An optimized algorithm with improved turnaround time for detection of carbapenemase-producing Enterobacterales using the NG Test CARBA 5 in a routine laboratory.

Introduction. Rapid and reliable detection of carbapenemase-producing Enterobacterales (CPE) from surveillance cultures is critical in supporting a good infection control programme. We implemented a new algorithm for CPE detection incorporating the NG Test CARBA 5 in January 2019.Aim. Our goals were to compare turnaround time (TAT), costs and staff requirements between the old and new algorithm, and to evaluate the performance of the CARBA 5 test directly on colonies grown on CARBA Smart agar.Methodology. We analysed and compared the TAT of CPE surveillance cultures processed using the old and new CPE screening algorithm. The total actual reagent costs and staff requirements for the new CPE algorithm were compared with the estimated costs and staff requirements of the old CPE algorithm.Results. Of 197 isolates included in the evaluation of the new algorithm, 64 were positive for carbapenemases by both CARBA 5 and Xpert Carba-R assay. Of the 133 that were negative, two were found to harbour NDM and IMI genotypes. Significant improvements in TAT were achieved with 88.7 % of cultures with CPE, reported on the same day as growth was observed on CARBA Smart agar compared to none in the old algorithm. The new algorithm incurred lower costs and, based on our workload, the new algorithm is estimated to save 28.9 man-hours annually.Conclusion. CARBA 5 performs well on colonies growing on CARBA Smart agar and significant improvements in TAT can be achieved without incurring additional costs or staff requirements.

Liquid based microbiological transport systems: conformity assessment of two commercial devices.

We compared two types of liquid-based microbiology devices for microorganism viability according to standardized quantitative elution method CLSI M40-A2. The eSwab® met CLSI acceptance criteria of viability maintenance for all microorganisms tested. The Σ-Transwab® failed to meet CLSI acceptance criteria for Peptostreptococcus anaerobius, Prevotella melaninogenica, Fusobacterium nucleatum and Haemophilus influenzae.

Cost-benefit analysis from the hospital perspective of universal active screening followed by contact precautions for methicillin-resistant Staphylococcus aureus carriers.

OBJECTIVE To explore the economic impact to a hospital of universal methicillin-resistant Staphylococcus aureus (MRSA) screening. METHODS We used a decision tree model to estimate the direct economic impact to an individual hospital of starting universal MRSA screening and contact precautions. Projected costs and benefits were based on literature-derived data. Our model examined outcomes of several strategies including non-nares MRSA screening and comparison of culture versus polymerase chain reaction-based screening. RESULTS Under baseline conditions, the costs of universal MRSA screening and contact precautions outweighed the projected benefits generated by preventing MRSA-related infections, resulting in economic costs of $104,000 per 10,000 admissions (95% CI, $83,000-$126,000). Cost-savings occurred only when the model used estimates at the extremes of our key parameters. Non-nares screening and polymerase chain reaction-based testing, both of which identified more MRSA-colonized persons, resulted in more MRSA infections averted but increased economic costs of the screening program. CONCLUSIONS We found that universal MRSA screening, although providing potential benefit in preventing MRSA infection, is relatively costly and may be economically burdensome for a hospital. Policy makers should consider the economic burden of MRSA screening and contact precautions in relation to other interventions when choosing programs to improve patient safety and outcomes.

Highly selective colorimetric bacteria sensing based on protein-capped nanoparticles.

A rapid and cost-effective colorimetric sensor has been developed for the detection of bacteria (Bacillus subtilis was selected as an example). The sensor was designed to rely on lysozyme-capped AuNPs with the advantages of effective amplification and high specificity. In the sensing system, lysozyme was able to bind strongly to Bacillus subtilis, which effectively induced a color change of the solution from light purple to purplish red. The lowest concentration of Bacillus subtilis detectable by the naked eye was 4.5 × 10(3) colony-forming units (CFU) mL(-1). Similar results were discernable from UV-Vis absorption measurements. A good specificity was observed through a statistical analysis method using the SPSS software (version 17.0). This simple colorimetric sensor may therefore be a rapid and specific method for a bacterial detection assay in complex samples.

A cost-effective solution for the reliable determination of cell numbers of microorganisms in liquid culture.

The concentration of microorganisms in growth medium is an important parameter in microbiological research. One of the approaches to determine this parameter is based on the physical interaction of small particles with light that results in light scattering. Table-top spectrophotometers can be used to determine the scattering properties of a sample as a change in light transmission. However, a portable, reliable, and maintenance-free instrument that can be built from inexpensive parts could provide new research opportunities. In this report, we show how to build such an instrument. This instrument consists of a low power monochromatic light-emitting diode, a monolithic photodiode, and a microcontroller. We demonstrate that this instrument facilitates the precise determination of cell concentrations for the bacteria Escherichia coli and Pseudomonas aeruginosa as well as the cyanobacterium Synechocystis sp. PCC 6803 and the green alga Chlamydomonas reinhardtii.

Vascular catheter tip cultures for suspected catheter-related blood stream infection in the intensive care unit: a tradition whose time has passed?

BACKGROUND: Catheter-related blood stream infections (CR-BSIs) are estimated to occur in 80,000 patients in intensive care units (ICUs) each year in the United States. We sought to determine the clinical utility of vascular catheter cultures in critically ill patients with suspected CR-BSI. METHODS: We reviewed retrospectively all positive (≥15 colony forming units/roll) vascular catheter tip cultures (CTCs) documented over a four-year period in the ICUs of two hospitals. A CR-BSI was defined as matching positive blood and catheter cultures. The time interval between catheter removal and blood culture was recorded. RESULTS: A total of 1,391 CTCs were obtained, of which 468 (34%) were positive and 143 (31% of the positive cultures) were associated with a diagnosis of CR-BSI. In 133 of these 143 cases (93%), the positive blood culture was obtained before or within 24 h after catheter removal and dictated antibiotic therapy. In only 10 of 143 cases (7%) did catheter removal and culture significantly (>1 day) precede the positive blood culture. In 55% of the CR-BSI cases, the catheter was removed empirically and close to the time of blood culture (-1.3±19.0 h). In the remaining 45%, the catheter was removed clinically (after a blood culture was positive), and this action was more remote in time (23.6±19.4 h; p<0.001 vs. empiric removal). Total microbiology laboratory costs for the CTCs were $75,300, and 600 microbiology technician hours were required. CONCLUSION: In an ICU patient population, only about one-third of vascular catheter cultures were positive, and only about one-third of the positive CTCs were associated with CR-BSI. Ninety-three percent of all CR-BSIs were identified by bacteremia either before or coinciding with catheter removal, and the results of the blood culture dictated antimicrobial therapy. Because CTCs rarely changed therapy, they may not be appropriate in the management of suspected CR-BSI in the ICU setting.

Profiling of recovery efficiencies for three standard protocols (FDA-BAM, ISO-11290, and modified USDA) on temperature-injured Listeria monocytogenes.

There have been a number of studies conducted in order to compare the efficiencies of recovery rates, utilizing different protocols, for the isolation of L. monocytogenes. However, the severity of multiple cell injury has not been included in these studies. In the current study, L. monocytogenes ATCC 19112 was injured by exposure to extreme temperatures (60°C and -20°C) for a one-step injury, and for a two-step injury the cells were transferred directly from a heat treatment to frozen state to induce a severe cell injury (up to 100% injury). The injured cells were then subjected to the US Food and Drug Administration (FDA), the ISO-11290, and the modified United States Department of Agriculture (mUSDA) protocols, and plated on TSAyeast (0.6% yeast), PALCAM agar, and CHROMAgar Listeria for 24 h or 48 h. The evaluation of the total recovery of injured cells was also calculated based on the costs involved in the preparation of media for each protocol. Results indicate that the mUSDA method is best able to aid the recovery of heat-injured, freeze-injured, and heat-freeze-injured cells and was shown to be the most cost effective for heat-freeze-injured cells.

A new cost-effective, selective and differential medium for the isolation of Cronobacter spp.

The objective of the present study was to develop a new selective, differential and cost-effective medium (Kim and Rhee - KR-medium) for the isolation of Cronobacter spp. In this new medium, which contained salicin as a differential agent, Cronobacter spp. generated typical colonies with characteristic violet-colored centers surrounded by a transparent to opalescent border, and the growth of other microorganisms (40 strains) was inhibited or produced visually distinguishable colonies. Using healthy and heat- and desiccation-injured cells, the quantity of nutrients was adjusted to determine the optimal recovery rate, selectivity, differentiation and cost-effectiveness. Peptone and salicin concentrations were established as 10 and 8 g/L, respectively. The KR medium was then validated using salicin fermenting organisms, including Cronobacter spp. (52 strains), Enterobacter cloacae (50 strains) and Klebsiella pneumonia (10 strains) isolated from clinical and food specimens. All strains of Cronobacter spp. produced typical colonies and other salicin fermenting organisms were easily distinguishable from Cronobacter spp. with the exception of 2 E. cloacae strains. The verification of KR medium was carried out in powdered infant formula artificially inoculated with healthy, heat-injured, and desiccation-injured Cronobacter spp. and the expected typical colonies were appeared. The KR medium had a high specificity (98%) and sensitivity (100%), with no false-negative results. Moreover, we show that the cost of the KR medium is much lower than that of other selective and differential media. The use of the KR medium for the selective isolation of Cronobacter spp. in laboratories and food industry settings may therefore lessen the financial burden of Cronobacter spp. detection.

The value of multiple surveillance cultures for methicillin-resistant Staphylococcus aureus.

BACKGROUND: We evaluated our experience in a low prevalence setting to determine the extent to which multiple swabs increased detection rates and the incremental costs of doing so. METHODS: Nasal and groin swabs submitted in pairs were cultured onto a single plate (Oxoid MRSA Denim Blue Agar; Oxoid Company, Napean, ON, Canada). We determined whether MRSA was detected when swabs submitted in the preceding 3 days were negative. We explored the costs associated with screening and of each additional colonized patient detected. RESULTS: In all, 60,049 paired nose and perineal swabs were submitted from 21,599 patients. In all, there were 12,750 duplicate, 1437 triplicate, and 112 instances when >4 swabs were processed within 3 days. The first culture was positive in 106 of 12,750 (0.83%%), 42 of 12,750 (0.33%) on the second when the first was negative, 7 of 1642 (0.43%) on the third or subsequent swab pair when the preceding 2 were negative. CONCLUSION: Overall, the sensitivity of the first of multiple cultures of a set was 74.3%. Had the 14,392 multiple samples not been submitted, 49 colonized patients would not have been identified. Additional laboratory costs associated with multiple samples equaled $2088 per patient identified.

Low-cost, high-throughput, automated counting of bacterial colonies.

Research involving bacterial pathogens often requires enumeration of bacteria colonies. Here, we present a low-cost, high-throughput colony counting system consisting of colony counting software and a consumer-grade digital camera or document scanner. We demonstrate that this software, called "NICE" (NIST's Integrated Colony Enumerator), can count bacterial colonies as part of a high-throughput multiplexed opsonophagocytic killing assay used to characterize pneumococcal vaccine efficacy. The results obtained with NICE correlate well with the results obtained from manual counting, with a mean difference of less than 3%. NICE is also rapid; it can count colonies from multiple reaction wells within minutes and export the results to a spreadsheet for data processing. As this program is freely available from NIST, NICE should be helpful in bacteria colony enumeration required in many microbiological studies, and in standardizing colony counting methods.

Evaluation of Petrifilm EC method for enumeration of E. coli from soil.

AIMS: To evaluate the suitability of commercially available Petrifilm EC plates for enumeration of Escherichia coli from soil. METHODS AND RESULTS: A confirmed E. coli strain isolated from liquid swine manure was inoculated into sterilized sandy clay loam and loam soils at the concentrations of 10(2), 10(3), 10(5) CFU g(-1) of soil. The efficiency of recovery on Petrifilm EC plates for soils spiked with E. coli was compared with standard membrane filtration techniques on m-FC basal medium supplemented with 3-bromo-4-chloro-5-indoyl-beta-D-glucopyranoside (BCIG) and most probable numbers (MPN) techniques in E. coli medium with 4-methylumbelliferyl-beta-D-glucuronide (EC-MUG) broth. Petrifilm EC and m-FC (BCIG) methods were then assessed for the ability to recover E. coli from field soils applied with swine manure. No significant differences (P > 0.05) were observed between Petrifilm EC, m-FC (BCIG) and MPN methods for the recovery of E. coli from spiked samples, irrespective of soil type. However, recovery of E. coli from manure-applied field soil samples showed a significant difference (P < 0.05) between the Petrifilm EC method and the m-FC method in enumerating E. coli possibly as a result of false positives on m-FC. CONCLUSION: The Petrifilm EC method is suitable for the enumeration of E. coli from soil with a detection limit of 10 CFU g(-1) soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The commercially available Petrifilm EC method is comparatively low cost, easy to use method for the enumeration of E. coli from soil without the need for further confirmation tests.

Novel flow cytometry-based screening for bacterial contamination of donor platelet preparations compared with other rapid screening methods.

BACKGROUND: Bacterial contamination is the major infectious hazard associated with transfusion of platelet preparations (PLTs). Routine testing for bacterial contamination in PLTs has become common, but transfusion-transmitted bacterial sepsis has not been eliminated. Here, we describe a novel flow cytometry-based method for point-of-issue screening of PLTs for bacterial contamination. METHODS: We used the BactiFlow flow cytometer to detect and count bacteria based on esterase activity in viable cells. We compared the assay to incubation (BacT/Alert culture system) and rapid nucleic acid-based or immunoassay (reverse transcription PCR, Pan Genera Detection) methods. RESULTS: We established a protocol for bacterial screening of PLTs consisting of enzymatic digestion and centrifugal filtration for the elimination of viable platelets and selective labeling of bacteria with fluorescent esterase substrate (ChemChrome V23). Results from the BactiFlow showed an excellent correlation (r = 0.9923 E. coli, r = 0.9736 S. epidermidis) to traditional plate count results. The lower detection limit of the assay was determined to be 150 CFU/mL, and the time to result was <1 h. CONCLUSIONS: Our study demonstrates that BactiFlow flow cytometry is suitable for rapid screening of PLTs for bacterial contamination and fulfils the requirements for a point-of-issue testing of PLTs with acceptable time to result, specificity, sensitivity, and cost.

DNA extraction and Escherichia coli quantification of anaerobically digested biosolids using the competitive touchdown PCR method.

Accurate enumeration of indicator organisms such as Escherichia coli is important for assessing the safety of water and wastewater samples. Recent research has shown that E. coli can enter a viable but non-culturable state; therefore, traditional cultivation methods could potentially underestimate the quantities of the organisms. The goals of the research were to develop and verify a DNA extraction protocol and a quantitative polymerase chained reaction (PCR) method for E. coli enumeration in digested biosolids. A solvent-based DNA extraction protocol with extensive cell lysis recovered approximately 78-84% of spiked DNA. In comparison, a commercial kit only recovered 28-42% of DNA, likely from inefficient cell lysis. The developed competitive touchdown PCR (cPCR) method for E. coli enumeration was comparable to both real-time PCR (rt-PCR) and cultivation methods with sensitivity of approximately 50,000-500,000 E. coli per gram dry solids (DS), which is suitable for Class B biosolids monitoring in the US and "conventional" biosolids in the European Union. The cPCR protocol provides a less expensive alternative than the rt-PCR as a culturing independent method for enumerating E. coli.

Economic impact of Candida colonization and Candida infection in the critically ill patient.

The objective of the study presented here was to assess the economic impact of Candida colonization and Candida infection in critically ill patients admitted to intensive care units (ICUs). For this purpose, a prospective, cohort, observational, and multicenter study was designed. A total of 1,765 patients over the age of 18 years who were admitted for at least 7 days to 73 medical-surgical ICUs in 70 Spanish hospitals between May 1998 and January 1999 were studied. From day 7 of ICU admission to ICU discharge, samples of tracheal aspirates, pharyngeal exudates, gastric aspirates and urine were collected every week for culture. Prolonged length of stay was associated with severity of illness, Candida colonization or infection, infection by other fungi, antifungal therapy, treatment with more than one antifungal agent, and toxicity associated with this therapy. Compared to non-colonized, non-infected patients (n=720), patients with Candida colonization (n=880) had an extended ICU stay of 6.2 days (OR, 1.69; 95%CI, 1.53-1.87; P<0.001) and an extended hospital stay of 8.6 days (OR, 1.27; 95%CI, 1.16-1.40; P<0.001). The corresponding figures for patients with Candida infection (n=105) were 12.7 days for ICU stay (OR, 2.13; 95%CI, 1.72-2.64; P<0.001) and 15.5 days for hospital stay (OR, 1.23; 95%CI, 0.99-1.52; P=0.060). Candida colonization resulted in an additional 8,000 EUR in direct costs and Candida infection almost 16,000 EUR. Both Candida colonization and Candida infection had an important economic impact in terms of cost increases due to longer stays in both the ICU and in the hospital.

Feasibility of introducing rejection criteria for stool cultures in a teaching hospital in Portugal.

The possible introduction of rejection criteria for stool cultures (

An alternative approach for enumeration of Escherichia coli in foods.

An assay to screen Escherichia coli in foods using MUG supplemented lauryl sulfate tryptose (LST) broth instead of tryptone water (TW) medium was evaluated. The results presented in this paper suggest that this method is more sensitive for lower levels of E. coli, faster (16-18 h vs. 6-10 days) and less expensive (2.454 vs. 2.887 EURO/sample) than the standard ISO procedure. Thus, this method may be beneficial for use when both fecal coliforms and E. coli analyses are required in food systems.

[Use of the colonization index]. / Utilisation pratique de l'index de colonisation.

In a retrospective study, 2,238 mycologic samples obtained in 1999 from 89 patients hospitalised in an intensive care unit dedicated to digestive diseases were analysed. Feasibility of monitoring fungal colonisation and implications for workload and costs were assessed. From this experience, we confirmed the ability of the Pittet index to identify patients at high risk for Candida infection. Monitoring of Pittet index required a high degree of cooperation between the intensive care unit and the laboratory of mycology, and a precise definition of the modalities of sampling. It entailed a significant increase in costs and workload. A treatment was started whenever a colonisation index > or = 0.5 was associated with severe clinical or biological signs. This involved an increase of the expense of antifungal drugs. The potential benefits could not be evaluated from our study. Direct observation of pseudomycelium in the samples and candiduria were significantly correlated to fungal colonisation.

Usefulness of bacteriological surveillance cultures for monitoring infection in hospitalized patients: a critical reappraisal.

Untargeted bacteriological surveillance of superficial and deep body sites is frequently performed routinely in various clinical settings. This practice is based on the assumption that early identification of surface microbial flora might be predictive of organisms that will later cause invasive disease and that it may consequently assist in guiding empirical antibiotic therapy. A comprehensive review of the literature however indicates that the clinical value and cost-effectiveness of such practices still remain debated and appear largely unproven in most conditions and situations where they are routinely advocated. The present article reviews and critically discusses the available body of evidence supporting or disproving the use of bacteriological surveillance cultures. It is also aimed to issue general recommendations, strategies and methodologies that could be applied in different hospital care settings including the neonatal or adult intensive care as well as the hematology-oncology units.

How to optimize the drop plate method for enumerating bacteria.

The drop plate (DP) method can be used to determine the number of viable suspended bacteria in a known beaker volume. The drop plate method has some advantages over the spread plate (SP) method. Less time and effort are required to dispense the drops onto an agar plate than to spread an equivalent total sample volume into the agar. By distributing the sample in drops, colony counting can be done faster and perhaps more accurately. Even though it has been present in the laboratory for many years, the drop plate method has not been standardized. Some technicians use 10-fold dilutions, others use twofold. Some technicians plate a total volume of 0.1 ml, others plate 0.2 ml. The optimal combination of such factors would be useful to know when performing the drop plate method. This investigation was conducted to determine (i) the standard deviation of the bacterial density estimate, (ii) the cost of performing the drop plate procedure, (iii) the optimal drop plate design, and (iv) the advantages of the drop plate method in comparison to the standard spread plate method. The optimal design is the combination of factor settings that achieves the smallest standard deviation for a fixed cost. Computer simulation techniques and regression analysis were used to express the standard deviation as a function of the beaker volume, dilution factor, and volume plated. The standard deviation expression is also applicable to the spread plate method.

Bacteriology of burn wounds in Enugu, Nigeria.

A retrospective study of bacterial infection in 71 burned patients over a 5-year period (1993-1997) was carried out. The commonest colonizing organism was Klebsiella species (26.7%) followed by Staph aureus (25.6%). There was a very high degree of resistance by these organisms to commonly available antibiotics in Nigeria, with the result that more expensive antibiotics such as the cephalosporins were required. The poor socioeconomic condition of most of the patients was a very important pre-disposing factor to burn wound infection, as only 25% of patients were able to afford the cost of wound microscopy and culture, thus leading to limited numbers of cultures being performed, the result being their prescription of antibiotics was made generally on an empirical basis. Restriction in the misuse of antibiotics and establishment of an infection control until will help to lower the incidence of infection.