Evaluation of four commercially available enzyme immunoassays for laboratory diagnosis of Clostridium difficile-associated diseases.

Four commercial enzyme immunoassays (EIAs) for the detection of Clostridium difficile toxin A have recently been developed and marketed (Premier, Meridian Diagnostics, Cincinnati, Ohio; VIDAS, bioMerierux Vitek, Inc., Hazelwood, Mo.; Tox-A-Test, TechLab, Blacksburg, Va.; and Bartels, Baxter Diagnostics, McGaw Park, Ill.). The performances of these EIAs were compared with those of the tissue culture cytotoxicity assay and a definition of C. difficile-associated disease based on both laboratory and clinical criteria for 329 clinical specimens. Two EIAs (Premier and VIDAS) showed good overall agreement (96 and 95%, respectively) with the cytotoxicity assay. However, they were less sensitive (84 and 71%, respectively) than the Bartels (94%) or Tox-A-Test (93%) EIAs. The Bartels and Tox-A-Test assays were much less specific, resulting in poor positive predictive values (56%) of the two assays when compared with that of the cytotoxicity assay. Tox-A-Test had the added drawback of having a significant number of indeterminate results (6.4%). These data indicate that the four EIAs all have specific shortcomings. When using these EIAs, testing strategies that take these shortcomings into consideration should be developed.     

Comparison of three commercial methods for rapid detection of Clostridium difficile toxins A and B from fecal specimens

Three rapid enzyme immunoassays (X/pect Clostridium difficile Toxin A/B test, Wampole Tox A/B Quik Chek, and ImmunoCard Toxins A&B) were compared for the diagnosis of Clostridium difficile infection. Of the 367 stool specimens tested, 102 (27.8%) were positive for toxigenic C. difficile when a combination of direct cytotoxicity assay and cytotoxic culture was used as the gold standard. Sensitivity/specificity values were 49.0%/95.8%, 54.9%/95.5%, and 66.7%/95.1%, respectively. The median times to test five stool specimens were 28, 30, and 24 min, respectively. The ImmunoCard test was the quickest and most sensitive test of the three enzyme immunoassays evaluated.     

Performance characteristics of four immunoassays for antiepileptic drugs on the IMMULITE 2000 automated analyzer

Carbamazepine, phenobarbital, phenytoin, and valproic acid are commonly used antiepileptic drugs that show complicated pharmacokinetic behavior Nonisotopic immunoassays are used routinely to monitor these drugs, and assay specificity is important to obtain accurate results. By using samples from subjects receiving each of these antiepileptic medications, competitive immunoassays for them were evaluated on an IMMULITE 2000 automated chemiluminescent analyzer (Diagnostic Products, Los Angeles, CA). Phenytoin assays were evaluated using an additional set of samples from patients with abnormal renal function. All 4 methods were linear, had imprecision of less than 10%, and compared well with other commercial immunoassays. A positive bias was observed for phenytoin measured in samples from uremic patients compared with a high-performance liquid chromatography reference method. The molar cross-reactivity of carbamazepine-10,11-epoxide was 12% in the carbamazepine assay. Phenytoin metabolites and fosphenytoin had substantial cross-reactivity in the phenytoin assay. All antiepileptic drug assays performed well and are suitable for use in monitoring patients receiving antiepileptic drug therapy. One possible exception is the phenytoin assay with samples from patients with renal insufficiency.     

Specificity of two-site immunoassays

When cross-reaction in two-site immunoassays was investigated both theoretically and experimentally it was found that such systems do not always result in enhanced specificity. Computer simulation studies indicated that substances which display negligible cross-reaction in a radioimmunoassay could produce an assay response identical to that of the analyte in a two-site immunoassay using excess antibody. Cross-reactivity in two differing two-site immunoassays was compared to that obtained in radioimmunoassays using the same monoclonal antibodies for human chorionic gonadotrophin. In addition to the effects of excess antibody, cross-reactivity was observed in one of the two-site immunoassays which could not have been predicted from the specificity of the antibodies or cross-reactivity in the radioimmunoassays. The unexpected cross-reaction of the beta subunit of human chorionic gonadotrophin in the assay resulted from an apparent alteration in the specificity of one of the antibodies following binding of the beta subunit to the second antibody. These studies emphasise the complexity of binding reactions in two-site immunoassays.     

Evaluation of eight anti-rubella virus immunoglobulin g immunoassays that report results in international units per milliliter

An evaluation of anti-rubella virus immunoglobulin G (IgG) immunoassays that report in international units per milliliter (IU/ml) was performed to determine their analytical performance and the degree of correlation of the test results. A total of 321 samples were characterized based on results from a hemagglutination inhibition assay. The 48 negative and 273 positive samples were used to determine the sensitivity and specificity of the assays. When equivocal results were interpreted as reactive, the sensitivity of the immunoassays ranged from 98.9 to 99.9% and the specificity ranged from 77.1 to 95.8%. All assays had positive and negative delta values of less than 2. A significant difference between the mean results of all assays was demonstrated by analysis of variance. However, post hoc analysis showed there was good correlation in the mean results expressed in IU/ml between some of the assays. Our results show the level of standardization between anti-rubella virus IgG immunoassays reporting results expressed as IU/ml has improved since a previous study in 1992, but further improvement is required.     

Current immunoassays and detection of antibodies elicited by Omicron SARS-CoV-2 infection

The present study aimed to determine whether current commercial immunoassays are adequate for detecting anti-Omicron antibodies. We analyzed the anti-SARS-CoV-2 antibody response of 23 unvaccinated individuals 1–2 months after an Omicron infection. All blood samples were tested with a live virus neutralization assay using a clinical Omicron BA.1 strain and four commercial SARS-CoV-2 immunoassays. We assessed three anti-Spike immunoassays (SARS-CoV-2 IgG II Quant [Abbott S], Wantaï anti-SARS-CoV-2 antibody ELISA [Wantaï], Elecsys Anti-SARS-CoV-2 S assay [Roche]) and one anti-Nucleocapsid immunoassay (Abbott SARS-CoV-2 IgG assay [Abbott N]). Omicron neutralizing antibodies were detected in all samples with the live virus neutralization assay. The detection rate of the Abbott S, Wantai, Roche, and Abbott N immunoassays were 65.2%, 69.6%, 86.9%, and 91.3%, respectively. The sensitivities of Abbott S and Wantai immunoassays were significantly lower than that of the live virus neutralization assay (p = 0.004, p = 0.009; Fisher's exact test). Antibody concentrations obtained with anti-S immunoassays were correlated with Omicron neutralizing antibody concentrations. These data provide clinical evidence of the loss of performance of some commercial immunoassays to detect antibodies elicited by Omicron infections. It highlights the need to optimize these assays by adapting antigens to the circulating SARS-CoV-2 strains.     

Comparison of four different methods for detection of Cryptosporidium species.

Newly available assays offer alternatives to conventional microscopic examination for Cryptosporidium spp. We compared two enzyme immunoassays, ProSpect Cryptosporidium microtiter assay (Alexon, Inc., Mountain View, Calif.) and Color Vue Cryptosporidium assay (Serady, Indianapolis, Ind.), and a direct immunofluorescent assay, Merifluor Cryptosporidium kit (Meridian Diagnostics, Cincinnati, Ohio), with acid-fast Kinyoun-staining for the detection of Cryptosporidium spp. Examinations were performed on 129 stool specimens received from patients during a recent waterborne outbreak. A specimen was considered positive when organisms could be identified visually by acid-fast and immunofluorescent stains or if organisms could be visualized by either acid-fast or immunofluorescent stain and detected by both enzyme immunoassays. The final number of positive specimens was 55. No single procedure detected all 55 positive specimens. Of these, ProSpect and Color Vue detected 52 (sensitivity, 94.5%), and the Kinyoun stain and Merifluor detected 53 (sensitivity, 96.4%). The final number of negative specimens was 74. One false-positive result was seen with both the Kinyoun stain and the ProSpect assay. The Color Vue and ProSpect assays required the most hands-on technologist time. The ProSpect assay and Merifluor kit were easiest to perform. The acid-fast stain was difficult to interpret. The Merifluor kit was easiest to read and was adaptable to both batch and single testing. Overall, the Kinyoun stain and the Merifluor test were preferable to both of the enzyme immunoassays because of the high reagent cost and hands-on time required for the enzyme immunoassays. The difficult interpretation of the Kinyoun stain smears made the Merifluor a more desirable test despite its higher cost.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the Bio-Rad Geenius HIV 1/2 Assay as an Alternative to the INNO-LIA HIV 1/2 Assay for Confirmation of HIV Infection

The Bio-Rad Geenius HIV 1/2 assay was evaluated as an alternative to the INNO-LIA HIV 1/2 assay for the confirmation of HIV infection in 198 serum samples reactive to 4th-generation HIV enzyme immunoassays (EIAs). The Geenius assay correctly identified 85% of the samples, compared to 75% identified by the INNO-LIA assay, reduced the number of indeterminate results, and shortened the overall turnaround time.     

Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhea.

Enzyme immunoassays (EIAs) based on monoclonal antibodies for the detection of Clostridium difficile toxins have recently been developed for clinical use. The aim of this study was to compare three commercially available EIAs, two for toxin A (Premier C. difficile Toxin A; Meridian, Osi, Elancourt, France; and Vidas C. difficile Toxin A; bioMérieux, Marcy l'Etoile, France) and one for toxins A and B (Cytoclone A + B EIA; Cambridge Biotech Corp., Codiapharm, Evian, France), with a cytotoxicity assay and toxigenic culture for the diagnosis of C. difficile-associated diarrhea (CDAD). The study was performed with 285 fresh stools from 285 patients with suspected CDAD. In case of disagreement, the tests were repeated on a frozen aliquot of the same stool sample, and the patient's chart was reviewed. CDAD diagnosis was established in 55 cases (incidence, 19.3%). The sensitivities and specificities of the methods were, respectively, 92.7 and 100% for the cytotoxicity assay, 96.4 and 99.1% for toxigenic culture, 75.5 and 97.8% for Cytoclone, 65.4 and 99.6% for Premier, and 65.4 and 100% for Vidas. The results were uninterpretable in 3.2% of cases with Cytoclone, 0.3% with Premier, and 2.5% with Vidas. We conclude that the cytotoxicity assay and toxigenic culture remain the best methods for the diagnosis of CDAD even though they lack standardization and require 48 to 96 h to obtain the result. Despite their rapidity and simplicity, EIAs are not sensitive enough to be relied on as the sole laboratory test.     

Analysis of false negative results in the immunoassay for anti-acetylcholine receptor antibodies in myasthenia gravis

Possible causes for the failure of immunoassays to detect anti-acetylcholine receptor activity in serum from confirmed myasthenia gravis (MG) patients were investigated. A more sensitive assay, using Protein A to trap immune complexes (ARIA), was applied to 65 MG sera which were negative in the usual assay and to 42 normal human sera. Normal and negative MG sera had antibody (Ab) activity in the same range (50-70 pM). Titers present in 70% of normal sera appeared to be specific antireceptor antibodies as defined by tests for antigen specificity. Thus, higher sensitivity assays did not improve discrimination of MG from normals. In a second group of 108 MG sera studied, 48 were negative by the usual assay criteria in a rat acetylcholine receptor immunoassay. Further detailed analysis of this negative group showed that 3/48 had IgG3 antibody not detectable in the test, 14/48 had Ab's recognizing human receptor determinants exclusively, 29/48 had toxin blocking Ab's not determined by immunoassays, and 6/48 were negative in all tests. The results indicate that the exclusive occurrence of toxin-blocking antibodies in MG subjects is a major factor contributing to false negatives in the ARIA test. Estimates of the amount of Ab's with this functionality indicated that they are present in very much smaller amounts than other classes of anti-receptor Ab's. Degree of blocking activity in patient serum showed a fair correlation with severity of disease. Thus, blocking antibodies appear capable of causing all degrees of disease severity in the absence of other types of antireceptor Ab's. The development of a sensitive and quantitative in vitro assay for blocking antibodies combined with the usual immunoassay would be a major improvement for a MG diagnostic test, with greater than 94% positivity predicted.     

Comparison of Six Automated Treponema-Specific Antibody Assays

Six different Treponema (TP)-specific immunoassays were compared to the fluorescent treponemal antibody absorption (FTA-ABS) test. A total of 615 samples were tested. The overall percent agreement, analytical sensitivity, and analytical specificity of each assay compared to the FTA-ABS test were as follows: Architect Syphilis TP, 99.2%, 96.8%, and 100%; Cobas Syphilis, 99.8%, 99.4%, and 100%; ADVIA Centaur Syphilis, 99.8%, 99.4%, and 100%; HISCL Anti-TP assay kit, 99.7%, 98.7%, and 100%; Immunoticles Auto3 TP, 99.0%, 97.5%, and 99.6%; Mediace TPLA, 98.0%, 98.1%, and 98.0%. All results that were discrepant between the TP-specific assays were associated with samples from noninfectious cases (11 immunoassay false positives and 7 from previous syphilis cases). Our study demonstrated that TP-specific immunoassays generally showed high sensitivities, specificities, and percentages of agreement compared to FTA-ABS, with rare cases of false-positive or false-negative results. Therefore, most TP-specific immunoassays are acceptable for use in screening for syphilis. However, it is important to perform a thorough review of a patient''s clinical and treatment history for interpreting the results of syphilis serology.     

Evaluations of commercial West Nile virus immunoglobulin G (IgG) and IgM enzyme immunoassays show the value of continuous validation

West Nile virus was introduced into the United States in 1999 and in only four seasons has become endemic east of the Rocky Mountains. Recently, immunoglobulin M (IgM)-capture enzyme immunoassays for the detection of West Nile virus-specific IgM and indirect IgG enzyme immunoassays for the detection of IgG antibodies against West Nile virus were made available from Focus Technologies and PANBIO, Inc. We evaluated these commercial IgG and IgM test systems and determined agreement, sensitivity, and specificity for the assays, compared to immunofluorescence assay and the Centers for Disease Control and Prevention's IgM-capture enzyme-linked immunosorbent assay (ELISA). Initially, the Focus and PANBIO IgM enzyme immunoassays had at least 95% agreement, sensitivity, and specificity, and, based on the 95% confidence intervals, both IgM-capture assays performed similarly. The IgG assays also performed well, although the Focus IgG assay demonstrated greater specificity (98.8%) and the PANBIO IgG assay demonstrated greater sensitivity (99.3%). However, for 400 samples consecutively submitted for West Nile virus antibody testing during 2 days of the 2003 West Nile virus season, agreement, clinical sensitivity, and clinical specificity were 93.1, 98.0, and 92.4%, respectively, for the PANBIO IgM assay and were 97.4, 100.0, and 97.1%, respectively, for the Focus IgM assay. The specificities observed in this second evaluation equates to an overall false-positivity rate of 6.3% in the PANBIO West Nile virus IgM-capture ELISA versus 2.5% with the Focus West Nile virus IgM-capture ELISA. This experience demonstrates the importance of continuously evaluating the performance of an assay in order to detect any changes in assay performance as the test population evolves.     

Differential measurement by ELISA of free and IgA bound alpha1-microglobulin in human serum without prior fractionation

Two solid phase enzyme immunoassays for the measurement of alpha1-microglobulin (alpha1m) in biological fluids are reported. These assays are complementary and allow the separate determination of free alpha1m and alpha1m bound to IgA without fractionation of the fluids. Specificity, reproducibility and sensitivity of the assays are reported. Differential measurements on normal or pathological sera were compared to the measurements obtained by chromatographic separation of free and bound alpha1m before assay. Results obtained with the enzyme immunoassays were significantly correlated with those obtained by radioimmunoassay.     

Evaluation of the Vitros Syphilis TPA Chemiluminescence Immunoassay as a First-Line Method for Reverse Syphilis Screening

We report here the results of the diagnostic performances of Vitros Syphilis TPA (a chemiluminescence treponemal assay) compared with those of two treponemal enzyme immunoassays and of traditional versus reverse syphilis algorithms. Ease of use, automation, and high throughput make the Vitros Syphilis TPA assay a good choice for syphilis screening in high-volume laboratories.     

Comparison among performances of a ligase chain reaction-based assay and two enzyme immunoassays in detecting Chlamydia trachomatis in urine specimens from men with nongonococcal urethritis.

We evaluated the performances of a ligase chain reaction (LCR)-based assay and two enzyme immunoassays (Chlamydiazyme and IDEIA) in the detection of Chlamydia trachomatis in urine specimens. We compared the results of testing urine specimens by these assays with those of urethral swab culture by examining samples from 131 men with nongonococcal urethritis. Discrepant results were analyzed by testing urethral swab specimens for C. trachomatis by a PCR-based assay. After the resolution of discrepant results, the sensitivity of urethral swab culture was 85.3%, whereas those of the LCR assay, Chlamydiazyme, and IDEIA with urine specimens were 94.1, 82.4, and 94.1%, respectively. The LCR assay and IDEIA were more sensitive than was urethral swab culture. In addition, the LCR assay, with a sensitivity equal to that of IDEIA, was more specific. Overall, the LCR assay proved to be superior to the enzyme immunoassays in detecting C. trachomatis in urine specimens. Testing urine specimens by LCR assay should be a helpful alternative method for diagnosing C. trachomatis urethral infection in men with nongonococcal urethritis.     

Laboratory diagnosis of Clostridium difficile disease

The laboratory diagnosis of Clostridium difficile -associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.     

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.     

Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM.     

Specific detection of Haemophilus influenzae type b lipooligosaccharide by immunoassay.

Three monoclonal antibody (MAb)-based immunoassays were developed for specific detection of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS). (i) Hib LOS was captured onto microtiter plates by polyclonal Hib-directed antibodies and detected with MAbs to the oligosaccharide component of Hib LOS in an enzyme-linked immunosorbent assay, (ii) The high affinity of polymyxin B for lipid A was used to bind Hib LOS to microtiter wells, and the oligosaccharide-specific MAbs were used as the detection system in the polymyxin B-MAb assay. (iii) Hib LOS solubilized in detergent was captured by MAbs, and the immobilized LOS was detected with a chromogenic Limulus amebocyte lysate method in the immunolimulus assay. Endotoxin concentrations were measured in in vitro samples and cerebrospinal fluid samples from rabbits with experimental Hib meningitis. The results were compared with those obtained with the standard chromogenic Limulus amebocyte lysate assay. There were significant correlations between the results of all four assays. These new immunoassays provide methods for specific detection of Hib LOS in laboratory fluids and in research involving quantification of Hib endotoxin in experimental animal models.     

Multicentre evaluation of the Elecsys<Superscript>®</Superscript> hepatitis B surface antigen II assay for detection of HBsAg in comparison with other commercially available assays

Screening for hepatitis B surface antigen (HBsAg) demands highly sensitive and specific immunoassays with the ability to detect clinically relevant surface gene mutants—the presence of which is an increasing concern and can compromise test results. The objective of the study was to compare the clinical and technical performance of the fully automated Elecsys HBsAg II assay with other widely used HBsAg immunoassays in China. This was a multicentre study in which eight Chinese laboratories compared the Elecsys HBsAg II assay with the Architect HBsAg assay, AxSYM HBsAg assay, or generic microtitre plate (MTP) enzyme-linked immunosorbent assays (manufactured in China) against preselected samples, including recombinant surface gene mutants, and routine clinical samples. Elecsys HBsAg II was the most sensitive assay for detecting positive results in seroconversion samples: up to 14, 11, and 22 days earlier than the Architect, AxSYM, and MTP HBsAg assays, respectively. It also detected all 211 preselected HBsAg-positive specimens and all 13 recombinant HBsAg mutants; the AxSYM and MTP HBsAg assays failed to detect 3 and 10 mutant samples, respectively. Sensitivity was 100% for Elecsys HBsAg II with routine samples, compared with 99.1, 98.9, and 95.2% for the Architect, AxSYM, and MTP HBsAg assays, respectively. In conclusion, it was demonstrated that the Elecsys HBsAg II assay is suitable for routine HBsAg screening in China.