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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 33  |  Issue : 1  |  Page : 22-26

Asymptomatic shedding of herpes simplex virus in oral cavity


1 Department of Oral Medicine and Radiology, PDU Dental College, Solapur, Maharashtra, India
2 Department of General Pathology, PDU Dental College, Solapur, Maharashtra, India

Date of Submission29-Oct-2020
Date of Decision30-Dec-2020
Date of Acceptance23-Jan-2021
Date of Web Publication26-Mar-2021

Correspondence Address:
Dr. Shruti Wadne
Department of Oral Medicine and Radiology, PDU Dental College, Solapur, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jiaomr.jiaomr_231_20

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   Abstract 


Herpes simplex virus (HSV) is a significant human pathogen. Dental surgeons and Dental Hygienists come across many patients which are not having clinical symptoms but are the carriers of the disease. The person working in the oral cavity is at high risk for getting infected and also the risk of cross-infection in the general population. Aim: In this study, we determine the prevalence of HSV qualitatively and quantitatively in the oral cavity in apparently asymptomatic patients. Study Setting: Institutional and PCR testing Labs. Methods & Materials: The study analyzed saliva samples derived from 220 individuals which were collected with sterile swabs. The traditional PCR method was done for qualitative analysis and real-time PCR for the quantitative analysis. Results: The results of qualitative analysis showed that HSV-2 was more commonly shed than HSV-1 in the oral cavity, while the infected persons showed more genomic copy numbers. Conclusion: The presence of asymptomatic shedding of HSV-2 may be an indicator of future immunosuppression which should be followed up.

Keywords: Herpes simplex virus, subclinical reactivation, viral genome copy number


How to cite this article:
Kulkarni A, Wadne S, Birangane R, Chaudhari R, Kole N, Sankpal S. Asymptomatic shedding of herpes simplex virus in oral cavity. J Indian Acad Oral Med Radiol 2021;33:22-6

How to cite this URL:
Kulkarni A, Wadne S, Birangane R, Chaudhari R, Kole N, Sankpal S. Asymptomatic shedding of herpes simplex virus in oral cavity. J Indian Acad Oral Med Radiol [serial online] 2021 [cited 2021 Apr 14];33:22-6. Available from: https://www.jiaomr.in/text.asp?2021/33/1/22/312206




   Introduction Top


Herpes Simplex Virus is a ubiquitous virus. They are the members of the Herpesviridae family belonging to the highly infectious alpha group of viruses. The seroprevalence of HSV-1 is 3.7 billion people under age 50 and the seroprevalence of HSV-2 is 491 million people aged 15-49, worldwide. In the developed countries, HSV-1 has become an increasing cause of genital herpes. Herpes infections are most contagious when symptoms are present but can still be transmitted to others in the absence of symptoms.[1] The presence of HSV in the absence of clinical lesions is known as Subclinical shedding. The ability of the virus to reactivate from latency is the key to its perpetuation. Asymptomatic release of the virus is not associated with systemic signs and symptoms. Subclinical shedding promotes the risk of cross-infection not only to other patients but also to the dental staff. This is especially important in immunocompromised patients and pregnant women.[2],[3]


   Aim and Objectives Top


In this study, we sought to determine the prevalence of subclinical reactivation of HSV qualitatively and quantitatively in apparently asymptomatic patients visiting for dental care.


   Methods Top


Study population and data collection

The study was conducted in the department of Oral Medicine and Radiology of a dental school in India. Ethical clearance was obtained from the Institutional Ethical Committee (MUHS/UDC/GFL/05/2016-17/1643/2017). The procedures followed were by the ethical standards of the Helsinki Declaration of 1964 and later versions. The study was carried out after obtaining written informed consent from individuals selected amongst the outpatients visiting the department.

The sample size was calculated using the formula:

n = 4Z2σ2/W2 and was estimated according to the amount of grant received.

220 individuals in the age range of 20 years to 70 years, individuals clinically asymptomatic for herpetic lesions, individuals having a negative history of immunosuppression were recruited in the study. The persons having a recent history of Herpes infections, immunocompromised status patients, the persons having a recent history of major illness and fever, pregnant and menstruating females, patients with severe dental pain, persons working in farms, and sun exposure were excluded from the study.

All participants had the standardized demographic data and were grouped 1:1 Male and Female age-matched. Each participant underwent a detailed clinical examination. The saliva was collected by aspiration method from the floor of the mouth. If needed, parotid glands were stimulated manually for saliva secretion. Then the collected saliva was added to aliquots containing viral transport media. Care was taken to avoid contamination with other anatomic areas during the collection of saliva (especially the lower lip). The aliquots were kept in a deep freezer till the end of the day and packed in dry ice. The aliquots were numbered and 2 groups were made viz. 160 and 60 samples. The samples were sent to laboratories for PCR testing. One group was subjected to real-time PCR for quantitative analysis and the other one was subjected to traditional PCR for qualitative analysis.

Laboratory methods

Method 1

The real-time PCR was performed on 160 salivary samples. Samples were tested by the DNA Master Hybridization Probes assay using a TaqMan probe. Taq Start antibody was added directly to the DNA Master solutions, and the mixtures were incubated at room temperature for 5 minutes. Then, MgCl2, primers, TaqMan probe, and water were added. Fifteen microliters of master mix and 5 ml of DNA template were added to each capillary. Sealed capillaries were centrifuged in a microcentrifuge and placed into the LC rotor. After denaturation for 2 min at 95°C, PCR cycles were run. Amplified signals were recorded digitally. So, with this process, genome copy number of HSV DNA was found.

Method 2

Sixty samples were subjected to DNA extraction procedure (Modified Proteinase-K method). Samples were transferred to the tube containing T.E. (Tris HCl and EDTA) buffer. Then the tube was centrifuged at 5,000 rpm for 5 min. The supernatant which was obtained was discarded. 500 microliters of fresh T.E. buffer was added to the tube and was centrifuged for 3-4 minutes. The above process was repeated 3-4 times with fresh T.E. buffer.

The supernatant was again discarded. 50 microliter lysis buffer I was added to the tube, Vortexed it, and was kept for 5 min. 50 microliter Lysis buffer II and 10 microliter proteinase–K (100 ugs/ml) were added to extracted DNA and were vortexed vigorously and DNA was stored at -20°C. Agarose gel electrophoresis was carried out for all samples and signals were recorded as 271 bp for HSV-1 and 231 bp for HSV-2 [Figure 1].
Figure 1: Recording of signal by Agarose gel electrophoresis as 271 bp for HSV-1 and 231 bp for HSV-2

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   Results Top


Quantifying the HSV DNA by real-time PCR

Frequency of subclinical HSV shedding at a single sampling

Saliva samples sent for quantitative analysis were 160. Out of those, male and female saliva samples were equally divided. From 160 saliva samples, 11 patients showed HSV DNA. Of which 3 were male samples and 8 were female samples.

Viral genome copy number

Real-time PCR measures quantitatively the viral genome copy number. In our study, 11 patients showed HSV DNA in saliva samples [Figure 2]. The highest and lowest genome copy number for positive samples of HSV was found to be 23,18,220 copies per ml of saliva and 23,210 copies per ml of saliva, respectively [Figure 3]. The mean genome copy number for HSV was 7,66,568 copies per ml of saliva [Figure 4] and [Figure 5].This data shows HSV reactivates frequently and replicates at a subclinical level in the oral cavity of asymptomatic individuals.
Figure 2: Comparing prevalence of HSV 1 and HSV 2 in males and fe

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Figure 3: Standard Curve. slope: -3.762 Y-Intercept: 48.717 R2: 0.919

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Figure 4: Amplification Plot (Rn vs. Cycle) showing fluorescence pickup above threshold level denoting positive genome copy numbers

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Figure 5: Amplification Plot (Rn vs. Cycle)

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Subclinical reactivation at multiple time points

Multiple samples were taken for 16 individuals (8 males and 8 females). The saliva samples were taken three times for each individual in a month with an interlude of 10 days. So out of them, 2 female samples showed HSV DNA. Subclinical reactivation at multiple time points was not found in any of the samples.

Traditional PCR-Agarose gel electrophoresis

Out of 60 Patients, 10 patients were positive for HSV (6 males and 4 females) [Figure 6]. From 10 HSV positive patients, 6 were positive for HSV-2 (5 male and 1 female) and 4 were positive for HSV-1 (2 male and 2 female) [Figure 2]. Out of 10 HSV positive patients, 1 was positive for both HSV-1 and HSV-2 (1 male). Periodontitis was associated with all HSV positive patients.
Figure 6: Assessment of HSV-1 and HSV-2

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   Discussion Top


The present study, which determined the prevalence of asymptomatic HSV shedding in the oral cavity, was undertaken for several reasons. First, there is growing evidence from human studies showing that the rate of HSV reactivation and viral shedding is more frequent than previously reported. Second to find out which type of Herpes Simplex Virus i.e., HSV-1 is more common or HSV-2 in the oral cavity. Third, the entity asymptomatic shedding is not highlighted in the textbooks but has got significant clinical importance.

The present study analyzed saliva samples derived from 220 individuals. Overall, 21 positive samples were found (16%). The results showed that by qualitative analysis HSV-2 was found to be more commonly shed than HSV-1 in the oral cavity. This observation is contradicting to the previous studies where HSV-1 was more prevalent than HSV-2. Craig S. Miller in his study found that HSV-1 was more likely to be detected than HSV-2 in the oral cavity of an asymptomatic person.[4] Van Velzen et al. in 2014 found that the shedding of HSV-1 occurs frequently.[5] Quantitative analysis results showed that the highest and lowest genome copy number for positive samples of HSV was found to be 23,18, 220 copies per ml of saliva, and 23,210 copies per ml of saliva, respectively. The mean genome copy number for HSV was 7,66,568 copies per ml of saliva. This data shows HSV reactivates frequently and replicates at a subclinical level in the oral cavity of asymptomatic individuals.

The change may be because of the change in sexual practices, so further studies should be carried out in finding the prevalence and the reasons if HSV-2 predominates. The prevalence was more common in males as compared to females. The older individuals shredded HSV-1 more commonly whereas younger individuals shredded HSV-2. One individual was positive for both HSV-1 and HSV-2. Periodontitis was associated with HSV positive individuals. This is supported by the previous studies.

Several of the findings from the present report impact on the practice of dentistry. Dentists should know HSV shedding in the oral cavity is frequent and common in the absence of oral lesions and those oral secretions commonly contain infectious HSV. Dentists are in a unique position to educate patients of the infectious nature of saliva and the risk of transmission during splash of saliva while talking, laughing in the dental office and outside.[6]

Also, these data reinforce the importance of implementing proper protective infection control measures when performing routine dental examinations and procedures. Efforts should be made to minimize splashes and splatter even in the absence of lesions by using mouth masks, hand gloves, and eyeglasses. Medical conditions (e.g., immunosuppression) and traumatic oral procedures increase the likelihood of HSV shedding in the oral cavity.[7] Although it is unclear if these asymptomatic periods of shedding contribute to oral disease, it has been purported that herpes viruses can contribute to the pathogenesis of periodontal disease and persistent mucosal ulcerations. The spread of HSV contributed to some extent or another to HSV-1 incidence. However, the oral-to-oral transmission will remain the dominant mode, but with an increasing role for the sexual modes of transmission. Though both orogenital (oral sex) and genitogenital (sexual intercourse) modes of transmission will steadily increase, the genitogenital mode will increase faster, as the pool of HSV-1 genital herpes infections increases.[8],[9] If studies continue to demonstrate important relationships between HSV shedding and specific oral diseases, targeted antiviral therapies may become more commonly used, because they have been shown to reduce both clinical and subclinical reactivation rates of HSV-1 and HSV-2. Finally, the high frequency of HSV shedding suggests that it's not only HSV-1 that is not as dormant during latency as previously believed and additional research is required to better understand the mechanisms that regulate reactivation.[10],[11]

Limitations and future prospects

Currently, <10% of HSV-1 genital herpes is because of genital-to-genital transmission, but this will increase up to 15% shortly after 2050. PCR-ELISA may be employed to evaluate HSV-1 shedding in the general population, identify basal shedding levels in known sufferers of RHL, and investigate the shedding of HSV-1 into saliva. These findings may have implications for cross-infection and clinical trials of antiviral drugs. The wide anatomical distribution of HSV shedding argues for limited protection at its best. Nonoxynol-9 has anti- HSV activity in animals, but no human data are supporting its use in the prevention of HSV transmission.[12]


   Conclusion Top


HSV-2 predominates over HSV-1 in the qualitative analysis which was contradicting to previous studies. Several patients with asymptomatic carriers were comparatively found less. While the infected persons showed more genome copy numbers which were in correlation with previous studies, the presence of asymptomatic shedding of HSV-2 may be an indicator of future immunosuppression which should be followed up. The efficacy of newer antiviral drugs can be checked by treating the viral load of an asymptomatic shedding of HSV.

Acknowledgements

Maharashtra University of Health Sciences, Nashik.

Financial support and sponsorship

Maharashtra University of Health Sciences, Nashik.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Koopaie M, Esmaily F. Assessment of oral shedding of herpes simplex virus before and after endodontic and fixed partial prosthodontics treatment in patients with recurrent herpes simplex. Ann Mil Health Sci Res 2018;16:82601.  Back to cited text no. 1
    
2.
Tsai J, Cohrs RJ, Nagel MA, Mahalingam R, Schmid DS, Choe A, et al. Reactivation of type 1 herpes simplex virus and varicella zoster virus in an immunosuppressed patient with acute peripheral facial weakness. J Neurol Sci 2012;313:193-5.  Back to cited text no. 2
    
3.
Kucera P, Gerber S, Marques-Vidal P, Meylan PR. Seroepidemiology of herpes simplex virus type 1 and 2 in pregnant women in Switzerland: An obstetric clinic based study. Eur J Obstet Gynecol Reprod Biol 2012;160:13-7.  Back to cited text no. 3
    
4.
Miller CS, Danaher RJ. Asymptomatic shedding of herpes simplex virus (HSV) in the oral cavity. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:43-50.  Back to cited text no. 4
    
5.
Tuokko H, Bloigu R, Hukkanen V. Herpes simplex virus type 1 genital herpes in young women: Current trend in Northern Finland. Sex Transm Infect 2014;90:160.  Back to cited text no. 5
    
6.
Ayoub H. Characterizing the transitioning epidemiology of herpes simplex virus type 1 in the USA: Model-based predictions. BMC Med 2019;17:57.  Back to cited text no. 6
    
7.
Herpes simplex virus. News Room World health Organization.who.int/news-room/fact sheets/detail.  Back to cited text no. 7
    
8.
Looker KJ, Magaret AS, May MT, Turner KM, Vickerman P, Gottlieb SL, et al. Global and regional estimates of prevalent and incident herpes simplex virus type 1 infections in 2012. PLoS One 2015;10:e0140765.  Back to cited text no. 8
    
9.
Regezi JA, Sciubba JJ, Jordan RCK. Oral pathology: Clinical pathologic correlations. Illustrated ed. Elsevier science; Google books 2016.  Back to cited text no. 9
    
10.
Gottlieb SL, Giersing BK, Hickling J, Jones R, Deal C, Kaslow DC, et al. Meeting report: Initial World health organization consultation on Herpes simplex virus (HSV) vaccine preferred product characteristics, March 2017. Vaccine 2019;37:7408-18.  Back to cited text no. 10
    
11.
Burket LW, Greenberg MS, Glick M. Burket's Oral Medicine: Diagnosis and Treatment., chapter 2. Vesiculobullous lesions PMPH-USA; 2003.  Back to cited text no. 11
    
12.
McGeoch DJ, Bowden RJ. Evolution of herpes simplex viruses. Herpes Simplex Viruses. CRC Press; 2017. p. 1-34.  Back to cited text no. 12
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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