Qualitative Microbiological Methods Research Articles

Non-inferiority testing on the accuracy of two qualitative microbiological methods on paired binary data

Before implementing a new microbiological test method in pharmaceutical industry, it is required to assess whether it matches or surpasses the detection of microorganisms of the current microbiological method. The United Stated Pharmacopoeia Chapter 1223 (USP < 1223>) proposes two statistical approaches for testing non-inferiority of two qualitative microbiological methods when they can both test the same samples (paired design). The first approach compares the positive rates of the two methods. The non-inferiority test is not influenced by the performance of the methods alone, it is also affected by the spiked bacterial density. The second approach compares most probable numbers (MPNs) between the two methods, but the MPN assumes that the microbiological methods are perfect. To address the two abovementioned flaws, a generalized MPN (gMPN) approach is being proposed. Estimators for the accuracy, detection proportion, and bacterial density parameter are being derived using the method of moments. It will be shown that estimation of the accuracy and detection proportion has limited to no bias, but the bias for the bacterial density can become substantial. A test statistic for non-inferiority of accuracy, along with a sample size formula, is presented, showing nominal type I error and appropriate power. The proposed approach is demonstrated using real data.

Non-inferiority testing for qualitative microbiological methods:Assessing and improving the approach in USP

ABSTRACT The United States Pharmacopoeia (USP) presents two approaches for showing non-inferiority of an alternate qualitative microbiological method versus a compendial method. One approach compares the positive rates for the alternate and compendial methods at one spike level, while the other one compares multiple most probable number (MPN) estimates from a multi-spike design using a t-test. In this paper, we discuss these approaches under certain assumptions and propose a third approach that can be used for both single and multiple dilutions, which we call the generalized MPN (gMPN) approach. Simulations, using Poisson distributed numbers of microorganisms in test samples, confirm that the USP approach based on rates is not suitable, that the USP approach based on MPNs is appropriate for non-inferiority, but the gMPN approach outperforms the MPN-based approach and is therefore recommended.

Optimal Spiking Experiment for Noninferiority of Qualitative Microbiological Methods on Accuracy With Multiple Microorganisms

The European and United States Pharmacopoeia demand a noninferiority study on the detection of microorganisms when an alternate qualitative microbiological method is intended to replace the compendial microbiological method. However, without imposing any modeling assumptions or constraints, noninferiority studies require large numbers of test samples for a proposed noninferiority criterion of 0.7 or higher for each microorganism. When we can assume that the accuracy of the alternate method with respect to the compendial method is homogeneous across microorganisms, a joint statistical analysis of the data from all microorganisms can be used to help reduce the sample size dramatically. For this situation, we provide a test statistic for noninferiority, an optimal spiking experiment, and a sample size calculation approach under only mild modeling assumptions of the microorganism-specific detection proportions. We illustrate our approach on a real dataset and demonstrate good performance of our method using simulation studies.

Идентификация антибиотиков в мясе. Возможности проточной цитометрии

The problem of the uncontrolled use of antibiotics currently remains unresolved. Step-by-step monitoring of meat using modern methods will reduce the risk of using contaminated meat raw materials for food production. Qualitative monitoring will identify samples containing residual antimicrobial substances. The use of methods for identifying groups of antibiotics will help narrow the search for antibiotics by expensive chromatographic methods. A study was carried out of beef, pork and poultry meat, which is used in meat processing plants in the production of raw smoked sausages, using two methods. At the first stage, using a qualitative microbiological method, the raw meat was evaluated for the presence of antimicrobial substances, then their group was determined using a NovoCyte flow cytometer. According to the results of a study on a flow cytometer, it was found that out of 10 groups of antibiotics that can be determined by the tested method, the group of lincosamides was not found in all meat samples. The most common groups of chemotherapy drugs in pork were sulfonamides – 29.6 %, tetracycline group – 18.5 % and beta-lactams – 14.8 %, and in beef aminoglycosides – 36.7 %, phenicols – 30 % and beta-lactams – 13.3 %. In poultry meat samples, the most common were sulfonamides – 23.2 %, fenicols – 23.2 %, and beta-lactams – 16 %. Five groups of antibiotics were found in all studied types of meat: fenicols, β-lactams, macrolides, polypeptide antibiotics, and quinolones. This indicates their widespread use in animal husbandry and poultry farming.

A comparison of spiking experiments to estimate the detection proportion of qualitative microbiological methods

ABSTRACTThe detection proportion of a qualitative microbiological test method is the probability to detect a single micro-organism. A general expression for the moment estimator of the detection proportion is provided. It depends on the distribution of the spikes used in a validation study through its moment-generating function. Several forms of spiking experiments are compared on their estimation performance using simulations and assuming a generalized Poisson distribution (GPD) for the spikes. The optimal design, which minimizes the mean squared error of our proposed moment estimator, depends on the dispersion parameter of the GPD. The design that uses just one spiked solution instead of multiple solutions is optimal for Poisson and overdispersed Poisson and it is robust against distributions for the spikes.

Efficient estimation of the limit of detection and the relative limit of detection along with their reproducibility in the validation of qualitative microbiological methods by means of generalized linear mixed models

The reproducibility of measurement results is a core performance characteristic for quantitative methods. However, in the validation of qualitative methods it is not clear how to characterize a method’s reproducibility. One approach for determining a qualitative method’s reproducibility is presented for microbiological methods, where the distribution of colony forming units (CFU) follows a Poisson distribution. The method’s reproducibility is defined in terms of the variability of the limit of detection (LOD) values. For a better estimation of reproducibility precision, our proposed approach is using an orthogonal factorial plan. Since an exact determination of absolute contamination levels is often not possible, following the ISO 16140-2:2016 [Microbiology of food and animal feed—method validation—part 2: protocol for the validation of alternative (proprietary) methods against a reference method, 2016], an approach is proposed which is based on the ratio of the LOD values of a reference and an alternative method. This approach is illustrated on the basis of an example.

Validation of Qualitative Microbiological Methods of Pharmaceutical Analysis

We present here the results of validation of qualitative microbiological methods, particularly a modified method for determining the microbiological purity of medicines. The parameters required for validating qualitative microbiological methods and criteria for evaluating them were identified. Experimental data from validation studies demonstrate the value of using improved methods for determining microbiological purity using smaller sample sizes, and confirm that this is possible.

Microbiological Screening Test Validation for Detection of Tylosin Excretion in Milk of Cows with Low and High Somatic Cell Counts

Antibiotic residues in milk above tolerance levels interfere with dairy product processing and pose potential health risks to consumers. Residue avoidance programmes include, among other components, the observance of withdrawal times indicated in label instructions. Persistence of antibiotics in milk following treatment is influenced by drug, dosage, route of administration, body weight and mammary gland health status. Compositional changes that take place during intramammary infection (IMI) can affect antibiotic excretion in milk, thus modifying milk withdrawal time. The objectives of this study were to validate sensitivity and specificity of a qualitative microbiological method (Charm AIM-96) to detect tylosin in bovine composite milk and to determine the influence of subclinical IMI in tylosin excretion following intramuscular administration. For test validation, two groups of approximately 120 cows were used; one received a single intramuscular injection of tylosin tartrate at a dose of 20 mg/kg, while the other group remained as untreated control. Test sensitivity and specificity were 100% and 94.1% respectively. To determine the influence of subclinical IMI in tylosin excretion, two groups of seven cows, one with somatic cell counts (SCC) < or =250 000 cells/ml and the other with SCC > or =900 000, were administered a single intramuscular injection of tylosin tartrate at a dose of 20 mg/kg. Milk samples were obtained every 12 h for 10 days following treatment. Milk tylosin excretion averaged between 5 and 9 days for cows with low and high SCC respectively (P < 0.0001). Compositional changes in cows with high SCC most likely affect the pharmacokinetic characteristics of tylosin, extending the presence of the antibiotic in milk, thus influencing milk withdrawal times.

Analysing collaborative trials for qualitative microbiological methods: accordance and concordance

In qualitative (detection) food microbiology, the usual measures of repeatability and reproducibility are inapplicable. For such studies, we introduce two new measures: accordance for within laboratory agreement and concordance for between laboratory agreement, and discuss their properties. These measures are based on the probability of finding the same test results for identical test materials within and between laboratories, respectively. The concordance odds ratio is introduced to present their relationship. A method to test whether accordance differs from concordance is discussed.

Validation of ISO method 11290 Part 1 — Detection of Listeria monocytogenes in foods

The European and International Standard method for the detection of Listeria monocytogenes, described in EN ISO 11290 Part 1: 1997 (International Organisation for Standardisation, Geneva) was validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098). Nineteen laboratories in 14 countries in Europe participated in a collaborative trial to determine the performance characteristics of the method, which are intended for publication in the corresponding standard. An additional objective of this project was to devise a new series of parameters to indicate the ‘precision’ of microbiological qualitative methods. The method was challenged with three food types, namely fresh cheese, minced beef and dried egg powder and a reference material. Inoculation levels ranged from 5 to 100 cfu/25 g. Each participant examined five replicates of each food type at three inoculum levels and five reference materials. Both PALCAM and Oxford media were assessed. All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial. The results demonstrated that the method prescribed in EN ISO 11290-1 had an overall sensitivity of 85.6% and a specificity of 97.4%. L. monocytogenes was detected in most cases after primary enrichment, although secondary enrichment often yielded further positives. However, a significant number of false-negative results were obtained with all food types when large numbers of L. innocua were present in the test materials. L. innocua tended to dominate L. monocytogenes during the selective enrichment stages and thus masked small numbers of colonies of L. monocytogenes on the isolation media. There was no evidence from this collaborative study to demonstrate a significant difference in performance between Oxford and PALCAM media. Due to the problem of false-negative results with this method as highlighted in this trial, recommendations have been made to ISO to launch a revision of the standard to improve the detection of low numbers of L. monocytogenes in foods. New statistical methods devised to advance the measurement of the performance of qualitative microbiological methods are also described.

Evaluating Presence/Absence of Target Microbes in Microbiological Tests

A typical qualitative microbiological method performance (collaborative) study gathers a data set of responses about a test for the presence or absence of a target microbe. We developed 2 models that estimate false-positive and false-negative rates. One model assumes a constant probability that the tests will indicate the target microbe is present for any positive concentration in the test portion. The other model assumes that this probability follows a logistic curve. Test results from several method performance studies illustrate these estimates.