Enzyme-linked Fluorescent Immunoassay Research Articles

Susceptibility to varicella zoster of children with cystic fibrosis

Varicella zoster virus (VZV) infection can result in significant morbidity in patients with cystic fibrosis (CF), and the risk of severe pulmonary complications rises with age [[1]MacDonald N.E. Morris R.F. Beaudry P.H. Varicella in children with cystic fibrosis.Pediatr Infect Dis J. 1987; 6: 414-416Crossref PubMed Scopus (10) Google Scholar]. This is a greater concern in those receiving systemic corticosteroids, which in the case of CF is most often given for acute allergic bronchopulmonary aspergillosis (ABPA). Severe complications such as pneumonitis, secondary bacterial infections, and liver failure may occur despite prompt treatment with antiviral agents. The seroprevalence of VZV antibodies in CF children is unknown, although in the general population >80% of children over 6 years of age have evidence of immunity [[2]Vyse A.J. Gay N.J. Hesketh L.M. Morgan-Capner P. Miller E. Seroprevalence of antibody to varicella zoster virus in England and Wales in children and young adults.Epidemiol Infect. 2004; 132: 1129-1134Crossref PubMed Scopus (60) Google Scholar]. We aimed to investigate the seroprevalence of VZV antibodies in children attending our large paediatric specialist CF centre, whether or not there was a history of chicken pox. In 2011 we started routine testing for VZV antibodies in 6 year olds at their annual review. This age was selected in view of the low prevalence of ABPA under that age and the chance that children would already have had chicken pox. Evidence of immunity to VZV was determined by VZV IgG antibodies ≥0.9 IU/L (measured by a VIDAS VZV IgG enzyme-linked fluorescent immunoassay). In 2012 the prevalence of ABPA in our CF clinic was 9%, 96% of which was aged 6 and above. As this was a new policy, we also tested older children in order to cover the whole clinic. We identified all children in our CF clinic aged ≥6 years at August 2012, and collected data on VZV immunity 12 months later, thus ensuring that children aged ≥6 years at the initial time-point would have had an annual review. We asked general practitioners to vaccinate patients with a negative result. There were 232 patients aged ≥6 years. 178 patients (77%) in this cohort had been tested for evidence of VZV immunity within the year. Of these, n = 22 (12%) had no evidence of VZV antibodies and were therefore susceptible to chickenpox (Fig. 1). In our paediatric CF population, the prevalence of VZV antibodies was similar to that reported for the general population. However, unlike the general population, children with CF are at greater risk of serious complications from varicella infection. In contrast to other groups of patients with chronic diseases whose treatment may result in immunosuppression, there is no UK-wide specific recommendation for VZV immunization in CF. Vaccination against VZV before immune compromise has been used as a strategy to prevent VZV complications in patients starting immunosuppressive therapy [[3]Levin M.J. Varicella vaccination of immunocompromised children.J Infect Dis. 2008; 197: S200-S206Crossref PubMed Scopus (47) Google Scholar]. We suggest screening for VZV antibodies in children with CF at 6 years and offering vaccination for those found to be seronegative.

Evaluation of the Diagnostic Performance of the XpertClostridium difficileAssay and Its Comparison With the Toxin A/B Enzyme-Linked Fluorescent Assay and In-House Real-Time PCR Assay Used for the Detection of ToxigenicC. difficile

Clostridium difficile genes or toxin can be detected using several laboratory techniques. In this study, we compared the performance of the Xpert C. difficile assay with that of a toxin A/B enzyme-linked fluorescent immunoassay (ELFA) and an in-house real-time PCR assay for the tcdB gene. From April 2011 through January 2012, 138 soft or liquid stool samples from 138 adult patients at Paik Hospital were tested using the toxin A/B ELFA, in-house real-time PCR assay, and Xpert C. difficile assay to detect toxigenic C. difficile. Specimens were considered true positives if results were positive in both the in-house real-time PCR for tcdB gene and Xpert C. difficile assays. Sensitivity of the toxin A/B ELFA, in-house tcdB gene real-time PCR, and Xpert C. difficile assay were 67.6%, 97.3%, and 100.0%, respectively. The specificity of the in-house tcdB gene real-time PCR assay was 100%, while the specificity was 98.0% for the other two methods. The turnaround time (TAT) was 50 min for the Xpert C. difficile assay, 75 min for the toxin A/B ELFA, and 160 min for the in-house real-time PCR assay. The Xpert C. difficile assay and the in-house real-time PCR assay had higher sensitivity than the toxin A/B ELFA; however, the specificities of the three assays were similar. Considering its rapid TAT and high sensitivity, use of the Xpert C. difficile assay is highly recommended for rapid and accurate diagnosis of C. difficile infection.

Evaluation of 4 methods for the serological diagnosis of Epstein-Barr virus infection using an immunofluorescence assay as the reference method.

Tests specific for VCA IgM, VCA IgG, and EBNA IgG are used to diagnose Epstein-Barr virus (EBV) infections and interpret disease status. The immunofluorescence assay (IFA) is accepted as the "gold standard" test. The purpose of this study was to evaluate the performance of 4 methods in comparison with IFA. In total, 101 serum samples were obtained from clinically suspected cases of EBV infection between May 2010 and May 2012 and evaluated by IFA. All serum samples were analyzed by an immunoblot assay, enzyme-linked fluorescent assay (ELFA), enzyme immunoassay (EIA), and immunochromatographic assay (ICA). ELFA and ICA results were in good agreement with IFA for the detection ofVCA IgM, VCA IgG, and EBNA IgG. The results of the immunoblot assay agreed less well with IFA for EBNA IgG, while EIA results were not in agreement with IFA for EBNA IgG or VCA IgM. Among the tests studied, ELFA and ICA appear to be suitable methods for the diagnosis and staging of EBV when considering cost-effectiveness, turnaround times, need for a specialist, and IFA concordance.

Comparative Evaluation of the VIDAS®Listeria monocytogenes Xpress (LMX) for the Detection of Listeria monocytogenes in a Variety of Foods

The VIDAS Listeria monocytogenes Xpress (LMX) test is an enzyme-linked fluorescent immunoassay designed for use with the automated VIDAS or mini-VIDAS instruments for the specific detection of L. monocytogenes using a 26 h proprietary enrichment broth. The VIDAS LMX method was validated according to harmonized AOAC Research Institute (RI) and Official Methods of Analysis guidelines in both the AOAC Performance Tested Method (PTM) and GovVal programs. In the PTM comparison studies, the VIDAS LMX method was compared to the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook, the U.S. Food and Drug Administration Bacteriological Analytical Manual, and AOAC Official Methods. The comparative food studies consisted of two main parts: internal testing and AOAC independent laboratory testing, which included seven food matrixes (deli ham, processed cheese, vanilla ice cream, cooked shrimp, smoked white fish, frozen spinach, and peanut butter). As part of the AOAC R1 GovVal program, the VIDAS LMX method was compared to the Health Canada MFHPB-30 method for the detection of L. monocytogenes in five ready-to-eat (RTE) meats (hot dogs, deli turkey, deli ham, fermented sausage, and liver paté). Twenty replicates of each inoculation level and five uninoculated controls were evaluated in each study. The LMX method also included the use ofchromogenic media, chromID Ottaviani Agosti agar and chromID L. mono. agar, for confirmation of LMX presumptive results. In both the PTM and GovVal evaluations, there were no significant differences in the Chi-square values for the LMX method when compared to reference methods. The additional parameters tested in the PTM evaluation (inclusivity, exclusivity, ruggedness, stability, and lot-to-lot) satisfied the AOAC RI performance requirements. In both the PTM and GovVal validation studies, the VIDAS LMX method demonstrated reliability as a rapid qualitative method for next-day detection of L. monocytogenes in a variety of foods, including RTE meats.

VIDAS®Listeria monocytogenes II (LMO2)

This AOAC GovVal study compared the VIDAS Listeria monocytogenes II (LMO2) to the Health Products and Food Branch MFHPB-30 reference method for detection of L. monocytogenes in ready-to-eat (RTE) meats. The VIDAS LMO2 test is an automated enzyme-linked fluorescent immunoassay for the detection of L. monocytogenes in foods. The LMO2 test, following the enrichment procedure from the MFLP-33 method, also included use of the chromogenic media, chromID Ottaviani Agosti Agar (OAA) and chromID Lmono for confirmation of LMO2 presumptive results. In previous AOAC validation studies comparing the VIDAS LMO2 method to the U.S. Food and Drug Administration Bacteriological Analytical Manual and U.S. Department of Agriculture-Food Safety and Inspection Service reference methods, LMO2 was approved as AOAC Official Method 2004.02 for the detection of L. monocytogenes in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry. The GovVal comparative study included 20 replicate test portions, each at two contamination levels for each matrix, where fractionally positive results (5-15 positive results/20 replicate portions tested) were obtained by at least one method at one level. Five uncontaminated controls were included. Chi-square analysis of the comparative data in this study indicates no statistical differences between the VIDAS LMO2 and the MFHPB-30 standard methods at the 5% level of significance. Confirmation of presumptive LMO2 results with the chromogenic OAA and Lmono media was shown to be equivalent to the appropriate reference method agars. The data demonstrate that the VIDAS LMO2 method is an acceptable alternative method to the MFHPB-30 standard culture method for the detection of L. monocytogenes in RTE meats, including liver paté, hot dogs, raw fermented sausage, sliced deli turkey, and sliced deli ham.

A Novel Solid Substrate Room Temperature Phosphorimetry for the Determination of Trace Cytochrome C and Forecast of Human Diseases

The reaction between fullerenol (F-ol) and cytochrome C (Cyt C) could be carried out to form a nonphosphorescence compound using tween-80 as photosensitizer which causes the sharp quenching of room temperature phosphorescence (RTP) of F-ol. Bearing this in mind, a novel solid substrate room temperature phosphorimetry (SSRTP) for the determination of trace Cyt C has been proposed in this study. Under the optimum conditions, the linear range of this method is 1.0×10-16 –2.4×10-14 g mL-1, which is directly proportional to ΔIp of Cyt C-F-ol–tween-80 system, and the detection limit (DL) is 3.0×10−17 g mL-1. It has been applied to the determination of Cyt C in human serum and forecast of diseases, and the result matches with the enzyme-linked fluorescence immunoassay (ELISA). Meanwhile, the reaction mechanism of SSRTP for the determination of Cyt C and the enhancing effect of tween-80 on RTP of F-ol were also discussed.

Improving ante mortem diagnosis of Erysipelothrix rhusiopathiae infection by use of oral fluids for bacterial, nucleic acid, and antibody detection

Swine erysipelas is an economically important disease caused by Erysipelothrix rhusiopathiae. Pen-based collection of oral fluids has recently been utilized for monitoring infection dynamics in swine operations. The diagnostic performance of bacterial isolation, real-time PCR, and antibody detection by enzyme-linked immunosorbent assay (ELISA) and fluorescent microbead-based immunoassay (FMIA) methods were evaluated on pen-based oral fluid samples from pigs experimentally infected with E. rhusiopathiae (n=112) and from negative controls (n=32). While real-time PCR was a sensitive method with an overall detection rate of 100% (7/7 pens) one day post inoculation (dpi), E. rhusiopathiae was successfully isolated in only 28.6% (2/7 pens). Anti-Erysipelothrix IgM and IgG antibodies in pen-based oral fluids were detected at 4 to 5 dpi by FMIA and at 5 and 8 dpi by ELISA. The number of infected animals per pen, and in particular the timing of antimicrobial treatment administration impacted bacterial isolation and ELISA results. In oral fluid field samples, E. rhusiopathiae DNA was found in 23.3% of the samples while anti-E. rhusiopathiae IgG and IgM antibodies were found in 59.6% and 5.5% of the samples, respectively. The results suggest that an algorithm integrating oral fluids as specimen and real-time PCR and FMIA as detection methods is effective for earlier detection of an erysipelas outbreak thereby allowing for a more effective treatment outcome.

Detection of Clostridium difficile toxin A/B genes by multiplex real-time PCR for the diagnosis of C. difficile infection

Toxigenic Clostridium difficile culture is considered to be the standard diagnostic method for the detection of C. difficile infection (CDI). Culture methods are time-consuming and although enzyme immunoassay is rapid and easy to use, it has low sensitivity. In the present study, the AdvanSure CD real-time (RT)-PCR kit (LG Life Sciences) was evaluated for its ability to detect C. difficile toxin A (tcdA) and B (tcdB) genes, simultaneously. A total of 127 fresh diarrhoeal stool specimens, submitted to the clinical microbiology laboratory for C. difficile culture, were tested. C. difficile toxins and toxin genes were detected with a VIDAS C. difficile A&B (VIDAS-CDAB) enzyme-linked fluorescent immunoassay (ELFA) and the AdvanSure RT-PCR kit, respectively, according to the manufacturers' instructions. Their performance was compared with a standard toxigenic culture method as a reference. The sensitivity, specificity and positive and negative predictive values using the AdvanSure RT-PCR kit were 100 %, 98.3 %, 84.6 % and 100 %, respectively, while those of the VIDAS-CDAB system were 63.6 %, 100 %, 100 % and 96.6 %, respectively. Four tcdA(+)/tcdB(+) strains of C. difficile were detected with the AdvanSure RT-PCR kit, which offers comparable sensitivity and specificity to the reference method with a turnaround time of ~3 hours.

Biosensors for the analysis of microbiological and chemical contaminants in food

Increases in food production and the ever-present threat of food contamination from microbiological and chemical sources have led the food industry and regulators to pursue rapid, inexpensive methods of analysis to safeguard the health and safety of the consumer. Although sophisticated techniques such as chromatography and spectrometry provide more accurate and conclusive results, screening tests allow a much higher throughput of samples at a lower cost and with less operator training, so larger numbers of samples can be analysed. Biosensors combine a biological recognition element (enzyme, antibody, receptor) with a transducer to produce a measurable signal proportional to the extent of interaction between the recognition element and the analyte. The different uses of the biosensing instrumentation available today are extremely varied, with food analysis as an emerging and growing application. The advantages offered by biosensors over other screening methods such as radioimmunoassay, enzyme-linked immunosorbent assay, fluorescence immunoassay and luminescence immunoassay, with respect to food analysis, include automation, improved reproducibility, speed of analysis and real-time analysis. This article will provide a brief footing in history before reviewing the latest developments in biosensor applications for analysis of food contaminants (January 2007 to December 2010), focusing on the detection of pathogens, toxins, pesticides and veterinary drug residues by biosensors, with emphasis on articles showing data in food matrices. The main areas of development common to these groups of contaminants include multiplexing, the ability to simultaneously analyse a sample for more than one contaminant and portability. Biosensors currently have an important role in food safety; further advances in the technology, reagents and sample handling will surely reinforce this position.

Effects of quetiapine on body mass index and reproductive hormonal levels in male schizophrenic patients

Objective: To asses the effects of quetiapine therapy on body mass index (BMI) and serum level of testosterone, prolactin, follicle stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) in newly diagnosed male schizophrenic patients in comparison to healthy controls. Patients and methods: Thirty male patients with schizophrenia were included in this study. The diagnosis of schizophrenia was made according to DSM-IV criteria of the American Psychiatric Association (APA). Another thirty apparently healthy male individuals were included in the study as a control group. Blood samples were taken initially from patients and controls and assay of serum testosterone, prolactin, FSH, LH, and E2 were done using enzyme –linked fluorescent immunoassay (ELFA) technique by vidas instrument. Later, after 6 months of quetiapine therapy, other blood samples were taken and assay of the same parameters were done. Calculation of BMI was done for the patients and controls using special equation. Results: There was an insignificant difference in the mean BMI, serum testosterone, prolactin, FSH, LH, and E2 levels between patients in the pre-therapy stage group and in both the post-therapy stage group and controls. Conclusion: Chronic quetiapine therapy might be regarded as a safe drug with regard to effects on BMI and serum levels of reproductive hormones in newly diagnosed male schizophrenic patients. Keywords: Schizophrenia, quetiapine, BMI, testosterone, prolactin, FSH, LH, E2.

Evaluation of VIDAS®Salmonella (SLM) Easy Salmonella Method for the Detection of Salmonella in a Variety of Foods: Collaborative Study

The VIDAS Salmonella (SLM) Easy Salmonella method is a specific enzyme-linked fluorescent immunoassay performed in the automated VIDAS instrument. The VIDAS Easy Salmonella method is a simple 2-step enrichment procedure, using pre-enrichment followed by selective enrichment in a newly formulated broth, SX2 broth. This new method was compared in a multilaboratory collaborative study to the U.S. Food and Drug Administration's Bacteriological Analytical Manual, Chapter 5 method for five food matrixes (liquid egg, vanilla ice cream, spinach, raw shrimp, and peanut butter) and the U.S. Department of Agriculture's Microbiology Laboratory Guidebook 4.04 method for deli turkey. Each food type was artificially contaminated with Salmonella at three inoculation levels. A total of 15 laboratories representing government, academia, and industry, throughout the United States, participated. In this study, 1583 samples were analyzed, of which 792 were paired replicates and 791 were unpaired replicates. Of the 792 paired replicates, 285 were positive by both the VIDAS and reference methods. Of the 791 unpaired replicates, 341 were positive by the VIDAS method and 325 were positive by the cultural reference method. A Chi-square analysis of each of the six food types was performed at the three inoculation levels tested. For all foods evaluated, the VIDAS Easy SLM method demonstrated results comparable to those of the reference methods for the detection of Salmonella.

Wild Boars as an Important Reservoir for Foodborne Pathogens

One hundred fifty-three wild boars shot in the canton of Geneva, Switzerland, were studied for the occurrence of foodborne pathogens. Tonsils and fecal samples of the animals were examined using real-time polymerase chain reaction, enzyme-linked fluorescent immunoassay, and cultural methods. The detection rate of Salmonella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, stx-positive Escherichia coli, and Listeria monocytogenes was 12%, 35%, 20%, 9%, and 17%, respectively, when tonsil samples were studied. Only Y. enterocolitica (5%) and L. monocytogenes (1%) were detected in fecal samples. None of the samples was positive for Campylobacter spp. Females (71%) and young animals (61%) carried more frequently one or more pathogens than males (53%) and older ones (44%). In total, 8 Salmonella spp., 14 Y. enterocolitica, 4 Y. pseudotuberculosis, and 26 L. monocytogenes strains were further characterized. Most of the Salmonella spp. strains were of serotype Salmonella Enteritidis (75%) followed by serotypes Salmonella Stourbridge (13%) and Salmonella Veneziana (13%). L. monocytogenes strains belonged to serotypes 1/2a (42%), 1/2b (19%), and 4b (38%). Serotypes O:3 (36%), O:5,27 (21%), and O:9 (29%) were identified among Y. enterocolitica strains and serotypes O:1 (75%) and O:2 (25%) among Y. pseudotuberculosis strains. This study shows that wild boars are frequent carriers of foodborne pathogens. High wild boar densities and increasing popularity of outdoor ranging of pigs may intensify the risk of transmission of these pathogens to fattening pigs.

Evaluation of an Enzyme-Linked Fluorescent Assay for the Detection of Listeria monocytogenes from Food

The VIDAS Listeria monocytogenes II (LMO2) method, which is an automated enzyme-linked fluorescent immunoassay (ELFA), was used for rapidly, specifically and sensitively detecting L. monocytogenes in food samples. All 31 L. monocytogenes strains examined gave positive results. The other bacterial species except Salmonella showed completely negative results, but it was suspected that Salmonella spp. had given a false-positive reaction to the assay. As the detectable limit of the assay was 10⁶ cfu/ml in a food suspension, food samples were required to be enriched in order to increase the number of the bacteria to the detectable limit. However, the ELFA was reconfirmed to be applied satisfactorily as a rapid and precise method for the detection of L. monocytogenes in various food samples, even if the culture had to be enriched for 12 h prior to the assay.

Comparison of a Rapid Molecular Method, the BD GeneOhm Cdiff Assay, to the Most Frequently Used Laboratory Tests for Detection of Toxin-Producing Clostridium difficile in Diarrheal Feces

Six hundred diarrheal stool specimens were collected from inpatients and outpatients at local university hospitals for the detection of toxigenic Clostridium difficile using three parallel methods, the BD GeneOhm Cdiff assay, the tissue culture cytotoxicity assay, and a commercially available enzyme-linked fluorescence immunoassay (ELFA) (Vidas C. difficile toxin A and B assay; bioMérieux). Toxigenic C. difficile culture was also performed to further clarify discordant results. During a 3-month study period, 58 (9.7%) of the 600 diarrheal samples examined were positive by the BD GeneOhm Cdiff assay, while the Vidas C. difficile toxin A and B assay and the cytotoxicity assay performed directly on stool samples gave 4.7% and 6.3% positivity rates, respectively. In the case of four samples, BD GeneOhm Cdiff assay results were not evaluable at first because of the presence of PCR inhibitors, but upon repeat testing from the frozen lysates, all of these samples proved to be negative. After resolution with toxigenic culture, the cytotoxicity assay proved to be positive in 55 samples (9.2%), while the ELFA was positive in 37 samples (6.2%). Results of culture and repeated cytotoxicity assays emphasized the importance of the culture method, because the use of ELFA or enzyme immunoassay without a culture method may lead to a substantial portion of toxigenic C. difficile strains being missed.

Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.

Faecal shedding and strain diversity of Listeria monocytogenesin healthy ruminants and swine in Northern Spain

BackgroundListeria monocytogenes is among the most important foodborne bacterial pathogens due to the high mortality rate and severity of the infection. L. monocytogenes is a ubiquitous organism occasionally present in the intestinal tract of various animal species and faecal shedding by asymptomatically infected livestock poses a risk for contamination of farm environments and raw food at the pre-harvest stages. The aim of this study was to determine the prevalence and strain diversity of L. monocytogenes in healthy ruminants and swine herds.ResultsFaecal samples from 30 animals per herd were collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country and screened in pools by an automated enzyme-linked fluorescent immunoassay (VIDAS®) to estimate the prevalence of positive herds. Positive samples were subcultured onto the selective and differential agar ALOA and biochemically confirmed. L. monocytogenes was isolated from 46.3% of dairy cattle, 30.6% beef cattle and 14.2% sheep herds, but not from swine. Within-herd prevalence investigated by individually analysing 197 sheep and 221 cattle detected 1.5% of faecal shedders in sheep and 21.3% in cattle. Serotyping of 114 isolates identified complex 4b as the most prevalent (84.2%), followed by 1/2a (13.2%), and PFGE analysis of 68 isolates showed a highly diverse L. monocytogenes population in ruminant herds.ConclusionThese results suggested that cattle represent a potentially important reservoir for L. monocytogenes in the Basque Country, and highlighted the complexity of pathogen control at the farm level.

Biomarker evidence for mild central nervous system injury after surgically-induced circulation arrest

Previously, we identified 14-3-3 β and ζ isoforms and proteolytic fragments of α-spectrin as proteins released from degenerating neurons that also rise markedly in cerebrospinal fluid (CSF) following experimental brain injury or ischemia in rodents, but these proteins have not been studied before as potential biomarkers for ischemic central nervous system injury in humans. Here we describe longitudinal analysis of these proteins along with the neuron-enriched hypophosphorylated neurofilament H (pNFH) and the deubiquitinating enzyme UCH-L1 in lumbar CSF samples from 19 surgical cases of aortic aneurysm repair, 7 involving cardiopulmonary bypass with deep hypothermic circulatory arrest (DHCA). CSF levels of the proteins were near the lower limit of detection by Western blot or enzyme-linked fluorescence immunoassay at the onset of surgical procedures, but increased substantially in a subset of cases, typically within 12–24 h. All cases involving DHCA were characterized by > 3-fold elevations in CSF levels of the two 14-3-3 isoforms, UCH-L1, and pNFH. Six of 7 also exhibited marked increases in α-spectrin fragments generated by calpain, a protease known to trigger necrotic neurodegeneration. Among cases involving aortic cross-clamping but not DHCA, the proteins rose in CSF preferentially in the subset experiencing acute neurological complications. Our results suggest the neuron-enriched 14-3-3β, 14-3-3ζ, pNFH, UCH-L1, and calpain-cleaved α-spectrin may serve as a panel of biomarkers with clinical potential for the detection and management of ischemic central nervous system injury, including for mild damage associated with surgically-induced circulation arrest.

Escherichia coli O157:H7 and Non‐O157 Shiga Toxin‐producing E. coli in Healthy Cattle, Sheep and Swine Herds in Northern Spain

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.

A rapid and sensitive immunoresonance scattering spectral assay for microalbumin

BackgroundMicroalbuminuria (MAU) is the earliest clinical finding for renal disease and a risk factor for hypertensive cardiovascular disease. Several methods, including enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoturbidimetry (IT), immunonephelometry (IN), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA) and time-resolved fluorescence (TRF) have been applied for detection of MAU. However, the resonance scattering (RS) spectral assay, based on the immunoreaction and its resonance scattering effect, has not been reported. MethodIn the presence of 75 mg/l polyethylene glycol (PEG), the immunoreaction of microalbumin (Malb) and its goat anti-human Malb antibody took place specifically in pH 4.4 buffer solution and aggregated to form immunocomplex particles that exhibit a strongest resonance scattering peak at 488 nm, and it was used to assay of Malb. ResultsThe RS intensity at 488 nm (ΔI) was proportional to the Malb concentration (C) in the range of 0.03–0.96 mg/l, the regression equation was ΔI=116.0C−2.1, the detection limit was 0.02 mg/l. Urine samples from 20 healthy subjects were assayed by this assay. The results were in agreement with those obtained with IT. ConclusionThis assay has been applied to detection of Malb in real samples, with simplicity, rapidity, high sensitivity and good selectivity.

Measles-Mumps-Rubella and Varicella Vaccine Responses in Extremely Preterm Infants

Extremely preterm infants mount lower antibody responses than term infants to several vaccines. The objective of this study was to measure the immunogenicity of measles-mumps-rubella and varicella vaccines in preterm and term children. Immune status before immunization and immune response after immunization with measles-mumps-rubella and varicella vaccines at 15 months of age were compared in 32 infants, 16 of whom were preterm (< 29 weeks' gestation) and 16 of whom were term (> or = 37 weeks' gestation) at birth. Blood was drawn before vaccination and 3 to 6 weeks thereafter. Measles antibody was measured by plaque reduction neutralization assay. Mumps and rubella immunoglobulin G were measured in available sera by enzyme-linked fluorescent immunoassay. Varicella immunoglobulin G was measured in available sera by glycoprotein enzyme-linked immunosorbent assay. Values that were above or below the assay limits were assigned values double or half those limits, respectively. The primary outcome was the geometric mean antibody titer. Preterm children had lower mumps and rubella geometric mean titers than did term children before vaccine, and nearly all children were seronegative for each of the 4 vaccine antigens before immunization. Measles, mumps, rubella, and varicella geometric mean titers were similar between groups after vaccine. All children were seropositive for measles after vaccine, whereas 13 of 14 preterm and 11 of 13 term children were seropositive for mumps, 13 of 14 preterm and 13 of 13 term children were seropositive for rubella, and 11 of 16 preterm and 9 of 15 term children were seropositive for varicella. Preterm children mounted antibody responses that were similar to those of term children after measles-mumps-rubella and varicella vaccines at 15 months of age.