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We describe an immunocompetent 61-year-old woman who was negative for human immunodeficiency virus and who had recurrent human herpesvirus 8 (HHV-8) infection associated with a relapsing systemic inflammatory syndrome characterized by fever, lymphadenopathy, splenomegaly, edema, arthrosynovitis, and rash. Kaposi's sarcoma developed 10 months after the initial clinical presentation. A correlation was documented between the recurrent clinical manifestations and the HHV-8 load in plasma and peripheral-blood mononuclear cells. Histologic examination of an enlarged lymph node heavily infected with HHV-8 revealed an atypical lymphoproliferative disorder characterized by paracortical hyperplasia and collapsed primary and secondary follicles.
We describe a previously healthy woman who was negative for human immunodeficiency virus (HIV) and who had recurrent episodes of active HHV-8 infection, in whom a relapsing systemic inflammatory syndrome, characterized by fever, lymphadenopathy, edema, arthrosynovitis, and erythematous rash, preceded and accompanied the development of KS. A correlation was observed between the clinical manifestations and the levels of HHV-8 replication.
Case Report
In February 2000, a 61-year-old woman was referred to our hospital with a one-year history of low-grade fever, lymphadenopathy, splenomegaly, arthralgia, edema, and rash involving the face and limbs. Her medical history was unremarkable. In February 1999 she had been hospitalized elsewhere. On that occasion, laboratory studies showed mild anemia, an elevated erythrocyte sedimentation rate (34 mm per hour; normal range, 2 to 15), and elevated levels of C-reactive protein (63 mg per liter; normal range, 2 to 6), lactate dehydrogenase (751 U per liter; normal range, 210 to 425), and beta2-microglobulin (6.6 mg per liter; normal, less than 3.0). To rule out a lymphoproliferative disorder, the patient underwent an axillary–lymph-node biopsy, which was described as showing "nonspecific inflammatory changes," and a bone marrow core biopsy, the results of which were reported to be normal. She was treated with antibiotics, acetaminophen, and nonsteroidal antiinflammatory drugs, with minimal benefit. In November 1999, multiple cutaneous nodules with normal overlying skin, Raynaud's phenomenon, and diffuse severe arthrosynovitis developed. A biopsy of a skin nodule was diagnostic of KS. No specific treatment was instituted.
At the time of admission to our hospital, in March 2000, the patient appeared fatigued but not in acute distress. Her body temperature was 37.5°C. A few lymph nodes, each less than 2 cm in diameter, were palpable in the cervical, axillary, and inguinal regions. Severe arthrosynovitis of the hands, wrists, elbows, and knees was present. A nonpitting edema and a nonpruritic, nonpalpable, erythematous rash that involved the face and limbs and blanched on diascopy, as well as multiple cutaneous nodules with normal overlying skin, were present (Figure 1). The spleen was palpable. The findings on physical examination were otherwise normal. Routine laboratory examination showed mild anemia and leukopenia with a normal differential count, increased levels of lactate dehydrogenase and C-reactive protein, and an increased erythrocyte sedimentation rate. Tests for rheumatoid factor, antinuclear antibodies, anti-DNA antibodies, and antibodies against extractable nuclear antigens were negative, as were direct and indirect Coombs' tests. The results of the immunologic studies are summarized in Table 1. Findings on examination of a skin-nodule biopsy specimen were diagnostic of hypodermic KS.
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In September 2000, 17 typical KS skin lesions appeared on the patient's left arm; they were associated with recurrence of edema and an erythematous rash on the face and legs, as well as diffuse arthrosynovitis. The patient was again treated with liposomal daunorubicin, with progressive improvement and regression of sarcoma nodules. In February 2001, the patient once again reported arthralgias, leg and face edema, diffuse rash, and the reappearance of multiple KS skin lesions. Her symptoms worsened over a three-week period, and a high-grade fever developed. Given the reappearance of HHV-8 viremia, the patient was treated with cidofovir (5 mg per kilogram, given intravenously every other week for five months), resulting in the disappearance of all clinical signs and symptoms and a concomitant sustained virologic response. The patient had no symptoms for six months after the termination of therapy, at which time a small KS lesion accompanied by local lymphedema appeared on her left ankle. Three months later, inguinal lymphadenopathy developed. A lymph-node biopsy showed localized KS.
Methods
Immunologic Studies
In vitro proliferation assays for recall antigens were performed according to published protocols.11 The natural killer cell activity was assessed by means of a standard chromium-51–release assay.
Sample Preparation and Quantification of HHV-8 DNA
DNA was extracted from plasma, peripheral-blood mononuclear cells, or tissue samples according to the phenol–chloroform protocol. The levels of HHV-8 DNA were determined by a quantitative, calibrated, real-time polymerase-chain-reaction (PCR) assay, as described previously.12 This assay is based on the use of a specific synthetic calibrator molecule, which allows one to adjust for the difference in the recovery of nucleic acids during extraction and to identify PCR artifacts. The limit of detection was 10 genome equivalents of HHV-8 DNA per milliliter of plasma.
Serologic Assays
To detect antibodies against lytic and latent antigens of HHV-8, we used immunofluorescence assays based on the HHV-8–positive BCBL-1 cell line and the BCP-1 cell line, respectively, as described previously.7,13,14
Histopathology and Immunohistochemistry
We used sequential sections of formalin-fixed, paraffin-embedded lymph-node tissue that were 3 µm thick. Monoclonal antibodies or antiserum against CD3 and CD21 (Ylem), CD20, and 
and 
immunoglobulin chains (Dako) and antibody against HHV-8 interleukin-6 (Advanced Biotechnologies) were used according to the manufacturers' instructions or as described previously.15 The antibody against HHV-8 interleukin-6 specifically recognizes viral interleukin-6 and does not cross-react with human interleukin-6. Paraffin-embedded sections of the lymph node were also tested for Epstein–Barr virus (EBV) by means of in situ hybridization with a fluorescein isothiocyanate–labeled probe specific for EBER-1, an EBV-encoded RNA transcript (Dako). Sections from normal lymph nodes with mild plasmacytosis were obtained from two age-matched patients and used as negative controls.
Measurement of Human Interleukin-6 and Interleukin-10 Levels
Levels of human interleukin-6 and interleukin-10 were measured in sequential plasma samples by means of an enzyme-linked immunosorbent assay (ELISA) (R&D Systems), according to the manufacturer's instructions. Plasma levels of human interleukin-6 and interleukin-10 were considered normal if they were lower than 10 pg per milliliter and 8 pg per milliliter, respectively.16
Results
Virologic and Serologic Findings
At the time of the first admission to our institution, the patient had a high viral load (4.2x107 genome equivalents of HHV-8 DNA per 106 cells) in the excised KS lesion; the levels were lower in peripheral-blood mononuclear cells and plasma (850 genome equivalents per 106 cells and 164 genome equivalents per milliliter of plasma, respectively). Treatment with liposomal daunorubicin was followed by clinical improvement associated with rapid abatement of the viremia (Figure 2). In June 2000, the patient had a recurrence of viremia and a clinical relapse, accompanied by massive and diffuse lymphadenopathy; PCR analysis revealed a very high HHV-8 DNA load in an enlarged lymph node (3.7x106 genome equivalents per 106 cells). The patient was treated for a short time with foscarnet sodium monotherapy, which resulted in a virologic and clinical improvement. During follow-up, the patient had two more peaks of viremia associated with clinical relapse. In March 2001, cidofovir monotherapy was begun; it resulted in clinical and virologic remission (Figure 2). Eventually, before the patient was lost to follow-up, reactivation of plasma viremia was confirmed, with the reappearance of a KS lesion and, shortly thereafter, the development of KS in a lymph node (64 and 254 genome equivalents per milliliter of plasma, respectively).
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Histologic Features of Lymph Nodes
An axillary lymph node removed during the first relapse was moderately enlarged (diameter, 2 cm) and showed substantial effacement of the architecture by a lymphoid infiltrate, which had a predominantly diffuse and focally vaguely nodular pattern of growth (Figure 3) and comprised small lymphocytes, large lymphoid cells (many with the appearance of immunoblasts), scattered eosinophils, and polyclonal plasma cells, as indicated by immunostaining for immunoglobulin light chains. There was an intense proliferation of postcapillary venules lined by high endothelium. Many of the sinuses were dilated. Lymphoid follicles were difficult to discern, and germinal centers were absent. There was no evidence of necrosis or hyalinized vessels, and mitotic activity was moderate. About 10 percent of plasma cells in the paracortical or interfollicular region were strongly positive for HHV-8 interleukin-6 (Figure 3D). EBV-encoded RNA transcripts were not detected. No foci of KS were identifiable. Reexamination of the axillary lymph node that had been excised approximately one year earlier showed similar characteristics, despite the fact that the architecture was more conserved.
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We describe a relapsing syndrome characterized by fever, lymphadenopathy, splenomegaly, edema, arthrosynovitis, and erythematous rash accompanied by leukopenia and anemia of chronic inflammatory disease in an HIV-negative, immunocompetent woman who had recurrent episodes of HHV-8 reactivation. Over a 20-month period of follow-up, we documented four recurrences of the same signs and symptoms, which closely correlated with the course and extent of HHV-8 replication. In accordance with descriptions of KS and multicentric Castleman's disease in the literature,9,10,17 exacerbations were always accompanied by spikes of viremia, whereas improvements after therapeutic interventions were consistently associated with an abatement of the viral load. Of particular interest was the finding that antiviral therapy with foscarnet or cidofovir led not only to the suppression of HHV-8 viremia, but also to the concomitant disappearance of both the KS nodules and the other clinical manifestations.
KS is extremely rare in women in the absence of concomitant HIV infection or other causes of immunosuppression. In our patient, however, an extensive search for infection with immunosuppressive retroviruses was unsuccessful, and no clinical or immunologic signs of immune dysfunction were observed.
The extensive lymph-node and splenic involvement suggested the presence of an underlying lymphoma or multicentric Castleman's disease, but both diagnoses were ruled out on the basis of histopathological analysis of two lymph nodes excised at different times. Indeed, the morphologic changes were consistent with the presence of an atypical immunoproliferative disorder characterized by reactive, nonspecific paracortical hyperplasia, which can occur in response to viral infections18 but to our knowledge has not previously been described in a patient with HHV-8 infection.
As far as Castleman's disease is concerned, the morphologic features of our patient did not satisfy the criteria for either the hyaline vascular or the plasma-cell type of that disorder.19 Regarding the former, follicles were not prominent, germinal centers were hardly discernible, and the hyaline vascular changes that characterize this entity were absent; regarding the plasma-cell variant, the number of plasma cells present in the excised lymph nodes was limited, and such cells were not arranged in the form of solid sheets. Moreover, we did not detect increased levels of both human interleukin-6 and interleukin-10, as are found during the active phases of HHV-8–associated multicentric Castleman's disease.10,20,21,22
Polyclonal plasma cells in the patient's lymph node were productively infected by HHV-8, as revealed by intense immunostaining for viral interleukin-6.15,23 This finding, together with the massive viral load in lymph-node–derived mononuclear cells and the presence of splenomegaly and diffuse lymphadenopathy, suggests that secondary lymphoid organs were important reservoirs for viral replication and that viral interleukin-6 may have played a role in some of the systemic manifestations in our patient.
The availability of molecular diagnostic techniques has permitted the association of HHV-8 infection with diseases or syndromes the causes of which were previously unknown.2,3,4,5,6,24,25 Although HHV-8 is apparently less prevalent in the general population than are other human herpesviruses, the assessment of serum antibody titers and even of cell-associated viral DNA may not provide clinically significant information, since this approach does not distinguish between latent and active infection. Only the integration of these data with measurement of the HHV-8 viral load in plasma and biologic fluids by means of reliable quantitative methods allows the accurate diagnosis of an active infection and thus makes it possible to establish an etiologic link.
Supported in part by grants from the Fourth National AIDS Project from the Ministry of Health, Rome (to Drs. Lusso and Malnati). Dr. Dagna is the recipient of a research fellowship from the Università Vita-Salute San Raffaele.
We are indebted to Drs. Juan Rosai and Massimo Freschi for their comments and critical review of the histologic findings, to Drs. Paolo Monini and Barbara Ensoli for their help in setting up the procedures for HHV-8 immunostaining, and to Sara Silva for performing the cytokine assays.
Source Information
From the Università Vita-Salute San Raffaele School of Medicine, Milan (L.D., M.G.S.); the Units of Human Virology (L.D., F.B., P.L., M.S.M.) and Rheumatology and Clinical Immunology (L.P., M.G.S.), the Department of Pathology (C.T.P.), and the Laboratory of Tumor Immunology (M.F.), San Raffaele Scientific Institute, Milan; the Department of Public Health, University Tor Vergata, Rome (L.S.); and the Department of Biomedical Sciences, University of Cagliari, Cagliari (P.L.) — all in Italy.
Address reprint requests to Dr. Dagna at the Università Vita-Salute San Raffaele School of Medicine, Unit of Human Virology, P2/P3 Labs — DIBIT, Via Olgettina 58, I-20132 Milan, Italy, or at lorenzo.dagna{at}hsr.it.
References
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Related Letters:
A Relapsing Inflammatory Syndrome and HHV-8
Dogan A., Du M.-Q., Isaacson P. G., Dagna L., Rosai J., Lusso P.
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N Engl J Med 2005;
353:1746-1747, Oct 20, 2005.
Correspondence
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