Measurement uncertainty evaluation and risk of false conformity assessment for microbial enumeration tests.

Microbial enumeration tests are widely used to assess the microbiological quality of non-sterile pharmaceutical products. Despite of all efforts to guarantee the reliability of microbial enumeration tests, there will always be an uncertainty associated with the measured values, which can lead to false conformity/non-conformity decisions. In this work, we evaluated the measurement uncertainty using a bottom-up approach and estimate the consumer's or producer's risk due to the measurement uncertainty. Three main sources of uncertainty were identified and quantified: dilution factor, plated volume, and microbial plate counts. The contribution of these sources of uncertainty depends on the measured value of microbial load in pharmaceutical products. The contribution of dilution factor and plated volume uncertainties increase with an increase of measured value, while the contribution of microbial plate count uncertainty decreases with an increase of measured value. The overall uncertainty values were expressed as uncertainty factors, which provide an asymmetric 95% level confidence level of microbial load in pharmaceutical products. In addition, the risk of false conformity/non-conformity decisions due to measurement uncertainty was assess using Monte Carlo method. When the measured value is close to the upper specification limit and/or the measurement uncertainty is large, the risk of false conformity/non-conformity decisions may be significantly high. Thus, we conclude that the use of uncertainty factor in the conformity/non-conformity assessment is important to guarantee the reliability of microbial enumeration test results and to support decision-making.

Comparative study of Legiolert with ISO 11731-1998 standard method-conclusions from a Public Health Laboratory.

BACKGROUND: Legionella pneumophila (L. pneumophila) is responsible for 96% of Legionnaires' disease (LD) and 10% of all worldwide pneumonia cases. Legiolert™, a liquid culture method for most probable number (MPN) enumeration of L. pneumophila, was developed by IDEXX Laboratories. The method detects all serogroups of L. pneumophila in potable and non-potable water samples. OBJECTIVE: The goal of this study is to establish that Legiolert is a suitable alternative method to meet testing requirements in Spain for the enumeration of Legionella in water samples. METHODOLOGY: The laboratory analyzed 118 environmental water samples from the Barcelona region (56 potable and 62 non-potable) in parallel by the Standard method for detection and enumeration of Legionella (ISO 11731:1998) and by Legiolert. Comparison of the recovery of the alternative method (Legiolert) and the Standard was made using ISO 17994:2014 and McNemar's binomial test statistical methods. RESULTS: 44 samples were positive for Legionella (36 potable and 8 non-potable). Legiolert and the Standard method detected a similar percentage of positive samples, with Legiolert being slightly higher (31 vs 30%) and detecting higher concentrations of Legionella within the samples. ISO 17994:2014 analysis of the potable water samples found Legiolert was more sensitive than the Standard at detecting Legionella, even when complete Legionella species (L. spp.) results were considered for both methods. The two methods also demonstrated equivalent detection of L. spp. according to the McNemar's test. The comparison is significantly more in favor of Legiolert when only L. pneumophila results are considered. Each confirmation run with material extracted from positive Legiolert wells contained L. pneumophila, giving the method a specificity of 100%. Although statistical results for non-potable waters are not included because of the low number of samples, the two methods trended towards equivalence. CONCLUSIONS: Relative to the Standard method, Legiolert has a greater sensitivity and selectivity, and appears to have higher recovery for L. pneumophila, and equivalent recovery when L. spp. is included in the comparison. Legiolert also has high specificity. The procedural advantages of Legiolert allow laboratories to save on resources, costs, and time and consequently to test more frequently. In conclusion, the study finds IDEXX Legiolert a suitable alternative to ISO 11731:1998.

A fully automated deep learning pipeline for high-throughput colony segmentation and classification.

Adenine auxotrophy is a commonly used non-selective genetic marker in yeast research. It allows investigators to easily visualize and quantify various genetic and epigenetic events by simply reading out colony color. However, manual counting of large numbers of colonies is extremely time-consuming, difficult to reproduce and possibly inaccurate. Using cutting-edge neural networks, we have developed a fully automated pipeline for colony segmentation and classification, which speeds up white/red colony quantification 100-fold over manual counting by an experienced researcher. Our approach uses readily available training data and can be smoothly integrated into existing protocols, vastly speeding up screening assays and increasing the statistical power of experiments that employ adenine auxotrophy.

Antimicrobial Effectiveness in Eye Drops: Limited Sterility versus Reduction in Microbial Count.

Eye drops are sterile preparations intended for instillation into the eye. All major pharmacopoeias require these products to pass the antimicrobial effectiveness test (AET). This test is similar to that used for an oral dosage form despite the fact that both product categories differ in their microbiological specifications. The eye drops might pass the official requirements of the AET, but in practice, contaminants introduced into the preparation might not be killed before its next use by the patient and this may compromise ocular health. The objective of this work was to investigate the possible application of a limited sterility testing in a multichallenge test that mimics more closely real life use of eye drops. The AET was performed on 12 brands of eye drops, and results were compared with the suggested pass criteria of various pharmacopoeias. The multichallenge test was designed and used to demonstrate the ability of each tested product to kill the entire challenge organism population within a few hours. The results demonstrated that all products investigated complied with the AET acceptance requirements of the USP <51> and the "B" criteria of the European Pharmacopoeia (Ph Eur) <5.1.3>. Only two of the tested products did not comply with the no recovery term of Ph Eur <5.1.3> "A" criteria. Products repeatedly challenged with Pseudomonas aeruginosa ATCC 9027 (103 CFU/mL) were found to be self-sterilizing within 2 h of each inoculation. In conclusion, all tested products passed the acceptance criteria of the USP <51>, class B of the Ph Eur <5.1.3>, and the multichallenge test. The size of the challenge organism population in the AET seems to be severe for eye drops, and the pass criteria of the British Pharmacopoeia Appendix XVI are the most stringent. The no recovery term given in the Ph Eur <5.1.3> should be defined to a specified range.

Fate of generic and Shiga toxin-producing Escherichia coli (STEC) in Central Florida surface waters and evaluation of EPA Worst Case water as standard medium.

Agricultural water is considered as one of the main contamination source for produce prior to harvest. The purpose of study was to evaluate the fate of Shiga toxin-producing Escherichia coli (STEC), and generic E. coli in Central Florida agricultural surface water at different temperatures and the potential use of EPA Worst Case water as a standardized media. Cocktails of STEC (O145, O104, O111, O103, O157), and generic E. coli K-12 were inoculated into agricultural surface water samples (non-sterile and sterilized) and EPA Worst Case water, and enumerated for up to 168 days. E. coli was held at 15 and 25 ±â€¯1 °C. Tested microorganisms decreased most rapidly in non-sterile surface water. At day 168, E. coli populations decreased to ≤2.5 log CFU/100 ml in non-sterile surface water and were 4.8 ≤ and ≤ 8.5 log CFU/100 ml in sterile surface water and EPA Worst Case water. Populations were significantly (P ≤ .05) higher in sterile surface water and EPA Worst Case water at all sampling points starting from Day 28. Rate of declines in non-sterile surface waters were between 32.8 and 50 days at both tested temperatures and microorganisms. Addition of cycloheximide to non-sterile surface waters resulted in no significant effect on behavior of E. coli populations. Monitoring generic E. coli (represented by K-12) population changes is a reasonable indicator of STEC survival in agricultural water. EPA Worst Case water is a suitable standard control for surface water microcosms.

Performance of yeast-like cell counting (YLCC) using the Sysmex UF-1000i for clinical candiduria screening.

Candiduria is common in clinical practice. However, an effective and convenient assay to screen for candiduria is still needed. This study aimed to evaluate the performance of the Sysmex UF-1000i urine analyzer for yeast-like cell counting (YLCC) to screen for candiduria prior to urine culture. We retrospectively analyzed data from 5233 urine samples from 1813 patients, including 837 males and 976 females. Urine culture and urinalysis-obtained YLCC data were used to estimate the performance of YLCC in diagnosing candiduria. Different cutoff values were used to calculate sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The YLCC-positive rates differed according to the Candida colony-forming units (CFU) counts in the urine samples. A sharp drop in YLCC-positive rate (from 64.3 to 22.0%) was observed between the urine groups with 104 CFUs and 103 CFUs. A cutoff value of 0 YLCs/µL results in the highest Youden index (0.71) with 77.04% sensitivity and 93.68% specificity. In a group of 34 hospitalized candiduria patients with serial urinalysis data, 25 were YLCC-positive before urine culture. In conclusion, YLCC with the Sysmax UF-1000i could serve as an auxiliary technique to exclude culture-negative specimens prior to urine culture. Positive YLCC results could imply candiduria, especially when persistent YLCC-positive results were observed.

Fluorescent Capillary Electrophoresis Is Superior to Culture in Detecting Candida Species from Samples of Urinary Catheters and Ureteral Stents with Mono- or Polyfungal Biofilm Growth.

Molecular techniques in fungal detection and identification represent an efficient complementary diagnostic tool which is increasingly used to overcome limitations of routinely used culture techniques. The aim of this study was to characterize Candida sp. representation in samples from urine, urinary catheter, and ureteral stent biofilm using ITS2 ribosomal DNA (rDNA) amplification followed by fluorescent capillary electrophoresis (f-ITS2-PCR-CE) and to compare the results with those obtained by culture. A total of 419 samples were analyzed, and 106 (25.2%) were found positive, out of which 17 (16%) were polyfungal. The positivity rate did not differ between samples from catheters and stents (23.6% versus 20.9%) or between catheter and stent corresponding urine samples (40.2% versus 30.2%). Ten different Candida species were detected, with Candida parapsilosis (31.4%), Candida albicans (26.5%), and Candida tropicalis (12.4%) predominating. f-ITS2-PCR-CE was evaluated as substantially less time-consuming and 8.3 times more sensitive than the routinely applied culture technique with 1 µl of urine/sonicated fluid inoculated, detecting 67 (19.9%) versus 8 (2.4%) positive samples out of 337 initially analyzed samples. The culture sensitivity considerably improved to 1.7 times lower than that of f-ITS2-PCR-CE after the inoculation volume was increased to 100 µl in the additional 82 samples. Moreover, the molecular technique, unlike routine cultivation, enabled precise pathogen composition determination in polymicrobial samples. In conclusion, the f-ITS2-PCR-CE method was shown to be a quick and efficient tool for culture-independent detection and identification of fungi in urinary tract-related samples, demonstrating a higher sensitivity than culture.

Comparative assessment of qPCR enumeration methods that discriminate between live and dead Escherichia coli O157:H7 on beef.

Quantitative Polymerase Chain Reaction (qPCR) is a molecular method commonly used to detect and quantify bacterial DNA on food but is limited by its inability to distinguish between live and dead cell DNA. To overcome this obstacle, propidium monoazide (PMA) alone or with deoxycholate (DC) was used to prevent dead cell detection in qPCR. qPCR methods were used to detect strains of Escherichia coli O157, which can cause infection in humans with an infectious dose of less than 10 cells. A 5 strain E. coli O157:H7 cocktail was inoculated onto beef steaks and treated with interventions used in meat facilities (lactic acid (5%), peroxyacetic acid (200 ppm) or hot water (80 °C for 10 s)). Treatment of PMA or PMA + DC was applied to samples followed by DNA extraction and quantification in qPCR. RNA was also quantified in addition to conventional plating. For lactic acid intervention, qPCR DNA quantification of E. coli O157:H7 yielded 6.59 ±â€¯0.21 and 6.30 ±â€¯0.11 log gene copy #/cm2 for control and lactic acid samples, respectively and after treatment with PMA or PMA + DC this was further reduced to 6.31 ± 0.21 and 5.58 ± 0.38, respectively. This trend was also observed for peroxyacetic acid and hot water interventions. In comparison, RNA quantification yielded 7.65 ± 0.13 and 7.02 ± 0.38 log reverse transcript/cm2 for rRNA control and lactic acid samples, respectively, and for plating (LB), 7.51 ±â€¯0.06 and 6.86 ±â€¯0.32 log CFU/cm2, respectively. Our research determined that treatment of PMA + DC in conjunction with qPCR prevented dead cell DNA detection. However, it also killed cells injured from intervention that may have otherwise recovered. RNA quantification was more laborious and results had higher variability. Overall, quantification with conventional plating proved to be the most robust and reliable method for live EHEC detection on beef.

Bordetella holmesii Contamination of Platelet Concentrates: Revisiting the Definition of a Positive Culture.

Bacterial contamination remains the most important infectious risk of platelet transfusion. After an initially positive result, a second test is performed on the blood products and the initial culture bottle to confirm the contamination. Based on the blood center's decision algorithm used, results can be either confirmed negative, positive, or indeterminate, or be unconfirmed or discordant. Here, we report the first cases of platelet concentrates contaminated with Bordetella holmesii The in vitro growth characteristics of this unusual contaminant in platelet concentrate were investigated. Two B. holmesii strains isolated from platelet concentrates, as well as a control strain (Serratia marcescens), were spiked into platelet concentrates (PCs) at 1 and 10 CFU/ml. PCs were stored at 20 to 24°C under agitation. Samples were collected on days 2, 3, 4, and 7 for colony count and for bacterial screening using the BacT/Alert 3D system. Two PCs were detected as being positive for B. holmesii However, recultures were negative. In vitro, B. holmesii did not grow but remained detectable in PCs. Its viability diminished rapidly in contact with human plasma. Upon screening using the BacT/Alert 3D system, the majority of products spiked with B. holmesii were negative. This is the first description of PCs contaminated with B. holmesii This bacterium survives in blood products and remains dormant at low concentrations in blood products stored at room temperature, thus making difficult its detection with the BacT/Alert 3D system. The present definition of a true-positive culture of PCs may be overly restrictive for certain bacterial strains.

Laboratory diagnosis of urinary tract infections: Towards a BILULU consensus guideline.

Urinary tract infections (UTI) are very common throughout life and account for the majority of the workload in the clinical microbiology laboratory. Clear instructions for the interpretation of urine cultures by the laboratory technicians are indispensable to obtain standardized, reliable, and clinically useful results. In literature, there is often a lack of evidence-based practice in processing urinary samples in the laboratory. In this consensus document, the BILULU Study Group presents a practical approach for the implementation of existing guidelines for the culture of urine in the microbiology laboratory and offers answers for issues where no clear solution is available in the guidelines.

Microbiological Control for Affinity Capture Chromatography Processing: An Industry Perspective.

The purpose of this paper is to provide a summary of a BPOG-led industry survey of the microbiological control aspects of affinity chromatography processing in the biopharmaceutical industry. The document provides a summary of historical microbiological control concerns, coupled with industry-derived best practices, for material, equipment, and storage controls required to mitigate the potential for microbial ingress and contamination of chromatography resin and equipment. These best practice guidelines, which are derived from the members of the BPOG Bioburden Working Group, are intended to assist biopharmaceutical manufacturers to enhance microbial control and monitoring strategies for chromatography systems.

Method Verification Requirements for an Advanced Imaging System for Microbial Plate Count Enumeration.

The Growth Direct™ System that automates the incubation and reading of membrane filtration microbial counts on soybean-casein digest, Sabouraud dextrose, and R2A agar differs only from the traditional method in that micro-colonies on the membrane are counted using an advanced imaging system up to 50% earlier in the incubation. Based on the recommendations in USP <1223> Validation of New Microbiological Testing Methods, the system may be implemented in a microbiology laboratory after simple method verification and not a full method validation.LAY ABSTRACT: The Growth Direct™ System that automates the incubation and reading of microbial counts on membranes on solid agar differs only from the traditional method in that micro-colonies on the membrane are counted using an advanced imaging system up to 50% earlier in the incubation time. Based on the recommendations in USP <1223> Validation of New Microbiological Testing Methods, the system may be implemented in a microbiology laboratory after simple method verification and not a full method validation.

What Urinary Colony Count Indicates a Urinary Tract Infection in Children?

Post-hoc analysis of the Randomized Intervention for Children with Vesicoureteral Reflux study suggests that, in concordance with European guidelines, using bacteriologic criterion of ≥10 000 colony forming units/mL of a single organism does not decrease diagnostic specificity of an urinary tract infection in children aged 2 months to 6 years in a properly collected urine if symptoms/fever and pyuria are present. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00405704.

Diagnóstico microbiológico de mastitis: ¿qué sabemos sobre recuentos y microorganismos significativos? / Diagnostic microbiology in mastitis. What do we know about counts and significant microorganisms?

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Quantification of colony-forming units for M. tuberculosis complex using gyrB-based real-time PCR assay.

BACKGROUND: It is inappropriate to select insertion sequence (IS) 6110 as the amplification target for the quantitative analysis of Mycobacterium tuberculosis complex (MTC). OBJECTIVE: To develop a novel Taqman real-time polymerase chain reaction (RT-PCR) for the quantitative analysis of MTC by amplifying a single-copy PCR target. DESIGN: Analytic sensitivity and specificity, repeatability and reproducibility, and standard curve were estimated by analysing 18 reference strains and 100 clinical isolates. Diagnostic sensitivity and specificity, positive predictive value (PPV) and negative predictive value (NPV) were evaluated by detecting 50 clinical specimens. Quantitative accuracies of commercial and in-house Taqman RT-PCR were also compared. RESULTS: Analytic sensitivity of this method was 30 colony-forming units/ml and analytic specificity was 100%. In comparison with commercial Taqman RT-PCR (reference), diagnostic sensitivity, diagnostic specificity, PPV and NPV of the novel Taqman RT-PCR were respectively 85.7%, 94.4%, 85.7% and 94.4%. There was no significant difference between the two methods (χ(2) = 0.25, P > 0.05) and there was strong agreement between them (κ = 0.802, P < 0.05). In-house Taqman RT-PCR was more accurate than commercial assay. CONCLUSIONS: The novel RT-PCR is sensitive and specific for the detection of MTC and it is also accurate for quantitative analysis of MTC.

The Application of Noninvasive Headspace Analysis to Media Fill Inspection.

The results of a proof-of-principle study demonstrating a new analytical technique for detecting microbial growth directly in pharmaceutical containers are described. This analytical technique, laser-based headspace analysis, uses tunable diode laser absorption spectroscopy to nondestructively determine gas concentrations in the headspace of a media-filled pharmaceutical container. For detecting microbial growth, the levels of headspace oxygen and carbon dioxide are measured. Once aerobic microorganisms begin to divide after the lag phase and enter the exponential growth phase, there will be significant consumption of oxygen and concomitant production of carbon dioxide in the sealed container. Laser-based headspace analysis can accurately measure these changes in the headspace gas composition. The carbon dioxide and oxygen measurement data for the representative microorganisms Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Aspergillus brasiliensis were modeled using the Baranyi-Roberts equation. The mathematical modeling allowed quantitative comparisons to be made between the data from the different microorganisms as well as to the known growth curves based on microbial count. Because laser-based headspace analysis is noninvasive and can be automated to analyze the headspace of pharmaceutical containers at inspection speeds of several hundred containers per minute on-line, some potential new applications are enabled. These include replacing the current manual human visual inspection with an automated analytical inspection machine to determine microbial contamination of media fill and pharmaceutical drug product vials. LAY ABSTRACT: A novel analytical technique has been demonstrated for detecting microbial growth in media-filled pharmaceutical containers. This analytical technique, laser-based headspace analysis, uses tunable diode laser absorption spectroscopy to determine gas concentrations in the headspace of a pharmaceutical container. For detecting microbial growth, the levels of headspace oxygen and carbon dioxide are measured. The study shows that once aerobic microorganisms begin to grow after the lag phase and enter the exponential growth phase there will be a significant consumption of oxygen in the sealed container as well as a corresponding production of carbon dioxide. Headspace analysis can accurately measure and monitor these changes in the headspace gas composition and could therefore be used to detect contaminated pharmaceutical containers. Because the technique can be automated to analyze hundreds of containers a minute on-line, there are opportunities for implementing a headspace inspection machine to perform automated inspection of media fills used to validate aseptic filling operations.

Preprostate Biopsy Rectal Culture and Postbiopsy Sepsis.

Transrectal ultrasound-guided biopsy of the prostate (TRUSP) remains the primary procedure for the accurate histologic diagnosis of prostate cancer. Fluoroquinolones (FQs) are still recommended as the agents of choice for antimicrobial prophylaxis for TRUSP despite the alarming increasing incidence of FQ-resistant organisms among men undergoing TRUSP. This article reviews the current TRUSP antimicrobial prophylaxis guidelines, antimicrobial resistance and its implications for these guidelines, the incidence of post-TRUSP infectious complications including urosepsis, the seminal data supporting pre-TRUSP rectal swab (RS), RS technique and protocol, and the current available literature surrounding the efficacy of RS in reducing post-TRUSP infectious complications.

Standardization of Operator-Dependent Variables Affecting Precision and Accuracy of the Disk Diffusion Method for Antibiotic Susceptibility Testing.

Parameters like zone reading, inoculum density, and plate streaking influence the precision and accuracy of disk diffusion antibiotic susceptibility testing (AST). While improved reading precision has been demonstrated using automated imaging systems, standardization of the inoculum and of plate streaking have not been systematically investigated yet. This study analyzed whether photometrically controlled inoculum preparation and/or automated inoculation could further improve the standardization of disk diffusion. Suspensions of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 of 0.5 McFarland standard were prepared by 10 operators using both visual comparison to turbidity standards and a Densichek photometer (bioMérieux), and the resulting CFU counts were determined. Furthermore, eight experienced operators each inoculated 10 Mueller-Hinton agar plates using a single 0.5 McFarland standard bacterial suspension of E. coli ATCC 25922 using regular cotton swabs, dry flocked swabs (Copan, Brescia, Italy), or an automated streaking device (BD-Kiestra, Drachten, Netherlands). The mean CFU counts obtained from 0.5 McFarland standard E. coli ATCC 25922 suspensions were significantly different for suspensions prepared by eye and by Densichek (P < 0.001). Preparation by eye resulted in counts that were closer to the CLSI/EUCAST target of 10(8) CFU/ml than those resulting from Densichek preparation. No significant differences in the standard deviations of the CFU counts were observed. The interoperator differences in standard deviations when dry flocked swabs were used decreased significantly compared to the differences when regular cotton swabs were used, whereas the mean of the standard deviations of all operators together was not significantly altered. In contrast, automated streaking significantly reduced both interoperator differences, i.e., the individual standard deviations, compared to the standard deviations for the manual method, and the mean of the standard deviations of all operators together, i.e., total methodological variation.

[Analysis on 2011 quality control results on aerobic plate count of microbiology laboratories in China].

OBJECTIVE: To test the aerobic plate count examining capability of microbiology laboratories, to ensure the accuracy and comparability of quantitative bacteria examination results, and to improve the quality of monitoring. METHODS: The 4 different concentration aerobic plate count piece samples were prepared and noted as I, II, III and IV. After homogeneity and stability tests, the samples were delivered to monitoring institutions. The results of I, II, III samples were logarithmic transformed, and evaluated with Z-score method using the robust average and standard deviation. The results of IV samples were evaluated as "satisfactory" when reported as < 10 CFU/piece or as "not satisfactory" otherwise. Pearson χ2 test was used to analyze the ratio results. RESULTS: 309 monitoring institutions, which was 99.04% of the total number, reported their results. 271 institutions reported a satisfactory result, and the satisfactory rate was 87.70%. There was no statistical difference in satisfactory rates of I, II and III samples which were 81.52%, 88.30% and 91.40% respectively. The satisfactory rate of IV samples was 93.33%. There was no statistical difference in satisfactory rates between provincial and municipal CDC. CONCLUSION: The quality control program has provided scientific data that the aerobic plate count capability of the laboratories meets the requirements of monitoring tasks.

Analysis of older adults' domestic kitchen storage practices in the United Kingdom: identification of risk factors associated with listeriosis.

Increased listeriosis incidence among older adults (≥ 60 years) has been reported internationally, with many cases reported to be sporadic and associated with ready-to-eat (RTE) food products with extended refrigerated shelf life. Given that the home kitchen is recognized as a significant location where foodborne illnesses are acquired, it is important that consumers implement safe food practices to minimize risks. This is crucial for vulnerable consumers, such as older adults. Consumer food safety recommendations in the United Kingdom to reduce the risk of listeriosis at home include (i) following "use-by" dates on unopened prepacked RTE food products, (ii) consuming RTE food products within 2 days of opening, and (iii) ensuring the safe operating temperatures of domestic refrigerators (≤ 5 °C). This study utilized observation, self-reporting, and microbiological analysis to determine actual food storage practices to identify behavioral risk factors. A domestic kitchen survey was conducted in older adult (≥ 60 years) consumers' domestic kitchens (n = 100) in South Wales, United Kingdom. Forty-one percent of foods in home refrigerators were beyond the use-by date, of which 11% were unopened RTE food products commonly associated with listeriosis. Sixty-six percent of opened RTE foods had been or were intended to be stored beyond the recommended 2 days after opening. Older adults failed to ensure safe refrigeration temperatures, with 50% of central storage and 85% of door storage areas operating at temperatures >5 °C. Older refrigerators operated at significantly (P < 0.05) higher temperatures. Given that Listeria monocytogenes was isolated in 2% of kitchens, these findings suggest that storage malpractices may have a greater effect on the potential risk of listeriosis than its presence alone. The study has determined that many older adults fail to adhere to recommendations and subject RTE foods associated with L. monocytogenes to prolonged storage at unsafe temperatures which may render food unsafe for consumption.