Romanian Journal of Medical and Dental Education Vol. 9, No. 4, July - August 2020

THE IMPORTANCE OF VIRAL DETECTION IN ORAL FLUIDS IN PATIENTS WITH . REVIEW.

Alexandru Flondor1, Irina-Georgeta Sufaru2*, Maria-Alexandra Martu2, Ionut Luchian2, Liliana Pasarin2, Vasilica Toma3, Silvia Martu2

1DMD, PhD, Private Practice, Iasi, Romania 2University of Medicine and Pharmacy “Grigore T. Popa”, Faculty of Dental Medicine, Department of Periodontology, Iasi, Romania 3University of Medicine and Pharmacy “Grigore T. Popa”, Faculty of Dental Medicine, Department of Pediatric , Iasi, Romania

*Corresponding authors: Sufaru Irina-Georgeta. E-mail: [email protected]

#All authors had equal contributions with the first author

Abstract The direct involvement of herpesviruses in the evolution of periodontitis can change the concepts of pathogenesis and disease management. A double periodontal of herpesviruses and pathogenic bacteria tends to be associated with more severe periodontitis than a periodontal infection involving only bacteria. Classical techniques in viral diagnosis use cell culture or antigenemia. These diagnostic techniques have been largely replaced by DNA and / or RNA-based tests, which possess superior technical performance and cost advantages. False-negative and false-positive PCR results are an inherent concern. Poor design of the PCR test, without optimization or validation of the PCR methodology, may lead to inaccurate findings and incompatible with those of other studies. The concept of viral of the diseased periodontium may introduce a new level of understanding of the importance of preventing and controlling periodontal disease for medical purposes. Keywords: viruses, periodontal bacteria, saliva, crevicular fluid, PCR

Introduction The etiopathogenesis of and periodontitis are periodontitis includes specific bacteria and infectious diseases that tend to be viruses [2], protective and destructive particularly severe in immune responses of the host [3], immunocompromised individuals [1]. modifiable and unchangeable Chronic gingivitis is relatively easy to environmental factors [4], and genetic and prevent and cure and virtually all types of epigenetic factors [5]. However, the periodontitis are controllable, but due to relative importance of different etiological delayed diagnosis of destructive determinants remains elusive due to the periodontal disease, therapy can be complex pathophysiology of periodontitis complex and expensive. Even after and divergent results in studies. Studies adequate periodontal treatment, the long- conducted since the mid-1990’s have term outcome may be unsatisfactory in identified a high prevalence and a large patients with poor adherence to an number of copies of cytomegalovirus, effective oral hygiene regimen or with Epstein-Barr virus and difficulty accessing professional virus type 1 in progressive periodontal periodontal care. The development of disease [6]. Evidence supporting a more effective therapies may require a periodontopathogenic role of better perspective on the aetiology of herpesviruses comes from association periodontal disease. studies and research based on

59 Romanian Journal of Medical and Dental Education Vol. 9, No. 4, July - August 2020 immunology, but the specific molecular viruses and varicella-zoster virus), B mechanisms by which herpesviruses can lymphocytes (Epstein-Barr virus, cause or exacerbate periodontitis have yet herpesvirus-8) or monocytes and T to be identified. The direct involvement of lymphocytes (cytomegalovirus and -6 and herpesviruses in the evolution of -7). periodontitis can change the concepts of Conversion of latent herpesvirus to pathogenesis and disease management. lytic replication may occur spontaneously or may be caused by environmental Herpesviruses stimuli, chemical agents, and events of Herpesvirus species comprise the physical and psychosocial stress, as found most widespread viral family in human in subjects under acute stress, astronauts in saliva and are important space flight, students before important periodontopathogenic agents. Eight academic exams, elite athletes in intensive species of herpesvirus, with distinct training and subjects with work-related biological and clinical characteristics, can fatigue. Reactivation of an oral herpesviral infect humans: herpes simplex virus-1 and infection can be estimated by an increase -2, varicella-zoster virus, Epstein-Barr in salivary herpesvirus counts or a virus, human cytomegalovirus, human significant increase in herpesvirus-specific herpesvirus-6, human herpesvirus-7 and salivary antibodies. Immunocompetent human herpesvirus 8 (Kaposi's sarcoma individuals usually experience the virus). Herpesviruses establish a persistent reactivation of herpesvirus that lasts only a infection throughout life, and some species few hours or days [9], which is probably of herpesvirus infect up to 90% of the too short a period of time to initiate or adult population [7]. exacerbate the clinical disease. However, The clinical outcome of a the release of herpesvirus virions into herpesvirus infection ranges from a saliva poses a risk for infection of subclinical or mild disease to encephalitis, individuals in intimate contact. pneumonia, and various cancers. In contrast, long-term Herpesviral infections of the oral cavity immunosuppressive conditions ⁄ diseases can lead to asymptomatic and and medications can lead to the unrecognized release of virions into saliva reactivation of oral herpesviruses that or diseases of the or continue for an extended period of time periodontium. One study looked at acute and may present a pathogenetic risk to the herpesviral infections in children's oral infected person. The immune system of cavities [8]. the elderly may not control a latent Herpesviruses have a cycle of varicella-zoster infection, resulting in biphasic infection that involves a lytic, outbreaks of , or may not replicative (productive) phase and a latent, effectively protect against Epstein-Barr unproductive phase. The replication phase virus and cytomegalovirus reactivation involves the expression of viral regulatory [10]. Herpesvirus infection in such people and structural proteins and the formation can be characterized as chronically of infectious virion particles [7]. The reactivated instead of latent. ability to switch between replicative and The vast majority of systemically latent states ensures viral transmissibility healthy adults continuously release between individuals, as well as a herpesvirus DNA into saliva. Herpes permanent host infection. Following the simplex-1 virus DNA was detected in initial infection, herpesviruses saliva in amounts up to 2.0-2.8 · 106 ⁄ ml preferentially exist in a latent state in [11]. DNA copies of Epstein-Barr virus in sensory ganglion cells (herpes simplex saliva can reach levels of 108 ⁄ ml, 1.6 ·

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109 ⁄ ml [12], 7.1 · 105 ⁄ ml [13] and 2.2 · between salivary and subgingival levels of 106 to 0.5 lg of DNA [14]. Because the Epstein-Barr virus in one study [21], but salivary number of Epstein-Barr virus has not in another study [22]. decreased moderately only after high- Active periodontal volume oral rinsing, or after normal cytomegalovirus infection is closely linked swallowing every 2 minutes, a large to [7]. amount of virus must enter the saliva Cytomegalovirus DNA was detected in the constantly [15]. However, the salivary saliva of 50% of periodontitis patients, but load of Epstein-Barr virus can vary over was not found in the saliva of gingivitis several months, which complicates the patients or total prosthesis wearers, classification of individuals as small, suggesting that salivary cytomegalovirus intermediate, or large viral release agents originates primarily from periodontitis [15]. Cytomegalovirus DNA was detected [12]. in the saliva of 61% of immunocompetent Cytomegalovirus DNA from and immunocompromised subjects (65) infected breast milk also appeared in the and was able to reach a number of copies saliva of infants at 4 months of age, of saliva DNA of 4.2 · 104 ⁄ ml [12]. peaked at 4-10 months after birth, and Herpesvirus-6 and herpesvirus-7 can occur subsequently decreased or became in saliva, with prevalences exceeding 95% undetectable [23]. and in amounts of several million copies In short, a large part of salivary of DNA ⁄ ml [16]. Varicella-zoster virus herpeviruses are released from the sites of DNA is present at a low prevalence and in periodontal disease. Because periodontal amounts of <1.100 children ⁄ ml in the treatment can significantly reduce the saliva of both healthy people and people number of subgingival [24] and salivary infected with HIV [17]. herpesvirus DNA, establishing a healthy A double periodontal infection of periodontium can reduce the risk of herpesviruses and pathogenic bacteria transmitting herpesvirus and herpesvirus- gives rise to increased cytokine release and related diseases. The close relationship immune signalling disorder [18] and tends between some species of herpesvirus and to be associated with more severe periodontitis also supports the examination periodontitis than a periodontal infection of the potential use of the salivary involving only bacteria. herpesvirus number to indicate the risk of Herpes simplex-1 virus may periodontal disease. contribute to periodontitis in a subgroup of Infectious mononucleosis is caused individuals, and the virus has been by a primary infection with Epstein-Barr identified in total saliva samples in 24% of virus and mainly by Epstein-Barr virus patients with . In the type 1 [25]. Approximately 10% of same group of patients, the DNA of the mononucleosis-like disease can be herpes simplex virus-1 was present in 16% attributed to cytomegalovirus. Epstein- of the subgingival samples and in 8% of Barr virus infects B lymphocytes, which the peripheral blood samples [7]. Herpes gives rise to a strong response of T simplex virus DNA was found in the lymphocytes, characteristic of saliva of 84% of patients with obvious mononucleosis. Clinical signs of infectious herpes lesions [19]. Epstein-Barr virus mononucleosis are long-term fever, DNA was detected in the complete saliva tonsillo-pharyngitis, lymphadenopathy, of 79% of periodontitis patients and 33% fatigue and occasionally splenomegaly, of gingivitis patients [12], and in 49% of liver damage and pericarditis [26]. Oral periodontitis patients and 15% of healthy signs are sore throat, cleft and individuals [20]. A correlation was found satellite lymphadenopathy. Epstein-Barr

61 Romanian Journal of Medical and Dental Education Vol. 9, No. 4, July - August 2020 virus is transmitted through direct contact to her young children [33], but despite the with virus-infected saliva, such as kissing, possibility of uterine infection, vertical and rarely through air or blood. Young transmission of the virus is less common adults with a primary infection with in infants born to an HIV-positive mother Epstein-Barr virus can quickly remove the [7]. virus from the blood, but not from the oropharynx [27]. However, people who Hepatitis viruses are already infected with Epstein-Barr Hepatitis viruses (designated A to virus (and cytomegalovirus) are not at risk G) cause most cases of acute and chronic of infectious mononucleosis, even when hepatitis and liver damage worldwide. exposed to people with the disease. Hepatitis varies pathologically from Relationships have been found asymptomatic or mild disease to fulminant between Bell's palsy (idiopathic peripheral liver failure. Hepatitis A and hepatitis E facial paralysis) and active infection with viruses are transmitted by water herpes simplex-1 virus [28], between contaminated with faeces (faecal-oral oropharyngeal lesions of Ramsay Hunt route), produce acute infections and do not syndrome and varicella-zoster virus and induce a chronic carrier condition. A high between HIV infection and Epstein-Barr incidence of viral infections with hepatitis virus and herpesvirus-8. Young children A and hepatitis E occurs in countries with with sudden rash acquired the disease from poor health standards. their mothers who excreted causal Hepatitis A virus RNA was herpesvirus-6 in saliva [7]. detected in the saliva of 50% of patients during a hepatitis A outbreak [34]. A study HIV of cynomolgus monkeys found that the Human immunodeficiency virus tonsils and salivary glands acted as infection is a potent reactor of herpesvirus, extrahepatic sites for early replication of demonstrated by a strong correlation the hepatitis A virus and were potential between decreased CD4 cell count in HIV- sources of saliva-borne infection [35]. The infected patients and increased rates of hepatitis B virus is transmitted parenterally herpesvirus reactivation [29]. HIV and is frequently associated with chronic infection is often associated with the viremia. Hepatitis B virus DNA was found salivary presence of several reactivated at concentrations of> 105 copies / ml of herpesvirus species. In synergism, saliva in 15% of patients with chronic herpesviruses [30], P. gingivalis [31] and hepatitis B. This concentration may be other periodontal bacteria [32] can also sufficient to allow horizontal transmission activate a latent HIV infection. People of the virus and probably some of the 20% infected with the human of patients with hepatitis B, who contract immunodeficiency virus who may or may the disease without a known origin of the not have received highly active infection, could have acquired the hepatitis antiretroviral therapy (HAART) have been B virus by salivary transfer [36]. shown to have a similar rate and amount of Chronic hepatitis C affects more oral shedding of herpes simplex virus, than 170 million people worldwide, and Epstein-Barr virus and cytomegalovirus. the hepatitis C virus persists in 80% of Subjects who did not receive HAART had infected people, where it can lead to liver a moderately greater elimination of oral , liver cirrhosis and herpesvirus-8 [7]. Herpesvirus-8 is found hepatocellular carcinoma [37] and in the oral epithelial cells of HIV-infected probably periodontitis, Sjögren's subjects and can be transmitted syndrome, oral lichen plan and horizontally from an HIV-infected mother syaladenitis [38]. Hepatitis C virus RNA

62 Romanian Journal of Medical and Dental Education Vol. 9, No. 4, July - August 2020 was present in the saliva of 39-72% of number of copies too small to have clinical subjects with chronic hepatitis [39] and significance. Because low herpesvirus was detected in 59% of gingival crevicular levels occur more frequently in healthy or fluid samples from viremic patients [40]. slightly inflamed periodontal sites than in Gingival fluid has been identified periodontal lesions [44], PCR-based as a major source of salivary hepatitis C studies may disproportionately virus. Twenty-seven percent of the overestimate the significance of husbands of people with chronic hepatitis herpesviruses in normal periodontal sites. C revealed antibodies to the virus, Real-time fluorescence-based PCR indicating a mode of transmission of the methodology provides quantification of virus within the family, but not necessarily nucleic acid copies. Real-time PCR is sexually. Toothbrushes used by patients particularly useful in testing slow-growing with hepatitis C may contain the virus and or difficult-to-grow infectious agents and should not be used by other family has become one of the basic technologies members. Hepatitis G virus RNA was for diagnosing infectious diseases. detected in 7% of salivary samples from Real-time PCR studies have individuals with chronic hepatitis G [7]. identified over one million herpesvirus copies in a single periodontal and Respiratory viruses have further linked herpesviruses to Respiratory disease viruses are destructive periodontal disease [45]. PCR usually transmitted through coughing or reverse transcription can detect and sneezing, which release large amounts of quantify RNA expression and can identify high-speed droplets into the air, and the active periodontal infections with risk of cross-infection through salivary herpesvirus. In a study of localized exchange is relatively low. Children with (juvenile) aggressive periodontitis, six respiratory disease revealed respiratory patients had transcription / activation of viruses (respiratory syncytial virus, cytomegalovirus at sites with radiographic influenza virus, parainfluenza virus, evidence of evolving periodontal adenovirus) in 74% of oral samples and destruction, while two patients with 77% of nasopharyngeal samples [41], and nontranscriptional / latent cytomegalovirus respiratory syncytial virus RNA in 76% of were periodontally stable [46]. In chronic salivary samples [42]. Although present in periodontitis, which has a slow evolution, saliva, influenza virions may not be cytomegalovirus reactivation was detected infectious due to the activity of the anti- in 2% and cytomegalovirus latency in 48% influenza virus of salivary glycoproteins of the study sites [47]. Reactivation of [43]. herpesvirus has also been linked to symptomatic and large, probably Methodological issues progressive, periapical lesions of Classical techniques in viral endodontic origin [48]. diagnosis use cell culture or antigenemia. False-negative and false-positive These diagnostic techniques have been PCR results are an inherent concern. Poor largely replaced by DNA and / or RNA- design of the PCR test, without based tests, which possess superior optimization or validation of the PCR technical performance and cost methodology, may lead to inaccurate advantages. PCR, since its establishment findings and incompatible with those of in 1983, has become the reference other studies. PCR test verification criteria methodology for detecting nucleic acids in include amplicon specificity, target herpesvirus. Due to the high sensitivity of sensitivity, and amplification efficiency. the test, PCR can detect herpesviruses in a False-negative PCR results may be caused

63 Romanian Journal of Medical and Dental Education Vol. 9, No. 4, July - August 2020 by failure of sampling of Epstein-Barr identify sites of disease. activate. Kamma virus-rich gingival epithelial cells or by an et al. [51] studied the presence of inhibitory effect on the components of the herpesviruses in 16 patients who had amplification process. The latter is best periodontal sites with active and stable identified and avoided by including a non- disease with similar probing depth and destined DNA sequence as an internal degree of gingival inflammation. control in the PCR reaction mixture. False- Cytomegalovirus was detected in 59% of positive results can be prevented by active sites and 13% of stable sites (P practicing good laboratory techniques to <0.001), Epstein-Barr virus in 44% of reduce the risk of cross-contamination active sites and 13% of stable sites (P = between samples, by using clean PCR 0.01), herpes virus simplex type 1 in 35% reagents and an appropriately balanced of active sites and 9% of stable sites (P = mixture of reagents, and by using the 0.03) and co-infection with any of the closed-tube format to eliminate the risk of three herpesviruses in 44% of active sites amplicon carry-over at subsequent testing and 3% of stable sites (P <0.001) [51]. stages. In 2006, a PCR-virochip study Regarding the effect of treatment, implicated a retrovirus related to several authors [52, 53] failed to detect xenotropic murine leukaemia virus in subgingival herpesviruses for several prostate cancer, which was later shown to weeks or months after treatment in 50- be an error caused by a laboratory artefact 90% of sites that had pre-treatment related to contamination [49]. viruses. Sunde et al. [54] demonstrated However, the finding of a that systemic antiviral treatment of significantly different prevalence of refractory periodontitis lesions, which herpesviruses in periodontal health and contained hundreds of thousands of disease and between gingivitis and Epstein-Barr virus copies pre-treatment, periodontitis is consistent with a low and led to the absence of the Epstein-Barr insignificant level of nonspecific PCR virus genome for at least 1 year. amplification / false positive results. Also, Consequently, disease-stable and because periodontal herpesviruses have newly treated periodontal lesions may been identified by a variety of primers and contain relatively few herpesviruses, even PCR methodologies and by histomolecular if these agents were numerous at the time techniques, the likelihood of a systematic of active tissue destruction. This type of misidentification of these viruses is pattern of appearance might actually be virtually non-existent. expected for a true periodontal pathogen. Different inclusion criteria from Other factors that influence the prevalence studies and sampling sites are probably the of herpesviruses are race / ethnicity, main reasons for differences in the rate of socioeconomic status and life in low- detection of periodontal herpesviruses. In income countries compared to richer order to elucidate the role of herpesviruses countries. in periodontal destruction, it is important A systematic analysis identified a to investigate lesions of periodontitis that higher frequency of cytomegalovirus and a are actively progressing or, at the very lower rate of herpes simplex virus type 1 least, that have not recently received among people in South Asia living in professional treatment. Lindhe et al. [50] socio-economically developed countries found that only 12% of untreated than in the majority European Caucasian periodontitis lesions showed additional population [55]. Levine et al. [56] found attachment loss over a 6-year period and that the level of serum cytomegalovirus therefore a single measurement of pocket antibodies in the US was higher in low- depth or attachment loss is unlikely to income individuals and higher in non-

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Hispanic black subjects than in white spread by saliva must be identified and subjects and was predictive of periodontal controlled. disease. The aetiological factors of a disease are best identified in patients with Perspectives and conclusions active disease, and this notion is important Knowledge about infectious agents in the study of viruses in the oral cavity. A in the human oral cavity has expanded better understanding of viral greatly in recent years, mainly due to immunobiology in periodontitis may lead molecular techniques that can identify and to more effective preventive measures of quantify oral bacteria and viruses with the disease, including vaccination. great accuracy. Antiviral pharmacotherapeutic agents may Several oral and medical pathogens be prescribed in the treatment of advanced occur in saliva at levels sufficient to infect periodontitis. close individuals, and contact with saliva In short, the viral pathogenic may be a more important way of significance is an open and promising transmitting the pathogen than previously frontier of research. The concept of viral achieved. Raising awareness of the infections of the diseased periodontium infectious potential of saliva raises may introduce a new level of challenging questions about the safety of understanding of the importance of interpersonal contact. The risk of cross- preventing and controlling periodontal infection by salivary transfer may not be disease for medical purposes. Increased trivial and should be further studied. The research in periodontal virology is type of pathogens that can retain encouraged, given the remarkable infectivity in saliva and that are effectively preventive and curative possibilities it can offer.

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