Viral infections in the mouth

Oral hairy leukoplakia (OHL) and Kaposi's sarcoma (KS) are commonly encountered in the HIV-infected patient. A unique feature of OHL is non-cytolytic high level of replication of Epstein–Barr virus (EBV) in the glossal epithelium. The expression of viral-encoded anti-apoptotic proteins concomitant to replicative proteins probably underlies this phenomenon. The question of whether OHL arises from activation of EBV latent in the tongue, or from superinfection by endogenous EBV shed via non-glossal sites or by exogenous EBV remains unresolved. Human herpesvirus 8 (HHV8) is now seen as necessary but not sufficient cause of KS. Expression of HHV8-encoded oncogenic proteins in endothelial cells probably explains the aberrant proliferation of these cells in KS lesions. Studies into why KS is so commonly observed at the palate in HIV-infected patients may provide important clues to its pathogenesis.     

Presence of human herpes virus‐8 in saliva and non‐lesional oral mucosa in HIV‐infected and oncologic immunocompromised patients

Background/Aim: Human Herpes Virus‐8 (HHV‐8) is a recently identified virus etiologically associated with Kaposi's sarcoma. Studies regarding its presence in the oral cavity have given variable results. This study attempted to determine the oral presence of HHV‐8 in an area where classic Kaposi's sarcoma is primarily found such as Greece. Methods: Three groups of patients were studied: 10 immunocompromised with hematologic malignancies, 10 immunocompromised with HIV infection and 20 immunocompetent as controls. Whole unstimulated saliva and scrapes from the lingual and the buccal mucosa were collected and polymerase chain reaction was applied to amplify HHV‐8 DNA. Results: None of the patients in any group had oral lesions. In the control group, all samples tested negative (0/60). HHV‐8 DNA was detected in 5/30 (17%) of all samples from HIV‐positive patients (the mean value of their CD4⁺ T‐lymphocytes being 385/mm³) and in 13/30 (43%) of all samples from oncologic patients (mean CD4⁺ T‐lymphocytes 51/mm³). HHV‐8 DNA was found in 10% of saliva samples and 40% of lingual and buccal scrapes both of HIV‐infected and of oncologic patients. Conclusion: HHV‐8 is present in the saliva and the non‐lesional oral mucosa (not simultaneously) of patients with impaired immunity, with or without HIV co‐infection. The oral epithelium seems to represent an independent location of viral residency and may be of viral replication; the clinical implications need further clarification.     

Detection of human herpesvirus-8 DNA in oral ulcer tissues of HIV-infected individuals

OBJECTIVE: To determine the frequency of detection of human herpesvirus-8 (HHV-8) in HIV-related oral ulcers. DESIGN: Analysis of archived biopsy material. METHODS: Nested polymerase chain reaction of DNA extracts. RESULTS: HHV-8 DNA was detected in six of 10 oral ulcers of HIV-positive patients without oral Kaposi's sarcoma (KS) lesions and five of 11 oral KS lesions. The positive non-KS samples were derived from various oral sites. CONCLUSIONS: In HIV-positive people, HHV-8 can infect oral tissues that are not affected by KS.     

Kaposi's sarcoma-associated herpesvirus-like DNA sequences (KSHV/HHV-8) in oral AIDS-Kaposi's sarcoma: A PCR and clinicopathologic study

Recently, a new human herpesvirus (KSHV/HHV-8) has been identified in classic, transplant, endemic, and AIDS Kaposi's sarcoma that may be involved in the pathogenesis of Kaposi's sarcoma. The purpose of this study was to evaluate oral AIDS-Kaposi's sarcoma for detection of KSHV/HHV-8 DNA. DNA extracted from 54 oral AIDS-Kaposi's sarcoma lesions (47 initial, 7 postvinblastine treated), 5 non-Kaposi's sarcoma HIV-positive lesions, and 3 non-Kaposi's sarcoma HIV-negative lesions was evaluated by polymerase chain reaction (KS330_233bp amplicon) for KSHV/HHV-8. The AIDS-Kaposi's sarcoma study population consisted of 52 patients (51:1, men:woman; 92% men having sex with men, 8% heterosexual; mean age, 38 years; mean, CD4 59/mm³) Opportunistic infections occurred in 88% (candidiasis, 65%; Pneumocystis carinii pneumonia, 31%; nonoral Kaposi's sarcoma, 25%, mycobacterium avium-intracellulare (MAI), 16%; cytomegalovirus, 14%; herpes simplex virus, 14%). Sexually transmitted diseases occurred in 73% (gonorrhea, 37%; syphilis, 23%; condyloma, 22%; HSV, 16%). Most frequent lesion sites were palate (74%) and gingiva (17%). Most common lesion types were purple nodular (48%) and macular (42%). Histopathologic subtypes were nodular (71%), plaque (27%), and patch (2%). Polymerase chain reaction analysis detected KSHV/HHV-8 DNA in 53 of 54 AIDS-Kaposi's sarcoma lesions (47 of 47 initial, 6 of 7 postvinblastine treatment). KSHV/HHV-8 DNA was not detected in non-Kaposi's sarcoma lesions in HIV-positive or HIV-negative persons. KSHV/HHV-8 DNA sequence is present in a high proportion of oral AIDS-Kaposi's sarcoma lesions. Whether KSHV/HHV-8 is an etiologic agent or a cofactor in the development of this vascular neoplasm is uncertain and remains to be proven. Polymerase chain reaction analysis for KSHV/HHV-8 DNA sequence detection may be helpful in identifying Kaposi's sarcoma in early vascular proliferations, when the characteristic histopathologic features are not present.     

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.     

The expression of the Epstein-Barr virus nuclear antigen (EBNA-I) in oral hairy leukoplakia

OBJECTIVE: Oral hairy leukoplakia (OHL) is characterised by the presence of a replicative Epstein-Barr virus (EBV) in the superficial layers of the epithelium. There is some doubt, however, whether this reflects activation of a latent infection of the basal epithelial cells. EBV latency is associated with the expression of the viral gene product EBNA-I and the aim of this study was to investigate EBNA-I expression in OHL. METHODS: 22 biopsies of clinically suspicious OHL and three cases of normal mucosa were available as fresh frozen or paraffin embedded material. EBNA-I was detected immunocytochemically using a rat monoclonal antibody (IH4-I) following microwave irradiation. Lytic EBV infection was confirmed by the identification of the BZLF-I protein. RESULTS: 16 of the 22 cases displayed focal replicative EBV meeting the criteria for OHL, and in 13 of these, EBNA-I expression was restricted to the nuclei of epithelial cells in the upper layers of the epithelium. EBNA-I expression was absent from the basal cells in all cases and in the nine BZLF-I negative mucosal biopsies. CONCLUSION: These findings suggest that lytic EBV infection in OHL is not the result of activation of a latent infection of basal cells and suggests a role for EBNA-I, not only in latent EBV infection, but also in virus replication.     

Possibility of a viral etiology in recurrent aphthous ulcers and Behçet's syndrome

Abstract The clinical signs, laboratory data, and histological features of recurrent aphthous ulcers (RAU) and Behçet's syndrome suggest a viral etiology. In fact, there are reports of adenovirus isolations in herpetiform oral ulcers and on the isolation of a filterable agent in sporadic cases of Behçet's syndrome. However, isolation studies on the major and minor aphthous ulcers and more recent studies on Behçet's syndrome have been negative. A review of the literature on the role of viruses and autoimmunity in RAU and Behçet's syndrome is presented. Biopsy specimens of ulcerative lesions were grown in vitro for up to 300 days. Those cultures, along with leukocytes and body fluids, were examined by a variety of techniques for the presence of virus or viral antigens. Although a persistent or latent virus was not detected, these negative studies cannot exclude a viral etiology. In fact, the hypothesis of an infectious and viral etiology is still reasonable.     

US1HIV – changing patterns in HAART era,patients’ quality of life and occupational risks

Oral manifestations are early and important indicators of HIV‐infection. Several lesions with strong association to HIV infection have been described: oral candidiasis (OC), oral hairy leukoplakia (OHL), Kaposi's sarcoma (KS), Non‐Hodgkin‐Lymphoma (NHL), necrotising ulcerative gingivitis and periodontitis. These lesions may be present in up to 50% of patients with HIV‐infection and up to 80% of those with AIDS. Changing patterns in HAART era: With the advent of highly active antiretroviral therapy (HAART) the prevalence of OC, OHL and HIV – associated periodontal disease has decreased in adults. The prevalence of KS has not changed. However, there has been an increase in HPV‐associated oral lesions (papillomas, condylomas and focal epithelial hyperplasia) and HIV‐related salivary gland disease. In children receiving HAART no change in the prevalence of HIV‐related oral lesions has been found. Quality of life: The presence of oral lesions has a marked impact on health related quality of life. HIV‐associated orofacial lesions may lead to facial disfigurement (KS, NHL) or may impair speech and swallowing. Consequently, weight loss and pain may be result. Studies have shown that patients with OC, angular cheilitis and OHL have a high score of decayed teeth (DMFT). Xerostomia and taste disturbances may also be factors with impact on quality of life. Occupational risks: Occupational exposure to HIV has resulted in 57 documented cases of HIV sero‐conversion among healthcare workers in the US (December 2001). Exposure to HBV and HCV carries a much higher risk of occupational infection than that for HIV‐exposure.     

Oral ulcers in AIDS patients frequently associated with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections

Oral ulcers are common in AIDS patients, with a wide spectrum of underlying causes, including different viruses. In the present study, the presence of cytomegalovirus (CMV), Epstein-Barr virus (EBV) and human herpesvirus-8 (HHV-8) DNA was analysed in 21 biopsies from oral ulcers of 17 male homosexual AIDS patients. The methods used were in situ hybridization (ISH) and the polymerase chain reaction (PCR) with subsequent non-radioactive Southern blot hybridization to confirm the specificity of PCR products. With ISH, 4 biopsies were CMV DNA-positive and 11 contained EBV-DNA. Using PCR, an additional 4 CMV- and 7 EBV-positive samples were detected, and HHV-8 DNA was present in three oral ulcers. Six of the patients (35%) had oral ulcers co-infected by two or three viruses. The overall figures for patients with the detectable EBV-, CMV-, and HHV-8 DNA were 82% (14/17), 35% (6/17) and 18% (3/17), respectively. This is the first study to show the frequent presence of EBV-DNA in oral ulcers of AIDS patients. Because ISH-positivity signifies active virus replication, these results implicate an etiological role of EBV in AIDS-associated oral ulcers. The causal role of HHV-8 has to be considered as well, because this virus was detected in three such ulcers, which were not associated with Kaposi's sarcoma. To conclude, three common members of the herpesvirus family (CMV, EBV, HHV-8) were detected in all but three ulcers in AIDS patients, warranting the inclusion of these viral analyses in the diagnosis of ulcerative lesions of the oral mucosa in all immunosuppressed individuals.     

Lack of evidence for local immune activity in oral hairy leukoplakia and oral wart lesions

BACKGROUND: Oral warts, caused by human papillomavirus (HPV), and oral hairy leukoplakia (OHL) caused by Epstein-Barr virus (EBV), are common oral manifestations in HIV-infected persons. Although both conditions occur most often with reduced blood CD4+ T-cell numbers, oral warts and OHL rarely occur simultaneously, suggesting that dysfunctions in other secondary local immune parameters are also involved. The present study evaluated tissue-associated proinflammatory and T-helper cytokine and chemokine mRNA expression and the presence of T cells in each lesion. METHODS: Biopsies were taken from lesion-positive and adjacent lesion-negative sites of HIV+ persons with oral warts or OHL and lesion-negative sites from HIV+ persons who were oral HPV or EBV DNA-positive (matched controls). Cytokine/chemokine mRNA expression was quantified by real-time polymerase chain reaction. CD3, CD4, and CD8 cells were identified by immunohistochemistry. RESULTS: No differences were detected in tissue-associated cytokine/chemokine mRNA expression in warts or OHL when compared to lesion-negative sites. Immunohistochemical analysis of T cells showed CD8+ cells exclusively, but few cells were present in either lesion. No differences were detected between lesion-positive and -negative control sites of each pathologic condition. CONCLUSION: Little evidence was found for local immune reactivity to either oral warts and OHL, suggesting that CD4+ T cells are a primary host defense against both oral warts and OHL, but with nonimmune factors potentially responsible for the divergent prevalence of each.     

Detection of Epstein-Barr virus in oral scrapes in HIV infection, in hairy leukoplakia, and in Healthy non-HIV-infected people

Epstein-barr Virus (EBV) has been implicated in the pathogenesis of oral hairy leukoplakia (OHL). EBV is normally detected by lesional biopsy. The objectives of this study were to examine oral scrapes containing squamous epithelial cells from healthy non-HIV-infected people with and without clincial lesions of OHL, and from healthy non-HIV-infected controls, for EBV-DNA by the polymerase chain reaction (PCR). EBV-DNA was detected in 65% of HIV-infected individuals it was found as frequently in those without OHL as in those with. Moreover, EBV-DNA was not detected in all HIV-infected individuals, nor in all OHL as in those with. Moreover, EBV-DNA was not detected in all Hiv-infected individuals, nor in all OHL> The results suggest that the presence or absenc e of detectable EBV-DNA in oral scrapes, though a guide, cannot be regarded as absolutely reliable in the diagnosis or exclusion of OHL.     

Extensive oral shedding of human herpesvirus 8 in a renal allograft recipient

Introduction: Studies were conducted to investigate changes in the extent of human herpesvirus 8 (HHV‐8) shedding and diversity of HHV‐8 strains in the mouth of a renal allograft recipient who developed cutaneous post‐transplantation Kaposi’s sarcoma. Methods: Matched oral and blood samples were obtained from a Saudi Arabian renal allograft recipient from 3 days before to 38 weeks after transplantation, and from his kidney donor. Polymerase chain reaction (PCR) protocols to amplify selected HHV‐8 sub‐genomic regions were applied to detect and quantify HHV‐8 DNA. Sequence diversity was determined by cloning the PCR products and subjecting them to denaturing gradient gel electrophoresis and to nucleotide sequencing. Results: Before transplantation, the recipient was seropositive for anti‐HHV‐8 immunoglobulin G, but the donor was seronegative; HHV‐8 DNA could be detected in the recipient’s blood, whole‐mouth saliva (WMS) and buccal exfoliates, and the salivary viral load was estimated as 2.6 million genome‐copies/ml. Post‐transplantation, the recipient’s salivary viral load initially increased to 4.1 million genome‐copies/ml, and thereafter declined precipitously, coinciding with an increase in the dosage of valaciclovir given; HHV‐8 DNA was detected most often in WMS compared with parotid saliva, and buccal and palatal exfoliates. Carriage of multiple HHV‐8 strains was evident in blood and oral samples; whereas before transplantation strains belonging to genotypes A1 and A5 were observed, after transplantation genotype A5 strains became dominant and A2 strains emerged. Conclusion: Immunosuppression and antiviral prophylaxis may interact to influence the spectrum of oral HHV‐8 strains and the extent of post‐transplantation HHV‐8 shedding into the mouth.     

Management of the oral mucosal lesions seen in association with HIV infection

Oral lesions cause considerable morbidity in association with HIV infection. Their successful management depends upon accurate diagnosis and the use of appropriate therapy. Various treatment approaches are described for some of the common oral lesions including Kaposi's sarcoma, oral candidiasis, hairy leukoplakia and recurrent oral ulcers associated with HIV disease. This paper will discuss the therapies available in the USA and UK. In other countries some of the drugs discussed will be available in different doses and preparations. In addition other drugs may be available in other parts of the world that are not licensed for use in the USA or UK, and their availability may vary.     

Human papillomavirus and Epstein Barr virus in oral hairy leukoplakia among HIV positive Venezuelan patients

Oral hairy leukoplakia (OHL) is commonly found in individuals infected with HIV and represents the most frequent oral manifestation. The purpose of this study was to detect the presence of Human Papillomavirus (HPV) and Epstein Barr Virus (EBV) in OHL of HIV+ Venezuelan patients. We evaluated 21 HIV+ adult patients with clinically present OHL lesions: 11 under antiretroviral therapy, 10 without therapy, and 10 oral mucosal samples as controls. Nested-PCR was used to detect EBV and HPV infection. The INNO-LiPA HPV Genotyping v2 was applied to determine the HPV genotype. The EBV genome was found in 16/21 (76%) of the HIV+ patients with OHL. No difference was observed in EBV+ and EBV- patients related to antiretroviral therapy viral load and CD4+ Tcell coant. HPV-DNA was observed in 7/21 HIV positive cases (33%). The HPV genotypes detected were: 6, 11, 31, 33, 52, and 56/74. The most frequently HPV found was genotype 6 in 7/7, while two cases were HPV-11 and two HPV-52. Of the positive cases, 5/7 (71%) presented co-infection with more than one HPV genotype and 4/7 (57%) had HPV coinfection with high and low risk types. No case was EBV or HPV positive in the control group. In this study, a higher EBV prevalence was observed in OHL-HIV+ patients, confirming the etiologic role in this entity. A considerable number of cases were positive for HPV infection, and many patients presented coinfection with more than one HPV genotype as well as the presence of high oncogenic risk HPV in OHL.     

In situ hybridization and the polymerase chain reaction (PCR) in the analysis of biopsies and exfoliative cytology specimens for definitve diagnosis of oral hairy leukoplakia (DHL)

The definitive diagnosis of oral hairy leukoplakia (OHL) demands that Epstein-Barr virus (EBV) is demonstrated in the lesional tissue, since the histopathological features on conventional light microscopy are not pathognomonic. We have investigated the possible use of polymerase chain reaction (PCR) technology in reaching a definitive diagnosis of this lesion by its application to ten biopsy specimens with definitive diagnoses of OHL determined by in situ hybridization. EBV DNA was demonstrated by PCR in all ten OHL biopsy specimens analysed, and none of ten control specimens. Furthermore, we have investigated the role of PCR in analysis of exfoliative cytology samples collected from the lateral border of the tongue by a minimally-invasive scraping technique. EBV DNA was not only detected in all OHL lesional scrapings but also in more than one-third of healthy controls, due to viral presence in saliva at the time of sampling. In this application, the highly sensitive PCR technique has low specificity and cannot be recommended.     

Bacterial diversity in aphthous ulcers

INTRODUCTION: Recurrent aphthous ulcers are common lesions of the oral mucosa of which the etiology is unknown. This study aimed to estimate the bacterial diversity in the lesions and in control mucosa in pooled samples using a culture-independent molecular approach. METHODS: Samples were collected from ten healthy individuals and ten individuals with a clinical history of recurrent aphthous ulcers. After DNA extraction, the 16S ribosomal RNA bacterial gene was amplified by polymerase chain reaction with universal primers; amplicons were cloned, sequenced and matched to the GenBank database. RESULTS: A total of 535 clones were analyzed, defining 95 bacterial species. We identified 62 putative novel phylotypes. In recurrent aphthous ulcer lesions 57 phylotypes were detected, of which 11 were known species. Control samples had 38 phylotypes, five of which were already known. Only three species or phylotypes were abundant and common to both groups (Gemella haemolysans, Streptococcus mitis strain 209 and Streptococcus pneumoniae R6). One genus was found only in recurrent aphthous ulcer samples (Prevotella) corresponding to 16% of all lesion-derived clones. CONCLUSION: The microbiota found in recurrent aphthous ulcers and in the control groups diverged markedly and the rich variety of genera found can provide a new starting point for individual qualitative and quantitative analyses of bacteria associated with this oral condition.     

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.     

Prevalence of Epstein-Barr virus and human papillomavirus in oral mucosa of HIV-Infected patients

Epstein-Barr virus (EBV) has been implicated in the genesis of oral hairy leukoplakia (OHL). Initially, OHL was also associated with human papillomavirus (HPV) as evidenced by staining with antiserum to papillomavirus common structural antigens and reports of two HPV-positive OHL as detected by in situ DNA hybridization. The aims of this study were to determine the prevalence of EBV and HPV DNA in OHL and normal oral mucosa and to explain the basis for the staining of OHL tissues with antibodies to papillomavirus common structural antigens. EBV DNA was detected by in situ hybridization in 47 of 47 cases of OHL from human immunodeficiency virus (HIV)-seropositive individuals and in 1 of 10 biopsies of clinically normal buccal mucosa from the same group of individuals. Twenty-five of 35 OHL specimens stained with antibody to papillomavirus common structural antigens. There was no staining of two EBV-containing lymphoblastoid lines, indicating that the staining with anti-papillomavirus antibody was not due to antigenic cross-reactivity with EBV antigens. HPV DNA was detected by polymerase chain reaction amplification in 10 of 18 OHL specimens and in 6 of 10 normal buccal mucosa specimens. Our results indicate that EBV and HPV are present frequently in OHL and that HPV can be found regularly in histologically normal mucosa.     

Detection of Epstein–Barr virus DNA in saliva of HIV‐1‐infected individuals with oral hairy leukoplakia

We present three cases of oral hairy leukoplakia (OHL) in whom the diagnosis was established by EBV DNA detection in whole saliva. Three HIV‐infected patients came to the Oral Medicine Clinic with similar chief complaints of asymptomatic white lesions on the tongue. All patients were diagnosed with suspected OHL and oral thrush also in the first patient. A multiplex PCR DNA microarray was performed to detect EBV DNA in saliva collected by spitting method. All saliva samples showed positive results for EBV DNA, and the definitive diagnosis of OHL was made. Resolution of lesions was found at 1‐ to 2‐month follow‐up after treatment with application of acyclovir 5% cream 5 times daily. Additionally, anti‐fungal treatment was given to the first patient and anti‐retroviral treatment to the first and second patients. EBV is mostly transmitted by asymptomatic shedding into saliva. Therefore, the detection of salivary EBV DNA is useful in establishing a definitive diagnosis of OHL allowing more effective treatment for both HIV‐infected patients receiving ART and treatment‐naïve patients at any CD4 + count.     

A simple and rapid technique for the detection of Epstein-Barr virus DNA in HIV-associated oral hairy leukoplakia biopsies

A method of generating nucleic acid probes by polymerase chain reaction (PCR) for the detection of Epstein-Barr virus (EBV)-DNA by in situ hybridization in oral hairy leukoplakia (OHL) lesions is described. This method has the advantage over older methods of being cheaper, quicker and retaining sensitivity and specificity. Purified PCR products of Epstein-Barr virus DNA of 110 bp and 328 bp were labelled with biotin by nick translation or random primer labelling and were compared in in situ hybridization experiments with probes prepared by incorporation of biotin-labelled nucleotides in the PCR reaction mixture, with EBV viral DNA as a template. These probes were applied to 18 OHL tongue biopsies known to be positive for EBV-DNA, using a commercially available biotin-labelled BamHI "V" fragment EBV-DNA probe. To determine the specificity of the probes, we applied them to 20 normal tongue tissue samples and to 12 biopsies taken from keratotic tongue lesions from patients without risk factors for HIV infection and known to be negative for EBV-DNA. Clear positive signals for EBV-DNA were detected in all 18 cases of OHL biopsies using the amplimer of 328 bp labelled by PCR and random primer labelling. However, nick translation labelling was less efficient and sensitive. All control specimens were negative for EBV-DNA.