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 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.

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.

Application of microbiological quantitative methods for evaluation of changes in the amount of bacteria in patients with wounds and purulent fistulas subjected to phage therapy and for assessment of phage preparation effectiveness (in vitro studies).

PURPOSE: Quantitative microbiological studies may provide important information required for successful phage therapy (PT), however methods for PT monitoring of purulent wounds and fistulas has never been reported before. Therefore our goal was to determine and apply microbiological quantitative methods (MQMs) for monitoring experimental PT. METHODS: Samples from agar plates with growing bacteria were collected using dry and wet sterile compresses, or swabs. After shaking the sample in saline the amount of bacteria in suspension was determined. The method was standardized. The MQM using compress was applied for comparison of in vitro activity of phage preparations with other agents for wound rinsing. The usefulness of this swabbing method was tested in the Phage Therapy Unit for monitoring of experimental PT of patients with chronic wounds or purulent fistulas. RESULTS: Minimum, maximum and standard deviation values used for standardization of the studied method showed that data repeatability was good; thus the method was used for quantitation of bacteria taken both from plates in vitro and patients samples. Effectiveness of phage preparations was compared to gentamicin in vitro. Phages were as effective as antibiotics in reducing the amount of bacteria on agar plates, and this effect was not only due to simple mechanical removal of bacteria, but dependent on their antibacterial activity. We have also observed that the results of bacteria quantitation may correlate with the local status of a wound/fistula in a particular stage of PT. CONCLUSION: The standardized swabbing method of bacteria quantitation can be used for PT monitoring. Presented MQMs are simple and may help to monitor the therapy process and to decide on its duration, frequency and a kind of the phage applied. They can also be applied in other antibacterial treatment strategies.

Microbial contamination of human milk purchased via the Internet.

OBJECTIVE: To quantify microbial contamination of human milk purchased via the Internet as an indicator of disease risk to recipient infants. METHODS: Cross-sectional sample of human milk purchased via a popular US milk-sharing Web site (2012). Individuals advertising milk were contacted to arrange purchase, and milk was shipped to a rented mailbox in Ohio. The Internet milk samples (n = 101) were compared with unpasteurized samples of milk donated to a milk bank (n = 20). RESULTS: Most (74%) Internet milk samples were colonized with Gram-negative bacteria or had >10(4) colony-forming units/mL total aerobic count. They exhibited higher mean total aerobic, total Gram-negative, coliform, and Staphylococcus sp counts than milk bank samples. Growth of most species was positively associated with days in transit (total aerobic count [log10 colony-forming units/mL] ß = 0.71 [95% confidence interval: 0.38-1.05]), and negatively associated with number of months since the milk was expressed (ß = -0.36 [95% confidence interval: -0.55 to -0.16]), per simple linear regression. No samples were HIV type 1 RNA-positive; 21% of Internet samples were cytomegalovirus DNA-positive. CONCLUSIONS: Human milk purchased via the Internet exhibited high overall bacterial growth and frequent contamination with pathogenic bacteria, reflecting poor collection, storage, or shipping practices. Infants consuming this milk are at risk for negative outcomes, particularly if born preterm or are medically compromised. Increased use of lactation support services may begin to address the milk supply gap for women who want to feed their child human milk but cannot meet his or her needs.

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.

Assessment of laboratory and biosafety practices associated with bacterial culture in veterinary clinics.

OBJECTIVE: To investigate bacterial culture practices in veterinary clinics, with an emphasis on laboratory biosafety and on quality of laboratory practices. DESIGN: Survey-based prospective study. SAMPLE POPULATION: 166 veterinarians. PROCEDURES: Veterinarians were recruited through the Veterinary Information Network (an Internet-based network restricted to veterinary personnel). All Network-registered veterinarians were eligible to participate. A standardized questionnaire regarding bacterial culture practices in veterinary clinics was completed electronically by study participants. RESULTS: 720 veterinarians completed the survey; 166 (23%) indicated that bacterial culture was performed in his or her clinic. Clinic practices ranged from preliminary aerobic bacterial culture only with submission of isolates to a diagnostic laboratory for further testing (93/160 [58%]) to bacterial culture, identification, and antimicrobial susceptibility testing (19/160 [12%]). Most commonly, urine samples were cultured (151/162 [93%] clinics). Several problematic practices were identified regarding quality and quality control, including inadequate facilities, equipment, supervision, interpretation of data, and culture methods. Biosafety infractions were also common, including inadequate laboratory location, lack of biosafety protocols, and dangerous disposal practices. Ninety-four percent of respondents stated that continuing education regarding culture practices and laboratory safety would be useful. CONCLUSIONS AND CLINICAL RELEVANCE: Data confirmed that bacterial culture was commonly performed in clinics, but that major deficiencies in laboratory methods were widespread. These could result in negative effects on testing quality and increased risk of laboratory-acquired infections among clinic personnel. Veterinary practices in which bacterial cultures are performed must ensure that adequate equipment, facilities, personnel, and training are provided to enable accurate and safe sample testing.

Quantitative risk assessment: is more complex always better? Simple is not stupid and complex is not always more correct.

In quantitative risk assessments a large variety of complexities can be found, from simple and deterministic to very extensive and stochastic. This publication advocates that both simple and complex approaches have their value and should be done in parallel. The simple analysis gives much insight and can help to detect main factors and potential errors in the complex analysis. Extensive analysis with increased complexity suggests better precision but might not increase the accuracy, due to the uncertainty in the additional parameters. However, complex analysis supplies more confidence in certain phenomena and might also increase insight. This is shown with two examples. The first is the effectiveness of sampling plans for powdered infant formula, for factories operating at various levels of contamination. The results of a simple determination, an analysis including a within batch variability and an analysis including both within batch and between batch variability will be compared. The last approach has as advantage that apart from determining the probability of rejection of a batch, it can determine also the reduction of the health risk in the population following a certain sampling plan; it is more complex but it also does bring additional information. However the conclusions still contain large uncertainty, due to the difficulty of obtaining realistic values of the within batch and between batch variability. The second example is dose-response relations comparing the exponential model (one parameter), the beta-Poisson model (two parameters) and the Weibull-gamma model (three parameters). The conclusion is not that simple is best, but that simple is not stupid, and provides valuable information. Complex, on the other hand, is not always by definition more correct, but also does have its merits.

How clean is clean? Proposed methods for hospital cleaning assessment.

SUMMARY: Reducing infection rates in hospitals depends on a variety of factors, including environmental measures. Although microbiological standards have been proposed for surface hygiene in hospitals, standard methods for environmental sampling have not been discussed. The aim of this study was to assess the effectiveness of cleaning/disinfection in critical care units using the wipe-rinse method to detect an indicator organism and dipslides to quantitatively determine the microbial load. Frequent-hand-touch surfaces from clinical and non-clinical areas were microbiologically surveyed, targeting both meticillin-susceptible (MSSA) and meticillin-resistant (MRSA) Staphylococcus aureus. A subset of the surfaces targeted was sampled quantitatively to determine the total aerobic count. MRSA was isolated from 9 (6.9%) and MSSA was isolated from 15 (11.5%) of the 130 samples collected. Seven of 81 (8.6%) samples collected from non-clinical areas grew MRSA, compared with two (4.1%) from 49 samples collected from clinical areas. Of 116 sites screened for the total aerobic count, 9 (7.7%) showed >5 cfu/cm(2) microbial growth. Bed frames, telephones and computer keyboards were among the surfaces that yielded a high total viable count. There was no direct correlation between the findings of total aerobic count and MRSA isolation. We suggest, however, that combining both standards will give a more effective method of assessing the efficacy of cleaning/disinfection strategy. Further work is required to evaluate and refine these standards in order to assess the frequency of cleaning required for a particular area, or for changing the protocol or materials used.

[Drinking water analysis for Legionella. Suggestions for sampling, laboratory analysis and assessment]. / Legionellenuntersuchung bei der Trinkwasseranalyse : Hinweise zur Probenahme, Durchführung im Labor und Bewertung.

Drinking water analysis for Legionella from building installations is done quite frequently. Some questions arise from experience with this analysis. They will be discussed to allow uniform and comparable execution. Application of DIN EN ISO 19458 will lead to changes in the sampling procedure. This may make changes necessary even in current sampling and assessment programs. Concerning laboratory investigation, quality control of membrane filters and media turned out to be crucial. The assessment of quantitative results requires knowledge of the drinking water distribution system and of other facts that may be relevant for hygiene. Therefore, the assessment ought to be conducted by somebody with the respective knowledge.

Assessment of measurement uncertainty for quantitative methods of analysis: comparative assessment of the precision (uncertainty) of bacterial colony counts.

An interlaboratory trial was made, by three analysts in each of the 19 laboratories, of three International Standards Organisation (ISO) colony count methods for aerobic organisms (ACC), Enterobacteriaceae (ECC) and Escherichia coli (EcCC). All tests were done in duplicate and were further replicated by plating both on culture media supplied by the organisers and on each laboratory's own choice of culture media. In order to avoid any influence of food matrix on the results, the inoculum for each test was a freeze-dried ampoule of a standardised mixed culture. After collation of test results, individual data sets were examined for obvious non-consistency, and colony counts for each individual test were determined both as simple and 'weighted' mean values. The derived colony counts were then log(10)-transformed and examined statistically. Estimates of repeatability and reproducibility for each set of results were derived and used to calculate the parameters for the uncertainty of measurement. Estimated values of the uncertainty of reproducibility and repeatability for the ACC ranged, respectively, from 9.3 to 12.1% and 2.0 to 3.9% of the mean log(10) colony count, depending on the specific culture media, the method of deriving the mean count and the statistical procedure employed. Similarly, estimates of the uncertainty of reproducibility and repeatability for the ECC ranged, respectively, from 14.0 to 17.4% and 4.1 to 6.7%. The estimates of uncertainty of reproducibility and repeatability for the EcCC data ranged, respectively, from 21.1 to 30.9% and 6.7 to 15.4%. Whilst the uncertainty estimates for the ACC data conform to long-held views on the repeatability and reproducibility of microbial count data, the estimates for the ECC and EcCC are considerably greater. It was notable that no benefits were seen in the use of the weighted mean as compared to simple mean colony count. The importance of uncertainty estimates of colony count data in the assessment of the microbiological quality of foods is discussed.

Practical experience of microbiological validation of sterilisation.

The addition of the VDmax method provides manufacturers with a choice of three microbiological validation methods to substantiate a 25 kGy sterilisation dose. This article reports on experiences of applying these methods and suggests ways to handle difficult products.

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

The possible introduction of rejection criteria for stool cultures (