HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Meer, S. Articles by Altini, M. Search for Related Content PubMed PubMed Citation Articles by Meer, S. Articles by Altini, M.

Cytomegalovirus Co-infection in AIDS-associated Oral Kaposi’s Sarcoma

S. Meer^*, and M. Altini

Division of Oral Pathology, School of Oral Health Sciences, Private Bag 3, University of the Witwatersrand, WITS, 2050, Johannesburg, South Africa

Correspondence: ^* corresponding author, shabnum.meer{at}nhls.ac.za or meersa{at}dentistry.wits.ac.za

	Abstract

TOP Abstract Introduction Aim Materials and Methods Results Discussion References

 
The increasing appearance of AIDS-associated oral Kaposi’s sarcoma (KS) in South Africa may be ascribed to the later start of the HIV epidemic, more patients reaching stages III and IV, and the inaccessibility of most patients to anti-retroviral therapy. The objective of this study was to demonstrate cytomegalovirus (CMV) co-infection in oral KS and to consider its possible significance. We reviewed 20 cases of oral KS in known HIV-positive patients without active CMV disease. HHV8 PCR and CMV immunohistochemistry were performed. HHV8 DNA was present in all cases. CMV inclusions were detected in five cases. The significance of CMV co-infection in oral KS is unclear. The inclusions suggest active infection, although there is no evidence to support CMV in the pathogenesis of KS. Nonetheless, it is vital that physicians be alerted to active CMV infection, so that timely intervention and careful observation can be instituted, ensuring early diagnosis and treatment.

KEY WORDS: Cytomegalovirus • CMV • AIDS • Kaposi’s sarcoma

	Introduction

TOP Abstract Introduction Aim Materials and Methods Results Discussion References

 
Through our biopsy service, we are currently encountering an increasing frequency in the number of cases of AIDS-associated oral Kaposi’s sarcoma (Lager et al., 2003). This increase is in keeping with the rapidly expanding HIV epidemic in South Africa and a three-fold increase in the general incidence of KS recorded between 1988 and 1996 (Sitas and Newton, 2001). This is in contrast to developed countries, where several centers have reported a steady decline in incidence (Flaitz et al., 1995; Lundgren et al., 1995; Biggar and Rabkin, 1996). This difference may be ascribed to the greater proportion of patients who are now reaching stages III and IV of the disease (Dorrington et al., 2002). In addition, the vast majority of our patients do not have access to anti-retroviral therapy, although this situation is being addressed. In one London clinic, the incidence of most AIDS-defining diseases has decreased dramatically over the preceding 9 years, with the most striking reduction being in the incidence of KS. The authors attribute this decline to the use of highly active anti-retroviral therapy, HAART, with either non-nucleoside reverse-transcriptase inhibitors or protease inhibitors, in combination with at least 2 nucleoside reverse-transcriptase inhibitors, as the standard of care (Ives et al., 2001).

	Aim

TOP Abstract Introduction Aim Materials and Methods Results Discussion References

 
The aim of this study was to demonstrate cytomegalovirus co-infection in oral KS and to consider its possible significance.

	Materials and Methods

TOP Abstract Introduction Aim Materials and Methods Results Discussion References

 
We retrieved 20 cases of oral KS from the files of the Division of Oral Pathology of the University of the Witwatersrand. Only those cases in which the patient was known to be HIV-positive were selected. All of these cases were part of our routine departmental diagnostic service and were histopathologically examined by the reporting pathologist.

In all cases, two 10-µM-thick sections were cut from the archived paraffin-wax-embedded tissue blocks, with the DNA extracted by a modified phenol chloroform method. Two rounds of nested polymerase chain-reaction (PCR) were performed on a Perkin-Elmer Gene Amp PCR system (Perkin-Elmer Corp., Norwalk, CT, USA). In the first round, a 2-µL quantity of re-suspended DNA was added to a reaction mix containing 0.2 µM each of the primers KS4 and KS5 (Roche, Basel, Switzerland). A second reaction with internal primers KS1 and KS2 (Roche) was performed with 4 µL of the amplimer obtained from the first round. The reaction mix was the same as that used in the first round. This yielded amplified DNA of 233 base pairs. Each sample was also subjected to PCR for demonstration of ß-globin gene DNA sequences as a confirmation of DNA integrity. If these sequences could not be detected, that specimen was excluded from the study.

Both negative and positive controls were included in each run. Agarose gel (3%) electrophoresis was performed on DNA that was stained with ethidium bromide and then visualized on a UV transilluminator operating at 320 nm. The presence of HHV-8 was established by comparison with the known molecular-weight marker: Marker V (Roche, Basel, Switzerland).

In each case, additional deparaffinized 4-µ sections were immunohistochemically stained with CMV antiserum from Zymed (Clone DDG9.CCH2; lot no. 20570754; South San Francisco, CA, USA) [Dilution 1:50; 1 hr/RT], with use of the Envision kit (Dako, Glostrup, Denmark). Prior to the sections being stained, antigen retrieval was accomplished with the use of pepsin for 10 min at 37°C. Following antigen retrieval, the sections were immersed in quench (3% H₂O₂ in H₂O) for 30 min and then incubated with the primary antibody for 1 hr at room temperature. Both positive and negative controls were used.

	Results

TOP Abstract Introduction Aim Materials and Methods Results Discussion References

 
The patients included in this study were all black, except for one male patient who was white, and consisted of 11 males and nine females. The average age was 33.3 yrs, and all patients had tested positive for HIV. The CD4 counts were not known. None exhibited any signs or symptoms of CMV infection, and some were not even aware of their oral KS lesions or HIV status. All 20 cases showed the presence of HHV-8 DNA sequences in their oral lesions. In addition, five patients showed co-infection with CMV, detected on the hematoxylin and eosin (H & E)-stained sections and confirmed with immunohistochemistry (Table). The patients were black males (three) and females (two), between the ages of 29 and 46 yrs, with an average age of 37.8 yrs. The palate was the most common site of occurrence (three cases), followed by the maxillary gingivae (two cases) (Figs. 1, 2). The CMV inclusion bodies appeared as enlarged cells with an "owl’s eye" appearance. The inclusions were identified in either macrophages or endothelial cells, or both, and occurred either at the periphery or within the KS lesional tissue. In one case, numerous inclusion bodies were present, but in the other four cases, relatively few inclusions could be seen, making CMV identification difficult (Figs. 3, 4). Use of CMV immunohistochemistry facilitates the identification of the inclusion bodies.

View larger version (80K): [in this window] [in a new window]   Fig. 1 - KS with concomitant CMV infection presenting as a large, erythematous nodular mass on the palate and gingiva, and as a poorly circumscribed red-blue macule on the posterior palate.

View larger version (90K): [in this window] [in a new window]   Fig. 2 - A red, nodular, friable lesion of KS and CMV infection with multiple minute superficial ulceration involving the posterior palate and extending into the interdental papillae.

View larger version (158K): [in this window] [in a new window]   Fig. 3 - Clusters of prominent CMV inclusion particles in endothelial cells deep within a KS spindle cell proliferation (hematoxylin and eosin, X40).

View larger version (136K): [in this window] [in a new window]   Fig. 4 - Positive CMV immunoreactivity of an inclusion body within a macrophage subepithelially within granulation tissue in a KS lesion (CMV antiserum; Zymed, X40).

	Discussion

TOP Abstract Introduction Aim Materials and Methods Results Discussion References

At the time of this study, although in excess of 80 cases of oral KS were recorded in our departmental archives, we decided to restrict this investigation to only those cases in which the patient was known to be HIV-positive. This resulted in only 20 cases being included. These 20 cases included the three cases showing CMV co-infection, to which we had referred in a previous study (Lager et al., 2003). The additional two cases were identified during the course of this study. It was not our intention to determine the frequency of CMV co-infection in oral KS; thus, we did not include all cases accessed in our department. This frequency of CMV co-infection (20%) should not be seen as the true frequency, since the scope of the study was limited and the selection of cases biased.

It is regrettable that, in South Africa at the present time, HIV testing is not compulsory but is largely at the patient’s discretion. Similarly, because of the large number of cases and the lack of funds, determination of CD4 counts is also not routinely performed. This unfortunately compromises our ability to include these meaningful data in our publications, and, as a result, valuable clinico-pathologic correlations are being lost.

CMV has previously been demonstrated, by PCR and IHC, in 7 of 26 specimens of KS of the skin in AIDS patients (Kempf et al., 1995). It was also simultaneously detected in other organs of these patients, indicating a disseminated viral infection. CMV in KS lesions has also frequently been detected by electron microscopy, but whether this represents active infection remains uncertain (Said et al., 1997). After reviewing the relevant literature, many authors (Kempf et al., 1995; Kempf and Adams, 1996; Hille et al., 2002) concluded that there was no new evidence to support an active role for CMV in the pathogenesis of KS.

CMV has also been reported as co-infecting KS lesions from other sites, including the thyroid, the ileocecal area, and the thymus, in both HIV-positive and HIV-negative patients (Moysset et al., 1997; Cohen et al., 2001; Zhang et al., 2003), but has not previously been reported in KS lesions from the oral cavity in AIDS patients. Other infectious agents, such as Cryptococcus, have also been reported as co-infecting KS (Glassman and Hale, 1995).

All of the cases we are reporting showed viral inclusion bodies with the typical "owl’s eye" appearance. These cytomegalic cells were found either at the periphery of the lesional tissue in the inflammatory infiltrate or within the typical spindle cell areas of KS. They represented either infected endothelial cells or macrophages. In one case, they were abundant and easily identifiable, but, for the most part, there were relatively few cytomegalic cells, which were detectable only with careful observation and could easily have been overlooked. It behooves the pathologist to examine all submitted tissue carefully in cases of oral KS, to ensure that CMV co-infection is not missed, to the possible detriment of the patient.

This report of five cases of CMV co-infection of oral KS in AIDS patients raises several important questions which cannot now be answered but which warrant further research. The questions include: What is the significance of this co-infection? Is the CMV a ‘passenger’, or is this an active infection? Is the infection isolated, or is it part of a widespread tissue involvement? Do the KS cells provide a suitable milieu for proliferation of the virus? Could the KS act as a reservoir for the CMV virus, thus providing a source for the virus to spread to other sites as immunosuppression worsens? and If the KS is treated, does the CMV viral co-infection disappear at the same time?

We have already referred to the fact that several authors have concluded that there is no new evidence implicating CMV in the pathogenesis of KS. However, this possibility cannot yet be excluded, and the not-infrequent presence of CMV in KS, in either latent or active form, does raise the possibility that CMV may act as a co-factor with a role in transactivation of the HIV or HHV8 viruses, facilitating the proliferation of the vasoformative cells by inducing a cascade of cytokines (Kempf and Adams, 1996), and thus development of lesions of KS.

The presence of viral inclusion bodies mitigates against CMV merely being a passenger. Sufficient numbers of viral copies must be produced before inclusion bodies will form, this suggesting an active infection (Syrjänen et al., 1999). It seems highly unlikely that this represents an isolated or chance infection, and the possibility of CMV infection of other tissues must be considered. It is indeed possible that the KS tissue provides a suitable milieu for proliferation of the virus, and that it may act as a reservoir for spread of the virus to other sites as immunosuppression worsens.

Whatever the answers to these questions might be, it is vital that the attending physicians be alerted to the presence of an active CMV infection, so that timely intervention and careful observation might be instituted, thus ensuring early diagnosis and treatment.

	Acknowledgments

 
We thank Professor Hedley Coleman (Griffith University, Gold Coast City) and Dr. Chris Rachanis and Mrs. Hasiena Ali (University of the Witwatersrand, Johannesburg), for their expert assistance and contribution to this study.

	References

TOP Abstract Introduction Aim Materials and Methods Results Discussion References

 
Biggar RJ, Rabkin CS (1996). The epidemiology of AIDS-related neoplasms. Hematol Oncol Clin North Am 10:997–1010.[Medline]

Cohen RL, Tepper RE, Urmacher C, Katz S (2001). Kaposi’s sarcoma and cytomegaloviral ileocolitis complicating long-standing Crohn’s disease in an HIV negative patient. Am J Gastroenterol 96:3028–3031.[Medline]

Dorrington R, Bradshaw D, Budlender D, Bourne D (2002). The current state and future projections of the HIV/AIDS epidemic in South Africa. S Afr Dent J 57:449–450.

Flaitz CM, Nichols CM, Hicks MJ (1995). An overview of the oral manifestations of AIDS-related Kaposi’s sarcoma. Compend Contin Educ Dent 16:136–142.[Medline]

Flaitz CM, Nichols CM, Hicks MJ (1996). Herpesviridae-associated persistent mucocutaneous ulcers in acquired immunodeficiency syndrome. A clinicopathologic study. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 81:433–441.[Medline]

Glassman SJ, Hale MJ (1995). Cutaneous cryptococcosis and Kaposi’s sarcoma occurring in the same lesions in a patient with the acquired immunodeficiency syndrome. Clin Exp Dermatol 20:480–486.[Medline]

Hille JJ, Webster-Cyriaque J, Palefski JM, Raab-Traub N (2002). Mechanisms of expression of HHV8, EBV and HPV in selected HIV-associated oral lesions. Oral Dis 8(Suppl 2):161–168.

Ives NJ, Gazzard BG, Easterbrook PJ (2001). The changing pattern of AIDS-defining illnesses with the introduction of highly active antiretroviral therapy (HAART) in a London clinic. J Infect 42:134–139.[Medline]

Kempf W, Adams V (1996). Viruses in the pathogenesis of Kaposi’s sarcoma—a review. Biochem Mol Med 58:1–12.[Medline]

Kempf W, Adams V, Pfaltz M, Briner J, Schmid M, Moos R, et al. (1995). Human herpesvirus type 6 and cytomegalovirus in AIDS-associated Kaposi’s sarcoma: no evidence for an etiological association. Hum Pathol 26:914–919.[Medline]

Lager I, Altini M, Coleman H, Ali H (2003). Oral Kaposi’s sarcoma: a clinicopathologic study from South Africa. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 96:701–710.[Medline]

Lundgren JD, Melbye M, Pedersen C, Rosenberg PS, Gerstoft J (1995). Changing patterns of Kaposi’s sarcoma in Danish acquired immunodeficiency syndrome patients with complete follow-up. The Danish Study Group for HIV infection (DASHI). Am J Epidemiol 141:652–658.[Abstract/Free Full Text]

Moysset I, Lloreta J, Miguel A, Vadell C, Ribalta T, Estrach T, et al. (1997). Thymoma associated with CD4+ lymphopenia, cytomegalovirus infection, and Kaposi’s sarcoma. Hum Pathol 28:1211–1213.[Medline]

Said JW, Chien K, Tasaka T, Koeffler HP (1997). Ultrastructural characterization of human herpesvirus 8 (Kaposi’s sarcoma-associated herpesvirus) in Kaposi’s sarcoma lesions: electron microscopy permits distinction from cytomegalovirus (CMV). J Pathol 182:273–281.[Medline]

Sitas F, Newton R (2001). Kaposi’s sarcoma in South Africa. J Natl Cancer Inst Monogr 28:1–4.

Syrjänen S, Leimola-Virtanen R, Schmidt-Westhausen A, Reichart PA (1999). Oral ulcers in AIDS patients frequently associated with cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections. J Oral Pathol Med 28:204–209.[Medline]

Zhang X, el-Sahrigy D, Elhosseiny A, Melamed MR (2003). Simultaneous cytomegalovirus infection and Kaposi’s sarcoma of the thyroid diagnosed by fine needle aspiration in an AIDS patient. A case report and first cytologic description of the two entities occurring together. Acta Cytol 47:645–648.[Medline]

This Article Abstract Full Text (PDF) Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Meer, S. Articles by Altini, M. Search for Related Content PubMed PubMed Citation Articles by Meer, S. Articles by Altini, M.