Relationship between herpesviruses and periodontal disease progression.

AIM: To investigate the role of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and anaerobic bacteria in the progression of periodontitis. METHODS: Eighty-one adults with generalized moderate to severe periodontitis were randomly assigned to: oral hygiene or scaling and root planning ± placebo or polyunsaturated fatty acids fish oil. Subgingival plaque samples collected from three healthy and three disease sites at weeks 0, 16, and 28 and from sites demonstrating disease progression were analysed for EBV, CMV, P. gingivalis (Pg), T. forsythia (Tf), and T. denticola (Td) DNA using quantitative polymerase chain reaction. RESULTS: Cytomegalovirus was detected in 0.3% (4/1454) sites. EBV was present in 12.2% of healthy sites (89/728) and 27.6% disease sites (201/726; p < .0001), but was in low copy number. Disease progression occurred in 28.4% of participants (23/81) and developed predominantly at sites identified as diseased (75/78; 96.2%). CMV and EBV were not associated with disease progression (p = .13) regardless of treatment. In contrast, disease sites were associated with higher levels of Pg, Td, Tf, and total bacteria, and sites that exhibited disease progression were associated with an abundance of Td and Tf (p < .04). CONCLUSION: Disease progression was associated with Gram-negative anaerobic bacteria; not EBV or CMV.

Histone deacetylase inhibitors prevent persistent hypersensitivity in an orofacial neuropathic pain model.

Chronic orofacial pain is a significant health problem requiring identification of regulating processes. Involvement of epigenetic modifications that is reported for hindlimb neuropathic pain experimental models, however, is less well studied in cranial nerve pain models. Three independent observations reported here are the (1) epigenetic profile in mouse trigeminal ganglia (TG) after trigeminal inflammatory compression (TIC) nerve injury mouse model determined by gene expression microarray, (2) H3K9 acetylation pattern in TG by immunohistochemistry, and (3) efficacy of histone deacetylase (HDAC) inhibitors to attenuate development of hypersensitivity. After TIC injury, ipsilateral whisker pad mechanical sensitization develops by day 3 and persists well beyond day 21 in contrast to sham surgery. Global acetylation of H3K9 decreases at day 21 in ipsilateral TG . Thirty-four genes are significantly ( p < 0.05) overexpressed in the ipsilateral TG by at least two-fold at either 3 or 21 days post-trigeminal inflammatory compression injury. The three genes most overexpressed three days post-trigeminal inflammatory compression nerve injury are nerve regeneration-associated gene ATF3, up 6.8-fold, and two of its regeneration-associated gene effector genes, Sprr1a and Gal, up 174- and 25-fold, respectively. Although transcription levels of 25 of 32 genes significantly overexpressed three days post-trigeminal inflammatory compression return to constitutive levels by day 21, these three regeneration-associated genes remain significantly overexpressed at the later time point. On day 21, when tissues are healed, other differentially expressed genes include 39 of the top 50 upregulated and downregulated genes. Remarkably, preemptive manipulation of gene expression with two HDAC inhibitors (HDACi's), suberanilohydroxamic acid (SAHA) and MS-275, reduces the magnitude and duration of whisker pad mechanical hypersensitivity and prevents the development of a persistent pain state. These findings suggest that trigeminal nerve injury leads to epigenetic modifications favoring overexpression of genes involved in nerve regeneration and that maintaining transcriptional homeostasis with epigenetic modifying drugs could help prevent the development of persistent pain.

HSV-1 clinical isolates with unique in vivo and in vitro phenotypes and insight into genomic differences.

Strain-specific factors contribute in significant but undefined ways to the variable incidence of herpes simplex virus (HSV) recrudescence. Studies that investigate these strain-specific factors are needed. Here, we used qPCR, in vitro assays, and genomic sequencing to identify important relationships between in vitro and clinical phenotypes of unique HSV-1 clinical isolates. Nine HSV-1 isolates from individuals displaying varying reactivation patterns were studied. Isolates associated with frequent recurrent herpes labialis (RHL) (1) displayed higher rates of viral shedding in the oral cavity than those associated with rare RHL and (2) tended to replicate more efficiently at 33 °C than 39 °C. HSV-1 isolates also displayed a more stable phenotype during propagation in U2OS cells than in Vero cells. Draft genome sequences of four isolates and one variant spanning 95.6 to 97.2 % of the genome were achieved, and whole-genome alignment demonstrated that the majority of these isolates clustered with known North American/European isolates. These findings revealed procedures that could help identify unique genotypes and phenotypes associated with HSV-1 isolates, which can be important for determining viral factors critical for regulating HSV-1 reactivation.

JC virus antibody status underestimates infection rates.

OBJECTIVE: JC virus (JCV) seropositivity is a risk factor for progressive multifocal leukoencephalopathy (PML) in patients on natalizumab. Accordingly, the JCV serological antibody test is of paramount importance in determining disease risk. METHODS: We tested the accuracy of the JCV serum antibody test by comparing the results of JCV serology to JCV viruria and viremia in 67 patients enrolled in a single-center, retrospective cohort study. Bodily fluids (urine and blood) were assessed for JCV DNA by real time quantitative polymerase chain reaction 6 to 47 months (mean = 26.1 months) before JCV antibody testing. In 10 individuals, blood and urine samples were obtained on 2 separate occasions at 6-month intervals. RESULTS: Forty (59.7%) of the 67 patients were JCV seropositive. Of 27 JCV seronegative patients, 10 (37%) had JCV viruria. Urine JCV DNA copy numbers were significantly higher in the seropositive group (mean log copy number = 5.93, range = 1.85-9.21) than the seronegative group (mean log copy number = 2.41, range = 1.85-5.43; p = 0.0026). Considering all body fluid test results, 50 (74.6%) of the 67 patients were previously infected with JCV. INTERPRETATION: The false-negative rate of the JCV serology in this study was 37%; therefore, JCV serostatus does not appear to identify all patients infected with JCV. Thus, a negative JCV antibody result should not be conflated with absence of JCV infection. This discordance may be important in understanding JCV biology, risk for PML, and PML pathogenesis.

C-terminal trans-activation sub-region of VP16 is uniquely required for forskolin-induced herpes simplex virus type 1 reactivation from quiescently infected-PC12 cells but not for replication in neuronally differentiated-PC12 cells.

The HSV-1 tegument protein VP16 contains a trans-activation domain (TAD) that is required for induction of immediate early (IE) genes during lytic infection and induced reactivation from latency. Here we report the differential contributions of the two sub-regions of the TAD in neuronal and non-neuronal cells during activation of IE gene expression, virus replication, and reactivation from quiescently infected (QIF)-PC12 cells. Our studies show that VP16- and chemical (hexamethylenebisacetamide)-induced IE gene activation is attenuated in neuronal cells. Irrespective of neuronal or non-neuronal cell backgrounds, IE gene activation demonstrated a greater requirement for the N-terminal sub-region of VP16 TAD (VP16N) than the C-terminal sub-region (VP16C). In surprising contrast to these findings, a recombinant virus (RP4) containing the VP16N deletion was capable of modest forskolin-induced reactivation whereas a recombinant (RP3) containing a deletion of VP16C was incapable of stress-induced reactivation from QIF-PC12 cells. These unique process-dependent functions of the VP16 TAD sub-regions may be important during particular stages of the virus life cycle (lytic, entrance, and maintenance of a quiescent state and reactivation) when viral DNA would be expected to be differentially modified.

Disease-modifying drugs for multiple sclerosis and JC virus expression.

Natalizumab-associated progressive multifocal leukoencephalopathy in multiple sclerosis (MS) occurred in two individuals also treated with interferon ß1a, raising concerns about the interaction of these disease-modifying agents and leading to the recommendation to avoid their concomitant administration. However, type I interferons are antiviral. Using a real-time quantitative polymerase chain reaction for the detection and quantification of the John Cunningham virus (JCV), DNA in peripheral blood mononuclear cells (PBMCs), and urine in MS patients, we tested the hypothesis that MS disease-modifying drugs (DMD) qualitatively and quantitatively alter JCV prevalence and viral copy numbers. Two hundred thirty-nine patients were enrolled in a cross-sectional study in which blood and urine specimens were collected at a single time and 37 newly diagnosed, treatment-naïve MS patients were enrolled in a longitudinal study in which specimens were obtained at diagnosis and 6 months after treatment initiation. JCV DNA was detected in PBMCs of only two patients (0.07 %), but was commonly detected in the urine (46.8 %) in this population. There was no effect of DMDs on blood or urinary JCV prevalence or viral copy numbers with either glatiramer acetate (Copaxone®) or interferon-ß therapy (Avonex®, Betaseron®, or Rebif®). The small number of patients on other therapies precluded meaningful comment about their effects. No obvious effect of the platform DMDs on JCV prevalence was observed even for the interferon-ßs.

Effect of chewing gum containing Xylitol and blackberry powder on oral bacteria: A randomized controlled crossover trial.

OBJECTIVE: The aim was to determine the effect of chewing gum containing xylitol and freeze-dried blackberry powder on oral bacteria. DESIGN: This was a randomized, controlled, cross-over study (RCT #: NCT05133557). Fifty participants chewed gum over an 8 h period, four times for 20 min at 2-hour intervals, containing 700 mg xylitol (CG) with or without 50 mg blackberry powder (BG), while wearing a stent containing a sterile enamel chip. After a 1 week washout, participants chewed gum from the other group following the same protocol. The primary outcome was the amount of nine oral bacteria in saliva as determined by quantitative PCR. The secondary outcome was bacteria formed on enamel chips. RESULTS: Chewing BG for four twenty-minute intervals reduced mean total bacteria load and the relative abundance of six of the nine bacteria studied in saliva (p < 0.05). In comparison, only four bacteria were reduced in abundance in the CG group. After gum chewing and regardless of group, S. sanguinis and A. naeslundii were the predominant bacteria adherent to enamel, with S. mutans representing < 1 % of the total bacteria on enamel. CONCLUSION: Bacterial loads in saliva were rapidly, differentially, and significantly reduced after one day of chewing BG.

Real-time polymerase chain reaction to determine the prevalence and copy number of epstein-barr virus and cytomegalovirus DNA in subgingival plaque at individual healthy and periodontal disease sites.

BACKGROUND: Detection of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in plaque from patients with periodontal disease provides support for the theory that these viruses play a role in the pathogenesis of periodontitis. This study sought to further define this relationship by determining the prevalence of these viruses at individual disease and healthy sites of patients with periodontal disease and to determine whether the presence and amount of viral DNA correlate with disease severity. METHODS: Subgingival plaque from three healthy and three disease sites of 65 patients who had chronic periodontitis were evaluated for the presence and amount of EBV, CMV, and Fusobacterium nucleatum DNA using real-time polymerase chain reaction. Patient serum was evaluated for antibodies against EBV and CMV using enzyme-linked immunosorbent assays. RESULTS: EBV DNA was detected in 18.5% of subgingival plaque samples (72/390) and in at least one of the six plaque samples in 44.6% (29/65) of the patients. CMV DNA was detected in one plaque sample (0.3%). EBV was significantly more prevalent in disease sites (28.2%; 55/195) than in healthy sites (8.7%; 17/195; P = 0.002). However, neither EBV prevalence nor its amount correlated with increased probing depth >5 mm or attachment loss >2 mm, whereas the amount of F. nucleatum DNA did. Sites positive for EBV had a median copy number of eight. Antibodies against EBV and CMV were detected in 85.7% and 78.6% of persons evaluated, respectively. CONCLUSION: EBV was infrequent and CMV was rarely present in individual subgingival sites affected by chronic periodontitis.

Combination Drug Therapy of Pioglitazone and D-cycloserine Attenuates Chronic Orofacial Neuropathic Pain and Anxiety by Improving Mitochondrial Function Following Trigeminal Nerve Injury.

OBJECTIVES: The study aim was to determine how peripheral trigeminal nerve injury affects mitochondrial respiration and to test efficacy of combined treatment with 2 Federal Drug Administration approved drugs with potential for improving mitochondrial bioenergetics, pain and anxiety-related behaviors in a chronic orofacial neuropathic pain mouse model. METHODS: Efficacy of (R)-(+)-4-amino-3-isoxazolidinone (D-cycloserine, DCS), an N-Methyl-D-aspartate antagonist/agonist, and Pioglitazone (PIO), a selective agonist of nuclear receptor peroxisome proliferator-activated receptor gamma was investigate in the trigeminal inflammatory compression (TIC) neuropathic nerve injury mouse model. Combined low doses of these drugs (80 mg/kg DCS and 100 mg/kg PIO) were given as a single bolus or daily for 7 days post-TIC to test ability to attenuate neuropathic nociceptive and associated cognitive dependent anxiety behaviors. In addition, beneficial effects of the DCS/PIO drug combination were explored ex vivo in isolated cortex/brainstem mitochondria at 28 weeks post-TIC. RESULTS: The DCS/PIO combination not only attenuated orofacial neuropathic pain and anxiety-related behaviors associated with trigeminal nerve injury, but it also improved mitochondrial bioenergetics. DISCUSSION: The DCS/PIO combination uncoupled mitochondrial respiration in the TIC model to improve cortical mitochondrial dysfunction, as well as reduced nociceptive and anxiety behaviors present in mice with centralized chronic neuropathic nerve injury. Combining these drugs could be a beneficial treatment for patients with depression, anxiety, or other psychological conditions due to their chronic pain status.

PPARγ Agonists Attenuate Trigeminal Neuropathic Pain.

OBJECTIVES: The aim of this study is to investigate the role of peroxisome proliferator-activated receptor-gamma isoform (PPARγ), in trigeminal neuropathic pain utilizing a novel mouse trigeminal inflammatory compression (TIC) injury model. RESULTS: The study determined that the PPARγ nuclear receptor plays a significant role in trigeminal nociception transmission, evidenced by: 1) Intense PPARγ immunoreactivity is expressed 3 weeks after TIC nerve injury in the spinal trigeminal caudalis, the termination site of trigeminal nociceptive nerve fibers. 2) Systemic administration of a PPARγ agonist, pioglitazone (PIO), attenuates whisker pad mechanical allodynia at doses of 300 mg/kg i.p. and 600 mg/kg p.o. 3) Administration of a PPARγ antagonist, GW9662 (30 mg/kg i.p.), prior to providing the optimal dose of PIO (300 mg/kg i.p.) blocked the analgesic effect of PIO. DISCUSSION: This is the first study localizing PPARγ immunoreactivity throughout the brainstem trigeminal sensory spinal nucleus (spV) and its increase three weeks after TIC nerve injury. This is also the first study to demonstrate that activation of PPARγ attenuates trigeminal hypersensitivity in the mouse TIC nerve injury model. The findings presented here suggest the possibility of utilizing the FDA approved diabetic treatment drug, PIO, as a new therapeutic that targets PPARγ for treatment of patients suffering from orofacial neuropathic pain.

Distribution and Quantity of Sites of John Cunningham Virus Persistence in Immunologically Healthy Patients: Correlation With John Cunningham Virus Antibody and Urine John Cunningham Virus DNA.

Importance: Although seroepidemiological studies indicate that greater than 50% of the population has been infected with John Cunningham virus (JCV), the sites of JCV persistence remain incompletely characterized. Objective: To determine sites of JCV persistence in immunologically healthy individuals. Design, Setting, and Participants: Tissue specimens from multiple sites including brain, renal, and nonrenal tissues were obtained at autopsy performed in the Department of Pathology at the University of Kentucky from 12 immunologically healthy patients between February 9, 2011, and November 27, 2012. Quantitative polymerase chain reaction was performed on the tissue specimens and urine. Serum JCV antibody status was determined by enzyme-linked immunosorbent assay. Main Outcomes and Measures: The detection and quantification of JCV from the tissues by quantitative polymerase chain reaction illuminated sites of viral persistence. These results were correlated with JCV antibody levels. Results: Autopsies were performed on 12 individuals, 10 men and 2 women, ranging in age from 25 to 75 years (mean, 55.3 years). Seven of 12 individuals were JCV antibody seropositive based on absorbance units. Serostatus was associated with amounts of JCV DNA in urine and its tissue distribution. John Cunningham virus DNA was found in 75% of genitourinary tissue samples from donors (18 of 24) with high JCV antibody levels, 13.3% of donors with low levels i(4 of 30), and 0% of seronegative persons (0 of 32). In nongenitourinary tissues, JCV DNA was detected in 45.1% of tissue samples of donors (32 of 71) with high JCV, 2.2% of donors with low JCV serostatus (2 of 93), and 0% of seronegative persons (0 of 43). Genitourinary tissues had higher copy numbers than other sites. John Cunningham virus DNA was detected in urine of seronegative individuals in a research-grade assay. Conclusions and Relevance: Persistent (latent or actively replicating) JCV infection mostly predominates in genitourinary tissues but distributes in other tissues at low copy number. The distribution and copy numbers of the virus appear to correlate with urinary JCV shedding and serostatus.

Efficacy of Herpes Simplex Virus Vector Encoding the Human Preproenkephalin Gene for Treatment of Facial Pain in Mice.

AIMS: To determine whether herpes simplex virus-based vectors can efficiently transduce mouse trigeminal ganglion (TG) neurons and attenuate preexisting nerve injury-induced whisker pad mechanical hypersensitivity in a trigeminal inflammatory compression (TIC) neuropathic pain model. METHODS: Tissue transduction efficiencies of replication-conditional and replication-defective vectors to mouse whisker pads after topical administration and subcutaneous injection were assessed using quantitative real-time PCR (qPCR). Tissue tropism and transgene expression were assessed using qPCR and reverse-transcriptase qPCR following topical application of the vectors. Whisker pad mechanical sensitivities of TIC-injured mice were determined using graduated von Frey fibers before and after application of human preproenkephalin expressing replication-conditional vector (KHPE). Data were analyzed using one-way analysis of variance (ANOVA) and post hoc tests. RESULTS: Transduction of target TGs was 8- to 50-fold greater after topical application than subcutaneous injection and ≥ 100-fold greater for replication-conditional than replication-defective vectors. Mean KHPE loads remained constant in TGs (4.5-9.8 × 10(4) copies/TG) over 3 weeks but were below quantifiable levels (10 copies/tissue) within 2 weeks of application in other nontarget cephalic tissues examined. Transgene expression in TGs was maximal during 2 weeks after topical application (100-200 cDNA copies/mL) and was below quantifiable levels (1 cDNA copy/mL) in all nontarget tissues. Topical KHPE administration reduced TIC-related mechanical hypersensitivity on whisker pads 4-fold (P < .05) for at least 1 week. CONCLUSION: Topically administered KHPE produced a significant antinociceptive effect in the TIC mouse model of chronic facial neuropathic pain. This is the first report in which a gene therapeutic approach reduced trigeminal pain-related behaviors in an established pain state in mice.

Persistent neuropathic pain influences persistence behavior in rats.

AIMS: To determine whether self-regulation can be studied successfully in a rodent model and whether persistent facial pain influences self-regulatory behavior. METHODS: Thirty male Sprague-Dawley rats, divided into two groups, (1) chronic constriction injury of the infraorbital nerve (CCI-ION) and (2) naïve, were used in a two-part behavioral paradigm of self-regulation. This paradigm consisted of both a cued go/no-go task (part one) and a persistence trial (part two). All animals were acclimated and trained for a period of 4 weeks prior to the experimental manipulation and then tested for a total of 5 weeks following experimental manipulation. Results were analyzed with t tests, one-way analysis of variance, and two-way, repeated measures analysis of variance. RESULTS: CCI-ION surgery induced significant mechanical hypersensitivity of the ipsilateral whisker pad that began 3 weeks postsurgery and persisted through the duration of the experiment (P < .001). At weeks 4 and 5 post-experimental manipulation, naïve animals demonstrated a significant decrease in lever presses during the persistence task (P < .05) compared to baseline, whereas CCI-ION animals did not (P = .55). CONCLUSION: These results suggest that persistent pain influences behavioral regulation and that animals experiencing persistent pain may have difficulty adapting to environmental demands.

Expression of miR-15/107 family microRNAs in human tissues and cultured rat brain cells.

The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs), sharing a 5' AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively). In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS). In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs). Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

Antiviral effects of blackberry extract against herpes simplex virus type 1.

OBJECTIVE: The aim of this study was to evaluate antiviral properties of blackberry extract against herpes simplex virus type 1 (HSV-1) in vitro. STUDY DESIGN: HSV-infected oral epithelial (OKF6) cells and cell-free virus suspensions were treated with blackberry extract (2.24-1,400 µg/mL), and virus yield and infectivity were quantified by direct plaque assay. RESULTS: Blackberry extract ≥56 µg/mL inhibited HSV-1 replication in oral epithelial cells by >99% (P < .005). Concentrations ≥280 µg/mL were antiviral when the extract was added after virus adsorption and entry. Exposure of cell-free virus to ≥280 µg/mL blackberry extract for 15 minutes at room temperature was virucidal (P = .0002). The virucidal effects were not due to pH changes at concentrations up to 1,500 µg/mL. CONCLUSIONS: Blackberry extract inhibited the early stages of HSV-1 replication and had potent virucidal activity. These properties suggest that this natural fruit extract could provide advantage as a topical prophylactic/therapeutic agent for HSV infections.

HIV protease inhibitors alter innate immune response signaling to double-stranded RNA in oral epithelial cells: implications for immune reconstitution inflammatory syndrome?

In this investigation, several HIV protease inhibitors altered the virally associated, double-stranded RNA (dsRNA)-stimulated, innate immune response. Lopinavir, the most potent inducer of interleukin (IL)-8 expression, also inhibited dsRNA-induced monocyte chemotactic protein 1 expression. Further analyses demonstrated that nuclear factor-κB is required for lopinavir's induction of IL-8. These findings demonstrate that protease inhibitors, such as lopinavir, differentially dysregulate innate immune signaling in a manner that could affect immune (reconstitution) inflammatory responses in oral epithelium.

HIV protease inhibitors block oral epithelial cell DNA synthesis.

OBJECTIVES: Anti-retroviral therapy regimens that include HIV protease inhibitors (PIs) are associated with diverse adverse effects including increased prevalence of oral warts, oral sensorial deficits and gastrointestinal toxicities suggesting that PIs may perturb epithelial cell biology. To test the hypothesis that PIs could affect specific biological processes of oral epithelium, the effects of these agents were evaluated in several oral epithelial cell-lines. DESIGN: Primary and immortalized oral keratinocytes and squamous carcinoma cells of oropharyngeal origin were cultured in the presence of pharmacologically relevant concentrations of PIs. Their affects on cell viability, cytotoxicity and DNA synthesis were assessed by enzymatic assays and incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA. RESULTS: Viability of primary and immortalized oral keratinocytes as well as squamous carcinoma cells of oropharyngeal origin was significantly reduced by select PIs at concentrations found in plasma. Of the seven PIs evaluated, nelfinavir was the most potent with a mean 50% inhibitory concentration [IC(50)] of 4.1 microM. Lopinavir and saquinavir also reduced epithelial cell viability (IC(50) of 10-20 microM). Atazanavir and ritonovir caused minor reductions in viability, while amprenavir and indinavir were not significant inhibitors. The reduced cell viability, as shown by BrdU incorporation assays, was due to inhibition of DNA synthesis rather than cell death due to cytotoxicity. CONCLUSION: Select PIs retard oral epithelial cell proliferation in a drug and dose-dependent manner by blocking DNA synthesis. This could account for some of their adverse effects on oral health.

Low prevalence of varicella zoster virus and herpes simplex virus type 2 in saliva from human immunodeficiency virus-infected persons in the era of highly active antiretroviral therapy.

OBJECTIVES: Human herpesviruses, e.g., herpes simplex virus (HSV) type 1, Epstein-Barr virus, and cytomegalovirus, appear in saliva at greater frequency in persons infected with human immunodeficiency virus (HIV) than in healthy individuals. However, it is not known if varicella zoster virus (VZV) and HSV-2 appear simultaneously during HIV infection at greater frequency in saliva in this era of highly active antiretroviral therapy (HAART). The aim of this study was to investigate the prevalence and amounts of VZV and HSV-2 in the saliva of HIV-infected orally asymptomatic patients. STUDY DESIGN: Quantitative polymerase chain reaction was used to investigate the prevalence, quantity, risk, and correlations of salivary VZV and HSV-2 from 59 HIV-seropositive individuals and 53 healthy control subjects in a case-control cross-sectional study. Seventy-eight percent of the HIV-seropositive patients (46 out of 59) were taking HAART. RESULTS: VZV DNA was detected in the saliva of 5.1% (3 out of 59) of the HIV-positive group and in only 1 healthy control 1.9% (1 out of 53; P = .62). The amount of VZV DNA in the expressors was low, generally <1,100 copies/mL, with no observed difference between the HIV-positive group and the control subjects (P = 1.0). HSV-2 DNA was not detected in either group. In the HIV-infected group, VZV shedding occurred in those on HAART, but was not associated with oral lesions, specific CD4(+) or CD8(+) T-cell levels, or demographic factors. CONCLUSIONS: Varicella zoster virus was detected at low prevalence in the saliva of HIV-infected persons, whereas HSV-2 was not detected in the saliva of this cohort. HAART does not appear to diminish the risk for asymptomatic VZV shedding.

Asymptomatic shedding of herpes simplex virus (HSV) in the oral cavity.

OBJECTIVE: The aim of this study was to investigate the rate of herpes simplex virus (HSV) shedding from the oral cavity, because recent studies suggest that shedding is more frequent than originally reported. Factors that could influence the rate and duration of shedding from the oral cavity were examined. METHODS: Existing epidemiologic data from 22 reports of HSV shedding from more than 3,500 individuals were analyzed with regard to demographics, frequency of sampling, and methodologic assays. RESULTS: HSV-1 was more likely to be detected than HSV-2 in the oral cavity of asymptomatic persons (7.5 odds ratio, 95% confidence interval 4.4-12.8; P < .0001). The rate of shedding was highly variable among individuals, ranging from none to 92% of days tested, and occurred in seropositive and seronegative individuals. In cell culture studies, the rate of detection on a single day was 6.3%. Polymerase chain reaction studies provided a different picture. HSV-1 DNA was present in 97 of 180 patients (53.9%) at multiple visits, with a rate of daily detection of 33.3%. The mean duration of shedding was between 1 and 3 days, but more than 3 days in about 10% of patients. CONCLUSIONS: At least 70% of the population shed HSV-1 asymptomatically at least once a month, and many individuals appear to shed HSV-1 more than 6 times per month. Shedding of HSV-1 is present at many intraoral sites, for brief periods, at copy numbers sufficient to be transmitted, and even in seronegative individuals. The dental implications of these findings are discussed.

Detection of human cytomegalovirus in dental plaque from individual periodontal sites by real-time polymerase chain reaction.

OBJECTIVE: The aim was to evaluate three primer-probe sets and real-time polymerase chain reaction (PCR) for the detection of human cytomegalovirus (HCMV) in dental plaque from individual periodontal sites. STUDY DESIGN: Fifty subgingival plaque specimens from 13 healthy subjects (on average at least 2 healthy and 2 periodontal disease sites per subject) and 50 saliva specimens from 24 subjects, including 16 controls, were assessed using 3 primer-probe sets (polymerase [POL], glycoprotein B [gB], and US14) and real-time PCR. Kappa statistics were performed to measure agreement between the primer-probe sets. RESULTS: There was excellent agreement between the gB and POL primers in the detection of HCMV (kappa statistic = 0.85 [95% confidence interval 0.71-0.99]), yielding a prevalence of 4% (2 out of 50) at individual periodontal disease sites and a similar rate of 8.8% (3 out of 34) in saliva. CONCLUSION: Human cytomegalovirus was infrequently detected in dental plaque. Of 3 primer-probe sets evaluated, those targeting the POL and gB genes were more accurate in the detection of HCMV than that targeting US14.