Oral manifestations of cytomegalovirus infection

oral medicine Editor H. DEAN MILLARD,

DDS, MS

1205 Glen Leven Road Ann Arbor, Michigan 48103

Oral manifestations of cytomegalovirus infection ./or1 B. Epstein, DMD, MSD,” Christopher H. Sherlock, MD, FRCPC,h and Robert A. Wolber, MD, FRCPC,’ Vancouver, British Columbia, Canada, and Seattle, Wash. HRITISH

COLUMBIA

GEKIFRAI-

CANCER

HOSPITAL,

AGENCY,

UNIVERSITY

UNILtKSITY

OF

WASHINGTOK.

Ot

BRITISH

S-T. PAUL.‘S

COLUMBIA,

VANCOUVER

HOSPITAL

Disease caused by cytomegalovirus is reported with increasing frequency. Cytomegalovirus is an Important pathogen in immunocompromised and immunosuppressed patients. The most common manifestation of cytomegalovirus infection of the gastrointestinal tract including the oral mucosa is ulceration. The role of cytomegalovirus in xerostomia, Sjdgren’s syndrome, and Kaposi’s sarcoma is continuing to be investigated. This article reviews the oral manifestations of cytomegalovirus, Including recently reported oral manifestations. I OR,\I

SI IN; OR \I. MED ORAL PATWX

1993;75:443-51)

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ytomegalovirus (CMV) is a herpes group virus that commonly infects human beings, with serologic evidence of infection in 40% to 80% of adultsm3 with higher incidence in economically disadvantaged populations and homosexual men.4-6Clinically apparent infection caused by CMV occurs more frequently in immunocompromised persons4 In immunocompetent hosts, most primary infections are asymptomatic. However, CMV hasbeen reported as the most common infectious complication in organ transplantation, as a cause of serious congenital diseasein the newborn, and as a significant cause ,lMedical/Dcntal

Staff,

British

Professor.

Universit)

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Medicineand Research Washington.

Clinical ,\ssociate,

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Agency:

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bMedical Microbiology, University of British Columbia; Department of Pathology, St. Paul’s Hospital, Vancouver. ‘Vancouver General Hospital, Departments of Dentistry, Anatomic Pathology, and Medical Microbiology and University of British Copyright

Columbia. 1993

0030~4’20/93/%1.00

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Moshy-Year

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Book,

7/13/44210

Inc.

of morbidity and mortality in immunocompromised patients in general. ‘, ‘-I4 CMV may be of greatest importance in immunocompromised patients, including those who receive immunosuppressive therapy, those with acquired immunodeficiency syndrome, and those who receive bone marrow or organ transplantation.‘. ‘. ‘1‘?-I8 CMV reactivation hasbeen reported in more than 75% of seropositive patients who receive bone marrow transplantation,t8. I’) and in patients who receive organ transplantation.‘?-“‘ The incidence of infection is increased in patients who are CMV seropositive and in patients who are CMV seronegative and their donor is seropositive.“. Ix Three possible patterns of infection occur: primary infection, reactivation, and superinfection.t3, I5 Infection may be a result of reactivation of latent virus, a primary infection from the transplant, or supportive hematologic care. Reactivation of latent herpesviruses commonly after transplantaresults in complications tion.8. ‘s-15,“. 20-25CMV is the single most common causeof morbidity and mortality after cardiac transplantation with active infection in up to 93% of 443

Fig.

1.

I!lceration

demonstrated

minimall\

in ( ae i involve> rolled horders

junctwn

01’ hard

patients.‘j In cardiac transplantation, primary- CM\; infection may produce a mononucleosissyndrome or progressive multisystem disease.” CMV pneumonia is a common causeof morbidity and mortality in cardiac transplantation. CMV and herpessimplex viru\ have been reported to cause one quarter of casesot esophagitis after boric marrow transplantation. and \vith the improved control of herpes simplex virus h> acyclovir. CMV may increase in proportion.“- “’ 01’ 30 patients who had received renal allografts 735 developed infection with CMV within the lirst 3 months after transplantation.” In none of thesecaseswas mucositis attributed to CMV. CMV has been implicated in the pathogenesis of graft-versus-host disease in bone marrow transplantation’” and in organ graft rcjection, although this remains controversial.“’ There is evidence that immunosuppressionresults in reactivation of CMV.“-‘-’ There is also evidcncc that herpesvirus infections including CMV may them. selvescause immunosuppressionand atl’ect both humoral and cellular immunity. ’ ’ Ii. I”. ‘X. ?‘) Specifically, suppressionof natural killer cell (‘unction and cytotoxic T lymphocyte- and monocytc-induced suppressionof lymphocyte function is seen. The purpose of this article i\ to prebent IWOcase> of CMV-associated oral lesionsand 10review the literature to allow discussionof the oral munifestationb of CMV infection that have been identified to date. CASE

REPORT

A 3%year-old May 1991 with

1 homosexual man. aith AIDS presented in a painful oral ulcer of more than &months

and wit

~alatc.

! Iccr

\‘ias 5 mtn

in diameter

and

duration. lie \\a identified 35 t1IV po5itibc In t+ebruar\ I S90. tic subsequently developed pneumocystis pneumonia in December 1990. He began taking lidovudine in November I990 when his CD3 lymphocyte count LZLIS 340. He continued taking Lidovudine and received pneumocystih pneumonia prophylaxis with aerosol pentamidine. P:I\;~ treatment trial\ of penicillin. nystatin rinse. topical her;tmeth~rsonc. and intralesional injection of dexamethasone ,lnd oral acyclw ir i 7OOmg~6 ~abs/day) did not affect tht: ,)raI leziun. When the patient ~13~ seen ill Ma> IY91 er>them~~tou~ candidiasis was present 2nd changes consistent with hair! Icukoplakia that involved the lateral borders i~f the tongue \+ere seen. The oral ulcer was ;I single round 5 m m lesion with minimally rolled borders that involve the boft palate (Fig I j. Because of the nonspecilic nawre of the ulcer and the lack of clinical diagnosis. ‘I hiopq W:I\ performed. The hiopq demonstrated epithelial ulceration. pcri\ascular Inllammation. and intranuclear inclusions that Involved vascular cndothelial cells consiant with CM\‘. In situ hybridiation WIS performed on paraffin-embedded tissue from the mucosal biopsies. Sections of 5 pm were cut on to 3-“niinopropyl-triettlox~silane-tre~~ted slides (SipW;I Chemical (‘I~:.. SC Louis. MO.) and dried overnight. Deparallini/ed. rch~tlrated sections were Incubated for 1 q mmutes in 3’;’ hydrogen peroxide in methanol, washed in phosphate buffered saline (PBS) solution, then digested in l’rehhlypreparedproteinaseK (0.2 mg/ml, Sigma Chemical (‘0.) in PBS solution at room temperature. A biotinl-Iated double-k,tranded DNA probe for CMV (L:nzo, Ne\n York, N .Y. J was used at a concentration of 0. I &ml. The probe diluent consisted of 509 deionized formamide (Sigma Chemical (‘o. ). I O’P dextran sulfate (Sigma Chemical (‘I).). O.Ol’r salmon sperm DNA (Sigma Chemical Co.). in 3 X SC‘<‘. .\ wlution of SO WLIof probe W;IS applied to each slide. \+hich L+;IX then covered with autoclavahle plastic and

ORAL

SURGERY

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P~~THoLoC\

Epstein, Sherlock,

and Wdber

445

Volume 75. Number 1

Fig. 2. Photomicrograph of epithelium at border of ulcer and intact tissue shows intense black markers in connective tissue after in situ hybridization for CMV confirming presence of massive CMV infection of connective tissue.

heated to 100” C for 15 minutes. Slides were transferred to a 38” c’ humidified chamber and incubated overnight. Negative controls were incubated with a nonhomologous DNA probe. Posthybridization steps were as follows: a 30-second wash in 0.05% Triton X-100 (Sigma Chemical Co.) in PBS solution, a IO-minute wash in 0.2 X SSC at 38” C. two 5-minute washes in 0.05% Triton X-l 00 in PBS solution. Sections were incubated for I hour in peroxidase conjugated streptocidin at a I:200 dilution (Jackson Immunochemicals, Gaithersburg, Md.), then washed in 0.1 m Tris-acetate buffer, pH 5.0, and developed with aminoethyl carbazole chromogen. Sections were then washed in water. counterstained in hematoxylin, mounted in aqueous medium, and examined. In situ hybridization results confirmed the presence of CMV in the connective tissue (Fig. 2). CASE REPORT 2 A 35-year-old man received a heart transplant on November 9, 1989, because of irreversible cardiomyopathy.30 Before the transplant he was CMV seronegative, whereas the donor was CMV seropositive. Posttransplantation immunosuppression was accomplished using cyclosporine A (275 mg twice a day), azathioprine (I 50 mg once a day), and prednisone (I 0 mg four times a day). Cyclosporine serum level was maintained at 348 to 468 pg/l. In December the patient became aware of gum enlargement and sensitivity. In January 1990 cervical lymphadenopathy was noted. There was no change in the immunosuppressive medications during this period. In February 1:he gum enlargement and tenderness became the chief complaint, interfering with oral hygiene and diet. Lymphadenopathy was identified in the preauricular area and in I:he right inguinal region. His immunosuppressive medications were changed to cyclosporine A 200 mg twice a day, prednisone reduced to 5 mg twice a day, and the dosage of a.zathioprine was unchanged.

Viral serology results had been documented on a weekly basis beginning when the patient was admitted for transplantation. The results were positive for Epstein-Barr virus (EBV) and HSV antibody before the transplant as well as CMV, IgG, and IgM negative. The results remained negative until seroconversion on January 4, 1990 when they became CMV IgM positive. The patient remained IgM seropositive until March 15, 1990, when he was found to be IgM seronegative. Blood, urine, and throat cultures were negative until February 15, 1990, when CMV was isolated from the throat and urine. Subsequently, CMV was isolated from his blood on March 15, 1990 and by tissue culture from a gingival biopsy on April 4. The results of a follow-up biopsy were negative for CMV on July 26. The persisting gingival complaints were investigated in mid-February. The gingival tissues were markedly enlarged, firm, pink, and extended in many areas up to the incisal edges and cusp tips (Fig. 3). The differential diagnosis included lymphoma and cyclosporine hyperplasia although the extent of the hyperplasia and the length of time the patient had been taking cyclosporine made cyclosporine-associated hyperplasia less likely. A gingival biopsy was performed on February 26, 1990. Histologically, the biopsy revealed extensive squamous epithelial hyperplasia with stromal and epithelial chronic inflammation. Numerous enlarged cells with nuclear and cytoplasmic inclusions characteristic of CMV were present in nubmucosal vascular endothelial cells and stromal tibroblasts. There were no histologic findings of lymphoma or other malignant conditions, no significant Gbroblast proliferation, and no significant librosis in the specimen. Subsequent in situ DNA hybridization for human CMV was performed as described above and numerous positive cells were seen in stromal tibroblasts and vascular endothelium. The epithelial cells were unaffected (Fig. 4). At I month follow-up, the lymphadenopathy had re-

solved. and the gingival enlargement had dimintshed in ail areas except the mandibular anterior region (Fig. 5). :2 further biops! and gingivoplastl \\ere carried out. The patient became CMV IgM seronegatiic on March 15. IWO A repeat biopsy of the gingival hyprrplasia on April 1. 1990 was CMV negative on the basis of the results of routine hi+ tolog). in Gtu hybridization. and tihsuc culture. .Acqclo\it (200 mg orally 5 times ;I day) had been iniliated on February 16 on empirical grounds by the attending physician and 1~~s continued for 2 months. At followup 6 months later no evidence of the previous hyperplasia remained.

C MV ria

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Ulcers mucosal Esophageal

comprise infection and

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present

in adjacent

be isolated

from

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ulcers

associated

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the

presence

of CMV.‘”

when

have

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also

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Biopsies intranuclear

CM\:

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”

10 CMV

I~nnittnohi~toctiemic~tl

the principle manifestations ,vit,, CMV.1’. 1 :, ii. ‘(I-??.

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gastrointestinal

attributed

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CMV csophagitis most odynophagia. liwphageal

btudy

DISCUSSION CMV-associated

been

varied

and

vi-

seen

in-

reported.“’

appears M ith in I1 patients

as large.

single,

shal-

with routine histologic inclusions in all patient\. positive

staining In the

in 8 of 14 patients. M;I\

used

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“I

and by To

CMV

arrive

Epstein,

Sherlock,

and Wolber

447

Fig. 4. A, Photomicrograph of gingiva shows portion of epithelium and connective tissue. Stroma is infiltrated by inflammatory cells and numerous cytomegalovirus infected stromal fibroblasts and endothelial cells. (Hematoxyiin-eosin stain; original magnification x40.) B, Cytomegalovirus infected stromal and endothelial cells with characteristic nuclear and cytoplasmic inclusions. (Hematoxylin-eosin stain; original magnificafor cytomegalovirus DNA shows intense labeling of nuclear inclusions, tion X 100.) C, In-situ hybridization (Hematoxylin with aminoethylcarbazole: original magnification x200.)

of CMV-induced tissue change, evidence of the presenceof the virus in tissue is needed. The techniques used may include electron microscopy, immunohistochemistry, in situ hybridization, and tissue culture. Oral mucosal ulcerations have been attributed to CMV in patients with AIDS.17. 2o The diagnosis of CMV ulceration in these caseswas made on the basis of intranuclear and cytoplasmic CMV inclusions on electron microscopy. Three other cases of CMVrelated ulcers of the oral mucosa have been reported: in a patient who received immunosuppressivechemotherapy for Wegener’s granulomatosis2’; in a patient after bone marrow transplantation”; and in an immunosuppressedpatient with lupus erythematosus.‘3 In these cases,inclusions were identified in the endothelium subjacent to the ulcer although direct infection of epithelium by CMV was not noted. Recently, at a diagnosis

four cases of CMV-associated oral ulcers were reported in HIV-infected patients who had disseminated CMV infection.17 CMV inclusionswere seenon light microscopy and confirmed in the tissue by immunohistochemistry and in situ hybridization. Activated T-lymphocytes were identified in the tissue and high titers of serumCMV antibodies were present in the caseswith disseminatedCMV.17 In these four casesCMV was detected principally in endothelial cells or perivascular and subepithelial connective tissue. CMV antigen was detected in occasional epithelial cells. These findings were confirmed in case 1 presented in this article. A study of aphthous stomatitis was carried out to look for a viral cause using immunofluorescent techniquesx7 CMV was not identified but varicella zoster virus was detected in I of 8 patients, and the symptoms were reduced, but recurrences were not elimi-

Fig. 5. ticul WI>

sted 1xir-

I I1 ill

nated with the useof acyclovir. More sensitive techniques remain to be usedin studies of this diseaseand nonspecific ulcers in HIV-positive persons. The oral ulcers reported in association with CMV to date are nonspecific in clinical presentation. The lesionsappear to involve either keratinized or nonkerutinized tissues. In immunosuppressedpatients oral ulcers may represent CMV-associated lesions and should be considered in the differential diagnosis. It is possiblethat oral ulcers in immunosuppressedpatients, which are currently diagnosedasnonspecific OI aphthous-like, may in somecasesby CMV-induced. Specific diagnosis may become more common when the differential diagnosis includes CMV. Future assessmentand treatment of these aphthous-like ulcers may require specific study for CMV

CMV and gingivitis In case 1. gingival hyperplasia developed in a person who was <‘i\/lV seronegative before cardiac transplantation, but who developed primary CMV infection after cardiac transplantation from a CMVseropositive donor. Infection was documented by servoconversion and by shedding of CMV in the uropharynx and urine and CMV viremia. The gingivae were grossly hyperplastic, firm with minimal erythema, and with localized areas of ulceration. These Lindingsled to a biopsy to rule out a secondary ljmphoma or infiltration of leukemic cells. The dramatic histologic and hybridization demonstration of CMV pathosis, plus the concomitant culture findings in the same time course suggesteda significant role played by CMV in this condition. These findings are

Epstein, Sherlock, and Wdher in contrast with an earlier report of a possible role of CMV in necrotizing gingivitis’; this hypothesis was based on evidence of depressed cell-mediated immunity, decrease in T-helper/suppressor ratios,38. j9 and reduced response to concanavalin A in both acute necrotizing gingivitis and in CMV infection. Additional epidemiologic evidence presented as support for this hypothesis includes the age of onset in developing and industrial countries and the higher incidence of both CMV infection and necrotizing gingivitis in homosexual men.’ Case 2, which was reported here, CMVassociated gingival hyperplasia, is in contrast to previous tindings of necrosis associated with CMV. The minor gingival ulcerations in this case may have been the result of limited vascular necrosis. The gingival hyperplasia may have been the result of epithelial hyperplasia. packing of the connective tissue with large numbers of CMV infected cells and inflammatory cell infiltration and edema. While the patient was on cyclosporine. the histologic findings were not those of cyclosporine-associated gingival hyperplasia, and the patient had been on cyclosporine for only I month at the time of onset of hyperplasia. A study of 41 HIV-infected hemophiliacs identified an association with elevated serum anti-CMV IgG and atypical gingivitis (HIV-G)? A 4-year longitudinal follow-up of 34 of these patients was then conducted.‘” In extended follow-up, CMV-IgG levels were found to be extremely variable; however, only 3 of 28 patients with gingivitis did not show elevation of IgG during follow-up.” Developing seropositivity W,JS associated with onset of HIV-G; in six patients, when gingivitis regressed, the titers of CMV IgG returned to normal; and six patients had no evidence of gingivitis. In Case 2 we documented improvement in the clinical manifestations of oral pain and gingival hyperplasia, elimination of viral replication at all sites iieluding oral mucosa, and conversion of CMV IgM iiter to negative after a reduction in immunosuppressive drug dosages and institution of acyclovir treatment. The recovery from CMV infection may have been due to a reduction in immunosuppressive medications. the role of acyclovir in the improvement is not clear. The improvement in gingival hyperplasia suggests that CMV was a cause of the clinical and histologic hyperplasia. CMV and Kaposi’s

sarcoma

Epidemiologic data suggest that Kaposi’s sarcoma (KS) in HIV disease may be associated with a transmissible agent other than HIV.40.4’ There is increasing evidence of a sexually transmitted factor in the cause of KS.Jo-“7 because KS is particularly common

449

in those with risk of sexually transmitted IHIV.30 Cofactors that may be involved in the cause of KS include a sexually transmitted virus, possible co-virus infection, and other nonviral factors that result in production of angiogenic growth factors.“‘-‘” The role of CMV in oral KS in AIDS is unclear.50 The findings of positive CMV serology and CMV-DNA, CMV-RNA, and CMV antigens in biopsies from KS suggest an association of CMV with KS. CMV was identitied in 2 of 5 cases in the nuclei of endothelial cells but not in spindle-shaped cells of the lesion.5’ However, CMV is not identified in all cases and can be latent in endothelium even in non-HIV-infected persons and may represent an opportunistic infection within the KS tissue. CMV and Sjijgren’s

syndrome

Because of the presence of CMV in salivary gland tissue, there has been continuing interest in a possible etiologic role of CMV in salivary gland disease, including Sjogren’s syndrome. High levels of complement-fixing antibodies against CMV with no increase in the prevalence of CMV antibody using enzymelinked immunosorbent assay (ELISA) have been described in patients with primary and secondary Sjiigren’s syndronle.5’ Another study found no difference in serum anti-CMV antibodies using enzymelinked immunosorbent assay between patients with secondary Sjogren’s syndrome and controls.‘” No correlation was seen between patients with primary Sjogren’s syndrome and antibodies to CMV. however, a relationship between anti-EBV nuclear antigen (EBV-NA) was suggested.5” In a study of lacrimal gland biopsies from patients with Sjogren’s syndrome, one of eight cases studied demonstrated CMV antigen, and three demonstrated EBV in B cells and T-helper cells (Leu-3+ cells).” The results of these studies suggest that a portion of CMV antibodies may be associated with the cause of Sjiigren’s syndrome in some patients.” CONCLUSION Oral diseases caused by CMV will become more common as transplant programs increase in number and scope and as the HIV epidemic continues. Given the frequency and multiple causes of oral lesions in immunocompromised patients, it is important to be aware of CMV-induced oral disease and to use the recent advances in the technology of CMV detection to make a specific diagnosis. This is important not only for instituting specific therapy but also for avoiding inappropriate empiric treatment of these lesions. The precise pathogenesis of CMV-induced oral le-

sions remains unclear but the casts described here ant! others in the literature support the hypothesis that the primary focus of infection lies in the vascular cndw thelium underlying the lesions rather than prim:lri infection of the epithelium itself, The secondary event is necrosis in most cases or hyperplasia of the oval!ing epithelium resulting in the clinical presentation 01 ulcers or. in the one case described here. pingival h> perplasia. Clearly. more work needs to bc done t(i clarify this process.

REFERENCES

Epstein, Sherlock.

41.

4’.

33.

14.

45.

16. 17.

4x.

49.

Friedman-Kien AE. SaltLman BR, Cao Y, et al. Kaposi’s sarcoma in IilV-negative homosexual men. Lancet 1990;335: 16X-9. Archibald CP. Schechter MT, Craib KJP, et al. Risk factors for Kaposi‘s sarcoma in the Vancouver IymphadenopathpAIDS stud!. J .4cquir Immune DeRc Syndr 1990;3(suppl I ):1X-23. Jacobson LP, Munoz A. Fox R. et al. Incidence of Kaposi’q sarcoma in n whorl of homosexual men infected with the human immunodeticiency virus type I. J 4cquir Immune Detic S) ndr I990:3( tranhl’eclion of Kaposi’s sarcoma DNA encodes a grouth lactor that is ‘I memher of the PSI: familq. Cell 1987:50:72937. Bayle! AC, Lucas SB. Knposi’s sarcoma or Kaposi‘s disease? In: Fletcher CDM. McKee PH. eds. Pathohiology of soft ti+ \uc tumour\. London: (‘hurchill Livingstone. 1990:32X-31.

50.

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53.

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and Wolher

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Epstein JB. Silverman S. Jr. tlead and neck malignancies associated with HIV infection. OK,~I SL I<(; 0~11 MI II ORZI P,\THOL 1992;73:) 93-300. Neuland JR. Adler-StorthL K. Cytomegalo\irus in Intraoral Kaposi’s sarcoma. ORAI &KG 0rt\1 Ml.11 OKAI. P.\rllol 1989;67:296-300. Thorn JJ. Oxholm P, Andersen HK. Iligh levels ol complement fixing antibodies against cytomegalovirus in patients with primary Sjiigren’c syndrome. Clin Exp Rheumatol 19XX;fIl: 7 I-4. Scull) C. Sjiigren’s syndrome: no demonstrahlc association hq serology of secondary SjBgren‘s syndrome wilh cytomegalowus. J Oral Pathol Med 1990:19:43-4. Bcrtetti E. Ahero R, D’Agostino P, Radclli L. Riboldi P. Antibodies to Epstein-Barr virus and cytomegalovirus in primary Sjiigren’s syndrome. Boll 1st Sieroter Milan 19X8:67:265-74. Pepose JS, .4kata RF, PRugfelder SC, Voigt W. Mononuclear cell phenotypes and immunoglobulin gene rearrangements in lacrimal gland biopsies from patients with SjQren’s avndrome. Ophthalmol 1990;97: I 599-605. reyursis:

Joel E. Epstein Departmen( of Dentistry British Columbia Cancer 600 West 10th 4venue Vancouver. BC. Canada

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