Epstein-Barr virus in lingual epithelium of liver transplant patients

Thirty-three liver transplant patients and 20 controls were examined for the presence of EBV in epithelium of the lateral border of the tongue by negative staining electron microscopy. Five of the specimens (15%) showed particles typical of the herpes virus family, while all controls were negative for EBV. In 3 of the 5 patients moderate oral hairy leukoplakia was observed clinically. Our results indicate that EBV may be expressed at the lateral border of the tongue of liver transplant patients, who in some cases show oral hairy leukoplakia clinically.     

Detection of Epstein-Barr virus DNA in tongue tissues from AIDS autopsies without clinical evidence of oral hairy leukoplakia

Epstein-Barr virus (EBV) DNA was detected by in situ hybridization at 3 sites of 30 samples taken from clinically normal lateral border of tongue mucosa from 15 AIDS autopsies and in none of 20 samples from 10 controls. The first positive case showed a thin layer of parakeratosis correlated with positive signals for EBV in one area and an adjacent area without obvious parakeratosis was also positive for EBV. These findings were present on both sides of the tongue. The second case was unilaterally positive for EBV and parakeratosis was absent. The hybridization signals were localised to koilocyte-like cells in the stratum spinosum, as in oral hairy leukoplakia (OHL). These observations suggest that the in situ hybridization technique can detect very early or subclinical OHL, and supports the role of EBV in the pathogenesis of this lesion.     

Occurrence of corrugated white patch lesions on lateral border of tongue in lymphoma patients during cytostatic treatment

The question of whether or not there was an association between immunosuppression and occurrence of corrugated white patch lesions on the lateral border of the tongue was studied in 79 patients being treated for non-Hodgkin lymphoma or Hodgkin's disease. The mouths of 55 patients (mean age 47.8 years, 34 males, 21 females) were examined during periods of chemotherapy. All patients were HIV-seronegative. White non-removable lesions on the lateral margins of the tongue were noted in 27 patients (42.8%) 74 days after commencement of chemotherapy and 10 days after termination of medication. In 12 cases (44.4%) the lesions were bilateral. Epstein-Barr virus (EBV) DNA was found by gene amplification using polymerase chain reaction (PCR) in one of the two biopsy samples taken. No white lesion on the lateral border of tongue had been seen in any patient before treatment, nor were any evident 1 year after treatment. Leucocyte counts were significantly (P=0.001) lower when the lesion was present than when it was not detected. Before chemotherapy, 70.4% of patients with lesions and 47.6% of patients without lesions had positive salivary yeast cultures. Yeasts could be cultued from the saliva of 80.5% of patients when the lesions were present. In 2 patients clinical oral candidiasis was diagnosed at the time of the lesion. The study revealed a correlation between the occurrence of corrugated white, non-removable lesions of the lateral borders of the tongue, high salivary yeast counts and leucocytopenia. Clinical diagnosis of the lesion was consistent with oral hairy leukoplakia (OHL) or pseudo oral hairy leukoplakia (pseudo OHL), but histological studies are needed to confirm the diagnosis of the lesion. However, the lesion may be an early clinical sign of immunosuppression.     

Negative staining EM for the detection of Epstein-Barr virus in oral hairy leukoplakia

The performance of two different EM techniques applied for the detection of Epstein-Barr Virus (EBV) in oral hairy leukoplakia (HL) was assessed, i.e. the conventional two-step method of negative staining (CNS) and negative staining after Airfuge enrichment (ANS). Scrape specimens from the lateral borders of tongue of 66 HIV-positive patients with or without HL, of 3 patients with infectious mononucleosis and of 10 HIV-negative patients were evaluated. While CNS resulted in virus detection only in 25% of clinically diagnosed HL cases, EBV was detected by ANS in 85% of clinically suspected cases of HL. Scrape specimens of individuals negative for HIV were negative in EM while 2 of 3 mononucleosis patients were positive without clinical evidence for HL. Due to this high sensitivity the method of negative staining after Airfuge enrichment appears to be useful in the diagnosis of HL. The finding of EBV in clinically normal oral mucosa in HIV-seropositive individuals is interesting and indicates that EBV expression may precede the clinical appearance of HL.     

Immunocytochemical detection of herpes viruses in oral smears of HIV-infected patients

Cytologic smears (CS) were taken from the lateral border of the tongue of HIV-seropositive patients (HIV+) (n = 39) and of seronegative controls (HIV-) (n = 19) and examined by immunocytochemistry (APAAP) and in situ hybridization (ISH) (biotinylated DNA probes) for the presence of viral antigens/DNA of EBV and CMV. While none of the HIV controls showed positive results for EBV antigen, 61% (APAAP) resp. 79% (ISH) of oral epithelial cells in the group of HIV+ patients were EBV-positive. While all CS taken from areas with the clinical diagnosis of hairy leukoplakia (HL) were EBV positive (APAAP and/or ISH), the detection of EBV in CS from uninvolved oral mucosa seemed to be associated with the later development of HL. In the group of HIV+ patients the detection rate for CMV was about five times (APAAP) resp. three times (ISH) higher than in HIV- persons. This non-invasive technique seems to be a valuable tool to screen for viral antigens/genomes.     

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.     

Oral hairy leukoplakia: An exfoliative cytology study

Oral hairy leukoplakia (OHL) is a white, hyperplastic, vertically corrugated lesion that occurs on the lateral border of the tongue, usually unilateral. Caused by the Epstein-Barr Virus (EBV), the lesion is said to be an early indicator of an immune deficiency status, thereby unmasking subclinical systemic conditions. OHL mimics many other white lesions of the oral cavity; therefore, it becomes imperative to identify the lesion. This study used exfoliative cytology, a noninvasive procedure, which helped in identifying the cellular changes brought about by the virus in the oral epithelium. The study revealed a subclinical phase of OHL, where the cellular changes were seen even before the appearance of the clinical lesion.     

Epstein-Barr virus in oral hairy leukoplakia scrapes: identification by PCR

Oral hairy leukoplakia (OHL) is a lesion associated with a compromised immune system, and its diagnosis is determined by the demonstration of the presence of Epstein-Barr virus (EBV) in lesional tissue. The purpose of this article was to develop a simple technique to help the diagnosis of OHL, using PCR as an alternative technique to evidence EBV in scrapings. DNA samples were obtained by scraping the lateral border of the tongue of 38 adult patients: 29 HIV-positive patients (4 with clinical evidence of OHL; 4 with history of OHL, but without lesion at the moment the samples were collected; and 21 without clinical evidence of OHL), and 9 healthy volunteers for the control group. DNA was extracted from scrapes and amplified by PCR using specific primers for EBV. Of the 29 cases of HIV-positive patients, 22 (75.86%) were positive for EBV: 2 patients with clinical evidence of OHL, 4 patients with history of OHL, but without lesion at the moment the samples were collected, and 16 patients without clinical evidence of OHL. In the control group, samples of 5 (55.56%) healthy volunteers presented amplification for EBV. We concluded that the use of PCR in oral scrapes suggests a high sensitivity but low specificity for the diagnosis of OHL.     

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.     

Epstein-Barr virus in oral diseases

Epstein-Barr virus (EBV), a B-lymphotropic gamma-herpesvirus, causes infectious mononucleosis and oral hairy leukoplakia, and is associated with various types of lymphoid and epithelial malignancies. Saliva is the main vehicle for EBV transmission from individual to individual. Recent studies have also implicated EBV in the pathogenesis of advanced types of periodontal disease. EBV DNA is detected in 60-80% of aggressive periodontitis lesions and in 15-20% of gingivitis lesions or normal periodontal sites. The periodontal presence of EBV is associated with an elevated occurrence of periodontopathic anaerobic bacteria. Moreover, EBV active infection occurs in approximately 70% of symptomatic and large-size periapical lesions. EBV and cytomegalovirus often co-exist in marginal and apical periodontitis. Periodontal therapy can markedly suppress the EBV load in periodontal pockets as well as in saliva, which has the potential to reduce the risk of viral transmission between close individuals. EBV proteins up-regulate cytokines and growth factors, which seem to play a central role in the proliferative response of tongue epithelial cells in oral hairy leukoplakia and in the cell-transformation process of EBV-associated malignancies. Further research is needed to identify the full range of EBV-related diseases in the human oral cavity.     

Bilateral white corrugated lesions on the lateral tongue surface

A patient with bilateral hairy leukoplakia and candidiasis of the tongue was diagnosed and described. Lesions of the tongue that should be considered in the differential diagnosis include: idiopathic clinical leukoplakia, tobacco-induced leukoplakia, frictional keratosis, edema, lichen planus, galvanic lesions, geographic tongue, maceration, and chronic hyperplastic candidiasis. Hairy leukoplakia occurs predominantly on the tongue as in the patient described. The typical presentation is easily diagnosed but atypical cases may be diagnosed by their lack of response to antifungal treatment and histological exclusion of other lesions. The natural history of hairy leukoplakia needs further study. Hairy leukoplakia has not been reported in patients not infected by HIV; therefore, patients with hairy leukoplakia should be considered highly suspect for subsequent development of AIDS.     

Oral hairy leukoplakia in HIV infection: a diagnostic pitfall.

Twenty-nine human immunodeficiency virus (HIV)-infected patients with white, nonremovable lesions on the lateral border of the tongue, clinically suggestive of oral hairy leukoplakia (HL), were studied. In particular, the value of local antifungal therapy in establishing the diagnosis of HL was investigated. In 15 patients (52%) the lesions could be ultimately attributed to a candidal infection of the tongue. In 10 of the remaining 14 patients, a biopsy was obtained from lesions persisting after local antifungal treatment. In all biopsy specimens, the diagnosis of HL was confirmed by histopathologic examination and the demonstration of Epstein-Barr virus DNA by polymerase chain reaction, Southern blot hybridization, and DNA in situ hybridization. The present data confirm that the diagnosis of HL in HIV-infected patients cannot be reliably made on clinical criteria alone, but requires histopathologic confirmation including the demonstration of Epstein-Barr virus DNA, preferably by DNA in situ hybridization. However, with regard to the differential diagnosis of white, nonremovable lesions on the lateral border of the tongue in HIV-infected patients, the present study suggests that persistence of lesions after local antifungal therapy is highly suggestive of HL.     

Macro- and microanatomy of the lateral border of the tongue with special reference to oral hairy leukoplakia

The macro- and microanatomy of the marginal tongue mucosa were studied. Tissues were harvested from 12 individuals at autopsy. Random sections from six different locations along the margin and serial sections representing three defined section planes to the margin of the tongue were evaluated. Vertical, parallel mucosal folds alternating with shallow grooves were a characteristic macroscopical findings on the lateral border of the tongue. The mucosa presented a non-keratinized epithelium with PAS-positive, lightly stained spinous cells and no or slight inflammatory reaction in the connective tissue. Changes in the epithelium mimicking hyperplasia, acanthosis, keratin projections, and focal parakeratosis could be produced by changing the direction of tissue sectioning. The macro- and microscopical parameters recorded in normal marginal tongue mucosa are among other included in criteria for diagnosing oral hairy leukoplakia. The results emphasize the importance of a thorough knowledge of the normal anatomy of a mucosal site to arrive at reliable diagnostic criteria.     

Oral hairy leukoplakia in an HIV-negative renal transplant recipient

Oral hairy leukoplakia (HL) has been seen exclusively in those infected with HIV or at risk for AIDS. This case report describes an example of HL seen in a renal transplant recipient who was negative for HIV on serology and culture. The diagnosis of HL was confirmed using in situ hybridization for EBV DNA.     

Oral hairy leukoplakia with extensive oral mucosal involvement. Report of two cases

Oral hairy leukoplakia (HL) is a lesion that occurs predominantly on the tongue in HIV-infected persons. Evidence strongly indicates that HL is related to the presence of Epstein-Barr virus in the epithelial cells. The lesion appears on the lateral border of the tongue as a painless, white plaque varying in size from a few millimeters to extensive lingual involvement. Histopathologically, the characteristic findings are hyperparakeratosis, hyperplasia, and ballooning of prickle cells resembling koilocytosis. HL is now considered a frequent, early, and specific sign of HIV infection and a strong indicator that AIDS will develop in the patient. We report on two cases of HL with marked oral mucosal involvement with extension to the pharyngeal mucosa.     

Altered patterns of keratin expression in oral hairy leukoplakia: prognostic implications

To establish why the lateral border of tongue is the site of predilection for the development of hairy leukoplakia (HL) and to understand its likely behavior, the pattern of keratin expression was compared in 8HL lesions with matched controls in an immunocytochemical study. Keratins 7, 8, 18 were absent in HL and normals; uniform basal keratin 19 was present in normals but much reduced in HL. Loss of conformationally sensitive epitopes of keratin 14 in lower epithelial layers was seen in HL. Overall expression of non-cornifying keratins 4/13 was reduced in HL and completely lost in the parakeratin zone. Expression of the high-turnover keratins 6/16 was reduced in HL. The HL keratin phenotype suggests that no dysplastic change is likely, but in contrast there is enhanced differentiation, which suggests a benign course for the condition.     

Oral hairy leukoplakia arising in an oral lichen planus lesion in an otherwise immune-competent patient

Oral hairy leukoplakia (OHL) presents clinically as thickened white plaques which cannot be rubbed off, and is most often found on the lateral margin of the tongue. The name is derived from its often hairy appearance. OHL has clinical significance because of its association with HIV infection, and it is rare in immune-competent people. We report on a case of unilateral OHL which arose in an oral lichen planus lesion in an immune-competent patient.     

Crystalline inclusions in epithelial cells of hairy leukoplakia: a new ultrastructural finding

Hairy leukoplakia is often the first manifestation of an HIV infection. In the present study we have investigated the ultrastructural features of hairy leukoplakia in two HIV seropositive male homosexual patients. Ultrastructurally EBV particles were found in the upper prickle and keratinized epithelial cells, whereas papilloma virus particles were not found. Candida albicans were also seen in the keratinized layer. In addition two types of inclusions which have not previously been reported in hairy leukoplakia were commonly seen in the epithelial cells containing the EBV. The first was a crystalline microtubular structure which may take the form of several arrays. The second was an elongated multivesicular structure consisting of membrane-bound rounded vesicles embedded in a background of fine filaments. The vesicles were similar to the empty looking EBV particles and their mean diameter was 100 nm. Further investigations are needed to elucidate the exact nature of these structures and their relationship to the Epstein-Barr virus.     

Oral hairy leukoplakia in healthy immunocompetent patients: a small case series

Background

Oral hairy leukoplakia (OHL) is caused by the Epstein-Barr virus (EBV) and usually presents in patients with human immunodeficiency virus (HIV) infection and systemic immunosuppression. It is rarely seen in patients who are immunocompetent. It is clinically characterised as an asymptomatic, soft, white and corrugated lesion that cannot be scraped from the surface it adheres to.

Methods

Immunocompetent patients with OHL attending Bristol Dental Hospital within the last 6 months were identified. EBV infection was demonstrated using EBV in situ hybridization. Clinical features and medical history were determined by reviewing medical records.

Case report

Four cases of OHL in immunocompetent individuals were identified. All lesions were located on the lateral borders of the tongue.

Discussion

OHL should be considered as a differential diagnosis for white patches on the lateral borders of the tongue in apparently healthy immunocompetent patients, even when they do not have a typical corrugated appearance. OHL should no longer be regarded as pathognomonic for HIV infection or systemic immunosuppression.

     

Hairy leukoplakia: Epstein-Barr virus receptors on oral keratinocyte plasma membranes

Hairy leukoplakia (HL) is an oral white lesion associated with, and probably caused by, the Epstein-Barr virus (EBV) among persons who are seropositive for infection with human immunodeficiency virus. A unique feature of HL is its localization to the lateral portion of the tongue. To determine site differences for EBV receptors according to epithelial phenotype, these receptors were mapped in oral mucosa with the use of monoclonal antibodies HB5 and B2(specific for the Complement Fraction 3d/EBV receptor on B lymphocytes). Immunoperoxidase and immunofluorescence techniques were employed with the use of both cytologic suspensions and frozen tissue sections of oral epithelium. Pericellular plasma membrane immunoreactants were localized to upper spinous layer cells of the parakeratin phenotype; basal and parabasilar layers as well as all strata of orthokeratinized epithelia were negative. Those cells harboring EBV DNA as detected by in situ hybridization corresponded to cells with C3d/EBV receptors.