Time to Seroconversion by WB
Median time to development of a full WB profile was 33 days for group 1 (Figure 1A), 40 days for group 2 (Figure 1B), and 47 days for group 3 (Figure 1C). Sixty percent of subjects with primary HSV-1 infection had full WB profiles to HSV-1 by 6 weeks. Full HSV-2 profiles developed by 6 weeks for 36% to 53% in groups 3 and 2, respectively (Table 1). Time to seroconversion, as determined by WB, did not differ among the three groups (P = 0.22; log rank test).
Positivity of a WB test is based on the development of antibody responses sufficient to show at least four bands. 18 However, seroconversion is a dynamic process, with antibodies to different HSV proteins developing at different rates. 17,21 Thus, when a progressive change from a seronegative blot to one with fewer than four specific bands is noted in paired testing of at least two sera, this “limited profile” can be highly predictive of recent HSV infection. 22 Therefore, we examined time to seroconversion on the basis of the first indication of a limited profile to the appropriate HSV type. Not surprisingly, median times were less than those required for a full WB profile to develop: 29 days for group 1 (vs. 33 days for a full HSV-1 profile), 19 days for group 2 (vs. 40 days for a full HSV-2 profile), and 34 days for group 3 (vs. 47 days for a full HSV-2 profile) (Figure 1A–C). A higher proportion showed limited WB profiles than full WB profiles at 6 weeks and 3 months (Table 1).
Differences Between Tests of Time to Seroconversion
Time to seroconversion by HerpeSelect versus time to development of a full WB profile was compared by the paired Prentice–Wilcoxon test. 20 No statistically significant difference in time to HSV-1 seroconversion was seen (group 1;P = 0.12). However, HSV-2 seroconversion was determined significantly faster by the HerpeSelect test than by WB (P < 0.001 for group 2 and P = 0.02 for group 3).
When HerpeSelect results were compared with results based on observing a limited WB profile, the WB was faster in demonstrating primary HSV-2 seroconversion (group 2;P = 0.01). However, no significant differences between the two tests were seen for subjects with primary HSV-1 infection (P = 0.45) or subjects with nonprimary first episodes of HSV-2 infection (P = 0.23).
Subjects Without Documented Seroconversion
Overall, 15% of subjects (17 of 113) did not show seroconversion by HerpeSelect during the study period: 7 of 31 (23%) in the HSV-1 primary group, 7 of 56 (13%) in the HSV-2 primary group, and 3 of 26 (12%) in the nonprimary HSV-2 group. The 17 HerpeSelect nonseroconverters were followed a median of 54 days (range, 15–208) from the date of primary onset, compared with a median of 97 days (range, 14–361) for subjects who did seroconvert.
Sixteen of 113 (14.2%) did not seroconvert by WB. The median time of follow-up for the 16 WB nonseroconverters was 37 days (range, 15–167), compared with 98 days (range, 14–361) for subjects who seroconverted. Among 89 subjects who seroconverted by both tests, the median time for follow-up was 99 days (range, 14–361).
Nine of 113 subjects—3 infected with HSV-1, 3 with HSV-2, and 3 with nonprimary HSV-2—did not seroconvert by either test (median follow-up time, 38 days; range, 15–167). Thus, for both HerpeSelect and WB tests, apparent failure to seroconvert may have been a function of short follow-up time.
Some patients failed to seroconvert by HerpeSelect but became WB-positive, whereas others seroconverted only by HerpeSelect. Of those with primary HSV-1 infection (group 1), 4 seroconverted by WB but not by HerpeSelect, whereas 2 seroconverted by HerpeSelect but not by WB; for those with primary HSV-2 infection (group 2), 4 showed seroconversion by WB but not by HerpeSelect, and another 4 seroconverted by HerpeSelect but not by WB. One subject with nonprimary HSV-2 infection seroconverted by HerpeSelect but not by WB.
Increasing Sensitivity by Combining Tests
Because of the differences in seroconversion times and prevalence between the two tests, we explored the use of combined HerpeSelect and WB results for the most effective identification of new infections. Using a definition of seroconversion time as the minimum observed time to a positive HerpeSelect test or an indication of a change in the WB profile or to a full WB profile, we found that only 1 (3%) of 31 subjects with primary HSV-1 infection, 3 (12%) of 26 subjects with nonprimary HSV-2 infection, and no patients with primary HSV-2 infection failed to seroconvert by any measure. Median time to seroconversion was reduced by about a week in each group when earliest time to positivity by either WB or HerpeSelect was compared with time to seroconversion by only HerpeSelect: 17 versus 25 days for primary HSV-1 infection, 17 versus 21 days for primary HSV-2 infection, and 13 versus 23 days for nonprimary first episodes of HSV-2 infection. The proportion of patients seroconverting by any test by 6 weeks was 88% for group 1, 93% for group 2, and 71% for group 3 (Table 1).
Some studies have indicted that gG-based tests could become falsely negative over time for men 23 and over the course of pregnancy for women. 24 While our study was designed to determine time to seroconversion in sera drawn early in the course of infection, we tested sera for as long as a year after onset of genital herpes to demonstrate seroconversion in patients who might require longer to become positive by either HerpeSelect or WB tests. We examined all follow-up sera for this analysis, including sera drawn more than 180 days apart and, for subjects appearing to serorevert, sera drawn more than 1 year later if available.
Of 31 primary HSV-1 patients, 3 (10%) became positive by HSV-1 ELISA and then had at least one HerpeSelect-negative result. One subject became negative on day 22 and remained negative in tests of eight samples drawn between days 22 and 376. However, WB became positive on day 8 and remained positive throughout follow-up for this subject. Another subject became positive by HSV-1 ELISA at 17 days but was negative in testing of a single serum drawn on day 158. WB was negative on days 17 and 158 for this subject. The third subject became positive on day 70 by HSV-1 ELISA and then was transiently negative in testing of one serum on day 372, before returning to positive on day 387. WB for this subject became positive on day 29 and remained positive.
Of 82 patients with genital HSV-2, two (2%) had negative test results following at least one positive HSV-2 result by HerpeSelect. One subject became positive on day 9, was negative on day 81, and was subsequently seropositive on days 91, 101, 168, 191, and 361. WB became positive for HSV-2 on day 91 for this patient and did not change thereafter. The second patient became positive on day 14 and then had an equivocal result on day 14 and a negative result on day 108. WB became positive for HSV-2 on day 14 and remained positive.
The FDA has approved three gG-based tests for diagnosing HSV infections: Focus Technologies’ HerpeSelect ELISA for HSV-1 and HSV-2 antibodies, HerpeSelect immunoblot for HSV-1 and HSV-2 antibodies, and Diagnology's POCkit-HSV-2 for HSV-2 antibodies. In addition, gG-based ELISAs from Gull Laboratories were FDA-approved for diagnosis of HSV-1 and HSV-2 infection and marketed briefly by Meridian Biosciences under the Premier brand name. 25 The Gull/Meridian tests are no longer available. WB testing is considered the “gold standard” for detecting HSV antibodies, but it is not FDA-approved and is not widely available.
Of these tests, HerpeSelect ELISA is the most cost-effective test for commercial laboratory testing. Published data on HerpeSelect ELISA indicate a sensitivity of 98% and specificity of 94% for HSV-1 antibodies 26 and sensitivity of 98% and specificity of 95% for HSV-2 antibodies. 27 However, sensitivity may be lower in newly infected persons who have not developed high antibody titers. Therefore, we selected 113 patients with culture-documented first episodes of genital herpes to test in parallel by HerpeSelect ELISA and WB to determine time to seroconversion. Median time from onset of symptoms to seroconversion by HerpeSelect ELISA was 25 days for patients with primary genital HSV-1 infection, 21 days for those with primary HSV-2 infection, and 23 days for those with prior HSV-1 antibodies and new (nonprimary) genital HSV-2 infections.
HerpeSelect was significantly faster for HSV-2 seroconversion than was development of full antibody profiles by WB. However, the time required for development of the first evidence of de novo, HSV-specific bands (a less stringent criterion than a full antibody profile) was significantly less than with HerpeSelect for patients with HSV-2 primary infection.
The practical applications of our study relate to diagnosis of suspected new genital herpes infections. A positive HerpeSelect test within 6 weeks of lesion onset is probably an accurate reflection of genital herpes. However, a negative test result occurred in our study for 38% of HSV-1-infected patients in the first 6 weeks and in 23% to 41% of HSV-2-infected patients. By 3 months, between 73% and 93% of patients had seroconverted to either HSV-1 or HSV-2 by HerpeSelect. Thus, retesting at a later date appears to help detect seroconversion. Alternatively, our data show that performing WB in addition to HerpeSelect can decrease the time to detection of seroconversion for some patients. A serum found to be negative or equivocal by HerpeSelect had a full WB profile in 37% of samples from HSV-1-infected patients and in 17% of samples from HSV-2-infected patients.
Our study did not test the sensitivity of HerpeSelect for patients with subtle or subclinical infections, and it is unclear whether such patients have the same kinetics of seroconversion to gG that we recorded for symptomatic patients. Up to 90% of patients with HSV-2 antibodies do not have a history of genital herpes 5; thus, type-specific serology is an important option to diagnose unrecognized recurrent herpes. 28,29 Studies are in progress to test the sensitivity and specificity of HerpeSelect for HSV-2 infections identified by WB and subsequently confirmed when shedding of virus from the genital tract is determined by culture or PCR. 30
It should be emphasized that the current data reflect only one aspect of test performance: time to seroconversion in the patient with symptoms severe enough to result in a clinic visit. Furthermore, because our study was based on infected patients (and thus lacks positive test findings for uninfected patients), we do not have data to identify the specificity of the HerpeSelect tests.
The occurrence of seroreversion to gG has been noted for other gG-based tests. 23,24 Our study was not specifically designed to determine the frequency or clinical correlates for HerpeSelect seroreversion. However, we identified transient reversion to negative test outcomes in 3 of 31 patients with primary HSV-1 infection and in 2 of 82 patients with nonprimary HSV-2 infection. WB profiles from seroreverting patients did not change over time. Although this observation suggests that decreases in gG antibody titer were not the major factor in seroreversion, WB is not a quantitative test, and the possibility of antibody titer changes cannot be definitely dismissed. The issue for most clinicians is not the possibility of seroreversion in a patient with diagnosed infection but what the observation of seroreversion in longitudinal studies means for the detection of antibodies in patients with no such diagnosis. Larger studies to test the sensitivity of HerpeSelect over longer periods of time would be helpful.
In summary, HerpeSelect ELISAs can detect seroconversion to HSV-1 and HSV-2 within a month of symptom onset in the majority of patients. Although careful correlation of laboratory results with history and clinical observation is always important, the availability of these tests should provide clinicians with an important diagnostic tool.
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© Copyright 2003 American Sexually Transmitted Diseases Association
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