Dentistry, Faculty of
DSpace
UBCV
Lan, Yolanda Haruka
Master of Science - MSc
University of British Columbia
Objectives: Herpes simplex virus (HSV-1, 2) can present as symptomatic oral lesions or asymptomatic shedding. The risk of viral spreading during dental procedures is a safety concern, which we hypothesized may be reduced using cyanoacrylate adhesive (CA). The study aimed to determine 1) the magnitude of asymptomatic reactivation compared to symptomatic and 2) the efficacy of CA in limiting the virus transmission in symptomatic oral lesions. Methods: Thirty-three participants were recruited from UBC-Faculty of Dentistry. Oral mucosal (OS) and lesion swabs (LS) were collected and tested for HSV-1 and 2 using polymerase chain reaction (PCR) assay. Asymptomatic participants with possible history of oral HSV lesions (n=17) were followed for three months. Symptomatic lesions (n=11) had OS and LS collected, CA applied and subjected to soft tissue manipulation (STM). CA’s efficacy to alleviate/cause pain was assessed by daily journaling using visual analogue scale (VAS: 0-10). Examiner was blinded to symptomatic LS results and statistical analyses were performed using SPSS 27. Results: Twenty-one participants completed the study. One case of asymptomatic reactivation was detected out of 17 asymptomatic participants (2.0% reactivation rate). Data on 11 lesions were collected. Correlations between various lesion clinical parameters were assessed. Both lesion size and lesion PCR cycle threshold value (Ct) (p=0.04) and lesion day and stage (p<0.001) were positively correlated. Lesion discomfort and lesion stage were negatively correlated (p<0.03). Mean of reported average discomfort was 1.16±1.11, out of 10. Significant differences were detected in Ct of OS and LS (p<0.02), OS and post-STM swab (p<0.03). LS was observed to have lower Ct than post-PA swab. PA was accepted by participants with mean inverse-VAS score of 1.61±1.52. PA remained intact for an average of 25.25±17.63 hours. No adverse reactions to PA were reported. Side-effects include feeling of localized tightness and restriction in mouth-opening. Conclusions: Magnitude of asymptomatic reactivation cannot be determined, as insufficient cases were detected in this study. Observation found majority of LS Ct were lower than post-PA Ct suggests efficacy of CA-PA in limiting HSV transmission. The results suggest CA-PA to be a safe option as oral herpetic lesion barrier in the dental settings.
DENTAL MANAGEMENT OF HERPES SIMPLEX VIRUS (HSV-1 & HSV-2) USING N-BUTYL AND 2-OCTYL CYANOACRYLATE AS A BARRIER: A PILOT STUDY by Yolanda Haruka Lan BDSc., The University of British Columbia, 2018 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Craniofacial Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2021 © Yolanda Haruka Lan, 2021 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled: Dental management of herpes simplex virus (HSV-1 & HSV-2) using n-butyl & 2-octyl cyanoacrylate as a barrier: a pilot study submitted by Yolanda Haruka Lan in partial fulfillment of the requirements for the degree of Master of Science in Craniofacial Science Examining Committee: Penny Hatzimanolakis, Clinical Associate Professor, Oral Biological & Medical Sciences, UBC Supervisor Dr. Lari Häkkinen, Professor, Oral Biological & Medical Sciences, UBC Supervisory Committee Member Dr. Denise Laronde, Associate Professor, Oral Biological & Medical Sciences, UBC Supervisory Committee Member Dr. Eli Whitney, Associate Professor of Teaching, Oral Biological & Medical Sciences, UBC Supervisory Committee Member Dr. Vincent Lee, Assisting Professor of Teaching, Oral Health Sciences, UBC Additional Examiner iii Abstract Objectives: Herpes simplex virus (HSV-1, 2) can present as symptomatic oral lesions or asymptomatic shedding. The risk of viral spreading during dental procedures is a safety concern, which we hypothesized may be reduced using cyanoacrylate adhesive (CA). The study aimed to determine 1) the magnitude of asymptomatic reactivation compared to symptomatic and 2) the efficacy of CA in limiting the virus transmission in symptomatic oral lesions. Methods: Thirty-three participants were recruited from UBC-Faculty of Dentistry. Oral mucosal (OS) and lesion swabs (LS) were collected and tested for HSV-1 and 2 using polymerase chain reaction (PCR) assay. Asymptomatic participants with possible history of oral HSV lesions (n=17) were followed for three months. Symptomatic lesions (n=11) had OS and LS collected, CA applied and subjected to soft tissue manipulation (STM). CA’s efficacy to alleviate/cause pain was assessed by daily journaling using visual analogue scale (VAS: 0-10). Examiner was blinded to symptomatic LS results and statistical analyses were performed using SPSS 27. Results: Twenty-one participants completed the study. One case of asymptomatic reactivation was detected out of 17 asymptomatic participants (2.0% reactivation rate). Data on 11 lesions were collected. Correlations between various lesion clinical parameters were assessed. Both lesion size and lesion PCR cycle threshold value (Ct) (p=0.04) and lesion day and stage (p<0.001) were positively correlated. Lesion discomfort and lesion stage were negatively correlated (p<0.03). Mean of reported average discomfort was 1.16±1.11, out of 10. Significant differences were detected in Ct of OS and LS (p<0.02), OS and post-STM swab (p<0.03). LS was observed to have lower Ct than post-PA swab. PA was accepted by participants with mean inverse-VAS score of 1.61±1.52. PA remained intact for an average of 25.25±17.63 hours. No iv adverse reactions to PA were reported. Side-effects include feeling of localized tightness and restriction in mouth-opening. Conclusions: Magnitude of asymptomatic reactivation cannot be determined, as insufficient cases were detected in this study. Observation found majority of LS Ct were lower than post-PA Ct suggests efficacy of CA-PA in limiting HSV transmission. The results suggest CA-PA to be a safe option as oral herpetic lesion barrier in the dental settings. v Lay Summary Herpes simplex virus can reactivate and shed through the cheeks in the mouth without showing symptoms (asymptomatic reactivation). The most common signs of this virus are cold sores on the lips (herpes labialis lesions) and open ulcerations inside the mouth (oral herpetic lesions), usually on the palate or gums. Based on current guidelines, the presence of these lesions results in delays in non-emergency dental treatments. The novel use of a medical grade cyanoacrylate adhesive as a barrier may allow patients to receive dental treatment safely without virus spreading concerns. We were unable to assess the amount of virus released in asymptomatic reactivations as only one case was detected. We demonstrated that the medical-grade adhesive may be safely used on cold sores with high acceptance from study participants. In addition, there was tendency for the adhesive to limit the spreading of the virus and protected the lesion leading to improved healing. vi Preface This thesis is an original intellectual product of the author Yolanda Lan. The Clinical Research Ethics Board at the University of British Columbia (UBC) approved the clinical trial involving human subjects with certificate number H18-03266. The author was involved in the protocol proposal, ethics obtainment, and data collection under the supervision of Clinical Associate Professor Penny Hatzimanolakis. The BC Centre for Disease Control (BCCDC) virology laboratory analyzed oral mucosa swab samples produced in this trial. The author received laboratory test results and performed statistical analysis. The author prepared the preliminary manuscript, then incorporated feedback from Prof. Penny Hatzimanolakis, Dr. Lari Häkkinen, Dr. Denise Laronde, Dr. Eli Whitney, and Dr. Vincent Lee. The author submitted the final manuscript. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ........................................................................................................................ vii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiii List of Abbreviations ...................................................................................................................xv Acknowledgements ................................................................................................................... xvii Dedication ................................................................................................................................... xix Chapter 1: Introduction ................................................................................................................1 Chapter 2: Literature Review .......................................................................................................4 2.1 Herpes Simplex Virus (HSV) ......................................................................................... 4 2.1.1 Introduction ................................................................................................................. 4 2.1.2 Symptomatic Oral Herpetic Infections ....................................................................... 5 2.1.3 Asymptomatic Reactivation ........................................................................................ 9 2.1.4 Dental Considerations and Implications ................................................................... 10 2.2 Therapy Management of Symptomatic Lesions ........................................................... 13 2.2.1 Antiviral Therapies ................................................................................................... 13 2.2.2 Alternative Therapies ................................................................................................ 16 2.2.3 Lesion Barriers .......................................................................................................... 18 2.2.4 Cyanoacrylate Adhesives (CA)................................................................................. 20 viii 2.2.4.1 Structure and Properties of Cyanoacrylates ...................................................... 20 2.2.4.2 Medical and Therapeutic Indications ................................................................ 24 2.2.4.3 Application and Limitations ............................................................................. 25 2.2.4.4 Summary ........................................................................................................... 26 2.3 Detection and Management of Asymptomatic Reaction .............................................. 27 2.3.1 Testing Methods........................................................................................................ 27 2.3.1.1 Viral Culture ..................................................................................................... 27 2.3.1.2 Serum Antigens/Antibodies .............................................................................. 28 2.3.1.3 Nucleic Acid Amplification Test ...................................................................... 29 Chapter 3: Rationale, Hypotheses, and Objectives...................................................................30 3.1 Rationale ....................................................................................................................... 30 3.2 Hypotheses .................................................................................................................... 32 3.3 Objectives ..................................................................................................................... 32 3.3.1 Primary endpoints: .................................................................................................... 33 3.3.2 Secondary endpoints: ................................................................................................ 33 Chapter 4: Materials and Methods ............................................................................................35 4.1 Data Collection Materials and Methods ....................................................................... 35 4.1.1 Study forms ............................................................................................................... 35 4.1.2 Equipment ................................................................................................................. 36 4.1.2.1 PCR Methods .................................................................................................... 38 4.2 Research Methods ......................................................................................................... 39 4.2.1 Study Design ............................................................................................................. 39 ix 4.2.1.1 Sample Size ....................................................................................................... 39 4.2.2 Blinding..................................................................................................................... 39 4.2.3 Study Enrolment ....................................................................................................... 40 4.2.3.1 Inclusion Criteria .............................................................................................. 40 4.2.3.2 Exclusion Criteria ............................................................................................. 40 4.2.3.3 Asymptomatic Arm (A1, and A2) Participants................................................. 41 4.2.3.4 Symptomatic Arm (Arm S) Participants ........................................................... 42 4.2.4 Study Visits Schedule and Procedures ...................................................................... 42 4.2.4.1 Visit 0: Baseline data collection (30 minutes) .................................................. 44 4.2.4.2 Visit 1: Lesion data collections and PA placement (30 minutes) ..................... 44 4.2.4.3 Visit 2: PA follow-up (15 minutes) .................................................................. 45 4.2.4.4 Visit 3: Healing stage follow-up (15 minutes) .................................................. 46 4.2.5 Data Management ..................................................................................................... 47 4.2.6 Data Analysis ............................................................................................................ 48 4.2.6.1 Statistical Tests Selected ................................................................................... 48 Chapter 5: Results........................................................................................................................51 5.1 PCR Results of Asymptomatic Reactivation and Symptomatic Recrudescence .......... 56 5.2 PA Application on Symptomatic Lesions ..................................................................... 58 5.2.1 Study Arms A2 vs. S ................................................................................................. 58 5.2.2 Variables Affecting Lesion Presentations ................................................................. 66 5.2.2.1 Correlations of Lesion Size to Ratio Parameters Measured During Visits ....... 66 5.2.2.2 Correlations of Lesion Stage to Ratio Parameters Measured During Visits ..... 72 x 5.2.2.3 Correlations to Average Reported Discomfort ................................................. 72 5.2.2.4 Correlation to Total Change in Lesion Size ...................................................... 74 5.2.2.5 Effect of Lesion Site on Lesion Ct Value and Lesion Size ............................... 76 5.2.3 PA Efficacy, Duration, Acceptability and Discomfort ............................................. 78 5.2.3.1 PA Efficacy ....................................................................................................... 78 5.2.3.2 PA Duration, Acceptability and Discomfort ..................................................... 81 5.2.3.3 Daily Journal Comments................................................................................... 84 Chapter 6: Discussions ................................................................................................................85 6.1 Asymptomatic Shedding in the Oral Cavity ................................................................. 85 6.2 Symptomatic Recrudescence in the Form of HLL and OHL ....................................... 88 6.2.1 Clinical Lesion Parameters (HLL) ............................................................................ 90 6.2.2 PCR Ct Values of Lesions ........................................................................................ 91 6.2.3 Participant-reported Factors ...................................................................................... 92 6.3 The Acceptance and Efficacy of PA as An Adhesive Barrier ...................................... 94 6.4 Limitations, Challenges and Summary ......................................................................... 95 Chapter 7: Conclusions and Future Directions.........................................................................98 7.1 Conclusions ................................................................................................................... 98 7.2 Future Directions ........................................................................................................ 101 Bibliography ...............................................................................................................................103 Appendices ..................................................................................................................................116 Appendix A Health Canada Approval for PeriAcryl® 90 Clear ............................................ 116 Appendix B Participant Recruitment Letter ........................................................................... 117 xi Appendix C Participant Information and Consent Form ........................................................ 119 Appendix D Baseline Intake Survey ....................................................................................... 136 Appendix E Visit 1 Clinical Form .......................................................................................... 139 Appendix F Visit 2 and 3 Follow-up Clinical Form ............................................................... 142 Appendix G Daily Journal ...................................................................................................... 144 xii List of Tables Table 1 Lesion progression of HLL versus OHL. ......................................................................... 8 Table 2 Recommendation of systemic antiviral agents for HLL and OHL. ................................ 14 Table 3 Study procedures during each visit. ................................................................................ 47 Table 4 Participant study arm assignments. ................................................................................. 53 Table 5 Participants Demographics. ............................................................................................ 54 Table 6 History of HLL and OHL among participants. ............................................................... 55 Table 7 Lesion parameters comparison of Arm A2 and Arm S. ................................................. 64 Table 8 Correlations of lesion stage to other lesion parameters. ................................................. 72 Table 9 Mean ranks of Ct values of the four swabs collected in Visit 1 for PCR assay. ............ 79 Table 10 Result of Wilcoxon signed ranks test and the p-value for each pair of Ct values. ....... 79 Table 11 Descriptive statistics of PA duration, PA acceptability, and average discomfort. ....... 81 xiii List of Figures Figure 1 Picture of HLL captured in this study. ............................................................................ 3 Figure 2 HSV Structure. ................................................................................................................ 4 Figure 3 Formation of cyanoacrylate monomer through condensation. ...................................... 23 Figure 4 Formation of cyanoacrylate polymers. .......................................................................... 23 Figure 5 Photo of PeryAcryl® 90 Clear. ..................................................................................... 37 Figure 6 Study flow chart. ........................................................................................................... 43 Figure 7 Consort flow chart. ........................................................................................................ 52 Figure 8 Bar graph of participants’ HLL/OHL outbreak frequencies. ........................................ 55 Figure 9 Bar graph of participants’ previous lesion management methods................................. 56 Figure 10 Distribution of Ct value for symptomatic lesions observed. ....................................... 57 Figure 11 Duration of lesion healing between study arms A2 and S........................................... 59 Figure 12 Total number of lesions between study arms A2 and S. ............................................. 59 Figure 13 Visit 1 lesion size between study arms A2 and S. ....................................................... 60 Figure 14 Visit 1 Ct value between study arms A2 and S. .......................................................... 60 Figure 15 Total change in lesion size between study arms A2 and S. ......................................... 61 Figure 16 Average reported discomfort between study arms A2 and S. ..................................... 61 Figure 17 Length of time PA remained intact between study arms A2 and S. ............................ 62 Figure 18 Average reported PA acceptability between study arms A2 and S. ............................ 62 Figure 19 Total change in lesion size, with outliers removed. .................................................... 65 Figure 20 Duration that PA remained intact, with outliers removed. .......................................... 65 Figure 21 Correlation between lesion size and lesion day. .......................................................... 66 xiv Figure 22 Correlation between lesion size and total number of lesions. ..................................... 67 Figure 23 Correlation between lesion size and lesion Ct value. .................................................. 68 Figure 24 Correlation between lesion size and reported stress. ................................................... 69 Figure 25 Correlation between lesion size and reported fatigue. ................................................. 70 Figure 26 Correlation between lesion size and reported lesion discomfort. ................................ 71 Figure 27 Correlation between average lesion discomfort and duration of lesion healing. ........ 73 Figure 28 Correlation between average lesion discomfort and total number of lesions. ............. 73 Figure 29 Correlation between total change in lesion size and duration of lesion healing. ........ 74 Figure 30 Correlation between total change in lesion size and total number of lesions. ............. 75 Figure 31 Boxplots showing distribution of lesion Ct value for each lesion site. ....................... 76 Figure 32 Boxplots showing distribution of lesion size for each lesion site. .............................. 77 Figure 33 Correlation between PA duration and total change in lesion size. .............................. 80 Figure 34 Correlation between average PA acceptability and PA duration. ............................... 82 Figure 35 Correlation between average lesion discomfort and PA duration. .............................. 82 Figure 36 Correlation between average PA acceptability and average lesion discomfort. ......... 83 xv List of Abbreviations BCCDC: BC Centre for Disease Control CA: cyanoacrylate adhesive CDC: The Centers for Disease Control and Prevention Ct: cycle threshold FOD: Faculty of Dentistry gG: glycoprotein G HLL: herpes labialis lesions HSV: herpes simplex viruses HSV-1: herpes simplex virus 1 HSV-2: herpes simplex virus 2 Ig: immunoglobulin LASER: light amplification by stimulated emission of radiation LOD: limit of detection LS: lesion swab NAAT: nucleic acid amplification tests OHL: oral herpetic lesions OS: oral mucosal swab PA: PeriAcryl®90 Clear PCR: polymerase chain reaction PPE: personal protective equipment STM: soft tissue manipulation xvi UBC: University of British Columbia UTM: universal transport medium VAS: visual analogue scales xvii Acknowledgements Foremost I would like to express my extreme gratitude toward my supervisor Professor Penny Hatzimanolakis for her continuous support throughout my education and this project. She has provided me guidance and has been extremely patient with me. I appreciate her encouragements when I encountered difficulties, and motivated me to advocate for myself. She has inspired me to continue pursuing research and further education. I am truly honoured to have worked with such a knowledgeable and enthusiastic supervisor. I would also like to express my thanks toward my supervisory committee members Dr. Lari Häkkinen, Dr. Denise Laronde, and Dr. Eli Whitney, as well as my external examiner Dr. Vincent Lee, for their encouragement, advice, and expertise. They have contributed their time to provide countless valuable feedback from the perspectives of their various expertise, which greatly helped improve my knowledge. I would like to offer my gratitude to Glustitch Inc. for providing the cyanoacrylate adhesive, PeriAcryl®90 Clear, and supporting the research. I would also like to thank Dr. Mel Krajden for authorizing the collaboration with BC Centre for Disease Control Public Health Laboratory, and the team at BCCDC: Kingsley Gunadasa, Ron Chow, and other technical staff for your help in ensuring smooth operations. Thank you to Grace Fan and Gary Lam for allowing the samples of this study to be dropped off at the UBC Hospital Laboratory and facilitating the delivery to BCCDC. xviii Special thanks to Dr. Andrea Esteves for providing and arranging clinic space for use during the clinical trial. Last but not least, I would like to especially extend my gratitude to my family, Bill, and my friends for cheering me on throughout my education journey, while reminding me to take breaks. Thank you for being there for me; I could not have done it without you. xix Dedication To Mom, Thank you very much for everything you have done for me! You have always encouraged me to seek answers to my curiosities. Even during the most crazy and chaotic times, you have always put me and my choice in pursuing higher education first. Words cannot express the gratitude I have for your selfless support. You are a strong woman, and you will always be my role model! 1 Chapter 1: Introduction Herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2) are DNA viruses belonging to Herpesviridae family infecting humans.1 The herpes simplex viruses (HSV) are among the most prevalent infectious disease, where 60% to 95% of adults worldwide are seropositive with one or both HSV.1-3 HSV-1 has a higher prevalence, infecting 37% to 89% of the population in Canada2,4-5, causing oral lesions, mucocutaneous orofacial disease, and ocular lesions. Primary oral herpes lesions may result from HSV-2 through orogenital contact.1,4,6 The usual presentation of recrudescence in immunocompetent hosts is herpes labialis lesions (HLL) (Figure 1), with 20-40% of Canadians experiencing HLL in their lifetime, and one in five having annual incidents.4,7-9 Reactivation (or recurrence) of the virus can also present as HSV-1 oral shedding from saliva without presenting any herpetic lesions and is referenced as asymptomatic, whereas visual, oral herpetic lesions (OHL) and HLL are symptomatic recrudescence.1,3-4,10-11 A concern with OHL and HLL is virus shedding via saliva even after the lesion appears healed, thus spreading the virus and causing self-transmission or transmission to clinicians during dental treatments.7,10,12-13 Canadian dental professional regulatory bodies in accordance with the national centre for disease centre recommends not treating a patient presenting with an active lesion, and to postpone non-emergency treatment until the lesion has healed and fully scabbed over, which can take 10-14 days.14-15 However, this does not address the risk management of asymptomatic patients, nor the risks present at each stage of herpetic lesion development, including post healing. 2 Psychosocial considerations are also a concern for patients suffering from HLL or OHL.2,16 The frequency of HLL recrudescence varies among individuals depending on the host's immune status and may range from occasional to more than 12 recrudescence per year.9 Given HSV's unpredictability, patients are challenged in planning their dental visits accordingly, creating inconvenience and adverse economic effects for patients and the dental practices, respectively. Short cancellations or having treatment deferred until the lesion heals can result in loss of time and revenue for the patients, given that many must schedule time away from work and incur the cost for transportation and parking. Lost appointments in dental offices also translate to loss of production. Therefore, safely managing HLL or OHL while providing dental therapy to patients could avoid these issues. The current best practice in managing herpetic lesions is systemic antiviral medications to decrease the viral load. Topical antiviral medications as an adjunct can alleviate the discomfort and other symptoms associated with the HLL outbreak, but neither systemic nor topical antiviral decrease the patient's risk of spreading the virus during dental treatment.1,4,6 A novel approach introduces cyanoacrylate adhesives (CA) to manage the symptomatic lesions during dental treatments and to potentially act as a barrier in reducing the virus's spreading. CA are medical-grade sealant materials safely used in surgical and treatment procedures in the digestive system, circulatory system, muscular system, skeletal system, and the oral cavity.17-22 Some studies have utilized CA as a microbial sealant on open wounds or suture sites to prevent post-surgery infections and concluded that groups treated with CA had lower infection rates.21,23 Therefore, the purpose of this human pilot study is to: 1) evaluate the efficacy of CA as an 3 adhesive barrier for the symptomatic herpetic lesions; and 2) identify a quantitative difference in HSV presence in asymptomatic and symptomatic individuals through the polymerase chain reaction (PCR) analysis. Figure 1 Picture of HLL captured in this study. 4 Chapter 2: Literature Review 2.1 Herpes Simplex Virus (HSV) 2.1.1 Introduction The Herpesviridae family consists of over 170 viruses divided into three subfamilies: Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae.24 Eight of these viruses are human pathogens, including HSV-1, HSV-2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, human herpesvirus 6, human herpesvirus 7, and Kaposi’s sarcoma virus.24-25 HSV have an icosahedral-shaped capsid enclosing the double-stranded DNA virus genome (Figure 2). A protein layer called tegument surrounds the nucleocapsid and is further protected by an envelope composed of a lipid bilayer containing highly virulent glycoproteins.24-25 Both HSV-1 and HSV-2 are part of Genus Simplexvirus under the Alphaherpesvirinae subfamily, and they are neurotropic.24 HSV-1 and HSV-2 are also the two most similar viruses from the Herpesviridae family, sharing 70% of the identical genomes.25 Figure 2 HSV Structure. Glycoproteins Lipid bilayer Double-stranded DNA Icosahedral-shaped capsid Envelope Tegument 5 HSV is transmitted to susceptible individuals by direct contact with active lesions or bodily secretions of people shedding the virus asymptomatically. Primary infections will occur at the site of contact, and often first happens in childhood. The most common site for primary infection is the oral mucosa, presenting as herpetic gingivostomatitis.26 Between the two sub-types of HSV, HSV-1 was traditionally known to cause primary oral lesions and HSV-2 was known to cause primary genital lesions. However, recent studies show both HSV types infect epithelial regions at any site, via orogenital contact.1,4,6,26 Both HSV-1 and 2 invade the host nervous system, replicate, and remain latent in sensory nerve ganglia (trigeminal ganglia or the sacral ganglia) and reactivate near the site of primary exposure.1,3,6 The neurons latently infected by the virus contain HSV genomes; however, these neurons do not express viral proteins since the viral DNA are not replicated and other genomes are transcribed silently. Thus, the immune system cannot detect and eliminate the infection. Another reason the HSV genomes are preserved in these latently infected neurons is that neurons do not proliferate; therefore, HSV cannot be cured and persists through the lifetime of its host. 2.1.2 Symptomatic Oral Herpetic Infections After initial transmission, HSV replicates in the epithelial cells during an incubation period of four to six days.25 The virus then lyses host cells, triggering an inflammatory response from the host immune system, where immune cells infiltrate the surrounding lymph nodes. The inflammatory response may lead to fever, malaise, irritability, headache, and cervical lymphadenopathy in the patient, and clinically presents as an acute herpetic gingivostomatitis in the oral cavity, where the gingiva and the rest of oral mucosa are affected.27-28 Clinical presentation of herpetic vesicles and erythema also occur at the site of infection.25 HSV then 6 proceeds to infect the peripheral sensory nerves and the dorsal root ganglia. HSV uses the peripheral sensory nerve pathways to further spread to other epithelial cells of surrounding mucosal and skin, reproducing the symptomatic lesions. These vesicles eventually rupture to form painful ulcerations, which will then coalesce to form larger ulcers throughout the buccal mucosa, palate, floor of mouth, tongue, gingiva, tonsillar pillars, and pharynx.27 After the host immune response contains the primary HSV infection, the virus stays latent in the trigeminal ganglia until reactivated. Although it is still unclear what triggers reactivation, some possibilities include exposure to ultraviolet radiation, stress, fatigue, menstruation, immunosuppression, trauma, compression of the trigeminal nerve, and irritation by dental instruments.1,7-8,12,27 The literature does suggest a decrease in the frequency of recurrence after the age of 35 years.1,6 Reactivation of the virus is in the latently infected ganglia, where HSV-1 is transported down the sensory neuron axons in a retrograde manner to infect and lyse epithelial cells, which most commonly occurs as HLL. The development of HLL includes 5 stages: the prodromal, papule/vesicles, ulcer, crust, and healing stages.1-2,16 During prodromal, localized soreness and burning sensation can occur with erythema. The lesion then develops into a papule, which will further develop into fluid-filled vesicles. In the next stage, the vesicles collapse or rupture to form ulcerations.16 The fluid that escapes from these vesicles is noted to have the highest viral load and is highly infectious.10 After the ulcer stage, the lesion progresses into the crust stage, where the viral load is still considerably high, and the risk of viral spreading is still present.10-16 7 Studies identify that even after complete clinical healing on day 10-14, HSV-1 can still shed via saliva and spread.7,10,12 Another eruption in the oral cavity region is OHL, which present as intraoral vesicles and ulcerations, specifically in areas of keratinized tissue (commonly on hard palate) in non-immunocompromised patients, and is usually self-contained and visually healed within 1-2 weeks.25 The pain, until healed, may affect the patient’s eating, swallowing, oral self-care routines, and speaking.27 The lesion progression of OHL differs slightly from that of HLL (Table 1), as crusts or scabs do not form on intraoral tissue due to the moist environment of the oral cavity. In the oral mucosal wounds (moist wound) keratinocytes migrate through the fibrin clot allowing fast re-epithelialization.29 The epithelial cell migration process starts with the release of cytokines, growth factors and other bioactive proteins from blood clot, damaged cells, and inflammatory cells.29 This activates epithelial cells at the wound edge and keratinocytes behind the migrating cells undergo cell proliferation.29 In skin (dry wound) keratinocytes migrate between the fibrin clot and connective tissue.29 This route is longer than in the oral mucosa.29 8 Table 1 Lesion progression of HLL versus OHL. HLL OHL - Prodromal stage - Papule/vesicles stage - Ulcer stage - Crust stage (dry wound) - Healing stage - Prodromal stage - Papule/vesicles stage - Ulcer stage - Moist wound (crust) stage - Healing stage HSV-1 has caused symptomatic lesions in other parts of the body, presenting as herpetic whitlow on fingers, herpes gladiatorum at sites of abrasion, herpes blepharitis on eyelids, herpes keratitis on the cornea of eyes, and most severely, herpes encephalitis in the central nervous system.26 Herpes keratitis causes scarring of the cornea, impairing vision and may lead to complete loss of sight. HSV-1 reactivation is unpleasant in immunocompetent patients, but being immunocompromised, such as in neonates and young children, the risk of herpes encephalitis is much higher. Herpes encephalitis is rare, with 4.3 cases of encephalitis due to any cause per 100,000 person/year in Canada30, and approximately 2000 cases per year in the United States.31 Canadian data on the incidence of encephalitis specifically due to HSV is not available, but cases related to encephalitis has a high morbidity and mortality rate; 70% of treated cases will live with significant neurological damage, while 30% will not survive one-year post-treatment.25 9 Pregnant women with active sexually transmitted genital herpes lesions caused by HSV-2 during childbirth are recommended to receive caesarean section, given the risk of neonatal herpes encephalitis and ocular keratitis.26 However, more than 90% of herpes encephalitis is caused by HSV-1.32 AK et al. proposed three routes HSV-1 infects the brain: infection through trigeminal or olfactory nerves from primary infection, infection through trigeminal or olfactory nerves from reactivation of virus at peripheral sites, and reactivation of latent in-situ HSV-1 in the central nervous system.32 Herpes encephalitis causes unilateral hemorrhagic necrosis of the temporal lobe, which can further spread contralaterally.25 This can present with signs and symptoms of irritability, altered behaviour, lethargy, sleepiness, fever, seizures, tremors, altered consciousness and neurological abnormalities.25,32 In immunocompromised adults, herpes encephalitis can have atypical or subtle signs and symptoms, therefore its diagnosis may be missed or delayed.32 2.1.3 Asymptomatic Reactivation Another reactivation pathway allowing HSV to spread is by asymptomatic shedding via mucosal tissues and bodily secretions, such as oral mucosa and tears. Reactivation triggers and mechanisms are identical to symptomatic recrudescence. In oral asymptomatic virus shedding, HSV-1 is detected more frequently than HSV-2 at a single time point, regardless of herpetic lesion history and serum antibody status (6.3% and 0.4%, respectively).11 Shedding of HSV-1 can be detected from multiple intraoral sites, including the oral buccal mucosa and gingival sulcus, via PCR even when the virus is not detectable through serology testing, albeit the reason is unknown based on current research.11,33 In the study by Knaup et al., 34% of 30 enrolled immunocompetent participants, recruited from a dental education institution, had asymptomatic reactivation of HSV, whereas 20% of all participants developed symptomatic herpetic lesions.34 10 This finding is supported by the meta-analysis by Miller et al., where they concluded that at least 70% of the population sheds HSV-1 asymptomatically in the oral cavity each month and that some individuals shed the virus more than 6 times per month.11 The finding that 20% of the population experiencing recurrent HLL annually9, further suggest higher rate of asymptomatic HSV reactivation than that of symptomatic lesions. However, it is of importance to note that many studies included in Miller et al.’s meta-analysis categorized the virus shedding detected from the oral buccal mucosa as asymptomatic, despite HLL being present at the same time; only samples collected directly at lesion site was categorized as symptomatic.11 Besides HSV-1 shedding asymptomatically, the virus can also be shed intraorally with the presence of a symptomatic lesion. The HSV-1 lesion DNA count is higher and has a longer shedding duration than when HSV-1 is only shedding asymptomatically.34-36 2.1.4 Dental Considerations and Implications According to Canadian health authorities for disease control and prevention, the management of HSV is to postpone elective non-emergency oral health treatment when patients present with symptomatic oral lesions as HSV is transmittable through contact with symptomatic lesions or infected saliva, cross-infection from dental tools or surfaces exposed to HSV, and aerosolization in the dental setting.8,14-15,37 The most common reported occupational hazard to dental professionals is herpetic whitlow, but the more severe herpes keratitis has also been reported.13,38 The case series by Browning et al. highlights the risk of HSV exposure in dental professionals via inadequate personal protective 11 equipment (PPE) use, accidental puncture with contaminated sharp instruments, and cross-infection through lack of mindfulness leading to breach of infection prevention and control.38 The risk of the dental professional-to-patient transmission of HSV is also concerning. The research in 1981 by Manzella et al. reported that 20 patients were infected with herpetic gingivostomatitis after a dental hygienist provided treatment with herpes whitlow, which the dental hygienist had contracted from treating a patient with active HLL without using gloves.13 Since the mandatory implementation of gloves during proper infection and prevention protocols, reported cases of oral herpetic infections contracted by oral health care providers have decreased since the time research by Manzella et al. was published, and illustrates the importance of proper PPE use for any infections, viral or other. The SARS-CoV-2 2019 pandemic re-emphasized the importance of comprehensive medical screening of patients and proper universal protocols. The additional requirements for splatter and aerosol-generating procedures are to include surgical mask, eye protection that covers the front and sides of the face, and a gown or protective clothing to cover personal clothing and skin of the forearms.39 HSV is infectious through direct contact and transmits through cross-contamination, and given asymptomatic shedding occurs, dental professionals must abide and stay attuned to established best practices in infection and prevention protocols. Patients with HLL or OHL infections are tasked with managing their oral health care needs, especially with the unforeseeable nature of HSV reactivations. Delaying elective treatments due to the presence of a lesion may impact their overall health, increase stress as a result of delaying 12 procedures and rescheduling appointments. Individuals residing in rural areas may have driving times of over an hour for dental appointments or have limited access to care and must adjust their working/life schedules around their dental appointments. An unknowing patient presenting with symptomatic lesion might need to be rescheduled, which will negatively impact both parties. A 2014 survey reports that approximately 10% of dental appointments are cancelled each year due to assorted reasons and that 5% of the no-show rate in the long term is estimated to equal the loss of one year's production for every 20 years.40-41 Although the nature of the assorted reasons was not defined, HSV’s high prevalence rate may impact the revenue loss of a dental clinic and patients. 13 2.2 Therapy Management of Symptomatic Lesions The pain and appearance of HLL or HOL in an immunocompetent patient are often a nuisance as the reactivations are unpredictable. Even though lesions are usually self-limiting, patients often seek various therapies to alleviate the pain and expedite the healing process.4,8,10 There is currently no treatment available to eradicate or cure HSV, therefore, only prevention and management of symptoms will be discussed.42 Antiviral medications, supplements, herbal and natural therapies, and physical barriers are some of the different methods used in managing symptomatic herpetic lesions. 2.2.1 Antiviral Therapies Antiviral medications for the management of symptomatic oral and peri-oral lesions include systemic and topical medicines. Common antiviral medications include acyclovir, famciclovir, penciclovir, and valacyclovir.42 These antiviral medications all prevent viral replications by inhibiting the DNA polymerase.42 Systemic famciclovir and valacyclovir have better absorption and therefore increased bioavailability than acyclovir in the human body, which means less frequent doses are required in symptom management of HLL.42-43 Stoopler et al. and Woo et al. suggest the following systemic antiviral regime (Table 2) for HLL and OHL8,44: 14 Table 2 Recommendation of systemic antiviral agents for HLL and OHL. Prophylaxis therapy - preventing recrudescence of oral herpetic lesions Acyclovir 400 mg 2-3x/day Valacyclovir 500-2000 mg 2x/day Abortive therapy - management of recurrent HLL at prodromal stage to prevent further lesion progression Valacyclovir 2000 mg 2x for one day at first sign of recrudescence Famciclovir 500 mg 2-3x for one day at first sign of recrudescence Active therapy - treatment of active lesions to reduce viral load Acyclovir 400 mg 3x/day for 5-7 days Valacyclovir 500-1000 mg 2x/day for 3-5 days Although there are many antiviral agents available for various purposes, these medications require prescriptions by health care professionals and may be costly when they have to be used long-term (>12 months).42 Additionally, there are differences in bioavailability of each antiviral agent, and coupled with patient compliance challenges maximum prophylactic effect.42 Immunocompromised individuals or those with persisting herpetic lesions, however, may benefit from using antiviral agents long-term to control HSV reactivations and minimize the symptoms of HLL or OHL.42 The long-term antiviral therapy demonstrated a statistically significant reduction in the recurrence of HLL or OHL, but the reduction has no meaningful clinical significance as there was a minimal extension of non-recurrent period by a few days.42 In addition, the meta-analysis performed by Chi et al. shows a lack of evidence supporting short-term use of systemic antiviral 15 medications, as there were no consistent associations reported between the dose of antiviral agent and recurrence prevention of HLL or OHL.42 On the other hand, some evidence demonstrates systemic antiviral agents’ ability to reduce the viral load of active HLL and OHL.1,4,6 Both long-term and short-term use of antiviral medications for HSV management are reported to be safe, with rare virus resistance reported in both immunocompetent and immunocompromised patients (<0.5% and 5%, respectively).45 The reduction in viral load, however, still will not allow dental treatments to be delivered safely to patients as the virus is still present in the oral and peri-oral environment. Another antiviral treatment modality is topical antiviral medicines. Common topical antiviral agents used to relieve HLL or OHL symptoms include penciclovir 1%, acyclovir 5%, and docosanol 10% creams or ointments. While the docosanol 10% is readily available over the counter in Canada, penciclovir and acyclovir topical medications require prescriptions.8 All topical antivirals require application every 2-4 hours, for up to 5 times each day from the prodromal stage to complete healing stage of lesion progression.8,42 Like their systemic counterparts, short-term use of topical antiviral medications may provide relieve from the symptoms and support healing but had no statistical or clinical significance in preventing recurrent HLL and OHL eruption.42 There is also not enough research evidence to conclude the long-term prophylaxis effect and ability to alleviate symptoms of topical medications related to herpetic lesions.42 Based on evidence from current research, the use of topical antiviral medication would not have a beneficial effect on preventing virus transmission in the dental settings. 16 2.2.2 Alternative Therapies A common method of HLL and OHL prevention is L-lysine supplements, which are easily accessible by consumers. Amino acid L-lysine has shown laboratory evidence of inhibiting replication of HSV in-vitro and animal models as it is a competitive inhibitor of arginine, an amino acid vital in the replication of HSV.46 Thus, slowing down the progression of herpes infection and decreasing the viral load.46-47 In studies with human participants, however, the L-lysine dose of 1000mg daily did not significantly promote lesion healing as measured by the rate of healing and appearance of lesions.42,47 On the contrary, higher dosage L-lysine at 1.2g daily and long-term intake, where the serum concentration of lysine exceeds the threshold of 165 nmol/mL, showed significant prophylaxis effect in decreasing recurrence rate of HLL and OHL.47-48 Therefore, many reviews have concluded that L-lysine supplements may effectively prevent HLL recurrence, but do not show evidence in alleviating signs and symptoms of erupted lesions.42,46-48 Another accessible prophylactic method to prevent HLL and OHL recurrence is the application of sunscreen. Studies have shown that ultraviolet rays from the sun are a stimulus for HSV replications.42,44 There are limited experimental studies on the efficacy of sunscreen use alone in preventing recrudescence of herpetic lesions, but the literature reviews by Woo et al. and Chi et al. concluded that sunscreen with SPF 15 and higher had shown significant efficacy in preventing recurrence of HLL when study subjects were exposed to experimental ultraviolet lights.42,44 However, it should be noted that the use of sunscreen as prophylaxis during exposure to natural sunlight showed an insignificant result. Therefore, the true efficacy of sunscreens as a prophylaxis method is inconclusive.6,42,49 17 Topical zinc oxide cream, zinc sulphate gel, and topical anesthetic solutions are other standard over-the-counter therapies advertised to alleviate the symptoms of HLL. These topical therapies do not have prophylactic properties as L-lysine or sunscreens; however, they can alleviate the associated symptoms of tingling and pain sensations at the early onset of HLL.6 An emerging HLL and OHL therapy is the use of light amplification by stimulated emission of radiation (LASER). LASERs in different wavelengths are used in some dental clinics to manage various conditions. Low-level LASER therapy (wavelength of 600-1070 nm) promotes cascade of cellular events to improve lesion healing, including ATP synthesis, extracellular matrix synthesis, and release of growth factors allowing new capillaries synthesis.50-51 In preventing and reducing recurrences of OHL and HLL, LASER increases host immune response to HSV by elevating the production of proinflammatory cytokines and mediators.50 Some literatures have theorized LASER’s ability to stabilize cellular membrane as the contributing factor for increasing pain threshold, leading to its analgesic effect.50 However, LASER did not alleviate pain associated with HLL and OHL, nor reduce the recurrence of future lesions.42 In addition, even though the LASER-treated lesions were statistically smaller in size and had less edema accumulation, the difference in size was minimal to indicate clinical significance.42 Emerging evidence is suggesting LASER assists in the proliferation of HSV in the vesicle stage, as LASER promotes nucleic acids and ATP synthesis in any cell.50 Overall, LASER treatments are not as accessible as other methods of HSV prophylaxis, and may even delay initiation of lesion healing process. Depending on the clinician’s scope of practice and National health authorities' regulations, LASER therapy is not standard care in the management of HSV lesions. 18 Other non-conventional methods of HLL and OHL prophylaxis were noted in the literature, including iontophoresis, herbal preparations of sage and rhubarb, butylated hydroxytoluene, idoxuridine, topical iodine, interferon, HSV-1 subunit vaccine, yellow fever vaccine, and hypnotherapy.42,47 Clinical trials showed efficacy in some therapies, such as iontophoresis of topical acyclovir reduced healing time, topical preparation of sage and rhubarb showed comparable healing time as topical acyclovir, idoxuridine and topical iodine reduced both pain and healing time, and hypnotherapy reduced the intensity of symptoms related to HLL and OHL.42,47 However, more research is needed to conclude the prophylactic properties of these modalities. 2.2.3 Lesion Barriers Recent studies suggest occlusive dressings can promote dry wound healing, as they prevent the lesion from drying out and allow re-epithelialization.16 Occlusive dressings are also ideal treatment for partial-thickness wounds such as HLL, where the histological damage extends past the cutaneious basement membrane into the dermis.52 Many over-the-counter HLL creams, ointments, and gels claim to form a moisture barrier that protects the lesion. However, they are not physical barriers and can be easily wiped away. This section only includes barriers in films or patch forms that cannot be removed by wiping or gentle touching. There are two types of topical therapies that are film-forming and contain active medical ingredients to manage herpetic lesion symptoms. Both therapies have also demonstrated efficacy in acting as barriers for oral mucosal ulcers. A 10% benzyl alcohol gel with hydroxypropyl cellulose, in which the hydroxypropyl cellulose forms a layer of bioadhesive over the HLL. In an 19 in-vitro trial by Rodu et al., the diluted benzyl alcohol gel showed virucidal activities against HSV-1 while maintaining cell monolayer integrity without showing cytotoxicity.53 The second topical therapy containing zinc sulphate, with addition of beta-glucan, also forms a physical barrier layer. As noted, zinc sulphate is effective in symptom alleviation of HLL and promotes wound healing. When comparing HLL treated with acyclovir 5% topical cream, to zinc sulphate with beta-glucan, the mean healing time showed statistically comparable results of 9.30-9.80 days.54 However, unlike the benzyl alcohol and hydroxypropyl cellulose gel, the zinc sulphate with beta-glucan topical therapy does not have virucidal effects.54 Hydrocolloid patches are occlusive dressings without therapeutic ingredients but are applied to HLL, acting as an HSV shield. These patches enhance wound re-epithelialization and reduce inflammation by preventing scab formation and providing the HLL with an aqueous environment.54-55 No statistical and clinical differences were observed compared to acyclovir 5% topical cream in the time required for HLL to heal.54-55 However, it was noted the margins of the hydrocolloid patches lifted easily, which causes patient dissatisfaction and requires replacement with new patches.54 In the clinical trial by Boes et al., zinc sulphate with beta-glucan is compared to hydrocolloid patches. The acceptance and satisfaction regarding the quality of therapy was rated higher in participants who received zinc sulphate and beta-glucan.54 The replacement of patches is also associated with other pain due to lesion scabs being torn off upon removal of the patch.54 In addition, unlike the topical barrier therapies with active ingredients, the hydrocolloid patches are only for extraoral applications, which limits its use to only HLL and not OHL. 20 In the dental setting, the film-forming solution of 10% benzyl alcohol gel with hydroxypropyl cellulose and a solution of zinc sulphate and beta-glucan may be useful to cover the HLL or OHL to prevent HSV transmission. However, the two solutions do not prevent the virus from spreading immediately at placement. In addition, the zinc sulphate and beta-glucan solution are currently not available in the North American nor is the evidence on hydrocolloid patches sufficient for use in dental management of HLL. In summary, varied materials demonstrate promising efficacy as barriers in the management of HLL or OHL, the evidence is limited. Therefore, further research of these materials is needed. 2.2.4 Cyanoacrylate Adhesives (CA) CA is a fibrin adhesive that has been safely administered in medicine and dentistry for many years to occlude open wounds.17-22 As evident in some research, occlusive, film-forming materials promote HLL and OHL healing.16,54 The same properties are found in CA. Therefore, the use of CA to manage the symptomatic herpetic lesion during dental treatments by acting as a barrier to potentially reduce viral transmission and improve healing, is a novel approach. 2.2.4.1 Structure and Properties of Cyanoacrylates Cyanoacrylates are organic compounds first discovered in 1949 and became recognized as an adhesive in 1951.56-57 Since then, the adhesive has gained popularity in the commercial industry as fast-drying glues in medicine and dentistry. Cyanoacrylates have a series of homologous monomers that differ in chemical structure and properties.22,56 The chemical structure of 21 cyanoacrylates differs at the alkyl side chain, where short-chain cyanoacrylates include methyl-cyanoacrylate, ethyl-cyanoacrylate, and the long-chain cyanoacrylates include isobutyl-cyanoacrylate, isohexyl-cyanoacrylate, and octyl-cyanoacrylate.56 CA is polymerized from cyanoacrylate monomers formed by condensation of formaldehyde and cyanoacetate molecules in the presence of a catalyst.56,58 The cyanoacetate forms an enolate anion and acts as a nucleophile to form a covalent bond with the carbon of the formaldehyde, which forces the same carbon to release the double bond with the -OH group to form a cyanoacrylate monomer (Figure 3).59-60 When an anion of a base (B-), or nucleophile, donates elections to the double-bonded carbons on a cyanoacrylate monomer, the polymerization process is initiated. The carbon anion formed from this process will then act as another nucleophile to break the double-bonded carbons on another monomer, forming a covalent bond and another carbon anion on the newly formed cyanoacrylate polymer.61 This means the propagation of electron transfers will occur to form a web of cyanoacrylate polymer structure (Figure 4). In clinical settings, water can act as the catalyst for the polymerization reaction as the hydroxyl ion will act as the nucleophile. Since biological tissue contains water or other base residuals, CA will polymerize rapidly over the tissue to form an occlusive film within 5-60 seconds.22,56 Even though the chemical reactions in cyanoacrylate monomer and polymer formation are similar in short and long-chain cyanoacrylates, the adhesive property and cytotoxicity differ between the two types of CA. Short-chain CA are used domestically as glues, and while short- chain CA have been used in medicine previously, recent medical-grade CA are all long-chain cyanoacrylates. Short-chain CA are more superior in bonding speed, strength, and tenacity than long-chain CA.22,62 However, evidence has shown alkyl side chain length to be directly related to 22 the degree of toxicity, which means short-chain CA is less histocompatible compared to long-chain CA.22,56,58,62 The histotoxicity might explain the reaction due to the heat release during the exothermic polymerization of CA and formaldehyde during degradation of CA polymers, which may be retained in the tissue.22,56,62 The histotoxicity presents clinically as acute and chronic inflammation, reduced healing, and tissue necrosis, and is affected by the degree of vascularity of tissues where CA is placed. 22,56,62 One study found greater incompatibility in well-vascularized soft tissues.56 Hence, by increasing the alkyl chain length, both the polymerization and degradation process slow down to release significantly less heat and fewer toxic chemicals to levels that are acceptable to biological tissues.56 23 Figure 3 Formation of cyanoacrylate monomer through condensation. Figure 4 Formation of cyanoacrylate polymers. O R O N R O O N B - R-cyanoacrylate polymer O R O N B N O R O - R-cyanoacrylate monomer R-cyanoacrylate monomer R-cyanoacrylate monomer anion Nucleophile reaction - B R O N O O H H Condensation Catalyst O N O -- R OH N O O R H H H - B O R N O H H R-cyanoacrylate Cyanoacetate enolate anion (R represents an alkyl group) Formaldehyde 24 2.2.4.2 Medical and Therapeutic Indications CA has been safely administered in medicine as sealant material for surgical procedures in the digestive system, circulatory system, respiratory system, nervous system, musculoskeletal system, skin, and craniofacial area.17-21,56 It gained approval for medical use from the United States Food and Drug Administration in 1998.56 In Canada, depending on the type of CA, it is labelled as a medical device with varying level of health risks. Class I representing lowest risk, and ranging to Class IV representing highest risk. CA have also been safely used intraorally in dentistry for gingival grafts and as alternatives to sutures post-surgery.22,56 Their primary functions include wound closure, wound repair, hemostasis, and as drug carriers.56 Studies have utilized CA as a microbial sealant to prevent post-surgery infections and concluded the groups treated with CA had fewer infections.21,23 When comparing the efficacy of short and long-chain CA, the in vitro study by Hida et al. shows that both types of CA are bacteriostatic, and the short-chain CA has higher bacteriostatic activity for a wider range of bacteria species.58 Similarly, an in vitro study by Romero et al. demonstrates comparable results where the short-chain CA had increased bactericidal effect, especially in actively polymerizing short-chain CA.63 However, the in vitro study by Rushbrook et al. examining long-chain CA alone suggests it has a bactericidal mechanism against Gram-positive bacteria after testing through swab cultures.64 The antibacterial property of CA is due to its occlusive property to prevent foreign material and organism entry to the surgical site, as well as the strong electronegative charge on its polymers.22,65 The electronegative charge affects the gram-positive bacteria, as their cell walls have a positive charge.65 CA’s occlusive and barrier-forming properties also allow it to be an effective hemostatic compound. The presence of a mechanical barrier encourages coagulation of clotting factors, which prevents hemorrhage.22 In terms of tissue regeneration ability, the in vivo 25 study by Sohn et al. in mice models showed that long-chain CA allowed proper bone regeneration, whereas the short-chain CA caused bone damage.62 In soft tissue, long-chain CA reduces the healing time of open wounds and expedites epithelial keratinization.22 Gumus et al. report preservation of the free gingival graft dimensions when long-chain CA was used in their randomized, controlled, in vivo trial.66 CA’s self-shed biodegradability provides convenience to clinicians and comfort to patients.22,66 2.2.4.3 Application and Limitations Although CA is versatile in medicine and dentistry, the safety of CA needs to be assessed. There are no carcinogenicity or allergic type I hypersensitivity, involving IgE mediated release of histamine and other mediators from mast cells and basophils, from long-chained CA.56,67 However, a type IV or delayed hypersensitivity is a cell-mediated versus antibody-mediated reaction occurring in response to contact with certain allergens and can result after days of the initial exposure.67-68 The exposures reported were from both long and short-chain CA, and the type IV hypersensitivity reactions included allergic contact dermatitis, erythematous rash or hives, pain, and swelling.56,67-68 Most hypersensitivity reactions only involve cutaneous reactions and are mild and self-limiting.56,67 Long-chain CA, as identified, is more histocompatible than the short-chain version, which explains the rationale behind a few cases of pulmonary hypersensitivity reactions reported after occupational exposure to short-chain CA.69 Even though limited evidence with only case reports of type IV hypersensitivities related to CA are available, it does appear that long-chain CA is safer for medical use.22,56,58,62 26 2.2.4.4 Summary Cyanoacrylates have been used in various dental and medical applications with the primary goals of attaining hemostasis, aiding wound healing and primary closure, support of secondary closure, stabilization of infections, and as a barrier to enable adequate healing to minimize dentine hypersensitivity. Various off-label uses of this adhesive have been successfully seen, such as retaining locally delivered antibiotics in periodontal pockets following their sub-gingival placement or controlling hemostasis at sites with inadvertent tissue damage following periodontal therapy procedures. Due to its high degree of clinical effectiveness and relative paucity safety concerns, clinical applications with HVS and more controlled studies are needed to establish its use as a standard of care practice in various settings. 27 2.3 Detection and Management of Asymptomatic Reaction Symptomatic herpetic lesions are usually visually detectable, especially in distinct stages of clinical representation, and their management is straightforward. Asymptomatic reactivation of HSV has similar triggers and virus replication mechanisms as HLL and OHL, but it would be challenging to prevent asymptomatic reactivations as compared to symptomatic recrudescence. Patients who are HSV carriers cannot determine when or if they are shedding the virus due to a lack of signs and symptoms. Without signs and symptoms, there lacks awareness of shedding and hence, no management of asymptomatic reactivation to prevent virus transmission. Currently, asymptomatic reactivations of HSV are only detected and diagnosed when professionals in experimental or clinical settings perform laboratory tests. 2.3.1 Testing Methods 2.3.1.1 Viral Culture Viral cultures, or cell cultures, have been recognized as the gold standard for detecting HSV on symptomatic lesions for the past two decades, as they are type-specific and highly specific.70-71 They are also less expensive in comparison to other diagnostic tests.72 The HSV specimen can be obtained using cotton/nylon flocked swabs or aspiration using a small gauge needle when vesicles of symptomatic lesions are present.71-72 These direct collection methods are ideal for patients as they do not inflict pain. However, viral cultures are not overly sensitive and have high frequencies of false negatives due to requirements on the quality of swabs collected before being cultured.70-72 The swabs collected have to contain a large amount of live HSV viable for replication in the culture.70 This would be difficult to obtain on healing HLL or OHL that are past the vesicle or ulcer phase, and even more so in asymptomatic reactivation detection since 28 the site with maximum virus shedding is not apparent. The HSV swabs need storage in a viral transport medium or universal transport medium (UTM) immediately, stored on ice to maintain maximum of 40°C and transported to the virology laboratory as soon as possible as HSV is highly sensitive to desiccation and heat exposure.71 Another disadvantage of viral cultures is that they take several days to weeks for the test to be complete.70 In the end, the sensitivity of viral cultures is less than 50% in ideal conditions, with the average being approximately 25%.70 2.3.1.2 Serum Antigens/Antibodies A specific yet sensitive method commonly used for HSV diagnosis is detecting HSV-specific antibodies in the serum71-72 Serum testing allows investigators to determine if a person is an HSV carrier initially.73 Serology assay can also be type-specific, where it would be able to detect the presence of HSV-specific glycoprotein G (gG), gG1 for HSV-1 and gG2 for HSV-2.71 Even though serology tests have high sensitivity and specificity in detecting the presence of antibodies, these antibodies provide limited information about the HSV infection status of a patient. The most common immunoglobulin (Ig) tested for HSV diagnostics are type-specific IgG; however, IgG will not be detected during early HSV infections.71 When an individual has a primary infection, the IgM will spike between two weeks to three months before the seroconversion to IgG production occurs. Serum testing of IgM is available; nevertheless, it cannot distinguish between IgM presence due to initial infection or reactivation and thus is not recommended in routine diagnostic tests.71 In addition, there has been evidence that asymptomatic reactivation diagnosed with other HSV tests showed seronegative results.11,34,72 For that reason, serum antibodies help detect the history of HSV infections but may not be sensitive enough to detect asymptomatic reactivations. 29 2.3.1.3 Nucleic Acid Amplification Test Nucleic acid amplification tests (NAAT) have gained popularity in recent years as growing evidence suggests higher sensitivity and specificity in detecting HSV DNA with less sample collection and transportation requirements than viral cultures. Strick et al. suggest PCR, the most common type of NAAT, to be the new gold standard of HSV diagnostic assay.70 All NAAT can detect HSV DNA by amplifying the genome from the sample collected and quantify the result by presenting the copies of DNA per millilitre of sample or cycle threshold (Ct) values.71 Ct is the number of cycles required for the fluorescence signal to exceed the background fluorescence set as the threshold.74-75 The limit of detection (LOD) for a PCR is usually set to 40 cycles (Ct = 40) of gene amplifications before the result is deemed negative or undetectable.76 Various types of NAAT are available, including end-point PCR and real-time PCR, but they are all based on the procedures of end-point PCR.71 The sample collection materials are similar to that of viral cultures, but for PCR, the sample does not need to contain live HSV and can be from different anatomical locations.70 Given that only the genetic material is tested in PCR, the temperature requirement during transportation to the laboratory is lenient. PCR detects and diagnoses HSV more accurately, as samples collected from healing symptomatic lesions or mucosal tissue with asymptomatic reactivation can be detected without compromise. In the study by Ramchandani et al., asymptomatic shedding of HSV-1 in tears, nasal, and oral mucosa are 3.3 times more likely to be detected by PCR than by viral culture.77 PCR sensitivity in detecting HSV-1 ranges from 96.7%-100% and specificity has been found to be 95.1-99.4%.71 In conclusion, PCR is a well-rounded diagnostic test for both symptomatic herpetic lesions and asymptomatic reactivations. 30 Chapter 3: Rationale, Hypotheses, and Objectives 3.1 Rationale With 37% to 89% of the Canadian adult population infected with HSV-1, oral health care providers will inevitably encounter HSV-positive cases.2,4-5 These cases will be predominately HLL in immunocompetent patients during symptomatic recurrences and as OHL in immunocompromised ones.25 The British Columbian oral health regulatory bodies, College of Dental Hygienists of British Columbia, and College of Dental Surgeons of British Columbia, govern their members and advise against providing elective therapy to patients with active symptomatic herpetic lesions. However, the advice does not address the 70% of the Canadian population who shed HSV-1 asymptomatically in the oral cavity each month11, nor that asymptomatic reactivations of HSV are more frequent than symptomatic lesions.8,14-15,37 The lack of support or guidance for an undetectable and frequent asymptomatic reactivation may pose a risk of viral contamination and transmission within the dental setting. This human pilot clinical trial aims to improve our understanding of the magnitude of asymptomatic HSV reactivations and compare the virus shed during asymptomatic reactivations to symptomatic recurrences. The detection of viral shedding during asymptomatic reactivations uses various laboratory diagnostic tests, including viral cultures, serum antibodies, and NAAT. Given that NAAT, or PCR, is the less invasive test with the highest sensitivity and specificity in detecting active HSV shedding despite the anatomical location of samples collected, type-specific quantitative PCR was selected. This study aimed to determine the Ct value of 31 asymptomatic reactivation compared to symptomatic lesions by collecting oral mucosal swab samples from participants once per month for three months and tested for HSV DNA using PCR. Providing oral health therapy to patients with symptomatic HLL or OHL is unavoidable when patients seek emergency treatments; however, high transmission risks to both parties are evident without the proper management of the herpetic lesions.13,38 Patients needing elective oral health care and challenged with access to care, demographics, socioeconomic issues bring forth inconveniences and potential hardships. Thus, a solution to avoid delays in both emergency and elective oral health care would provide better patient-centred care. The current standard of using antiviral medicine to control the viral load of active herpetic lesions does not address the lack of evidence to support their efficacy to reduce the viral load to a safe level for dental treatment, which means the risk of transmission cannot be dismissed.1,4,6 Other treatment modalities for symptomatic recrudescence, primarily for HLL, are available as occlusive barriers, but not all are recommended for intraoral use.53-55 All treatments available to consumers are not suitable as a barrier in the dental setting due to their mobility and ease of removal. Therefore, it is imperative to search for an occlusive barrier that is safe to use intra and extra-orally, reduces viral transmission, and stays intact during a dental procedure. Literature has demonstrated CA as a safe, barrier-forming medical device that provides occlusive properties and promotes tissue regeneration in open wounds. Currently, CA is not applied on lesions with viral origins, but its properties are analogous to the film-forming barriers currently used on HLL and OHL. Therefore, as a novel method to consider therapy with HLL and OHL lesion and potentially limit virus transmission, this study tested the application of CA on HLL 32 and OHL sites. The CA used in this study is n-butyl and 2-octyl cyanoacrylate combination, PeriAcryl®90 Clear (PA) (GluStitch Inc., British Columbia, Canada). PA has Health Canada approval as a Class I medical device under the Medical Devices Regulations (Appendix A) and is approved for intraoral use. This formulation, PA, was tested in clinical trials for extraction socket healing and as an adjunct for non-surgical periodontal debridement.78-79 PA self-sheds within 5-7 days, has a simple and atraumatic application, and has a rapid set time, expedited by saliva or water. Hence, this pilot study tested the safety and efficacy of PA as an adhesive barrier when simulating dental management with participants presenting with HLL or OHL. 3.2 Hypotheses The PCR Ct value for HSV-1 and HSV-2 will be similar for asymptomatic reactivations and those of symptomatic lesions, and that the application of PA to OHL or HLL will reduce HSV shedding and spreading during elective dental procedures. 3.3 Objectives The purpose of this study is two-fold: 1) To determine the mean Ct value of viral shedding during asymptomatic HSV reactivation as compared to symptomatic OHL or HLL; and 2) To determine the efficacy of PA as an adhesive barrier in managing dental patients who present with either HLL or OHL, by limiting the HSV viral shedding and spreading. 33 3.3.1 Primary endpoints: - To determine the HSV PCR Ct value of asymptomatic HSV in oral shedding from the oral buccal mucosa. - To determine the HSV PCR Ct value of symptomatic HSV in oral shedding and transmission. - To determine the HSV PCR Ct value in HLL and OHL after the application of PA via PCR assay. 3.3.2 Secondary endpoints: - Determine the parameters of OHL and HLL over 3 weeks (approximately 20 days): o Lesion size o Lesion site o Lesion stage o Reported stress o Reported fatigue o Reported discomfort o Duration of lesion healing o Total number of lesions present - Determine the acceptability and discomfort of PA placement on symptomatic lesions using visual analogue scales (VAS). o Length of time PA was intact o Acceptability of PA 34 - Determine any side effects of the PA placement on OHL or HLL. 35 Chapter 4: Materials and Methods 4.1 Data Collection Materials and Methods PPE included gloves, level-3 surgical masks, eye protection, and face shield* were donned by the examiner as the first step of any visit. *Added during the SARS-CoV-2 pandemic after March 2020. 4.1.1 Study forms The participants completed the study forms in sequence prior to lesion assessment or sample collection: - Discussed the study with eligible participants and answered any questions or concerns prior to the participants reading and signing the Informed Consent Form (Appendix C). - The participants’ demographics, medical and HSV history information were collected on the Baseline Intake Survey (Appendix D). The examiner then collected lesions data on the following study forms: - During initial lesion assessment (Visit 1), lesion information was recorded on Visit 1 Clinical Form (Appendix E). - During lesion follow-up visits, (Visits 2 and 3), lesion information was recorded on Visit 2 and 3 Follow-up Clinical Form (Appendix F). - All participants that developed symptomatic lesions were given Daily Journal (Appendix G) for participants to record their VAS. 36 4.1.2 Equipment 1) Size 4 metal dental mirror and 2x2 gauze A dental mirror and gauze were used to examine the peri-oral area and the oral cavity to identify any HLL or OHL present. If tissue manipulation was indicated for the appointment, dental mirror and investigator’s gloved fingers were used to stretch and retract the participants’ cheeks and lips to simulate a dental examination. 2) Marquis probe The size of each lesion identified was measured using a Marquis probe and recorded to the nearest millimeter with visual examination. 3) Single-packaged PeriAcryl®90 Clear (manufactured by GluStitch Inc., British Columbia, Canada), single-use cups, and cotton-tipped applicators PA in a clear solution was used following manufacturer directions. A micropipette (Figure 5) facilitated infection prevention and control. PA was dispensed from the micropipette in minimal amounts, enough to cover approximately 3mm beyond the lesion margin to ensure a good seal. To expedite PA’s polymerization and drying process, water was transported in single-use cups, and applied with cotton-tipped applicators. Any excess PA that flowed away from the lesion area was immediately removed using dry gauze. Set time was between 40-60 seconds for each layer. The same process was repeated for an additional layer of PA after the base layer was set, resulting in a total of two PA layered seal. 37 Figure 5 Photo of PeryAcryl® 90 Clear. 4) 12MP, ƒ/1.8 camera All lesion photos were collected using 12MP, ƒ/1.8 camera on an iPhone 8 for documentation purposes. Photos were taken with participants’ consent. Each photo was captured with the bridge of nose and chin as the borders, ensuring calibration yet the photo did not contain identifiable markers. 5) Viral collection kit: AlereTM cotton-tipped swab and UTMTM viral transport media (YY3C004N) The viral collection kit was used to collect the swab samples for HSV PCR testing. For the lesion swabs, the post-PA swabs, and the post soft tissue manipulation (STM) swabs, the cotton-tip was directly placed on the lesion or the PA site and rolled five times, so that all surfaces of the cotton-tip contacted the site. 38 For the oral buccal mucosal swabs, all the samples collected from both the left and right buccal mucosa: the left buccal mucosa was first swabbed with left-half of the cotton-tip with five up-down strokes, followed with the right buccal mucosa swab with the right-half of the cotton-tip using the same method and number of strokes. 4.1.2.1 PCR Methods All HSV viral samples collected were sent to BC Centre for Disease Control (BCCDC) Public Health Laboratory for PCR testing. The BCCDC used a lab-developed assay with an ABI 7500 real-time PCR instrument. The primers used included:  HSV-1 o F CGGCCGTGTGACACTATCG o R CTCGTAAAATGGCCCCTCC  HSV-2 o F CGCTCTCGTAAATGCTTCCCT o R TCTACCCACAACAGACCCACG Probes used included:  HSV-1P 6-FAM™ IBFQ CCATACCGA /ZEN/ CCACACCGACGAACC -IBFQ  HSV-2P MAX™ IBFQ MAX- CGCGGAGAC /ZEN/ ATTCGAGTACCA GATCG- IBFQ 39 4.2 Research Methods 4.2.1 Study Design The study was a pilot, in vivo clinical trial conducted in the Faculty of Dentistry (FOD) at the University of British Columbia (UBC). The ethical approval was obtained from the Clinical Research Ethics Board at UBC for clinical trial involving human subjects with certificate number H18-03266. 4.2.1.1 Sample Size The aim was to have data collected from 15 HLL or OHL and 15 HSV asymptomatic participants. Due to lack of similar clinical trial performed on the research questions explored by this study, no sample size calculation could be performed. Instead, this sample size was determined based on the recommendation of 12 participants for each study arm in medical and pharmaceutical pilot studies by van Belle and Julious.80-81 In the end, data on 11 HLL or OHL and 17 asymptomatic participants were collected for the study. 4.2.2 Blinding The unblinded investigating student (YL) collected all the clinical data from all participants and assigned the study arms based on the PCR results of their baseline swabs. YL was blinded from all the PCR results of symptomatic lesions until completion of the clinical trial to prevent performance bias. 40 4.2.3 Study Enrolment An invitation to participate in the pilot study was sent to all faculty, staff, and students in the FOD at UBC (Appendix B). Eligible participants were assigned to either the asymptomatic arm or the symptomatic arm. 4.2.3.1 Inclusion Criteria - Participants can give informed consent and are 18 years of age or older. - Participants can communicate, read, and write in English. - Participants are generally healthy with ASA classifications I & II.82 - Participants are willing to complete the daily journals post-application of PA and take part in follow-up visits. 4.2.3.2 Exclusion Criteria - Participants with a negative PCR result or Ct value of zero, and no history of symptomatic lesions. - Participants with significant health history or condition where the investigator identifies him/her/them to be immunocompromised, such as AIDS, active cancer, uncontrolled diabetes, malnutrition, and genetic disorders that affect the immune system.83 - Participants with an unhealthy lifestyle inducing chronic inflammatory responses in the body, such as tobacco use, heavy alcohol use, unhealthy diet, obesogenic behaviours.84-85 - Participants who are pregnant. - Participants who have known allergies to CA. 41 - Participants with asthma or other respiratory disorder or distress. - Participants with oral lesions not caused by HSV, such as herpetiform aphthae, herpes zoster, erythema multiforme, traumatic ulcers, and vesiculobullous disease.28 - Participants taking systemic or topical antiviral medication. - Participants taking prescribed anti-inflammatory or immunosuppression medication, including corticosteroids, cytotoxic drugs, cyclosporine, tacrolimus and sirolimus.86 4.2.3.3 Asymptomatic Arm (A1, and A2) Participants Participants were assigned to the asymptomatic arm if at screening/baseline did not visually present with HLL or OHL. Participants in this arm were subdivided according to the PCR Ct value used to detect HSV DNA, history of past lesions, and the onset of HLL or OHL in the duration of the study. A negative PCR result at screening (Visit 0) and the participant’s certainty of having no history of HLL or OHL, led to the participant being excluded from the study and thus no study arm assigned. Definition of Arm A1 Participants: If PCR result was 1. Positive; 2. Negative, but the participant previously had HLL or OHL; or 42 3. Negative and uncertain regarding his/her/their history of HLL or OHL, they were swabbed from the oral buccal mucosa once a month for three months to monitor for asymptomatic reactivations. Definition of Arm A2 Participants: If the participants in the asymptomatic group (A1) developed HLL or OHL. 4.2.3.4 Symptomatic Arm (Arm S) Participants Definition of Arm S Participants: Participants were assigned to the symptomatic arm if they presented with a visible HLL or OHL at the screening visit (Visit 0). 4.2.4 Study Visits Schedule and Procedures The study comprised of 4 visits. Please refer to Figure 6 for the visits in each study arm, while Table 3 describes the detailed procedures for each visit. During recruitment, the participants were instructed to notify the examiner when they felt a OHL or HLL developing. The goal was to assess each participant within the first 4 days of a lesion development; however, with restrictions and limitations, participants at all lesion stages were examined, and swab samples were collected. 43 Figure 6 Study flow chart. Baseline and oral mucous membrane swab collectionVisit 0Asymptomatic Arm(no lesion present)PCR negative; no Hx of HSVExclude from study1) PCR positive; or2) PCR negative & known Hx of HSV; or3) PCR negative & unknown Hx of HSVMonitor monthly (oral mucous membrane swab collection)Lesion developsBaseline data, oral mucous membrane swab, PA placementVisit 13-4 days post visit 1 follow-up, oral mucous membrane swabVisit 214 days post visit 2 healing stage follow-up, oral mucous membrane swabVisit 3Symptomatic Arm(lesion present)Baseline data, oral mucous membrane swab, PA placementVisit 13-4 days post visit 1 follow-up, oral mucous membrane swabVisit 214 days post visit 2 healing stage follow-up, oral mucous membrane swabVisit 3Arm A1 Arm S Arm A2 44 4.2.4.1 Visit 0: Baseline data collection (30 minutes) A 30-minute appointment was provided for eligible participants on a walk-in basis. After reviewing the research protocol with participants, they were allowed to review any questions about the study. Informed consent was obtained from willing participants. Baseline data were collected, followed by extraoral and intraoral examinations for the visual presence of OHL or HLL. All findings were recorded using Visit 1 Clinical Form (Appendix E). Baseline HSV oral buccal mucosa swabs were collected to evaluate the baseline Ct value and assess if asymptomatic viral shedding was present. The swab samples were collected from both the left and right buccal mucosa: the left buccal mucosa was first swabbed with left-half of the cotton-tip with five up-down strokes, then the right buccal mucosa was swabbed with the right-half of the cotton-tip using the same method and number of strokes. All viral swab samples collected were then sent to BCCDC for PCR testing. The PCR results from this visit determined the participants’ assigned study arms: asymptomatic (A1 or A2) or symptomatic (S). 4.2.4.2 Visit 1: Lesion data collections and PA placement (30 minutes) The participants presenting with symptomatic OHL or HLL during the screening at Visit 0 and after consent were asked to stay for the additional 30 minutes to collect further data for Visit 1. Visit 1 immediately followed Visit 0 on the same day if participants presented with a visual HLL or OHL. Step 1) The extraoral and intraoral findings of the participants were updated from visit 0, and further information about the OHL or HLL was recorded using Appendix E. 45 Step 2) The first HSV swab sample was collected was from the presenting lesion/s. The cotton-tip of the sample collection kit was directly placed on the and rolled five times, so that all surfaces of the cotton-tip contacted the lesion. Step 3) The second swab sample was collected intraorally from the oral buccal mucosa, using the same method as in Visit 0, to determine the HSV DNA present. Step 4) The PA was placed on the entire OHL or HLL and allowed to dried completely and harden. Step 5) The third HSV swab collected was the PA and along the PA margins by rotating the cotton-tip of the sample collection kit five times to ensure all surfaces of PA was swabbed. Step 6) STM was performed to simulate dental treatment by stretching the participants’ lips and cheeks. Step 7) The final HSV swab on the PA/lesions post-STM, using the same method as the third post-PA swab, to determine HSV transmission. Step 8) Participants were provided with a journal (Appendix G) to complete daily during lesion healing until their Visit 3 follow-up appointments. To ensure participants can correctly self-identify the lesion stage, the presentation of lesion in each lesion stage was explained and participants were given opportunities to ask questions and clarifications. 4.2.4.3 Visit 2: PA follow-up (15 minutes) The first follow-up was approximately 3-4 days after Visit 1. Participants were reminded of their follow-up date via their preferred method of contact, via email or text. 46 Step 1) the OHL or HLL were swabbed using the same method as in Visit 1, then the lesion size was assessed and recorded along with stage of healing in Visit 2 and 3 Follow-up Clinical Form (Appendix F). Step 2) Collected oral buccal mucosa swab using method described in Visit 0. Step 3) The presence or absence of PA on the lesion was noted. Step 4) Remind to participants to continue entering their daily findings in the daily journal (Appendix G). 4.2.4.4 Visit 3: Healing stage follow-up (15 minutes) The second follow-up took place between 14-21 days after Visit 2. Participants were reminded of their follow-up date via their preferred method of contact, bring the completed daily journals to the appointment, or electronically submit via email. During this final follow-up visit, the OHL or HLL was swabbed and assessed as repeated in Visit 2: Step 1) the OHL or HLL were swabbed using the same method as in Visit 1, then the lesion size was assessed and recorded along with stage of healing in Appendix F. Step 2) Collected oral buccal mucosa swab. Step 3) The presence or absence of PA on the lesion was noted. All daily journals were collected at the end of the appointment or confirmed from electronic submissions. 47 Table 3 Study procedures during each visit. Procedures Visit 0 Visit 1 Visit 2 Visit 3 A S A S A S A S Arm A1 Arm A2 Arm S Arm A2 Arm S Arm A2 Arm S Arm A2 Arm S Obtain informed consent    Obtain medical and dental history          Baseline Intake Survey (Appendix D)    Oral buccal mucosa swab for PCR          Follow up for consecutive three months  Visit 1 Clinical Form (Appendix E)   HLL or OHL swab for PCR       Placement of PA   Post PA application swab   STM   Post STM swab   Give participant Daily Journal (Appendix G)   Visit 2 & 3 Clinical Form (Appendix F)     Daily Journal collection   A: Asymptomatic (no visual lesion) S: Symptomatic (visual lesion) 4.2.5 Data Management The investigators assigned unique anonymized identifiers specific to this study to each participant. All data were entered, analyzed, and reported using these unique anonymized study identifiers only. The list of participants' names and study identifiers, and other paper-based data were kept in the secured, locked research office of the principal investigator and her delegates within the UBC FOD Department of Oral Biological and Medical Sciences. The electronic data were housed on a secure server with the BCCDC's laboratory result reporting platform, 48 Excelleris, which can only be accessed on authorized computers with electronic certifications and logged in using user name and password only known to the primary investigator (PH) and the YL. 4.2.6 Data Analysis Data analysis was completed with IBM® SPSS statistics 27. Given that the data collected for the study does not meet the sample size requirement of n ≥ 30 for the Central Limit Theorem, nonparametric statistical analyses were performed. 4.2.6.1 Statistical Tests Selected The HSV Ct values from the PCR assays were quantitative ratio data, with absolute zero. Thus, to compare the Ct value between the asymptomatic reactivation and symptomatic recrudescence, a Mann-Whitney U test would be used. In determining PA's efficacy in preventing the shedding and spreading of HSV, the differences in means of Ct values from the swabs collected in Visit 1 were determined using Friedman’s ANOVA. Since this test was significant (p = 0.023), a Wilcoxon signed-rank test was indicated and performed based on a 5% significant value and 95% confidence interval. The parameters measured from the OHL and HLL from participants of arms A2 and S contained both numerical and categorical data. The numerical ratio parameters included maximum lesion size (mm2), the total number of lesions present, PCR Ct value, reported stress, reported fatigue, reported discomfort, duration of lesion healing (days), and change in lesion size (mm2). 49 Categorical ordinal parameter included lesion stage and categorical nominal parameter included lesion site. Other ratio data collected to evaluate PA included the length of time PA was intact (hours) and reported acceptability of PA. All self-reported data on stress, fatigue, discomfort, and acceptability of PA collected from the participants were recorded using VAS with scores ranging from 0-10. A score of 0 represented no stress, no fatigue, no discomfort, or acceptable, respectively, whereas a score of 10 represented extreme stress, extreme fatigue, worst possible pain, or not tolerable, respectively. To compare the distributions of lesion healing, numbers of lesions, maximum lesion size, lesion Ct value, total change in lesion size, average reported discomfort, length of time PA was intact, and average reported acceptability of PA between arms A2 and S, Mann-Whitney U tests were performed. At each visit, to evaluate the different effect variables had on the presentations of OHL or HLL, correlation analyses were conducted. Spearman correlation analyses were performed to determine the effects of PCR Ct value, reported stress, reported fatigue, and reported discomfort on lesion size observed at each visit. Ordinal logistic regressions were used to determine the effects these same parameters had on each visit's lesion stage. Furthermore, to determine the effects duration of lesion healing and the total number of lesions present had on reported discomfort and the total change in lesion size, Spearman correlation analyses were again selected. 50 During the clinical trial, it was observed that all participants' lesions presented at three general sites: vermillion border, lip, and philtrum. Thus, we determined the mean rank PCR Ct value and lesion size differences at each site. The comparisons of means were made through the Kruskal-Wallis tests. The acceptability and discomfort of PA placement on symptomatic lesions were assessed by Spearman analyses, and determined the effects on the length of time PA were reported to be intact on the lesions. The duration of time PA remained intact was also tested for its effect on the change in lesion size using the same statistical method. Any side effects of the PA placement were noted from the comments provided by the participants in their daily journals, and common themes were transcribed and analyzed. 51 Chapter 5: Results Between the clinical trial period of September 2019 to December 2020, 33 participants were recruited from UBC FOD; 12 were excluded because they either did not meet the inclusion criteria (n = 11) or voluntarily dropped out (n = 1). (Figure 7) Of the remaining 21 participants, 17 were assigned to the asymptomatic arm of the study (including arms A1 and A2). Five of those participants later developed HLL or OHL in the duration of the study and was assigned to study arm A2. Four participants joined the study with symptomatic lesions and were assigned to study arm S. (Table 4) 52 Figure 7 Consort flow chart. Participants (aged >/= 18 and ASA I/II health) recruited from UBC FOD n=33Exclude from study Study Arm A(Asymptomatic)Arm A1n=11Arm A2n=22Dropped outn=1Study Arm S(Symptomatic)Arm Sn=4n=18n=17n=5 53 Table 4 Participant study arm assignments. Of the remaining 21 participants, 19 (90.5%) identified as female and two were male. Participants’ age range were 18-25 years (28.6%), followed by 46-55 years (23.8%). 61.9% of the participants self-identified as Caucasians, 28.6% were Asians, 4.8% were Black or African Canadians, and 4.8% were South Asians (Table 5). Ten participants (47.6%) reported a history of HLL without a history of OHL (Table 6), and 28.6% had a lesion recurrence once a year (Figure 8). The most common lesion management method reported was the use of topical antivirals at 33.3%, with additional participants (23.8% total) reporting use of topical antivirals in conjunction with other remedies, such as systemic antiviral medications (9.5%), cold sore patches (9.5%), and lysine supplements (4.8%) (Figure 9). All participants from the asymptomatic arm had negative HSV PCR at baseline (Visit 0). 54 Table 5 Participants Demographics. Demographics n % Sex Female 19 90.5% Male 2 9.5% Age 18-25 yo 6 28.6% 26-35 yo 4 19.1% 36-45 yo 4 19.1% 46-55 yo 5 23.8% 56-65 yo 2 9.5% Race Asian 6 28.6% Black/African Canadian 1 4.8% South Asian 1 4.8% White/Caucasian 13 61.9% 55 Table 6 History of HLL and OHL among participants. History of OHL Total No Yes Unsure History of HLL No 0 (0.0%) 1 (4.8%) 0 (0.0%) 1 (4.8%) Yes 10 (47.6%) 2 (9.5%) 4 (19.1%) 16 (76.2%) Unsure 2 (9.5%) 1 (4.8%) 1 (4.8%) 4 (19.0%) Total 12 (57.1%) 4 (19.1%) 5 (23.8%) 21 (100.0%) Figure 8 Bar graph of participants’ HLL/OHL outbreak frequencies. 56 Figure 9 Bar graph of participants’ previous lesion management methods. One participant from arm A2 and one participant from arm S had two symptomatic recrudescence during the course of this study, therefore, data on 11 lesions from nine participants were collected. All 11 of these lesions received PA applications. 5.1 PCR Results of Asymptomatic Reactivation and Symptomatic Recrudescence One participant resulted with an asymptomatic reactivation (Ct value = 28.11) at second-month follow-up. The rest of 50 oral buccal mucosa swabs from 17 participants in Arm A1, including two swabs from the participant with one incidence of detectable asymptomatic reactivation, all had negative HSV results meaning they were undetectable after 40 cycles of PCR DNA amplification. 57 In participants that developed symptomatic lesions (arms A2 and S), all 11 lesions were HLL. The mean lesion HSV Ct value was 19.27 (SD = 13.07). The range was 33.03, and the median was 24.94. No outliers were detected. Figure 10 shows the distribution of lesion Ct value for all HLL lesions on a boxplot. Due to the lack of PCR Ct value data in Arm A1, we were unable to compare between the asymptomatic and symptomatic study arms to test the first hypothesis. Figure 10 Distribution of Ct value for symptomatic lesions observed. 58 5.2 PA Application on Symptomatic Lesions 5.2.1 Study Arms A2 vs. S The differences in mean ranks between lesion parameters measured from lesions of study arms A2 and S were first analyzed to evaluate the efficacy of PA on HLL. The parameters compared included duration of lesion healing, total number of lesions, lesion size at Visit 1, Ct value at Visit 1, total change in lesion size, average reported discomfort, length of time PA remained intact, and average reported PA acceptability. The distributions of each dependent variable for the two study arms are shown in Figures 11-18. It should be noted that only lesion size and Ct value measured in Visit 1 were analyzed, as they are the largest lesion size observed overall and have the highest Ct values. Change in lesion size distribution is shown in Figure 19. 59 Figure 11 Duration of lesion healing between study arms A2 and S. Figure 12 Total number of lesions between study arms A2 and S. Count Count (number of lesions) (number of lesions) 60 Figure 13 Visit 1 lesion size between study arms A2 and S. Figure 14 Visit 1 Ct value between study arms A2 and S. Count Count (number of lesions) (number of lesions) 61 Figure 15 Total change in lesion size between study arms A2 and S. Figure 16 Average reported discomfort between study arms A2 and S. Count Count (number of lesions) (number of lesions) 62 Figure 17 Length of time PA remained intact between study arms A2 and S. Figure 18 Average reported PA acceptability between study arms A2 and S. Count Count (number of lesions) (number of lesions) 63 Table 7 shows the results of lesion parameters for arms A2 and S. There was no difference in the mean duration of lesion healing time (p=1.00), mean number of lesions (p=0.25), mean lesion size (p=0.79), mean lesion HSV Ct value (p=0.79), mean change in lesion size (p=0.35), mean reported discomfort average (p=0.13), mean duration of time PA was intact (p=0.71), and mean reported PA acceptance average (p=0.33) between study arms A2 and S. Due to similarities in the lesion data between the two groups, they were collated for each parameter and then graphed to identify any outliers using boxplots. In the collated data, outliers identified for the total change in lesion size and length of time PA remained intact. For the change in lesion size, the interquartile range (IQR) was 19, and outliers identified were lesion size changes of 42 and 64 mm2. For the length of time, PA was intact, the IQR was 76, and three outliers were identified at 96, 192, and 264 hours. The adjusted distributions, with the outliers removed, are shown in Figure 19 for the change in lesion size and Figure 20 for the PA duration. Outliers are also removed for the inferential analyses from here on forward. 64 Table 7 Lesion parameters comparison of Arm A2 and Arm S. Parameters Arm A2 Arm S p value Mean (SD) Median Range Mean (SD) Median Range Duration of lesion healing 12.67 (4.50) 11.00 12.00 12.00 (2.24) 12.00 6.00 1.00 Total number of lesions 1.33 (0.82) 1.00 2.00 2.40 (1.34) 3.00 3.00 0.25 Lesion size 27.83 (22.46) 22.50 63.00 34.40 (34.99) 20.00 77.00 0.79 Lesion HSV Ct value 18.90 (14.87) 25.66 32.34 19.72 (12.27) 22.37 33.03 0.79 Total change in lesion size 18.83 (13.26) 16.00 37.00 21.40 (24.86) 15.00 62.00 0.35 Average reported discomfort 1.66 (1.24) 1.60 3.11 0.56 (0.58) 0.50 1.18 0.13 Length of time PA was intact 37.33 (32.29) 27.00 94.00 106.00 (115.43) 50.00 260.00 0.71 Average reported PA acceptability 2.17 (1.72) 2.00 4.00 0.95 (1.01) 1.00 2.50 0.33 65 Figure 19 Total change in lesion size, with outliers removed. Figure 20 Duration that PA remained intact, with outliers removed. 66 5.2.2 Variables Affecting Lesion Presentations To understand how different variables affected the HLL presentations, some correlations were evaluated. 5.2.2.1 Correlations of Lesion Size to Ratio Parameters Measured During Visits Correlations analyses were conducted to determine the correlations of lesion size to lesion day, number of lesions, lesion Ct value, reported stress, reported fatigue, and reported lesion discomfort. The correlation between lesion size and lesion day was inconclusive, as it was not statistically significant (rs = -0.25, p = 0.16) (Figure 21). Figure 21 Correlation between lesion size and lesion day. 67 The correlation between lesion size and total number of lesions was also not statistically significant (rs = -0.30, p = 0.38) (Figure 22). Figure 22 Correlation between lesion size and total number of lesions. 68 A moderate, positive correlation between lesion size and lesion Ct value (Figure 23), was found to be statistically significant (rs = 0.35, p = 0.04). Figure 23 Correlation between lesion size and lesion Ct value. 69 Correlation between lesion size and reported stress was inconclusive, as it was not statistically significant (rs = -0.14, p = 0.43) (Figure 24). Figure 24 Correlation between lesion size and reported stress. 70 The correlation between lesion size and reported fatigue was also inconclusive, as the result was not statistically significant (rs = -0.16, p = 0.37) (Figure 25). Figure 25 Correlation between lesion size and reported fatigue. 71 The correlation between lesion size and reported lesion discomfort was not statistically significant (p = 0.14), thus the correlation was inconclusive (Figure 26). Figure 26 Correlation between lesion size and reported lesion discomfort. 72 5.2.2.2 Correlations of Lesion Stage to Ratio Parameters Measured During Visits The correlations between lesion stage and other ratio lesion parameters (Table 8), found that lesion days increased as the lesion progressed through the lesion stages, with an odds ratio of 1.47 (p < 0.001); in addition, lesion discomfort decreased as the lesion progressed through the stages towards healing, with an odds ratio of 0.63 (p < 0.03). Self-reported stress, reported fatigue, and lesion Ct value were not associated with lesion progression. Table 8 Correlations of lesion stage to other lesion parameters. Lesion Parameters Odds Ratio 95% Confidence Interval Wald χ2 p value Lesion day & lesion stage* 1.47 [1.18, 1.83] 11.79 < 0.001 Lesion discomfort & lesion stage* 0.63 [0.42, 0.95] 4.81 < 0.03 Reported stress & lesion stage 1.36 [0.89, 2.08] 1.99 0.16 Reported fatigue & lesion stage 1.28 [0.76, 2.14] 0.85 0.36 Lesion Ct value & lesion stage 0.97 [0.90, 1.05] 0.49 0.48 * Statistically significant (p < 0.05) 5.2.2.3 Correlations to Average Reported Discomfort The correlation between average reported lesion discomfort and duration of lesion healing cannot be concluded, as the p-value was not statistically significant (rs = 0.15, p = 0.66) (Figure 27). The correlation between lesion discomfort and the total number of lesions was also inconclusive, as the correlational analysis did not demonstrate statistical significance (rs = -0.18, p = 0.59) (Figure 28). 73 Figure 27 Correlation between average lesion discomfort and duration of lesion healing. Figure 28 Correlation between average lesion discomfort and total number of lesions. 74 5.2.2.4 Correlation to Total Change in Lesion Size The correlation between total change in lesion size and duration of lesion healing was not statistically significant (rs = 0.21, p = 0.58) (Figure 29). The correlation between total change in lesion size and the total number of lesions present was also not statistically significant (rs = -0.44, p = 0.24) (Figure 30). Figure 29 Correlation between total change in lesion size and duration of lesion healing. 75 Figure 30 Correlation between total change in lesion size and total number of lesions. 76 5.2.2.5 Effect of Lesion Site on Lesion Ct Value and Lesion Size During the clinical trial, it was observed that the HLL developed were concentrated to three different sites: the vermillion borders, the lips, and the philtrum. To examine whether these lesion sites effected the lesion PCR Ct value and lesion size, Kruskal-Wallis tests were utilized to assess the differences in mean ranks between each site. Lesion site was not significantly associated with lesion PCR Ct value (p = 0.34). The mean rank lesion Ct value was lowest at the vermillion border (16.44), as compared to the lip (18.65), and the philtrum (21.83) (Figure 31). Figure 31 Boxplots showing distribution of lesion Ct value for each lesion site. mean=16.44 mean=18.65 mean=21.83 0 0 22.37 77 Lesion site was associated with lesion size (p = 0.003), with lesions at the philtrum (29.83 mm2) being larger than lesions on the vermillion border (18.41 mm2), and the lip (16.50 mm2) (Figure 32). Figure 32 Boxplots showing distribution of lesion size for each lesion site. mean=18.41mm2 mean=16.50mm2 mean=29.83mm2 16 14 63 78 5.2.3 PA Efficacy, Duration, Acceptability and Discomfort 5.2.3.1 PA Efficacy The second hypothesis, the efficacy of PA on preventing the shedding and spreading of HSV, compared PCR assay results from before and after PA was applied. Analyses identified the mean ranks between the Ct value of the four swabs collected in Visit 1. The Friedman’s ANOVA showed the mean rank for lesion Ct value was 2.68, the mean rank for oral buccal mucosa Ct value was 1.77, the mean rank for Ct value after PA was applied on lesion was 2.50, and the mean rank for lesion Ct value after STM simulation post-PA was 3.05 (Table 9). This statistical analysis detected statistical significance between the Ct values for at least one of the pairs amongst these four different Ct values (χ2(3) = 8.91, p = 0.03). The Wilcoxon signed ranks test showed two pairs of Ct values with statistically significant mean ranks (Table 10). There was a statistically significant difference in the mean ranks between Ct values of the lesion and the oral buccal mucosa (p < 0.02); also, a statistically significant difference in mean ranks between Ct values of oral buccal mucosa and lesion after STM (p < 0.03). 79 Table 9 Mean ranks of Ct values of the four swabs collected in Visit 1 for PCR assay. Table 10 Result of Wilcoxon signed ranks test and the p-value for each pair of Ct values. 80 The efficacy of PA on promoting tissue healing was examined to assess the effect PA duration had on the total change in lesion size. The analysis showed the correlation between the duration of time PA stayed intact and the total change in lesion size was not statistically significant (rs = 0.17, p = 0.71) (Figure 33). Figure 33 Correlation between PA duration and total change in lesion size. 81 5.2.3.2 PA Duration, Acceptability and Discomfort The duration, acceptability and discomfort of PA were assessed. First, the descriptive statistics of these PA parameters were determined (Table 11). The mean duration of time PA stayed intact on the HLL reported by participants was 25.25 hours (SD = 17.63, n = 8 with outliers removed). The mean reported PA acceptability was 1.61 out of a total score of 10 (SD = 1.52, n = 11), and the mean reported average discomfort was 1.16 out of the total score of 10 (SD = 1.11, n = 11). Table 11 Descriptive statistics of PA duration, PA acceptability, and average discomfort. Participant Reported Parameters Mean (SD) Median Range PA Duration 25.25 (17.63) 24.00 48.00 PA Acceptability 1.61 (1.52) 1.00 4.00 Average Discomfort 1.16 (1.11) 1.14 3.40 The correlations between PA duration and average PA acceptability and average lesion discomfort were assessed. The correlation between average reported PA acceptability and PA duration was not statistically significant (rs = -0.57, p = 0.15) (Figure 34). The correlation between average reported lesion discomfort and PA duration was not statistically significant (rs = 0.28, p = 0.51) (Figure 35). 82 Figure 34 Correlation between average PA acceptability and PA duration. Figure 35 Correlation between average lesion discomfort and PA duration. 83 Correlation between average PA acceptability and average lesion discomfort were also examined, however, the correlation was also not statistically significant (rs = 0.43, p = 0.19) (Figure 36). Figure 36 Correlation between average PA acceptability and average lesion discomfort. 84 5.2.3.3 Daily Journal Comments During the duration of symptomatic lesion healing, participants were instructed to complete the daily journal (Appendix G), allowing a ranking of the acceptability of PA and the lesion discomfort they felt for the day. The daily journal also contained a comment section where the participants could report any adverse reactions or side effects they experienced. The participants were also encouraged to provide any feedback they had about the PA. Through the qualitative analysis method of coding, common themes from participants' comments were extracted. No adverse reactions or effects were reported among all participants, and no adverse reactions were clinically observed by investigators during participants’ follow-up visits. The most reported (5/11; 45%) experience by the participants was the feeling of tightness at the site of the PA application. Three (27%) of the same participants reported feeling restricted upon opening their mouth for daily activities such as smiling or yawning. It was also noted that four out of five (80%) lesions with perceived tightness were located at the vermillion border and one (20%) was on the lip. Therefore, the feeling of tightness may be due to the amount of movement required at the lesion location when opening the mouth. One (9%) participant reported that PA fluid had overflowed from the lesion site to their incisor, and another participant reported a non-painful sensation when PA was placed. 85 Chapter 6: Discussions According to the current health authority’s best practices, the recommended medical management of symptomatic HSV recrudescence are systemic antiviral medications for lesions in the oral and perioral areas and topical antiviral medications for extraoral lesions to alleviate symptoms and reduce the amount of virus shed.8,42-44) The use of antiviral medications, however, does not prevent the transmission of HSV. Given the method of transmission is through direct contact with active lesions and infected bodily secretions, current best practice guidelines advise a delay of elective dental treatment for a patient with HLL or OHL.8,14-15,37 Nevertheless, there is a dearth of guidance for clinicians regarding newly healed symptomatic lesions and the ability of asymptomatic HSV reactivation to shed through mucous membranes without presenting any clinical lesions. Both symptomatic lesions and asymptomatic reactivation have similar triggers and mechanisms. Asymptomatic patients are unaware they are actively shedding the virus, increasing the potential risk of transmission.11,33 Therefore, the pilot study's objectives were two-fold: 1) to determine the mean Ct value of viral shedding during asymptomatic HSV reactivation as compared to visible symptomatic lesions, OHL or HLL; and 2) to determine the efficacy of PA as an adhesive barrier in managing dental patients who present with either HLL or OHL, by limiting the HSV viral shedding. 6.1 Asymptomatic Shedding in the Oral Cavity In this study, PCR was adopted as the laboratory test to assess the presence of HSV and quantify the result. The gold standard for detecting virus activities had been the use of virus cultures, but newer studies have shown greater sensitivity and specificity in detecting asymptomatic HSV shedding using PCR assay.70-71,77 The quantitative results of the PCR assays allowed a 86 comparison on the amount of virus shed in asymptomatic reactivation and symptomatic recrudescence. The most current evidence comparing incidence of asymptomatic HSV reactivations to symptomatic recrudescence in the oral cavity using the same PCR method showed contradicting results. Forty-three percent out of 30 study participants experienced asymptomatic HSV reactivations, while 20% experienced symptomatic recrudescence within 58–167 days in the study by Knaup et al.34 However, results by Ramchandani et al. showed the asymptomatic shedding rate was 27.1% out of 240 non-lesion days for the eight subjects in their study compared to the lesion shedding rate of 36.4% of 11 lesion days.77 The subjects all had histories of HLL and were followed up for a total of five weeks with daily swabbing.77 The findings by Miller et al. also indicate a lower frequency of asymptomatic reactivation, which found 9.5% of 63 participants had HSV detected through PCR without presentation of OHL or HLL; whereas 20.6% of participants developed symptomatic lesions.87 The oral mucosa swab samples in this study were collected on day three/four and day seven.87 In a study that followed up their participants for 8-11.5 months, collecting samples periodically every 30-60 days, da Silva et al. report that all 25 participants, with either history of HLL or tested HSV seropositive, had tested positive at least once via PCR; specifically, 21.7% of 249 samples (n=7) were asymptomatic, and ten out of 25 participants developed HLL at least once with 24.1% of 249 samples collected were from HLL.88 In summary, most clinical trials have indicated the incidence of asymptomatic reactivations detected in the oral cavity to be lower than incidence of the HLL development. 87 The incidence comparison studies of asymptomatic and symptomatic HSV-1 shedding are advantageous in assessing the impact of asymptomatic shedding in the dental settings. Quantifying and comparing the amount of virus shed between asymptomatic reactivations and symptomatic recrudescence can contribute to a broader knowledge of inherently detrimental effects, especially with vulnerable populations. The study by Kaufman et al. examined the HSV DNA count detected from the asymptomatic reactivations by following the participant for 30 days and collecting oral buccal mucosa swabs 2x/day.89 Twenty percent out of 1020 positive samples had 10-14.9 copies/10 μL (nearly undetectable HSV viral count); 14% had 15-239.9 copies/10 μL (low HSV viral count); 34% had 240-24,000 copies/10 μL (moderate HSV viral count); 32% had >24,000 copies of HSV DNA/10 μL (high HSV viral count). Ninety-two percent of 48 subjects had shed HSV DNA at least once during the 30-day study period.89 Although the PCR result of the current study was not based on the copies of virus DNA detected, the Ct value is inversely related to the copies of virus DNA.74-75 This means that when there is a lower amount of viral HSV DNA in a sample, the PCR result may show a large Ct value approaching the LOD at 40 cycles. In this study, the swab samples for PCR were collected at the oral buccal mucosa once a month for three months to test for HSV DNA to confirm asymptomatic reactivations. Only one sample of asymptomatic reactivation was detected out of 51 samples tested from study arm A1 (asymptomatic arm). The Ct value was 28.11, and the rest of the 50 samples tested with PCR all had negative HSV results (reached LOD of 40 cycles without detection of HSV DNA). 88 Although this study could not conclude the quantity and incidence of asymptomatic HSV reactivations compared to symptomatic recrudescence using inferential analysis due to insufficient data, the available data illustrated that HSV can shed through oral buccal mucosa during asymptomatic reactivations. In comparing the available data to the mean symptomatic lesion HSV Ct value, it was observed that less amplification cycles were required in PCR to detect the HSV DNA of symptomatic lesions on average at any stage than of asymptomatic reactivation. This finding aligns with the result concluded by Ramchandani et al., Miller et al., and da Silva et al., that although asymptomatic shedding in the oral cavity was observed at a lesser frequency than symptomatic recrudescence in the oral cavity and perioral area, shedding may still occur. PCR Ct values cannot inform how severe an infection is or the likelihood of developing a lesion, but it indicates approximately how much virus was likely in the sample collected. In conjunction with this study's smaller sample size, the virus loads at the time of swab visits were not detectable, and only taking one swab sample per month for three months was not enough to quantify the frequency of asymptomatic shedding rate. Another possibility is that different samples from the same person can result in different Ct values, which may suggest the need to collect multiple samples for accuracy. 6.2 Symptomatic Recrudescence in the Form of HLL and OHL The current CDC recommendations for managing HLL or OHL during elective dental procedures addressed the risks of the symptomatic type of HSV transmissions. Nonetheless, antiviral medications will not prevent the virus from shedding via the developed lesion when 89 patients require emergency oral health treatment. In addition, the movements in a dental procedure may also break the vesicles or crack the crust formed over the HLL, which increases the risk of transmission and may also cause more pain to the patients. The novel approach of applying a safe medical-grade CA – in the form of n-butyl and 2-octyl combination (PA) – on an HLL or OHL to prevent further transmission and spreading of the HSV during a dental appointment setting, while protecting the lesion and improving comfort to patients, was assessed in this pilot study. To our knowledge, this is the first study to apply a CA as a barrier to herpetic lesions to test its efficacy and safety. In clinical trials by Boes et al. and Karlsmark et al., the effectiveness of the different types of barriers (non-CA) and other lesion therapies were assessed using a ten-point scale known as the global assessment of therapy scale (GAT), where 0 represents no response, and 10 represents an excellent response.54-55 The subjects and assessors scored using GAT to evaluate the speed of lesion healing and the quality of the therapy provided to achieve healing.54-55 The current study adopted the GAT concept and used a similar scale, the VAS, while adding other parameters to evaluate participants' stress levels, fatigue level, lesion discomfort, and PA acceptability. However, given the subjective nature of scales rated by participants, PCR testing and other HLL or HOL clinical parameters were also assessed for PA's safety, speed of lesion healing, and efficacy. 90 6.2.1 Clinical Lesion Parameters (HLL) All 11 lesions from nine participants that erupted within the duration of this study were HLL; six lesions were from study arm A2, and five were from study arm S. The clinical parameters measured from these lesions included the total number of lesions present, maximum lesion size, lesion site, lesion stage, duration of lesion healing, and total change in lesion size. No statistical differences in mean ranks were detected for these clinical parameters and participant-reported factors (stress, fatigue, pain) between study arms A2 and S; thus, the data for each parameter were combined, and outliers removed to improve accuracy in the analyses. The correlation analyses supported understanding for each clinical parameter's effect on the lesion characteristics, such as size, stage, site, and duration of lesion healing. As expected, an increase in lesion day was statistically significantly associated with an increase in the odds of a more progressed lesion stage, as lesion stage progresses over time. Other expected correlations included the decrease in lesion size as lesion progressed towards healing, and the increased duration of lesion healing time as the total change in lesion size increased. However, both correlations were not significant. Whitley et al. noted that the severity of the HSV infection is demonstrated by the size, number, and extent of the lesions.24 An interesting finding in the current study had an increased number of lesions correlating to less change in lesion size. Although the correlation was not significant, it sheds light on the possibility that the more lesions there are from one episode of HSV recurrence, the smaller the size of each lesion. The results of this study support this hypothesis, as an increased number of lesions was correlated to decreased lesion size measured at Visit 1. Based 91 on these findings, it appears the patterns are similar, which was further confirmed based on the Ct values from PCR. 6.2.2 PCR Ct Values of Lesions The distribution of PCR Ct values between study arms A2 and S were also not statistically different. Once combined for analysis of distribution, no outliers were identified and the quantity of virus shed between the HLL tested in this study were similar. This study showed a slightly negative association between the Ct value and the lesion stages. This association was statistically insignificant and was unexpected. However, since many lesion HSV samples collected in Visits 2 and 3 tested negative, the data were entered to have the value of as “0”, which decreased the overall mean of the Ct value leading to the negative correlation. Therefore, the clinical observation of an increased number of negative data points in Visits 2 and 3 should be considered instead: as the lesions were healing and progressed through the lesion stages, the chances of PCR result being negative increased. Another unexpected analysis result was that decrease in the Ct value was moderately correlated to the decrease in lesion size, with statistical significance. However, when considering the samples showing negative PCR results, it can be concluded that as lesion size decreased, there was a higher chance of PCR result being negative. In analyzing the effect lesion site had on the Ct value, no statistically significant difference was observed between the three lesion sites categorized in this study: the vermillion border, lip, and philtrum. This finding concurs with the study by da Silva et al., where they compared the rate of 92 positive swabs detected from various intraoral sites and found the following: 29.5% were collected from buccal mucosa, 30.7% from the lower lip, and 33.1% from the tongue.88 However, in the current study, the results indicated statistically significant differences in the mean ranks of lesion size observed at the three sites. Specifically, the philtrum had the largest mean size, followed by the vermillion border and lip. In combination with the possible skew in data due to the sample size of the study, this finding may have also been influenced by the ease of swab collection and the amount of pressure that can be applied during sample collection, which increased the amount of virus picked up by the sample collecting cotton-tipped swab. 6.2.3 Participant-reported Factors Participant-reported stress, fatigue, lesion discomfort, and PA acceptability were measured using VAS during follow-up visits and recorded daily in the participant daily journal. VAS has been used to assess pain and patient satisfaction alongside other measurement scales in numerous studies. The review by Chiarotto et al. concluded there was no significant advantage in using VAS compared to other measurements; on the contrary, Voutilainen et al. indicated VAS was less prone to bias and had recorded ratings higher than options available on the scale itself.90-91 The VAS scale (in Appendices E-G) used in this study had a line with ten marks that were equal width apart but did not have identification numbers to reduce bias. This allowed for quantitative VAS analyses. Some results of correlation analyses on reported discomfort aligned with the expected results: an increase in lesion size was correlated to higher reported discomfort rating; a decrease in reported lesion discomfort was significantly associated with an increased odd of more progressed lesion 93 stage. Higher reported discomfort was correlated to longer duration of lesion healing, which is not surprising as the HOL can causes pain and other discomfort through the lesion progression, until the lesion enters the healing stage. The low, negative correlation between reported discomfort and the number of lesions meant that higher discomfort was reported with fewer lesions, which was unexpected. However, when examined further by comparing to the moderate, negative correlation between the number of lesions and lesion size, the result of this correlation analysis was explained. Since the decrease in the number of lesions was correlated to larger lesion size, the increase in discomfort was explained by the increased lesion size, which was confirmed to be positively correlated. Other participant factors measured using VAS (reported stress and fatigue) resulted in correlations that were not anticipated. Increase in lesion size was correlated to lower self-reported stress, as well as lower self-reported fatigue. The result of reported stress and fatigue could be skewed due to the sample population being students, staff and faculty members who were actively studying/working when the samples were collected, therefore, their stress and fatigue levels may have stayed consistent throughout their lesion healing period. It should also be noted that the sample size was small (n = 11) for all participant-reported factors, in addition to possible self-reporting bias. Thus, with the combination of sample population and the samples size factors, meaningful conclusions on these correlations cannot be drawn. 94 6.3 The Acceptance and Efficacy of PA as An Adhesive Barrier The reported PA participant acceptability was high. The inversed VAS measurement for PA acceptability indicates lower scores as higher acceptability and higher scores indicate unacceptability. The mean reported average discomfort was 1.16 out of ten for the duration of lesion healing. The mean duration of time PA remained intact was 25.25 hours after three outliers were removed. A plausible explanation of the reported higher duration time by the three outliers may be linked to mistaking the naturally forming crust of the lesions remaining clear PA. The correlation analyses concluded that the longer duration PA stayed intact was correlated to higher PA acceptability from participants, and the longer PA duration was also correlated to increasing reported average discomfort. This finding of increased discomfort may be explained using the themes extracted from participants' comments recorded in their daily journals. The complaints of feeling tightness at the site of PA application and feeling restricted when opening mouth when smiling and yawning may have contributed to the increased discomfort rating. Interestingly, 80% of lesions that reported tightness after PA application were located at the vermillion border and 20% on the lip. This may suggest the tightness felt by participants was due to increased tension and movement required at the lesion location when opening mouth, rather than the side-effect of PA alone. On the other hand, a decrease in lesion discomfort was correlated to higher PA acceptability. The PA acceptance among participants of this study was high. The Ct values showed statistically significant differences in mean ranks between the lesion Ct value and oral buccal mucosa Ct value and between oral buccal mucosa Ct value and the post- 95 STM Ct value obtained in Visit 1. Even though the mean rank of lesion Ct value was higher than the Ct value after PA was applied and statistical significance was not observed, there was an observable increase in Ct value from PCR after PA application in those with positive results. The mean rank was lowered in the post-PA Ct value because 45.5% of the lesions had negative PCR results. The negative PCR results were interpreted with “0” for the Ct value, which affected this mean rank. When looking at the data from individual lesions, only 16.7% of lesions with detectable HSV after PA application had a lower Ct value than the lesion Ct value, with a 0.3% decrease. This percent of decrease was minute, therefore the conclusion that there was a decrease in the amount of HSV DNA detected after PA was applied could be plausible. Cyanoacrylates have found acceptance amongst clinicians due to their cost, straightforward application, and their ability to support ideal healing in tissue wounds. It is hypothesized that the adhesive forms a barrier on the wound surface, preventing any seepage of fluids, thereby providing an isolated environment for undisturbed healing, and preventing secondary infection. The superior surface of the adhesive serves as a scab, providing a moist surface which would promote faster epithelial migration and wound closure.92 6.4 Limitations, Challenges and Summary Due to this study being the first of its kind to 1) compare the quantity of both asymptomatic HSV shedding and symptomatic lesions in the oral cavity or the perioral area, and 2) evaluate the efficacy of PA as an occlusive barrier in managing HLL or OHL in the dental settings, references were not available to calculate the sample size. Therefore, the recommended sample size for medical and pharmaceutical pilot trials was determined from literature by van Belle and Julius.80- 96 81 The original aim was to have data from 30 participants to achieve 15 cases of each arm; asymptomatic reactivations through the oral buccal mucosa and symptomatic HLL or HOL; however, considering the current COVID-19 global pandemic and the nation-wide lockdown beginning March 2020, the sample collection part of this study was put on hold. This affected the number of samples available, as the sample collection was not possible when participants developed HLL or HOL during the first part of the pandemic and placement of the self-quarantine requirement. The pandemic has also extended the time required to obtain the results of PCR assays, as the BCCDC had prioritized PCR testing of the SARS-CoV-2. This restricted the number of samples sent to the laboratory, limiting the number of samples collected from each participant. Another factor leading to undetected asymptomatic reactivation may be the swab collection technique and frequency. The sole examiner, although trained, was inexperienced and may have technical challenges such as not applying enough pressure, staying consistent with location swabbed intraorally, or time spent when swabbing for samples. The study design of only one swab sample per month for three months in the asymptomatic arm might have misrepresented the reactivation frequency and Ct values. Another consideration was participants were recruited from only one faculty within one institution, which limited the participant pool. The faculty is comprised of various populations in age, gender, professionals/students, and ethnicity. The sampling method utilized was voluntary response sampling, and given the nature of HSV latency and the study's eligibility criteria, random sampling would not be possible. 97 In summary, the results of this study reinforced the crucial significance of proper PPE and ongoing evaluation of infection protocols in providing universal precaution in reducing the risk of virus transmissions. Due to the unpredictable nature of HSV reactivation, the current guidelines from the CDC on the management of HLL are still best practices, though this draws attention to the importance of comprehensive screening and questioning patients about their HSV level exposure and HLL or OHL recurrences. PCR testing, although supportive, is only an adjunct to analyzing other factors such as type of test used, history of exposure, symptoms, and individual characteristics of the patients. Questions such as, "have you ever experienced a sore/bumps in your mouth or around your lips, eyes? When was your last tingling, or HLL/OHL episode?" would allow professional judgement in determining the safety in providing oral health care therapy, given transmission is possible from a recently healed HSV lesion. The novel use of PA as a barrier has shown promising results in limiting the spreading of the virus while promoting tissue healing. However, more defined research is needed to confirm this pilot study's findings and implement them into safe practice. 98 Chapter 7: Conclusions and Future Directions 7.1 Conclusions HSV can present through two different methods, asymptomatic shedding of the virus through mucosal tissues and symptomatic recrudescence of herpetic lesions. The most common asymptomatic shedding in the orofacial area is through the oral buccal mucosa, and in comparison, the most common presentation of symptomatic recurrence in this area is HLL. HSV may also reactivate and shed through OHL within the oral cavity, although less common in immunocompetent patients. Current guidelines and literature only highlight the importance of delaying elective dental treatment when symptomatic HLL or OHL are present due to the risk of virus transmission and spread. To assess the risks of HSV transmission by asymptomatically shedding through oral buccal mucosa, its magnitude, in this study was compared to HLL. The asymptomatic reactivation rate was 2.0% in the present study with participant follow-up of the asymptomatic arm for three months. A comparison of means was not possible due to limited asymptomatic reactivation detected. However, when the PCR result of the asymptomatic reactivation cohort was detected and compared to the mean result of HLL from the symptomatic arm, the Ct value pointed to the possibility of a lower amount of HSV DNA detectable in asymptomatic virus shedding through oral buccal mucosa, than in HLL. The second objective of this study involves finding a solution to the inconveniences symptomatic recrudescence cause patients seeking oral health care who develop HLL or OHL at inopportune times. In search of an ideal barrier for HLL and OHL, the novel use of PA – a medical-grade long-chain CA safely used for intra- and extraoral use – was proposed for this study. The 99 efficacy of PA in preventing the transmission of HSV was evaluated via PCR. Statistically significant differences in mean ranks were observed between the lesion Ct value and oral buccal mucosa Ct value and between the oral buccal mucosa Ct value and the Ct value of the lesion area after STM simulation post-PA application. Further observations noted that the Ct value after PA was applied was higher than the lesion Ct value in 83.3% of cases with detectable HSV DNA and that 45.5% of all cases had undetectable viral count. These results indicated a general decrease in the number of HSV DNA detectable after PA was applied. Participants reported no adverse reactions. PA remained intact for approximately one full day. Patient acceptance of PA was high, with low reported discomfort throughout the duration of follow-up. Some reported side-effects of PA included a feeling of localized tightness and restriction when opening mouths. On the other hand, PA demonstrated lesion healing properties by decreasing the size of HLL when PA was intact for longer durations. In closing, even though the measurement of asymptomatic shedding through the oral buccal mucosa cannot be concluded due to the limited data available from the present study, the presence of one asymptomatic reaction case in a small sample size such as this study’s brings attention to the importance for clinicians to ask patients about history of HSV lesions before dental appointments. Based on this pilot study and its limitations, PA application as a medical adhesive barrier, appears as a safe option for placing on HLL lesions to support patients. Current data suggest PA is well tolerated and might assist in tissue healing, and may reduce the spreading of HSV. 100 However, future studies with a larger sample size, confirming the efficacy of PA from this study is needed and clinicians must continue with current and best practice guidelines in managing HSV patients in the dental setting. 101 7.2 Future Directions Given the pilot nature of this study and the lack of existing literature on similar objectives, sample size calculation could not be done for the current study. However, future studies can determine the appropriate samples size based on a power analysis with the data now available from this study. Larger sample size may confirm the statistical and clinical significance of this study's findings. The use of parametric statistical analysis will also be recommended if parametric distribution and sample size assumptions are met, as parametric analysis will allow direct comparison of means instead of comparison of mean ranks. When recruiting participants, expanding the target population will allow a larger pool of participants. The best sampling method would be purposive sampling, given the specific eligibility criteria to meet the study objectives. To further increase the accuracy of herpetic lesion history or observe its effect on reactivation frequencies, HSV serum antigen testing would be recommended if research funding allows. The study design of the current study was unique as the study arm assignments for the participants were based on their lesion status. Randomization of study arm/group assignments would avoid possible confounding variables. Designing future studies to be randomized controlled trials would assess the significance of PA's effectiveness in preventing HSV transmission and promoting lesion healing, and adding a comparison arm of another comparable barrier might also strengthen the results. 102 Ct values are influenced by several factors, including the PCR test kit, when the sample was collected, the machine used for testing, the technique of the health professional obtaining the sample, and the selected sampling method. As HSV reactivations are unpredictable, increasing the swab collection frequency for PCR could increase the detection of asymptomatic and symptomatic reactivations, although different samples from the same person may result in different Ct values. However, with advancements in technology, current PCR research studies examine the link between Ct values and severity of disease and infectiousness, which may lead to including participants' self-administering of PCR testing. 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WHAT: Have you ever had a cold sore (herpes simplex virus-HSV) or wondered how to manage if a lesion presented at the most inconvenient times, such as dental procedures? Ever wondered if you might have the HSV, even if you never had the signs and symptoms of a cold sore lesion? Did you know that HSV may not only reactivate on the lips, where it is most commonly found, but can also shed through the cheek and released into saliva without any symptoms? WHO: As a Masters student in the Faculty of Dentistry, I need your support to answer my research questions: “What is the proportion of HSV presented as lip sores (labialis lesions) versus those shed through the cheek (oral buccal mucosa)”; and “Will an adhesive (cyanoacrylate adhesive), similar to a clear band-aid, allow dental procedures without rescheduling”. HOW: Please help by simply participating in a quick, easy, and painless salivary swab collection. All results will remain confidential. 118 WHEN: We welcome all participants (Faculty, Staff, and Students) any time during the weekday starting September 1, 2019 to July 31, 2020. WHERE: All visits are at the Nobel Biocare Oral Health Centre (OHC) at the Faculty of Dentistry. You can participate as a walk-in or schedule a visit. Please contact lyolanda@dentistry.ubc.ca. WHY: You will be remunerated for your expenses to maximum of $50 CAD. For more detailed information, please click here. If you have any question, please feel free to email lyolanda@dentistry.ubc.ca. Thank you for supporting me with advancing research in HSV and determining ideal patient-centered care plans for dental therapy. 119 Appendix C Participant Information and Consent Form The Efficacy of Cyanoacrylate Adhesive in the Dental Management of Herpes Simplex Virus (1 & 2): A Pilot Study. Principal Investigator: Ms. Penny Hatzimanolakis, DipDH, BDSc, MSc, DHP(c) Clinical Associate Professor Department of Oral Biological & Medical Sciences University of British Columbia Tel: 778-998-0924 Co-Investigators: Ms. Yolanda Lan, BDSc, DHP(c) Graduate Student Master of Science in Craniofacial Sciences University of British Columbia Invitation You are being invited to participate in a research study to determine best practices in managing Herpes Simplex Virus (HSV-1 and 2), in a dental setting. This virus is also referred to as cold sores which can present inside or outside the mouth. You are invited to take part in this research study because you are a staff, faculty, or student in the Faculty of Dentistry at the University of British Columbia. Your participation is voluntary 120 Your participation is voluntary. You have the right to refuse to participate in this study. If you decide to participate, you may still choose to withdraw from the study at any time without any negative consequences to the medical care, education or other services to which you are entitled or are presently receiving. You should be aware that there is a difference for both you and your study investigator between being a patient and being a research participant. As a patient all medical procedures and treatments are carried out for your benefit only according to standard accepted practice. As a research participant you and your study investigator also must take into account the requirements for the research study. These may include procedures and treatments that are not part of standard practice or are not yet proven. This consent form describes the diagnostic and treatment procedures that are being carried out for research purposes. Please review the consent document carefully when deciding whether or not you wish to be part of the research and sign this consent only if you accept being a research participant. If you wish to participate in this study, you will be asked to sign this form. Please take time to read the following information carefully and to discuss it with your family, friends, and family doctor before you decide. Who is conducting this study? This study is being conducted by Clinical Associate Professor, Penny Hatzimanolakis, with the University of British Columbia in the Department of Oral Biological & Medical Sciences. All participant communication and data collection is done by Master of Craniofacial Sciences student, Yolanda Lan, with the University of British Columbia in the Faculty of Dentistry. The study uses cyanoacrylate adhesive (PeriAcryl®90 Clear) that has been provided by Blacklock Medical Products and GluStitch, Inc. as a gift in kind. 121 A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required by U.S. Law. This website will not include information that can identify you. At most, the website will include a summary of the results. You can search this website at any time. Background Cold sores (herpes labialis) and oral herpetic sores are caused by the herpes simplex virus. Herpes simplex virus is a DNA virus with two subtypes: herpes simplex virus 1 (HSV-1) and herpes simplex virus 2 (HSV-2). Both subtypes are common and infectious worldwide. HSV-1 is the most common viral strain and usually, presents as a sore (lesion) on the lip or in localized areas in the mouth. However, HSV-2 which is mostly associated with genital herpes may also cause oral (inside mouth) herpetic lesions. Cold sores and oral herpetic lesions usually heal within 10-14 days; however, during this time, they can be painful, and often bring inconvenience for patients seeking dental care. The Centers for Disease Control and Prevention protocol is to withhold non-emergency dental procedures with patients who present with a lesion, creating hurdles for both patients and practitioners in rescheduling appointments, loss of time and revenue. Recent studies suggest adhesive dressings which completely covers the cold sore lesion can prevent them from drying out, and support the healing process. Cyanoacrylate adhesive (PeriAcryl®90 Clear) in this study is a Class I device and is Health Canada approved. The use of cyanoacrylate adhesive has been used in medicine to close open wounds in diverse types of surgeries, including those in the mouth. Applying this adhesive on cold sores and oral herpetic lesions may prevent spreading of the virus, allow safe dental treatments and reduce barriers to oral care. This study is a pilot trial at UBC, and we intend to enroll 30 participants. What is the purpose of the study? 122 This is a pilot study to determine the effectiveness of PeriAcryl®90 Clear in limiting the viral shedding and spreading of the herpes simplex virus from cold sores and oral herpetic lesions in the dental setting. A “pilot study” or “feasibility study” is done to test the study plan and to find out whether enough participants will join a larger study and accept the study procedures. This type of study involves a small number of participants and so it is not expected to prove safety or effectiveness. The results may be used as a guide for larger studies, although there is no guarantee that they will be conducted. Participation in a pilot study does not mean that you will be eligible to participate in a future larger study. Knowledge gained from pilot or feasibility studies may be used to develop future studies that may benefit others. There is currently no barrier device that can be placed on herpetic oral sores or cold sores to prevent spreading of the herpes simplex virus during dental treatments. This study will help discover a potential solution for patients to receive elective dental treatment when they present with cold sores or oral herpes and help determine the safety of treating such patients for dental clinicians. Who can participate in this study? You may be eligible to participate in this study if:  You have the capability to give informed consent and are 18 years of age or older;  You are able to communicate, read and write in English;  You are generally healthy;  You are willing to complete the daily journals after application of cyanoacrylate adhesive and take part in the follow-ups;  You are willing to return for additional appointments if a cold sore or oral herpetic lesion starts to develop during the course of this research; 123  You are willing to participate in this study for maximum of 3 months (dependent on the study arm each participant will enter, see details below), plus the additional month after lesion develops. Who should not participate in this study? You will not be eligible to participate in this study if:  You have significant health conditions, where the investigator may identify you to be immunocompromised;  You smoke, use recreational drugs, or drink alcohol excessively (>15 drinks/week for men, >8 drinks/week for women);  You are pregnant, or planning to become pregnant over the course of the study;  You have known allergies to cyanoacrylate adhesive;  You are taking any antiviral medication (systemic or topical);  You are taking any prescribed anti-inflammatory medication;  Your baseline viral swab test does not contain herpes simplex virus DNA, and you do not have history of herpes lesions previously. What does the study involve? When you agree to participate in this study and sign this consent form, you agree to have your medical history reviewed. You will receive swabs inside your mouth and on the lesion (if present). The oral mucous membrane swabs and lesion swabs are painless procedures that will help investigators determine the level of herpes simplex virus present in your saliva or on the lesion. The time commitment in each arm is as follows: 124  Arm A1: total of 30 minutes  Arm A2: total of 45 minutes (over 3 months) + additional time in Arm A3 if a lesion later develops  Arm A3: total of 3 hours 10 minutes o 1 hour 30 minutes (over 20 days) + o 1 hour 40 minutes (dedicated for daily journal writing for 20 days)  Arm S: total of 3 hours 10 minutes o 1 hour 30 minutes (over 20 days) + o 1 hour 40 minutes (dedicated for daily journal writing for 20 days) What does each arm of the study entail? If you agree to take part in this study, you will be screened (Visit 0) but the remaining procedures and number of visits you will you take part in, is depend if you have a cold sore/mouth lesion (Arm A) or do not have a sore present (Arm S). Visit 0: Baseline data and swab collection (30 minutes) You will review this consent form and have your eligibility assessed. With your consent, you will have the following procedures:  Collection of your medical history and vital signs;  Complete Baseline Intake Survey;  Complete oral examination for presence of any lesion;  Collection of a baseline herpes virus swab from saliva. All virus swabs collected in this study will be sent to BC Centre of Disease Control Public Health Laboratory for testing. 125 Arm A1: If the virus swab result from Visit 0 returns as zero and you have no known history of having cold sores or oral herpetic sores, you will be excluded from the study. Arm A2: If the virus swab result from Visit 0: a) is positive; b) is zero but you have known history of having cold sores or oral herpetic sores; or c) is zero but you are uncertain about the history of having cold sores or oral herpetic sores, you will be asked to return for monthly follow-ups for three consecutive months. You will have the following procedures during each follow-up (15 minutes):  Update your medical history;  Complete oral examination for presence of any lesion;  Collection of herpes virus swab from saliva. 126 Arm A3: If you experience cold sores or oral herpetic sores at any time within the study period, you will be asked to attend additional visits. This series of visits have the same procedures as Visits 1, 2, and 3 for the symptomatic arm (Arm S), in the same sequence. Arm S: Visit 1: Herpetic lesion data, swab collection, and cyanoacrylate placement (30 minutes) When you experience cold sores or oral herpes sores, you will be asked to come for visit 2. Ideally, the investigators would like to swab the lesion within 2-4 days development; however, you are free to attend this visit as soon as you possibly can. You will have the following procedures:  Update your medical history;  Complete oral examination;  Collection of photographs on the lesion;  Collection of herpes virus swab sample from saliva;  Collection of second swab on the cold sore or oral herpes sore;  Placement of cyanoacrylate adhesive on the lesion;  Collection of the third swab along margins of cyanoacrylate placed;  Lip retraction near cold sore to stimulate dental treatment;  Collection of final HSV swab after lip retraction;  You will be given a journal to record lesion status, discomfort and cyanoacrylate adhesion status each day during the course of lesion healing until the first follow-up visit. Visit 2: Cyanoacrylate follow-up (15 minutes) 127 The study team will assess you in follow-up. You will have the following procedures:  Update your medical history;  Complete oral examination: assessment of lesion healing stage and any presentation of new lesions;  Assessment on presence of cyanoacrylate adhesive left on lesion;  Collection of photographs on the lesion;  Collection of herpes virus swab sample from saliva;  Collection of second swab on the cold sore or oral herpes sore;  Collection of your 3- or 4-day journals;  You will be asked to continue recording in the daily journal for 2 weeks until the next follow-up visit. Visit 3: Healing stage follow-up (15 minutes) The study team will assess you in follow-up. You will have the following procedures:  Update your medical history;  Complete oral examination: assessment of lesion healing stage and any presentation of new lesions;  Collection of photographs on the lesion;  Collection of herpes virus swab sample from saliva;  Collection of second swab on the cold sore or oral herpes sore;  Collection of your 2-week journals. What are my responsibilities? The participants in the study are responsible for:  Reporting to the study team if cold sores or oral herpes sores appear; 128  Notifying the study team about possible pregnancy;  Reporting any updates in health, appearance of new symptoms, side-effects and medication changes over the course of the study;  Notifying the study team about any antibiotics and anti-inflammatory medication you may use or be prescribed; and  Not purposely touching or manipulating the area where cyanoacrylate adhesive has been applied. What are the possible harms and discomforts? In rare cases, the side-effects of cyanoacrylate include skin reactions (such as allergic skin reaction) and airway reactions (such as asthma) have been reported in a different formulation of cyanoacrylate (short-chained cyanoacrylate) used in early medical research. However, there have been no reported side effects with the use of cyanoacrylate product PeriAcryl®90 Clear (long-chained cyanoacrylate) we will use in this study. What are the potential benefits of participating? You may benefit from the use of cyanoacrylate as a barrier device to prevent the spreading of herpes simplex virus. However, no one knows whether or not you will benefit from this study. There may or may not be direct benefits to you from taking part in this study. We hope that the information learned from this study can be used in the future to benefit other people with a similar disease. What are the alternatives to the study treatment? If you choose not to participate in this study or to withdraw at a later date, conventional cold sore treatments or anti-viral medications may be available to you as prescribed by your family physician. You 129 can discuss these options with your family doctor before deciding whether or not to participate in this research project. What if new information becomes available that may affect my decision to participate? If you choose to enter this study and at a later date a more effective treatment becomes available, it will be discussed with you. You will also be advised of any new information that becomes available that may affect your willingness to remain in this study. What happens if I decide to withdraw my consent to participate? You may withdraw from this study at any time without giving reasons. If you choose to enter the study and then decide to withdraw at a later time, you have the right to request the withdrawal of your information and samples collected during the study. This request will be respected to the extent possible. Please note however that there may be exceptions where the data or samples will not be able to be withdrawn for example where the data or sample is no longer identifiable (meaning it cannot be linked in any way back to your identity) or where the data has been merged with other data. If you would like to request the withdrawal of your data and samples, please let your study investigator know. Can I be asked to leave the study? If you are not able to follow the requirements of the study or for any other reason, the investigator may withdraw you from the study and will arrange for your care to continue. On receiving new information about the treatment, your research investigator might consider it to be in your best interests to withdraw you from the study without your consent if they judge that it would be better for your health. If you are asked to leave the study, the reasons for this will be explained to you and you will have the opportunity to ask questions about this decision. How will my taking part in this study be kept confidential? 130 Your confidentiality will be respected. However, research records and health or other source records identifying you may be inspected in the presence of the Investigator or his or her designate by representatives of Health Canada, and UBC Clinical Research Ethics Board for the purpose of monitoring the research. No information or records that disclose your identity will be published without your consent, nor will any information or records that disclose your identity be removed or released without your consent unless required by law. You will be assigned a unique study number as a participant in this study. This number will not include any personal information that could identify you (e.g., it will not include your Personal Health Number, SIN, or your initials, etc.). Only this number will be used on any research-related information collected about you during the course of this study, so that your identity will be kept confidential. Information that contains your identity will remain only with the Principal Investigator and/or designate. The list that matches your name to the unique study number that is used on your research-related information will not be removed or released without your consent unless required by law. Studies involving humans now routinely collect information on race and ethnic origin as well as other characteristics of individuals because these characteristics may influence how people respond to different medications. Providing information on your race or ethnic origin is voluntary. Your rights to privacy are legally protected by federal and provincial laws that require safeguards to ensure that your privacy is respected. You also have the legal right of access to the information about you that has been provided to the sponsor and, if need be, an opportunity to correct any errors in this information. Further details about these laws are available on request to your study investigator. The results of virus swab tests may be disclosed to you on an individual base upon your request through your preferred contact method you have provided upon study enrollment. Only compiled results (with personal identifiers removed) will be published at the completion of this study. A description of this clinical trial will be available on ClinicalTrials.gov, as required by U.S. Law. This website will not 131 include information that can identify you. At most, the website will include a summary of the results. You can search this website at any time. What happens if something goes wrong? By signing this form, you do not give up any of your legal rights and you do not release the study investigator, participating institutions, or anyone else from their legal and professional duties. If you become ill or physically injured as a result of participation in this study, medical treatment will be provided at no additional cost to you. The costs of your medical treatment will be paid by your provincial medical plan. In case of a serious medical event, please report to an emergency room and inform them that you are participating in a clinical study and that the following person can then be contacted for further information: Ms. Penny Hatzimanolakis at telephone number: 778-998-0924. What will the study cost me? All research-related adhesive application and related tests that you will receive during your participation in this study will be provided at no cost to you. You will not be paid for participating. However, you may be provided reasonable reimbursement for your extra costs, such as parking and public transportation, up to $50 that you may need due to participation after having completed the trial at your last visit. Your study team will discuss how this will be done. Original receipts (except for meter parking) are required. Please give your receipts to a member of the study team. Who do I contact if I have questions about the study during my participation? If you have any questions or desire further information about this study before or during participation, or if you experience any adverse effects, you can contact Ms. Penny Hatzimanolakis at 778-998-0924. Who do I contact if I have any questions or concerns about my rights as a participant? 132 If you have any concerns or complaints about your rights as a research participant and/or your experiences while participating in this study, contact the Research Participant Complaint Line in the University of British Columbia Office of Research Ethics by e-mail at RSIL@ors.ubc.ca or by phone at 604-822-8598 (Toll Free: 1-877-822-8598). Please reference the study number [H18-03266] when calling so the Complaint Line staff can better assist you. After the study is finished You may not be able to receive the study treatment after your participation in the study is completed. There are several possible reasons for this, some of which are:  The treatment may not turn out to be effective or safe.  Your caregivers may not feel it is the best option for you.  You may decide it is too expensive and insurance coverage may not be available.  The treatment may not be available free of charge. Future Contact It is possible a future study similar to the current study may be conducted to expand our understanding on the effectiveness of cyanoacrylate adhesives on oral sores caused by herpes simplex virus. Please indicate, by checking the applicable box, whether you would like us to invite you for participation in further studies. This is not a consent to the future study.  Yes, I want the study investigator to contact me for future studies. My phone number is: My email address is: Participant Initials: 133  No, I do not want the study investigator to contact me for future studies. Participant Initials: 134 Signatures: Study Title: The Efficacy of Cyanoacrylate Adhesive in the Dental Management of Herpes Simplex Virus (1 & 2): A Pilot Study. Participant Consent My signature on this consent form means:  I have read and understood the information in this consent form.  I have had enough time to think about the information provided.  I have been able to ask for advice if needed.  I have been able to ask questions and have had satisfactory responses to my questions.  I understand that all of the information collected will be kept confidential and that the results will only be used for scientific purposes.  I understand that my participation in this study is voluntary.  I understand that I am completely free at any time to refuse to participate or to withdraw from this study at any time, and that this will not change the quality of care that I receive.  I authorize access to my health records [insert if applicable and samples] as described in this consent form.  I understand that I am not waiving any of my legal rights as a result of signing this consent form.  I understand that there is no guarantee that this study will provide any benefits to me. I will receive a signed copy of this consent form for my own records. I consent to participate in this study. 135 Participant’s Signature Printed Name Date Signature of Person Printed Name Study Role Date Obtaining Consent Investigator Signature Printed Name Date My signature above signifies that the study has been reviewed with the study participant by me and/or by my delegated staff. My signature may have been added at a later date, as I may not have been present at the time the participant’s signature was obtained. 136 Appendix D Baseline Intake Survey Study Number: _____________________ Please check the answer that best describes you. For any question, please email lyolanda@dentistry.ubc.ca 1. What is your sex?  Male  Female 2. How old are you?  Less than 18  18-25  26-35  36-45  46-55  56-65  66 and above 3. What race do you identify as?  Aboriginal/North American Indian  Asian  Black/African American  Hispanic or Latino  South Asian  White/Caucasian  Other (please specify) ______________________ 4. Do you have an unmanaged health condition(s) that may compromise your immune system?  None  Autoimmune disease  Diabetes  Heart disease  HIV/AIDs  Organ transplant  Respiratory disease  Other (please specify) _______________________ 137 5. Have you ever experienced cold sores/fever blisters/herpes labialis on the lips, skin around the mouth or around the eyes, chin or cheek, similar to the pictures below? https://commons.wikimedia.org/wiki/File:Herpes_labialis_-_opryszczka_wargowa.jpg https://commons.wikimedia.org/wiki/File:Herpes_labialis.jpg  Yes  No  Cannot recall 6. Have you ever experienced or have been informed you had herpes lesions, such as those shown in pictures below the first time you had an outbreak? https://commons.wikimedia.org/wiki/File:Herpes_Gingivalis.tiff  Yes  No  Cannot recall 138 7. On average, how frequently do you experience an outbreak?  1x/year  2x/year  3x/year  4x/year  5x/year  6x/year  More than 6x/year  Never had symptoms  Other (please specify) _______________________ 8. If you have had a lesion before, how do you usually manage the lesions?  Topical antiviral medication  Oral antiviral medication  Cold sore patches  Do not do anything and leave them alone  Never had cold sores before  LASERS Thank you for your time and supporting our research! ***Investigator use only*** Laboratory Results: Oral mucous membrane swab: 139 Appendix E Visit 1 Clinical Form Date: _________________________ Study Number: _____________________ Health History: 1. Please list any medication you are currently taking. 2. Please indicate on the line below your current stress level. Not at all stressed Extremely stressed 3. Please indicate on the line below your current fatigue level. Not at all tired Extremely fatigued Herpes Lesion History: 1. When did you start noticing the tingling/burning sensation of the lesion? 2. When did you notice the first blister on the lesion? 140 3. Have you applied topical antiviral agents or taken oral antiviral medications for the cold sore/oral herpes? If yes, please specify the type. 4. Please indicate on the line below the discomfort you are currently experiencing with the lesion. No pain Worst possible pain 5. How many cold sores or oral herpes have you had within one year from today’s date? 141 ***Investigator use only*** Lesion Location Please lable the location on the diagrams below. lesion stage size shape color appearance margin texture http://6weeks.co.uk/female-head-side-diagram.html https://www.researchgate.net/figure/Map-of-the-oral-cavity_fig1_237095390 Laboratory Results Lesion swab: Oral mucous membrane swab: Post-PA swab: Post-manipulation swab: 142 Appendix F Visit 2 and 3 Follow-up Clinical Form Visit 2 or 3 (circle) Date: _________________________ Study Number: _____________________ Health History: 1. Please note down any changes in your medical health since the last visit. 2. Please indicate on the line below your current stress level. Not at all stressed Extremely stressed 3. Please indicate on the line below your current fatigue level. Not at all tired Extremely fatigued Herpes Labialis Lesion History: 1. When did the lesion barrier start to fall off, if it did? 2. Please indicate on the line below the discomfort you are currently experiencing with the lesion. No pain Worst possible pain 3. Did additional lesions appear since the initial appointment? If so, please write down the date when they appeared and let the investigator know. 143 ***Investigator use only*** Has PA fallen off? Completely lost Partial loss Still intact Lesion Location Please lable the location on the diagrams below. lesion stage size shape color appearance margin texture http://6weeks.co.uk/female-head-side-diagram.html https://www.researchgate.net/figure/Map-of-the-oral-cavity_fig1_237095390 Laboratory Results Lesion swab: Oral mucous membrane swab: 144 Appendix G Daily Journal Date: _________________________ Study Number: _____________________ 1. What stage is your cold sore in?  Multiple blisters (small fluid bumps)  Blister has coalesced (all blisters formed into one large blister)  Ulcer (open wound)  Crusted (scab)  Healed (no sign of any lesion: normal skin) 2. Please indicate on the line below the discomfort you are currently experiencing with the cold sore. No pain Worst possible pain 3. Is the barrier placed on your cold sore still intact?  Completely intact  Edges have lifted  Parts of barrier has fallen off  Completely lost (please skip question 4) 4. Please indicate on the line below your acceptance level of the barrier placed on the cold sore. Acceptable Not tolerable 5. Any additional comments or adverse events you would like to share with the investigators?
Thesis/Dissertation
eng
Craniofacial Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Hatzimanolakis, Penny
Dental management of herpes simplex virus (HSV-1 & HSV-2) using n-butyl and 2-octyl cyanoacrylate as a barrier : a pilot study
Text