A novel bead-based assay to detect specific antibody responses against Toxoplasma gondii and Trichinella spiralis simultaneously in sera of experimentally infected swine

<p>Abstract</p> <p>Background</p> <p>A novel, bead-based flow cytometric assay was developed for simultaneous determination of antibody responses against <it>Toxoplasma gondii </it>and <it>Trichinella spiralis </it>in pig serum. This high throughput screening assay could be an alternative for well known indirect tests like ELISA. One of the advantages of a bead-based assay over ELISA is the possibility to determine multiple specific antibody responses per single sample run facilitated by a series of antigens coupled to identifiable bead-levels. Furthermore, inclusion of a non-coupled bead-level in the same run facilitates the determination of, and correction for non-specific binding. The performance of this bead-based assay was compared to one <it>T. spiralis </it>and three <it>T. gondii </it>ELISAs. For this purpose, sera from <it>T. gondii </it>and <it>T. spiralis </it>experimentally infected pigs were used. With the experimental infection status as gold standard, the area under the curve, Youden Index, sensitivity and specificity were determined through receiver operator curve analysis. Marginal homogeneity and inter-rater agreement between bead-based assay and ELISAs were evaluated using McNemar's Test and Cohen's kappa, respectively.</p> <p>Results</p> <p>Results indicated that the areas under the curve of the bead-based assay were 0.911 and 0.885 for <it>T. gondii </it>and <it>T. spiralis</it>, respectively, while that of the <it>T. gondii </it>ELISAs ranged between 0.837 and 0.930 and the <it>T. spiralis </it>ELISA was 0.879. Bead-based <it>T. gondii </it>assay had a sensitivity of 86% and specificity of 96%, while the ELISAs ranged between 64-84% and 93-99%, respectively. The bead-based <it>T. spiralis </it>assay had a sensitivity of 68% and specificity of 100% while the ELISA scored 72% and 95%, respectively. Marginal homogeneity was found between the <it>T. gondii </it>bead-based test and one of the <it>T. gondii </it>ELISAs. Moreover, in this test combination and between <it>T. spiralis </it>bead-based assay and respective ELISA, an excellent inter-rater agreement was found. When results of samples before expected seroconversion were removed from evaluation, notably higher test specifications were found.</p> <p>Conclusions</p> <p>This new bead-based test, which detects <it>T. gondii </it>and <it>T. spiralis </it>antibodies simultaneously within each sample, can replace two indirect tests for the determination of respective antibodies separately, while performing equally well or better.</p

Modernization of Control of Pathogenic Micro-Organisms in the Food-Chain Requires a Durable Role for Immunoaffinity-Based Detection Methodology—A Review

Food microbiology is deluged by a vastly growing plethora of analytical methods. This review endeavors to color the context into which methodology has to fit and underlines the importance of sampling and sample treatment. The context is that the highest risk of food contamination is through the animal and human fecal route with a majority of foodborne infections originating from sources in mass and domestic kitchens at the end of the food-chain. Containment requires easy-to-use, failsafe, single-use tests giving an overall risk score in situ. Conversely, progressive food-safety systems are relying increasingly on early assessment of batches and groups involving risk-based sampling, monitoring environment and herd/flock health status, and (historic) food-chain information. Accordingly, responsible field laboratories prefer specificity, multi-analyte, and high-throughput procedures. Under certain etiological and epidemiological circumstances, indirect antigen immunoaffinity assays outperform the diagnostic sensitivity and diagnostic specificity of e.g., nucleic acid sequence-based assays. The current bulk of testing involves therefore ante- and post-mortem probing of humoral response to several pathogens. In this review, the inclusion of immunoglobulins against additional invasive micro-organisms indicating the level of hygiene and ergo public health risks in tests is advocated. Immunomagnetic separation, immunochromatography, immunosensor, microsphere array, lab-on-a-chip/disc platforms increasingly in combination with nanotechnologies, are discussed. The heuristic development of portable and ambulant microfluidic devices is intriguing and promising. Tant pis, many new platforms seem unattainable as the industry standard. Comparability of results with those of reference methods hinders the implementation of new technologies. Whatever the scientific and technological excellence and incentives, the decision-maker determines this implementation after weighing mainly costs and business risks

Detection of egg yolk antibodies reflecting Salmonella enteritidis infections using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor assay was developed on the basis of a lipopolysaccharide antigen of Salmonella enterica serovar enteritidis (S. enterica serovar enteritidis) to detect egg yolk antibodies against S. enterica serovar enteritidis. This biosensor assay was compared to two commercial ELISA kits based on LPS antigen and flagellar antigen. A number of 163 egg yolk and combined egg white and yolk samples from chickens experimentally infected with S. enterica serovar enteritidis and 90 egg yolk and combined egg white and yolk samples from uninfected chickens were analyzed. Receiver operating characteristic analysis of the data calculated a diagnostic sensitivity of 82% and a diagnostic specificity of 100%. The within-day coefficient of variation of a positive internal-control egg yolk was 1%. The SPR biosensor assay was able to detect antibodies in a significantly higher percentage of known positive samples than the commercial ELISA's. The anticipated use of the SPR biosensor assay is to determine the S. enterica serovar enteritidis serostatus of non-vaccinated layer hen

Bayesian estimation of diagnostic accuracy of a new bead-based antibody detection test to reveal Toxoplasma gondii infections in pig populations

The success of a Toxoplasma gondii surveillance program in European pig production systems depends partly on the quality of the test to detect infection in the population. The test accuracy of a recently developed serological bead-based assay (BBA) was investigated earlier using sera from experimentally infected animals. In this study, the accuracy of the BBA was determined by the use of sera from animals from two field subpopulations. As no T. gondii infection information of these animals was available, test accuracy was determined through a Bayesian approach allowing for conditional dependency between BBA and an ELISA test. The priors for prevalence were based on available information from literature, whereas for specificity vague non-informative priors were used. Priors for sensitivity were based either on available information or specified as non-informative. Posterior estimates for BBA sensitivity and specificity were (mode) 0.855 (Bayesian 95% credibility interval (bCI) 0.702-0.960) and 0.913 (bCI 0.893-0.931), respectively. Comparing the results of BBA and ELISA, sensitivity was higher for the BBA while specificity was higher for ELISA. Alternative priors for the sensitivity affected posterior estimates for sensitivity of both BBA and ELISA, but not for specificity. Because the difference in prevalence between the two subpopulations is small, and the number of infected animals is small as well, the precision of the posterior estimates for sensitivity may be less accurate in comparison to the estimates for specificity. The estimated value for specificity of BBA is at least optimally defined for testing pigs from conventional and organic Dutch farms

Bayesian estimation of diagnostic accuracy of a new bead-based antibody detection test to reveal Toxoplasma gondii infections in pig populations

The success of a Toxoplasma gondii surveillance program in European pig production systems depends partly on the quality of the test to detect infection in the population. The test accuracy of a recently developed serological bead-based assay (BBA) was investigated earlier using sera from experimentally infected animals. In this study, the accuracy of the BBA was determined by the use of sera from animals from two field subpopulations. As no T. gondii infection information of these animals was available, test accuracy was determined through a Bayesian approach allowing for conditional dependency between BBA and an ELISA test. The priors for prevalence were based on available information from literature, whereas for specificity vague non-informative priors were used. Priors for sensitivity were based either on available information or specified as non-informative. Posterior estimates for BBA sensitivity and specificity were (mode) 0.855 (Bayesian 95% credibility interval (bCI) 0.702-0.960) and 0.913 (bCI 0.893-0.931), respectively. Comparing the results of BBA and ELISA, sensitivity was higher for the BBA while specificity was higher for ELISA. Alternative priors for the sensitivity affected posterior estimates for sensitivity of both BBA and ELISA, but not for specificity. Because the difference in prevalence between the two subpopulations is small, and the number of infected animals is small as well, the precision of the posterior estimates for sensitivity may be less accurate in comparison to the estimates for specificity. The estimated value for specificity of BBA is at least optimally defined for testing pigs from conventional and organic Dutch farms

Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078

Since 2005, an increase in the prevalence of Clostridium difficile infection (CDI) due to polymerase chain reaction ribotype 078 has been noticed in The Netherlands. This strain has also been identified as the predominant strain in pigs and calves. CDI caused by type 078 was studied in relation to CDI caused by the hypervirulent type 027 and by types other than 027 and 078. Human and porcine isolates were further investigated and characterized by multilocus variable number tandem repeat analysis. From February 2005 through February 2008, the incidence of type 078 among isolates obtained from 1687 patients increased from 3% to 13%. Compared with patients infected with type 027, patients infected with type 078 were younger (67.4 vs. 73.5 years; P <.01) and more frequently had community-associated disease (17.5% vs. 6.7%; odds ratio, 2.98; 95% confidence interval, 2.11-8.02); rates of severe diarrhea (38.9% vs. 40.0%) and attributable mortality (3.8% vs. 4.0%) were similar in both groups. Compared with patients infected with other types, patients infected with type 078 more frequently received fluoroquinolone therapy (29.4% vs. 19.8%; odds ratio, 2.17; 95% confidence interval, 1.06-4.44). Type 078 isolates contained genes for toxin A, toxin B, binary toxin, and a 39-base pair deletion in toxin regulator gene (tcdC), as well as a point mutation at position 184, resulting in a stop codon. Multilocus variable number tandem repeat analysis of 54 human and 11 porcine isolates revealed 4 clonal complexes containing both porcine and human isolates. CDI due to type 078 and CDI due to type 027 present with similar severity, but CDI due to type 078 affects a younger population and is more frequently community associated. C. difficile type 078 isolates from humans and pigs are highly genetically relate