Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

A comparison of viral microneutralization and haemagglutination inhibition assays as measures of seasonal inactivated influenza vaccine immunogenicity in the first year after reduced intensity conditioning, lymphocyte depleted allogeneic haematopoietic stem cell transplant.

Traditionally, immune response to influenza vaccines has been measured using the haemagglutination inhibition (HAI) assay. A broader repertoire of techniques including the sensitive viral microneutralization (VMN) assay is now recommended by the European Medicines Agency (EMA). Comparing HAI and VMN, we determined immune response to a trivalent 2015-2016 seasonal inactivated influenza vaccine (SIIV) administered to 28 recipients of allogeneic haematopoietic stem cell transplant (HSCT). Vaccination was within the first-year post-transplant at a median of 78.5 (24-363) days. The proportion of patients with baseline and post-vaccination HAI titres ≥ 1:40 were 28.6% and 25% for A(H1N1)pdm09, 14.3% at both timepoints for A(H3N2), and 32.1% and 25% for B(Phuket). Pre and Post-vaccination geometric mean titres(GMT) were higher by VMN than HAI for A(H1N1)pdm09 and A(H3N2), but lower for B(Phuket)(p=<0.05). Geometric mean ratios(GMR) of baseline and post-vaccination titres were similar by HAI and VMN(p > 0.05) for all components. A single seroconversion to A(H1N1) was detected by ELISA-VMN. None of patient age, lymphocyte count, days from transplant to vaccination, donor type, or graft-versus-host disease (GVHD) or immunosuppressive therapy (IST) at vaccination correlated with baseline or post-vaccination titres by either assay. This absence of seroresponse to SIIV in the first-year post HSCT highlights the need for novel immunogenic vaccination formulations and schedules in this high-risk population.

Quality control assessment for the serological diagnosis of dengue virus infections.

BACKGROUND: A major drawback of modern society's rapidly increasing mobility is the ease with which dangerous infections can be imported into Europe. Often these infections are not diagnosed because physicians are not familiar with the symptoms and laboratory tests are not always available in local diagnostic centres. Improving diagnostics is the most important step in detecting and dealing with these pathogens and quality control measures are, therefore, essential tools. OBJECTIVES: To assess the diagnosis of imported dengue virus infections in Europe by (1) running a pre-evaluation panel (four serum samples, sent out in 1999) and optimising sample preparation and shipping procedures and (2) initiating an External Quality Assurance (EQA) program (20 serum samples, sent out in 2002). STUDY DESIGN: All serum samples sent out were to be tested for the presence of dengue virus-specific IgM and IgG. For the pre-evaluation panel, four samples were distributed (one sample IgM+/IgG+, one sample IgM-/IgG+, two samples IgM-/IgG-) and for the EQA 20 samples (12 samples IgM+/IgG+, five samples IgM-/lgG+, one sample lgM+/IgG- two samples IgM-/IgG-). 13 laboratories took part in the pre-evaluation panel and 18 laboratories participated in the first EQA run. RESULTS: For the pre-evaluation panel, the participants reported concurrent and correct results for 88% of the IgG-positive samples and for 100% of the IgG-negative samples. The results for the IgM-positive sample were correct in 91% of the reported tests and in 97% of the IgM-negative samples. For the EQA, the participants reported concurrent and correct results for 71% of the IgG-positive samples and 89% of the IgG-negative samples. 58% concurrent and correct results were reported for the IgM-positive samples and 97% for the IgM-negative samples. CONCLUSIONS: The results presented here demonstrate the importance of quality measures for imported viral pathogens like dengue viruses and clearly indicate the need for improving the existing test systems.

A comparative study of serological techniques for detection of West Nile virus antibodies.

This study was conducted to compare different serological techniques namely enzyme-linked immunosorbent assay (ELISA), hemagglutination inhibition (HI), indirect immunofluorescence (IFT) and plaque reduction neutralization (PRNT), for detection of West Nile virus (WNV) antibodies in sera collected from inhabitants of a flooded village (Begiram, Minufiya Governorate). ELISA showed 45% while HI and IFT indicated 37.6 and 26.4% positive sera among the tested 178 sera taken from the flooded village, respectively. The results obtained by ELISA, HI and IFT of only 55 randomly chosen sera were compared with their PRNT results. ELISA was found to be more sensitive (83.8%) while IFT and HI were more specific (88.9% and 83.3%, respectively). The positive predictive values for the 3 tests were more than 80% while the negative predictive values were different for these tests: 66.7% for ELISA, 44.1% and 37% for HI and IFT, respectively. From this study it is concluded that for screening of population in an endemic area, two serological tests in combination can be used starting with ELISA (the more sensitive) followed by HI and/or IFT (the more specific) to exclude the false positive results.

Multicenter evaluation of five commercial rubella virus immunoglobulin G kits which report in international units per milliliter.

In a multicenter study, the consistency of international units expressed by five commercially available rubella virus immunoglobulin G kits was evaluated. The linearity and within-run and between-run precision were determined for each kit. All kits demonstrated good linearity and had within-run and between-run precision coefficients of variation ranging from 5.1 to 21.7% and from 9.5 to 51.0%, respectively. To compare the international units expressed, the results from 40 samples tested in duplicate were compared with the results of a reference enzyme immunoassay calibrated with World Health Organization international standard serum and a hemagglutination inhibition test. The results of the kits were plotted against those of the reference tests, and linear regression analysis was applied. The Pearson correlation coefficient ranged from 0.64 to 0.75 when the commercial kit results were compared with those of the reference enzyme immunoassay, indicating only a moderate degree of correlation. Therefore, the international units expressed by the commercial kits are insufficiently consistent to be of practical use in diagnostic clinical microbiology.

Rubella virus antibodies in women of childbearing age.

Rubella is a common contagious disease with mild constitutional symptoms, but when it occurs during pregnancy there is significant risk of severe damage to the fetus. A study was undertaken to determine the level of rubella virus antibodies in females in the childbearing age (from 15-40). A total of one hundred and thirty sera were examined for rubella-specific IgG antibodies by hemagglutination inhibition (HAI) test. Ninety out of these sera were examined also by ELISA, for comparative purposes. It was found that, by HAI test, the percentage of antibody positive sera in the females was 72.2% (HAI titer greater than 1:16), while by the more sensitive ELISA test, the percentage of antibody positive sera was 92.2% (50 IU/ml antirubella IgG). The most susceptible females were in the age group of 20-25 years. The need to ensure the protection of seronegative susceptible women of childbearing age by immunization before marriage or pregnancy is emphasized. ELISA is more sensitive, rapid, specific, reproducible and easily adaptable test to large scale screening than the conventional HAI test.

Evaluation of the beta-Neocept test for early pregnancy.

We tested the validity of the beta-Neocept test for early pregnancy against that of the plasma human chorionic gonadotropin beta-subunit radioimmunoassay (beta-HCG RIA). The beta-Neocept test had a sensitivity of 88%, a specificity of 93%, a positive predictive value of 95%, a negative predictive value of 84% and an accuracy of 90%. In view of these performance characteristics, its low cost and its ease of use, the beta-Neocept test could be used as the initial pregnancy test when there is a high probability of pregnancy, as there was in this study population, which consisted of 111 women attending endocrine infertility clinics. The more expensive beta-HCG RIA could be reserved for special indications and for patients in whom the results of the urinary hemagglutination inhibition tests are inconsistent with the clinical signs and symptoms.

PIP: Paired urine and blood samples from 111 women attending endocrine infertility clinics in Vancouver, Canada, were used to compare the validity of the beta-Neocept test for early pregnancy against that of the plasma human chorionic gonadotropin beta-subunit radioimmunoassay (beta-HCG-RIA). None of the urine specimens contained glucose, 1 specimen contained 1 + protein measured by dipstick, and 1 contained a large amount of blood. 67 patients were pregnant and 44 were not pregnant. Beta-Neocept test results were positive for 59 pregnant patients, negative for 8 pregnant patients, positive for 3 nonpregnant patients, and negative for 41 nonpregnant patients. There were 62 positive and 49 negative tests. Results with the Beta-Neocept test became positive as early as the 28th day after the onset of the last menstrual period and negative results occurred with plasma beta-HCG levels ranging between 5 and 157 IU/1. The sensitivity of the Beta-Neocept test was 88%, the specificity 93%, the positive predictive value 95%, the negative predictive value 84%, and the overall accuracy 90%. The 8 false-negative results all occurred at low plasma beta-HCG levels. The false-negatives occurred in 1 patient on no medication, 1 taking clomiphene citrate and prednisone, 1 taking gonadotropin releasing hormone (GRH), 1 taking human menopausal gonadotropin and human chorionic gonadotropin, 1 taking GRH and bromocriptine mesylate, 1 taking clomiphene citrate alone, and 2 taking clomiphene citrate and progesterone vaginal inserts. The 3 false-positive results occurred in 2 patients using progesterone vaginal inserts for luteal-phase support and 1 who was taking clomiphene citrate. The results indicate that the Beta-Neocept test is sensitive and specific, and would be an appropriate initial test for patients with a high probability of pregnancy. The beta-HCG-RIA could be reserved for patients in whom the results of the less expensive and less difficult Beta-Neocept were inconsistent with clinical signs and symptoms.

Clinical evaluation of the Ortho Rubella ELISA Test System.

The Ortho Rubella ELISA Test System (Ortho Diagnostics Canada Ltd., Toronto, Ontario) is an enzyme-linked immunosorbent assay (ELISA) for the determination of IgG antibodies to rubella virus. Comparison of the Ortho Rubella ELISA with a commercial hemagglutination inhibition (HAI) test revealed that the ELISA is well suited for routine use as a qualitative screening test for rubella immune status or the demonstration of seroconversions. Quantitation of rubella antibodies by the Ortho Rubella ELISA, however, requires that ELISA endpoint determination be made on serial serum dilutions.

Evaluation of assays for the detection of antibodies to rubella. A report based on data from the College of American Pathologists Surveys of 1982.

Data from laboratories participating in the College of American Pathologists Surveys in 1982 provided information on the trends in testing for antibodies to rubella. Methods used by participants included: passive hemagglutination, 29%; latex agglutination card assay test, 25%; hemagglutination inhibition, 19%; enzyme immunosorbent assays, 13%; indirect fluorescent antibody assays, 11%; and radioimmunoassay, 3%. The results from these methods generally agreed well with the standard HI test, particularly for detection of immunity in negative and strongly positive samples. Laboratories should use caution, however, that the tests they use give satisfactory results with low titered sera. Compared to laboratories using the one to three-day-old chick cells with Heparin-MnCl2 for HI, laboratories using Human O Cells tended to get higher titers, and laboratories using Fixed Chick cell or Kaolin methods tended to get lower titers.

Procedures for measuring accuracy and sensitivity of immunochemical pregnancy test kits.

Analytical procedures were used to measure the accuracy and sensitivity of immunochemical pregnancy test kits. Performances of all currently marketed hemagglutination inhibition, latex agglutination inhibition, and direct latex agglutination pregnancy kits were evaluated.

PIP: 7 immunochemical methods are currently available for measuring human chorionic gonadotropin (HCG) which is secreted during pregnancy, reaching its highest level during the 1st trimester: 1) latex agglutination inhibition (LAI), 2) hemagglutination inhibition (HAI), 3) direct latex agglutination (DLA), and 4) radioimmunoassay (RIA). In a study of 5 commercial immunochemical pregnancy kits, it was concluded that the HAI kits were more accurate and sensitive than the LAI or DLA kits. The need for a standardized, transferrable analytical method for evaluating kit performance was expressed by the Food and Drug Administration and by increased over-the-counter sales of these products. This paper presents protocols developed for testing the performance of these kits including the following aspects: accuracy and sensitivity determination, test procedures for HAI, LAI, and DLA, apparatus, and reagents. A test was performed on 206 assorted lots of kits with the following results: 1) none of the 98 HAI kits gave false positive or false negative results, 2) the LAI kits gave 24 false and 3 inconclusive results, 3) the mean level of HCG detected by HAI kits was below that obtained with LAI and DLA kits; thus the HAI detects pregnancy at an earlier stage, 4) average sensitivity of all kits tested was greater than or equal to that claimed by the manufacturer, 5) denaturation of the antisera may enhance the sensitivity and given an increase of false positive reactions, 6) low concentrations or HCG standard in dilute solutions were unstable due to absorption of HCG on glass surfaces, 7) erroneous results can be caused by HCG secreted in hydatidiform moles, choriocarcinoma and certain tumors, ectopic pregnancy, and protein in the urine, 8) data from repeat analyses could be misleading, and 9) reagents should be visually inspected before use to detect bad reagents.

Center for disease control diagnostic immunology proficiency testing program results for 1976.

Over 900 laboratories participated in the Diagnostic Immunology portion of the 1976 Proficiency Testing Program, which was provided by the Center of Disease Control under the authority of the Clinical Laboratories Improvement Act of 1967. One hundred specimens prepared by the Center for Disease Control for analysis were distributed on a quarterly schedule or in special surveys. Feedback from participating laboratories included over 37,500 qualitative and 33,000 quantitative responses, which were analyzed to determine individual laboratory proficiency levels. In addition, information supplied by participants in each survey helped to delineate trends in testing protocols. The specimens chosen for analysis called for a broad range of tests commonly performed in diagnostic immunology laboratories, including those for rubella antibodies, hepatitis B surface antigen, bacterial antibodies, rheumatoid factor, immunoglobulins and other serum-specific proteins, and carcinoembryonic antigen. A summary of the data analysis is provided so that the laboratories can improve their overall performance levels.

Standardized viral hemagglutination and hemagglutination-inhibition tests. II. Description and statistical evaluation.

Standardized hemagglutination and hemagglutination-inhibition procedures are described and statistically evaluated for all animal viruses where applicable, except for rubella and the arbovirus group. The standardized tests employ a constant phosphate-buffered saline diluent and constant volumes of serum, antigen, and standardized erythrocyte suspension. The standardized hemagglutination test has a reproducibility of 84 to 96% with adenoviruses, rubeola, and the myxoviruses, and 78 to 93% with reoviruses; the standardized hemagglutination-inhibition test has a reproducibility of 95 to 100% with all viruses tested.