Recurrent HSV infection often is preceded by emotional stress, trauma or both.^2,8 Dental procedures present unique triggers of recurrent HSV infection because of the emotional and physical stress endured by some patients. Researchers have reported HSV outbreaks following dental procedures and orofacial fractures.^9–11 However, data are lacking on the prevalence of HSV outbreaks after routine dental procedures, as well as on the efficacy of anti-herpetic agents in the prevention of cold sores induced by these procedures.

Valacyclovir (Valtrex, GlaxoSmithKline, Research Triangle Park, N.C.) is the L-valine ester prodrug of acyclovir. It is well-absorbed and rapidly converted to acyclovir after oral administration, which results in increased bioavailability compared with acyclovir.^12–14 Recent clinical trials have demonstrated the efficiency of valacyclovir in the prevention and treatment of oral cold sores.^15,16 This led us to hypothesize that valacyclovir could prevent dentally induced recurrent HSV infections.

Accordingly, we performed a prospective, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of oral valacyclovir administered during the first two days of a dental procedure for the suppression of HSV recrudescence (that is, cold sore outbreak and HSV shedding). This article is the first, to our knowledge, to report the incidence of HSV recrudescence after routine dental treatment and the benefit of oral valacyclovir in the suppression of dentally related HSV disease.

Recent clinical trials have demonstrated the efficiency of valacyclovir in the prevention and treatment of oral cold sores.

	PATIENTS AND METHODS

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Patients. We enrolled 150 patients in the trial. Men and women 12 years of age or older who were in good general health and scheduled to receive routine dental care at the University of Kentucky College of Dentistry, Lexington, were included. Eligible patients had a history of oral HSV that recurred at least once per year, and had experienced at least one clinical recurrence within the previous year. A nurse showed patients clinical photographs of recurrent HSV lesions and aphthous ulcers for them to distinguish between the disorders, and we tested patients for HSV seropositivity.

We excluded patients who were immunosuppressed or who were taking immunosuppressant medication, had liver or kidney dysfunction, or were pregnant. Patients who were HSV-seronegative or had clinical evidence of an active oral HSV lesion at the beginning of the study also were excluded. Use of antiviral therapy within one week before the study enrollment date was prohibited. All patients provided written informed consent that was approved by the institutional review board.

Assay for HSV antibodies. Personnel in the University of Kentucky Hospital laboratory tested serum samples from all patients for HSV antibodies using a herpes 1 and 2 immunoglobulin G solid-phase chemiluminescent enzyme immunoassay kit according to the manufacturer’s directions (IMMULITE, Diagnostic Products, Los Angeles). This laboratory is certified and meets Clinical Laboratory Improvement Amendments requirements.

Dental procedures. We planned treatment for all patients according to standard procedures used by the college of dentistry. Based on the treatment plan sequence, we began the study for each subject on the first available day of his or her scheduled dental procedure. Dental procedures included periodontal, restorative, endodontic, orthodontic and oral surgical procedures. We excluded diagnostic (for example, clinical examinations, radiographic procedures) and prosthodontic procedures.

Study medication. The study medication was valacyclovir (500-milligram tablets) or a matching placebo. At enrollment, we randomly assigned patients to double-blind treatment and either the oral valacyclovir group (2 g to be taken within one hour of the dental procedure) or the placebo group. We gave patients a second 2-g dose of valacyclovir to be taken the evening of the dental procedure, as well as two 1-g doses to be taken 12 hours apart the next day, or a matching placebo. We determined compliance via oral confirmation by the patient that all medication had been taken according to the prescribed schedule and with the return of the empty pill bottle.

Blinding and assignment. For the double-blinded study medications, we packaged 12 pills per identical white bottle. We assigned patients sequentially to the study medication, which was numbered according to a computer-generated randomized code. Three randomization codes were used per treatment group based on lesion frequency categories. Category 1 was composed of patients with a history of one lesion per year; category 2, patients with a history of two to four lesions per year; and category 3, patients who had a history of more than four lesions per year. The treatment blind was maintained throughout the trial and was not broken for any subject.

Study design and procedures. This trial was a prospective, randomized, double-blinded, placebo-controlled study. After being recruited and receiving randomized study medication and dental treatment, patients were asked to report to the clinic on day 3 and day 7 (± 24 hours) after the dental appointment (day 0). At each clinical visit, we performed a thorough oral examination, collected expectorated whole saliva (5 milliliters) and received accounts of compliance and any adverse events.

On day 3, we swabbed the mouth and any clinically evident lesions with sterile culturettes for virus isolation. We immediately transported the culturettes on ice to the University of Kentucky Hospital microbiology laboratory where technologists determined the presence or absence of HSV via viral culture. All positive specimens were confirmed via immunofluorescence. In addition, we instructed patients in how to assess their lesions and provided them with diaries in which to record information regarding lesion presence and stage, pain level, adverse events and study compliance twice daily for eight days.

The authors gave patients diaries in which to record information regarding lesion presence and stage, pain level, adverse events and study compliance twice daily for eight days.

Measurements and evaluations. Clinical measures of efficacy included the following:

– percentage of patients who experienced a recurrence after the dental procedure;

– percentage of patients who shed HSV in saliva;

– evaluation of lesion severity (1 = papule, 2 = vesicle, 3 = ulcer);

– duration of lesion healing and episode;

– time to pain cessation for treatment and placebo groups.

Real-time polymerase chain reaction, or RT-PCR. We used RT-PCR for the detection and quantification of HSV-1 and HSV-2 DNA in saliva. We isolated DNA from 1 mL of saliva using the QIAamp DNA Mini Kit (Qiagen, Valencia, Calif.) according to the manufacturer’s directions. We performed RT-PCR on an ABI Prism 7700 Sequence Detection system (Applied Biosystems, Foster City, Calif.) in a 50-microliter reaction volume consisting of final concentrations of 1x TaqMan Universal PCR master mix (Applied Biosystems), 900-nanomolar primers and a 250-nanomolar TaqMan probe (Applied Biosystems).

We used primers and the probe specific to the gene-encoding glycoprotein G for HSV-1 and HSV-2 and followed the cycling parameters described by Ryncarz and colleagues.¹⁷ Probes were labeled at the 5' end with the reporter fluorochrome, 6-carboxy-fluorescein, or 6-FAM, for HSV-2 or tetrachlorinated analogue of 6-FAM for HSV-1 and at the 3' end with quencher fluorochrome, 6-carboxy-tetramethyl-rhodamine (Applied Biosystems).

We performed standards in duplicate that consisted of 10-fold serial dilutions of a plasmid containing the HSV-1 VP16 gene ranging from 2 x 10² to 2 x 10⁶ genome equivalents per reaction. HSV-1 and HSV-2 culture-positive specimens were used as positive controls. For negative controls, the template was substituted with water. We performed all reactions in duplicate. We scored the results as positive if both reactions yielded a threshold cycle value above 10 copies per milliliter. Reactions that yielded one positive and one negative result were repeated in duplicate. We scored samples as being positive only when both repeated reactions yielded positive results.

Statistical analysis. We used a ² statistic or Fisher exact test to compare differences in percentages between groups, and used two-sample t tests to compare means between groups. Safety was assessed via analysis of the incidence of adverse events.

	RESULTS

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Demographics of study population. A total of 150 patients were enrolled in the trial. We excluded 23 patients who failed to return to the clinic and two patients who were HSV-seronegative. Sixty-three patients in the placebo group and 62 patients in the valacyclovir group had evaluable efficacy data. The demographic characteristics of the two groups were similar (Table 1). The majority of patients were white women, with a mean age of 38.8 years. The mean number of historical episodes of herpes labialis per year was similar for both groups.

View larger version (40K): [in this window] [in a new window]   Figure 1. Percentage of patients who had evidence of herpes simplex virus 1 infection in their mouths after undergoing a dental procedure (days 3 and 7).

Figure 2 shows the percentage of patients who shed HSV-1 in saliva. On the day of dental treatment, 10 patients (8 percent) had detectable levels of HSV-1 in saliva. Valacyclovir treatment decreased the percentage of patients who shed HSV-1 in saliva 72 hours after the dental procedure to 1.6 percent (one of 62 patients). The percentage of patients who had clinically evident lesions and who tested positive for HSV-1 according to salivary PCR analysis at the 72-hour follow-up visit decreased significantly (P = .026) in the valacyclovir group (seven [11.3 percent] of 62 patients) compared with the placebo group (17 [27 percent] of 63 patients). Similarly, patients in the valacyclovir group had fewer (P = .06) detectable recurrent HSV infections and fewer HSV-positive saliva samples at days 3 and 7 after the dental visit (10 [16.1 percent] of 62 patients) compared with patients in the placebo group (19 [30.2 percent] of 63 patients). Overall, the percentage of patients whose saliva samples remained free of HSV up to one week after the dental procedure was higher in the valacyclovir group (58 [93.5 percent] of 62 patients) than in the placebo group (54 [85.7 percent] of 63 patients). HSV-1 shedding in saliva on consecutive visits was rare (1.6 percent) in both groups.

View larger version (46K): [in this window] [in a new window]   Figure 2. Percentage of patients who expressed herpes simplex virus, or HSV, 1 in their mouths. The asterisk indicates a statistically significant difference between placebo- and valacyclovir-treated patients at the indicated time point.

Lesion severity, time to healing and cessation of pain. The mean time to observation of a recurrent HSV lesion was similar in both groups (placebo, four days; valacyclovir, 3.3 days), with a range of one to seven days (Figure 3). We did not observe a significant difference in the mean clinical lesion severity score (1 = papule, 2 = vesicle, 3 = ulcer) between the groups (placebo, 1.9; valacyclovir, 1.7). However, valacyclovir treatment correlated with a significantly shorter time to cessation of pain (3.2 days) in comparison with the placebo group (6.2 days; P = .006). In addition, only one patient in the valacyclovir group experienced pain on day 7 after the dental treatment compared with four patients in the placebo group.

View larger version (42K): [in this window] [in a new window]   Figure 3. Time course for the development of recurrent herpes simplex virus, or HSV, infections.

	DISCUSSION

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Valacyclovir has proven to be effective in preventing recurrent herpes simplex virus infections when given as a daily prophylaxis and when given during the early prodromal period.

Prophylactic antiviral agents such as acyclovir suppress orofacial HSV recurrences with reasonable success.^7,18,19 Valacyclovir, which is three to five times more bioavailable than is acyclovir, has proven to be effective in preventing recurrent HSV infections when given as a daily prophylaxis¹⁶ and when given during the early prodromal period.¹⁵ In facial resurfacing studies, researchers have reported that valacyclovir was effective as a prophylactic drug.^20–22 However, many facial resurfacing studies are not blinded or placebo-controlled, and the HSV serostatus of enrolled patients has not been well-documented.

In contrast, all patients enrolled in our study were HSV-seropositive and at risk of experiencing a recurrence because of the stress involved and the historical evidence of their susceptibility to recurrent HSV infection. Under these conditions, we detected clinical lesions in 20.6 percent of placebo-treated and 11.3 percent of valacyclovir-treated patients within one week after dental treatment. This represents a 46 percent reduction in the number of clinical recurrences in patients who received valacyclovir prophylaxis.

HSV-1 recrudescence also is measured by asymptomatic viral shedding. The rate of viral recovery from saliva samples of healthy patients during asymptomatic lesion-free periods ranges from 0 to 20 percent depending on the patient population and the method of collection, storage and detection (culture versus PCR).^23–27 Most researchers would agree that HSV-1 can be detected in the mouth of 4 to 10 percent of immunocompetent adults who are free of oral lesions.^25,28,29 Consistent with previous reports, our results demonstrate an 8 percent rate of detection of HSV-1 in the saliva of dental patients, as well as an average duration of shedding of less than three to four days.

Interestingly, we did not detect an upward spike in the incidence of HSV-1 in the saliva of placebo-treated patients on days 3 or 7 after the dental treatment visits. However, during the course of the seven-day postoperative period, the percentage of patients in the placebo group who shed HSV-1 increased to 14.3 percent. In contrast, the percentage of patients in the valacyclovir group decreased on day 3 and returned to baseline by day 7 after the dental treatment. Based on reports that viral shedding increases during the prodromal period,³⁰ after oral trauma^9,29 and over time,³¹ our data suggest that valacyclovir prophylaxis was effective in reducing viral shedding in this study population. This is consistent with the ability of valacyclovir and acyclovir to suppress subclinical shedding of HSV-2.^32–34

Studies indicate that recurrent HSV lesions often develop one to seven days after oral trauma, with the majority of lesions appearing on days 3 and 4 after treatment.^29,35 Consistent with this observation, the incidence of lesions in this study was highest on postoperative day 3 (Figure 3). Although valacyclovir appeared to have little effect on the time to lesion appearance, once lesions developed, their duration was significantly shorter with valacyclovir treatment. This finding is encouraging because valacyclovir was administered only on the day of dental treatment and on the next day. This suggests that the beneficial effect of valacyclovir may extend one or two days beyond the last day of administration.

An explanation for these clinical effects could be that valacyclovir targets viral replication at both the neuronal and epithelial sites. Thus, virions projecting from the neuron during valacyclovir therapy may initiate epithelial replication (after valacyclovir therapy) with reduced numbers of viral particles that, in turn, may reduce the severity of the episode.

Consistent with previous studies,^34,36 the results of our study show that valacyclovir appeared to be safe and efficacious at the dosage used. Adverse effects reported were minimal and comparable between groups. Headache was the most common adverse effect, occuring in a higher percentage of patients in the valacyclovir-treatment group (14.5 percent) than in the placebo group (4.8 percent) (P = .06). All patients reported that their headaches were mild, which likely were attributable to the dosage administered (4 g of valacyclovir on the day of dental treatment and 2 g the next day). We did not discontinue therapy for any patient because of adverse effects, suggesting that most patients could comply with the dosing regimen during routine dental visits. However, we did not explore the relationship between adverse effects and the reasons why some patients dropped out of the study (23 [15.5 percent] of 148).

	CONCLUSION

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Data from this clinical trial suggest that 20 percent of HSV-1–seropositive patients develop clinical HSV-1 lesions and 30 percent develop clinical signs of HSV-1 infection within one week after dental treatment. We found that valacyclovir significantly reduced viral recrudescence and shortened the duration of symptoms of recurrent HSV after dental treatment. Theoretically, valacyclovir’s ability to reduce the rate of asymptomatic HSV-1 shedding could reduce transmission of this contagion. Practitioners should be aware of the utility of valacyclovir prophylaxis for patients at risk of developing oral HSV recurrences after dental procedures.