Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay for Detection of Serum Immunoglobulin G Response to Human Herpesvirus 6

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1996, p Vol. 34, No /96/$ Copyright 1996, American Society for Microbiology Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay for Detection of Serum Immunoglobulin G Response to Human Herpesvirus 6 THEO P. SLOOTS, 1 JOHN P. KAPELERIS, 2 IAN M. MACKAY, 1 MARISSA BATHAM, 2 AND PETER L. DEVINE 2 * Clinical Virology Research Unit, Sir Albert Sakzewski Virus Research Centre, Royal Children s Hospital, Brisbane, 1 and PanBio Pty. Ltd., Windsor, 2 Queensland, Australia Received 31 July 1995/Returned for modification 29 August 1995/Accepted 27 December 1995 A rapid (60-min) commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G (IgG) class antibodies to human herpesvirus 6 (HHV-6) was evaluated. The specificity of the ELISA for HHV-6 was confirmed by absorption studies, with the reactivities of HHV-6-positive sera being unaffected by other herpesviruses (cytomegalovirus, herpes simplex virus, and varicella-zoster virus) or the HSB 2 cell line used to culture HHV-6. HHV-6 IgG antibody levels in a panel of 502 serum samples were determined by ELISA and an indirect immunofluorescence assay (IFA). Results obtained by the two methods were in close agreement, suggesting that the ELISA provides a suitable test method for the determination of HHV-6 IgG antibodies in a routine clinical laboratory. Both tests were positive in 398 cases (79%), and both were negative in 71 cases (14%), with a different result obtained by IFA and ELISA in only 33 cases (7%). Furthermore, absorption of sera with HHV-6 prior to assay revealed that the majority of these results were false positive (n 8) or false negative (n 23) in the IFA (true positives or negatives in the ELISA). Subsequently, the ELISA showed a sensitivity of 99.76% and a specificity of 98.75%. HHV-6-specific IgG levels were also determined in paired serum samples collected from 49 donors 14 with exanthem subitum (ES), 15 with ES which was complicated with central nervous system involvement, and 20 undergoing bone marrow transplantation in whom HHV-6 infection had been demonstrated by virus isolation and/or PCR. All patients with ES or central nervous system complications showed an increase in HHV-6-specific IgG, indicating that this ELISA may be a useful aid in the diagnosis of these conditions. Furthermore, 14 of 20 patients undergoing bone marrow transplantation showed an increase in HHV-6-specific IgG levels, possibly reflecting a reactivation of HHV-6 in these patients. Downloaded from Human herpesvirus 6 (HHV-6) was first isolated in 1986 from patients with lymphoproliferative disease or immunosuppressive disorders (39). Subsequently, HHV-6 was isolated from the lymphocytes of persons with various illnesses and from the saliva of several healthy adults (2, 17, 29, 31, 37). The virus is ubiquitous in the adult human population, with the majority of subjects over 2 years of age showing an antibody response to the virus (7). HHV-6 was confirmed as the cause of exanthem subitum (ES) (roseola infantum or sixth disease), a common disease in infants (incidence, 30%) characterized by high fever and the appearance of a rash, lasting 5 to 15 days, that coincides with subsidence of the fever (50). HHV-6 has also been implicated in a number of other childhood diseases and has been reported to be a major cause of febrile seizures, hospitalizations, and visits by infants and young children to hospital emergency departments (6, 38). In adults, primary infection is less common, with HHV-6 implicated in cases of infectious mononucleosis, hepatitis, chronic fatigue syndrome, certain human malignancies, and AIDS (1, 4, 9, 11, 12, 16, 20, 22, 23, 26, 28 30, 32, 39, 42, 43, 45, 48). Furthermore, many clinically diagnosed cases of measles, rubella, or parvovirus B19 in young children are due to primary HHV-6 infection (46), so a test to aid in the correct diagnosis of HHV-6 is important. The seroprevalence of HHV-6 immunoglobulin G (IgG) * Corresponding author. Mailing address: PanBio Pty. Ltd., P.O. Box 7269, East Brisbane, Qld 4169, Australia. Phone: Fax: antibodies in children and adults has been reported by many researchers, using the indirect immunofluorescence assay (IFA) (1, 7, 27). Although these rates have varied widely (20 to 95%) (35, 40, 49), they have provided useful information on HHV-6 infection in several populations. The subjective nature of IFA makes variation in the results obtained between laboratories almost inevitable, and the reported differences in HHV-6 seroprevalence are probably due to a lack of uniformity in discriminating between negative and weak-positive reactions. Despite the high prevalence of HHV-6 antibody levels in the general population, the titer of antibody diminishes following infection, and high or increasing levels of anti- HHV-6 IgG antibodies in serum may act as an indicator of the patient s recent exposure to HHV-6. Alternatively, HHV-6 infection can be diagnosed by the isolation of virus or the detection of viral products by PCR. The enzyme-linked immunosorbent assay (ELISA) has previously been shown to be a sensitive and reliable alternative to IFA, without the inherent problem of nonspecific reactivity often found with IFA (31). Also, IFA is insensitive and requires an experienced operator to interpret results. A number of different ELISAs have been described and used in seroepidemiology studies and in the serological detection of HHV-6 infection (5, 15, 36). However, these ELISAs have used a number of different antigen preparations, including control antigens, have been technically demanding, and have required extended incubation times. We describe an ELISA for the rapid detection of HHV-6- specific IgG in human serum that should be a valuable aid in on October 4, 2018 by guest 675

2 676 SLOOTS ET AL. J. CLIN. MICROBIOL. the serodiagnosis of HHV-6-associated infection and provide a more sensitive and reliable alternative to IFA. The results of this ELISA were compared with the results of IFA, and this ELISA was used in the clinical testing of various patient groups. MATERIALS AND METHODS Serum samples. Blood was collected by venipuncture and allowed to clot. Samples were centrifuged, and the serum was collected, divided into aliquots, and stored at 70 C until the assay was performed. Serum samples (n 502) randomly selected from hospital patients in different age groups (18 to 45 years), consisting of blood donors, transplant recipients, and patients undergoing surgical procedures, were used to compare IFA and ELISA results. In addition, serum samples were also collected from 14 children with ES, 15 patients with ES and central nervous system (CNS) complications (which included febrile convulsions, impairment of motor function, and bulging of the anterior fontanelle), and 20 patients undergoing bone marrow transplantation (BMT). An additional serum sample was collected from each of these patients during the course of illness (7 to 42 days later; mean, 16 days) for further serological investigation. Isolation of HHV-6. Infection with HHV-6 was confirmed by isolation of the virus from peripheral blood mononuclear cells by coculture with phytohemagglutinin-stimulated umbilical cord blood mononuclear cells (38, 39). Cell morphology was examined daily over a 4-week period for cytopathic changes. HHV-6 isolation was demonstrated by specific staining of cultured cells in the IFA, with human serum positive for HHV-6 antibody staining and known negative-control serum not staining. Extraction of viral DNA. Peripheral blood mononuclear cells obtained from each patient s blood were washed twice in phosphate-buffered saline (PBS) and then resuspended in PCR buffer (50 mm KCl 10 mm Tris-HCl [ph 8.3] 2.5 mm MgCl % [wt/vol] gelatin 0.45% [vol/vol] Nonidet P % [vol/vol] Tween 20) containing 0.006% (wt/vol) proteinase K. After incubation at 55 C for 1 h, the mixture was heated at 95 C for 10 min to inactivate the proteinase K. A 10- l volume of this extract was used for DNA amplification. PCR for the detection of HHV-6 DNA. PCR was used to confirm virus isolation results or to detect HHV-6 DNA in PBMCs from patients with suspected HHV-6 infection. Amplification was performed with primers and conditions specific for the detection of HHV-6 DNA as described previously, with minor modifications (13). Briefly, DNA was amplified in a 50- l reaction volume containing 0.1 M each primer. The specimens were preheated for 3 min at 95 C and then were heated for 3 min at 55 C over three cycles. Amplification was carried out for 35 cycles (1 min at 95 C, 1 min at 55 C, and 2 min at 72 C), and this was followed by a 7-min extension step at 72 C after the final cycle. The amplification products were analyzed by electrophoresis in a 1.5% agarose gel followed by ethidium bromide staining and visualization under UV light. The presence of HHV-6 DNA was indicated by the detection of an 830-bp amplicon. The identity of the amplicon was confirmed by hybridization with an oligonucleotide probe homologous to a region of the DNA sequence flanked by the primers used in the PCR. Antigen preparation. The virus used in this study was HHV-6 variant A, obtained as a local isolate from a chronic fatigue syndrome patient. This isolate was cultured in HSB 2 cells until more than 90% of cells showed cytopathic effects (4 to 5 days). The infected cells were harvested by centrifugation, washed twice in PBS, and sonicated for 30 s to release the virus particles. HHV-6 virions were harvested and concentrated from both the sonicated cell suspension and the media of infected cell cultures and further purified by sucrose gradient centrifugation. The purified virus preparation was used for absorption studies. ELISA. The commercially available HHV-6 IgG ELISA kit (PanBio, Brisbane, Australia) was used as described in the manufacturer s instructions. Briefly, serum was diluted 1/100 and incubated in HHV-6-coated Microwell strips for 20 min at 37 C (100 l per well). After being washed with PBS containing 0.05% Tween 20, bound IgG was detected via a 20-min incubation with anti-human IgG peroxidase (100 l per well) and, after being washed again, a 10-min incubation with tetramethylbenzidine substrate (100 l per well). The reaction was stopped by the addition of 100 l of 1 M phosphoric acid per well, and the A 450 softhe strips were read with a microtiter plate reader. Positivity was determined by comparison with a reference serum (cutoff calibrator) based on the upper limit of samples showing no IgG response to HHV-6. A positive sample was defined as having a sample/calibrator absorbance ratio of 1.0, and a negative sample was defined as having a sample/calibrator absorbance ratio of 1.0. The percentage coefficient of variation (%CV) was determined by using the formula %CV (SD/mean) 100, where SD is the standard deviation. Intra-assay variation was determined by running 16 replicates of both calibrator serum and strongly positive serum in a single assay. Interassay variation was determined by comparing the sample/calibrator absorbance ratios determined in six assays run on different days. IFA. Antibodies to HHV-6 were measured in serum by indirect immunofluorescence as previously described, with minor modification (38). Briefly, smears of HHV-6-infected HSB 2 cells were dried onto microscope slides, fixed in acetone, and used as substrate for the IFA. Sera were diluted in PBS at a 1:10 dilution for IgM and a 1:40 dilution for IgG antibody detection. A 20- l volume was applied in duplicate to each well of a slide and incubated for 30 min at 37 C. Excess Sample serum positive for: TABLE 1. Specificity of HHV-6 ELISA a Absorbance for unabsorbed sample Absorbance for sample absorbed with: HSB 2 HHV-6 CMV HSV-1 VZV HHV CMV HSV VZV a Absorbance is expressed as a ratio of test optical density to cutoff optical density. antibody was removed by three 10-min washes in PBS. The appropriate fluorescein isothiocyanate-conjugated secondary antibody [anti-human IgG F(ab ) 2 or anti-human IgM F(ab ) 2 ; Silenus, Melbourne, Australia] diluted 1:40 in PBS was applied to each slide for 30 min at 37 C. The slides were rinsed with three 10-min washes in PBS, mounted, and examined with a fluorescence microscope. Fluorescence was scored according to an arbitrary four-point scale of 0, 1, 2, and 3, where 0 represents no reactivity and 3 represents strong reactivity. A positive result was defined as a score greater than or equal to 1. Absorption studies. Sera that gave discordant results in the ELISA and IFA were absorbed with HHV-6-infected HSB 2 cells, uninfected HSB 2 cells, or purified HHV-6 and retested by ELISA and IFA to further determine the specificity of the result. In addition, serum samples with known antibody reactivities to herpes simplex virus type 1 (HSV-1), varicella-zoster virus (VZV), and cytomegalovirus (CMV) were absorbed with HHV-6 antigen to examine whether antibody to other human herpesviruses could cross-react in the ELISA (36). Following absorption, the sera were tested by the HHV-6 IgG ELISA as well as ELISA for each corresponding herpesvirus to determine reactivities to HHV-6 and changes in reactivity to the other herpesviruses. Each serum sample was also absorbed with antigen preparations corresponding to each antibody reactivity and reassayed. Data analysis. The proportion of patients with reactivity levels above the designated cutoff for ELISA and IFA was determined. Analysis of variance and the Tukey-Kramer multiple-comparison test were used to compare the mean ELISA ratios for different IFA scores. The chi-square test was used to compare the proportion of patients with an elevated ELISA ratio for different IFA scores. Statistical analyses were performed with Instat software (Graphpad Software Inc., San Diego, Calif.). RESULTS Performance characteristics of HHV-6 ELISA. The HHV-6 IgG ELISA showed good reproducibility, with intra-assay coefficients of variation for the calibrator and positive-control sera (n 16) of 7.9% (mean optical density at 450 nm, 0.355) and 5.2% (mean optical density at 450 nm, 1.203), respectively. Similarly, the interassay coefficient of variation of 4.1% (mean positive/calibrator ratio, 3.49) in six assays was very acceptable. Specificities of HHV-6 ELISA and IFA. Preabsorption of positive sera with other herpesviruses (CMV, HSV, VZV) and HSB 2 cells had no effect on binding to the HHV-6-coated plate, while pretreatment with HHV-6 led to a loss of activity (Table 1). HHV-6 had no effect on the binding of CMV-, HSV-, or VZV-positive sera to plates coated with these viruses (negative controls), while CMV, HSV, or VZV inhibited binding of CMV-, HSV-, or VZV-positive sera to each of the respective antigen-coated plates (positive controls) (Table 1). Similar results were observed in the HHV-6 IFA, in which reactivity was inhibited by HHV-6 but not the other herpesviruses mentioned above (results not shown). Comparison of IFA and HHV-6 ELISA. The results of IFA and ELISA for the 502 serum samples tested were in close agreement, with both the mean ELISA ratio and the percentage of patients with a positive ELISA result increasing with increasing IFA score (Tables 2 and 3). Individual results obtained by the two methods were in close agreement (Table 3), with both tests positive in 398 cases (79%), both tests negative in 71 cases (14%), and each test yielding a different result (one positive and one negative) in only 33 cases (7%). Furthermore, when IFA-positive ELISA-negative samples were absorbed

3 VOL. 34, 1996 ELISA FOR HUMAN HERPESVIRUS IFA score TABLE 2. IFA scores and ELISA ratios a n Mean SD ELISA ratio Range a One-way analysis of variance showed that variation among ELISA means was significant (P ). The Tukey-Kramer multiple-comparison test showed that all ELISA means were significantly different from each other (P for all other pairings except groups with IFA scores of 2 and 3, for which P 0.05). The linear-trend test showed a significant linear trend between ELISA means and IFA scores (P ). with HHV-6-infected HSB 2 cells prior to assay, eight of these nine samples remained IFA positive, indicating that these were false-positive samples in the IFA (true negatives in the ELISA). When IFA-negative ELISA-positive samples were treated similarly, 23 of the 24 samples became ELISA negative, suggesting that these were true positive samples in the ELISA (false-negative samples in the IFA test). Similar treatment with uninfected HSB 2 cells did not alter the reactivities of these sera as judged by ELISA, indicating a specific reaction with HHV-6. Subsequently, the ELISA appeared to correctly diagnose 421 of 422 HHV-6-positive serum samples (sensitivity, 99.76%) and 79 of 80 HHV-6-negative serum samples (specificity, 98.75%). HHV-6-specific IgG responses in paired serum samples. IgG ELISA ratios for paired serum samples from patients with ES, CNS complications, and BMT are shown in Fig. 1 and summarized in Table 4. Although HHV-6 was isolated from the majority of these patients, it is of interest that a lower level of HHV-6 DNA detection by PCR than by virus isolation occurred with ES patients. This may be due to insufficient peripheral blood mononuclear cells for PCR or the presence of inhibitory serum factors (e.g., heparin) in the specimens tested. All 14 patients with ES showed an IgM response and increased HHV-6-specific IgG response in paired samples, with the ELISA ratio more than doubling for all but one of these patients. Furthermore, in 11 of 14 patients the ELISA ratio rose from 1.0 in the first serum sample to 2.0 in the second sample. Similarly, all 15 patients with CNS complications showed large increases in IgG response, while only 10 of these patients showed an IgM response by IFA. Only 4 patients who had undergone BMT (n 20) had detectable IgM responses to HHV-6, while 14 of these patients showed increased IgG responses in paired serum samples. TABLE 3. IFA scores and proportions of donors with positive ELISA ratios a IFA score No. of samples with ELISA ratio/no. with IFA score (%) /95 (75) 24 b /95 (25) 1, 2, or 3 9 c /407 (2) 398/407 (98) a An IFA score of 1 was considered positive, while an ELISA ratio of 1.0 was considered positive. Fisher s exact test showed a significant association between the proportion of patients positive by IFA and ELISA (P ). b Of 24 ELISA-positive IFA-negative serum samples absorbed with HHV-6, 23 became negative by ELISA (ELISA true positive; IFA false negative). c Of nine IFA-positive ELISA-negative serum samples absorbed with HHV-6, eight remained positive by IFA (IFA false positive; ELISA true negative). FIG. 1. HHV-6-specific IgG expression (determined by ELISA) in paired serum samples collected from 14 children with ES (a), 15 patients with ES including CNS complications (b), and 20 patients undergoing BMT (c). The ELISA ratio for the first serum is represented with an open bar, while the ELISA ratio for the second serum is represented with a filled bar. The ELISA ratio of 1.0 is shown by a broken line. DISCUSSION The commercially available ELISA described in this report (H6G kit; PanBio) is highly specific for HHV-6, shows excel-

4 678 SLOOTS ET AL. J. CLIN. MICROBIOL. TABLE 4. HHV-6-specific IgG response in paired serum samples Condition (n) No. of patients with result HHV-6 isolated HHV-6 DNA detected Elevated HHV-6 IgM a Elevated HHV-6 IgG b ES (14) CNS complication (15) c BMT (20) a IgM, anti-igm antibody (detected by IFA). b IgG, anti-igg antibody (detected by ELISA). c PCR was performed on samples from only 13 of the 15 patients in the CNS complication group, and HHV-6 DNA was detected in each sample. lent reproducibility, and yields results well correlated with those by IFA, suggesting that this ELISA provides a suitable method for the determination of HHV-6 IgG antibodies in a routine clinical laboratory. ELISA and IFA gave the same result in 93% of the 502 serum samples tested. Furthermore, absorption studies showed that anomalous results with nearly all other sera were the results of an incorrect IFA result, indicating that the ELISA has very high levels of sensitivity and specificity for HHV-6- specific IgG in human serum. The poorer performance of the IFA may be a reflection of the subjective nature of interpretation of this test. The lack of cross-reactivity between HHV-6 and other herpesviruses is not surprising and has also been noted by other investigators (8, 25, 34, 35, 44). As HHV-6 has been confirmed as the cause of ES in children (24, 50, 51), it is not surprising that all patients with this disease showed elevated IgM responses by IFA. The ELISA could also be used as an aid to diagnosis of this condition, as IgG titers in paired serum samples showed large increases in all cases tested. Also, as many clinically diagnosed cases of measles, rubella, or parvovirus B19 in young children are due to primary human HHV-6 infection (46), this test should be a valuable aid in the correct diagnosis of HHV-6 infection. HHV-6 has also been implicated in a number of other childhood diseases and has been reported as a major cause of febrile seizures and hospitalizations for infants and young children (6, 38). In addition, HHV-6 has been associated with neurological disorders in adults (9, 41). The IgG ELISA described here should also be a diagnostic aid in these cases, since the IgG titer rose considerably for all patients with complications of the CNS, and this ELISA was superior to IFA for detection of anti-hhv-6 IgM (positive in only 57% of cases); the lack of IgM response is possibly indicative of reactivation of a latent infection. A significant increase in anti-hhv-6 IgG antibody titer has been reported for recipients of BMTs, and the virus has been isolated from the bone marrow of a number of these patients (3, 10, 14, 18, 19, 23, 47). Furthermore, recent studies have shown a correlation between HHV-6 reactivation, patient survival, and clinical complications following BMT (21, 33), and it has been suggested that transplant recipients should be monitored closely in order to diagnose any HHV-6 infection, avoid undue complications, and manage graft rejection (26). The IgG ELISA utilized in this study showed a significant increase in IgG titer for over half of the patients tested, while an IgM response was detected by IFA in only 20% of these patients, illustrating that this assay will be a useful adjunct in the monitoring of BMT recipients. These studies have illustrated the potential utility of the HHV-6 IgG ELISA to ensure the correct diagnosis of ES with and without CNS complications, and this ELISA should be a valuable aid in differentiating between other infections presenting with similar symptoms (measles, rubella, parvovirus B19). Furthermore, the assay can be used to detect HHV-6 reactivation in the majority of patients undergoing BMT, leading to early intervention with appropriate antiviral therapy. ACKNOWLEDGMENT This work was supported by a grant from the Queensland Government Research and Development Scheme (QGRAD). REFERENCES 1. Ablashi, D. V., S. F. Josephs, A. Buchbinder, K. Hellman, S. Nakamura, T. Llana, P. Lusso, M. Kaplan, J. Dahlberg, S. Memon, F. Imam, K. L. Ablashi, P. D. Markham, B. Kramarsky, G. R. F. Krueger, P. Biberfeld, F. Wong- Staal, S. Z. Salahuddin, and R. C. Gallo Human B-lymphotropic virus (human herpesvirus-6). J. Virol. Methods 21: Agut, H., D. Guetard, H. Collandre, C. Dauguet, L. Montagnier, J. Miclea, H. Baurmann, and A. Gessain Concomitant infection by human herpesvirus-6, HTLV-1 and HIV-2. Lancet i: Asano, Y., T. Yoshikawa, S. Suga, T. Nakashima, T. Yazaki, M. Fukuda, S. Kojima, and T. Matsuyama Reactivation of herpesvirus type 6 in children receiving bone marrow transplants for leukemia. N. Engl. J. Med. 324: Asano, Y., T. Yoshikawa, S. Suga, T. Yazaki, K. Kondo, and K. Yamanishi Fatal fulminant hepatitis in an infant with human herpesvirus-6 infection. Lancet 335: Asano, Y., T. Yoshikawa, S. Suga, T. Yazaki, T. Ozaki, Y. Saito, Y. Hatano, and M. Takahashi Enzyme-linked immunosorbent assay for detection of IgG antibody to human herpesvirus-6. J. Med. Virol. 32: Breese Hall, C., C. E. Long, K. C. Schnabel, M. T. Caserta, K. M. Mcintyre, M. A. Constanzo, A. Knott, S. Dewhurst, R. A. Insel, and L. G. Epstein Human herpesvirus-6 infection in children. A prospective study of complications and reactivations. N. Engl. J. Med. 331: Briggs, M., J. Fox, and R. S. Tedder Age prevalence of antibody to human herpesvirus-6. Lancet i: Buchbinder, A., D. V. Ablashi, C. Saxinger, S. F. Josephs, S. Z. Salahuddin, R. C. Gallo, P. Biberfeld, and A. Linde Human herpesvirus-6 and cross-reactivity with other herpesviruses. Lancet i: Buchwald, D., P. R. Cheney, D. L. Peterson, B. Henry, S. B. Wormsley, A. Geiger, D. V. Ablashi, S. Z. Salahuddin, C. Saxinger, R. Biddle, R. Kikinis, F. A. Jolesz, T. Folks, N. Balachandran, J. B. Peter, R. C. Gallo, and A. L. Komaroff A chronic illness characterised by fatigue, neurologic and immunologic disorders, and active human herpesvirus type 6 infection. Ann. Intern. Med. 116: Carrigan, D. R., and K. K. Knox Human herpesvirus 6 (HHV-6) isolation from bone marrow: HHV-6-associated bone marrow suppression in bone marrow transplant patients. Blood 84: Chen, M., N. Popescu, C. Woodworth, Z. Berneman, M. Corbellino, P. Lusso, D. V. Ablashi, and J. A. Dipaolo Human herpesvirus 6 infects cervical epithelial cells and transactivates human papillomavirus gene expression. J. Virol. 68: Clark, D. A., F. E. Alexander, P. A. Mckinney, B. E. Roberts, C. O Brien, R. F. Jarrett, R. A. Cartwright, and D. E. Onions The seroepidemiology of human herpesvirus-6 (HHV-6) from a case control study of leukemia and lymphoma. Int. J. Cancer 45: Collandre, H., J. T. Aubin, H. Agut, J. Bechet, and L. Montagnier Detection of HHV-6 by the polymerase chain reaction. J. Virol. Methods 31: Cone, R. W., R. C. Hackman, M. L. W. Huang, R. A. Bowden, J. D. Meyers, M. Metcalf, J. Zeh, R. Ashley, and L. Corey Human herpesvirus-6 in lung tissue from patients with pneumonitis after bone marrow transplantation. N. Engl. J. Med. 329: Dahl, H., A. Linde, V. Sundqvist, and B. Wahren An enzyme-linked immunosorbent assay for IgG antibodies to human herpesvirus 6. J. Virol. Methods 29: Di Luca, D., M. Zorzenon, P. Mirandola, R. Colle, G. A. Botta, and E. Cassai Human herpesvirus 6 and human herpesvirus 7 in chronic

5 VOL. 34, 1996 ELISA FOR HUMAN HERPESVIRUS fatigue syndrome. J. Clin. Microbiol. 33: Downing, R. G., N. Sewankambo, D. Serwadda, R. Honess, D. Crawford, R. Jarrett, and B. E. Griffin Isolation of human lymphotropic herpesviruses from Uganda. Lancet ii: Drobyski, W. R., W. M. Dunne, E. M. Burd, K. K. Knox, R. C. Ash, M. M. Horowitz, N. Flomenberg, and D. R. Carrigan Human herpesvirus-6 (HHV-6) infection in allogeneic bone marrow transplant recipients: evidence of a marrow suppressive role for HHV-6 in vivo. J. Infect. Dis. 167: Drobyski, W. R., K. K. Knox, D. Majewski, and D. R. Carrigan Brief report: fatal encephalitis due to variant B human herpesvirus-6 infection in a bone marrow transplant recipient. N. Engl. J. Med. 330: Holmes, G. P., J. E. Kaplan, J. A. Stewart, B. Hunt, P. F. Pinsky, and L. B. Schonberger A cluster of patients with a chronic mononucleosis-like syndrome: is Epstein-Barr virus the cause? JAMA 257: Horvat, R. T., E. Goldstein, M. Amare, A. M. Meis, P. C. Chiang, C. Penn, and A. Rabe Detection of human herpesvirus 6 and cytomegalovirus following bone marrow transplantation. In Proceedings of the International Conference on HHV-6/HHV-7, Atlanta. 22. Horvat, R. T., C. Wood, and N. Balachandran Transactivation of human immunodeficiency virus promoter by human herpesvirus 6. J. Virol. 63: Irving, W. L., and A. L. Cunningham Serological diagnosis of infection with human herpesvirus type 6. Br. Med. J. 300: Kikuta, H Human herpesvirus-6 (HHV-6) and exanthum subitum, p In D. V. Ablashi, G. R. F. Kreuger, and S. Z. Salahuddin (ed.), Human herpesvirus-6: epidemiology, molecular biology, and clinical pathology. Elsevier, Amsterdam. 25. Kirchesch, H., T. Mertens, U. Burkhardt, J. P. Kruppenbacher, A. Hoffken, and H. J. Eggers Seroconversion against human herpesvirus-6 (and other herpesviruses) and clinical illness. Lancet ii: Krueger, G. R. F., U. Klueppelberg, A. Hoffmann, and D. V. Ablashi Clinical correlates of infection with human herpesvirus-6. In Vivo 8: Levine, P. H., R. Jarrett, and D. A. Clark Epidemiology of human herpesvirus-6. Perspect. Med. Virol. 4: Levine, P. H., G. R. F. Krueger, M. Kaplan, D. Bell, R. E. DuBois, A. Huang, A. Quinlan, D. Buchwald, L. Archard, S. Gupta, J. Jones, A. Straus, and G. Tostao The post-infectious chronic fatigue syndrome, p In D. V. Ablashi (ed.), Epstein-Barr virus and human diseases. Humana Press, Clifton, N. J. 29. Levy, J. A., F. Ferro, D. Greenspan, and E. T. Lennette Frequent isolation of HHV-6 from saliva and high seroprevalence of the virus in the population. Lancet 335: Lopez, C., and R. W. Honess Human herpesvirus-6, p In B. N. Fields and D. M. Knipe (ed.), Virology, 2nd ed. Raven Press, New York. 31. Lopez, C., P. Pellett, J. Stewart, C. Goldsmith, K. Sanderlin, J. Black, D. Warfield, and P. Feorino Characteristics of human herpesvirus-6. J. Infect. Dis. 157: Lusso, P., P. D. Markham, E. Tschachler, F. Veronese, S. Z. Salahuddin, D. V. Ablashi, S. Pahwa, K. Krohn, and R. C. Gallo In vitro cellular tropism of human B-lymphotropic virus (human herpesvirus-6). J. Exp. Med. 167: Miyoshi, H., K. Tanaka-Taya, J. Hara, A. Tawa, S. Okada, and K. Yamanishi Human herpesvirus-6 and human herpesvirus-7 infection in bone marrow transplant recipients. In Proceedings of the International Conference on HHV-6/HHV-7, Atlanta. 34. Morris, D. J., E. Littler, D. Jordan, and J. R. Arrand Antibody responses to human herpesvirus 6 and other herpesviruses. Lancet ii: Okuno, T., K. Takahashi, K. Balachandra, K. Shiraki, K. Yamanishi, M. Takahashi, and K. Baba Seroepidemiology of human herpesvirus 6 infection in normal children and adults. J. Clin. Microbiol. 27: Parker, C. A., and J. M. Weber An enzyme-linked immunosorbent assay for the detection of IgG and IgM antibodies to human herpesvirus type 6. J. Virol. Methods 41: Pietroboni, G. R., G. B. Harnett, M. R. Bucens, and R. W. Honess Antibody to human herpesvirus-6 in saliva. Lancet i: Pruksananonda, P., C. Breese Hall, R. A. Insel, K. Mcintyre, P. E. Pellett, C. E. Long, K. C. Schnabel, P. H. Pincus, F. R. Stamey, T. R. Dambaugh, and J. A. Stewart Primary human herpesvirus 6 infection in young children. N. Engl. J. Med. 326: Salahuddin, S. Z., D. V. Ablashi, P. D. Markham, S. F. Josephs, S. Sturzenegger, M. Kaplan, G. Halligan, P. Biberfeld, F. Wong-Staal, B. Kramarsky, and R. C. Gallo Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders. Science 234: Saxinger, C., H. Polesky, N. Eby, S. Grufferman, R. Murphy, G. Tegtmeir, V. Parekh, S. Memon, and C. Hung Antibody reactivity with HBLV (HHV-6) in U.S. populations. J. Virol. Methods 21: Sloots, T. P., I. M. Mackay, and J. H. Pope Diagnosis of human herpesvirus-6 infection in two patients with central nervous system complications. Clin. Diagn. Virol. 3: Spira, T. J., L. H. Bozeman, K. C. Sanderlin, D. T. Warfield, P. M. Feorino, R. C. Holman, J. E. Kaplan, D. B. Fishbein, and C. Lopez Lack of correlation between human herpesvirus-6 infection and the course of human immunodeficiency virus infection. J. Infect. Dis. 161: Steeper, T. A., C. A. Horwitz, D. V. Ablashi, S. Z. Salahuddin, C. Saxinger, R. Saltzman, and B. Schwartz The spectrum of clinical and laboratory findings resulting from human herpesvirus-6 (HHV-6) in patients with mononucleosis-like illnesses not resulting from Epstein-Barr virus or cytomegalovirus. Am. J. Clin. Pathol. 93: Torigoe, S., T. Kumamoto, W. Koide, K. Taya, and K. Yamanishi Clinical manifestations associated with human herpesvirus 7 infection. Arch. Dis. Child. 72: Ward, K. N., J. J. Gray, and S. Efstathiou Brief report: primary human herpesvirus-6 infection in a patient following liver transplantation from a seropositive donor. J. Med. Virol. 28: Ward, K. N., D. R. Tait, D. W. G. Brown, and E. Miller Many cases diagnosed clinically as measles and rubella in young children are due to primary human herpesvirus-6 infection. In Proceedings of the International Conference on HHV-6/HHV-7, Atlanta. 47. Wilborn, F., V. Brinkman, C. A. Schmidt, F. Neipel, H. Gelderblom, and W. Siegert Herpesvirus type 6 in patients undergoing bone marrow transplantation: serologic features and detection by polymerase chain reaction. Blood 83: Yadav, M., A. Chandrashekran, D. M. Vasudevan, and D. V. Ablashi Frequent detection of human herpesvirus 6 in oral carcinoma. J. Natl. Cancer Inst. 86: Yadav, M., S. Umamaheswari, and D. V. Ablashi Antibody reactivity with two strains of human herpesvirus-6 in Malaysians. J. Med. Virol. 33: Yamanishi, K., T. Okuno, K. Shiraki, M. Takahashi, T. Kondo, Y. Asano, and T. Kurata Identification of human herpesvirus-6 as a causal agent for exanthem subitum. Lancet i: Yamanishi, K., T. Okuno, K. Takahashi, K. Balachandra, K. Kondo, T. Kondo, and T. Mukai Exanthem subitum and HHV-6 infection, p In D. V. Ablashi, G. R. F. Krueger, and S. Z. Salahuddin (ed.), Human herpesvirus-6: epidemiology, molecular biology and clinical pathology, 1st ed. Elsevier, Amsterdam.