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Evaluation of the specificity and sensitivity of indirect immunofluorescence tests for IgG to human herpesviruses-6 and -7
Journal of Virological Methods 106 (2002) 107 /113 www.elsevier.com/locate/jviromet
Evaluation of the specificity and sensitivity of indirect immunofluorescence tests for IgG to human herpesviruses-6 and -7 K.N. Ward *, X. Couto Parada, J. Passas, A.D. Thiruchelvam Department of Virology, Royal Free and University College Medical School, Windeyer Institute of Medical Sciences, 46 Cleveland Street, London W1T 4JF, UK Received 11 April 2002; received in revised form 17 June 2002; accepted 20 June 2002
Abstract Sera from 118 children aged up to 4 years were tested by indirect immunofluorescence for human herpesvirus-6 and 7 (HHV-6 and HHV-7) antibodies. Antibody results were confirmed as true positives if the relevant viral DNA was detected in saliva or, in some cases of primary infection, by the finding of the relevant DNA in cerebrospinal fluid or serum. Results from samples taken from the 15 children less than 6 months old showed that HHV-6 and/or HHV-7 antibody was either absent or present at low titre suggesting persistent maternal antibody rather than true infection. The sensitivity, specificity, positive and negative predictive values of the HHV-6 IgG test were therefore based on the data from the 103 children older than 6 months and the results were 95, 84, 91 and 90%, respectively. Likewise, the sensitivity, specificity, positive and negative predictive values of the HHV-7 IgG test were 95, 76, 84 and 93%, respectively. There was limited cross-reactivity between HHV-6 and HHV-7 antibodies; where both HHV-6 and HHV-7 antibodies are detected, titres above 32 may be accepted as true positives but lower titres require confirmation by detection of the relevant viral DNA or, in the case of primary infection, by a rising antibody titre. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Human herpesvirus-6; Human herpesvirus-7; Immunofluorescence test specificity; Immunofluorescence test sensitivity
1. Introduction The b human herpesviruses-6 and -7 (HHV-6 and HHV-7), discovered, respectively, in 1986 (Salahuddin et al., 1986) and 1990 (Frenkel et
* Corresponding author. Tel.: /44-20-7679-9490/9137; fax: /44-20-7580-5896
al., 1990), are closely related, with nucleic acid sequence identity ranging from 20.7 to 75.7% in various genes, and share some common antigenic epitopes (reviewed Braun et al., 1997; Black and Pellett, 1999). Primary infection with each virus usually occurs in early childhood, often without symptoms, and subsequently both viruses persist in the host and are shed in saliva throughout life (Jarrett et al., 1990; Kido et al., 1990; Wyatt and
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Frenkel, 1992; Hidaka et al., 1993; Tanaka-Taya et al., 1996). As regards disease, primary infection with either virus may sometimes cause exanthem subitum, a classical childhood illness with a rash (Yamanishi et al., 1988; Tanaka et al., 1994), and primary infection has also been associated with neurological symptoms (Hall et al., 1994; Ward and Gray, 1994; Torigoe et al., 1996). Study of the relationship between primary infection and disease requires reliable antibody tests able to distinguish between the two viruses. Yamanishi et al. (1988) used an indirect immunofluorescence test for HHV-6 IgG to detect seroconversion and hence demonstrate that primary HHV-6 infection can cause exanthem subitum. Upon the discovery of HHV-7 an indirect immunofluorescence test for HHV-7 IgG was developed (Wyatt et al., 1991). Subsequently, other tests were developed for HHV-6 and HHV-7 antibody (Black et al., 1996) but the indirect immunofluorescence test remains the most valuable; it allows evaluation of antibody development and titres in sequential serum samples, and when modified to detect antibody avidity can distinguish primary from long standing infection where only a single convalescent sample is available (Ward et al., 1993a,b, 2001). Equally importantly, antibody avidity can be used to tell primary HHV-6 and HHV-7 infections apart (Ward et al., 2001). Notwithstanding this capability of distinguishing between HHV-6 and -7 primary infections, limited antibody cross-reactivity has been reported between the two viruses, as measured by indirect immunofluorescence in sequential serum samples from children undergoing primary infection (Wyatt et al., 1991; Huang et al., 1997). In the present paper we evaluated the extent of antibody cross-reactivity in a large panel of sera from children aged from 6 months to 4 years by comparing HHV-6 and -7 antibody titres with the presence or absence of viral DNA in saliva and in some cases serum and/or cerebrospinal fluid (CSF). Thus, if antibody was detected, confirmation of specificity was sought by the detection of viral DNA, and the sensitivity and specificity of the HHV-6 and HHV-7 IgG indirect immunofluorescence tests were calculated.
2. Materials and methods 2.1. Patients The patients were 118 children whose samples were referred to Dr. K.N. Ward, Department of Virology, University College Hospital, London, UK, for diagnosis of HHV-6 and HHV-7 infection as part of a British Isles-wide Survey of encephalitis in young children which she and Professor E.M. Ross, Community Paediatrics, Guy’s, King’s and St Thomas’ School of Medicine, London, UK, conducted using the network of the British Paediatric Surveillance Unit. Ethical approval for the Survey was given by the Public Health Laboratory Service Ethics Committee. Saliva was collected from all the children with a sponge swab (Mortimer and Parry, 1991), extracted from the swab (Brown et al., 1994), and stored at /20 8C until tested. At the same time, blood was collected on filter paper after finger prick, and serum extracted at a dilution of 1 in 16 (Farzadegan et al., 1987). In total there were 140 serum/saliva pairs from the 118 children. For some of the children there were additional serum samples which had been collected by venepuncture but not at the same time as the serum/saliva pairs. Thus, acute and convalescent serum samples were available from 70% of the children (83/118) but only a single serum sample from the remainder. CSF was also collected from some of the children. 2.2. Indirect immunofluorescence tests for HHV-6 and HHV-7 antibodies HHV-6 and -7 IgG antibody titre and avidity were detected as described previously (Ward et al., 1993a, 2001). The sera were tested in doubling dilutions starting at 1 in 16 and those with a titre of B/16 were defined as seronegative. Seropositive samples with an antibody titre which was reduced 8-fold or greater by urea were defined as having low avidity IgG and conversely sera whose titre was reduced 4-fold or less were defined as having high avidity (Ward et al., 1993a, 2001). Each test run included positive control sera of low and high avidity.
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2.3. Polymerase chain reaction for HHV-6 and HHV-7 DNA The saliva extract was centrifuged at 15 000 /g for 5 min. Fifty microlitres of supernatant was removed, boiled for 10 min, cooled immediately on ice and held briefly at 4 8C until required. Nucleic acid (both RNA and DNA) was prepared from 140 ml of serum or CSF using QIAmp Viral RNA Mini Kit (Qiagen Ltd, Crawley, UK) according to the manufacturers’ instructions. Ten microlitres of the boiled saliva, or nucleic acid from serum or CSF was tested for HHV-6 and -7 DNA by the polymerase chain reaction (Kidd et al., 1998). All positive results were confirmed by a repeat test. For saliva, a further boiled aliquot of saliva was used. If sufficient of the serum or CSF fluid remained, a second extraction of nucleic acid was prepared, but, if not, an aliquot of the previously prepared nucleic acid was used. To avoid contamination, DNA extraction, PCR and gel electrophoresis were done in separate locations using separate dedicated equipment. 2.4. Definitions 2.4.1. HHV-6 and HHV-7 primary infections Primary infection was identified where (a) there was only a single serum sample available, which was either seronegative or had low avidity antibodies, and which contained the relevant viral DNA (Chiu et al., 1998; Ward et al., 2001) or (b) there were paired serum samples showing seroconversion to low avidity IgG antibody to the particular virus and also DNA of the same virus in the acute serum, or a saliva or CSF sample (Ward et al., 1993a, 2001). 2.4.2. True and false positive and negative antibody results 1) Positive antibody results on a particular serum were defined as true positives if (a) the corresponding viral DNA was detected in a saliva sample taken on the same day, or (b) if viral DNA was absent from the paired saliva and the serum under test had been taken during a primary infection too soon for
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DNA to be detected in saliva but the relevant DNA was detected in serum or CSF. 2) Positive antibody results were defined as false positives where viral DNA was not detected in the corresponding saliva and there was no evidence of a primary infection. 3) Negative antibody results were defined as true negatives if (a) the corresponding viral DNA was absent from saliva or (b) there was DNA in a saliva sample taken on the same day which was early during a primary infection before enough time had elapsed for antibody to develop. 4) Negative antibody results were considered as false negatives where viral DNA was detected in saliva but there was no evidence of a primary infection.
3. Results 3.1. HHV-6 and -7 DNA in saliva There were 140 saliva samples from 118 children up to 4 years old; sequential pairs of saliva were available from 22 children. The age distribution of viral shedding as determined by the presence of viral DNA in the 140 saliva samples is shown in Table 1. The proportion of saliva samples containing HHV-6 DNA was lowest in children less than 6 months old at 15% (3/20) and thereafter steadily increased with the overall prevalence in children aged 6 months up to 4 years being 63% (76/120). No HHV-7 DNA was detected in the saliva of a child less than 6 months old (0/20) but thereafter the proportion increased with time and the overall prevalence in children aged 6 months up to 4 years was 60% (72/120). 3.2. Primary HHV-6 and Primary HHV-7 Infections There were 7 primary HHV-6 infections, 11 primary HHV-7 infections and 2 dual primary infections, i.e. 7.6% of the children (9/118) had a primary HHV-6 infection and 11% (13/118) a primary HHV-7 infection (see Section 2.4); only one of these primary infections (an HHV-6) was in
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Table 1 Prevalence of HHV-6 and -7 DNA in children’s saliva in relation to age Age (months old)
0 /5 6 /11 12 /23 24 /35 36 /47
Viral DNA No. HHV-6 positive/total
% HHV-6 positive
No. HHV-7 positive/total
% HHV-7 positive
3/20 4/16 38/61 23/30 11/13
15 25 62 77 85
0/20 5/16 33/61 24/30 9/13
0 31 54 80 69
a child of less than 6 months old. A further four children, all older than 6 months, had evidence of recent primary infection (1 HHV-6 and 3 HHV-7) as demonstrated by a single low avidity antibody result. 3.3. Comparison of HHV-6 and -7 IgG antibody in sera with HHV-6 and -7 DNA in saliva Of all the serum samples from the 118 children, 140 had been taken on the same day as a sample of saliva; sequential serum/saliva pairs were available from 22 children. 3.3.1. Children B/6 months old Twenty of the serum/saliva pairs were from 15 children under 6 months old (sequential pairs were available from 5 children). Only three of the saliva samples contained HHV-6 DNA (two were paired with sequential negative sera from a child with primary HHV-6 infection and one was paired with a serum from a child with an HHV-6 IgG titre of 128 of high avidity). The remaining 17 saliva samples contained neither HHV-6 nor HHV-7 DNA; the corresponding sera either contained no antibody (9 were HHV-6 seronegative and 12 HHV-7 seronegative) or low levels of antibody titres (8 HHV-6 IgG geometric mean titre 19, and 8 HHV-7 IgG geometric mean titre 21). 3.3.2. Children 6 months to 4 years old For HHV-6, 120 serum/saliva pairs were available from 103 children aged between 6 months and 4 years old (sequential serum/saliva pairs had been taken from 17 children). For HHV-7, 118 serum/ saliva pairs were available from 101 children as it
was not possible to test for HHV-7 antibodies in 2 samples because of non-specific immunofluorescence. 3.3.2.1. Sensitivity, specificity, positive and negative predictive values of the HHV-6 IgG test. The results of the HHV-6 IgG tests on the sera are shown in Table 2. As regards the 71 true positives (geometric mean HHV-6 IgG titre 191; 95% confidence limits 138 to 263), 67 were confirmed by the presence of HHV-6 DNA in the paired saliva and the remaining 4 had been taken from 3 children during primary HHV-6 infection. The other 7 sera gave a false positive HHV-6 antibody result; 5 of these sera (geometric mean titre 37) had been taken from 4 children all seropositive for HHV-7 IgG of high avidity (geometric mean titre 169) and the other 2 sera (HHV-6 titres both 8192) from 2 children who were undergoing primary HHV-7 infection. In the case of the 38 true antibody negative results, 33 were confirmed by the absence of HHVTable 2 HHV-6 IgG results Serum HHV-6 IgG
True False
Positive
Negative
71* 7***
38** 4****
* 67 with HHV-6 DNA in saliva and 4 during primary HHV-6 infection. ** 33 with no HHV-6 DNA in saliva and 5 early in primary HHV-6 infection. *** HHV-6 DNA not detected in saliva. **** HHV-6 DNA detected in saliva.
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6 DNA in saliva and the remaining 5 had been taken from 4 children early in a primary HHV-6 infection. The other 4 sera gave a false negative antibody result and were from children for whom convalescent sera were not available to test for seroconversion and hence primary HHV-6 infection could not be excluded. The sensitivity, specificity, positive and negative predictive values of the HHV-6 IgG test were 95% (71/75), 84% (38/45), 91% (71/78) and 90% (38/42), respectively. 3.3.2.2. Sensitivity, specificity, positive and negative predictive values of the HHV-7 IgG test. The results of the HHV-7 IgG tests on the sera are shown in Table 3. As regards the 64 true positives (geometric mean HHV-7 IgG titre 302; 95% confidence limits 218 to 416), 63 were confirmed by the presence of HHV-7 DNA in the paired saliva and the remaining 1 had been taken from a child during primary HHV-7 infection. The other 12 sera which gave a false positive HHV-7 antibody result (geometric mean titre 32) had been taken from 11 children all seropositive for HHV-6 IgG of high avidity (geometric mean titre 242). In the case of the 39 true antibody negative results, 35 were confirmed by the absence of HHV7 DNA in saliva and the remaining 4 had been taken from 4 children early in a primary HHV-7 infection. The other 3 sera gave a false negative antibody result, 2 of which were from children for whom convalescent sera were not available to test for seroconversion and hence primary HHV-7 infection could not be excluded. Table 3 HHV-7 IgG results Serum HHV-7 IgG
True False
Positive
Negative
64* 12***
39** 3****
* 63 with HHV-6 DNA in saliva and 1 during primary HHV-7 infection. ** 35 with no HHV-6 DNA in saliva and 4 early in primary HHV-7 infection. *** HHV-7 DNA not detected in saliva. **** HHV-7 DNA detected in saliva.
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The sensitivity, specificity, positive and negative predictive values of the HHV-7 IgG test were 95% (64/67), 76% (39/51), 84% (64/76) and 93% (39/42), respectively.
4. Discussion Although indirect immunofluorescence tests for HHV-6 and HHV-7 IgG have been in use for over a decade and we ourselves have recently used these tests to show that antibody avidity can distinguish primary HHV-6 and -7 infections, some doubts remain about the extent of cross-reactivity between the two viruses and hence about the value of the tests for diagnosis. To investigate the extent of cross-reactivity, a panel of samples from both infected and uninfected individuals is required and such a population is only to be found amongst very young children as primary infection almost always occurs in early childhood. We therefore chose to analyse a panel of samples from children aged up to 4 years old. It should be noted there was an especially high prevalence of primary HHV-6 and -7 infections in this group because samples had been submitted to identify illness caused by the two viruses. For this reason account was taken of primary infections in the definition of true and false antibody results laid out in the methods. On this basis the sensitivity and specificity of the tests and their positive and negative predictive values were calculated. Samples taken in the first 6 months of life were excluded from the comparison since HHV-6 and/ or HHV-7 antibody was either absent or present at low titre suggesting the persistence of maternal antibody rather than true infection; this impression was confirmed by the fact that in this age group only 3 of the 20 saliva samples contained HHV-6 DNA and none contained HHV-7 DNA. Such persistence of maternal antibody early in life is to be expected and had been noted previously in various studies including our own (Ward et al., 2001). Considering the sensitivity and specificity of the HHV-6 and HHV-7 antibody tests, high sensitivity was found (95% in each case) but the specificity was lower (84 and 76%, respectively). The corre-
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sponding negative predictive values (90 and 93%, respectively) are consistent (Tijssen, 1985) with the high prevalence of infection in the children under study (Table 1). However, the positive predictive values were surprisingly low (91 and 84%, respectively) (Tijssen, 1985) considering the high prevalence of infection in this population (Table 1). As regards the seven HHV-6 false positive antibody results, all were defined as such because there was no evidence of viral infection, i.e. no viral DNA. However, in two cases the HHV-6 antibody was of very high titre and high avidity and accompanied by primary HHV-7 infection; such very high titres of high avidity are triggered in primary HHV-7 infection in children previously infected with HHV-6 (Ward et al., 2001). The surprising lack of HHV-6 DNA in the saliva of a child with pre-existing HHV-6 antibody has been noted previously during a primary HHV-7 infection (Asano et al., 1995). The five remaining false positive HHV-6 antibody results occurred when HHV-7 antibody was also present suggesting a limited cross-reaction between HHV-7 and HHV6; in each instance the HHV-6 antibody titre was low and below the 95% confidence limits for the antibody titre of the confirmed HHV-6 positive sera. The evidence for limited cross-reaction was more striking in the case of HHV-7 as the 12 false positive HHV-7 antibody results all showed weakly staining fluorescence of low titre and below the 95% confidence limits for the titre of the true HHV-7 positive sera; in each case HHV-6 antibody of high titre was also present. Such limited cross reactivity between naturally induced antibodies to HHV-6 and -7 has indeed been noted in earlier investigations (Black et al., 1996; Huang et al., 1997). Limited antibody cross-reaction is the most likely explanation for the five HHV-6 false positive antibody results of low titre, but another explanation might be that shedding of virus in saliva after primary infection is intermittent rather than continuous. Salivary shedding of HHV-6 is known to be irregular in adults (Tanaka-Taya et al., 1996) and Hall et al. (1998) showed that, 6 months to 1 year after primary HHV-6 infection, HHV-6 DNA was detected in the saliva of only 70% of children. In contrast, we did not find evidence suggestive of
intermittent shedding in our population of young children since saliva samples were invariably positive where there was a high titre of high avidity antibody except in the two cases of primary HHV7 infection. As for HHV-7, intermittent salivary shedding seems even more unlikely since this virus is always present in human saliva (Wyatt and Frenkel, 1992; Hidaka et al., 1993), with HHV-7 DNA being detected in 96% of adults (Kidd et al., 1996). In summary, HHV-6 and HHV-7 IgG indirect immunofluorescence tests are both sensitive and specific although there is some evidence of limited cross-reactivity. Nevertheless, if the tests are used with proper safeguards they are thoroughly reliable. Where HHV-6 and/or HHV-7 antibody is present in titres above 32, they may be accepted as true positives without further investigation whereas the rare cases of lower antibody titres require confirmation by detection of the relevant viral DNA or in the case of primary infection by a rising antibody titre. Thus the present work has further validated the indirect immunofluorescence tests for HHV-6 and HHV-7 and confirmed their usefulness for the exploration of the clinical consequences of infection by these viruses.
Acknowledgements The authors thank the Wellcome Trust (project grant 051350/Z) for generous support for the Survey of encephalitis in young children, and the British Paediatric Surveillance Unit for access to its British Isles-wide network. They are also most grateful to the many paediatricians and laboratories who have sent samples from patients for specialised HHV-6 and HHV-7 diagnosis, and to Ms Gloria Charter for excellent assistance as data co-ordinator for the Survey.
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K.N. Ward et al. / Journal of Virological Methods 106 (2002) 107 /113 Black, J.B., Pellett, P.E., 1999. Human herpesvirus 7. Rev. Med. Virol. 9, 245 /262. Black, J.B., Schwarz, T.F., Patton, J.L., Kite-Powell, K., Pellett, P.E., Wiersbitzky, S., Bruns, R., Muller, C., Jager, G., Stewart, J.A., 1996. Evaluation of immunoassays for detection of antibodies to human herpesvirus 7. Clin. Diagn. Lab. Immunol. 3, 79 /83. Braun, D.K., Dominguez, G., Pellett, P.E., 1997. Human herpesvirus 6. Clin. Microbiol. Rev. 10, 521 /567. Brown, D.W., Ramsay, M.E., Richards, A.F., Miller, E., 1994. Salivary diagnosis of measles: a study of notified cases in the United Kingdom, 1991 /3. Br. Med. J. 308, 1015 /1017. Chiu, S.S., Cheung, C.Y., Tse, C.Y., Peiris, M., 1998. Early diagnosis of primary human herpesvirus 6 infection in childhood: serology, polymerase chain reaction, and virus load. J. Infect. Dis. 178, 1250 /1256. Farzadegan, H., Quinn, T., Polk, B.F., 1987. Detecting antibodies to human immunodeficiency virus in dried blood on filter papers. J. Infect. Dis. 155, 1073 /1074. Frenkel, N., Schirmer, E.C., Wyatt, L.S., Katsafanas, G., Roffman, E., Danovich, R.M., June, C.H., 1990. Isolation of a new herpesvirus from human CD4/ T cells. Proc. Natl. Acad. Sci. USA 87, 748 /752. Hall, C.B., Caserta, M.T., Schnabel, K.C., Long, C., Epstein, L.G., Insel, R.A., Dewhurst, S., 1998. Persistence of human herpesvirus 6 according to site and variant: possible greater neurotropism of variant A. Clin. Infect. Dis. 26, 132 /137. Hall, C.B., Long, C.E., Schnabel, K.C., Caserta, M.T., McIntyre, K.M., Costanzo, M.A., Knott, A., Dewhurst, S., Insel, R.A., Epstein, L.G., 1994. Human herpesvirus-6 infection in children. A prospective study of complications and reactivation. New Engl. J. Med. 331, 432 /438. Hidaka, Y., Liu, Y., Yamamoto, M., Mori, R., Miyazaki, C., Kusuhara, K., Okada, K., Ueda, K., 1993. Frequent isolation of human herpesvirus 7 from saliva samples. J. Med. Virol. 40, 343 /346. Huang, L.M., Lee, C.Y., Liu, M.Y., Lee, P.I., 1997. Primary infections of human herpesvirus-7 and herpesvirus-6: a comparative, longitudinal study up to 6 years of age. Acta Paediatr. 86, 604 /608. Jarrett, R.F., Clark, D.A., Josephs, S.F., Onions, D.E., 1990. Detection of human herpesvirus-6 DNA in peripheral blood and saliva. J. Med. Virol. 32, 73 /76. Kidd, I.M., Clark, D.A., Ait, K.M., Griffiths, P.D., Emery, V.C., 1996. Measurement of human herpesvirus 7 load in peripheral blood and saliva of healthy subjects by quantitative polymerase chain reaction. J. Infect. Dis. 174, 396 / 401. Kidd, I.M., Clark, D.A., Bremner, J.A., Pillay, D., Griffiths, P.D., Emery, V.C., 1998. A multiplex PCR assay for the simultaneous detection of human herpesvirus 6 and human herpesvirus 7, with typing of HHV-6 by enzyme cleavage of PCR products. J. Virol. Methods 70, 29 /36. Kido, S., Kondo, K., Kondo, T., Morishima, T., Takahashi, M., Yamanishi, K., 1990. Detection of human herpesvirus 6
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DNA in throat swabs by polymerase chain reaction. J. Med. Virol. 32, 139 /142. Mortimer, P.P., Parry, J.V., 1991. Non invasive virological diagnosis: are saliva and urine specimens adequate for blood? Rev. Med. Virol. 1, 73 /78. Salahuddin, S.Z., Ablashi, D.V., Markham, P.D., Josephs, S.F., Sturzenegger, S., Kaplan, M., Halligan, G., Biberfeld, P., Wong, S.F., Kramarsky, B., et al., 1986. Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders. Science 234, 596 /601. Tanaka, K., Kondo, T., Torigoe, S., Okada, S., Mukai, T., Yamanishi, K., 1994. Human herpesvirus 7: another causal agent for roseola (exanthem subitum). J. Pediatr. 125, 1 /5. Tanaka-Taya, K., Kondo, T., Mukai, T., Miyoshi, H., Yamamoto, Y., Okada, S., Yamanishi, K., 1996. Seroepidemiological study of human herpesvirus-6 and -7 in children of different ages and detection of these two viruses in throat swabs by polymerase chain reaction. J. Med. Virol. 48, 88 / 94. Tijssen, P.J., 1985. Practice and theory of enzyme immunoassays. In: Burdon, R.H., van Knippenberg, P.H. (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, vol. 15. Elsevier, Amsterdam, New York, Oxford, pp. 389 /390. Torigoe, S., Koide, W., Yamada, M., Miyashiro, E., Tanaka, T.K., Yamanishi, K., 1996. Human herpesvirus 7 infection associated with central nervous system manifestations. J. Pediatr. 129, 301 /305. Ward, K.N., Gray, J.J., 1994. Primary human herpesvirus-6 infection is frequently overlooked as a cause of febrile fits in young children. J. Med. Virol. 42, 119 /123. Ward, K.N., Gray, J.J., Fotheringham, M.W., Sheldon, M.J., 1993a. IgG antibodies to human herpesvirus-6 in young children: changes in avidity of antibody correlate with time after infection. J. Med. Virol. 39, 131 /138. Ward, K.N., Gray, J.J., Joslin, M.E., Sheldon, M.J., 1993b. Avidity of IgG antibodies to human herpesvirus-6 distinguishes primary from recurrent infection in organ transplant recipients and excludes cross-reactivity with other herpesviruses. J. Med. Virol. 39, 44 /49. Ward, K.N., Turner, D.J., Couto Parada, X., Thiruchelvam, A.D., 2001. Use of immunoglobulin G antibody avidity for differentiation of primary human herpesvirus 6 and 7 infections. J. Clin. Microbiol. 39, 959 /963. Wyatt, L.S., Frenkel, N., 1992. Human herpesvirus 7 is a constitutive inhabitant of adult human saliva. J. Virol. 66, 3206 /3209. Wyatt, L.S., Rodriguez, W.J., Balachandran, N., Frenkel, N., 1991. Human herpesvirus 7: antigenic properties and prevalence in children and adults. J. Virol. 65, 6260 /6265. Yamanishi, K., Okuno, T., Shiraki, K., Takahashi, M., Kondo, T., Asano, Y., Kurata, T., 1988. Identification of human herpesvirus-6 as a causal agent for exanthem subitum. Lancet 1, 1065 /1067.
Evaluation of the specificity and sensitivity of indirect immunofluorescence tests for IgG to human herpesviruses-6 and -7 K.N. Ward *, X. Couto Parada, J. Passas, A.D. Thiruchelvam Department of Virology, Royal Free and University College Medical School, Windeyer Institute of Medical Sciences, 46 Cleveland Street, London W1T 4JF, UK Received 11 April 2002; received in revised form 17 June 2002; accepted 20 June 2002
Abstract Sera from 118 children aged up to 4 years were tested by indirect immunofluorescence for human herpesvirus-6 and 7 (HHV-6 and HHV-7) antibodies. Antibody results were confirmed as true positives if the relevant viral DNA was detected in saliva or, in some cases of primary infection, by the finding of the relevant DNA in cerebrospinal fluid or serum. Results from samples taken from the 15 children less than 6 months old showed that HHV-6 and/or HHV-7 antibody was either absent or present at low titre suggesting persistent maternal antibody rather than true infection. The sensitivity, specificity, positive and negative predictive values of the HHV-6 IgG test were therefore based on the data from the 103 children older than 6 months and the results were 95, 84, 91 and 90%, respectively. Likewise, the sensitivity, specificity, positive and negative predictive values of the HHV-7 IgG test were 95, 76, 84 and 93%, respectively. There was limited cross-reactivity between HHV-6 and HHV-7 antibodies; where both HHV-6 and HHV-7 antibodies are detected, titres above 32 may be accepted as true positives but lower titres require confirmation by detection of the relevant viral DNA or, in the case of primary infection, by a rising antibody titre. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Human herpesvirus-6; Human herpesvirus-7; Immunofluorescence test specificity; Immunofluorescence test sensitivity
1. Introduction The b human herpesviruses-6 and -7 (HHV-6 and HHV-7), discovered, respectively, in 1986 (Salahuddin et al., 1986) and 1990 (Frenkel et
* Corresponding author. Tel.: /44-20-7679-9490/9137; fax: /44-20-7580-5896
al., 1990), are closely related, with nucleic acid sequence identity ranging from 20.7 to 75.7% in various genes, and share some common antigenic epitopes (reviewed Braun et al., 1997; Black and Pellett, 1999). Primary infection with each virus usually occurs in early childhood, often without symptoms, and subsequently both viruses persist in the host and are shed in saliva throughout life (Jarrett et al., 1990; Kido et al., 1990; Wyatt and
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Frenkel, 1992; Hidaka et al., 1993; Tanaka-Taya et al., 1996). As regards disease, primary infection with either virus may sometimes cause exanthem subitum, a classical childhood illness with a rash (Yamanishi et al., 1988; Tanaka et al., 1994), and primary infection has also been associated with neurological symptoms (Hall et al., 1994; Ward and Gray, 1994; Torigoe et al., 1996). Study of the relationship between primary infection and disease requires reliable antibody tests able to distinguish between the two viruses. Yamanishi et al. (1988) used an indirect immunofluorescence test for HHV-6 IgG to detect seroconversion and hence demonstrate that primary HHV-6 infection can cause exanthem subitum. Upon the discovery of HHV-7 an indirect immunofluorescence test for HHV-7 IgG was developed (Wyatt et al., 1991). Subsequently, other tests were developed for HHV-6 and HHV-7 antibody (Black et al., 1996) but the indirect immunofluorescence test remains the most valuable; it allows evaluation of antibody development and titres in sequential serum samples, and when modified to detect antibody avidity can distinguish primary from long standing infection where only a single convalescent sample is available (Ward et al., 1993a,b, 2001). Equally importantly, antibody avidity can be used to tell primary HHV-6 and HHV-7 infections apart (Ward et al., 2001). Notwithstanding this capability of distinguishing between HHV-6 and -7 primary infections, limited antibody cross-reactivity has been reported between the two viruses, as measured by indirect immunofluorescence in sequential serum samples from children undergoing primary infection (Wyatt et al., 1991; Huang et al., 1997). In the present paper we evaluated the extent of antibody cross-reactivity in a large panel of sera from children aged from 6 months to 4 years by comparing HHV-6 and -7 antibody titres with the presence or absence of viral DNA in saliva and in some cases serum and/or cerebrospinal fluid (CSF). Thus, if antibody was detected, confirmation of specificity was sought by the detection of viral DNA, and the sensitivity and specificity of the HHV-6 and HHV-7 IgG indirect immunofluorescence tests were calculated.
2. Materials and methods 2.1. Patients The patients were 118 children whose samples were referred to Dr. K.N. Ward, Department of Virology, University College Hospital, London, UK, for diagnosis of HHV-6 and HHV-7 infection as part of a British Isles-wide Survey of encephalitis in young children which she and Professor E.M. Ross, Community Paediatrics, Guy’s, King’s and St Thomas’ School of Medicine, London, UK, conducted using the network of the British Paediatric Surveillance Unit. Ethical approval for the Survey was given by the Public Health Laboratory Service Ethics Committee. Saliva was collected from all the children with a sponge swab (Mortimer and Parry, 1991), extracted from the swab (Brown et al., 1994), and stored at /20 8C until tested. At the same time, blood was collected on filter paper after finger prick, and serum extracted at a dilution of 1 in 16 (Farzadegan et al., 1987). In total there were 140 serum/saliva pairs from the 118 children. For some of the children there were additional serum samples which had been collected by venepuncture but not at the same time as the serum/saliva pairs. Thus, acute and convalescent serum samples were available from 70% of the children (83/118) but only a single serum sample from the remainder. CSF was also collected from some of the children. 2.2. Indirect immunofluorescence tests for HHV-6 and HHV-7 antibodies HHV-6 and -7 IgG antibody titre and avidity were detected as described previously (Ward et al., 1993a, 2001). The sera were tested in doubling dilutions starting at 1 in 16 and those with a titre of B/16 were defined as seronegative. Seropositive samples with an antibody titre which was reduced 8-fold or greater by urea were defined as having low avidity IgG and conversely sera whose titre was reduced 4-fold or less were defined as having high avidity (Ward et al., 1993a, 2001). Each test run included positive control sera of low and high avidity.
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2.3. Polymerase chain reaction for HHV-6 and HHV-7 DNA The saliva extract was centrifuged at 15 000 /g for 5 min. Fifty microlitres of supernatant was removed, boiled for 10 min, cooled immediately on ice and held briefly at 4 8C until required. Nucleic acid (both RNA and DNA) was prepared from 140 ml of serum or CSF using QIAmp Viral RNA Mini Kit (Qiagen Ltd, Crawley, UK) according to the manufacturers’ instructions. Ten microlitres of the boiled saliva, or nucleic acid from serum or CSF was tested for HHV-6 and -7 DNA by the polymerase chain reaction (Kidd et al., 1998). All positive results were confirmed by a repeat test. For saliva, a further boiled aliquot of saliva was used. If sufficient of the serum or CSF fluid remained, a second extraction of nucleic acid was prepared, but, if not, an aliquot of the previously prepared nucleic acid was used. To avoid contamination, DNA extraction, PCR and gel electrophoresis were done in separate locations using separate dedicated equipment. 2.4. Definitions 2.4.1. HHV-6 and HHV-7 primary infections Primary infection was identified where (a) there was only a single serum sample available, which was either seronegative or had low avidity antibodies, and which contained the relevant viral DNA (Chiu et al., 1998; Ward et al., 2001) or (b) there were paired serum samples showing seroconversion to low avidity IgG antibody to the particular virus and also DNA of the same virus in the acute serum, or a saliva or CSF sample (Ward et al., 1993a, 2001). 2.4.2. True and false positive and negative antibody results 1) Positive antibody results on a particular serum were defined as true positives if (a) the corresponding viral DNA was detected in a saliva sample taken on the same day, or (b) if viral DNA was absent from the paired saliva and the serum under test had been taken during a primary infection too soon for
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DNA to be detected in saliva but the relevant DNA was detected in serum or CSF. 2) Positive antibody results were defined as false positives where viral DNA was not detected in the corresponding saliva and there was no evidence of a primary infection. 3) Negative antibody results were defined as true negatives if (a) the corresponding viral DNA was absent from saliva or (b) there was DNA in a saliva sample taken on the same day which was early during a primary infection before enough time had elapsed for antibody to develop. 4) Negative antibody results were considered as false negatives where viral DNA was detected in saliva but there was no evidence of a primary infection.
3. Results 3.1. HHV-6 and -7 DNA in saliva There were 140 saliva samples from 118 children up to 4 years old; sequential pairs of saliva were available from 22 children. The age distribution of viral shedding as determined by the presence of viral DNA in the 140 saliva samples is shown in Table 1. The proportion of saliva samples containing HHV-6 DNA was lowest in children less than 6 months old at 15% (3/20) and thereafter steadily increased with the overall prevalence in children aged 6 months up to 4 years being 63% (76/120). No HHV-7 DNA was detected in the saliva of a child less than 6 months old (0/20) but thereafter the proportion increased with time and the overall prevalence in children aged 6 months up to 4 years was 60% (72/120). 3.2. Primary HHV-6 and Primary HHV-7 Infections There were 7 primary HHV-6 infections, 11 primary HHV-7 infections and 2 dual primary infections, i.e. 7.6% of the children (9/118) had a primary HHV-6 infection and 11% (13/118) a primary HHV-7 infection (see Section 2.4); only one of these primary infections (an HHV-6) was in
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Table 1 Prevalence of HHV-6 and -7 DNA in children’s saliva in relation to age Age (months old)
0 /5 6 /11 12 /23 24 /35 36 /47
Viral DNA No. HHV-6 positive/total
% HHV-6 positive
No. HHV-7 positive/total
% HHV-7 positive
3/20 4/16 38/61 23/30 11/13
15 25 62 77 85
0/20 5/16 33/61 24/30 9/13
0 31 54 80 69
a child of less than 6 months old. A further four children, all older than 6 months, had evidence of recent primary infection (1 HHV-6 and 3 HHV-7) as demonstrated by a single low avidity antibody result. 3.3. Comparison of HHV-6 and -7 IgG antibody in sera with HHV-6 and -7 DNA in saliva Of all the serum samples from the 118 children, 140 had been taken on the same day as a sample of saliva; sequential serum/saliva pairs were available from 22 children. 3.3.1. Children B/6 months old Twenty of the serum/saliva pairs were from 15 children under 6 months old (sequential pairs were available from 5 children). Only three of the saliva samples contained HHV-6 DNA (two were paired with sequential negative sera from a child with primary HHV-6 infection and one was paired with a serum from a child with an HHV-6 IgG titre of 128 of high avidity). The remaining 17 saliva samples contained neither HHV-6 nor HHV-7 DNA; the corresponding sera either contained no antibody (9 were HHV-6 seronegative and 12 HHV-7 seronegative) or low levels of antibody titres (8 HHV-6 IgG geometric mean titre 19, and 8 HHV-7 IgG geometric mean titre 21). 3.3.2. Children 6 months to 4 years old For HHV-6, 120 serum/saliva pairs were available from 103 children aged between 6 months and 4 years old (sequential serum/saliva pairs had been taken from 17 children). For HHV-7, 118 serum/ saliva pairs were available from 101 children as it
was not possible to test for HHV-7 antibodies in 2 samples because of non-specific immunofluorescence. 3.3.2.1. Sensitivity, specificity, positive and negative predictive values of the HHV-6 IgG test. The results of the HHV-6 IgG tests on the sera are shown in Table 2. As regards the 71 true positives (geometric mean HHV-6 IgG titre 191; 95% confidence limits 138 to 263), 67 were confirmed by the presence of HHV-6 DNA in the paired saliva and the remaining 4 had been taken from 3 children during primary HHV-6 infection. The other 7 sera gave a false positive HHV-6 antibody result; 5 of these sera (geometric mean titre 37) had been taken from 4 children all seropositive for HHV-7 IgG of high avidity (geometric mean titre 169) and the other 2 sera (HHV-6 titres both 8192) from 2 children who were undergoing primary HHV-7 infection. In the case of the 38 true antibody negative results, 33 were confirmed by the absence of HHVTable 2 HHV-6 IgG results Serum HHV-6 IgG
True False
Positive
Negative
71* 7***
38** 4****
* 67 with HHV-6 DNA in saliva and 4 during primary HHV-6 infection. ** 33 with no HHV-6 DNA in saliva and 5 early in primary HHV-6 infection. *** HHV-6 DNA not detected in saliva. **** HHV-6 DNA detected in saliva.
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6 DNA in saliva and the remaining 5 had been taken from 4 children early in a primary HHV-6 infection. The other 4 sera gave a false negative antibody result and were from children for whom convalescent sera were not available to test for seroconversion and hence primary HHV-6 infection could not be excluded. The sensitivity, specificity, positive and negative predictive values of the HHV-6 IgG test were 95% (71/75), 84% (38/45), 91% (71/78) and 90% (38/42), respectively. 3.3.2.2. Sensitivity, specificity, positive and negative predictive values of the HHV-7 IgG test. The results of the HHV-7 IgG tests on the sera are shown in Table 3. As regards the 64 true positives (geometric mean HHV-7 IgG titre 302; 95% confidence limits 218 to 416), 63 were confirmed by the presence of HHV-7 DNA in the paired saliva and the remaining 1 had been taken from a child during primary HHV-7 infection. The other 12 sera which gave a false positive HHV-7 antibody result (geometric mean titre 32) had been taken from 11 children all seropositive for HHV-6 IgG of high avidity (geometric mean titre 242). In the case of the 39 true antibody negative results, 35 were confirmed by the absence of HHV7 DNA in saliva and the remaining 4 had been taken from 4 children early in a primary HHV-7 infection. The other 3 sera gave a false negative antibody result, 2 of which were from children for whom convalescent sera were not available to test for seroconversion and hence primary HHV-7 infection could not be excluded. Table 3 HHV-7 IgG results Serum HHV-7 IgG
True False
Positive
Negative
64* 12***
39** 3****
* 63 with HHV-6 DNA in saliva and 1 during primary HHV-7 infection. ** 35 with no HHV-6 DNA in saliva and 4 early in primary HHV-7 infection. *** HHV-7 DNA not detected in saliva. **** HHV-7 DNA detected in saliva.
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The sensitivity, specificity, positive and negative predictive values of the HHV-7 IgG test were 95% (64/67), 76% (39/51), 84% (64/76) and 93% (39/42), respectively.
4. Discussion Although indirect immunofluorescence tests for HHV-6 and HHV-7 IgG have been in use for over a decade and we ourselves have recently used these tests to show that antibody avidity can distinguish primary HHV-6 and -7 infections, some doubts remain about the extent of cross-reactivity between the two viruses and hence about the value of the tests for diagnosis. To investigate the extent of cross-reactivity, a panel of samples from both infected and uninfected individuals is required and such a population is only to be found amongst very young children as primary infection almost always occurs in early childhood. We therefore chose to analyse a panel of samples from children aged up to 4 years old. It should be noted there was an especially high prevalence of primary HHV-6 and -7 infections in this group because samples had been submitted to identify illness caused by the two viruses. For this reason account was taken of primary infections in the definition of true and false antibody results laid out in the methods. On this basis the sensitivity and specificity of the tests and their positive and negative predictive values were calculated. Samples taken in the first 6 months of life were excluded from the comparison since HHV-6 and/ or HHV-7 antibody was either absent or present at low titre suggesting the persistence of maternal antibody rather than true infection; this impression was confirmed by the fact that in this age group only 3 of the 20 saliva samples contained HHV-6 DNA and none contained HHV-7 DNA. Such persistence of maternal antibody early in life is to be expected and had been noted previously in various studies including our own (Ward et al., 2001). Considering the sensitivity and specificity of the HHV-6 and HHV-7 antibody tests, high sensitivity was found (95% in each case) but the specificity was lower (84 and 76%, respectively). The corre-
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sponding negative predictive values (90 and 93%, respectively) are consistent (Tijssen, 1985) with the high prevalence of infection in the children under study (Table 1). However, the positive predictive values were surprisingly low (91 and 84%, respectively) (Tijssen, 1985) considering the high prevalence of infection in this population (Table 1). As regards the seven HHV-6 false positive antibody results, all were defined as such because there was no evidence of viral infection, i.e. no viral DNA. However, in two cases the HHV-6 antibody was of very high titre and high avidity and accompanied by primary HHV-7 infection; such very high titres of high avidity are triggered in primary HHV-7 infection in children previously infected with HHV-6 (Ward et al., 2001). The surprising lack of HHV-6 DNA in the saliva of a child with pre-existing HHV-6 antibody has been noted previously during a primary HHV-7 infection (Asano et al., 1995). The five remaining false positive HHV-6 antibody results occurred when HHV-7 antibody was also present suggesting a limited cross-reaction between HHV-7 and HHV6; in each instance the HHV-6 antibody titre was low and below the 95% confidence limits for the antibody titre of the confirmed HHV-6 positive sera. The evidence for limited cross-reaction was more striking in the case of HHV-7 as the 12 false positive HHV-7 antibody results all showed weakly staining fluorescence of low titre and below the 95% confidence limits for the titre of the true HHV-7 positive sera; in each case HHV-6 antibody of high titre was also present. Such limited cross reactivity between naturally induced antibodies to HHV-6 and -7 has indeed been noted in earlier investigations (Black et al., 1996; Huang et al., 1997). Limited antibody cross-reaction is the most likely explanation for the five HHV-6 false positive antibody results of low titre, but another explanation might be that shedding of virus in saliva after primary infection is intermittent rather than continuous. Salivary shedding of HHV-6 is known to be irregular in adults (Tanaka-Taya et al., 1996) and Hall et al. (1998) showed that, 6 months to 1 year after primary HHV-6 infection, HHV-6 DNA was detected in the saliva of only 70% of children. In contrast, we did not find evidence suggestive of
intermittent shedding in our population of young children since saliva samples were invariably positive where there was a high titre of high avidity antibody except in the two cases of primary HHV7 infection. As for HHV-7, intermittent salivary shedding seems even more unlikely since this virus is always present in human saliva (Wyatt and Frenkel, 1992; Hidaka et al., 1993), with HHV-7 DNA being detected in 96% of adults (Kidd et al., 1996). In summary, HHV-6 and HHV-7 IgG indirect immunofluorescence tests are both sensitive and specific although there is some evidence of limited cross-reactivity. Nevertheless, if the tests are used with proper safeguards they are thoroughly reliable. Where HHV-6 and/or HHV-7 antibody is present in titres above 32, they may be accepted as true positives without further investigation whereas the rare cases of lower antibody titres require confirmation by detection of the relevant viral DNA or in the case of primary infection by a rising antibody titre. Thus the present work has further validated the indirect immunofluorescence tests for HHV-6 and HHV-7 and confirmed their usefulness for the exploration of the clinical consequences of infection by these viruses.
Acknowledgements The authors thank the Wellcome Trust (project grant 051350/Z) for generous support for the Survey of encephalitis in young children, and the British Paediatric Surveillance Unit for access to its British Isles-wide network. They are also most grateful to the many paediatricians and laboratories who have sent samples from patients for specialised HHV-6 and HHV-7 diagnosis, and to Ms Gloria Charter for excellent assistance as data co-ordinator for the Survey.
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