Kaposi's sarcoma associated with previous human herpesvirus 8 infection in heart transplant recipients

The aim of this study was to evaluate the seroprevalence of human herpesvirus 8 (HHV-8) in a group of 150 patients awaiting heart transplants and to detect HHV-8 seroconversion after transplantation. Four patients were HHV-8 seropositive before transplantation, and one of them developed Kaposi's sarcoma. One patient converted to seropositive HHV-8 status after transplantation.     

Salivary human herpesvirus 8 shedding in renal allograft recipients with Kaposi's sarcoma

Renal allograft recipients in the Middle East are at high risk of developing Kaposi's sarcoma. This report describes the extent of oral human herpesvirus 8 shedding and the genomic diversity of the virus in five Saudi Arabian kidney transplantation patients in whom Kaposi's sarcoma had developed. PCR protocols were applied to amplify three fragments of the viral genome from whole-mouth saliva, parotid saliva, buccal and palatal exfoliates, plasma, peripheral blood leukocytes and biopsy of the Kaposi's sarcoma lesion, and to quantify the viral load in whole-mouth saliva. Viral DNA was detected in all plasma and biopsy samples, 80% of whole-mouth saliva, 20% of each of the other oral samples, and none of the leukocyte samples. The viral load in the cell-free fraction of whole-mouth saliva ranged between approximately 1.2 x 10(3) and 2.2 x 10(6) genome-copies/ml. Genotypically distinct viral strains were evident: intra-lesionally in 1 patient; intra-orally in one patient; between an oral sample and biopsy in two patients; and in four patients, between an oral sample and plasma, and between plasma and biopsy. Thus, in the patients studied, salivary shedding of human herpesvirus 8 was frequent and could be extensive, and they were prone to multiple infections. Measures to curtail salivary viral transmission to pre- and post-transplantation patients might reduce the incidence of post-transplantation Kaposi's sarcoma.     

Spontaneously regressed Kaposi's sarcoma and human herpesvirus 8 infection in a human immunodeficiency virus-negative patient

Kaposi's sarcoma occurring in a 78-year-old woman, with the absence of the human immunodeficiency virus infection, was correctly diagnosed by immunohistochemistry using anti-human herpesvirus 8 (HHV8) antibody (PA1-73N) for the first time. The patient suffered from chronic respiratory failure and was treated with a low dose of steroids for 2.5 years. After her medication dosage was increased for the exacerbation of the respiratory failure, multiple skin tumors in her feet and legs suddenly developed. Histopathologically, skin tumors were suspected as Kaposi's sarcoma at the first biopsy and reactive angiomatosis at the second biopsy. Polymerase chain reaction and immunohistochemistry, however, revealed the presence of HHV8 DNA fragment and positive staining in the majority of spindle cells in the skin tumors. Serological examination confirmed the positivity of anti-HHV8 antibodies. HHV8 infection and steroid-induced immunosuppression, as well as environmental factors played a role in the development of Kaposi's sarcoma in this patient, because she was born in Okinawa, which is a well-known endemic area of Kaposi's sarcoma in Japan. As her general condition improved, the skin lesions regressed without any specific treatment, and disappeared completely 8 months later, in which regression may be associated with evidence of numerous CD8 cell infiltration in the second biopsy tissues. No recurrence was observed during the following 6 month follow up.     

Classic type of Kaposi's sarcoma and human herpesvirus 8 infection in Xinjiang, China

We report 17 cases of the classic type of Kaposi's sarcoma in Xinjiang, which is located in the north-western area of China surrounded by Mongolia in the east, Russia in the north and Kazakhstan in the west. Fifteen of the patients were of the Uygur people. All patients were male and did not have acquired immunodeficiency syndrome. Most of the lesions were found in the lower and/or upper extremities, with 16 patients showing multiple lesions. Immunohistochemical examination of the lesions revealed that human herpesvirus 8 (HHV-8)-encoded latency-associated nuclear antigen was expressed in the nuclei of spindle-shaped tumor cells. HHV-8 DNA was detected by polymerase chain reaction in all seven cases examined. Phylogenetic tree analysis revealed that DNA sequences of the HHV-8-encoded K1 gene in the seven Kaposi's sarcoma cases were classified as subtype C that was common in the Mediterranean, the Middle East and East Asian countries. In addition, using immunofluorescence we investigated the seroprevalence of HHV-8 in 73 Uygur patients with diseases other than Kaposi's sarcoma. Surprisingly, the serological study revealed that 34 of the patients (46.6%) were positive for antibodies against HHV-8, suggesting that HHV-8 infection is widespread in Xinjiang area. The occurrence of the classic type of Kaposi's sarcoma with a high seropositivity rate implies that Xinjiang is the most endemic area for HHV-8 infection in the world known to date. Considering that Xinjiang is located at the middle point of the Silk Road that used to extend from Rome to China, these data imply that the virus may have been in circulation in this area due to the migration of the people via the Silk Road.     

Kaposi's sarcoma in Uganda: risk factors for human herpesvirus 8 infection among blood donors

Human herpesvirus 8 (HHV-8) is etiologically linked to Kaposi's sarcoma, a common cancer in Uganda. The authors assessed HHV-8 seroprevalence, risk factors for infection, and HHV-8 assays in a cross-sectional study of Ugandan blood donors. Of 3,736 specimens, the authors selected 203 reactive for HIV, hepatitis B surface antigen (HBsAg), or syphilis, and, randomly, 203 nonreactive specimens. For HHV-8 testing, the authors used two peptide-based enzyme-linked immunosorbent assays (EIAs), ORFK8.1 and ORF65, and an immunofluorescence assay (IFA). Specimens reactive in at least two assays or on IFA alone were considered HHV-8-seropositive. Prevalence estimates were weighted to account for the sampling scheme. Overall HHV-8 seroprevalence was 40%. HHV-8 seroprevalence was higher among HBsAg-positive donors (53%) than HBsAg-negative donors (39%; p =.02) and higher among HIV-positive donors (63%) than HIV-negative donors (39%; p <.001). HHV-8 seroreactivity showed no trend with age. Kappa values for assay concordances were 0.68 (ORFK8.1 EIA and IFA), 0.37 (ORF65 EIA and K8.1 EIA), and 0.29 (ORF65 EIA and IFA). The association between HHV-8 and HBsAg positivity and the lack of association between HHV-8 and age point to primarily nonsexual HHV-8 transmission during childhood. The association with HIV indicates sexual transmission may also occur. The role of ORF65 EIA in testing specimens from Africa warrants further evaluation.     

Human herpesvirus 8 in Kaposi's sarcoma and Kaposi's sarcoma-mimicking vascular tumors.

Kaposi''s sarcoma (KS) had been a rare and unusual vascular tumor until a recent epidemic of a disseminated and fulminant form of KS in AIDS patients. Infectious agents have been suspected of causing KS, and recently partial genomic DNA sequences of human herpesvirus 8 (HHV8) have been identified in AIDS-associated KS lesions. Since then, genomic DNA sequences of HHV8 have been isolated in other forms of KS. Although the partial genomic DNA sequence of HHV8 was reported to be, if rare, identified in vascular tumors other than Kaposi''s sarcoma (KS), the presence of HHV8 in a very large fraction of KS indicates that detection of HHV8 by PCR is a useful auxiliary tool in differentiating KS from other KS-mimicking vascular tumors. We examined whether the 233-bp segment of the viral DNA was detected in Korean patients with KS and other KS-mimicking vascular tumors. HHV8 sequences were identified in all of nine classic type of KS but not in three epithelioid hemangioendotheliomas and seven angiosarcomas. Our results confirm the relatively restricted distribution of HHV8 and also argue against the likelihood of secondary colonization of KS cells by HHV8.     

Dominant human herpesvirus type 8 RNA transcripts in classical and AIDS-related Kaposi's sarcoma

Skin biopsy sections of Kaposi's sarcoma (KS) from 25 patients (5 AIDS-related, 20 classical cases) were histologically staged and hybridized in situ with oligonucleotide probes for constitutively transcribed human herpesvirus 8 (HHV-8) mRNA T0.7 and T1.1 using a colourimetric technique. T1.1 increases during experimental induction of the viral lytic phase in the HHV-8-infected lymphocytes of primary effusion lymphoma and its colourimetric detection in KS cells presumably corresponds to virion production. Immunostaining with anti-CD20, CD45RO, MAC 387, and α-smooth muscle actin was performed following T1.1 in situ hybridization (ISH). When the amount of T0.7 was above the detection threshold, the signal was made up of multiple coarse intranuclear dots in most spindle cells. Of the six early-stage lesions, none produced a T1.1 hybridization signal. Two of four AIDS-related and two of eight classical lesions with incipient spindle cell growth produced rare but distinct dense intranuclear T1.1 signals in endothelial cells lining narrow tubes. In contrast, eight of ten (all classical KS) mature spindle cell lesions displayed a signal, scattered in up to 2 per cent of spindled endothelial cells. Cell types other than endothelium produced no T1.1 hybridization signal in double stains. The results are consistent with other published data indicating latent HHV-8 infection in endothelium and its tumour cell progeny, with simultaneous virion production in a small subset of cells. Immunodeficiency may not influence the number of cells lytically infected with HHV-8 in early KS, in contradistinction to other herpesviruses with latent-lytic cycles. Copyright © 1999 John Wiley & Sons, Ltd.     

Molecular epidemiology of human herpesvirus 8 variants in Kaposi's sarcoma from Iranian patients

Kaposi's sarcoma (KS) is a rare cancer in Iran and there is no epidemiological and molecular information about HHV-8 variants circulating among the Iranian population. In this study HHV-8 sequences have been analyzed in 43 cutaneous KS biopsies from Iranian patients mainly affected by classic KS. DNA samples were subjected to PCR amplification of HHV-8 ORF26, T0.7 and K1 followed by direct nucleotide sequencing and phylogenetic analysis. The analysis of ORF26 showed that 30 (69.8%) and 13 (30.2%) samples belonged to subtypes A/C and K, respectively. In general, the clustering of HHV-8 T0.7 variants paralleled that of ORF26. Genotyping of K1 sequences showed that the majority of samples (39 out of 41) fall into the large C clade with only 2 belonging to the A clade. In conclusion, HHV-8 variants identified among classic Iranian KS are largely related to Eurasian genotypes previously identified in KS from Mediterranean, Middle East, and East Asian regions.     

Ultrastructural characterization of human herpesvirus 8 (Kaposi's sarcoma- associated herpesvirus) in Kaposi's sarcoma lesions: electron microscopy permits distinction from cytomegalovirus (CMV)

Kaposi's sarcoma (KS) has been shown by molecular techniques to be associated with infection with human herpesvirus 8 (HHV8/KSHV), but specific ultrastructural characterization of the virus has been impaired by the frequent presence in these lesions of other herpesviruses, particularly cytomegalovirus (CMV). Since the ultrastructural appearance of HHV8/KSHV has been studied in the cell line KS-1 uninfected with other viruses including CMV, it was possible to undertake a comparative study of CMV and HHV8/KSHV in KS lesions. HHV8/KSHV was sparsely present and lytic infection was restricted to endothelial cells. The following specific ultrastructural features allowed distinction between HHV8/KSHV and CMV: the viral particles were more delicate and less numerous in cases of HHV8/KSHV infection; the viral tegument was more electron-dense in CMV than in HHV8/KSHV; dense bodies characteristic of CMV were absent in HHV/KSHV; complete CMV viral particles were more variable in size and generally larger (150–200 nm) than HHV8/KSHV (120–150 nm); and finally, the viral envelope was more pleomorphic in CMV than in KSHV/HHV8. Similarities between CMV and HHV8/KSHV included the basic structure of the nucleocapsids and the presence of capsids lacking central DNA cores (so-called non-infectious enveloped particles). These observations show that electron microscopy can be used to identify HHV8/KSHV and confirm the relationship between HHV8/KSHV and KS. © 1997 John Wiley & Sons, Ltd.     

Molecular epidemiology of Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 strains from Russian patients with classic, posttransplant, and AIDS-associated Kaposi's sarcoma

We report the molecular characterization of 38 new Kaposi's sarcoma-associated herpesvirus (KSHV) strains from Russian patients with either classic (25 cases), epidemic/AIDS-associated (7 cases), or posttransplant/immunosuppressed patients (6 cases), or Kaposi's sarcoma (KS). While a complete sequence of the K1 gene (870 bp) was obtained from 30 strains, only partial sequences of the hypervariable regions VR1 (372 bp) and/or VR2 (381 bp) of the K1 gene were obtained from eight strains of KS paraffin blocks. Sequence comparison and phylogenetic studies indicate that the novel KSHV strains belong to either the A subtype (28 cases) or the C subtype (10 cases). Within the 28 strains of A subtype, 24 (86%) belong to the large A' subgroup, mostly A1 and A1' clades, and 4 belong to the A" subgroup, mostly A3 clade. Within the 10 strains of subtype C, 4 were of C' subgroup, and 6 of the C". Some molecular variants of subtype A' were observed, with 3 strains exhibiting an insertion of a single amino acid at the position 65 and 2 strains (both from AIDS-KS) with an unique deletion of 17 amino acids in the VR2 region. Polymerase chain reaction-based subtyping of the K14.1 genomic region indicated that most (23/32) of the novel strains belonged to the P subtype. The results indicate that despite a wide genetic diversity of A and C K1 subtypes of KSHV strains present in Russia, most are closely related and belong to the A1 or A1' molecular clades suggesting a common origin. This study also expands the data regarding the absence of any correlation between a K1 molecular subtype and a specific KS type (classic, epidemic, or posttransplant), as well as between the K1 and K14.1 molecular subtypes.     

JC病毒在肾移植受者中的感染状况

目的 探究多瘤JC病毒(JC virus,JCV)在肾移植受者中的感染情况及其感染对移植肾功能的影响,并初步探索JCV感染的影响因素.方法 采用巢式PCR方法检测49例肾移植病人术后尿液标本中的JCV DNA,并以24例健康体检者为对照;定性PCR方法检测病人尿液标本中的巨细胞病毒(cytomegalovirus,CMV)DNA;Binary Logistic回归方法分析JCV感染的可能影响因素:病人的年龄、性别、免疫抑制方案、CMV感染;以肾小球滤过率(glomerular filtration rate,GFR)作为肾功能的指标,t检验方法比较JCV感染和非感染者GFR有无差别.结果 JCV在肾移植病人中的感染率为42.9%,健康体检者为4.2%.移植后CMV感染和强的松+MMF+环孢素三联免疫抑制方案是JCV感染的危险因素(OR=10.101,P=0.049;OR=6.286,P=0.008).JCV感染者和非感染者的GFR分别为86.470±29.990和84.060±33.729,两者间的差异无统计学意义(t=0.259,P=0.797).结论 JCV在肾移植病人中有很高的感染率,CMV感染和使用强的松+MMF+环孢素三联免疫抑制方案可明显增加JCV感染的危险性;JCV感染对移植肾功能无影响.     

Human herpesvirus type 8 infection in an area of Northern Italy with high incidence of classical Kaposi's sarcoma

Previous studies have reported a large variation in the incidence of classical Kaposi's sarcoma across different Districts of the province of Mantua (Northern Italy). To assess whether such differences might be explained by different anti-HHV8 antibody prevalence, a serological study was conducted in 343 healthy elderly individuals resident in two adjacent Districts, at the highest and the lowest classical Kaposi's sarcoma incidence rate, respectively. Qualitative and quantitative determinations of IgG antibodies against both latent and lytic HHV-8 antigens were performed by indirect immunofluorescence assay. The assay's sensitivity was studied in 26 patients with classical Kaposi's sarcoma. Overall, anti-HHV8 antibodies were detected in 25 out of 26 patients (96%), confirming the high sensitivity of this assay. The prevalence of anti-HHV-8 antibodies was higher among individuals living in the District had a high incidence of classical Kaposi's sarcoma compared to those living in the District with low incidence (19.4% vs 9.8%, and 15.9% vs 8%; P<0.05, for latent and lytic antibodies, respectively). Anti-lytic antibody GMT was higher in people living in the District at high incidence rate compared to those of the other area (328.9 vs. 180.4; P<0.01). A higher prevalence of HHV-8 infection was found among persons living in municipalities surrounded by watercourses (OR 2.2, 95% CI: 1.10-4.32). In conclusion, variation in HHV-8 prevalence appears to explain differences in the incidence rates of classical Kaposi's sarcoma observed in different areas of the province.     

Human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) DNA in Kaposi's sarcoma lesions,AIDS Kaposi's sarcoma cell lines,endothelial Kaposi's sarcoma simulators,and the skin of immunosuppressed patients.

We used the polymerase chain reaction on 63 tissue specimens of histologically staged classic Kaposi''s sarcoma (KS) from 40 patients, 14 specimens from 14 acquired immune deficiency syndrome (AIDS)-KS cases (all from the same geographic area over a 10-year period), and peripheral blood mononuclear cells from 1 of the non-AIDS KS patients to amplify a specific 210-bp genomic sequence of the newly discovered KS-associated herpesvirus (KSHV). Also tested were 86 benign and malignant endothelial lesions, which potentially simulated each KS histological stage and were further matched by age approximation and by sex with a classical KS specimen. The lesions included hemangioma, lymphangioma, pyogenic granuloma, and angiosarcoma. KSHV was also sought in multiple well characterized vascular endothelial cell lines from AIDS-KS lesions and in 20 mainly cutaneous benign and malignant lesions from 15 immunosuppressed transplant patients. Overall, 92% of KS tissue specimens, representing 88% of classical KS and 100% of AIDS-KS patients, and in addition the sample of peripheral blood mononuclear cell DNA, were positive as visualized on ethidium bromide gels and confirmed by Southern blot hybridization (only 1 case was negative on gell visualization but positive on Southern blot), thus confirming the close association of KSHV with KS of different clinical forms. None of the various other endothelial lesion, skin lesions in immunosuppressed patients, or AIDS-KS endothelial cell lines contained amplifiable KSHV DNA, which indicates that reactivation of KSHV is not present in the skin lesions of immunosuppressed patients and probably is not a ubiquitous agent that secondarily infects proliferative endothelium. The absence of amplifiable virus DNA in the cultured endothelium of KS suggests that the stimulus for angioproliferation originates in another host cell or under conditions not reproduced in culture. The polymerase chain reaction is a specific and sensitive means of verifying KS in the differential diagnosis of angioproliferative lessons.     

Human herpesvirus 8 genoprevalence in populations at disparate risks of Kaposi's sarcoma

The prevalence of human herpesvirus 8 (HHV-8) in populations at different risks of developing Kaposi's sarcoma (KS) was assessed using a protocol involving immunomagnetic fractionation of CD45+ blood cells prior to detection of the HHV-8 genome by nested PCR. Preliminary studies using blood of eight gay men infected by human immunodeficiency virus-1 (HIV-1) revealed that, for the detection of HHV-8 DNA derived from open reading frame (ORF) 26 of the HHV-8 genome, this protocol provided substantially higher rates (100%) compared to one involving red blood cell (RBC) lysis (0%) and to another requiring double density gradient centrifugation (DDGC) of leukocytes (13%). When the CD45+ fractionation protocol was applied to samples from 103 other HIV-1-infected patients (the vast majority of whom were gay men) and 100 blood donors, the ORF 26 DNA detection rates obtained were 37% and 8%, respectively. When DNA from the variable region 1 of ORF K1 was additionally amplified from samples of the blood donors, a detection rate of 9% was achieved. This rate was highly concordant with the ORF 26 DNA detection rate. Furthermore, the ORF K1 sequences were predominantly unique, assignable to genotypes A1, A4, and C3. When assays for anti-HHV-8 and anti-herpes simplex viruses (HSV) 1 and 2 were applied, significant concordances between HHV-8 DNA detection rates and those relating to anti-HHV-8 and to anti-HSV 1 and 2 were more frequently observed for HIV-1-infected patients than for blood donors. The higher-than-expected HHV-8 genoprevalence rate in blood donors requires further confirmation in view of its implications for post-transfusion HHV-8 transmission.     

Expression and antigenicity of human herpesvirus 8 encoded ORF59 protein in AIDS-associated Kaposi's sarcoma.

Human herpesvirus 8 (HHV-8, Kaposi's sarcoma-associated herpesvirus, KSHV) is a new herpes virus isolated from patients with AIDS-associated Kaposi's sarcoma (AIDS-KS). The ORF59 protein of HHV-8 has recently been shown to encode a processivity factor (PF-8) for HHV-8-encoded DNA polymerase. By immunoscreening a cDNA library derived from the HHV-8-infected cell line TY-1, ORF59 antigen was identified in AIDS-KS patients. Immunoblotting revealed that recombinant ORF59 protein reacted with sera from patients with AIDS-KS. Enzyme-linked immunosorbent assay (ELISA) using ORF59-recombinant protein as the antigen revealed that 7 of 22 (31. 8%) AIDS-KS patients and 6 of 263 (2.2%) Japanese HIV-negative patients or healthy blood donors were positive for anti-ORF59 antibodies. Immunohistochemistry using anti-ORF59 rabbit antibodies revealed that this protein was expressed in some of the tumor cells found in KS tissues and that ORF59 protein was detected in 11 of 22 (50%) AIDS-KS tissues. In situ hybridization indicated that some of KS tumor cells were positive for HHV-8 T1.1 mRNA in the same specimen. These data suggest that ORF59 is one of the HHV-8 encoded antigens in patients with AIDS-KS and also indicated that viral replication occurred in some of KS tumor cells.     

Seroreactivity to Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) latent nuclear antigen in AIDS-associated Kaposi's sarcoma patients depends on CD4+ T-cell count

In AIDS/Kaposi's sarcoma (KS) patients, the sensitivity of immunofluorescence assays for detecting antibodies against latent nuclear antigen ranges from 52% to 93%. However, in classic and African KS, sensitivities above 90% have been reported systematically. This study evaluates whether CD4+ T-cell count affects seroreactivity to KSHV LANA and to lytic antigens in AIDS/KS patients. Kaposi's sarcoma-associated herpesvirus (KSHV) latent (IFA-LANA) and lytic (IFA-Lytic and ORF65/K8.1 EIA) antibodies were screened in 184 consecutive samples taken from 36 AIDS/KS patients grouped according to their CD4+ counts as follows: <100 (group A), 100-300 (group B), and >300 (group C) cells/mm(3). At enrollment, the immunofluorescence assay for the detection of antibodies against latent nuclear antigen (IFA-LANA) was positive in 3/11(27.2%) group A patients, in 10/11 (90.9%) group B patients, and in 14/14 (100%) group C patients (P < 0.01). Seropositivity to lytic antigens did not differ according to CD4+ T-cell count. Considering IFA-Lytic and ORF65/K8.1 EIA, seropositivity for lytic antigens was 100% in all three patient groups. In patients whose CD4+ count improved during follow-up, IFA-LANA seroconversion occurred; unstable counts resulted in a decrease in LANA antibody titers while the persistence of high counts resulted in unchanged, elevated antibody titers. In conclusion, LANA seroreactivity in AIDS/KS patients, as assessed by an immunofluorescence assay, depends on CD4+ T-cell count, rendering this evaluation important in the interpretation of seroepidemiological studies of KSHV infection in AIDS patients. To evaluate future serological tests based on latency-associated antigens, the selection of sera from KS patients with CD4+ cell count >300 cells/mm(3) as a positive gold standard is recommended.