Oral Hairy Leukoplakia

Background

This interesting disease is found primarily in patients with a severe immunodeficiency such as in patients with AIDS. It presents with flat to hairy white lesions on the lateral or ventral borders of the tounge. It is asymptomatic and related to a patient's immune status.

EPIDEMIOLOGY CHARACTERIZATION

INCIDENCE 20% of asymptomatic HIV infected patients
Incidence grows as CD4 count decreases

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

Ammatuna P, Capone F, Giambelluca D, Pizzo I, D'Alia G, Margiotta V.

Department of Hygiene and Microbiology, University of Palermo, Italy.
J Oral Pathol Med 1998 Oct;27(9):420-7 Abstract quote
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 saliva samples of 16 transplant patients (40%) and three healthy 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.

Presence of Epstein-Barr virus, cytomegalovirus and human papillomavirus in normal oral mucosa of HIV-infected and renal transplant patients.

Ammatuna P, Campisi G, Giovannelli L, Giambelluca D, Alaimo C, Mancuso S, Margiotta V.

Departments of Hygiene and Microbiology, University of Palermo, Palermo, Italy.
Oral Dis 2001 Jan;7(1):34-40 Abstract quote
OBJECTIVES: To determine the prevalence of EBV-DNA, CMV-DNA and HPV-DNA in oral healthy mucosa of HIV-infected and renal transplant patients. To associate the detection of viral genomes with laboratory parameters of immunodeficiency, gender, antiretroviral and immunosuppressive therapy.

DESIGN: A cross-sectional analysis of lingual and buccal cytobrushings from HIV-infected and renal transplant patients.

SUBJECTS AND METHODS: Lingual and buccal cytobrushings were obtained from clinically normal oral mucosa of 57 HIV+, 40 renal transplant patients and 30 healthy uninfected controls, all matched for age at baseline of examination. Presence of EBV-, CMV- and HPV-DNA was assessed by polymerase chain reaction (PCR). We evaluated their association, in HIV+ subjects, with gender, CD4+ cell count, HIV-RNA load, and antiretroviral therapy; and in renal transplant patients, with gender, CD4/CD8 ratio, and immunosuppressive therapy. Data were managed and analysed by Epi-Info 6.0.

RESULTS: EBV-DNA was detected in 42.1% of HIV+ (24/57), in 65.0% of transplant patients (26/40), and in 16.6% of controls (5/30) (P = 0.03 and P = 0.0001, respectively). Furthermore, male gender in HIV+ group was found to be significantly associated with the presence of EBV-DNA (P = 0.02) vs females, after adjusting for CD4+ cell count and HIV-RNA load. CMV- and HPV-DNA were detected in 3.5% and 7.0% of HIV+, and in none and 20.0% of transplant patients, respectively. No relationship was found between the epithelial detection of these two viruses and any parameter evaluated.

CONCLUSIONS: EBV genome was significantly detected in clinically normal oral mucosa of renal transplant and HIV+ patients. A significant gender association was found among HIV+, suggesting that oral hairy leukoplakia (OHL) is more likely to occur in HIV+ men than women.

DISEASE ASSOCIATIONS CHARACTERIZATION

IMMUNE STATUS

Oral hairy leukoplakia in nonimmunosuppressed patients. Report of four cases.

Lozada-Nur F, Robinson J, Regezi JA.

Department of Stomatology, University of California San Francisco.
Oral Surg Oral Med Oral Pathol 1994 Nov;78(5):599-602 Abstract quote
Hairy leukoplakia was first described in association with HIV infection. Today hairy leukoplakia has come to represent a sign of immunosuppression and not just of HIV infection. Although molecular mechanisms have not been fully elucidated, Epstein-Barr virus appears to play a significant role in its etiopathogenesis.

We present four cases that illustrate that HL may be seen in non-HIV and nonimmunocompromised patients. The use of high potency topical steroids for the treatment of oral vesiculoerosive diseases seems to have been a contributing factor in two of these cases.

LEUKEMIA

Oral hairy leukoplakia in a patient with acute lymphocytic leukemia.

Nicolatou O, Nikolatos G, Fisfis M, Belegrati M, Papadaki T, Oikonomaki E, Kalmantis T.

Department of Oral Pathology and Surgery, School of Dentistry, University of Athens, Greece.
Oral Dis 1999 Jan;5(1):76-9 Abstract quote
The first case of oral hairy leukoplakia (OHL) in an HIV-negative 56-year-old patient with acute lymphocytic leukemia (ALL) is reported. A white plaque was observed while the patient was in complete remission which followed the chemotherapeutic scheme.

The clinical and histopathologic findings were typical for OHL and the polymerase chain reaction method was positive for Epstein-Barr virus DNA. Underdiagnosis and underreporting of OHL in patients with a malignant haematological disease and the apparent different environmental factors to which these non-AIDS patients have been exposed, probably constitute some of the reasons for the very few OHL cases reported in these patients.

MULTIPLE MYELOMA

Oral hairy leukoplakia in a patient with multiple myeloma.

Blomgren J, Back H.

Clinic of Oral Medicine, Ostra University Hospital, Goteborg, Sweden.
Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996 Oct;82(4):408-10 Abstract quote
The first case of oral hairy leukoplakia in an HIV-negative patient with multiple myeloma is reported.

The patient is a 56-year old man who has had monoclonal gammopathy of undetermined significance since 1986 and has been treated for a symptomatic multiple myeloma since 1993. The clinical and histopathologic findings are typical for oral hairy leukoplakia, and Epstein-Barr virus was demonstrated with polymerase chain reaction technique.

Although a relatively large number of cases of oral hairy leukoplakia has been reported in HIV-negative patients, both immunocompromised and immunocompetent, only a few of these patients have had a malignant hematologic disease.

RENAL TRANSPLANT

Prevalence and risk factors associated with leukoplakia, hairy leukoplakia, erythematous candidiasis, and gingival hyperplasia in renal transplant recipients.

King GN, Healy CM, Glover MT, Kwan JT, Williams DM, Leigh IM, Thornhill MH.

Department of Oral Medicine and Periodontology, London Hospital Medical College, University of London, England.

Oral Surg Oral Med Oral Pathol 1994 Dec;78(6):718-26 Abstract quote
The aims of this study were to determine the prevalence of intraoral lesions in renal transplant recipients and to identify possible risk factors.

The oral mucosa of 159 renal transplant recipients and 160 control patients was examined. The most common lesion in renal transplant recipients was cyclosporin-induced gingival hyperplasia (prevalence 22%) and patients with gingival hyperplasia were found to be taking significantly more cyclosporin-A than those without (p < 0.001). The prevalence of hairy leukoplakia and leukoplakia in renal transplant recipients was 11.3% and 10.7%, respectively, compared with 0% and 5.6% in the controls. Oral candidiasis was observed in 9.4% of renal transplant recipients compared with 2.5% of the controls; 3.8% of renal transplant recipients exhibited erythematous candidiasis, but this was not seen in the controls.

Renal transplant recipients had a significantly increased risk of developing gingival hyperplasia (p < 0.0001), oral candidiasis (p < 0.0005), and two other conditions that have a well-established association with the immune suppression accompanying HIV infection, hairy leukoplakia (p < 0.0001) and erythematous candidiasis (p < 0.01).

SMOKING

Effect of receptive oral sex and smoking on the incidence of hairy leukoplakia in HIV-positive gay men.

Shiboski CH, Neuhaus JM, Greenspan D, Greenspan JS.

Oral AIDS Center, Department of Stomatology, University of California, San Francisco, 94143-0422, USA
J Acquir Immune Defic Syndr 1999 Jul 1;21(3):236-42 Abstract quote
We sought to determine whether hairy leukoplakia (HL), an Epstein-Barr virus-related oral lesion, is associated with receptive oral sex activity and cigarette smoking among HIV-positive gay men. Oral examinations were conducted every 6 months among San Francisco Men's Health Study participants over a 6-year period.

We fitted time-to-lesion regression models to compare the incidence of HL among men who had mouth-to-penis contact with various numbers of partners, while controlling for cigarette smoking and CD4 count. The 6-year incidence of HL was 32% among 291 HIV-positive men. We found no significant increase in the hazard of developing HL for each additional insertive-oral-sex male partner in the past 6 months (relative hazard = 1.01; 95% confidence interval [CI], 0.99, 1.02), and a similar lack of association when number of sex partners was categorized.

However, the hazard of developing HL doubled with any 300-unit decrease in CD4 count (95% CI, 1.4, 2.7), or if men smoked > or =20 cigarettes/day compared with nonsmokers (95% CI, 1.2, 3.9). This finding, which may suggest one effect that smoking produces on the oral mucosa's local immune response, merits further investigation.

PATHOGENESIS CHARACTERIZATION

EPSTEIN-BARR VIRUS INFECTION

The Epstein-Barr virus EBNA-2 gene in oral hairy leukoplakia: strain variation, genetic recombination, and transcriptional expression.

Walling DM, Perkins AG, Webster-Cyriaque J, Resnick L, Raab-Traub N.

Division of Infectious Diseases, University of North Carolina, Chapel Hill 27599.
J Virol 1994 Dec;68(12):7918-26 Abstract quote
Oral hairy leukoplakia (HLP) lesions frequently contain defective Epstein-Barr virus (EBV) genomes with deletions in the EBNA-2 gene that abundantly replicate and persist within the lesion.

To characterize these viral strains and recombinant variants, the EBNA-2 gene in EBV DNA from several different HLP biopsy specimens was analyzed. Amplification of EBNA-2 coding sequences by PCR demonstrated the presence in HLP of intact EBNA-2 genes as well as a variety of internally deleted variants of both EBNA-2A and EBNA-2B. Some of the deletion variants evolved within the HLP lesion from intact EBNA-2 genes, while other variants appeared to be transmissible strains that directly infected the lesion. Intrastrain recombination within the HLP lesion also generated variation within the EBNA-2 polyproline region. Cloning and sequencing of HLP cDNA demonstrated transcription from the internally deleted EBNA-2 open reading frame, indicating that these variant genes are expressed in HLP. Comparative analysis of the HLP EBNA-2 sequences confirmed previous findings of EBV coinfection with multiple types and strains. Sequence variation of these wild-type genes demonstrated that EBNA-2A sequences distinguish at least two separate strains and a variety of substrains of EBV type 1. Two of the HLP EBNA-2A sequences contained amino acid changes in a cytotoxic T-cell epitope within an otherwise highly conserved region of the gene.

These data indicate that EBV coinfection, strain variation, and recombination within the EBNA-2 gene are common features of HLP and suggest that the expression of internally deleted EBNA-2 variants could contribute to EBV pathogenesis in permissive infection.

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

Mabruk MJ, Flint SR, Toner M, Leonard N, Sheils O, Coleman DC, Atkins GJ.

Department of Microbiology, Moyne Institute, Trinity College, Dublin, Republic of Ireland.

J Oral Pathol Med 1995 Mar;24(3):109-12 Abstract quote
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.

Epstein-Barr virus coinfection and recombination in non-human immunodeficiency virus-associated oral hairy leukoplakia.

Walling DM, Clark NM, Markovitz DM, Frank TS, Braun DK, Eisenberg E, Krutchkoff DJ, Felix DH, Raab-Traub N.

Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7295, USA.
J Infect Dis 1995 May;171(5):1122-30 Abstract quote
Human immunodeficiency virus-associated oral hairy leukoplakia (HLP) is characterized by coinfection with multiple types and strains of Epstein-Barr virus (EBV) and recombination within the EBV genome.

HIV-seronegative immunosuppressed and immunocompetent patients with HLP were examined to determine the pathogenic contribution of EBV coinfection and recombination to the development of HLP. Multiple coinfecting EBV strains were detected in both HLP specimens and peripheral blood lymphocytes (PBL) of HIV-seronegative persons with HLP. One specific EBV strain was detected in HLP specimens from 3 of 4 patients. Also, viral recombination during productive replication within HLP generated variants of the latent membrane protein-1 (LMP-1) and nuclear antigen-2 (EBNA-2) genes. Some variants were also detected within PBL.

Thus, EBV coinfection and recombination are consistent findings in persons with HLP regardless of immune status. Virally mediated determinants may be important features of EBV pathogenesis.

Epstein-Barr virus (EBV) DNA in saliva and EBV serology of HIV-1-infected persons with and without hairy leukoplakia.

Lucht E, Biberfeld P, Linde A.

Department of Clinical Virology, Swedish Institute for Infectious Disease Control, Stockholm, Sweden.
J Infect 1995 Nov;31(3):189-94 Abstract quote
Secretion of Epstein-Barr virus (EBV) in saliva, as well as serum antibody titres against various EBV antigens, were analyzed in respect of (1) 15 HIV-1-infected patients with oral hairy leukoplakia proven to contain EBV by in situ hybridization, (2) 45 HIV-1 infected patients without hairy leukoplakia, (3) 10 HIV-1 infected patients treated with acyclovir or foscarnet and (4) 21 healthy controls. The numbers of CD4+ cells in the peripheral blood were also recorded.

The HIV-1 infected patients were at various stages of HIV-1-associated disease. Excretion of EBV DNA in the saliva was determined by means of the polymerase chain reaction (PCR) while the amount of EBV DNA in positive samples was estimated by repeated titrations. The frequency of shedding of EBV DNA increased from 33% in healthy controls to 78% in asymptomatic HIV-1 infected persons, but did not increase significantly with progression of HIV-1-associated disease. The titres of EBV DNA in saliva correlated inversely and significantly with the number of CD4+ cells in the peripheral blood.

All patients with hairy leukoplakia shed by EBV DNA in their saliva but the titres were not significantly higher than those of other HIV-1 infected persons. The serum titres of antibodies against EBV nuclear antigen 1 (EBNA-1) correlated positively and significantly with the CD4+ cell count in the peripheral blood. EBNA-1 IgG antibody in the serum was also significantly lower in symptomatic than in asymptomatic HIV-1 infected persons. There were, however, no significant differences in serum antibodies to various EBV antigens between patients with and without hairy leukoplakia.

Epstein-Barr virus reactivation in hairy leukoplakia.

Brandwein M, Nuovo G, Ramer M, Orlowski W, Miller L.

Department of Pathology and Oral Pathology, Mount Sinai School of Medicine, New York 10021, USA.
Mod Pathol 1996 Mar;9(3):298-303 Abstract quote
Hairy leukoplakia, often seen in patients with acquired immunodeficiency syndrome, is strongly associated with Epstein-Barr virus (EBV) infection and questionably associated with human papillomavirus (HPV) infection.

To date, most in situ hybridization (ISH) studies suggest that the EBV is localized only to the superficial squamous layers, favoring the theory of lingual infection by saliva rather than by reactivation of latent lingual infection. We describe 11 formalin-fixed, paraffin-embedded specimens from patients with lingual hairy leukoplakia that we examined for the presence of the EBV, HPV, cytomegalovirus, and human immunodeficiency virus. We used standard DNA ISH for the EBV and cytomegalovirus and polymerase chain reaction ISH for the EBV, HPV, and human immunodeficiency virus. The EBV was present in all 11 specimens according to polymerase chain reaction ISH studies but in only seven specimens according to conventional DNA ISH. Polymerase chain reaction ISH localized the EBV to the basal and parabasal layers in addition to the strong localization in the upper epithelial layers. No evidence for HPV or cytomegalovirus DNA was found. The human immunodeficiency virus was focally localized to rare superficial squamous cells in seven specimens.

The presence of EBV DNA in basal and parabasal lingual cells, as well as localization of latency-associated proteins in these layers, suggests that hairy leukoplakia in patients with acquired immunodeficiency syndrome might represent a reactivation of latent lingual infection accompanied by a dramatic increase in viral copy number in the more mature, superficial, squamous cells. The human immunodeficiency virus was also found in seven specimens, but the significance of this new finding is uncertain and requires further study. There is no evidence to suggest that the HPV is involved in the development of hairy leukoplakia.

Expression of Epstein-Barr virus BMRF-2 and BDLF-3 genes in hairy leukoplakia.

Penaranda ME, Lagenaur LA, Pierik LT, Berline JW, MacPhail LA, Greenspan D, Greenspan JS, Palefsky JM.

Department of Stomatology, University of California, San Francisco 94143, USA.
J Gen Virol 1997 Dec;78 ( Pt 12):3361-70 Abstract quote
The high level of Epstein-Barr virus (EBV) replication found in hairy leukoplakia (HL) provides a unique opportunity to study EBV expression in the oral epithelium. Screening of a cDNA library from an HL biopsy revealed expression of two genes not previously described in vivo: BMRF-2 and BDLF-3. Sequence analysis of the cDNAs demonstrated several nucleotide changes from the B95-8 sequence.

In all six different HL strains studied, only one amino acid change was found in BMRF-2 relative to B95-8 and two amino acid changes were found in the BDLF-3 ORF. mRNA expression of both genes was localized to the lower prickle cell layer of the tongue epithelium. BMRF-2 protein expression was primarily detected in the cell nuclei of the upper prickle cell layer; immunoelectron microscopy revealed that BMRF-2 was associated with the nuclear chromatin. BDLF-3 protein expression was observed in the perinuclear space and cytoplasm of the prickle cells. BDLF-3 has recently been identified as a virion-associated protein, but the functions of BMRF-2 and BDLF-3 have not been elucidated.

Transcription of Epstein-Barr virus latent cycle genes in oral hairy leukoplakia.

Webster-Cyriaque J, Raab-Traub N.

University of North Carolina Hospitals, University of North Carolina, Chapel Hill, North Carolina, 27599, USA.
Virology 1998 Aug 15;248(1):53-65 Abstract quote
The hairy leukoplakia lesion (HLP) is a unique example of a permissive infection with Epstein-Barr virus (EBV) in the tongue epithelium. HLP contains abundant replicating viral DNA and may be coinfected with multiple EBV strains.

In this study, characterization of viral gene transcription within HLP biopsy specimens revealed that several genes, usually expressed in latently infected lymphocytes, are also transcribed in the HLP lesion. The BamHI W and C promoters, (Wp and Cp) are consistently active in the HLP lesion, resulting in transcription and processing of mRNAs that encode the Epstein-Barr nuclear antigens (EBNAs) EBNA-LP, EBNA1, EBNA2, EBNA3B, and EBNA3C. The EBNA2 protein has been shown to activate expression of the EBV receptor, CD21. In HLP, CD21 transcription is also detected, usually in samples that contain transcripts for EBNA2. Transcripts encoding the LMP1 gene, the LMP2 gene, and rightward transcripts from the BamHI A fragment of the EBV genome are also detected in HLP. These gene products are invariably expressed in latently infected lymphocytes. This pattern of transcription suggests that genes characteristic of latent infection are also expressed in HLP.

The activation of Wp and expression of EBNA2 and CD21 may contribute to the unique ability of the HLP lesion to permit superinfection and viral replication of multiple EBV strains.

Human immunodeficiency virus-positive individuals with oral hairy leukoplakia are able to mount cytotoxic T lymphocyte responses to Epstein-Barr virus.

de Jong A, Palefsky JM, Stites DP, Nakagawa M.

Department of Laboratory Medicine, School of Medicine, University of California San Francisco, San Francisco, California 94143, USA.
Oral Dis 2000 Jan;6(1):40-7 Abstract quote
OBJECTIVE: Oral hairy leukoplakia (OHL) is a white lesion of the tongue that is caused by Epstein-Barr virus (EBV) and occurs mainly in people infected with human immunodeficiency virus (HIV). The aim of this study was to determine whether the presence of OHL reflects the absence of EBV-specific cytotoxic T lymphocyte (CTL) activity.

SUBJECTS AND METHODS: EBV-specific CTL responses were measured in HIV-positive homosexual men with OHL, HIV-positive homosexual men without OHL, and HIV-negative homosexual men. Also, the phenotypes of cells responsible for EBV-specific responses were studied.

RESULTS: Eighty percent (8/10) of HIV-positive subjects with OHL, 52% (12/23) of HIV-positive subjects without OHL, and 83% (15/18) HIV-negative subjects had a positive anti-EBV CTL response (P = 0.004, Kruskal-Wallis test). Two HIV-positive subjects showed a greater anti-EBV CTL response after developing OHL than before the appearance of OHL Additional experiments showed that CD8-positive T cells and CD4-positive T cells were responsible for the EBV-specific CTL responses.

CONCLUSION: Our data show more EBV-specific CTL activities in HIV-positive individuals with OHL than in HIV-positive individuals without OHL. Whether the presence of EBV-specific CTL contributes to resolution of OHL remains to be clarified.

LABORATORY/RADIOLOGY CHARACTERIZATION

LABORATORY

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

Mabruk MJ, Antonio M, Flint SR, Coleman DC, Toner M, Kay E, Leader M, Atkins GJ.

Department of Pathology, Royal College of Surgeons in Ireland and Beaumont Hospital, Dublin.
J Oral Pathol Med 2000 Mar;29(3):118-22 Abstract quote
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.

CD4 LEVELS

Immunologic status in patients infected with HIV with oral candidiasis and hairy leukoplakia.

Kolokotronis A, Kioses V, Antoniades D, Mandraveli K, Doutsos I, Papanayotou P.

School of Dentistry, Aristotle University of Thessaloniki, Specific Infectious Disease Unit, Greece.
Oral Surg Oral Med Oral Pathol 1994 Jul;78(1):41-6 Abstract quote
Although numerous studies of oral manifestations associated with HIV have been reported, only a few refer to the correlation of these lesions with laboratory parameters. In this study we investigated the relationships between the two most common HIV-associated oral lesions, oral candidiasis and hairy leukoplakia, with the stage of the disease, circulating CD4+ cell counts, and the presence of anti-p24 antibodies in serum and stimulated whole saliva in 43 known HIV-1-infected persons.

Although oral candidiasis and hairy leukoplakia were exclusively observed in subjects who were classified as Centers for Disease Control and Prevention group IV, only the prevalence of oral candidiasis is strongly associated with circulating CD4+ counts less than 200/mm3 (p < 0.02). The prevalence of oral candidiasis and hairy leukoplakia was significantly related to the absence of anti-p24 antibodies in serum (p < 0.01 and p < 0.01, respectively), but was only statistically significant for hairy leukoplakia in stimulated whole saliva (p < 0.02). The results suggest that oral candidiasis and hairy leukoplakia in correlation with immunologic status as indicated by low circulating CD4+ cell counts and the absence of anti-p24 antibodies in serum and the loss of secretory anti-p24 antibodies in subjects with hairy leukoplakia, may constitute prognostic markers for the progression of HIV-infection to AIDS.

Our results also indicate that the absence of anti-p24 antibodies is not only influenced by the low levels of circulating CD4+ cells but probably by the presence of oral candidiasis or hairy leukoplakia as well.

Relationship of circulating CD4+ T-lymphocytes and p24 antigenemia to the risk of developing AIDS in HIV-infected subjects with oral hairy leukoplakia.

Ravina A, Ficarra G, Chiodo M, Mazzetti M, Romagnani S.

Division of Allergy and Clinical Immunology, Polyclinic of Careggi, Florence, Italy.
J Oral Pathol Med 1996 Mar;25(3):108-11 Abstract quote
To investigate the relationship of circulating CD4+ T-lymphocytes and p24 antigenemia to the development of AIDS in HIV-infected subjects with hairy leukoplakia (HL), we have followed over eight years 173 HIV-positive patients, all asymptomatic at the start of the study, and compared those who developed HL (n=55) to those who did not (n=118). Both groups included injection drug users (IDUs), homosexual men, and hemophiliacs. At the time of their first visit, both HL+ and HL- patients had a normal value of CD4+ cells and a low frequency of p24 antigenemia.

During the years of follow-up, patients in the HL+ group showed a greater reduction in CD4+ cells, a significant increase in p24 antigenemia, and a higher rate of progression to AIDS.

Our study demonstrates that in HIV-positive patients, HL is associated with more compromised immunological parameters and a higher viral replication and that its appearance has a negative prognostic value in relation to AIDS progression.

CLINICAL VARIANTS CHARACTERIZATION

GENERAL

Oral hairy leukoplakia: clinicopathologic features, pathogenesis, diagnosis, and clinical significance.

Triantos D, Porter SR, Scully C, Teo CG.

Department of Oral Medicine, Eastman Dental Institute for Oral Health Care Sciences, University of London, United Kingdom.
Clin Infect Dis 1997 Dec;25(6):1392-6 Abstract quote
Oral hairy leukoplakia (OHL) is a lesion frequently, although not exclusively, observed in patients infected by human immunodeficiency viruses (HIV). OHL is clinically characterized by bilateral, often elevated, white patches of the lateral borders and dorsum of the tongue.

Histologically, there is profound acanthosis, sometimes with koilocytic changes, and a lack of a notable inflammatory infiltrate. The koilocytic changes are due to intense replication of Epstein-Barr virus (EBV), while epithelial hyperplasia and acanthosis are likely to result from the combined action of the EBV-encoded proteins, latent membrane protein-1, and antiapoptotic BHRF1.

How OHL is initiated and whether it develops after EBV reactivation from latency or superinfection remain unresolved; nevertheless, definitive diagnosis requires the demonstration of EBV replicating vegetatively in histological or cytological specimens. In patients with HIV infection, the development of OHL may herald severe HIV disease and the rapid onset of AIDS, but despite its title, OHL is not regarded as premalignant and is unlikely to give rise to oral squamous cell carcinoma.

PEDIATRIC

Oral hairy leukoplakia in two children with perinatally acquired HIV-1 infection.

Mannelli F, Galli L, Ficarra G, Romagnoli P, Vierucci S, de Martino M.

Department of Pediatrics, University of Florence, Italy.
Pediatr AIDS HIV Infect 1995 Feb;6(1):21-3 Abstract quote
We report on two children with acquired immunodeficiency syndrome (AIDS), a 5-year-old girl and a 9-year-old boy, who presented with oral hairy leukoplakia (OHL) as a late clinical manifestation of perinatally acquired human immunodeficiency virus type 1 (HIV-1) infection.

The reasons why OHL is a rare symptom in HIV-1-infected children, compared with HIV-1-infected adults, are discussed.

HISTOLOGICAL TYPES CHARACTERIZATION

GENERAL Ballooning degeneration of the keratinocytes of the upper stratum spinosum
Hyperparakeratosis
Mild or absent subepithelial inflammation

CYTOLOGY Oral cytology may reveal keratinocytes with intranuclear inclusions with ground-glass appearance

Use of exfoliative cytology in the diagnosis of oral hairy leukoplakia.

Migliorati CA, Jones AC, Baughman PA.

Department of Oral Diagnostic Sciences, University of Florida College of Dentistry
Oral Surg Oral Med Oral Pathol 1993 Dec;76(6):704-10 Abstract quote
The possibility of diagnosing oral hairy leukoplakia by means of exfoliative cytology and the Papanicolaou stain was investigated.

Exfoliative cytology and punch biopsy specimens were obtained from 10 lesions that demonstrated clinical features of hairy leukoplakia. All biopsy specimens demonstrated the characteristic histopathologic features of hairy leukoplakia whereas all Papanicolaou-stained cytologic smears demonstrated condensation and margination of the nuclear chromatin (nuclear beading). All biopsy specimens and cytologic smears displayed positive Epstein-Barr virus deoxyribonucleic acid in situ hybridization.

We conclude that routine exfoliative cytology may be a reliable, noninvasive, and inexpensive technique for the diagnosis of hairy leukoplakia.

Oral hairy leukoplakia. Histopathologic and cytopathologic features of a subclinical phase.

Dias EP, Rocha ML, Silva JuniorA, Spyrides KS, Ferreira SM, Polignano GA, Feijo EC, Da Fonseca EC.

Department of Pathology, Universidade Federal Fluminense, Niteroi/RJ, Brazil.
Am J Clin Pathol 2000 Sep;114(3):395-401 Abstract quote
Accurate diagnosis of oral hairy leukoplakia (OHL) is important because it may be an early indicator of undiagnosed HIV infection; moreover, it may be a prognostic indicator.

Our purpose was to investigate the histopathologic features of subclinical OHL and to evaluate and support the rationale of detecting subclinical OHL with cytopathology. The Epstein-Barr virus (EBV) was detected by immunohistochemistry and in situ hybridization in 4 cases of macroscopically normal lateral borders of tongue mucosa from 8 AIDS necropsies and in none of 8 controls. The histopathologic features were specific when based on nuclear changes: Cowdry type A inclusion, ground glass, and nuclear beading. Smears were obtained from 50 patients with AIDS, without OHL, from the scraping of lateral borders of the tongue. Numerous clusters of the cells were associated with Candida organisms (30% of cases). Nuclear changes were observed in 12 patients (24%) on both sides of the tongue.

We describe the histopathologic features of subclinical OHL, and our observations suggest that cytopathology can detect OHL in the subclinical phase.

SPECIAL STAINS/IMMUNOHISTOCHEMISTRY CHARACTERIZATION

IMMUNOHISTOCHEMISTRY

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

Cruchley AT, Murray PG, Niedobitek G, Reynolds GM, Williams DM, Young LS.

Department of Oral Pathology, St Bartholomew's, London, UK.
Oral Dis 1997 May;3 Suppl 1:S177-9 Abstract quote
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.

IN-SITU HYBRIDIZATION

A rapid microwave-in situ hybridization method for the definitive diagnosis of oral hairy leukoplakia: comparison with immunohistochemistry.

Mabruk MJ, Flint SR, Coleman DC, Shiels O, Toner M, Atkins GJ.

University of Dublin, Moyne Institute of Preventive Medicine, Department of Microbiology, Republic of Ireland.
J Oral Pathol Med 1996 Apr;25(4):170-6 Abstract quote
As a diagnostic technique, in situ hybridization requires a long processing time, a degree of expertise and may be difficult to handle routinely in some laboratories.

To simplify the in situ hybridization method, we have modified a microwave in situ hybridization technique and applied it to oral hairy leukoplakia (OHL) biopsies obtained from 10 HIV-seropositive patients (definitively diagnosed by a conventional in situ hybridization technique) with appropriate controls. It was necessary to design a novel chamber to avoid drying of sections during the hybridization step. This modified microwave in situ hybridization technique was equispecific and equisensitive to the conventional technique and it shortens the hybridization time from overnight incubation to 14 minutes.

To determine the sensitivity of our microwave in situ hybridization method we applied it to previously documented tongue tissue obtained from an AIDS autopsy without clinical evidence of OHL, but found to contain Epstein-Barr virus (EBV) by conventional in situ hybridization. This tissue specimen acted as a low EBV copy number, positive control. The sensitivity of immunohistochemistry using three different commercial detection kits was compared to that of in situ hybridization on the same tissues, following optimisation steps. This included the use of 2 cycles of primary and biotinylated secondary antibodies (antibody double cycling). Clearly positive signals for EBV were detected in all OHL biopsies with the Vectastain Elite ABC and the Histostain-SP kits. The sensitivity of the three commercial detection kits was evaluated at immunohistochemistry level by their application to the low-EBV copy number positive control specimen. Signals for EBV antigen in the low copy number positive control specimen were obtained only with the Vectastain Elite ABC kit.

This indicates that, in this application, use of the Vectastain Elite ABC kit gives comparable sensitivity for immunohistochemistry to that found by in situ hybridiation.

CYTOKERATIN

Expression of keratin 14 and 19 mRNA and protein in normal oral epithelia, hairy leukoplakia, tongue biting and white sponge nevus.

Su L, Morgan PR, Thomas JA, Lane EB.

Department of Oral Medicine, UMDS, London, England.
J Oral Pathol Med 1993 Apr;22(4):183-9 Abstract quote
This study was undertaken to analyze keratin gene expression at both the mRNA and protein level in oral hairy leukoplakia (OHL). Comparisons were made with normal lingual epithelium from a similar site, tongue biting, normal buccal mucosa and another condition which disturbs oral epithelial differentiation, white sponge nevus.

Combined immunocytochemical and in situ hybridization studies for keratins 14 and 19 were carried out on 2 specimens of OHL from HIV-positive males and one sample each of the other cases. Keratin 14 protein expression was uniform throughout all the epithelia. In normal epithelia and in lesions other than OHL, keratin 14 mRNA was most strongly expressed in basal cells with weaker but still significant amounts in the spinous cell layer. In both cases of OHL there was weaker basal cell expression of keratin 14 mRNA and frequent absence in koilocytoid cells. Keratin 19 protein expression was heterogeneous in the basal layer of all specimens with suprabasal staining of occasional groups of cells. Its mRNA was uniformly distributed in all cases.

The findings indicate the keratin mRNA expression does not always parallel that of protein and that, in the case of keratin 14, expression may be influenced by the presence of EBV.

ELECTRON MICROSCOPY

Oral hairy leukoplakia with ultrastructural evidence of Merkel-like cells in human tongue epithelium.

Cobb M, MacNeill R, Tobin J.

Department of Periodontics, School of Dentistry, University of Missouri-Kansas City, USA
Bull Group Int Rech Sci Stomatol Odontol 1998 Jan-Mar;40(1):24-37 Abstract quote
The purpose of this investigation was to examine cells in the stratum basale and subjacent lamina propria of oral hairy leukoplakia (OHL) specimens for previously unreported morphological changes.

Tongue biopsy specimens were obtained from ten HIV-positive and five healthy male patients and examined by light (LM) and transmission electron microscopy (TEM).

Observations made during the investigation revealed the following: (1) LM comparisons of control and OHL specimens indicated that clear cells in the stratum basale ranged from 3 to 7 per 100 basal epithelial cells in healthy tongue mucosa vs. 6 to 15 in OHL. (2) Merkel-like cells were noted in the stratum basale of control biopsies from the dorsal-lateral tongue surface.However, the frequency of observation was so rare as to imply that their presence was an exception rather than the norm. (3) In contrast, Merkel-like cells in OHL specimens were commonly encountered, indicating a possible unique relationship to the pathology. (4) Merkel-like cells associated with OHL lesions were morphologically similar, in all respects, to that of control specimens except for the presence of large, membrane bound, dense granules that ranged in size from 150-300 nm. (5) All biopsies of OHL exhibited evidence of Epstein Barr virus in the stratum corneum and superficial layers of the stratum spinosum. (6) A mild inflammatory infiltrate associated with OHL specimens revealed cytopathic changes in fibroblasts that appeared related to the presence of lymphoid cell types; indicating a possible cytolytic lymphocyte-mediated degradation of antigen altered host cells.

Oral hairy leukoplakia: an ultrastructural study and review of the literature.

Guccion JG, Redman RS.

Department of Veterans Affairs Medical Center, Washington, DC, USA.
Ultrastruct Pathol 1999 May-Jun;23(3):181-7 Abstract quote
A 39-year-old, homosexual, Caucasian man with a 9-month history of acquired immunodeficiency syndrome by reduced CD4 lymphocyte count alone was found to have extensive, asymptomatic, nonremovable, corrugated, white patches on the lateral borders and ventral aspects of the tongue typical of oral hairy leukoplakia (OHL).

Histologically, irregular hyperparakeratosis, acanthosis, and clusters of ballooned keratinocytes in the stratum spinosum were present in the biopsied lateral tongue. Some of the superficial ballooned keratinocytes had peripherally beaded nuclei, whereas others had ground glass intranuclear inclusions.

Ultrastructurally, the ballooned keratinocytes had three important findings of diagnostic significance. First, frequent herpesvirus nucleocapsids were largely confined to superficial ballooned keratinocytes having marginated and condensed chromatin. In searching for herpesvirus nucleocapsids, the marginated and condensed chromatin was an invaluable marker for cells harboring the virions. Second, the marginated and condensed chromatin frequently had a distinctive punched-out or cribriform appearance. Third, the ground glass intranuclear inclusion bodies consisted of central, medium electron-dense, finely granular material containing frequent herpesvirus nucleocapsids and partially surrounded or capped by prominent, clumped chromatin.

The patient died with progressive multifocal leukoencephalopathy 24 months after OHL was diagnosed.

DIFFERENTIAL DIAGNOSIS KEY DIFFERENTIATING FEATURES

PSEUDO-ORAL HAIRY LEUKOPLAKIA

Pseudo oral hairy leukoplakia in a renal allograft recipient.

Euvrard S, Kanitakis J, Pouteil-Noble C, Chardonnet Y, Touraine JL, Thivolet J.

Department of Dermatology, Hopital Edouard Herriot, Lyon, France.
J Am Acad Dermatol 1994 Feb;30(2 Pt 2):300-3 Abstract quote
Oral hairy leukoplakia (OHL) is a disorder of the tongue associated with Epstein-Barr virus (EBV). OHL is seen mainly in HIV infection but is also rarely seen in the course of iatrogenic immunosuppression, especially in kidney transplantation; OHL is even more rarely seen in immunocompetent hosts. Lesions that clinically and histologically mimicked OHL but were not associated with EBV were recently characterized as pseudo hairy leukoplakia.

We present such a case that occurred in a renal allograft recipient; light and electron microscopy, immunohistochemistry, and in situ hybridization were used to examine the patient for the presence of EBV and human papillomavirus. Two independent treatments with topical retinoid and oral amoxicillin resulted in complete remission.

Pseudo hairy leukoplakia may correspond, at least in some cases, to the conditions known as leukoedema and white sponge nevus; the distinction of these diseases from OHL is of importance because OHL is a hallmark of severe immunosuppression.

PROGNOSIS AND TREATMENT CHARACTERIZATION

PROGNOSTIC FACTORS
30% of persons with HL progressed to late-stage HIV disease characterized by CDC-defined AIDS within 36 months

47% of a group developed CDC-defined AIDS within 2 years and 67% within four years

Progression to CDC-defined AIDS was more rapid in those HIV-infected persons with HL than in those without HL, even after adjustment for CD4+ T-cell count

Time from HIV seroconversion to oral candidiasis or hairy leukoplakia among homosexual and bisexual men enrolled in three prospective cohorts.

Lifson AR, Hilton JF, Westenhouse JL, Canchola AJ, Samuel MC, Katz MH, Buchbinder SP, Hessol NA, Osmond DH, Shiboski S, et al.

Department of Epidemiology and Biostatistics, University of California, San Francisco.
AIDS 1994 Jan;8(1):73-9 Abstract quote
OBJECTIVES: We evaluated time from HIV seroconversion to diagnosis of two common oral lesions associated with HIV infection and disease progression. DESIGN: Oral examinations were performed on homosexual and bisexual men enrolled in prospective cohorts.

SETTING: Homosexual and bisexual men were followed in three epidemiologic cohort studies in San Francisco, California, USA.

PARTICIPANTS: Data were evaluated from 80 men with well-defined dates of HIV seroconversion from 1984 through 1991.

MAIN OUTCOME MEASURES: We determined the cumulative incidence of oral candidiasis and hairy leukoplakia after HIV seroconversion.

RESULTS: Four per cent of men developed oral candidiasis within 1 year after HIV seroconversion, 8% within 2, 15% within 3, 18% within 4, and 26% within 5 years. Nine per cent developed hairy leukoplakia within 1 year, 16% within 2, 25% within 3, 35% within 4, and 42% within 5 years. The median CD4+ count was 391 x 10(6)/l when oral candidiasis was first reported and 468 x 10(6)/l when hairy leukoplakia was first reported.

CONCLUSIONS: Oral candidiasis or hairy leukoplakia appeared in a significant proportion of HIV-infected homosexual and bisexual men. These lesions occurred relatively soon after HIV seroconversion, typically before AIDS. Evaluation of HIV-infected individuals for these lesions has many potential clinical and research benefits, including the possible use of oral lesions as primary end-points in clinical trials.

Oral hairy leukoplakia in 71 HIV-seropositive patients: clinical symptoms, relation to immunologic status, and prognostic significance.

Husak R, Garbe C, Orfanos CE.

Department of Dermatology, University Medical Center Benjamin Franklin, Free University of Berlin, Germany.
J Am Acad Dermatol 1996 Dec;35(6):928-34 Abstract quote
BACKGROUND: Oral hairy leukoplakia (OHL) is a benign hyperplasia of the oral mucosa that is induced by Epstein-Barr virus. It occurs nearly exclusively in men infected with HIV. OHL is a marker of moderate to advanced immunodeficiency and disease progression in patients with HIV infection.

OBJECTIVE: We attempted to determine the clinical characteristics of OHL in a large group of patients infected with HIV and to analyze its relation to immune status and prognosis.

METHODS: A total of 456 patients with HIV-associated skin disorders were evaluated during the years 1982 through 1992. All patients had an oral examination. CD4+ cell counts were obtained within 3 months of the examination.

RESULTS: OHL was diagnosed in 15.6% of 456 patients. The median age of the patients was 35 years. OHL was found most often on the lateral aspect of the tongue; in one patient the lesion covered the entire dorsal surface of the tongue. Significant immunosuppression was present in the majority of patients at the time of OHL diagnosis (median CD4+ T-lymphocyte count, 235/microliter; median CD4+/CD8+ ratio, 0.3). The median survival time was 20 months in patients with OHL. In patients with a higher CD4 cell count (CD4+ T lymphocyte count, > or = 300/microliter) the diagnosis of OHL was associated with shorter survival times (median survival time, 25 months) compared with other patients with HIV (median survival time, 52 months).

CONCLUSION: OHL is a frequent finding in patients with HIV and indicates advanced immunosuppression. Even in patients with more than 300/microliter CD4+ T lymphocytes, OHL is associated with a poor prognosis.

TREATMENT
Asymptomatic and does not require treatment

Disappeared in patients receiving high-dose acyclovir for herpes zoster, presumably because of the anti-EBV activity of acyclovir-lesion usually recurs with cessation of treatment

Am J Clin Pathol 2000;114:395-401.

Commonly Used Terms

Mouth and Throat

Internet Links

Gross or Clinical Photo

Last Updated 6/18/2002

EPIDEMIOLOGY	CHARACTERIZATION
INCIDENCE	20% of asymptomatic HIV infected patients Incidence grows as CD4 count decreases
Detection of Epstein-Barr virus (EBV) DNA and antigens in oral mucosa of renal transplant patients without clinical evidence of oral hairy leukoplakia (OHL). Ammatuna P, Capone F, Giambelluca D, Pizzo I, D'Alia G, Margiotta V. Department of Hygiene and Microbiology, University of Palermo, Italy.	J Oral Pathol Med 1998 Oct;27(9):420-7 Abstract quote 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 saliva samples of 16 transplant patients (40%) and three healthy 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.
Presence of Epstein-Barr virus, cytomegalovirus and human papillomavirus in normal oral mucosa of HIV-infected and renal transplant patients. Ammatuna P, Campisi G, Giovannelli L, Giambelluca D, Alaimo C, Mancuso S, Margiotta V. Departments of Hygiene and Microbiology, University of Palermo, Palermo, Italy.	Oral Dis 2001 Jan;7(1):34-40 Abstract quote OBJECTIVES: To determine the prevalence of EBV-DNA, CMV-DNA and HPV-DNA in oral healthy mucosa of HIV-infected and renal transplant patients. To associate the detection of viral genomes with laboratory parameters of immunodeficiency, gender, antiretroviral and immunosuppressive therapy. DESIGN: A cross-sectional analysis of lingual and buccal cytobrushings from HIV-infected and renal transplant patients. SUBJECTS AND METHODS: Lingual and buccal cytobrushings were obtained from clinically normal oral mucosa of 57 HIV+, 40 renal transplant patients and 30 healthy uninfected controls, all matched for age at baseline of examination. Presence of EBV-, CMV- and HPV-DNA was assessed by polymerase chain reaction (PCR). We evaluated their association, in HIV+ subjects, with gender, CD4+ cell count, HIV-RNA load, and antiretroviral therapy; and in renal transplant patients, with gender, CD4/CD8 ratio, and immunosuppressive therapy. Data were managed and analysed by Epi-Info 6.0. RESULTS: EBV-DNA was detected in 42.1% of HIV+ (24/57), in 65.0% of transplant patients (26/40), and in 16.6% of controls (5/30) (P = 0.03 and P = 0.0001, respectively). Furthermore, male gender in HIV+ group was found to be significantly associated with the presence of EBV-DNA (P = 0.02) vs females, after adjusting for CD4+ cell count and HIV-RNA load. CMV- and HPV-DNA were detected in 3.5% and 7.0% of HIV+, and in none and 20.0% of transplant patients, respectively. No relationship was found between the epithelial detection of these two viruses and any parameter evaluated. CONCLUSIONS: EBV genome was significantly detected in clinically normal oral mucosa of renal transplant and HIV+ patients. A significant gender association was found among HIV+, suggesting that oral hairy leukoplakia (OHL) is more likely to occur in HIV+ men than women.

DISEASE ASSOCIATIONS	CHARACTERIZATION
IMMUNE STATUS
Oral hairy leukoplakia in nonimmunosuppressed patients. Report of four cases. Lozada-Nur F, Robinson J, Regezi JA. Department of Stomatology, University of California San Francisco.	Oral Surg Oral Med Oral Pathol 1994 Nov;78(5):599-602 Abstract quote Hairy leukoplakia was first described in association with HIV infection. Today hairy leukoplakia has come to represent a sign of immunosuppression and not just of HIV infection. Although molecular mechanisms have not been fully elucidated, Epstein-Barr virus appears to play a significant role in its etiopathogenesis. We present four cases that illustrate that HL may be seen in non-HIV and nonimmunocompromised patients. The use of high potency topical steroids for the treatment of oral vesiculoerosive diseases seems to have been a contributing factor in two of these cases.
LEUKEMIA
Oral hairy leukoplakia in a patient with acute lymphocytic leukemia. Nicolatou O, Nikolatos G, Fisfis M, Belegrati M, Papadaki T, Oikonomaki E, Kalmantis T. Department of Oral Pathology and Surgery, School of Dentistry, University of Athens, Greece.	Oral Dis 1999 Jan;5(1):76-9 Abstract quote The first case of oral hairy leukoplakia (OHL) in an HIV-negative 56-year-old patient with acute lymphocytic leukemia (ALL) is reported. A white plaque was observed while the patient was in complete remission which followed the chemotherapeutic scheme. The clinical and histopathologic findings were typical for OHL and the polymerase chain reaction method was positive for Epstein-Barr virus DNA. Underdiagnosis and underreporting of OHL in patients with a malignant haematological disease and the apparent different environmental factors to which these non-AIDS patients have been exposed, probably constitute some of the reasons for the very few OHL cases reported in these patients.
MULTIPLE MYELOMA
Oral hairy leukoplakia in a patient with multiple myeloma. Blomgren J, Back H. Clinic of Oral Medicine, Ostra University Hospital, Goteborg, Sweden.	Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1996 Oct;82(4):408-10 Abstract quote The first case of oral hairy leukoplakia in an HIV-negative patient with multiple myeloma is reported. The patient is a 56-year old man who has had monoclonal gammopathy of undetermined significance since 1986 and has been treated for a symptomatic multiple myeloma since 1993. The clinical and histopathologic findings are typical for oral hairy leukoplakia, and Epstein-Barr virus was demonstrated with polymerase chain reaction technique. Although a relatively large number of cases of oral hairy leukoplakia has been reported in HIV-negative patients, both immunocompromised and immunocompetent, only a few of these patients have had a malignant hematologic disease.
RENAL TRANSPLANT
Prevalence and risk factors associated with leukoplakia, hairy leukoplakia, erythematous candidiasis, and gingival hyperplasia in renal transplant recipients. King GN, Healy CM, Glover MT, Kwan JT, Williams DM, Leigh IM, Thornhill MH. Department of Oral Medicine and Periodontology, London Hospital Medical College, University of London, England.	Oral Surg Oral Med Oral Pathol 1994 Dec;78(6):718-26 Abstract quote The aims of this study were to determine the prevalence of intraoral lesions in renal transplant recipients and to identify possible risk factors. The oral mucosa of 159 renal transplant recipients and 160 control patients was examined. The most common lesion in renal transplant recipients was cyclosporin-induced gingival hyperplasia (prevalence 22%) and patients with gingival hyperplasia were found to be taking significantly more cyclosporin-A than those without (p < 0.001). The prevalence of hairy leukoplakia and leukoplakia in renal transplant recipients was 11.3% and 10.7%, respectively, compared with 0% and 5.6% in the controls. Oral candidiasis was observed in 9.4% of renal transplant recipients compared with 2.5% of the controls; 3.8% of renal transplant recipients exhibited erythematous candidiasis, but this was not seen in the controls. Renal transplant recipients had a significantly increased risk of developing gingival hyperplasia (p < 0.0001), oral candidiasis (p < 0.0005), and two other conditions that have a well-established association with the immune suppression accompanying HIV infection, hairy leukoplakia (p < 0.0001) and erythematous candidiasis (p < 0.01).
SMOKING
Effect of receptive oral sex and smoking on the incidence of hairy leukoplakia in HIV-positive gay men. Shiboski CH, Neuhaus JM, Greenspan D, Greenspan JS. Oral AIDS Center, Department of Stomatology, University of California, San Francisco, 94143-0422, USA	J Acquir Immune Defic Syndr 1999 Jul 1;21(3):236-42 Abstract quote We sought to determine whether hairy leukoplakia (HL), an Epstein-Barr virus-related oral lesion, is associated with receptive oral sex activity and cigarette smoking among HIV-positive gay men. Oral examinations were conducted every 6 months among San Francisco Men's Health Study participants over a 6-year period. We fitted time-to-lesion regression models to compare the incidence of HL among men who had mouth-to-penis contact with various numbers of partners, while controlling for cigarette smoking and CD4 count. The 6-year incidence of HL was 32% among 291 HIV-positive men. We found no significant increase in the hazard of developing HL for each additional insertive-oral-sex male partner in the past 6 months (relative hazard = 1.01; 95% confidence interval [CI], 0.99, 1.02), and a similar lack of association when number of sex partners was categorized. However, the hazard of developing HL doubled with any 300-unit decrease in CD4 count (95% CI, 1.4, 2.7), or if men smoked > or =20 cigarettes/day compared with nonsmokers (95% CI, 1.2, 3.9). This finding, which may suggest one effect that smoking produces on the oral mucosa's local immune response, merits further investigation.

LABORATORY/RADIOLOGY	CHARACTERIZATION
LABORATORY
A simple and rapid technique for the detection of Epstein-Barr virus DNA in HIV-associated oral hairy leukoplakia biopsies. Mabruk MJ, Antonio M, Flint SR, Coleman DC, Toner M, Kay E, Leader M, Atkins GJ. Department of Pathology, Royal College of Surgeons in Ireland and Beaumont Hospital, Dublin.	J Oral Pathol Med 2000 Mar;29(3):118-22 Abstract quote 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.
CD4 LEVELS
Immunologic status in patients infected with HIV with oral candidiasis and hairy leukoplakia. Kolokotronis A, Kioses V, Antoniades D, Mandraveli K, Doutsos I, Papanayotou P. School of Dentistry, Aristotle University of Thessaloniki, Specific Infectious Disease Unit, Greece.	Oral Surg Oral Med Oral Pathol 1994 Jul;78(1):41-6 Abstract quote Although numerous studies of oral manifestations associated with HIV have been reported, only a few refer to the correlation of these lesions with laboratory parameters. In this study we investigated the relationships between the two most common HIV-associated oral lesions, oral candidiasis and hairy leukoplakia, with the stage of the disease, circulating CD4+ cell counts, and the presence of anti-p24 antibodies in serum and stimulated whole saliva in 43 known HIV-1-infected persons. Although oral candidiasis and hairy leukoplakia were exclusively observed in subjects who were classified as Centers for Disease Control and Prevention group IV, only the prevalence of oral candidiasis is strongly associated with circulating CD4+ counts less than 200/mm3 (p < 0.02). The prevalence of oral candidiasis and hairy leukoplakia was significantly related to the absence of anti-p24 antibodies in serum (p < 0.01 and p < 0.01, respectively), but was only statistically significant for hairy leukoplakia in stimulated whole saliva (p < 0.02). The results suggest that oral candidiasis and hairy leukoplakia in correlation with immunologic status as indicated by low circulating CD4+ cell counts and the absence of anti-p24 antibodies in serum and the loss of secretory anti-p24 antibodies in subjects with hairy leukoplakia, may constitute prognostic markers for the progression of HIV-infection to AIDS. Our results also indicate that the absence of anti-p24 antibodies is not only influenced by the low levels of circulating CD4+ cells but probably by the presence of oral candidiasis or hairy leukoplakia as well.
Relationship of circulating CD4+ T-lymphocytes and p24 antigenemia to the risk of developing AIDS in HIV-infected subjects with oral hairy leukoplakia. Ravina A, Ficarra G, Chiodo M, Mazzetti M, Romagnani S. Division of Allergy and Clinical Immunology, Polyclinic of Careggi, Florence, Italy.	J Oral Pathol Med 1996 Mar;25(3):108-11 Abstract quote To investigate the relationship of circulating CD4+ T-lymphocytes and p24 antigenemia to the development of AIDS in HIV-infected subjects with hairy leukoplakia (HL), we have followed over eight years 173 HIV-positive patients, all asymptomatic at the start of the study, and compared those who developed HL (n=55) to those who did not (n=118). Both groups included injection drug users (IDUs), homosexual men, and hemophiliacs. At the time of their first visit, both HL+ and HL- patients had a normal value of CD4+ cells and a low frequency of p24 antigenemia. During the years of follow-up, patients in the HL+ group showed a greater reduction in CD4+ cells, a significant increase in p24 antigenemia, and a higher rate of progression to AIDS. Our study demonstrates that in HIV-positive patients, HL is associated with more compromised immunological parameters and a higher viral replication and that its appearance has a negative prognostic value in relation to AIDS progression.

SPECIAL STAINS/IMMUNOHISTOCHEMISTRY	CHARACTERIZATION
IMMUNOHISTOCHEMISTRY
The expression of the Epstein-Barr virus nuclear antigen (EBNA-I) in oral hairy leukoplakia. Cruchley AT, Murray PG, Niedobitek G, Reynolds GM, Williams DM, Young LS. Department of Oral Pathology, St Bartholomew's, London, UK.	Oral Dis 1997 May;3 Suppl 1:S177-9 Abstract quote 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.
IN-SITU HYBRIDIZATION
A rapid microwave-in situ hybridization method for the definitive diagnosis of oral hairy leukoplakia: comparison with immunohistochemistry. Mabruk MJ, Flint SR, Coleman DC, Shiels O, Toner M, Atkins GJ. University of Dublin, Moyne Institute of Preventive Medicine, Department of Microbiology, Republic of Ireland.	J Oral Pathol Med 1996 Apr;25(4):170-6 Abstract quote As a diagnostic technique, in situ hybridization requires a long processing time, a degree of expertise and may be difficult to handle routinely in some laboratories. To simplify the in situ hybridization method, we have modified a microwave in situ hybridization technique and applied it to oral hairy leukoplakia (OHL) biopsies obtained from 10 HIV-seropositive patients (definitively diagnosed by a conventional in situ hybridization technique) with appropriate controls. It was necessary to design a novel chamber to avoid drying of sections during the hybridization step. This modified microwave in situ hybridization technique was equispecific and equisensitive to the conventional technique and it shortens the hybridization time from overnight incubation to 14 minutes. To determine the sensitivity of our microwave in situ hybridization method we applied it to previously documented tongue tissue obtained from an AIDS autopsy without clinical evidence of OHL, but found to contain Epstein-Barr virus (EBV) by conventional in situ hybridization. This tissue specimen acted as a low EBV copy number, positive control. The sensitivity of immunohistochemistry using three different commercial detection kits was compared to that of in situ hybridization on the same tissues, following optimisation steps. This included the use of 2 cycles of primary and biotinylated secondary antibodies (antibody double cycling). Clearly positive signals for EBV were detected in all OHL biopsies with the Vectastain Elite ABC and the Histostain-SP kits. The sensitivity of the three commercial detection kits was evaluated at immunohistochemistry level by their application to the low-EBV copy number positive control specimen. Signals for EBV antigen in the low copy number positive control specimen were obtained only with the Vectastain Elite ABC kit. This indicates that, in this application, use of the Vectastain Elite ABC kit gives comparable sensitivity for immunohistochemistry to that found by in situ hybridiation.
CYTOKERATIN
Expression of keratin 14 and 19 mRNA and protein in normal oral epithelia, hairy leukoplakia, tongue biting and white sponge nevus. Su L, Morgan PR, Thomas JA, Lane EB. Department of Oral Medicine, UMDS, London, England.	J Oral Pathol Med 1993 Apr;22(4):183-9 Abstract quote This study was undertaken to analyze keratin gene expression at both the mRNA and protein level in oral hairy leukoplakia (OHL). Comparisons were made with normal lingual epithelium from a similar site, tongue biting, normal buccal mucosa and another condition which disturbs oral epithelial differentiation, white sponge nevus. Combined immunocytochemical and in situ hybridization studies for keratins 14 and 19 were carried out on 2 specimens of OHL from HIV-positive males and one sample each of the other cases. Keratin 14 protein expression was uniform throughout all the epithelia. In normal epithelia and in lesions other than OHL, keratin 14 mRNA was most strongly expressed in basal cells with weaker but still significant amounts in the spinous cell layer. In both cases of OHL there was weaker basal cell expression of keratin 14 mRNA and frequent absence in koilocytoid cells. Keratin 19 protein expression was heterogeneous in the basal layer of all specimens with suprabasal staining of occasional groups of cells. Its mRNA was uniformly distributed in all cases. The findings indicate the keratin mRNA expression does not always parallel that of protein and that, in the case of keratin 14, expression may be influenced by the presence of EBV.
ELECTRON MICROSCOPY
Oral hairy leukoplakia with ultrastructural evidence of Merkel-like cells in human tongue epithelium. Cobb M, MacNeill R, Tobin J. Department of Periodontics, School of Dentistry, University of Missouri-Kansas City, USA	Bull Group Int Rech Sci Stomatol Odontol 1998 Jan-Mar;40(1):24-37 Abstract quote The purpose of this investigation was to examine cells in the stratum basale and subjacent lamina propria of oral hairy leukoplakia (OHL) specimens for previously unreported morphological changes. Tongue biopsy specimens were obtained from ten HIV-positive and five healthy male patients and examined by light (LM) and transmission electron microscopy (TEM). Observations made during the investigation revealed the following: (1) LM comparisons of control and OHL specimens indicated that clear cells in the stratum basale ranged from 3 to 7 per 100 basal epithelial cells in healthy tongue mucosa vs. 6 to 15 in OHL. (2) Merkel-like cells were noted in the stratum basale of control biopsies from the dorsal-lateral tongue surface.However, the frequency of observation was so rare as to imply that their presence was an exception rather than the norm. (3) In contrast, Merkel-like cells in OHL specimens were commonly encountered, indicating a possible unique relationship to the pathology. (4) Merkel-like cells associated with OHL lesions were morphologically similar, in all respects, to that of control specimens except for the presence of large, membrane bound, dense granules that ranged in size from 150-300 nm. (5) All biopsies of OHL exhibited evidence of Epstein Barr virus in the stratum corneum and superficial layers of the stratum spinosum. (6) A mild inflammatory infiltrate associated with OHL specimens revealed cytopathic changes in fibroblasts that appeared related to the presence of lymphoid cell types; indicating a possible cytolytic lymphocyte-mediated degradation of antigen altered host cells.
Oral hairy leukoplakia: an ultrastructural study and review of the literature. Guccion JG, Redman RS. Department of Veterans Affairs Medical Center, Washington, DC, USA.	Ultrastruct Pathol 1999 May-Jun;23(3):181-7 Abstract quote A 39-year-old, homosexual, Caucasian man with a 9-month history of acquired immunodeficiency syndrome by reduced CD4 lymphocyte count alone was found to have extensive, asymptomatic, nonremovable, corrugated, white patches on the lateral borders and ventral aspects of the tongue typical of oral hairy leukoplakia (OHL). Histologically, irregular hyperparakeratosis, acanthosis, and clusters of ballooned keratinocytes in the stratum spinosum were present in the biopsied lateral tongue. Some of the superficial ballooned keratinocytes had peripherally beaded nuclei, whereas others had ground glass intranuclear inclusions. Ultrastructurally, the ballooned keratinocytes had three important findings of diagnostic significance. First, frequent herpesvirus nucleocapsids were largely confined to superficial ballooned keratinocytes having marginated and condensed chromatin. In searching for herpesvirus nucleocapsids, the marginated and condensed chromatin was an invaluable marker for cells harboring the virions. Second, the marginated and condensed chromatin frequently had a distinctive punched-out or cribriform appearance. Third, the ground glass intranuclear inclusion bodies consisted of central, medium electron-dense, finely granular material containing frequent herpesvirus nucleocapsids and partially surrounded or capped by prominent, clumped chromatin. The patient died with progressive multifocal leukoencephalopathy 24 months after OHL was diagnosed.

CLINICAL VARIANTS	CHARACTERIZATION
GENERAL
Oral hairy leukoplakia: clinicopathologic features, pathogenesis, diagnosis, and clinical significance. Triantos D, Porter SR, Scully C, Teo CG. Department of Oral Medicine, Eastman Dental Institute for Oral Health Care Sciences, University of London, United Kingdom.	Clin Infect Dis 1997 Dec;25(6):1392-6 Abstract quote Oral hairy leukoplakia (OHL) is a lesion frequently, although not exclusively, observed in patients infected by human immunodeficiency viruses (HIV). OHL is clinically characterized by bilateral, often elevated, white patches of the lateral borders and dorsum of the tongue. Histologically, there is profound acanthosis, sometimes with koilocytic changes, and a lack of a notable inflammatory infiltrate. The koilocytic changes are due to intense replication of Epstein-Barr virus (EBV), while epithelial hyperplasia and acanthosis are likely to result from the combined action of the EBV-encoded proteins, latent membrane protein-1, and antiapoptotic BHRF1. How OHL is initiated and whether it develops after EBV reactivation from latency or superinfection remain unresolved; nevertheless, definitive diagnosis requires the demonstration of EBV replicating vegetatively in histological or cytological specimens. In patients with HIV infection, the development of OHL may herald severe HIV disease and the rapid onset of AIDS, but despite its title, OHL is not regarded as premalignant and is unlikely to give rise to oral squamous cell carcinoma.
PEDIATRIC
Oral hairy leukoplakia in two children with perinatally acquired HIV-1 infection. Mannelli F, Galli L, Ficarra G, Romagnoli P, Vierucci S, de Martino M. Department of Pediatrics, University of Florence, Italy.	Pediatr AIDS HIV Infect 1995 Feb;6(1):21-3 Abstract quote We report on two children with acquired immunodeficiency syndrome (AIDS), a 5-year-old girl and a 9-year-old boy, who presented with oral hairy leukoplakia (OHL) as a late clinical manifestation of perinatally acquired human immunodeficiency virus type 1 (HIV-1) infection. The reasons why OHL is a rare symptom in HIV-1-infected children, compared with HIV-1-infected adults, are discussed.

HISTOLOGICAL TYPES	CHARACTERIZATION
GENERAL	Ballooning degeneration of the keratinocytes of the upper stratum spinosum Hyperparakeratosis Mild or absent subepithelial inflammation
CYTOLOGY	Oral cytology may reveal keratinocytes with intranuclear inclusions with ground-glass appearance
Use of exfoliative cytology in the diagnosis of oral hairy leukoplakia. Migliorati CA, Jones AC, Baughman PA. Department of Oral Diagnostic Sciences, University of Florida College of Dentistry	Oral Surg Oral Med Oral Pathol 1993 Dec;76(6):704-10 Abstract quote The possibility of diagnosing oral hairy leukoplakia by means of exfoliative cytology and the Papanicolaou stain was investigated. Exfoliative cytology and punch biopsy specimens were obtained from 10 lesions that demonstrated clinical features of hairy leukoplakia. All biopsy specimens demonstrated the characteristic histopathologic features of hairy leukoplakia whereas all Papanicolaou-stained cytologic smears demonstrated condensation and margination of the nuclear chromatin (nuclear beading). All biopsy specimens and cytologic smears displayed positive Epstein-Barr virus deoxyribonucleic acid in situ hybridization. We conclude that routine exfoliative cytology may be a reliable, noninvasive, and inexpensive technique for the diagnosis of hairy leukoplakia.
Oral hairy leukoplakia. Histopathologic and cytopathologic features of a subclinical phase. Dias EP, Rocha ML, Silva JuniorA, Spyrides KS, Ferreira SM, Polignano GA, Feijo EC, Da Fonseca EC. Department of Pathology, Universidade Federal Fluminense, Niteroi/RJ, Brazil.	Am J Clin Pathol 2000 Sep;114(3):395-401 Abstract quote Accurate diagnosis of oral hairy leukoplakia (OHL) is important because it may be an early indicator of undiagnosed HIV infection; moreover, it may be a prognostic indicator. Our purpose was to investigate the histopathologic features of subclinical OHL and to evaluate and support the rationale of detecting subclinical OHL with cytopathology. The Epstein-Barr virus (EBV) was detected by immunohistochemistry and in situ hybridization in 4 cases of macroscopically normal lateral borders of tongue mucosa from 8 AIDS necropsies and in none of 8 controls. The histopathologic features were specific when based on nuclear changes: Cowdry type A inclusion, ground glass, and nuclear beading. Smears were obtained from 50 patients with AIDS, without OHL, from the scraping of lateral borders of the tongue. Numerous clusters of the cells were associated with Candida organisms (30% of cases). Nuclear changes were observed in 12 patients (24%) on both sides of the tongue. We describe the histopathologic features of subclinical OHL, and our observations suggest that cytopathology can detect OHL in the subclinical phase.