Estimation of the POD Function and the LOD of a Binary Microbiological Measurement Method from an Interlaboratory Experiment.

Background: We deal with interlaboratory experiments (collaborative studies) in which k participating laboratories, selected randomly from a population of laboratories, use samples from one and the same material or matrix. They perform binary microbiological measurements for which the measurement results are either "0" (target microorganisms not detected) or "1" (target microorganisms detected). The performance of such a measurement method is described by its probability of detection (POD) function, i.e., the POD as a function of the contamination of the sample (CFU per gram or CFU per milliliter), or by the level of detection (LODp), i.e., the contamination level of the sample that is detected (measurement result "1") with a specified probability p. Objective: We derive an approximate statistical analysis that is simple enough to be implemented in a spreadsheet application. Methods: Under the assumption of a Poisson distribution of the number of CFU in the samples, we estimate the mean POD function of the laboratories and the SD of the laboratory effect based on a complementary log-log model, a special case of the Generalized Linear Model in the special situation in which the contamination level is known by means other than the POD. The estimates are obtained by maximization of the Laplace approximation of the likelihood function. By simulation, a bias correction factor for the estimate of the SD is obtained. With the estimated POD function, LODs can be estimated. The model can also be used to evaluate the relative LOD of an alternative method with repect to a reference method. Results: The EXCEL program PODLOD-interlab_ver1.xls for this method of statistical analysis can be downloaded from http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich. Highlights: A simple approximate statistical method for the estimation of the POD and LOD is derived. The method also allows the estimation of the RLOD of an alternative method with respect to reference method. The method is implemented in an EXCEL program that can be downloaded from http://www.wiwiss.fu-berlin.de/fachbereich/vwl/iso/ehemalige/wilrich.

Inter-laboratory validation of bioaccessibility testing for metals.

Bioelution assays are fast, simple alternatives to in vivo testing. In this study, the intra- and inter-laboratory variability in bioaccessibility data generated by bioelution tests were evaluated in synthetic fluids relevant to oral, inhalation, and dermal exposure. Using one defined protocol, five laboratories measured metal release from cobalt oxide, cobalt powder, copper concentrate, Inconel alloy, leaded brass alloy, and nickel sulfate hexahydrate. Standard deviations of repeatability (sr) and reproducibility (sR) were used to evaluate the intra- and inter-laboratory variability, respectively. Examination of the sR:sr ratios demonstrated that, while gastric and lysosomal fluids had reasonably good reproducibility, other fluids did not show as good concordance between laboratories. Relative standard deviation (RSD) analysis showed more favorable reproducibility outcomes for some data sets; overall results varied more between- than within-laboratories. RSD analysis of sr showed good within-laboratory variability for all conditions except some metals in interstitial fluid. In general, these findings indicate that absolute bioaccessibility results in some biological fluids may vary between different laboratories. However, for most applications, measures of relative bioaccessibility are needed, diminishing the requirement for high inter-laboratory reproducibility in absolute metal releases. The inter-laboratory exercise suggests that the degrees of freedom within the protocol need to be addressed.

Determination of the relative level of detection of a Qualitative microbiological measurement method with respect to a reference measurement method.

Performance of qualitative microbiological measurement methods where the results are either "O" (microorganism not detected) or "1" (microorganism detected) is described by their probability of detection (POD) function, i.e., the POD as a function of the level of contamination of the sample, expressed as CFU/g or CFU/mL, or by the level of detection (LODp), i.e., the contamination of the sample that is detected (measurement result "1") with a specified probability p. When it is impossible to obtain samples of known contamination, estimation of the POD and LOD is impossible. However, it may not be the LOD of the method that is of interest, but its LOD with respect to the LOD of a reference method. Hence, an intralaboratory experiment is performed with a reference method, R, and an alternative method, A, at different levels of unknown contamination. A complementary loglog model is used to statistically estimate the relative LOD (RLOD) of A with respect to R that is equal for all chosen values p of the POD. An intralaboratory experiment for the detection of Listeria monocytogenes in fish and eggs illustrates the method. In a simulation study, the bias of the estimate of the RLOD was investigated. This bias is due to the small number of repeated measurements in intralaboratory studies; the relative bias increases with increasing true values of the RLOD from 0 for true RLOD = 1 to about 20% for true RLOD = 3. If the number of CFUs in the test portions does not follow a Poisson distribution, but instead follows a negative binomial distribution, e.g., because of overdispersion, the bias of the estimate of the RLOD decreases. An EXCEL program RLOD_ver1. xlsm for this method of statistical analysis can be downloaded from http://www.wiwiss.fu-berlin.de/ instituteliso/mitarbeiterlwilrichlindex.html.

Reduced detectability of Listeria monocytogenes in the presence of Listeria innocua.

Recent foodborne crises have demonstrated the importance of monitoring food safety. In terms of microbiological criteria, food safety requires the reliable detection of pathogens such as Listeria monocytogenes along the food chain by appropriate analytical methods. However, indications exist that accompanying Listeria innocua strains suppress the growth of L. monocytogenes during selective enrichment, which may cause reduced or even inhibited detection. To study these effects, the limit of detection of L. monocytogenes was investigated in the presence of L. innocua using the International Organization for Standardization standard method ISO 11290-1 and the VIDAS LDUO system, an automated method based on enzyme-linked fluorescence technology. The challenge was to provide low initial Listeria concentrations at sufficient precision to quantify the influence on the probability of detection of L. monocytogenes. The application of reference materials appropriate for quantitative test methods and a standardized dilution procedure were necessary to ensure accurate CFU levels of defined proportions of mixtures of both Listeria species. During selective enrichment, overgrowth of L. monocytogenes by L. innocua could be confirmed, leading to high rates of false-negative results. Moreover, with both methods, a significant decrease in the detectability of L. monocytogenes could be quantified at ratios of 2:1 at very low concentrations representative of natural contamination levels often found in foods and environments. It is concluded that there is a need to improve existing procedures with respect to selective enrichment, as well as the detection techniques.

Estimation of the POD function and the LOD of a qualitative microbiological measurement method.

Qualitative microbiological measurement methods in which the measurement results are either 0 (microorganism not detected) or 1 (microorganism detected) are discussed. The performance of such a measurement method is described by its probability of detection as a function of the contamination (CFU/g or CFU/mL) of the test material, or by the LOD(p), i.e., the contamination that is detected (measurement result 1) with a specified probability p. A complementary log-log model was used to statistically estimate these performance characteristics. An intralaboratory experiment for the detection of Listeria monocytogenes in various food matrixes illustrates the method. The estimate of LOD50% is compared with the Spearman-Kaerber method.

[Estimation of reproducibility and repeatability in microbiological ring trials--robust versus conservative methods]. / Schätzung von Reproduzierbarkeit und Wiederholbarkeit bei mikrobiologischen Ringversuchen--robuste versus konservative Verfahren.

During the last years there was a lot of progress to be seen in the development of standardized methods for microbiological ring trials. The statistical analyzing strategies, in particular the calculation of estimations for the parameters repeatability and reproducibility, will be considered in this paper. Apart from the conservative method of the variance analysis robust methods are increasingly discussed. We will compare and discuss these methods using data of recently realized ring trials. If we can assume a normal distribution of our data, then all estimations are theoretically precise and efficient. But up to now, we know very little about the character of the robust estimations, if the normal distribution cannot be assumed. In addition to this, we have to mention once more, that the use of robust estimators is unreasonable without taking a critical look on the data themselves. Thus, we will show the possibilities of graphical presentation of all data to identify laboratories with critical results.