Detection of Epstein-Barr virus (EBV) DNA and antigens in oral mucosa of renal transplant patients without clinical evidence of oral hairy leukoplakia (OHL)

The use of the polymerase chain reaction (PCR) to detect the presence of Epstein-Barr virus (EBV) DNA in oral mucosa in the absence of specific lesions gives rise to the problem of identifying the real viral replication sites. To verify whether the detection of EBV is due to salivary contamination or its true replicative capacity in oral mucosa, saliva samples and exfoliated cells from four different oral mucosa sites were taken from 40 renal transplant patients and 20 normal subjects for examination by PCR using two pairs of primers specific for the BamHI-L and BamHI-K genomic regions. EBV-specific sequences were detected in one or more of the oral mucosa samples from 29 transplant patients (72.5%) and six healthy controls (30%), and in the sliva samples of 16 transplant patients (40%) and three healty controls (15%). A total of 89 oral mucosa smears from 29 transplant patients, and 13 from healthy subjects, were EBV-positive. The positive samples were also investigated by means of in situ hybridization in order to confirm the intracellular presence of the viral genome, and by means of immunofluorescence testing with monoclonal antibodies to assess the possible expression of viral antigens. Hybridization with the EBV-specific probe was observed in 40/89 and 2/13 samples, respectively. Latent antigens (with or without lytic antigens) were detected in only 23 of the 40 samples (collected from eight different transplant patients) that were positive by in situ hybridization. Our data show that EBV is more frequently present in the oral mucosa of immunodeficient patients (where it can efficiently replicate) than in normal subjects.     

Human cytomegalovirus and Epstein-Barr virus type 1 in periodontal abscesses

OBJECTIVES: Recent studies have linked herpesviruses to severe types of periodontal disease, but no information exists on their relationship to periodontal abscesses. The present study determined the presence of human cytomegalovirus (HCMV) and Epstein-Barr virus type 1 (EBV-1) in periodontal abscesses and the effect of treatment on the subgingival occurrence of these viruses. MATERIAL AND METHODS: Eighteen adults with periodontal abscesses participated in the study. Subgingival samples were collected from each patient with sterile curettes from an abscess-affected site and a healthy control site. HCMV and EBV-1 were identified by polymerase chain reaction at the time of the abscess and at 4 months after surgical and systemic doxycycline therapy. RESULTS: HCMV was detected in 66.7% of periodontal abscess sites and in 5.6% of healthy sites (P=0.002). EBV-1 occurred in 72.2% of abscess sites but not in any healthy site (P<0.001). HCMV and EBV-1 co-infection was identified in 55.6% of the abscess sites. Posttreatment, HCMV and EBV-1 were not found in any study site. CONCLUSIONS: HCMV and EBV-1 genomes are commonly found in periodontal abscesses. These data favor a model in which a herpesvirus infection of the periodontium impairs the host defense and serves as a platform for the entrance of bacterial pathogens into gingival tissue with subsequent risk of abscess development.     

Epstein-Barr virus: Biology and disease

Epstein—Barr virus is a human herpes virus which, whilst found as a widespread asymptomatic infection, is also associated with certain tumours of lymphoid and epithelial origin including Burkitt's lymphoma (BL), immunoblastic lymphoma (IBL), Hodgkin's Disease (HD) and nasopharyngeal carcinoma (NPC). A unique characteristic of EBV is its ability to infect and transform primary resting B lymphocytes in vitro into permanently growing lymphoblastoid cell lines (LCLs); this effect is associated with constitutive expression of a limited set of viral genes. Interestingly, the pattern of EBV gene expression observed in LCLs in vitro is also a feature of IBLs, a tumour associated with immunosuppression. The other EBV associated tumours display a more restricted pattern of EBV latent protein expression. B cell lines can be activated in vitro into the virus replicative cycle, where a large number of viral genes associated with EBV DNA replication and virus assembly are synthesised. Whilst EBV can be detected in throat washings from seropositive individuals, the only in vivo situation where full virus replication can be reliably observed is hairy leukoplakia (HL), a benign lesion of lingual epithelium frequently found in AIDS patients. Thus, the relative contribution of lymphoid cells and epithelial cells to latent EBV infection/persistence vs replication in vivo remains controversial. Recent studies suggest that HL represents a focus of EBV replication in the absence of a truly latent infection and this supports the contention that EBV persistence resides in the lymphoid compartment. These aspects together with the role of EBV in oral diseases and the effect of certain EBV genes on the control of epithelial cell growth and differentiation will be discussed.     

Absence of a reservoir of Epstein-Barr virus (EBV) in normal tongue epithelium

We examined human tongue epithelium and serum samples at autopsy for evidence of latent Epstein-Barr virus (FBV) infection. Although clinical serology revealed anti-HBV antibodies in most sera indicating past FBV infection, we found no Epstein-Barr nuclear antigen (EBNA-coding sequences in tongue tissue-by polymerase chain reaction (PCR). or Epstein-Barr-encoded RNA (EBERI) by in situ hybridization. Tongue epithelium does not appear to be a natural reservoir for latent by EBV in immunocompetent hosts     

Epstein-Barr virus in tobacco-induced oral cancers and oral lesions in patients from India

D'Costa J, Saranath D, Sanghvi V, Mehta AR: Epstein-Barr virus in tobacco-induced oral cancers and oral lesions in patients from India. J Oral Pathol Med 1998; 27: 78–82. © Munksgaard, 1998.
We examined 103 oral squamous cell carcinomas (OSCC), 100 oral lesions consisting primarily of leukoplakia (82 cases), and 76 clinically normal mucosa specimens from the contralateral site in the oral cavity of individuals with oral lesions, for the presence of Epstein-Barr virus (EBV). Polymerase chain reaction (PCR) was used to amplify a 239 bp fragment of the BamHIL region of the EBV genome, followed by Southern blot hybridization with EBV oligonucleotide probe to increase further the specificity and sensitivity of the assay system. Since EBV seropositivity is frequent in populations, we also examined the peripheral blood cells (PBC) from 141 patients (50 oral cancer patients, 91 patients with oral lesions) for the presence of EBV We detected EBV in 25 of 103 (25%) OSCC, 13 of 100 (13%) oral lesions, 3 of 76 (4%) clinically normal mucosa samples and 10 of 141 (7%) PBC. Our results indicate that EBV may contribute as one of the multiple factors in oral cancers, in a certain proportion of Indian patients.     

Detection of Epstein-Barr virus (EBV) DNA by the polymerase chain reaction (PCR) in oral smears from healthy individuals and patients with squamous cell carcinoma

To assess the presence of Epstein-Barr virus (EBV) DNA in normal oral mucosa, as well as its relationship to age, sex and different sites in the oral cavity, oral smears from healthy adults were investigated using the polymerase chain reaction (PCR). Smears taken from oral cancer patients were also examined using the same method. Sixty healthy volunteers (30 men and 30 women) were selected and divided equally into three age groups. Four cytologic samples were taken from each subject using a cytobrush. Smears from 20 patients with oral cancer were taken from similar sites and from the lesion. The Bam W region of EBV DNA was chosen as the specific genome for PCR amplification. Fifteen out of 60 healthy individuals (25%) showed EBV positivity. Of these, seven were men and eight were women. There were no significant differences between the three age groups nor between the four sites of oral mucosa. Our results also showed that EBV DNA could be identified in 10 out of 20 oral cancer patients (50%), though in only 7 (35%) of the lesions. Taken into account with the age of the patients, these findings indicate that EBV infection in the oral cavity does not appear to be directly associated with the pathogenesis of oral squamous cell carcinoma.     

Correlation between different genotypes of human cytomegalovirus and Epstein-Barr virus and peri-implant tissue status

Background: The purpose of this study was to estimate the prevalence of different genotypes of human cytomegalovirus (HCMV) and Epstein‐Barr virus (EBV) in peri‐implantitis and mucositis sites, and to evaluate the correlation between herpesvirus presence and clinical parameters. Methods: A total of 80 dental implants (mean time of loading, 4.16 ± 1.8 years) were evaluated during the course of the study (30 peri‐implantitis, 25 mucositis and 25 healthy peri‐implant sites). The following clinical parameters were assessed: visible plaque index, bleeding on probing, suppuration and probing depth. A polymerase chain reaction (PCR) assay was used to identify the presence of different HCMV and EBV genotypes in peri‐implant tissue plaque samples. Results: HCMV‐2 was detected in 53.3% and EBV‐1 in 46.6% of the 30 peri‐implantitis sites evaluated. By contrast, HCMV‐2 was not detected in healthy periodontal sites and EBV‐1 was detected in one healthy site. A statistically significant correlation was found between the presence of HCMV‐2 and EBV‐1 genotypes and clinical parameters of peri‐implantitis. Conclusions: The results from the present study confirmed the high prevalence of HCMV‐2 and EBV‐1 in the peri‐implant tissue plaque of peri‐implantitis sites and suggests a possible active pathogenic role of the viruses in peri‐implantitis.     

Oral lesions and conditions associated with human immunodeficiency virus infection in 300 south Indian patients

BACKGROUND: Human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) is a major health problem in India. The National AIDS Control Organisation (NACO) of India reports a seropositivity of 25.03 per thousand for the whole country, as of October 1999. In spite of this high prevalence there are very few reports of oral lesions and conditions in Indian HIV/AIDS patients, which are important in early diagnosis and management of these patients. OBJECTIVE AND SETTING: The present report describes the oral lesions in 300 HIV positive symptomatic patients presenting to us at RAGAS-YRG CARE, a non-governmental organisation in Chennai, South India, over a period of 9 months in 1998. METHOD: Lesions were diagnosed on clinical appearance using international criteria. RESULTS: Of the 300 patients 89% had acquired the infection through heterosexual contact. There were 205 males and 95 females, aged from 7 months to 72 years. Forty-seven percent of the patients were in the age group 21-30 years. CD4 counts were ascertained for 105 patients, 64 (62%) had CD4 counts < or = 200. A total of 217 (72%) of the 300 patients had some oral lesion when examined. Gingivitis (47%) and pseudomembranous candidiasis (33%) were the most common oral lesions. The other oral lesions seen were oral mucosal pigmentation (23%), erythematous candidiasis (14%), periodontitis (9%), angular cheilitis (8%), oral ulcers (3%), oral hairy leukoplakia (3%), hyperplastic candidiasis (1%), oral submucous fibrosis (2%) and one case of leukoplakia. CONCLUSIONS: Oral lesions occur commonly in HIV infection. A comprehensive oral examination may not only suggest HIV disease but may also be useful in monitoring the disease progression. This is a cost-effective procedure, which may be useful in screening large populations in developing countries like India.     

Preliminary evidence for an association of Epstein-Barr virus with pre-ulcerative oral lesions in patients with recurrent aphthous ulcers or Behiçet's disease

In this study we used the polymerase chain reaction (PCR), slot blot and Southern blot hybridization, direct sequencing and in situ hybridization (ISH) to show the possible presence of EBV-DNA in pre-ulcerative oral aphthous lesions of patients with recurrent aphthous ulcers (RAU) or Behet's disease (BD). For this purpose, formalin-fixed biopsy specimens were obtained from 13 pre-ulcerative oral aphthous lesions of nine RAU and four BD patients. Five specimens of normal oral mucosa (NOM) from five normal control subjects and 10 specimens of oral erosive or ulcerative lesions from 10 patients with erosive lichen planus (ELP) were also included. EBV-DNA was detected by PCR in 5 of the 13 (38.5%) pre-ulcerative oral aphthous lesions, two from RAU patients and three from BD patients. However, no EBV-DNA was demonstrated in five NOM specimens from normal control subjects and in 10 specimens of oral lesions from ELP patients. EBV-DNA was also demonstrated in patients'peripheral blood lymphocytes and/ or plasma, suggesting that the lymphocytes may be the reservoir of latent EBV infection and there is EBV shedding in the plasma. EBV-DNA was detected by ISH in only one PCR-positive case; the reaction product was found to deposit on the nuclei of some of the epithelial cells and lymphocytes. By immunohistochemistry, expression of Epstein-Barr nuclear antigen and EBV/C3d receptors was also noted in some of the epithelial cells and lymphocytes in this ISH-positive case. Therefore, we suggest that the epithelial cells of pre-ulcerative oral aphthous lesions may be infected by EBV through EBV-infected lymphocytes; also, the cytotoxic T lymphocyte-induced lysis of the EBV-infected epithelial cells, but not the virus-induced cytolysis, may be the main mechanism causing oral ulcer formation. Our data provide preliminary evidence for an association of EBV with pre-ulcerative oral aphthous lesions in RAU and BD patients.     

Oral ulcers in HIV-infected patients: An update on epidemiology and diagnosis

Oral ulcers observed during the course of HIV infection may be very severe. Such manifestations may interfere with oral functions and alter the patients' quality of life. It is important to stress that when HIV-infected individuals present with ulcerative lesions of the oral cavity, neoplastic processes and rare infections must be included in the differential diagnosis. Nontumefactive oral ulcers in HIV-positive patients may be a source of diagnostic difficulties because of the diverse array of underlying pathologic entities and multiplicity of etiologic agents. Biopsy should always be performed on long-standing ulcers, since either infection or a neoplastic process may be present. In the absence of infection or neoplasm, such lesions are then designated ulcers not otherwise specified.     

Human papillomavirus (HPV) infections and their associations with oral disease

More than 65 distinct types of human papillomavirus (HPV) have been identified to date. Several of the HPV types have been proposed as etiologic agents of squamous cell carcinoma. In the oral cavity, HPVs have been found associated with several benign squamous cell proliferations. Evidence from histology and DNA hybridization studies suggests that HPV is also involved in oral carcinogenesis. It is apparent, however, that substantial amount of confusion exists in the diagnosis of oral HPV infections. The keratotic, papillary lesions in the oral cavity are usually small and easily overlooked. The gross appearance of these viral lesions is not distinct enough to be readily diagnosed by the clinicians. Degenerative changes found on oral mucosa frequently simulate koilocytosis. Thus, caution should be exercised to avoid overdiagnosis of HPV infection in the oral cavity. The present review summarizes the current evidence available on HPV infections in general and on oral HPV infections in particular. The diagnostic techniques available as well as the problems encountered in the distinction of these lesions are also discussed in short.     

Epstein-Barr virus infection and expression in oral lesions

OBJECTIVE: The prevalence of Epstein-Barr virus (EBV) and the recently discovered Kaposi's sarcoma associated herpes virus, human herpesvirus 8 (KSHV or HHV8), was determined within oral lesions common to HIV infection including OHL, pseudoOHL (PHL), oral lymphoma, oral aphthous ulcers, and an oral Kaposi's sarcoma. METHODS: DNA and RNA were extracted from oral lesions. EBV and HHV8 genomes were detected by Southern blot and polymerase chain reaction (PCR), and viral expression was analyzed using PCR amplification of cDNA. RESULTS: Multiple EBV strains were detected within OHL with recombination across repeat sequences generating new viral variants. EBV expression in OHL included expression of some viral genes, usually expressed in latent infections, that induce the EBV receptor. EBV replication was detected only within OHL lesions but not within adjacent Kaposi's tissue or oral aphthous ulcers while HHV8 was only detected within the Kaposi's lesions. CONCLUSIONS: These findings indicate that the OHL lesion is unique with viral replication and superinfection with additional EBV strains. Expression of the EBV receptor within the OHL lesion may promote superinfection which then activates EBV replication. The consistent detection of EBV replication only within OHL lesions and the detection of HHV8 only within Kaposi's sarcoma, strengthens the etiologic link between EBV and HHV8 infection to these specific pathologies.     

Oral findings in Mexican AIDS patients with cancer

Oral findings of 42 Mexican AIDS patients with cancer were reviewed. Kaposi's sarcoma (KS) was the most frequent malignancy (81%) followed by non-Hodgkin's lymphoma (NHL) (12%). All cases of NHL were of high or intermediate grade and most of them were extranodal. Out of the 34 individuals with KS, 22 (65%) showed oral KS and in 21 of them the palate was involved. The clinical features of oral KS including site, appearance and size are described. Pseudo-membranous candidosis (PC), hairy leukoplakia (HL) and exfoliative cheilitis (ECh) were also found in these patients. There was no association of these lesions with any type of cancer. A strong association of oral candidosis and history of this infection was found, RR = 7.0 (1.3–4.1). There was evidence of severe immunosuppression in most patients, with mean average CD4 counts of 116 mm³ (range 4–841 /mm³). Oral KS, ECh, PC and HL were more common in patients with lower CD4 counts. Our findings illustrate the most frequent oral lesions associated with HIV-1 infection in patients with AIDS and cancer, and further support the importance of oral examination in HIV infected patients.     

Human papilloma virus in erosive oral lichen planus

Several types of human papilloma viruses (HPV) have been associated with benign and malignant squamous cell tumours of mucosal epithelium. To identify HPV in erosive oral lichen planus (OLPe), considered as a premalignant lesion, tissues from 20 patients were examined by Southern blot hybridization with 32P-labeled HPV DNA probes. Type 11 was found in 6 of the lesions while HPV types 6, 16 and 18 were not detected in any of the tissues examined. Using a type-specific polymerase chain reaction (PCR) assay for HPV-6, 11, 16 and 18, HPV-11 was detected in 8 of the samples (all of those positive by Southern blot), and, in addition, HPV-6 was found in 5 samples and HPV-16 in 3 samples. Overall, by the more sensitive PCR assay, 65% of samples were positive for HPV DNA. The finding of HPV DNA in many of the samples using two different techniques indicates a high prevalence of HPV in the OLPe afflicted oral mucosa. However, the role of HPV in the pathogenesis of OLPe has yet to be determined.     

Epstein-Barr virus (EBV) genomes and c-myc oncogene in oral Burkitt's lymphomas

In addition to Burkitt's lymphomas, tentative evidence suggests the involvement of Epstein-Barr virus (EBV) in malignant lymphomas of T-cell origin. The c-myc proto-oncogene is strongly associated with the development of lymphoid neoplasias. In the present study, a series of 38 biopsies of oral lymphomas (29 Burkitt's lymphomas, 9 malignant lymphomas of other type) obtained from patients in Tanzania were studied using in situ hybridization (ISH) and polymerase chain reaction (PCR) for detection of EBV DNA and c-myc oncogene. In ISH applied on formalin-fixed, paraffin wax-embedded biopsies, the Bam HI W fragment of EBV DNA was used as the probe. Amplification of c-myc oncogene was studied by PCR with a primer set from Exon II area. As an internal standard (β-globin gene was simultaneously amplified. EBV DNA was disclosed by ISH in five Burkitt's lymphomas only. Using the PCR, 20 of the 29 cases (70%) of Burkitt's lymphomas showed amplification for EBV DNA. Of the other EBV-positive lymphomas, two were of the lymphocytic type (large non-cleaved cell), one histiocytic and one Burkitt's-like lymphoma. All EBV-positive cases found on the agarose gel were positive also with the dot blot, when hybridized with the ³²P-labeled EBV Bam HI W-fragment probe. All lymphomas showed similar bands on the gel for c-myc and β-globin indicating that no amplification of c-myc was present.     

Detection of cytomegalovirus and Epstein-Barr virus in labial salivary glands in Sjogren's syndrome and non-specific sialadenitis

To investigate the role of herpes viruses in Sjogren's syndrome, minor (labial) salivary gland tissues from Sjogren's syndrome and from non-specific sialadenitis were examined for Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) DNA by the polymerase chain reaction. Almost half of all salivary glands studied contained EBV and/or HCMV. There was, however, no significant difference between the detection of EBV or HCMV in salivary glands from patients with Sjogren's syndrome or non-specific sialadenitis. The findings are consistent with the persistence of EBV and HCMV in minor salivary glands following primary infection, but do not indicate a direct role for either virus in the aetiology of Sjogren's syndrome, and do not exclude reactivation of the viruses in this disease.