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For H. Whole-cell antigen from a sonicated pool Ultrasonic Disruptor QRW, Ultronique, Brazil of 56 strains was obtained as previously described. Detection of anti- H.
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Standardization was performed as previously described by Camorlinga-Ponce et al. ELISA plates were prepared as previously described. Polyclonal and monoclonal antibodies were used to evaluate the best detection test. Polyclonal antibodies comprise collections of antibodies from different B cells that recognize multiple epitopes on the same antigen. For this reason, they provide greater sensitivity, even for detecting proteins that are present in low quantities in a sample.
In contrast, monoclonal antibodies comprise antibodies from a single antibody-producing B cell. Thus, they bind with a single epitope. This is highly specific, with only a small risk of cross-reactivity, and it can provide better results in assays requiring quantification of the protein levels. Absorbance was read at nm Multiskan FC microplate photometer. All reactions were performed in triplicate, and the mean of three optical density OD measurements was used. For monoclonal antibodies, the following were the optimal concentrations: for antigen preparation, 3.
The best reading time for monoclonal antibodies was 25 minutes. ELISA tests were run for four consecutive days to evaluate interoperability and reproducibility. These reactions were performed through competitive inhibition assays, as previously described. The optical density OD cutoffs for the monoclonal and polyclonal tests were determined based on the serum antibody responses of samples 50 adults and 50 children and adolescents from H.
Thus, a pool of these serum samples was used as the negative serum control. The threshold for positivity was defined as the mean value plus three standard deviations of the optical density, as previously described. These values were compared with the values from a collection of 82 serum samples 33 adults and 49 children and adolescents from H. A pool of these 82 samples was used as the positive serum control.
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During the testing of the unknown samples, a positive serum pool was included in quadruplicate in every plate and the mean of the four OD values was used to calculate the threshold for that plate. Peripheral blood samples 10 ml were collected by means of venous puncture from patients on whom esophagogastroduodenoscopy had been performed. Six gastric biopsies were taken for the rapid urease test and for histological evaluation one from the gastric body and one from the antrum, for each of these , and for culturing two from the antrum.
The procedures were performed as previously described. Qualitative variables were described in terms of their proportions, and quantitative variables were described in terms of their means and standard deviations. Analyses on continuous variables were based on the negative gold standard negative rapid urease test, histological evaluation and culturing.
The threshold for positivity was defined as the mean value plus three standard deviations of the optical density. OD over the established cutoff value. The OD for each serum sample was determined in triplicate.
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The optimal OD cutoff was determined based on receiver operating characteristic ROC curves using different sensitivity and specificity values, and the area under the ROC curve AUC was estimated taking the significance level to be an accuracy value of 0. The desirable AUC was considered to be 0.
Thus, for this study, the sample size was estimated to be 78 adults and 95 children. The sensitivity, specificity, accuracy and positive and negative predictive values of the in-house ELISA serological tests were evaluated by comparing OD values, based on cutoffs that were determined using the ROC curve, against the gold standard rapid urease test, histological evaluation and culturing. Negative and positive likelihood ratios were evaluated. To validate our in-house serological test, consecutive dyspeptic patients, for whom the H. Pylori infection status was unknown were chosen: 96 children and adolescents age range years; mean: The serum samples that were negative in the monoclonal test presented OD values ranging from 0.
The threshold for positivity was 0. The serum samples that were negative in the polyclonal test presented OD values ranging from 0. The serum samples 82 samples that were positive in the monoclonal test presented OD values ranging from 0.
The serum samples 82 samples that were positive in the polyclonal test presented OD values ranging from 0. Despite the threshold for positivity based on the mean for the negative results plus 3 SD in the monoclonal test 0. The optimal OD cutoff value was 0. When age was considered, the monoclonal test presented a better result for sensitivity among adults, but specificity was better among children and adolescents. In contrast, the polyclonal test presented a better result for sensitivity among children and adolescents.
The accuracy was slightly better among children and adolescents in the monoclonal test and among adults in the polyclonal test Table 1. The sensitivity and specificity of the in-house serological tests for both polyclonal antibodies The in-house ELISA serological test that we developed did not present any cross-reactivity to heterologous bacterial species.
Selectivity in relation to gastroduodenal diseases can influence the sensitivity and specificity results. A study conducted by Aziz et al.
In the present study, adults, children and adolescents were evaluated together, and the OD results presented little variation. However, other studies conducted on Brazilian populations have shown that age had an influence on the sensitivity and specificity of commercial tests. Both commercial and in-house serological tests have presented large ranges of sensitivity and specificity results.
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Leal et al. There were 33 studies using 19 different commercial tests and nine studies using in-house tests. They observed that the mean sensitivity was The accuracy of serological tests can also be affected by the choice of antigen that is used to prepare the in-house ELISA assay. In some studies, H. The genotypic distribution in our study was similar to what has been found in other studies in Brazil. The diagnostic accuracy of a serological test is enhanced when the antigen pool is prepared based on multiple strains.
Widmer et al. Serological tests based on whole-cell antigen or whole-cell antigen lysate have also shown good performance among both children and adult patients. In particular, the optimal antigen concentration, serum sample dilution and antibody conjugation need to be determined.
These parameters should be optimized and adjusted to local conditions, to the population studied and to the method developed. It is very important to make these determinations because of false-positive titration results caused by high concentrations of antigens, which promote nonspecific bonds, even in negative serum. Moreover, low antigen concentrations can result in false negatives. In addition, high concentrations of conjugated antibodies may result in nonspecific reading, while low concentrations cause decreased OD.
The serum concentration can also interfere with the OD results. This study was the first on our population to evaluate the serological response to anti- H.
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Ideal tests for screening purposes should present high sensitivity and good specificity. This indicates that these tests are suitable for population-based screening, especially the polyclonal test, which is characterized by greater sensitivity. On the other hand, although the higher specificity among adults in the polyclonal test, with low occurrence of false-negative results, is desirable for a diagnostic method, it is not necessary for a screening test. The in-house polyclonal serological test that was developed using local strains in-house presented better diagnostic performance than did the monoclonal test for children and adolescents.
In contrast, the monoclonal test was better among adults.
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The results relating to accuracy, sensitivity, specificity and likelihood ratios from both tests suggest that these in-house serological tests could be used to detect anti- H. Go MF. Review article: natural history and epidemiology of Helicobacter pylori infection. Aliment Pharmacol Ther. PMID: Epidemiology of Helicobacter pylori infection. Crit Care. Catheter related blood stream infections in critically ill patients with continuous haemo dia filtration and temporary non-tunnelled vascular access.
Clostridium difficile infection in Europe: a hospital-based survey. Comparison of the DiversiLab repetitive element PCR system with spa typing and pulsed-field gel electrophoresis for clonal characterization of methicillin-resistant Staphylococcus aureus. Emergence of glutaraldehyde-resistant Pseudomonas aeruginosa. Improved detection of microbial ureteral stent colonisation by sonication.
Infection control--a European research perspective for the next decade. Lack of evidence for attributing chlorhexidine as the main active ingredient in skin antiseptics preventing surgical site infections. Letter 2: Systematic review and meta-analysis of preoperative anti- sepsis with chlorhexidine versus povidone-iodine in clean-contaminated surgery Br J Surg ; Br J Surg.
Livestock-associated methicillin-resistant Staphylococcus aureus in humans, Europe. Overall burden of healthcare-associated infections among surgical patients.