Comparative evaluation of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains for rapid detection of chemical mutagens and carcinogens

The aim of the present study was to evaluate the usefulness of different pairs of DNA repair-deficient and DNA repair-proficient bacterial tester strains in a mutagenicity/carcinogenicity screen, possibly as complements to the Ames test. 70 carcinogenic and non-carcinogenic compounds, representing a variety of chemical structures, were tested for their DNA-damaging effects, using 6 different DNA-repair-deficient bacterial strains. 2 Bacillus subtilis systems, H17/M45 and HLL3g/HJ-15, were used. The susceptibility of Escherichia coli AB1157 was compared with the susceptibility of 4 recombination-deficient mutants, JC5547, JC2921, JC2926 and JC5519. The test compounds were applied onto paper disks (spot test, ST), or incorporated into a top agar layer (agar-incorporation test, AT). The 2 B. subtilis systems were generally found to be more sensitive and reliable than the assays using E coli. The incorporation of the test compounds in the agar increased the sensitivity of the test for polycyclic aromatic hydrocarbons and other poorly water-soluble compounds. Hydrazines and several other highly polar chemicals could be tested more efficiently when applied onto paper disks. About 30% of the test compounds did not induce any growth inhibition and so could not be tested properly. In order to evaluate the ability of these DNA-repair tests to complement the Ames Salmonella mutagenicity test in a genetic toxicology screening program, results from this study were compared with published data both on mutagenicity in the Ames test and on carcinogenicity. 8 carcinogens generally found to be non-mutagenic for Salmonella were tested: 2 showed DNA-damaging properties (mitomycin C, 1,2-dimethylhydrazine), 5 failed to do so (actinomycin D, griseofulvin, thioacetamide, diethylstilbestrol, safrole), and one (thiourea) was not toxic, so that no classification was possible. 2 non-carcinogenic bacterial mutagens were examined; one, sodium azide, was equitoxic for repair-proficient and -deficient strains, while the other, nitrofurantoin, primarily inhibited repair-deficient strains. The DNA-repair tests failed to indicate the mutagenic and carcinogenic properties of acridine orange. Nalidixic acid, a non-mutagenic DNA synthesis inhibitor, damaged bacterial DNA. Apart from the differences summarized above, carcinogenicity was indicated correctly by the Salmonella S9 assay and most sets of DNA-repair-deficient and DNA-repair-proficient tester strains evaluated in this study. Thus, several more carcinogens could be detected by performing the Ames test and the bacterial DNA-repair tests in tandem than by using either test alone. Nevertheless, the use of both bacterial in vitro systems in a battery of short-term tests for mutagenicity/carcinogenicity evaluation is not considered to be ideal, since the Ames test and the pairs of DNA-repair-deficient and DNA-repair-proficient tester strains used had several shortcomings in common under the conditions of this study.     

Commercially available histoplasmin sensitized latex particles in an agglutination test for histoplasmosis

Summary A commercial preparation of histoplasmin sensitized latex particles was tested in an agglutination test with sera from 50 culturally confirmed cases of histoplasmosis in varying stages of the infection. The reactions were superior to those obtained with collodion agglutination and complement fixation tests in which the antigen histoplasmin was also used. The latex agglutination test with the commercially available antigen is easy to do, and can be done in any laboratory equipped to carry out agglutination tests with the common bacterial antigens. It warrants more extensive trial in the general hospital laboratory as a screening test for histoplasmosis, especially the primary, pulmonary type.     

Hemagglutination Inhibition for Serogrouping of Neisseria meningitidis

A hemagglutination inhibition (HI) test has been studied as an alternative to bacterial agglutination (BA) for serogrouping strains of Neisseria meningitidis isolated from clinical specimens. The HI test consists of polysaccharide antigens adsorbed to sheep red blood cells which were then agglutinated by group-specific antisera. Supernatant fluids from suspensions of meningococci were used to inhibit the agglutination. Results of the two tests agreed for 381 (80%) carrier strains. Of the remaining 95 strains, 82 (86%) were identified by HI although they were nongroupable by BA. Thus, the HI test has been shown to be more highly specific and sensitive and to be more economical of reagents and time than the BA test.     

A Systematic Review on the Diagnosis of Pediatric Bacterial Pneumonia: When Gold Is Bronze

Background

In developing countries, pneumonia is one of the leading causes of death in children under five years of age and hence timely and accurate diagnosis is critical. In North America, pneumonia is also a common source of childhood morbidity and occasionally mortality. Clinicians traditionally have used the chest radiograph as the gold standard in the diagnosis of pneumonia, but they are becoming increasingly aware that it is not ideal. Numerous studies have shown that chest radiography findings lack precision in defining the etiology of childhood pneumonia. There is no single test that reliably distinguishes bacterial from non-bacterial causes. These factors have resulted in clinicians historically using a combination of physical signs and chest radiographs as a ‘gold standard’, though this combination of tests has been shown to be imperfect for diagnosis and assigning treatment. The objectives of this systematic review are to: 1) identify and categorize studies that have used single or multiple tests as a gold standard for assessing accuracy of other tests, and 2) given the ‘gold standard’ used, determine the accuracy of these other tests for diagnosing childhood bacterial pneumonia.

Methods and Findings

Search strategies were developed using a combination of subject headings and keywords adapted for 18 electronic bibliographic databases from inception to May 2008. Published studies were included if they: 1) included children one month to 18 years of age, 2) provided sufficient data regarding diagnostic accuracy to construct a 2×2 table, and 3) assessed the accuracy of one or more index tests as compared with other test(s) used as a ‘gold standard’. The literature search revealed 5,989 references of which 256 were screened for inclusion, resulting in 25 studies that satisfied all inclusion criteria. The studies examined a range of bacterium types and assessed the accuracy of several combinations of diagnostic tests. Eleven different gold standards were studied in the 25 included studies. Criterion validity was calculated for fourteen different index tests using eleven different gold standards. The most common gold standard utilized was blood culture tests used in six studies. Fourteen different tests were measured as index tests. PCT was the most common measured in five studies each with a different gold standard.

Conclusions

We have found that studies assessing the diagnostic accuracy of clinical, radiological, and laboratory tests for bacterial childhood pneumonia have used a heterogeneous group of gold standards, and found, at least in part because of this, that index tests have widely different accuracies. These findings highlight the need for identifying a widely accepted gold standard for diagnosis of bacterial pneumonia in children.     

Evaluation of the applicability of the bacterial endotoxin test to antibiotic products.

Fundamental conditions for applying the bacterial endotoxin test to antibiotic products were investigated so as not to affect the level of regulation by the rabbit pyrogen test. According to accuracy evaluation of test methods, the kinetic-turbidimetric and kinetic-colourimetric assays were shown to allow more accurate measurement and, therefore, more sensitive detection of interference to the bacterial endotoxin test than the gel-clot method. In total, 102 antibiotic products were evaluated on their interfering effect to show that the antibiotics could be categorized into three groups depending on intensity of the interference. Although the test was shown to be applicable even to the group showing the strongest interference, it was assumed to be crucial to use appropriate reagents and an accurate test method for avoiding approval of a pyrogenic product. Accordingly, lists of antibiotics are presented to provide limits of concentration for eliminating interference and endotoxin limits for approval to facilitate effective bacterial endotoxin tests.     

Sensitivity of bacterial vs. acute Daphnia magna toxicity tests to metals

The objectives of this study were to evaluate the sensitivity of two bacterial tests commonly used in metal toxicity screening — the Vibrio fischeri bioluminescence inhibition test and the Pseudomonas putida growth inhibition test — in comparison to the standard acute Daphnia magna test, and to estimate applicability of the selected methods to the toxicity testing of environmental samples. The D. magna acute test proved to be more sensitive to cadmium (Cd), zinc (Zn) and manganese (Mn) than the two bacterial assays, whereas P. putida seems to be the most sensitive species to lead (Pb). Manganese appears to be slightly toxic to D. magna and non-toxic to the two selected bacteria. This leads to the conclusion that even in regions with high background concentrations, manganese would not act as a confounding factor. Low sensitivity of V. fischeri to heavy metals questions its applicability as the first screening method in assessing various environmental samples. Therefore, it is not advisable to replace D. magna with bacterial species for metal screening tests. P. putida, V. fischeri and/or other bacterial tests should rather be applied in a complex battery of ecotoxicological tests, as their tolerance to heavy metals can unravel other potentially present toxic substances and mixtures, undetectable by metal-sensitive species.     

Die-Away Kinetic Analysis of the Capacity of Epilithic and Planktonic Bacteria from Clean and Polluted River Water To Biodegrade Sodium Dodecyl Sulfate

The capacities of epilithic and planktonic river bacterial populations to degrade sodium dodecyl sulfate (SDS) in samples taken at two times during 1987 from one clean and four polluted sites in a South Wales river were estimated in die-away tests under simulated environmental conditions. There was a relatively slow disappearance of SDS in die-away tests for both planktonic and epilithic populations taken from the clean source site, as compared with those taken from the downstream polluted sites, for which the rate of biodegradation was accelerated, sometimes after an apparent initial lag period. The kinetic components contributing to the die-away curves were quantified by nonlinear regression analysis in which the experimental data were fitted to a variety of possible kinetic models. All samples except for one from the polluted sites best fitted a model which describes the biodegradation of SDS at concentrations well below its K_m by bacteria whose growth is exponential and unaffected by the addition of a test substrate. Each sample from the clean source site fitted a different model, but there was generally little or no growth on endogenous carbon. A consideration of the numerical values of constants derived from the modeling of epilithic and planktonic populations from polluted sites indicated clearly that the biodegradative capacity of epilithic bacterial populations towards SDS is more stable than that of planktonic bacterial populations.     

A new proposal for a principal component-based test for high-dimensional data applied to the analysis of PhyloChip data

A modification of the principal component test is presented. It uses a weighted combination of the sums of squares for different principal components and is thus more powerful in high-dimensional settings with small sample sizes. Under usual normality assumptions, a rotation test is proposed which enables an exact conditional parametric test. The procedure is demonstrated with microarray data for the bacterial composition in the rhizosphere of different potato cultivars. In simulation studies, the power of the proposed statistic is compared with the competing multivariate parametric tests.     

The mouse spot test. Evaluation of its performance in identifying chemical mutagens and carcinogens

The published results on 60 chemicals and X-rays investigated in the mouse spot test were compared with data on the same chemicals tested in the bacterial mutation assay (Ames test) and lifetime rodent bioassays. The performance of the spot test as an in vivo complementary assay to the in vitro bacterial mutagenesis test reveals that of 60 agents, 38 were positive in both systems, 6 were positive only in the spot test, 10 were positive only in the bacterial test and 6 were negative in both assays. The spot test was also considered as a predictor of carcinogenesis; 45 chemicals were carcinogenic of which 35 were detected as positive by the spot test and 3 out of 6 non-carcinogens were correctly identified as negative. If the results are regarded in sequence, i.e. that a positive result in a bacterial mutagenicity test reveals potential that may or may not be realized in vivo, then 48 chemicals were mutagenic in the bacterial mutation assay of which 38 were active in the spot test and 31 were confirmed as carcinogens in bioassays. 12 chemicals were non-mutagenic to bacteria of which 6 gave positive responses in the spot test and 5 were confirmed as carcinogens. These results provide strong evidence that the mouse coat spot test is an effective complementary test to the bacterial mutagenesis assay for the detection of genotoxic chemicals and as a confirmatory test for the identification of carcinogens. The main deficiency at present is the paucity of data from the testing of non-carcinogens. With further development and improvement of the test it is probable that the predictive performance of the assay in identifying carcinogens should improve, since many of the false negative responses may be due to inadequate testing.     

Avoiding erroneously high levels of detection in combinations of semi-independent tests

A randomization procedure is proposed which allows statistical tests to be combined into a single test to maintain specified and acceptable levels of false detection. This method was applied to the problem of detecting density dependence in 135 unpublished time-series (of 10 generations) from insect populations, and to simulated density-dependent and density-independent data, so that the correctness of observed levels of detection from the published data could be verified. To allow the application of the randomization procedure to Bulmer's (1975) tests and Varley and Gradwell's (1960) test, these were recast as randomization tests. The randomization procedure was tested with 39 combinations of tests for density dependence (and limitation/attraction); it generally producedcombined tests with levels of detection that were intermediate between detection levels of the constituent tests (and hence was limite by these). The specified rate of false detection (5%) was never exceeded (by more than 1%) when combined tests were applied to time-series from a random-walk model. Two different combinations of tests produced levels of detection from the published time-series which were slightly greater than their constituent tests when they were combined into single tests. These were the randomized form of Bulmer's (1975) first test with the tests of Pollard et al. (1987) and Reddingius and den Boer (1989) with the randomized form of Bulmer's second test. The combination of Bulmer's first and Pollard et al.'s test produced a greater level of detection (21.5%) than any other single test or combination of tests. These results were confirmed by the analysis of modelled density dependent data. Although the increase in power of combinations of tests over single tests is small with the data we used, the combined tests (listed above) had rates of detection that were less influenced by the form of data (of two forms of density-dependent data) than were their constituent tests. Hence, it appears that the combined tests are of greater generality than single test statistics. The method presented here for combining several statistical tests into a single randomization test is applicable in many other areas of ecology where we wish to apply several tests and take the most probable result of these; and if the tests being conducted are, or can be expressed as, randomization tests.     

Evaluation of Two PCR Tests for Coxiella burnetii Detection in Dairy Cattle Farms Using Latent Class Analysis

Different tests performed on bulk tank milk samples (BTM) are available to determine the C. burnetii status of herds. However, these tests, which are based on the detection of either antibodies directed against C. burnetii (ELISA) or bacterial DNA (PCR), have limitations. A currently disease-free herd infected in the past may continue to test positive with ELISA due to the persistence of antibodies in animals that were infected and that subsequently cleared the infection. Infectious herds can also be misclassified using PCR because of the absence of bacteria in the BTM when the test is performed. Recently, PCR has been used for bacterial DNA detection in the farm environment, which constitutes the main reservoir of C. burnetii. The objectives of this study were to assess and compare the sensitivities and specificities of one commonly used PCR test in BTM (PCR BTM) and of a PCR applied to environmental samples (PCR DUST) in dairy cattle farms. BTM and dust samples were collected (using environmental swabs) in 95 herds. The evaluation of the performance of the 2 tests was conducted using latent class models accounting for within herd disease dynamics. Parameter estimation was carried out using MCMC, within a Bayesian framework. Two types of priors were used for the specificity of PCR DUST. A model with a uniform prior on 0–1 fitted the data better than a model with a uniform prior on 0.95–1. With the best model PCR DUST had a lower sensitivity than PCR BTM (0.75 versus 0.83) and a specificity of 0.72. The moderately low value for the specificity of PCR DUST suggests that the presence of bacteria on farm is not always associated with persistent infections and shedding of bacteria in milk.     

Correlation between humification and the oxidase activity of the causative micro-organisms

Summary Decomposition of finely ground barley straw at 28°C was followed for 250 days in vessels which had been inoculated with mixed bacterial isolates of known oxidase activity derived from a sample of barley field soil. The artificial bacterial populations selected consisted of either 1) isolates which reacted positively in a number of oxidase tests, 2) isolates which gave negative responses in the same tests, or 3) these two groups in combination. For comparison, a suspension of the soil from which the bacterial isolates were derived was also used as an alternative inoculum. It was shown that the oxidase-negative bacteria were more effective than the oxidase-positive ones in the production of humic substance, and in decreasing the carbohydrate content of the straw.     

Testing for segregation distortion in genetic scoring data from backcross or doubled haploid populations

It is important that breeders have the means to assess genetic scoring data for segregation distortion because of its probable effect on the design of efficient breeding strategies. Scoring data is usually assessed for segregation distortion by separate nonindependent chi2 tests at each locus in a set of marker loci. This analysis gives the loci most affected by selection if it exists, but it cannot give a statistically correct test for the presence or absence of selection in a linkage group as a whole. I have used a combined test based on the statistic, which is the most significant P-value from the above tests, called the single locus test. I have also derived mathematically a new combined statistical test, the overall test, for segregation distortion that requires genetic scoring data for a single linkage group. This test also takes genetic linkage into account. Using a range of marker densities and population sizes, simulations were carried out, to compare the power of these two statistical tests to detect the effect of selection at one or two loci. The single locus test was always found to be more powerful than the overall test, but the single locus test required a more complicated P-value correction. For the single locus test, approximate correction factors for the P-values are given for a range of marker densities and genetic lengths.     

Direct-acting mutagenic properties of some hair dyes used in New Zealand

Mutagenicity or carcinogenicity data are not publicly available on many hair dyes or dye components commonly used within New Zealand. Representative mid- to dark-warm brown hair dyes of 12 brands supplying more than 1% of the New Zealand market were tested for direct-acting mutagenicity using the bacterial 'Ames' test. Despite recent scientific advances in the development of non-mutagenic dyes, 23 of the 40 products tested gave positive results in one or both of the tester strains used. There appeared to be differences between distributors in the proportion of their hair dyes which were mutagenic. In the case of 6 out of 10 of the above dyes which had tested positive, in vitro mutagenicity or toxicity was enhanced in the presence of verapamil, suggesting that risks from hair-dye exposure may change in the case of persons using this or similar drugs. It is recognised that there are uncertainties regarding human risks from dyes which are shown to be mutagenic in in vitro tests. However, from the above results, it seems possible to produce non-mutagenic hair dyes in this color range; and in the interests of public reassurance, it may be prudent to require that such dyes be used.     

Defining the True Sensitivity of Culture for the Diagnosis of Melioidosis Using Bayesian Latent Class Models

Background

Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests.

Methods/Principal Findings

Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard.

Conclusions/Significance

Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections.     

Genotoxicity of six pesticides by Salmonella mutagenicity test and SOS chromotest

Two in vitro tests (Ames test and SOS chromotest), one for bacterial mutagenicity and one for primary DNA damage, were assayed to determine the genotoxic activity of 6 pesticides (atrazine, captafol, captan, chlorpyrifosmethyl, molinate and tetrachlorvinphos). Assays were carried out both in the absence and presence of S9 fractions of liver homogenate from rat (Sprague–Dawley) pretreated with Aroclor 1254. Captan and captafol were genotoxic on both the Ames test and the SOS chromotest. Comparisons with mutagenesis data in Salmonella indicated that the SOS assay detected as genotoxic the pesticides that were mutagenic on the Salmonella test. Non-genotoxic effects were not detected in vitro either in the Salmonella/microsome assay nor in the SOS chromotest when bacterial tester strains were exposed to atrazine, molinate, chlorpyrifosmethyl and tetrachlorvinphos in the absence or presence of S9 mix.     

Estimation and Identification of Streptococcus dysgalactiae Protective Antibody in Sera of Rabbits and Cattle

Rabbit and cow anti-Streptococcus dysgalactiae sera were tested by bacterial agglutination, complement fixation, hemagglutination, and immunodiffusion for the presence of antibody. The results of these tests were compared with mouse-protection studies on the same serum to estimate which in vitro test would best reflect the in vivo protective capacity of serum. Identification of the antibody constituents responsible for the mouse protection, hemagglutination, and complement fixation titers were established by reacting whole and diluted antisera with mercaptoethanol before and after testing. Results indicate that the complement fixation test may be a more accurate indicator of IgG protective bovine and rabbit antibody, whereas the hemagglutination test may more readily reflect a wider range of protective antibody levels and IgM. The complement fixation test showed some shared responses to IgG and IgM in both the rabbit and cow, whereas the IgM components seemed to be the predominant factor influencing hemagglutination titers in the rabbit and more so in the bovine. Mouse protection tests with mercaptoethanol-treated cow and rabbit sera indicate that the protective capacity of these antisera is shared between IgM and IgG components.     

Diagnostic accuracy of serological tests for the diagnosis of Chikungunya virus infection: A systematic review and meta-analysis

BackgroundChikungunya virus (CHIKV) causes febrile illnesses and has always been misdiagnosed as other viral infections, such as dengue and Zika; thus, a laboratory test is needed. Serological tests are commonly used to diagnose CHIKV infection, but their accuracy is questionable due to varying degrees of reported sensitivities and specificities. Herein, we conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of serological tests currently available for CHIKV.Methodology and principal findingsA literature search was performed in PubMed, CINAHL Complete, and Scopus databases from the 1^st December 2020 until 22^nd April 2021. Studies reporting sensitivity and specificity of serological tests against CHIKV that used whole blood, serum, or plasma were included. QUADAS-2 tool was used to assess the risk of bias and applicability, while R software was used for statistical analyses.Thirty-five studies were included in this meta-analysis; 72 index test data were extracted and analysed. Rapid and ELISA-based antigen tests had a pooled sensitivity of 85.8% and 82.2%, respectively, and a pooled specificity of 96.1% and 96.0%, respectively. According to our meta-analysis, antigen detection tests serve as a good diagnostic test for acute-phase samples. The IgM detection tests had more than 90% diagnostic accuracy for ELISA-based tests, immunofluorescence assays, in-house developed tests, and samples collected after seven days of symptom onset. Conversely, low sensitivity was found for the IgM rapid test (42.3%), commercial test (78.6%), and for samples collected less than seven of symptom onset (26.2%). Although IgM antibodies start to develop on day 2 of CHIKV infection, our meta-analysis revealed that the IgM detection test is not recommended for acute-phase samples. The diagnostic performance of the IgG detection tests was more than 93% regardless of the test formats and whether the test was commercially available or developed in-house. The use of samples collected after seven days of symptom onset for the IgG detection test suggests that IgG antibodies can be detected in the convalescent-phase samples. Additionally, we evaluated commercial IgM and IgG tests for CHIKV and found that ELISA-based and IFA commercial tests manufactured by Euroimmun (Lübeck, Germany), Abcam (Cambridge, UK), and Inbios (Seattle, WA) had diagnostic accuracy of above 90%, which was similar to the manufacturers’ claim.ConclusionBased on our meta-analysis, antigen or antibody-based serological tests can be used to diagnose CHIKV reliably, depending on the time of sample collection. The antigen detection tests serve as a good diagnostic test for samples collected during the acute phase (≤7 days post symptom onset) of CHIKV infection. Likewise, IgM and IgG detection tests can be used for samples collected in the convalescent phase (>7 days post symptom onset). In correlation to the clinical presentation of the patients, the combination of the IgM and IgG tests can differentiate recent and past infections.     

Improving data interpretation of fragmentary data-sets on invertebrate dispersal with permutation tests

Biological data often tend to have heterogeneous, discontinuous non-normal distributions. Statistical non-parametric tests, like the Mann–Whitney U-test or the extension for more than two samples, the Kruskal–Wallis test, are often used in these cases, although they assume certain preconditions which are often ignored. We developed a permutation test procedure that uses the ratio of the interquartile distances and the median differences of the original non-classified data to assess the properties of the real distribution more appropriately than the classical methods. We used this test on a heterogeneous, skewed biological data set on invertebrate dispersal and showed how different the reactions of the Kruskal–Wallis test and the permutation approach are. We then evaluated the new testing procedure with reproducible data that were generated from the normal distribution. Here, we tested the influence of four different experimental trials on the new testing procedure in comparison to the Kruskal–Wallis test. These trials showed the impact of data that were varying in terms of (a) negative correlation between variances and means of the samples, (b) changing variances that were not correlated with the means of the samples, (c) constant variances and means, but different sample sizes and in trials (d) we evaluated the testing power of the new procedure. Due to the different test statistics, the permutation test reacted more sensibly to the data presented in trials (a) and c) and non-uniformly in trial (b). In the evaluation of the testing power, no significant differences between the Kruskal–Wallis test and the new permutation testing procedure could be detected. We consider this test to be an alternative for working on heterogeneous data where the preconditions of the classical non-parametric tests are not met.