Morrow, Rhoda Ashley PhD*§; Friedrich, David BS§; Krantz, Elizabeth MS*; Wald, Anna MD, MPH†‡
AS MANY AS 70% OF patients who present with their first episode of genital herpes simplex virus type 2 (HSV-2) infection have antibodies to HSV-2 by Western blot (WB).1,2 Because the development of antibody bands takes weeks to months,3 these persons almost certainly acquired HSV-2 in the past without recognized symptoms. These patients can be an enigma to clinicians, because the epidemiologic setting of the symptomatic episode understandably might not support new acquisition. As such, issues of infidelity often arise in these situations. Equally perplexing are situations in which a person tests as HSV-2-seropositive, sometimes after transmitting HSV-2 to a partner, despite lack of history or symptoms of genital herpes. Given the availability of serologic testing for HSV-2 and recommendations for their use in various clinical settings,4 this scenario is likely to become more common.
Initial genital HSV infection, regardless of clinical findings, can be operationally defined as recovery of HSV from the genital tract at a time that serum antibody tests are negative for the type of the virus found in genital secretions. Thus, in some patients, newly acquired HSV can be accurately defined using a combination of virologic and serologic tests. However, the duration of HSV infection becomes difficult to deduce once antibodies are detected. Currently, there are no laboratory tests to distinguish longstanding HSV-2 infection from recently acquired HSV-2 that has already resulted in the development of antibodies. In contrast to many infections, IgM tests are not useful because IgM could appear sporadically months to years after acquisition of genital herpes. Over 30% of patients with recurrences develop IgM to HSV-2 and 28% of patients with new infection have both IgG and IgM.5 Western blot can reveal profiles that are limited and likely to be early responses, in contrast to more complete profiles typical of infection of several months’ duration.3,6,7 Because WB is expensive and not widely accessible, it is not a practical method for inferring duration of HSV-2 infection.
During maturation of the immune response, antibodies develop more efficient binding capability (“avidity”). A number of studies of humoral responses to viral infections confirm that the avidity of virus-specific antibodies increases with time after infection.8–11 We developed a type-specific avidity assay based on the widely available, U.S. Food and Drug Administration-approved HerpeSelect gG-2 enzyme-linked immunosorbent assay (ELISA) from Focus Technologies and used it to test sera from persons with new genital HSV-2 infections, with established HSV-2 infection, with first clinical episodes of HSV-2 in the presence of fully developed HSV-2 antibody and with unrecognized HSV-2 infection.
Materials and Methods
Sera were obtained, with informed consent, from subjects at the University of Washington Virology Research Clinic, Seattle, WA. Groups of patients for the study were defined by clinical presentation, viral culture, and WB serology.12 Group 1 comprised 168 sera from 71 patients with initial infection with HSV-2, either “primary HSV-2 infection” as defined by acquisition of genital HSV-2 in a person previously seronegative for HSV-1 and HSV-2 or “nonprimary initial HSV-2” as defined by acquisition of genital HSV-2 in a person with prior HSV-1 antibody. Group 2 contained 45 sera from 21 patients presenting with episodes of recurrent genital herpes as confirmed by history, clinical examination, and viral culture. Group 3 consisted of 24 sera from 24 patients with first-recognized clinical episodes of HSV-2 as defined by new-onset genital lesions in history-negative persons who at the time of presentation had fully developed HSV-2 WB profiles consistent with established infection.1,2 Group 4 was 28 sera from 25 patients with HSV-2 antibody by WB and without a history of genital herpes at the time of serum draw. These patients were followed prospectively for viral shedding from the genital area as previously described.13
All sera were held at -20°C until testing. All had been thawed once and refrozen for prior testing by WB, Focus ELISA, or both.3 Sera were tested by Focus HerpeSelect HSV-2 ELISA IgG according to kit instructions (Focus Technologies, Cypress, CA) with the following modifications: After thawing, each sample was vortexed and 2.5 μL was diluted 1:101 into 2 dilution wells containing 250 μL sample diluent provided in the kit. The diluted samples were added in 100-μL portions to duplicate wells in Focus HSV-2 ELISA plates using an 8-multichannel pipetter. Samples were processed following the manufacturer’s instructions with 1 additional wash step before the addition of the conjugate. Wash buffer (200 μL) (provided in the kit) was added to 1 of the duplicate wells and 200μL of 0.75 mol/L sodium thiocyanate (NaSCN) in distilled water was added to the other duplicate well. After 5 minutes, the plate was washed twice with 1× wash buffer and the conjugate added according to kit instructions. At the completion of the test, plates were read with a EL-340 plate reader with KC-4 software Bio-Tek Instruments. Optical density (OD) values were exported to Microsoft Excel files. Standard index values for the untreated samples were obtained by dividing the OD by the mean of the cutoff calibrator OD. Samples with index values greater than 1.1 were considered positive, those with index values below 0.9 were considered negative, and those between 0.9 and 1.1 (inclusive) were recorded as equivocal.
Each sample had 2 recorded OD values: 1 from the NaSCN-treated well and one from the untreated well. The treated OD value was divided by the untreated OD value and multiplied by 100 to obtain an avidity value. Only data from samples with HerpeSelect HSV-2 index values >1.1 in the untreated well were included in avidity analyses.
To facilitate hypothesis testing, avidity values from the first positive sample per person, per time period or episode phase, were used for subjects with multiple samples in a given time period. Subsequently, Wilcoxon paired tests were used for paired comparisons and 2 group comparisons used Mann-Whitney tests. Linear mixed effects (LME) models with random intercept effects for each individual were used to assess trends in avidity values over time while accounting for the dependence among observations belonging to the same individual. The fixed effects examined in these models included log-transformed days elapsed since onset of initial infection, presence of recurrent lesions versus not, HSV-1 antibody status, and demographic characteristics such as age, gender, and race.
Half of the individuals (“training sample”) contributing information concerning avidity over time were randomly selected to develop an avidity-based definition of recent infection (≤6 weeks from initial acquisition) versus established infection (>6 weeks from initial acquisition). The other half (“validation sample”) was reserved to test the avidity rule on a sample set not used in the development process. This method of cross-validation is less likely to produce biased estimates of predicted values and more likely to give an avidity prediction rule generalizable to other populations.14
A receiver operating characteristic (ROC) curve was constructed using the training dataset to establish a rule to discriminate between recent and established HSV-2 infection. The resulting rule was tested on the validation dataset, calculating sensitivity as the percent of samples from recently infected people whose avidity was less than or equal to the threshold value and specificity as the percent of samples from people with established infection whose avidity fell above the threshold. S-PLUS 6.1 was used for modeling, Stata 8.0 was used for ROC analysis, and SPSS 11.5 was used for descriptive statistics and simple testing.
Description of Subjects
The study population was comprised of 141 persons and 265 samples that tested positive for the presence of HSV-2 antibody by Focus HerpeSelect HSV-2 ELISA. The demographic and clinical characteristics of the participants are summarized in Table 1. Group 1 included subjects with laboratory-documented new acquisition of genital HSV-2 infection who had at least 1 serum sample positive for HSV-2 by the Focus ELISA within 6 months of acquisition. One hundred twenty sera were collected from 51 patients with documented primary HSV-2 genital herpes. An additional 48 sera were collected from 20 patients with nonprimary first episodes of genital HSV-2. Forty-five sera were collected from 21 patients with established HSV-2 infection who presented with recurrent episodes of HSV-2 confirmed by viral culture (“group 2”). Median time since acquisition of HSV-2 in the 19 patients with recurrent genital HSV-2 in whom the time of acquisition was known was 6.1 year (range, 193 days to 19.2 years). Group 3 had 24 persons without a history of genital herpes who presented with their first clinically recognized episode of genital HSV-2. Each contributed a single serum sample drawn within 14 days of the start of the symptomatic episode and each had a fully developed WB profile. Group 4 was 28 sera from 25 persons who were HSV-2-positive by WB but who lacked a history of genital herpes and were not symptomatic. Despite a lack of history of genital herpes, all 25 subsequently had HSV-2 detected from the genital area at least once by HSV polymerase chain reaction or culture.13 Groups 3 and 4, whose members lacked history of genital herpes but had serologic evidence of established HSV-2 infections, had much higher seroprevalence of HSV-1 than the groups with newly acquired or established, diagnosed HSV-2 (69% vs. 26% respectively, P <0.001 by chi-squared test).
Avidity Changes After Primary Infection
Avidity data were available from 168 sera that were HSV-2-seropositive from 71 patients with primary or nonprimary first-episode HSV-2 infections. We compared avidity of samples taken within 6 weeks of onset with those taken more than 6 weeks after onset. Thirty-eight subjects (28 with primary HSV-2 and 10 with nonprimary first episode) had at least 1 sample from both time periods and, thus, were available for a paired analysis. Median avidity 6 weeks or more postinfection was 54.9 compared with median avidity of 30.2 for samples within the first 6 weeks of infection (P <0.001). This result was seen both in primary HSV-2 and nonprimary HSV-2 first infections. The rise in avidity after acquisition of HSV-2 is shown in Figure 1.
Avidity in Newly Acquired versus Established HSV-2 Infection
We compared avidity of positive sera obtained within 6 months of HSV-2 acquisition (group 1 initial samples) with avidity of sera obtained after a recurrent episode that occurred 6 months or later after HSV-2 acquisition (group 2). As shown in Table 1 and Figure 1, avidities were significantly lower in sera obtained from persons with newly acquired HSV-2 infection than in sera obtained from persons with established recurrent HSV-2 (median 33.7 versus 92.7, respectively; P <0.001).
Effect of Recurrent Episodes of Genital Herpes on Avidity
Twenty of 21 subjects from group 2 had at least 2 samples drawn during or after recurrent episodes with a first sample obtained in the first 7 days of the recurrence onset and a second sample drawn 8 to 42 days after onset of the recurrent episode (Fig. 2). Median avidity was 92.9 in the samples obtained within 7 days of a start of a recurrence and 93.2 in samples drawn 8 to 42 days after the start of the recurrence (P = 0.80). Thus, clinical episodes in subjects with a history of 6 months or more of genital herpes appeared to have little effect on antibody avidity. Sera drawn within 42 days of an initial episode had distinctly lower avidity than sera drawn after recurrent episodes (Fig. 2).
Avidity of Antibodies in Persons With a First Recognized Recurrence of Genital Herpes
We tested sera from 24 subjects from group 3 who presented with a first genital HSV-2 episode and who had fully developed HSV WB profiles at the initial visit, indicating infection for at least 3 months.6,7 Median avidity was 73.6 (range, 36.2–100), significantly higher than the median avidity during initial episodes in 21 persons with newly acquired HSV-2 infection (32.6; range, 6.4–57.6; P <0.001). However, the avidity of antibodies in persons with a first clinical recurrence of genital HSV-2 was significantly lower than the avidities of antibodies in patients with wide-ranging duration of established infection (median, 92.7; range, 55.1–100, P = 0.001), but comparable to the median avidity of 86.6 (range, 55.1–95.9) found in a subset of 9 patients with established HSV-2 infection of duration ranging from 6 months to 5 years from initial onset (P = 0.15). Again, little difference was seen in the antibody avidities of persons who had their sera drawn in the first 7 days versus 8 to 14 days after the first recognized episode (median 73.6; range, 36.7–100 vs. 71.2; range, 36.2–90.9, respectively).
Avidity of Antibody in HSV-2 Seropositive Persons Without a History of Genital Herpes
Twenty-five persons without a history of genital herpes who had HSV-2 detected in the genital tract had sera tested for antibody avidity. Because most persons were identified during a random survey of primary care patients,15 the duration of their infections is unknown. In this group, the median avidity was 75.8 (range, 53.4–100). Like group 3 subjects, these people had avidities significantly higher than those of persons with newly acquired infection (P <0.001) but lower than the entire established infection group (P = 0.06) and comparable to those of persons with established infection ranging from 6 months to 5 years from initial onset (P = 0.71).
Modeling Avidity of HSV-2 Antibodies
To formally take advantage of the longitudinal data available, we used a linear mixed-effects model to describe the pattern in avidity over time and tests for the effects of other covariates. In addition to samples collected prospectively from initial episode, we included samples from the subjects with recurrent herpes episodes whose date of disease onset (initial infection) was known for this portion of the analyses to give estimates of avidity for longer periods of time postinfection. The model showed that avidity increases over time after a cubic polynomial pattern with log-transformed time since initial infection (P <0.001). Thus, with time on the log scale, avidity shows a slight dip, followed by an increase, and then eventually tends to plateau (Fig. 3). Gender, race, age, and HSV-1 antibody status were not significantly associated with avidity and its pattern over time. Of interest, although avidity was significantly predicted by time from acquisition, it was not related to presence or absence of recurrent lesions at the time of the blood draw of the patients, when we adjusted for time.
Performance Characteristics of Avidity Test to Discriminate Between Newly Acquired and Established HSV-2 Infections
Using the training dataset, we created an ROC curve to examine various possibilities of avidity thresholds to differentiate between recent and established HSV-2 infection (Fig. 4). The area under the curve (AUC) was 0.92, indicating that our avidity test performed well. In choosing an avidity threshold for our rule, we prioritized specificity over sensitivity, because misclassifying a person with established infection as having recently acquired HSV-2 could potentially have a greater negative impact than misclassifying a person who has recently acquired the infection as having established infection. Table 2 shows 3 potential candidates for avidity thresholds that result in 80% or greater specificity in this training sample. We chose an avidity threshold of 40 to define recent infection, and proposed that avidity greater than 40 would indicate established infection and an avidity of ≤40 would define recently acquired infection. On the training sample, this rule had 81% sensitivity and 92% specificity.
The validation sample had 42 samples that were drawn within 6 weeks of initial onset of HSV-2 infection and 56 samples drawn more than 6 weeks after acquisition. Of the 42 samples that represent recent infection, 34 had an avidity less than or equal to 40. Of the 56 established infection samples, 48 had avidity greater than 40. Thus, when tested on our validation sample, our proposed avidity rule had a sensitivity of 81% and a specificity of 86%. We expect these values to be slightly lower than those from the training dataset, because these data were not used to develop the rule.
We describe a novel test for type-specific HSV-2 antibody avidity based on a widely available commercial ELISA (HerpeSelect HSV-2). This avidity test can be performed in tandem with the standard ELISA or in a second procedure after determining that a sample contains detectable antibodies against HSV-2. The results offer a test with potential clinical use to differentiate between early genital HSV-2 infection and more established infections of more than a few weeks’ duration in sera that test positive by HerpeSelect HSV-2 ELISA. Using a threshold of 40 on a scale of 0 to 100 (with 100 being the highest measured avidity), the test correctly identified 81% of newly infected patients as being within 6 weeks of HSV-2 acquisition, whereas only 14% of those with infections of greater than 6 weeks’ duration had antibody avidity <40. This test allows for defining persons with early infection who have already mounted a sufficient antibody response to test positive for HSV-2 antibodies on the HerpeSelect assay.3
Use of a gG-2-based test allowed discrimination of early versus established HSV-2 infection even among patients with prior HSV-1 antibodies. Such patients are problematic for tests based on crossreactive antigens. Hashido and colleagues16 found no significant difference between avidities of antibodies from patients with prior HSV-1 and new HSV-2 episodes and those from recurrent HSV-2 episodes with a test based on mixed antigen extracts from HSV-1- or HSV-2-infected cells. These tests are not effective in identifying newly arising HSV-2 antibodies in patients with preexisting HSV-1 antibodies (“nonprimary” first episodes) because of the extensive crossreactivity of the 2 viruses.17,18 The kinetics of HSV-2 gG-2 antibody avidity development are not affected by preexisting HSV-1 antibodies, and the threshold avidity value worked equally well in patients with nonprimary HSV-2 infections as in those with true primary HSV-2 infections.
Data from clinically and serologically defined groups of subjects in this study led to 2 general conclusions on HSV-2 antibody avidity. First, avidity of antibody increased over time after HSV-2 genital infection. Others have reported the same trend after infections with rubella, a virus that does not ordinarily recur,9,19 as well as after infections with viruses that become latent and can reactivate at a later time, including varicella zoster,20 cytomegalovirus,21,22 Epstein-Barr virus,23 and human herpes viruses 6 and 7.8 Antibody avidity development over time has been well established for other pathogens as well, including toxoplasma.24 Interactions of antibodies with their target antigens involve “affinity” or the balance of the antibody binding rate (“on-rate”) and the dissociation rate (“off-rate”) of the complex. Affinity maturation involves progressive increase in antibody affinity during the immune response. Hybridomas have been used to analyze heavy and light chain genes from early or late responses.25 Sequences of the late response genes in these studies revealed accumulation of point mutations that correlated with increased affinity and improved structural complementarily between the antigen and antibody binding site.26 Improved binding would result in higher avidity as operationally defined by the ability of a bound antibody to remain complexed with its antigen in the presence of a chaotropic agent such as urea or sodium thiocyanate.27–29
Our second observation relates to the role of antigen presence versus time in the increase of avidity after first-episode infections. We found that the appearance of HSV-2 lesions was not sufficient to increase HSV-2 antibody avidity. Sera drawn 8 to 42 days after the start of a recurrent episode did not have higher avidity antibodies than those drawn within 7 days of the episode start. This was confirmed in our model that showed no increase in avidity in the presence of recurrent lesions when we adjust for the effect of time on avidity. As mentioned previously, in viruses that do not ordinarily recur, others have found evidence of increasing avidity after infection. Thus, changes in the binding characteristics of serum antibodies are not necessarily dependent on repeated antigenic stimulation.
The practical applications of a type-specific HSV-2 avidity test include ruling out early HSV-2 infection in women who present with new lesions or with a first positive HSV-2 serology late in pregnancy. Women who have HSV-2 in the genital tract at term are at higher risk of transmitting HSV-2 to their newborn if they have recently acquired HSV-2 than if they have full antibody profiles by WB.30 In persons with newly acquired HSV-2, lack of type-specific antibodies is a clear indication of new infection. However, once antibodies develop, it is not possible to determine the duration of infection based on clinical history.
Avidity testing could be helpful in this setting. In addition to risk assessment in pregnancy, accurately identifying newly infected patients can be important for appropriate counseling and partner notification. Although definitive timing of infection is not feasible based on avidity alone, values above 70 were rare (1 of 110 samples) in subjects with infections of less than 2 months’ duration, whereas values below 35 were observed in only 1 of 99 samples from patients with infections of greater than 2 months’ duration.
This study is unique in including a cohort of subjects who presented with first clinical episodes of genital lesions and fully developed WB profiles despite having no history of confirmed genital herpes. Sera obtained during these episodes had avidity values similar to those of recurrent episodes. Only 2 of 24 had antibody avidity values less than 40, indicating that the majority of this group had had infections for longer than 6 weeks’ duration. This distinction could be important from a psychosocial standpoint because patients with lesions commonly assume their source of infection was a sexual partner from within the previous 1 to 2 weeks.
Patients who lacked symptoms and a history of genital herpes yet were HSV-2-seropositive at presentation also had high avidity values (all over 40), suggesting that subclinical, unrecognized infection can exist for months to years before detection. Serologic testing could be the only way to recognize such subjects. With the advent of licensed therapeutic regimens for reducing the risk of HSV-2 transmission to heterosexual partners, it is important for both symptomatic patients and their asymptomatic partners to be tested.31 As many as 25% of couples who report having 1 member with genital herpes and 1 member without genital herpes are actually concordant with both having HSV-2 infection. These couples are not at risk of transmitting HSV-2 within their relationship.32
In summary, we used a simple modification of a widely available type-specific serology on sera from well-characterized symptomatic patients to document that HSV-2 gG-2 antibody avidity rises over time, but not as a direct result of symptomatic recurrences. An avidity index of 40, as suggested by ROC curves, predicted with >80% sensitivity and specificity the status of an episode as being a new infection (≤40) versus an established infection (>40).
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