Comparison of Three Latex Agglutination Kits and a Commercial EIA for the Detection of Cryptococcal Antigen

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1 Scientific review

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3 Scientific review Comparison of Three Latex Agglutination Kits and a Commercial EIA for the Detection of Cryptococcal Antigen J.E. Bestrom, D.J. Jespersen, and N.L. Wengenack Mayo Clinic, Rochester, MN Evaluation of the Clostridium difficile Immunocard Toxin A and B Test K. J. Brown, D. J. Jespersen, M. F. Jones, S. K. Schneider, T.F. Smith, Ph.D. and J.E. Rosenblatt, M.D. Mayo Clinic, Rochester, MN Comparison of Focus Technologies, Inc. and Panbio, Inc. Elisa Assays for Detection of Antibodies to West Nile Virus DJ Jespersen, MF Jones, EM Beito, TF Smith Division of Clinical Microbiology, Mayo Clinic College of Medicine, Rochester, MN Comparative Evaluation of the Triturus Analyzer and the Eti-Max 3000 for Performance of Enzyme Immunoassays on Viral Hepatitis Markers D. Granger, D. Jespersen, T. Rasmussen, P.S. Mitchell, J.D.C. Yao Mayo Clinic, Rochester, MN An Evaluation Of Three Serologic Commercial EIA Kits for The Laboratory Diagnosis of Mycoplasma Pneumoniae Infections D. J. Jespersen, J. A. Harring, M. F. Jones, T.F. Smith, Ph.D. Mayo Clinic, Rochester, MN Validity of Enzyme Immunoassay (Eia) Serologic Results for The Laboratory Diagnosis of Epstein-barr Virus (Ebv) Infections. Julie A. Helgason, Deborah J. Jespersen, Mary F. Jones, Elaine M. Beito, and Thomas F. Smith. Mayo Clinic and Foundation, Rochester, MN 55905

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5 Comparison of Three Latex Agglutination Kits and a Commercial EIA for the Detection of Cryptococcal Antigen J.E. Bestrom, D.J. Jespersen, and N.L. Wengenack Mayo Clinic, Rochester, MN Abstract Latex agglutination tests have been used for >40 years for the rapid, sensitive, and specific identification of cryptococcal capsular antigen. However no comparison of latex agglutination kits has been reported in the last 10 years.we therefore compared the performance of the Meridian CALAS,Wampole Crypto-LA, and Murex Cryptococcus Test kits for the detection of cryptococcal antigen from serum and CSF. In addition, the Meridian EIA for cryptococcal antigen was adapted to the Triturus autoanalyzer and compared to the Meridian latex kit.fifty archived antigen-positive and 50 archived antigen negative sera and CSF specimens were examined using the 3 latex kits and the EIA. The manufacturer s instructions were followed for each kit, including the use of pronase to remove potential nonspecific interference with the Meridian and Murex kits. The percent concordance of the 100 specimens for each pair of kits was as follows: Meridian and Wampole latex = 96%,Meridian and Murex latex = 93%,Wampole and Murex latex = 93%,Meridian latex and Meridian EIA = 98%. The endpoint titers were also compared between the 3 latex kits. Agreement between the titers was defined as ±1 two-fold dilution and was found to occur 60% and 48% of the time respectively for the Wampole and Murex kits relative to the Meridian kit. Endpoint titer agreement between the Wampole and Murex kits was 48%. There were no false positive results among the 50 negative specimens tested by any of the latex kits or the EIA.We conclude that the three latex kits as available today have similar performance characteristics based on concordance values 93%. The Meridian latex kit endpoint was felt to be the easiest to read. The endpoint titers from the various kits should not be compared when following individual patients since the absolute titer value varied widely between the kits. The Meridian EIA performed as well as the latex kit and has the added advantage of permitting automated specimen processing in high-volume laboratories. Background C. neoformans is an opportunistic fungal pathogen that causes systemic disease in immunocompromised persons (eg., those with AIDS, diabetes, malignancies). Detection of C. neoformans antigen in serum or CSF is often more sensitive and rapid than culture of the organism. Latex agglutination tests have been widely used for the detection of cryptococcal antigen for the last 40 years but no comparison of commercials kits has been reported for almost a decade 1-2. The object of this study was to compare the performance of three commercially-available latex agglutination kits and a commercial EIA kit for the detection of cryptococcal antigen. Materials And Methods Latex agglutination kits evaluated Sample Number CALAS Cryptococcal Antigen Latex Agglutination System (Meridian Bioscience, Inc., Cincinnati, OH) Murex Cryptooccus Test (Remel, Lenexa, KS) Crypto-LA Test (Wampole Laboratories, Cranbury, NJ) EIA kit evaluated Meridian Premier Cryptococcal Antigen A side-by-side comparison of the 3 latex agglutination kits and the EIA kit was conducted using 50 archived, previously positive and 50 archived, previously negative serum and CSF specimens. The archived specimens had been tested initially using the Meridian CALAS latex kit prior to archiving. All latex agglutination tests were performed according to the manufacturer s package insert and the endpoint was read by two experienced technologists. Any endpoint titer, including 1:1, was considered positive for cryptococcal antigen. The EIA kit testing was performed according to the manufacturer s instructions using the automated Triturus EIA Analyzer (Grifols-Quest, Miami, FL). Triturus Table 1. C. neoformans Latex Agglutination Endpoint Titers and EIA results Meridian Latex Titer Wampole Latex Titer Results Murex Latex Titer Meridian EIA Result Sample number Meridian Latex Titer Wampole Latex Titer Murex Latex Titer Meridian EIA Result 1 1:128 1:16 1:256 POS 51 NEG NEG NEG NEG 2 1:256 1:8 1:8 POS 52 NEG NEG NEG NEG 3 1:2048 1:1024 1:2048 POS 53 NEG NEG NEG NEG 4 1:1024 1:256 1:256 POS 54 NEG NEG NEG NEG 5 1:2048 1:256 1:256 POS 55 NEG NEG NEG NEG 6 >1:8192 >1:8192 1:512 POS 56 NEG NEG NEG NEG 7 1:128 1:32 1:128 POS 57 NEG NEG NEG NEG 8 1:512 1:512 1:1024 POS 58 NEG NEG NEG NEG 9 1:8 1:2 1:8 POS 59 NEG NEG NEG NEG 10 1:16 1:2 1:4 POS 60 NEG NEG NEG NEG 11 >1:8192 >1:8192 >1:8192 POS 61 NEG NEG NEG NEG 12 1:16 1:8 1:4 POS 62 NEG NEG NEG NEG 13 1:512 1:128 1:512 POS 63 NEG NEG NEG NEG 14 1:1024 1:256 1:256 POS 64 NEG NEG NEG NEG 15 1:2 NEG 1:2 POS 65 NEG NEG NEG NEG 16 1:1024 1:512 1:32 POS 66 NEG NEG NEG NEG 17 1:2 1:1 NEG POS 67 NEG NEG NEG NEG 18 1:2048 1:2048 1:2048 POS 68 NEG NEG NEG NEG 19 1:16 1:2 NEG POS 69 NEG NEG NEG NEG 20 1:128 1:8 1:256 POS 70 NEG NEG NEG NEG 21 NEG NEG 1:256 POS 71 NEG NEG NEG NEG 22 1:2048 1:512 1:1024 POS 72 NEG NEG NEG NEG 23 1:4 NEG NEG NEG 73 NEG NEG NEG NEG 24 1:1024 1:512 1:2048 POS 74 NEG NEG NEG NEG 25 1:1024 1:512 1:2048 POS 75 NEG NEG NEG NEG 26 1:16 1:4 NEG GZ 76 NEG NEG NEG NEG 27 1:64 1:32 1:128 POS 77 NEG NEG NEG NEG 28 1:512 1:512 1:1024 POS 78 NEG NEG NEG NEG 29 1:512 1:128 1:32 POS 79 NEG NEG NEG NEG 30 1:32 1:16 1:8 POS 80 NEG NEG NEG NEG 31 1:8192 1:4096 1:2048 POS 81 NEG NEG NEG NEG 32 1:16 1:16 1:16 POS 82 NEG NEG NEG NEG 33 1:128 1:64 1:64 POS 83 NEG NEG NEG NEG 34 >1:8192 >1:8192 >1:8192 POS 84 NEG NEG NEG NEG 35 1:512 1:256 1:2048 POS 85 NEG NEG NEG NEG 36 >1:8192 >1:8192 1:4096 POS 86 NEG NEG NEG NEG 37 1:128 1:128 1:2048 POS 87 NEG NEG NEG NEG 38 1:1024 1:1024 1:4096 POS 88 NEG NEG NEG NEG 39 1:8 1:4 NEG POS 89 NEG NEG NEG NEG 40 1:512 1:512 1:2048 POS 90 NEG NEG NEG NEG 41 1:8 1:4 1:8 POS 91 NEG NEG NEG NEG 42 1:16 NEG 1:32 POS 92 NEG NEG NEG NEG 43 NEG NEG NEG NEG 93 NEG NEG NEG NEG 44 1:512 1:256 1:1024 POS 94 NEG NEG NEG NEG 45 1:1024 1:512 1:8192 POS 95 NEG NEG NEG NEG 46 1:1024 1:4096 1:4096 POS 96 NEG NEG NEG NEG 47 1:16 NEG NEG POS 97 NEG NEG NEG NEG 48 1:512 1:512 1:2048 POS 98 NEG NEG NEG NEG 49 1:1024 1:2048 1:4096 POS 99 NEG NEG NEG NEG 50 1:4096 1:1024 1:8192 POS 100 NEG NEG NEG NEG Table 2. Concordance of Endpoint Titers for Three Commercial C. neoformans Latex Agglutination Kits (Meridian, Wampole, and Murex) and the Meridian Premier EIA Test Meridian Latex Wampole Latex Positive Negative Positive 44 0 Negative 4 52 Meridian Latex Murex Latex Positive Negative Positive 42 1 Negative 6 51 Wampole Latex Murex Latex Positive Negative Positive 40 3 Negative 4 53 Meridian Latex Meridian EIA Positive Negative Positive 46 1 Negative 1 51 % concordance = 98% n=99 because 1 EIA result was "equivocal" and was not repeated In order to determine concordance for the latex agglutination tests, any titer including undiluted (1:1) was considered positive Table 3. Enpoint Titer Agreement for the Latex Agglutination Kits Kits Endpoint Agreement (±1 two-fold dilution) Wampole and Meridian 60% Murex and Meridian 48% Wampole and Murex 48% Conclusions No false positive results were found among the 50 negative specimens. The three C. neoformans latex agglutination kits as available today have similar performance characteristics based on concordances of s93%. The Meridian latex kit endpoint was felt to be easiest to read by the technologists performing the testing. The endpoint titers from the latex kits should not be compared when following individual patients since the absolute titer varies widely between the kits. The Meridian Premier EIA kit performed as well as the latex kits and has the added advantage of permitting automated specimen processing using the Triturus analyzer for highvolume laboratories. References 1. Tanner TC, Meinstein MP, Fedorciw B, Joho KL,Thorpe JJ, Reller LB Comparison of Commercial Kits for Detection of Cryptococcal Antigen. J Clin Microbiol. 32: Jaye DL,Waites KB, Parker B, Bragg SL, Moser SA Comparison of two rapid latex agglutination tests for detection of cryptococcal capsular polysaccharide. 109:

6 Evaluation of the Clostridium difficile Immunocard Toxin A and B Test K. J. Brown, D. J. Jespersen, M. F. Jones, S. K. Schneider, T.F. Smith, Ph.D. and J.E. Rosenblatt, M.D. Mayo Clinic, Rochester, MN Abstract We compared the Immunocard Toxin A and B assay with the Premier Toxin A and B assay (both manufactured by Meridian Bioscience, Inc, Cincinnati, Ohio) for the detection of C.difficile toxins A and B. Additionally, all samples were tested with the Clearview C. Diff A (Wampole Laboratories, Princeton, NJ) to determine how many positive samples were likely to contain Toxin B since this test detects only Toxin A. 210 consecutive stool samples submitted to the laboratory for C.difficile toxin testing were equally aliquotted, coded, and tested according to the product inserts. Samples were tested by the Premier kit on the Triturus (Grifols USA, Miami, FL) automated EIA analyzer. Both the Immunocard and Clearview assays are horizontal-flow EIAs and were tested manually. For the detection of Toxin A, discrepant results were resolved by agreement of two out of three different method results (extended gold standard). The Immunocard had a sensitivity of 100% and a specificity of 98%. There were three additional positive samples (p=0.25) with the Immunocard that were confirmed by the Clearview test indicating that the Immunocard was slightly more sensitive than the Premier assay for detection of Toxin A. Since the Clearview Assay detects only Toxin A and 5 of 62 (8%) positive samples were negative only by Clearview, this suggests that these samples contained Toxin B. The Immunocard and Premier assays appear to be comparable for the detection of C.difficile toxins A and B. The Immunocard can potentially improve turn around time (analytical time of 15 minutes vs. 60 minutes) and allows for on-demand single sample testing. Objectives Toxigenic C.difficile produces two distinct toxins. Toxin A is an enterotoxin and Toxin B is a cytotoxin: These toxins are responsible for a variety of diseases including antibiotic associated diarrhea, and pseudomembranous colitis. Our goal in this study was to compare the Immunocard Toxin A and B assay with the Premier Toxin A and B EIA assay. Additionally, we tested all of the samples with the Clearview C. Diff Kit to determine how many of the positive samples were likely to contain Toxin B since this assay only detects Toxin A. Triturus The Triturus is an open platform, fully automated, multi-test, multibatch analyzer. Results For the detection of Toxin A, discrepant results were resolved by agreement of two out of three different method results (extended gold standard). The Immunocard had a sensitivity of 100% and a specificity of 98%. There were three additional positive samples (p=0.25) with the Immunocard that were confirmed by the Clearview test indicating that the Immunocard was slightly more sensitive than the Premier assay for detection of Toxin A. Since the Clearview Assay detects only Toxin A and 5 of 62 (8%) positive samples were negative only by Clearview, this suggests that these samples contained Toxin B. The Immunocard and Premier assays appear to be comparable for the detection of C. difficile toxins A and B. Premier Toxin A and B Sensitivity = 98% + - Immunocard Toxin A and B Premier and Immunocard + - Clearview C. Diff A Sensitivity = 91% The Premier Toxin A and B assay was programmed on the Triturus according to the product insert. Conclusions The Immunocard and Premier assays appear to be comparable for detecting C.difficile Toxins A and B. The Immunocard would improve analytical turn around time (15 minutes vs 60 minutes). Immunocard Use of the Immunocard would allow for single sample, STAT testing, ensuring rapid institution of proper treatment and isolation precautions. 8% of the C.difficile positive specimens were not detected using the Clearview C. Diff A kit, suggesting that these samples contained Toxin B.

7 Comparison of Focus Technologies, Inc. and Panbio, Inc. Elisa Assays for Detection of Antibodies to West Nile Virus DJ Jespersen, MF Jones, EM Beito, TF Smith Division of Clinical Microbiology, Mayo Clinic College of Medicine, Rochester, MN Abstract Laboratory diagnosis of West Nile Virus [WNV] is best achieved by demonstration of specific IgG and IgM class antibodies in serum or CSF specimens; amplified target nucleic acid of the virus by PCR has been demonstrable only in approximately 55% of CSF and 10% of serum specimens from known WNV infected patients. Two test methods for detection of antibodies to WNV were compared using a panel of 100 sera from human cases; 75 specimens were from patients with signs and symptoms compatible with WNV infection. These antibodies were specific for this virus based on results of the plaque reduction neutralization test [PRNT] performed by the Centers for Disease Control [CDC] or the California State Department of Health. All 75 PRNT positives in the panel had IgM class antibody. FOCUS Technologies, Inc. [Cypress, California] and PanBio, Inc. [Columbia, Maryland] have developed Flavivirus (WNV) ELISA assays for detecting both IgG and IgM antibodies in human serum. These assays in conjunction with exposure history and local epidemiology have been used as an indication of early WNV infections. Both assays were also formatted and tested on the Triturus (Grifols-Quest), an automated open platform ELISA analyzer. IgM antibody is generally detected by the eighth day of illness while IgG antibody is usually found within three weeks post infection. Of the 75 PRNT positive sera, IgG antibodies were detected by the FOCUS test in 31 [41%] of the samples while the PanBio assay detected IgG in 38 [51%] of the samples. Both assays detected IgG antibodies in the same 31 specimens while 7 specimens were positive exclusively with the PanBio test. Not all samples that contained IgM antibody were positive for IgG antibody detection. Using both manual and automated formats, all sera with IgM antibodies to WNV [n=75] were detected with the FOCUS assay [sensitivity 100%] whereas the PanBio assay failed to detect 4 [5%] of the positive IgM samples [sensitivity 95%]. These two FDA approved kits were sensitive [95% - 100%] and specific, technically simple, and both assays can be formatted on the Triturus with 100% agreement between manual and automated testing necessary for testing high volumes. Objective Focus Technologies, Inc. and PanBio, Inc. both have FDA approved ELISAS for WNV. Our goal in this study was to compare the IgM results from both kits with samples that were known to be PRNT positive. Additionally, we wanted to see if the Focus kit performed as well on an automated system (Triturus) as it did with the manual method. Triturus The Triturus is an open, fully automated EIA analyzer Results For the detection of Toxin A, discrepant results were resolved by agreement of two out of three different method results (extended gold standard). The Immunocard had a sensitivity of 100% and a specificity of 98%. There were three additional positive samples (p=0.25) with the Immunocard that were confirmed by the Clearview test indicating that the Immunocard was slightly more sensitive than the Premier assay for detection of Toxin A. Since the Clearview Assay detects only Toxin A and 5 of 62 (8%) positive samples were negative only by Clearview, this suggests that these samples contained Toxin B. The Immunocard and Premier assays appear to be comparable for the detection of C. difficile toxins A and B. Results for IgM Antibodies CDC + - FOCUS (manual) CDC + - FOCUS (Automated Triturus) CDC + - PanBio (manual) Sensitivity = 95% Conclusions Both kits provided high sensitivity and specificity (95% -100%) Both assays can be formatted on the Triturus While both assays are technically simple, the PanBio assay does have shorter incubation steps than the Focus test

8 Comparative Evaluation of the Triturus Analyzer and the Eti-Max 3000 for Performance of Enzyme Immunoassays on Viral Hepatitis Markers D. Granger, D. Jespersen, T. Rasmussen, P.S. Mitchell, J.D.C. Yao Mayo Clinic, Rochester, MN Abstract Very few automated commercial systems are available for the processing and testing of clinical specimens for serologic markers of viral hepatitis that are done commonly in diagnostic laboratories. An accurate and labor-saving process for such an operation is essential for an efficient laboratory to produce test results with rapid turn-around time. In this study, we compared the performance characteristics, specimen throughput, and material costs of two automated systems, Triturus Analyzer (Triturus; Grifols-Quest Inc.,Miami, FL) and ETI-MAX 3000 (ETI-MAX; DiaSorin Inc., Stillwater, MN), on seven enzyme immunoassays (EIAs) for viral hepatitis markers. For each EIA, 50 known-positive, 50 knownnegative, and 100 prospectively collected clinical serum specimens were tested on both the Triturus and the ETI-MAX. Specimens yielding discrepant results were retested in duplicate by each system, with the final result of each specimen being the best two of three results. Each system was programmed to process and test these specimens according to the assay manufacturer s instructions. The following EIAs manufactured by DiaSorin Inc. were compared: anti-hav Total antibody, anti-hav IgM antibody, HBsAg, anti-hbs antibody, anti-hbc IgM antibody, HBeAg, and anti-hbe antibody. Total time duration (including handson and hands-off times) for each EIA was compared between the two systems in processing and testing 48 specimens. Direct material costs for consumables were also compared. When compared to ETI-MAX, the diagnostic sensitivity / specificity and difference in total time duration for each EIA on Triturus were as follows: Assay Sensitivity (%) Specificity (%) Total time difference (hrs) Anti-HAV Total Anti-HAV IgM HBsAg Anti-HBs Anti-HBc IgM HBeAg Anti-HBe Significant labor savings were noted for all EIAs performed on the Triturus with hands on times ranging from 0.2 to 0.3 hr for each assay, compared to 1.2 to 1.3 hr on ETIMAX. Based on current specimen test volumes in our laboratory, use of the Triturus for these seven EIAs would result in direct material cost savings of $17,500 annually. Although the performance characteristics of the two automated systems are comparable, the Triturus has several significant advantages over the ETI-MAX. In addition to the shorter hands-on times and labor savings, Triturus offered user-friendly instrument software, improved manual sample tube barcode reader, simple loading process of specimens for continuous batch testing, minimal time delay between batch testing, dual sampling probes with non-disposable tips, and capability of detecting clots in specimens. Objective This study evaluated seven DiaSorin hepatitis EIAs on both the Triturus and the ETIMAX Accuracy of test results, labor savings, test turn-around-time, and specimen throughput were compared between the two systems. Triturus The Triturus is an open, fully-automated, multi-test, multi-batch analyzer. Results Anti-IgM ETI-MAX Triturus Specificity = 100% ETI-MAX: 5.7 hr Triturus: 4.1 hr Anti-HBs ETI-MAX Triturus Specificity = 100% ETI-MAX: 5.3 hr Triturus: 3.9 hr Anti-HBeAg ETI-MAX Triturus Specificity = 98.5% ETI-MAX: 5.1 hr Triturus: 4.2 hr Anti-HAV Total ETI-MAX Triturus Sensitivity = 97.5% Specificity = 100% ETI-MAX: 5.3 hr Triturus: 3.9 hr Anti-HBsAg ETI-MAX Triturus Specificity = 99.3% ETI-MAX: 5.3 hr Triturus: 4.1 hr Anti-HBc IgM ETI-MAX Triturus Specificity = 100% ETI-MAX: 5.7 hr Triturus: 4.8 hr Anti-HBe ETI-MAX Triturus Sensitivity = 98.7% Specificity = 100% ETI-MAX: 5.1 hr Triturus: 3.4 hr ETI-MAX 3000 ETI-MAX 3000 is a fully automated walk-away microtiter plate analyzer. Conclusions Triturus demonstrated the following advantages over ETI-MAX 3000: Labor savings due to decreased hands-on time Higher specimen throughput due to greater flexibility in continuous batch testing More efficient software Easier sample loading process Large cost savings on consumables (disposable tips are not necessary) Improved manual barcode reader Dual testing probes (shorter test turn-around time)

9 An Evaluation Of Three Serologic Commercial EIA Kits For The Laboratory Diagnosis Of Mycoplasma Pneumoniae Infections D. J. Jespersen, J. A. Harring, M. F. Jones, T.F. Smith, Ph.D. Mayo Clinic, Rochester, MN Abstract Mycoplasma pneumoniae is a common cause of primary atypical pneumonia in both adults and children. An estimated 2 million cases with 100,000 pneumonia-related hospitalizations occur in the United States annually. Because M. pneumoniae is fastidious and grows very slowly, a definitive diagnosis is often based on the results of serological testing. Although indirect immunofluorescence () testing has historically been considered the gold standard, many laboratories perform an enzyme immunoassay (EIA) for the diagnosis of these infections. Automated EIA analyzers are often used to facilitate serologic testing and eliminate the subjectivity involved in processing and interpreting results. Our goal was to compare commercially-available EIA kits to the standard method for the laboratory diagnosis of M. pneumoniae when tested by the Triturus instrument (Grifols, USA) (Miami, FL.) Two-hundred samples representing different patterns of serological results by were coded and analyzed by three commercial kits for both IgG and IgM class antibodies. Kits from Wampole Laboratories (Princeton, NJ), Diamedix (Miami, FL), and Savyon (Afhdod, Israel) were tested. Discordant IgM results were resolved using the Meridian Immunocard (Cincinnati, Ohio). Sensitivities and specificities for IgG were 52% and 98% with the Diamedix assay, 70% and 94% with the Savyon assay, and 93% and 96% with the Wampole kit, respectively. IgM results were similar with 69% and 94% with the Diamedix kit, 86% and 92% with the Savyon assay and 100% and 82% with the Wampole assay. IgM samples that were negative by but positive by EIA were retested with the Immunocard and 77% of Savyon, 100% of Diamedix, and 90% of the Wampole results retested as positive. Although serology may be helpful, these results indicate a significant variability between FDA approved kits. We recommend that the diagnosis of M. pneumoniae infection not be based on serology alone, but should be correlated with clinical findings. Objective Laboratory diagnosis of M. pneumoniae is often established through serological testing since cultivating the organism is slow and difficult. Many commercial assays are available for the serologic evaluation of infection due to M. pneumoniae. Our goal was to determine which of three FDA approved IgG and IgM EIA kits performed best on the Triturus, an automated EIA analyzer, when compared to. Triturus Results Wampole IgG and IgM The Wampole EIA is a qualitative system for determining IgG and IgM Antibodies to M. pneumoniae using plates coated with partially purified inactivated antigen. This assay takes approximately one hour to perform on the Triturus. IgG 1: sens = 93% spec = 96% IgM 1: sens =100% spec = 82% Savyon IgG and IgM The Savyon EIA is a qualitative assay used for determining IgG and IgM antibodies to M. pneumoniae using plates coated with a purified fraction of M. pneumoniae membrane proteins containing the P1 protein. This assay takes approximately 2 hours and 40 minutes on the Triturus. IgG 1: sens = 70% spec = 94% Diamedix IgG and IgM The Diamedix EIA is a Qualitative Assay used for determining IgG and IgM antibodies to M. pneumoniae using plates coated with purified detergent treated M. pneumoniae extracts as antigens. This assay takes approximately 1 hour and 40 minutes to perform on the Triturus. IgG 1: sens = 52% spec = 98% IgM 1: sens =86% spec = 92% IgM 1: sens =69% spec = 94% Meridian IgM Immunocard 26 of 29 [90%] negative, Wampole IgM EIA positive samples tested positive with the Meridian IgM Immunocard 10 of 10 [100%] negative, Diamedix IgM EIA positive samples tested positive with the Meridian IgM Immunocard 10 of 13 [77%] negative, Savyon IgM EIA positive samples tested positive with the Meridian IgM Immunocard The Triturus is an open, Fully-automated, Multi-test, Multi-Batch analyzer that can Accommodate any EIA Methods All three of the EIAs were programmed on the Triturus and performed according to the manufacturer s product inserts for each kit. Conclusions There was a significant variability between commercially available FDA-approved kits. All three kits were easily adapted to an automated system. The Wampole kit provided the shorted testing time. The Wampole IgG kit provided the best sensitivity and specificity. The Savyon and Wampole IgM kits yielded similar sensitivities and specificities [86% and 91%, and 100% and 82%, respectively]. Collectively correlation of EIA with was poor

10 VALIDITY OF ENZYME IMMUNOASSAY (EIA) SEROLOGIC RESULTS FOR THE LABORATORY DIAGNOSIS OF EPSTEIN-BARR VIRUS (EBV) INFECTIONS. Julie A. Helgason, Deborah J. Jespersen, Mary F. Jones, Elaine M. Beito, and Thomas F. Smith. Mayo Clinic and Foundation, Rochester, MN Abstract Epstein-Barr Virus is a member of the herpesvirus group. EBV is the causative agent of infectious mononucleosis; however, this virus can produce persistent infections and neoplastic conditions in immunocompromised hosts. Although indirect fluorescent antibody () testing has historically been considered the gold standard, most laboratories perform enzyme immunoassay for the diagnosis of these infections. Automated EIA analyzers are often used to eliminate the subjectivity involved in processing and interpreting serological results. Our goal was to determine which commercially-available EBV EIA kit had the best performance characteristics when tested by the Triturus instrument (Diagnostic Grifols, S.A.). Based on predominant use reported in proficiency test surveys, we selected Diamedix (Diamedix Corporation, Miami, FL), DiaSorin (DiaSorin Inc., Stillwater, MN), SeraQuest, (SeraQuest, North Miami, FL), Wampole (Wampole Laboratories, Dist., Cranbury NJ) and Zeus (Zeus Scientific, Inc., Raritan,NJ) EIA kits to compare with. One hundred-fifty samples that represented the different patterns of serological results (VCA, IgG, VCA IgM, EBNA IgG) were analyzed. Discordant results with wide ranges of sensitivity and specificity for EBV markers were obtained with all assay kits, although the SeraQuest assay demonstrated the best overall performance. Commercial Supplier Sensitivity vs. Specificity vs. SeraQuest VCA IgG 82.6% 100% VCA IgM 100% 55.7% EBNA IgG 73% 97.1% A well-defined panel of sera [Anti-EBV Mixed Titer Performance Panel (PME201)] obtained from Boston Biomedica, Inc. [(BBI); Bridgewater, CT] was used to further compare the SeraQuest assay with the method. Results of this panel analysis demonstrated the same overall lack of sensitivity previously found with the other assays. SeraQuest vs. BBI Panel Sensitivity vs. Specificity vs. VCA IgG 83.3% 100% VCA IgM 77.8% 100% EBNA IgG 87.5% 87.5% With the Triturus automated instrument for serologic testing, all evaluated kits performed similarly. All current generation EIA assays were easily adapted to the Triturus instrument; however, all commercial EBV EIA kits produced variable results as compared to serology. We recommend that the diagnosis of EBV infections not be based on EIA results alone. Rather, interpretation of serologic results together with clinical features and other laboratory findings [hematological evaluation and other serological markers (e.g. rapid screening for infectious. Introduction Epstein-Barr virus (EBV) is the etiological agent of infectious mononucleosis (IM), Burkitt s lymphoma and nasopharyngeal carcinoma. The virus occurs worldwide and most people become infected with EBV in early childhood. Symptomatic infection, commonly in young adults, produces fever, sore throat and swollen lymph glands. IM (primary infection with EBV) is usually diagnosed in the laboratory by rapid latex tests based on detection of heterophile antibodies. Persistant, active EBV infection can occur as primary or reactivated latent virus in immunocompromised hosts. These infections manifest as posttransplantation lymphomas and smooth muscle cell tumors. These EBV associated conditions are diagnosed by detection of antibodies to specific antigens of the virus. Although indirect fluorescent antibody () testing has historically been considered the gold standard and is most often used to detect antibodies to EBV antigens, the assays are labor-intensive and require the experience and subjective judgment of an expert technologist. In addition, measurements of serum specimens may be misleading due to non-specific reactions for patients with autoimmune diseases. Serologic results of enzyme immunoassays (EIA) correlate with measurements and interpretation is standardized. The EIA procedure allows an objective determination of antibody status to be made from a single dilution of. Method Based on predominant use reported in proficiency test surveys, we selected five manufacturers kits to evaluate on the Triturus continuous batch analyzer; Diamedix (Diamedix Corporation, Miami, FL), DiaSorin (DiaSorin Inc., Stillwater, MN), SeraQuest (SeraQuest, North Miami, FL), Wampole (Wampole Laboratories, Dist., Cranbury, NJ) and Zeus (Zeus Scientific, Inc., Raritan, NJ). One hundred-fifty samples representing the different patterns of serological results (VCA IgG,VCA IgM and EBNA IgG) were analyzed and compared with. Discordant results with wide ranges of sensitivity and specificity for EBV markers were obtained with all assay kits, although the SeraQuest assay demonstrated the best overall performance. A well-defined panel of sera [Anti-EBV Mixed Titer Performance Panel (PME201)] obtained from Boston Biomedica, Inc. [(BBI); Bridgewater, CT] was used to further compare the SeraQuest assay with the method. Results of analysis of this panel demonstrated the same overall lack of sensitivity previously found with the other assays. Triturus Conclusions Antibody Responses to EBV-specific Antigens Diseases VCA-IgG VCA-IgM Anti-EBNA-IgG Sensitivity vs. Specificity vs. VCA IgG 51 % 100 % Diamedix VCA IgM 94.9 % 75.8 % EBNA IgG 79.5 % 97.1 % Sensitivity vs. Specificity vs. VCA IgG 89.8 % 95.7 % DiaSorin VCA IgM 90.5 % 54.2 % EBNA IgG 71.1 % 92.9 % Sensitivity vs. Specificity vs. VCA IgG 82.6 % 100 % SeraQuest VCA IgM 100 % 55.7 % EBNA IgG 73 % 97.1 % Sensitivity vs. Specificity vs. VCA IgG 55.1 % 100 % Wampole VCA IgM 76.3 % 33.8 % EBNA IgG 81.6 % 95.7 % Sensitivity vs. Specificity vs. VCA IgG 82.9 % 95.2 % Zeus VCA IgM 92.7 % 75.2 % EBNA IgG 74.7 % 94 % PHASE I. BEST CORRELATION COMPARED TO WAS OBTAINED WITH THE SERAQUEST ASSAY. Sensitivity vs. Specificity vs. VCA IgG 83.3 % 100 % SeraQuest vs VCA IgM 77.8 % 100 % BBI Panel EBNA IgG 87.5 % 87.5 % PHASE II. THE SERAQUEST ASSAY WAS THEN COMPARED TO WITH A CASE DEFINED PANEL OF SERA OBTAINED FROM BBI. 1. All current generation EIA assays were easily adapted to the Triturus continuous batch analyzer; however, our results indicated that all commercial EBV EIA kits produce variable results as compared to serology. 2. Discordant results were obtained with all assay kits; however, the SeraQuest EIA kit had the best correlation compared with results. 3. We recommend that the diagnosis of persistent EBV infection not be based on EIA results alone. Rather, interpretation of serologic results, together with clinical features and other laboratory findings, be collectively considered for the diagnosis of EBV infections.

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