densities are above or below RWQC-recommended values) from CCE results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples., (Published by Elsevier B.V.)

Ventilator-associated pneumonia (VAP) is a leading cause of health care-associated infection. It has a high rate of attributed mortality, and this mortality is increased in patients who do not receive appropriate empirical antimicrobial therapy. As a result of the overuse of broad-spectrum antimicrobials such as the carbapenems, strains of Acinetobacter, Enterobacteriaceae, and Pseudomonas aeruginosa susceptible only to polymyxins and tigecycline have emerged as important causes of VAP. The need to accurately diagnose VAP so that appropriate discontinuation or de-escalation of antimicrobial therapy can be initiated to reduce this antimicrobial pressure is essential. Practice guidelines for the diagnosis of VAP advocate the use of bronchoalveolar lavage (BAL) fluid obtained either bronchoscopically or by the use of a catheter passed through the endotracheal tube. The CDC recommends that quantitative cultures be performed on these specimens, using ≥ 10(4) CFU/ml to designate a positive culture (http://www.cdc.gov/nhsn/TOC&#95;PSCManual.html, accessed 30 October 2012). However, there is no consensus in the clinical microbiology community as to whether these specimens should be cultured quantitatively, using the aforementioned designated bacterial cell count to designate infection, or by a semiquantitative approach. We have asked Vickie Baselski, University of Tennessee Health Science Center, who was the lead author on one of the seminal papers on quantitative BAL fluid culture, to explain why she believes that quantitative BAL fluid cultures are the optimal strategy for VAP diagnosis. We have Stacey Klutts, University of Iowa, to advocate the semiquantitative approach.

The U.S. Environmental Protection Agency (EPA), in collaboration with an industry work group, spearheaded a collaborative study designed to further enhance the AOAC use-dilution method (UDM). Based on feedback from laboratories that routinely conduct the UDM, improvements to the test culture preparation steps were prioritized. A set of modifications, largely based on culturing the test microbes on agar as specified in the AOAC hard surface carrier test method, were evaluated in a five-laboratory trial. The modifications targeted the preparation of the Pseudomonas aeruginosa test culture due to the difficulty in separating the pellicle from the broth in the current UDM. The proposed modifications (i.e., the modified UDM) were compared to the current UDM methodology for P. aeruginosa and Staphylococcus aureus. Salmonella choleraesuis was not included in the study. The goal was to determine if the modifications reduced method variability. Three efficacy response variables were statistically analyzed: the number of positive carriers, the log reduction, and the pass/fail outcome. The scope of the collaborative study was limited to testing one liquid disinfectant (an EPA-registered quaternary ammonium product) at two levels of presumed product efficacies, high and low. Test conditions included use of 400 ppm hard water as the product diluent and a 5% organic soil load (horse serum) added to the inoculum. Unfortunately, the study failed to support the adoption of the major modification (use of an agar-based approach to grow the test cultures) based on an analysis of method's variability. The repeatability and reproducibility standard deviations for the modified method were equal to or greater than those for the current method across the various test variables. However, the authors propose retaining the frozen stock preparation step of the modified method, and based on the statistical equivalency of the control log densities, support its adoption as a procedural change to the current UDM. The current UDM displayed acceptable responsiveness to changes in product efficacy; acceptable repeatability across multiple tests in each laboratory for the control counts and log reductions; and acceptable reproducibility across multiple laboratories for the control log density values and log reductions. Although the data do not support the adoption of all modifications, the UDM collaborative study data are valuable for assessing sources of method variability and a reassessment of the performance standard for the UDM.

This study compared recovery of fecal coliform bacteria from sewage by Colilert-18 and Standard Methods 9222D (membrane-Fecal Coliform medium) in accordance with the U.S. Environmental Protection Agency (EPA) Alternative Test Protocol (ATP). Samples were collected from 10 different wastewater treatment plants in the northeastern United States and tested in a single laboratory. Twenty replicates of each sample were analyzed by each method, and 200 positive and 200 negative responses were confirmed for each method. Recovery of fecal coliforms by Colilert-18 was significantly higher than (8 of 10 sites) or statistically equivalent to (1 of 10 sites) recovery by the reference method (Standard Methods 9222D) for samples from all but one site. Both methods had low false-positive rates (< 2%); however, the false-negative rate observed with Standard Methods 9222D (21.5%) was substantially higher than that observed with Colilert-18 (7%). The accuracy rates of the two methods were calculated as 96.5 and 88.9% for Colilert-18 and Standard Methods 9222D, respectively. The results of this study demonstrate that Colilert-18 meets the acceptance criteria for alternative methods specified in the EPA ATP.

Three pathogens, Campylobacter, Salmonella, and Shiga-toxin-producing Escherichia coli, are leading causes of bacterial gastroenteritis in the United States and worldwide. Although these three bacteria are typically considered food-borne pathogens, outbreaks have been reported due to contaminated drinking water and irrigation water. The aim of this research was to develop two types of PCR assays that could detect and quantify three pathogens, Campylobacter spp., E. coli O157:H7, and Salmonella spp., in watershed samples. In conventional PCR, three target strains were detected by multiplex PCR (m-PCR) using each specific primer pair simultaneously. Under optimized m-PCR conditions, the assay produced a 90-bp product for Campylobacter jejuni, a 150-bp product for E. coli O157:H7, and a 262-bp product for Salmonella Typhimurium, and the limitation of detection was approximately 700 copies for all three bacteria. In addition, real-time PCR was performed to quantify the three pathogens using SYBR green fluorescence. The assay was designed so that each target had a different melting temperature [C. jejuni (80.1 °C), E. coli O157:H7 (83.3 °C), and S. Typhimurium (85.9 °C)]. Therefore, this system could quantify and distinguish three pathogens simultaneously in a single reaction., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)