Unlike HSV-1 seroprevalence, little is known about asymptomatic shedding of HSV-1 in tears. To the best of our knowledge, there have been only three studies to date in which the presence of HSV-1 was assessed in tears of healthy individuals.11–13
Kaye et al.12
found no shedding of HSV-1, and Kaufman et al.11
found 0.8% and 0.05%, respectively, but these studies used relatively insensitive culturing techniques. In comparison to those findings, our present tear data showed significantly higher HSV-DNA–positive results. However, as is the case in assessing HSV seropositivity, the present study results (33.5% and 37.5% positive eye and mouth specimens, respectively) are in part attributable to the sensitivity of the real-time PCR diagnostic technique used, in contrast to earlier studies that relied on culturing of viable HSV. In addition, shedding appears to be intermittent, and so the number of individuals with detectable virus depends on an adequate duration of the study.
In contrast, asymptomatic shedding of HSV-1 (infectious virus and DNA) in saliva has been more widely analyzed. Reported results range from 0.45% to 76.0%.11–13,23–27
Our findings are in accordance with the report of Knaup et al.,23
in which 25 (83.3%) of 30 healthy individuals were HSV seropositive by ELISA. Of the 25 seropositive individuals, 19 (76.0%) shed HSV-1 DNA at least once in 8 to 12 oral swabs collected over a period of 58 to 167 days, as shown by nested PCR. None of the five seronegative individuals had positive oral swabs at any time. In our present study, 37 (74%) of the 50 volunteers were antibody positive, and all the antibody-positive and 11 of the 13 antibody-negative subjects shed HSV-1 DNA. The discrepancy between the results obtained from serum analysis by ELISA and tear and saliva analysis by real-time PCR in our study could be due to the different sensitivities of the assays. Serum antibodies for HSV IgG may have been present in the 11 seronegative subjects that shed virus, but not in sufficiently high titers to be detected by ELISA. Real-time PCR, however, has been shown to be more sensitive, and it detects HSV DNA irrespective of the presence of infectious virus.
A report from Posavad et al.28
may offer one explanation as to how an individual could be seronegative and still shed HSV-1 DNA. In the course of testing 24 immunocompetent subjects with no history of oral, genital, or labial herpes for HSV-specific T-cell immunity, they found that 6 of the 24 individuals exhibited T-cell immunity to HSV, but were seronegative to both HSV-1 and -2. The investigators concluded, “The identification of persistent T-cell responses to HSV in seronegative subjects is novel to the herpesvirus field and suggests either undetected infection or acquired immunity in the absence of infection.” We believe that “undetected infection” is the more likely explanation. In our study, the ELISA determination of seropositivity was general for both HSV-1 and -2. That we were unable to repeat the serologies with a type-specific assay, due to a lack of serum from the subjects, is a limitation of this work.
Real-time PCR offers numerous advantages over conventional methods used to detect HSV DNA. It requires less hands-on involvement; therefore, it is faster, and, more important, there is a reduced possibility of cross-contamination. Also, real-time PCR has consistently been reported to be significantly more sensitive than viral culture and antigen detection.14–21
A 43.9% increase in HSV-1 detection and a 62.5% increase in HSV-2 detection were observed in comparison with shell vial culture.29
In addition, real-time PCR is not hindered by the quality of the sample.17,30
However, its detection capability can be affected by the sample origin.29
The most important difference between real-time PCR and other methods used for detection of HSV in clinical specimens and probably the reason that 96% of our subjects were positive is that the PCR assay does not rely on the presence of infectious virus or on the quality and presence of appropriately infected cells.31
Although the effect of storage time of the collected samples is not known, the presence of HSV-1 DNA in high concentrations in approximately one third of these samples is sufficient to be striking and important. Some, of course, may have degraded, but that does not detract from the findings in this study.
Shedding is clearly intermittent, and a longer-term study would be expected to show an overall higher number of positive subjects, as noted in this study in comparison with other shorter studies that have focused on asymptomatic HSV-1 DNA shedding in tears and saliva.11–13,23
The probability of detecting HSV DNA is directly proportional to the duration of the study and the number of samples collected. In our study, 33.5% of assayed tear samples and 37.5% of assayed saliva samples were positive, but 98% of the 50 subjects secreted viral DNA at some time over the course of 30 days. The number of episodes of intermittent shedding (defined as one or more days of shedding separated by one or more days of not shedding) ranged from 0 to 11 for the eye swabs and 0 to 10 for the mouth swabs. The averages were 3.98 (eye) and 4 (mouth).
As mentioned previously, population demographics play a fundamental role in the prevalence of HSV infections. Consequently, the percentage of individuals shedding HSV-1 DNA noted in the present study may be in part a function of our population sample and indicative only of the incidence of HSV-1 in this particular region of the United States. The predominantly African-American female (54%) cohort is representative of the demographics of this southeastern city, the population of which comprises 67.3% African Americans and 53.1% women—two significant predictors of HSV-1 infection.32
Other documented significant predictors are age, stress, socioeconomic status, level of education, age of first intercourse, and total years of sexual activity.
Also, natural stress factors, such as sunlight exposure, may have been a contributing factor to HSV-1 DNA shedding. The present study was conducted during the summer, and although there is no consensus on whether seasonal changes affect HSV-1 shedding in humans, in animals, UV exposure is a known method for reactivation of latent HSV. Viral DNA shedding, as well as antibody titers, may have also been affected by the subject’s age. Several studies have noted an increase in HSV-1 and -2 antibody titers and frequency of herpetic disease with increased age. This is probably a result of repeated infection and/or reactivation of the primary HSV infection.
Control of virus excretion could well limit transmission, especially of more virulent strains of virus. More important, if such a high proportion of adults excrete virus, the reduction and/or prevention of virus excretion may be a simple, cost-effective way to evaluate new antiviral drugs.