Acute parvovirus B19 infection frequently causes false positive results in the

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1 CVI Accepts, published online ahead of print on 0 December 00 Clin. Vaccine Immunol. doi:0./cvi Copyright 00, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. 0 0 Acute parvovirus B infection frequently causes false positive results in the Epstein-Barr virus immunoglobulin M and herpes simplex virus immunoglobulin M determinations done on the Liaison platform. Mario Berth*, Eugene Bosmans Algemeen Medisch Laboratorium, Immunology Department, Desguinlei, 0 Antwerpen, Belgium * Corresponding author. Mailing address: Algemeen Medisch Laboratorium Immunology Department Desguinlei 0 Antwerpen Belgium Phone: + 0 Fax: mario.berth@aml-lab.be Downloaded from on October, 0 by guest

2 ABSTRACT During an outbreak of parvovirus B we collected serum samples from non-pregnant 0 patients in the region of Antwerp (Belgium). Fifty seven (%) of the parvovirus B IgM positive sera had a positive result for Epstein-Barr virus (EBV) immunoglobulin M on Liaison, (0%) had a positive result for herpes simplex virus (HSV) IgM, 0 (%) samples were positive for cytomegalovirus IgM and (%) had a positive result for Borrelia burgdorferi sensu lato IgM. As assay interference was suspected, sera were further investigated by using additional infectious-disease serology tests and by performing various interference elimination procedures. We could show that the EBV IgM and HSV IgM results were false positive due to aspecific IgM reactions with the solid phase. All samples were also analyzed using a modified Liaison EBV IgM assay, based on the addition of polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA) to the dilution buffer, which partially eliminated this type of assay interference. Although the Liaison is a very convenient, automated immunoassay platform, this study demonstrates the potential for improvement of mainly the EBV IgM and HSV IgM tests. Downloaded from on October, 0 by guest

3 0 0 INTRODUCTION Previously we described that % of the positive Epstein-Barr virus (EBV) IgM results on the Liaison platform are falsely elevated, but we could only demonstrate the interference mechanism but not identify a concrete underlying (infectious) cause (). Recently an outbreak of B occurred in the region of Antwerp (Belgium) and we noticed that patients with a recent B infection frequently had positive, sometimes very high, titers of Epstein-Barr virus (EBV) immunoglobulin M (IgM) on Liaison. As we suspected these EBV IgM results to be false positive, we investigated sera from recently B infected patients by using alternative infectious- disease serology tests and by performing various interference elimination procedures. MATERIALS AND METHODS Sample selection. From March 00 until the end of July 00 we collected serum samples from adult, non-pregnant patients that were sent in by general practitioners. From patients (0 males / females, aged years) detailed clinical information was available and an acute parvovirus B infection could be biologically ascertained or was very probable. In this group, at the time of presentation B IgM could be shown and these patients all had a reticulocytopenia (< 0.%), which is typical for an acute B infection. Moreover, in 0 patients from this group a subsequent B immunoglobulin G (IgG) seroconversion could be shown, but in this study we only used the sample from the first presentation to the general practitioner. Nonspecific symptoms (i.e. fatigue, fever) were present in patients. Two patients presented with erythema infectiosum, arthralgias were present in patients and patients had an asymptomatic infection. Downloaded from on October, 0 by guest

4 0 0 For the other patients in which B IgM could be shown, exact timing of infection was not possible and/or no clinical information was available. Infectious serology assays. Parvovirus B specific antibodies were determined by enzyme- linked immunosorbent assay (ELISA) according to the manufacturer s instructions (Parvovirus B EIA, th Generation; Biotrin International, Dublin, Ireland). This ELISA has been shown to be highly specific (). Assays performed on the Liaison platform (DiaSorin, Saluggia, Italy), with their cut-offs for positivity, were: Borrelia burgdorferi sensu lato IgM (cut-off:., index), cytomegalovirus IgM (cut-off: 0 mu/liter), EBV IgM (cut-off: 0 mu/liter), varicella zoster IgM (cut-off:., index) and herpes simplex virus (HSV) IgM (cut-off:., index). On all samples HSV IgM and EBV IgM was determined using an ELISA (Enzygnost anti- HSV/IgM and Enzygnost anti-ebv/igm II; Dade Behring / Siemens Medical Solutions Diagnostics, Marburg, Germany). Sera positive for Borrelia burgdorferi sensu lato IgM on Liaison were also examined using an immunoblot (Borrelia afzelii Western blot; Euroimmun, Lübeck, Germany). Sera with positive cytomegalovirus IgM results on Liaison were also analyzed on mini VIDAS (biomérieux, Marcy l Etoile, France). Interference elimination studies. On 0 samples with enough serum available and showing positive EBV IgM and HSV IgM results on Liaison, we performed various interference elimination studies. In these methods, appropriate positive and negative control samples were used to detect any unexpected effects of the procedures. For statistical comparison of the three different sample pre-treatment methods, the Wilcoxon test for paired samples was applied using Medcalc software (version.; Mariakerke, Belgium). Downloaded from on October, 0 by guest

5 0 0 Two different commercial reagents for interference elimination were used. ) Heterophilic antibody interference was excluded by treating the sample, according to the manufacturer s instructions, with a nonspecific antibody-blocking tube (Scantibodies Laboratory, Santee, CA). ) RF-Absorbent (Dade Behring / Siemens Medical Solutions Diagnostics, Marburg, Germany), which contains sheep IgM antibodies targeted against human IgG Fc fragments: 0 µl of RF- Absorbent was added to 0 µl of serum, and the mixture was briefly vortexed and incubated for h at room temperature. Results obtained after pre-treatment with RF-Absorbent were multiplied by to account for the dilution, except for the B IgM ELISA since this is a µ-capture method. To confirm the presence of solid phase reactive antibodies, 00 microliters of serum was added to approximately 0. x 0 M-0 tosyl-activated beads (Dynabeads; Dynal Biotech, Oslo, Norway), vortexed, and incubated for min at room temperature. After centrifugation ( min;,000 x g), the supernatant was used for further analysis. Modified Liaison EBV IgM assay. All samples were analyzed by using a modified Liaison EBV IgM assay. This modification partially eliminates false positive results in the Liaison EBV IgM assay () and is based on inhibiting aspecific IgM reactivity by blocking nonspecific binding sites on the solid phase (, -). It is performed by adding polyvinylpyrrolidone (PVP-0; Sigma-Aldrich) and polyvinyl alcohol (P; Sigma-Aldrich) to the EBV IgM dilution buffer (buffer A) at a final concentration of 0. % and 0.00 % respectively, as described previously (). The results obtained with the original EBV IgM assay were compared to the modified EBV IgM assay. Discrepant results were defined as differing more than % (three times the interassay coefficient of variation) between the original and modified assay. RESULTS Downloaded from on October, 0 by guest

6 0 0 Fifty seven (%) of the B IgM positive sera had a positive result for EBV IgM on Liaison (range: 0 mu/liter 0 mu/liter, median: 0 mu/liter). One of these samples gave a borderline result using the Dade Behring EBV IgM ELISA. Sixty one (0%) of the B IgM positive sera had a positive result for HSV IgM on Liaison (range:.., median:.). Seven of these samples gave borderline results using the Dade Behring HSV IgM ELISA; were positive. There were no significant differences in IgM positivity rate for EBV and HSV between the group in which an acute B was highly probable (% positive EBV IgM / % positive HSV IgM) compared to the entire group of B positive IgM samples (% positive EBV IgM / 0% positive HSV IgM). Similar results were seen in the 0 patients with a B IgG seroconversion (0% positive EBV IgM / 0% positive HSV IgM). Figure shows the correlation between the Liaison EBV IgM and HSV IgM titers. The significant correlation (Pearson r = 0.; p<0.00) illustrates the aspecificity of both tests in the context of an acute B infection. As can be observed, two apparent outliers are present. One of these two samples was the one that showed the borderline positive result on EBV IgM ELISA, but it was also strongly positive on HSV IgM ELISA, suggesting a correct high titer of HSV IgM antibodies. For the other sample we could not find a straight forward explanation for the discrepancy. Twenty (%) samples were positive for cytomegalovirus IgM on Liaison (range: 0 mu/liter), from which were positive and gave borderline results on mini VIDAS. The four positive samples on mini VIDAS had results of,, and mu/liter on Liaison. The two borderline samples had results of 0 and mu/liter on Liaison. Downloaded from on October, 0 by guest

7 0 0 Fifteen (%) of the B IgM positive sera had a positive result for Borrelia burgdorferi sensu lato IgM on Liaison (index range:..0), from which two could be confirmed as positive on immunoblotting and two gave borderline results. The two confirmed samples had results of.0 and. on Liaison. The two borderline results had results of. and. on Liaison. After exclusion of these four samples, the index range narrowed down to... Two samples were positive for varicella zoster IgM on Liaison (index. and.0). These two samples were also positive for EBV IgM and HSV IgM on Liaison. Interference elimination studies. When pre-incubating the 0 selected samples with unlabeled beads, a strong reduction in the EBV IgM and HSV IgM titers could be obtained, compared with pre-treatment using a nonspecific antibody-blocking tube or RF-Absorbent (p<0.00). These three pre-treatments did not have significantly different effect on the B IgM titers. Figure shows the box-and-whisker plots from the different sample pre-treatments. These results confirm the presence of solid phase reactive antibodies as the cause of the false positive EBV IgM and HSV IgM titers. Modified Liaison EBV IgM assay. For of the EBV IgM positive samples (%), markedly different results were obtained with the original and modified EBV IgM assay. An average titer reduction of % was obtained, and although no positive EBV IgM results became negative, results became borderline (i.e. between 0 and 0 mu/liter). DISCUSSION Parvovirus B infection has been reported to produce false positive reactions in various infectious-disease serology assays (-0). In the present study we observed a very high frequency of false positive Liaison EBV IgM and HSV IgM results, which we showed to be caused by Downloaded from on October, 0 by guest

8 0 0 aspecific IgM reactions with the solid phase. Also, false positive results in the cytomegalovirus IgM and Borrelia burgdorferi sensu lato IgM tests were seen, albeit at lower frequencies. Although no diagnostic system is free from false positive results, the strikingly high frequency of false positive results in the Liaison EBV IgM and HSV IgM assays during an acute B infection raises questions about the overall specificity of these two tests. In 00 an initial analytical evaluation of the EBV IgM on Liaison was published (). Unfortunately in this study only healthy controls were used and sera from other infectious diseases (e.g. B) were not evaluated. Further information on assay specificity can be found in the Liaison EBV IgM and HSV IgM assay inserts which state that as a rule, the presence of potentially cross-reactive antibodies does not interfere in the assay. This statement is correct in the sense that specific B IgM antibodies will probably not cross-react in the EBV IgM and HSV IgM assays, but it is misleading considering the data presented here. This occurrence of false positive EBV IgM results has apparently also been noticed by the manufacturer since a recent adaptation of the Liaison EBV IgM assay insert (version June, 00) mentions a warning for possible false positive results in acute rubella virus infections. It is likely that the same type of interference previously described by us and reconfirmed in this study, is also causing false positive EBV IgM results in acute rubella virus infections (unpublished observations by us, on 0 rubella IgM strongly positive samples from which were EBV IgM positive). After having noticed that B frequently causes false positive results, we analyzed the samples with false positive EBV IgM results from our previous comparative study () and found that only of these samples had a high titer of B IgM antibodies. Rubella IgM antibodies were negative in these patients. It is probable that other (infectious) causes might induce false positive EBV IgM results on Liaison. Downloaded from on October, 0 by guest

9 0 0 The consequences of these false positive results may be important: 0 patients (%) with a certainly high probability of acute B infection had a positive EBV IgM result at the time of presentation. On the other hand, only patients from this EBV IgM positive group had no detectable EBV nuclear antigen IgG, which means that in the majority (0%) of these patients an acute EBV infection was unlikely. Generally, EBV IgM should always be interpreted in conjunction with EBV nuclear antigen IgG (). Preferably, a follow-up sample to show an IgG seroconversion or significant IgG titer change should be taken. The latter advice was recently added to the new assay insert from the Liaison EBV IgM (version June, 00). Prevention of these false positive results could be achieved by including various blocking reagents in the assays which compete with nonspecific adsorption of proteins to the solid phase (,, ). Unfortunately, the modification of the Liaison EBV IgM assay that we previously proposed, i.e. adding polyvinylpyrrolidone and polyvinyl alcohol to the dilution buffer (), could only partially decrease the false positivity rate in acute B infections. Either further work needs to be done on this aspect of assay modification or more fundamental changes by Diasorin (e.g. change of solid phase) are needed to improve the performance of especially the EBV IgM and HSV IgM assays. In conclusion we can say that, although the Liaison is a very convenient, automated immunoassay platform, this study demonstrates that there is still a major opportunity for improvement of mainly the EBV IgM and HSV IgM tests. ACKNOWLEDGMENTS We would like to thank A. Vereecken and G. Salembier for their support to this study. Downloaded from on October, 0 by guest

10 0 0 0 REFERENCES. Barrett, D. A., M. S. Hartshome, M. A. Hussain, P. N. Shaw, and M. C. Davies. 00. Resistance to nonspecific protein adsorption by poly(vinyl alcohol) thin films adsorbed to a poly(styrene) support matrix studied using surface plasmon resonance. Anal. Chem. :-.. Berth, M., and E. Bosmans. 00. Prevention of assay interference in infectious-disease serology tests done on the liaison platform. Clin. Vaccine Immunol. :-.. Enders, M., S. Helbig, A. Hunjet, H. Pfister, C. Reichhuber, and M. Motz. 00. Comparative evaluation of two commercial enzyme immunoassays for serodiagnosis of human parvovirus B infection. J. Virol. Methods :0-.. Feng, Z., Z. Li, B. Sui, G. Xu, and T. Xia. 00. Serological diagnosis of infectious mononucleosis by chemiluminescent immunoassay using capsid antigen p of Epstein-Barr virus. Clin. Chim. Acta :-.. Haycock, J. W.. Polyvinylpyrrolidone as a blocking agent in immunochemical studies. Anal. Biochem. 0:-.. Hess, R. D. 00. Routine Epstein-Barr virus diagnostics from the laboratory perspective: still challenging after years. J. Clin. Microbiol. :-.. Jenkerson, S. A., M. Beller, J. P. Middaugh, and D. D. Erdman.. False positive rubeola IgM tests. N. Engl. J. Med. :0-0.. Jones, J. W., J. V. Pether, and R. W. Frost.. Human parvovirus B. Hard to differentiate from infectious mononucleosis. BMJ. 0:.. Navalpotro, D., C. Gimeno, and D. Navarro. 00. Concurrent detection of human herpesvirus type and measles-specific IgMs during acute exanthematic human parvovirus B infection. J. Med. Virol. :-. Downloaded from on October, 0 by guest

11 0 0. Ratnam, S., G. Tipples, C. Head, M. Fauvel, M. Fearon, and B. J. Ward Performance of indirect immunoglobulin M (IgM) serology tests and IgM capture assays for laboratory diagnosis of measles. J. Clin. Microbiol. :-0.. Rodda, D. J., and H. Yamazaki.. Poly(vinyl alcohol) as a blocking agent in enzyme immunoassays. Immunol. Invest. :-.. Studentsov, Y. Y., M. Schiffman, H. D. Strickler, G. Y. Ho, Y. Y. Pang, J. Schiller, R. Herrero, and R. D. Burk. 00. Enhanced enzyme-linked immunosorbent assay for detection of antibodies to virus-like particles of human papillomavirus. J. Clin. Microbiol. 0:-0.. Waterboer, T., P. Sehr, and M. Pawlita. 00. Suppression of non-specific binding in serological Luminex assays. J. Immunol. Methods 0:00-0. Downloaded from on October, 0 by guest

12 0 FIG.. A significant correlation (Pearson s r = 0.) between the HSV IgM and EBV IgM titers on Liaison can be seen. Only parvovirus B IgM positive samples which were positive for both EBV IgM and HSV IgM are shown (n = ). FIG.. Box-and-whisker plots comparing the three different sample pre-treatment methods. Significant effects can be seen from the pre-incubation with unlabeled beads on EBV IgM titers (A) and HSV IgM titers (B). These three pre-treatments did not have significantly different effect on the B IgM titers (C). The slight, but statistically significant lower results (p<0.0) of the pretreated samples (beads, NSBT and RFAbs) compared to the untreated samples in panel C were not considered relevant in this experiment. Abbreviations used: NSBT = nonspecific antibody-blocking tube; RFAbs = RF-Absorbent. Downloaded from on October, 0 by guest

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