In the viral capsid, HHV-8 DNA is linear and double stranded, but upon infection of the host cell and release from the viral capsid, it circularizes. Reports of the length of the HHV-8 genome have been complicated by its numerous, hard-to-sequence, terminal repeats. Renne et al. 17 reported a length of 170 kilobases (Kb) but Moore et al. 18 suggested a length of 270 Kb after analysis with clamped homogeneous electric field (CHEF) gel electrophoresis. Base pair composition on average across the HHV-8 genome is 59% G/C; however, this content can vary in specific areas across the genome 2. HHV-8 possesses a long unique region (LUR) at approximately 145 Kb, with at least 87 genes, flanked by terminal repeats (TRs). Varying amounts of TR lengths have been observed in the different virus isolates. These repeats are 801 base pairs in length with 85% G/C content, and have putative packaging and cleavage signals 19. The LUR is similar to HVS and the HHV-8 genes are named after their HVS counterparts. New genes are still being discovered through transcription experiments with alternative splicing; the initial annotation by Russo et al. 19 was purposely conservative. A "K" prefix denotes no genetic homology to any HVS genes (K1–K15).

HHV-8 possesses approximately 26 core genes, shared and highly conserved across the alpha-, beta-, and gammaherpesviruses. These genes are in seven basic gene blocks, but the order and orientation can differ between subfamilies. These genes include those for gene regulation, nucleotide metabolism, DNA replication, and virion maturation and structure (capsid, tegument, and envelope). HHV-8, being a gammaherpesvirus, encodes more cellular genes than other subfamily viruses. HHV-8 in particular, has a large arrangement of human host gene homologs (at least 12) not shared by other human herpesviruses 19. These genes seemed to have been acquired from human cellular cDNA as evidenced by the lack of introns. Some retain host function or have been modified to be constitutively active; an example of this is the viral cyclin-D gene 20. Cellular homologs related to known oncogenes have been identified in HHV-8, including genes encoding viral Bcl-2, cyclin D, interleukin-6, G-protein-coupled receptor, and ribonucleotide reductase 19. Other genes, such as the chemokine receptor ORF 74, have homologues in other members of the RDV genera 19. A number of other genes derived from the capsid of HHV-8 have been identified, including Orf 25, Orf 26, and Orf 65 19. In addition to virion structural proteins and genes involved in virus replication, HHV-8, typical of a herpesvirus, has genes and regulatory components that interact with the host immune system, presumably as an antidote against cellular host defenses 21.

HHV-8 gene expression has been classified into three stages by current investigators, unlike the four stages of other herpesviruses described above 22. Class I genes are those that are expressed without the need for chemical induction of the viral lytic phase. Class II genes are induced to increased levels after chemical induction. However, Class III genes, are only expressed after chemical induction.

HSV infection is optimally detected through direct culture of tissues or secretions with observation of cytopathic effect (CPE) usually occurring in animal embryo cells after 1 – 3 days. Sensitivity of detection of infection is dependent upon the stage of the clinical illness with an average sensitivity of approximately 80%. The shell vial technique, a modified immunofluorescent assay, is also used. VZV grows with more difficulty in culture and it takes 4 to 8 days until CPE is evident, but shell vial techniques can improve the ability to detect VZV infection. Immunofluorescent assay detection (IFA) using monoclonal antibodies (mAb) and using samples taken from the lesions is much quicker than culture methods. However, serology has not been employed conventionally due to the successful culturing techniques. Also, for a successful serological diagnosis, serology requires acute and convalescent samples. Neither culture nor serology has shown optimal sensitivity. Detection of specific glycolsylated proteins can distinguish HSV-1 from HSV-2 infection 2.

These viruses (HCMV, HHV-6 & 7) have a more restricted host range than the alpha herpesviruses and exhibit slower growth in culture. They are ubiquitous in the general population but cause serious disease in immunocompromised patients. Diagnosis is difficult due to the absence of clinical disease in healthy persons; virus can be present without pathological effect in humans 2.

Current diagnosis of HCMV is complicated by the intrinsic labiality of the virus and that CPE is not seen in human fibroblast culture cells until after one to three weeks of growth. However, shell vial assays can give results in 24 – 48 hours 2. The presence of HCMV in peripheral blood is diagnostic for infection even if found in otherwise healthy patients without clinical symptoms. Detection of the HCMV protein, pp65, by an antigen assay is commercially available and can be used for rapid diagnosis of HCMV infection. The pp65 antigen comes from the HCMV lower matrix phosphoprotein customarily found in white blood cells. This antigen test has better sensitivity than culture and can provide positive laboratory results in a few hours. A mAb is used to detect pp65, but the antigen is labile and laboratory tests need to be run within 24 hours of the blood collection 2. HCMV IgM antibody is diagnostic for HCMV infection in the context of mononucleosis-like disease where the patient is EBV negative. However, acute EBV infection can produce a false positive HCMV IgM test result 31.

For HHV-6 and 7, asymptomatic viral shedding is common in the benign carrier state. Culture of these viruses has been successful with umbilical cord lymphocytes, but there is high background. There are a lack of diagnostic criteria to interpret serologic test results in immunocompromised patients, although the finding of seroconversion in infants is diagnostic 2. The IFA test using virally infected cells has been commonly used with success 32.

EBV replicates in vivo in lymphoid and epithelial cells and can be cultured in immortalized umbilical cord lymphocytes; EBV antigen is found within the cells. Serology is used for diagnosis of infectious mononucleosis (IM) by detecting IgM heterophile antibodies that agglutinate with red blood cells of horses. Serologic assays can also measure antibodies to the EBV viral capsid antigen (VCA) that is composed of four different proteins, the early antigens (EA) of which there are five proteins, and the nuclear antigens (NA). Testing for IgM against VCA defines acute infection and corresponds to clinical sequelae but lasts only a few months; however, IgG remains for the life of the patient 33. Anti-EA antibodies arise within a few weeks but are not detectable in all patients with mononucleosis 33. Anti-NA antibodies arise after the advent of EA antibodies and persist for life 33. In contrast to acute infection, serology is not useful for post-transplant lymphoproliferative disorder (PTLD) and antigen detection or detection by PCR of viral nucleic acids is required 2. Antibody production might be compromised due to the host's immunocompromised state or the rapid growth of the polyclonal tumor prior to reactivation of the memory immune response. Antigenic cross reactivity between EBV and other human herpesviruses is rare 2. This is demonstrated in one study of 42 patients with nasopharyngeal carcinoma, known to be associated with EBV and of all persons positive for EBV VCA, only two showed reactivity to HHV-8 lytic proteins 34.

The humoral antibody response to EBV infection is against four serologically defined antigens 2:

1. Epstein – Barr virus NA (EBNA) in latently infected cells.

2. EA either in its diffuse (methanol resistant) or restricted (methanol sensitive) compartments, expressed early in the viral lytic cycle.

3. VCA found during the late lytic cycle.

4. Membrane antigen (MA; gp350) as part of the viral envelope and is found on the surface of cells in the lytic phase. Anti-MA antibody levels correlate well with neutralization of the virus.

These EBV antigens are composites of several distinct proteins; e.g. EBNA = EBNA 1, 2, 3A, 3B, 3C. LP and EBNA1 are the most antigenic. The detection of EBV in IM is based upon the use of an enzyme-linked immunosorbant assay (ELISA) to detect IgM specific to BALF2 and BMRF1, the EA antigens, or against VCA components BFRF3 and BLRF2; combinations of these antigens are still recommended 3536. Diagnostics of HHV-8 will be discussed at length in Section 8, HHV-8 Diagnostics.

Transmission of other herpesviruses (e.g., HCMV and HHV-6) has been documented 88 and the body of evidence is growing that HHV-8 disease after organ transplantation is a concern for the transplant physician. Most reports in the literature have presented data describing the prevalence and the possible ramifications of HHV-8 infection on donor kidney recipients.

However, the concern of HHV-8 transmission in the context of organ transplantation has two problems. First, there are no large studies of the donor's and the recipient's HHV-8 serostatus and presence of HHV-8 in donor blood and organ. Properly done, both antibody prevalence and a determination of infectious virus by PCR would be necessary. Follow up measuring possible seroreactivity every few months after transplant would be critical. Second, even once the problem is defined, there are no current establish procedures or parameters to monitor the patients both diagnostically and clinically; seemingly, both problems would have to be addressed in tandem.

In areas where endemic KS is not found and in normally healthy people, HHV-8 infection has not been shown to be a life threatening infection. However, in the context of immunosuppression, as with organ transplants, both primary infection and reactivation become a proven concern. Post-transplant immunosuppression can cause iatrogenic KS to appear 89. The clinical significance of post-transplant KS can be rejection of the graft and death of the patient. In a study of 356 post-transplant patients with KS, 40% had visceral involvement, a manifestation of KS with poor prognosis, and 17% of those with visceral KS died from the tumor 89. The KS tumor can recede after withdrawal of immunosuppressive therapy, but with immunological recovery, graft loss or organ impairment often emerges as a unwanted condition 89. In an early study, Parravicini et al. 90 suggest that post-transplant KS is caused by emergence of latent HHV-8 after previously infected but clinically well transplant patients are immunosuppressed. Immunosuppression, such that occurs in transplant recipients, is known to facilitate reactivation of herpesviruses, (e.g., disseminated herpes zoster) and is associated with an increased incidence of herpesvirus associated lymphoproliferative malignancies 91.

Of importance, seroprevalence to HHV-8 increased from 6.4% to 17.7% overall one year after renal transplantation. In addition, seroconversion to HHV-8 occurred within the first year after renal transplantation in 25 of 220 patients and KS developed in two of the 25 within 26 months after transplantation 92. KS developed within 20 months in two renal transplant recipients from the same cadaveric donor; Orf 73 genotyping confirmed that the virus was transmitted from the donor 93. Detection of HHV-8 in the allograft kidneys or increases in antibody titer can be prognostic indicators of increased risk for KS 94. Other studies have found the median time to KS from transplantation to be between 7 months 90 and 24 months 95.

In another study, the increased risk of acquiring HHV-8 infection was shown by 10% of 100 transplant patients who seroconverted to HHV-8, however, there was no pattern associated with the type of organ donated, and none of the donors that could be tested were seropositive 96. Therefore the investigators concluded that the infection came from sources other than the transplanted organ; however this conclusion is lacking because healthy infected individuals (i.e., healthy organ donors) in the USA are less likely to exhibit antibodies, similar to blood donors, however, the organ might still harbor infectious virus or KS precursor cells 9394.

In a comparison of kidney and liver transplants, seroconversion was observed in 12% of transplant patients, combined. The incidence of KS in kidney patients was higher than in liver recipients 97. Importantly, patients already infected with HHV-8 had a greater chance to develop KS from viral reactivation than from primary infections 97. In a large study of solid organ transplant recipients in Spain (n = 1,328), Munoz et al. 95 reported that the overall KS incidence was 1 in 200 with more males diagnosed with KS than females (6:1 ratio). High HHV-8 antibody titers or seroconversions were prognostic indicators of possible KS development.

Because increased prevalence in transplant patients might be due to reactivation of HHV-8 and the subsequent increase of antibody tiers 98, molecular methods, although normally less sensitive, would be better indicators of transmission. Another possibility would be the use of antibody avidity assays to detect highly avid antibodies that would be indicative of reactivation events 99.

Post-transplant KS can develop in the recipient from transmission of the virus from the donor to the recipient 9394, and from KS progenitor cells seeded along with the donor organ, which undergo neoplastic change, and progress into KS 100. HHV-8 DNA can be detected in the KS lesions from patients suffering from post-transplant cutaneous and visceral KS. Other organs without evidence of KS involvement can test positive for HHV-8 sequences 101, as can circulating spindle cells infected with HHV-8 102. Disease entities associated with HHV-8 in the context of transplantation continue to be discovered. In at least one report, investigators have suggested that EBV-negative post-transplant lymphoproliferative disorders (PTLD) might be caused by HHV-8 103.

Non-neoplastic disease associated with HHV-8 has been documented 49104. Bone marrow failure was observed after a kidney transplant and after an autologous peripheral blood stem cell (PBSC) transplant for non-Hodgkin's lymphoma (NHL). HHV-8 produced a syndrome of fever, marrow aplasia and plasmacytosis; these occurred after primary infection and reactivation, respectively 104. Neither patient presented with KS, but both had detectable HHV-8 sequences by PCR after transplantation and at the presentation of symptoms – both patients died. Another case report 49 showed reactivation of HHV-8 in a seropositive patient and documented nonmalignant disease 17 days after PBSC transplantation in the context of NHL. The patient presented with fever, cutaneous rash, diarrhea, and hepatitis; here too HHV-8 DNA was detected in the serum by PCR with higher viral loads with exacerbation of symptoms. Therefore, transplant patients who are HHV-8 positive could benefit from close clinical follow-up to preempt the occurrence of KS with judicious use of immune suppressive therapy or antiviral drugs, or to begin the early and therefore more effective treatment of the tumor once detected.

The classical or sporadic form of KS (CKS) is an indolent tumor affecting the elderly, preferentially men, in Mediterranean countries such as Italy, Israel, and Turkey 116. The lesions tend to be found in the lower extremities and the disease, due to its non-aggressive course, usually does not kill those afflicted. HIV infection, unlike HHV-8, is not typically associated with CKS 117.

The older the age of the patient, the greater the risk of CKS disease progression; dissemination of KS lesions is more likely if immunosuppression also exists 118. Certain behaviors, such as corticosteroid use and infrequent bathing were found to be risk factors for greater incidence of CKS but surprisingly, increased cigarette smoking actually lowered the risk 119. The increased prevalence in Sardina of HHV-8 and CKS among family members of KS patients indicates that transmission of HHV-8 is probably by asexual routes 61.

In the context of the acquired immunodeficiency syndrome (AIDS), KS is the most common malignancy and is an AIDS defining illness 120. AIDS-KS is a more aggressive tumor than CKS and can disseminate into the viscera with a greater likelihood of death 121. Unlike CKS, it presents more often multifocally and more frequently on the upper body and head regions 117.

In those with HIV infection, HHV-8 prevalence increases with higher risk of KS, and in patients with HHV-8 seroconversion there is a greater likelihood of KS development 74. KS was more likely to develop when HHV-8 seroconversion occurred after the patient already had HIV 122123. An increased slope of CD4+ cell decline and higher HIV viral loads also suggested increased chances of KS development 122.

However, HIV infection alone might not be enough to increase the risk of KS. In a study of Ethiopians who had immigrated to Israel, only 0.85% of them with AIDS developed KS, as compared with 12.5% of non-Ethiopian AIDS patients (P < 0.001). The low risk of KS exists in the face of high HHV-8 prevalence (above 39%) in HIV+ and HIV- Ethiopian populations 124. Clearly, other factors are necessary for KS development and ethnic or genetic protective factors might be involved.

HHV-8 was prevalent in Africa prior to the HIV epidemic, and therefore, was responsible for the large prevalence of KS seen on the continent before HIV changed the scope of KS presentations 125. Prior to HIV coinfections, endemic KS affected men with an average age of 35 and very young children 126. In Africa, endemic KS is found more often in women and children than in other areas of the world 125. It presents in four clinical forms with one form similar to CKS, but found in younger adults; the other three forms are more aggressive, similar to AIDS-KS 117. They vary in the age of presentation and the sites of involvement.

HIV coinfection has raised the prevalence of KS significantly in Africa. In Uganda, for example, prior to 1970, KS was diagnosed in no more than 7% of the male cancer population and in none of the female cancer population. However, by 1991, KS prevalence had risen to 49% in male cancer patients and to 18% in females 126. The KS prevalence has increased in Africa, even in HIV negative populations, for unknown reasons 125.

Despite different clinical KS presentations, all forms of KS are associated with HHV-8 infection 111127. Paralleling the endemic KS pattern in children, HHV-8 infection in children is also high with seroprevalence reaching adult levels by the age of 20 and in certain locations even earlier 128. This occurrence of horizontal infection in the young is similar to that seen with EBV in other continents 128. Despite equal prevalences of HHV-8 in HIV-1 and HIV-2 patients, KS is found almost exclusively in persons infected with HIV-1 129.

More extensive information on transplant-associated KS and the involvement of HHV-8 can be found in the Literature Review: Section 4, Transmission of HHV-8. Briefly, iatrogenic KS can present either as a chronic condition or with a more rapid course 117. Immunosuppression, such that occurs in transplant recipients, is known to facilitate reactivation of herpesviruses 91 and so too with HHV-8, transplant patients under immunosuppressive therapy can present with KS. Withdrawal of the therapy can cause the KS to regress 117.

Iatrogenic KS seems to vary in its geographic prevalences, perhaps reflecting the varying HHV-8 prevalence in the general populations of different countries 125. KS appears most frequently in renal transplant patients 116 and in conjunction with cyclosporine treatment, used frequently in kidney transplant patients as an immunosuppressive drug; this steroid has been shown to reactivate HHV-8 in vitro 130.

Sarcoidosis is a multisystemic granulomatous disease of unknown etiology that can involve many different organs such as the lungs, lymph nodes, and skin. Currently, a diagnosis can be established when clinical and radiological findings are confirmed by histological tests showing noncaseous granulomas in more than one tissue 142.

Di Alberti et al. reported that HHV-8 DNA was significantly more prevalent in pulmonary tissues, lymph nodes, skin and oral tissues in 17 Italian patients with sarcoidosis than in tissues from 96 control specimens 143. However, a study by Belec et al. did not detect HHV-8 sequences in sarcoid tissues from French patients with systemic sarcoidosis 144. Very little diagnostic HHV-8 serology has been reported on sarcoid patients. In one report, 18% of patients were seropositive, but the investigators concluded that this was not different from the observed prevalences in the patients' respective geographic regions 145.

There is debate concerning the etiology of MM. MM is the most common lymphoid cancer found in Blacks and the second most common in Caucasians 146. It is a B cell malignancy of clonal origin in which the cancer cells, considered to be plasma cells, secrete monotypic immunoglobin. The pathogenesis of MM has been thought to include an initial antigenic stimulus of B cells followed by further mutagenic events. Studies have shown that autocrine and paracrine loops involving cytokines such as IL-6 147, TNF, and IL-1β 148 are important as stimuli for growth of the MM cells. It has been believed that T cells and the bone marrow stroma are the sources of these cytokines. Three oncogenes have been implicated in MM; ras, c-myc, and p53 with prevalences of 30%, 25%, and 15–45%, respectively 146.

The possible role of HHV-8 in MM has been debated and a full report of the evidence is beyond the scope of this review. In brief, Rettig et al. 149 who originally reported that there was an association between the virus and the disease, investigated 15 MM patients along with eight patients presenting with monoclonal gammopathy of unknown significance (MGUS). They used PCR to amplify the KS330₂₃₃ sequence of HHV-8 from bone marrow (BM) mononuclear and stromal cells of the MM patients. Southern blotting of the PCR fragments using an internal fragment confirmed the PCR results. They were able to amplify HHV-8 sequences from cultured BM stromal cells from 15/15 MM patients. However, none of the 23 non-cultured BM mononuclear preparations amplified. Said et al. 150 supported Rettig et al.'s claim that MM and HHV-8 were closely associated by finding 17 out of 20 BM biopsies from MM patients exhibiting HHV-8 positive cells. Gao et al. 151, provided important supportive serological evidence; of 27 MM patients, 81% and 52% possessed lytic and latent antibodies, respectively. All eleven patients with progressive MM were HHV-8 positive. The increased presence of lytic antibodies as opposed to latent antibodies was indicative of past or currently active viral infection in the MM patients.

Contrary to these findings, other groups have found a lack of supporting evidence. Whitby et al. 152 found latent antibodies in only 4/37 MM and in only 2/36 MGUS patients, but these prevalences were not significantly different from patients with Hodgkin's lymphoma, NHL, or normal blood donors. Additionally, whereas Rettig et al. postulated that MGUS might be the precursor of MM through infection with HHV-8 149, Whitby and colleagues found that 4 persons with MGUS who developed MM were HHV-8 negative, in contrast to two patients with antibodies to HHV-8 who had not exhibited MM symptoms after 36 and 48 months. MacKenzie et al. 153 and Parravicini et al. 154 found only 2/78 and 1/20 MM patients to be seropositive to latent antigen, respectively. The presence of lytic antibodies in MM patients has also been difficult to find by other investigators. Utilizing recombinant ORF 65 antigen in ELISA and Western blot formats, MacKenzie et al. 153 and Parravicini et al. 154 found lytic antibodies in only 2/78 and 1/20 MM patients, respectively. Masood et al. 155 using a lytic IFA and a whole virus lysate ELISA found that only 2/28 MM sera were positive. Perhaps as the pathogenesis of HHV-8 becomes better understood this etiological question will be answered.

Although there are many reports for other diseases and their possible associations with HHV-8, the data are sometimes circumstantial and weak, and many have not been confirmed by extensive investigation in large numbers of patients. Only a few selected diseases or conditions variably associated with HHV-8 are summarized below.

Bone marrow failure is a non-neoplastic disease possibly associated with HHV-8 observed after kidney and autologous peripheral blood stem cell transplants. HHV-8 produced a syndrome of fever, marrow aplasia and plasmacytosis; these occurred after primary infection and reactivation, respectively 104. Neither patient presented with KS, but both had HHV-8 sequences detected by PCR after transplantation and at the presentation of symptoms.

HHV-8 infection has been associated with congestive heart failure in both KS and PEL patients 138. Serological evidence has also indicated that Italian patients with cardiovascular disease have a higher prevalence of HHV-8 and HHV-8 DNA has been found in atheromatous plaques 156. Other studies have suggested possible associations with HHV-8 and pemphigus vulgaris and pemphigus foliaceus 157 and germinotropic lymphoproliferative disorder 158, but not primary central nervous system lymphomas 159.

Studies from populations from the North American continent have revealed large differences in HHV-8 prevalence between specific populations. Blood donors (BD) have been found to exhibit different levels of infection ranging from no detected infection 167 to as high as 15% 168, with more intermediate levels (~5%) found in most studies 345979. Individuals infected with HIV infection or having AIDS had more elevated prevalences of 30%–48% 3474167, although one study found no evidence of HHV-8 infection in their small HIV cohort 167. Homosexual men showed prevalences ranging from 20%–38% 74169170. In contrast, the highest prevalences, between 88% and 100%, were found in those patients with KS 3479167. Other miscellaneous populations, such as healthy individuals, the elderly, and those infected with EBV showed a range of 0%–8.6% 74167171. IVDUs had relatively higher prevalences of 10% in both heterosexual men and women; the longer the patient's injected drug use, the higher was the risk of HHV-8 infection, which was not dependent upon sexual behavior or demographic differences 169. Of note is the exceptionally high level of infection found in children in south Texas, 26% 172. One report from Quebec, Canada, did not find evidence of HHV-8 infection in 150 renal transplant patients 173.

The prevalence of HHV-8 in BDs from Jamaica, Trinidad, and Cuba was 3.6%, 1.2%, and 1.2%, respectively 34174175. Persons with HIV infection from Trinidad, Honduras, and Cuba possessed prevalences of infection at 0%, 24%, and 21%, respectively 34175176. Compared to other studies in KS patients, a relatively low prevalence of HHV-8 infection was found in AIDS KS samples from Cuba (78%) 175. A very low level of infection was found in attendees of a gynecology clinic in Jamaica (0.7%) 174, but an elevated prevalence was seen in healthy individuals in Honduras (11%) 176. Commercial sex workers in Honduras showed 19% infection 176.

Evidence of HHV-8 infection has been discovered in South America in at least four countries. In indigenous populations, those without specific risk factors, prevalences of 53% were found among Brazilian Amerindians 177, 16% in northern Brazil 178, and 36% in Amerindians of Ecuador 179; the prevalences in Ecuador ranged from 20%–100% depending upon the tribe tested 179. The HHV-8 prevalence was much less in BDs in Brazil (2.8%), Chile (3.0%), and Argentina (4.0%); although in Argentina the prevalence in BDs ranged between 2.4% – 4.3% in three different locales 180. In contrast, Sosa et al. 181 reported that in Argentinean HIV+ IVDUs, 17.4% showed HHV-8 seropositivity; where as, in HIV negative IVDUs the prevalence was lower at 11.1%. Still lower, HIV negative heterosexuals with no IVDU behavior had a prevalence of 5.7%, similar to that found by Perez et al. 180. AIDS-KS patients in Brazil had a prevalence of 80% 182.

In Europe, excluding Italy and its surrounding islands, the prevalence of HHV-8 in BDs was not above 6.5% in six countries: Hungary 0.83%–1.6%, Switzerland 5%, the United Kingdom 1.7%, France 2%, Spain 6.5%, and Germany 3% 5983183184185186. In healthy individuals in Switzerland, Greece, and Albania, evidence of HHV-8 infection was 13%, 12%, and 20% 59184187. Persons infected with HIV ranged from a low of 16% in women in Germany to a high of 31% in homosexuals in the United Kingdom 59184186. Homosexuals in Spain however, had an 87% prevalence 185. IVDUs and persons with STDs in the United Kingdom, Spain, and France showed prevalences of 3.2%–8.4%, 12%–17%, and 13%, respectively 59185188. Similar to North America, the HHV-8 prevalence in patients with classic or endemic KS was 75%, 94%, and 100% in Hungary, Greece, and France, respectively 5983183. The HHV-8 prevalence in AIDS-KS patients in Switzerland (92%), the United Kingdom (81%), France (80%), and Germany (100%) were similar to the prevalence of HHV-8 in classic KS in Europe 5983184186. IVDUs in the United Kingdom and Spain had prevalences of 0.0%–3.2% and 12%, respectively 59185.

Estimations of seroprevalence in Italian BDs were confounded by the variable geographic prevalences and the type of antibodies being detected. Whitby et al. 189 showed that the overall prevalence in 747 BDs in Italy was 14%. However, when these individuals were segregated by North/Central Italy and Southern Italy, the levels of HHV-8 infectivity dispersed to 7.3% and 24.6%, respectively. Even in Rome, centrally located in the country, the prevalence in BDs varied from 2% of people with latent antibodies to 28% with reactivity to lytic antigens 190. Other reports found prevalences in BDs to be between 3.5% to 18.7% 167191192. In the general population of Sardinia 61, Sicily 193, and for the elderly in Malta 194, antibodies to HHV-8 were found in 11%, 20%, and as high as 54%, respectively. In Italy, those infected with HIV showed a 14% prevalence for latent antibodies, but as high as 61% for lytic antibodies 190; an intermediate rate (25%) in HIV+ persons was observed by Calabro et al. 192. In Sicily, 34.6% of HIV+ patients had HHV-8 infection 193. In regards to other STDs, infections with syphilis were accompanied by HHV-8 infections with 37%–76% showing coinfection, whereas those free from syphilis infection only showed 11%–46% prevalence 190. No significant differences were seen in persons with or without HCV infections, 10%–50% and 16%–47%, respectively 190. Perna et al. suggested that the relatively low prevalence of HHV-8 in drug addicts in Sicily (16.6%) was indicative of the poor transmission of HHV-8 parenteraly 193. Calabro et al. 192 observed 61.5% prevalence in HIV+ homosexuals in Italy, but this rate might have been confounded by the coinfection of HIV because Perna et al. found a lower rate in homosexual men, 32.6% in Sicily 193. Even healthy adults in Sicily had an elevated prevalence beyond that found in BDs with 36.2% observed with HHV-8 infection 193. For this central region of the Mediterranean, the prevalence of HHV-8 in CKS normally exceeded that of AIDS-KS. CKS in Italy and Sardinia showed evidence of infection in 95%–100% of patients. However, AIDS-KS were reported to have a much wider range of reactivity in HHV-8 tests: 71%–79% 167, 57.1%–100% 191, 67%–83% 190, and 100% 192 in Italy, and 100% in Sicily 193.

Healthy individuals in Israel were found to have a HHV-8 seroprevalence of 4.8% 195, whereas individuals with HBV infection seemed to be at an increased risk of infection (22% prevalence) 81. Family members from these hepatitis patients also had increased prevalence of HHV-8 at 9.9% 81. When Ethiopian immigrants to Israel were tested for antibodies against HHV-8, this unique cohort possessed an elevated presence of antibodies against HHV-8 124. Fifty seven percent of Ethiopians with HIV infection showed HHV-8 infection, whereas those without HIV had a lower prevalence of 39.1% (P = 0.03). Interestingly, despite the high prevalence of HHV-8 in the HIV+ individuals, in those with AIDS, the occurrence of KS was almost nonexistent (0.85%) compared to non-Ethiopian immigrants with AIDS (12.5%) 124. Reports on HHV-8 prevalence from Egypt are scarce. Andreoni et al. showed data that in teenagers and young adults, 29% possessed lytic antibodies against HHV-8, but only 5% had latent antibodies 191.

Blood donors and healthy individuals in five Asian countries have shown a 3-fold range in HHV-8 prevalence. In healthy Indian individuals 34, only 3.7% had antibodies, with Thailand, Malaysia, and Sri Lanka exhibiting prevalences no higher than 4.4% 34. In Taiwan, lytic antibodies were found in 11.7% and 13% of the blood donors tested 196197. However, a much higher presence of prior infection was found in the general population of the Uygur people in northwestern China, 47% 198. The prevalence of infection in HIV positive individuals in Asia varied widely, as well. Prevalences of HHV-8 infection of 0.6% to 11.2%, 2.4%, and 40% where found in Thailand, India, and Taiwan, respectively 34197199. Classical KS still had the highest rate of infection, with 83% of patients in Taiwan 197 and 100% in China 198 showing positivity for HHV-8 antibodies.

There have been few studies on the seroprevalence of HVV-8 antibodies in the Pacific region. Despite this, the viral infection has been found in both Japan and New Guinea 200201. Fujii et al. 200 found a very low prevalence of HHV-8 infection in Japan in BDs where only 0.2% showed reactivity to latent antigen. Comparatively, persons with HIV infection had an elevated prevalence of between 9.8% and 11.6%. In New Guinea, Rezza et al. found a much higher prevalence in the indigenous general population with approximately 25% of the 150 people tested showing prior infection 201.

In sub-Saharan Africa, the seroprevalence of HHV-8 was above 36% in every population reported. In the southern part of the continent, healthy individuals showed a HHV-8 prevalence of 37.5% in Zambia 34, and 54.7%–90% in Botswana, depending upon the test used 179194. In Zambia, the HHV-8 prevalence was comparable for HIV+ persons (44%) 34 and 51.1% in HIV+ pregnant women 202. Cancer patients, in general, in South Africa also had a high prevalence of 36.3% 203. In comparison, patients with AIDS-KS exhibited a prevalence of 83% in South Africa 203 and 92.3% in Zambia 202.

Central African nations also had HHV-8 prevalences in keeping with those observed in the south. In the Congo, a high prevalence in healthy individuals, 69%–79%, showed prior HHV-8 infection 194. Somewhat lower percentages were found in healthy individuals in Ghana (41.9%) 34, in Uganda (38.7%) 34, 51%–62% 167), and 55.5% in Cameroonian pregnant women 83. Similar HHV-8 prevalences were found for HIV+ persons in Uganda with between 45.7% and 71% HHV-8 prevalence reported depending upon the study and the test used 3459167. The prevalence of HHV-8 infection in AIDS KS patients was relatively higher but did not reach 100%; in Uganda, Gao et al. reported 78% and 89% 167 and Simpson et al. found 82% prevalence 59.

In conclusion, prevalence rates varied depending on the geographic origin of the sera tested and the specific tests used to determine these prevalences; in particular, whether antibodies against latent or lytic antigens were detected could make a difference in the results. Additionally, it is unclear whether these differences were truly due to varying prevalence rates, or perhaps to a lack of sensitivity and specificity of the serologic assays, as has been shown for HIV 204 and HCV 205. Because most reports indicated high rates of HHV-8 infection in persons with KS, regardless of their origin, it is probable that the assays possess reasonable ability to detect true infection.

Regamey et al. reported that there was a trend of increasing HHV-8 antibody prevalence to Orf 65 antigen with increasing age in HIV negative individuals in Switzerland 184. Below 30 years of age, the prevalence increased from 15% to 23% and then to 50% in the next three decades. A similar effect was observed in BDs in Hungary 183. As age increased from 19 until 25 years of age and then for every decade afterwards, the distribution of seropositivity to LANA increased moderately, but significantly (P = 0.048). A similar association was observed with Orf 65 peptide reactivity but the numbers of subjects were too small to calculate statistical significance 183. In Taiwan, increased progression of antibody response against HHV-8 lytic antigens was observed, starting with a low of 3% in children under five years of age and peaking between age 31 and 40 (19.2%) 196. Many more examples of this have been reported in Africa 83128203, Sardinia 61, and Italy 192. Perna et al. 193 and others 172183185192 have shown that there most likely exists non-sexual routes of HHV-8 transmission because children worldwide have been infected by HHV-8.

PEL cell lines have been important sources of antigen mainly for use in IFAs, but also in the form of cell lysates for Western blotting and tools for investigations into HHV-8 pathogenesis 59210211212215224226. Over 12 PEL cell lines have been established and they each contain 50–150 episomal copies of HHV-8 per cell 8132133134135136. About half are coinfected with EBV (e.g., BC-1, BC-2, BCBL-2), but others have only latent HHV-8 infection (e.g., BCBL-1, BC-3, KS-1) 135. Induction of viral replication can be initiated by sodium butyrate (butyrate 228, 12-O-tetradecanoylphorbol-13-acetate (TPA), a phorbal ester 229, or less commonly hydrocortisone 130. Cell cultures derived from KS spindle cells are not good material for HHV-8 diagnostics because they lose the virus after 2–6 passages 230.

Other sources of antigen have been whole virus lysate, which has been used successfully in the ELISA format 231. After induction of a PEL cell line, the whole virus is usually purified over a sucrose gradient. The drawback of this method is that it preferentially selects for lytic antigens and does not allow detection of latent antibodies such as LANA1 112. In contrast, individual HHV-8 proteins have been incorporated into tests by either expressing them as recombinant proteins or as synthesized peptides. Recombinant proteins such as Orf 65, K8.1, Orf 25, and Orf 26 have been expressed in easy to grow bacterial systems 59221223. Antigenic proteins have also been expressed in more difficult to grow baculovirus systems (insect cells) 232233, but they have the added benefit of protein glycosylation which bacterial cells can not perform. It has also been reported that LANA1 (Orf73), because of its large size (>200 kDa) is expressed better in insect cells (personal communication, Dr. D. Whitby, NCI-Frederick). Synthesized peptides of immunodominant portions of antigenic proteins (e.g., K8.1, Orf 65) have been developed as a strategy to streamline the production process and to reduce non-specific reactions 183234.

ELISA tests are easier to manipulate and technically are the test of choice for large-scale seroprevalence studies. ELISAs based on recombinant antigens of HHV-8 have shown that a specific humoral response is produced against capsid proteins of HHV-8, allowing identification of HHV-8 infection 5992223. Recombinant proteins derived from a truncated Orf 65 minor capsid gene have been used with a relatively high degree of success to differentiate populations of KS patients from BDs 214. Similarly, recombinant proteins derived from the Orf 25 and Orf 26 genes (major and minor capsid proteins) have been used in ELISA assays to detect IgG and IgM antibodies, but with a lesser degree of success 223. Seroconversion against capsid proteins has been shown to occur in less than one year after infection using an Orf 65 ELISA 92.

An ELISA based on viral lysate antigens of HHV-8 has also produced encouraging results 231. Although this assay demonstrated a good sensitivity for detecting infection in patients with classical KS (CKS) and AIDS-KS (80%–90%), normal healthy blood donors had 2–11% prevalence. This ELISA also possessed the ability to differentiate populations based on antibody titer; the mean titer in blood donors was 1:30, while titers ranged from 1:6000 to 1:15000 in AIDS-KS and CKS patients.

Encouraging results have come from a recombinant ELISA based upon the K8.1 gene product 235 and has been considered one of the more sensitive tests with acceptable specificity. Immunodominant peptides from the Orf 65 and K8.1 antigens were incorporated in an ELISA format and used successfully to measure the risk factors in women 76 and to identify HHV-8 infection in allogeneic stem cell transplant patients who are at risk of KS because of their immunocompromised status 236.

Initially, the primary method of detection of latent antibodies was using the LANA IFA, however, subsequent cloning of Orf 73 (LANA) and its application in the ELISA format has begun to replace the LANA IFA. The Orf 73 ELISA has been found to possess the same high specificity, but with a 10% increase in sensitivity 107. The Orf 73 ELISA has found utility in gauging the progression to KS in HIV+, HHV-8 infected persons 43. In that study, increasing titers to Orf 73 over time were associated with HIV+ patients acquiring KS.

IFAs are a common method to identify antibodies to HHV-8. To detect latent antibodies, an HHV-8 infected PEL cell line (e.g., BCP-1, BCBL-1, BC-3, KS-1) is used to measure antibodies to the primary latent antigen, LANA1 or ORF 73 107. This latent antigen corresponds to a ~234 kDa nuclear antigen, which has been shown to be recognized by sera from KS patients 211, and is characterized by its speckled nuclear fluorescent signature in 95% of PEL cells 107. With this assay, seroprevalences have ranged from 2%–27% in several studies of blood donors where KS is endemic, but lower (0%–15%) for those geographic regions where KS is mainly associated with AIDS and transplant patients 227. However, the LANA1 assay has been shown to be relatively insensitive and therefore might not be the best choice of assay to screen low titered populations 235.

Lytic antigens can be expressed by these cells following induction with a TPA or with butyrate 237 and have produced encouraging results. The number of induced cells is dependent upon the cell line used, the time of induction, and the chemical used to induce the cells 107238. Studies using induced PEL cell lines point to much higher frequencies of infection than have been suggested by serology based on latent proteins in populations not at risk for sexually transmitted diseases 16239. However, other studies using lytic IFAs have also indicated that there are higher levels of HHV-8 infection in otherwise healthy individuals 227 and infection would be spread by non-sexual routes in these cases. As with the ELISA, the IFA has been used to determine antibody titers, with sera from HIV-positive persons with KS demonstrating higher titers to lytic and latent antigens as compared to individuals without KS 214231. This test method is relatively more sensitive to serum dilutions that are not extensive enough; the correct serum dilution is important to correctly differentiate true positive reactions from those that are non-specific 112214.

Western blots using purified viral lysates of HHV-8 have been used to identify immunodominant proteins using sera from pre- and post-KS patients 114221240. This method has shown utility in the diagnosis and prognosis of KS, but it is more cumbersome and expensive than other serologic assays. A 35–37 kDa glycoprotein has been a protein most frequently and intensively detected, and corresponded to the K8.1 Orf of HHV-8 220.

In a review of articles that used Western blot in their investigations of HHV-8, only four dealt with antigen identification or expression. These reports could influence the development of a confirmatory Western blot as they showed: 1) There are different antigen profiles in diseases associated with HHV-8 140. 2) HHV-8 possesses the glycoprotein, gB, found in other herpesviruses and might be a candidate antigen 241. 3) That different risk groups and different stages of disease could exhibit different antibody profiles 242. 4) Patients undergoing antiviral therapy might not produce certain antibodies due to a decreased expression of HHV-8 antigens 238. These findings suggest that it might be necessary to identify specific antigens for use at specific times of infection and even for different disease states.

Nine reports involved the use of Western blots for screening purposes 6194210219221226243244245. Recombinant Orf 65 was used most often followed by K8.1, Orf 59 and Orf 73, and finally vIL-6 and Orf 47; however, there was no utility in using vIL-6 or Orf 47. In these reports, KS sera were detected by K8.1 with the greatest sensitivity, followed by Orf 65 and then Orf 73. In these studies, there were not sufficient HIV+ sera examined to draw conclusions as to which antigen was best in that specific population. Sera/plasma from healthy controls varied from a low of 0% for Orf 59 and Orf 73 to 6.5% for K8.1 and 8.3% for Orf 65.

Eleven research reports utilized Western blots as tools to confirm the results of previously run serological assays 5992183232246247248249250251252. Most of these authors used the same antigen found in the ELISA as the confirmatory antigen in the Western blot; however, two reports had the Western blot confirm IFA results. In seven instances, the authors used the Western blot to confirm a single screening assay and in four reports, they used the Western blot it to resolve a disagreement between two screening assays or in duplicate samples.

More recent reports have continued to use the Western blot as both a primary assay and as a confirmatory test 175253. The Western blot method has the benefit of allowing identification to one or more antigens. With accessibility to multiple recombinant proteins now possible, several researchers have developed recombinant Western blot utilizing more than one protein 197254. In those reports, they accepted reactivity to one of three antigens to be a marker of HHV-8 infection. In this manner, Wang et al. proposed a new antigen, Orf 57, for use in asymptomatic populations 197. Clearly, despite the technical difficulties in producing Western blots, they are a useful, multitasking serological method in HHV-8 diagnostics.

Estimates of the prevalence of HHV-8 by different ELISAs have varied. This variance has been shown in reports of multicenter or multitest studies. Spira et al. 255 found a range of concordance between 69% and 94% using seven serologic tests with Kappa values as low as 0.387 (fair agreement) and as high as 0.909 (almost perfect). Rabkin et al. 256 also evaluated seven serologic tests and found a range of concordance between 50% and 94%, with Kappa statistics ranging from -0.08 to 0.86, indicating that the interassay correlation between the assays was less than favorable. The tests frequently disagreed on individual sera, particularly from blood donors. It was concluded that current antibody tests for HHV-8 have uncertain accuracy in asymptomatic HHV-8 infection and that additional tests to define the actual prevalence may be required. Poor correlation for positive results has been observed in other studies 257. Second generation tests seem to provide better concordances, although the best results came from IFA tests rather than ELISAs in one multicenter study 258. Even with more optimized assays, sensitivity and specificity can be insufficient for clinical use 235. As with the detection of infection by many viruses (e.g., HIV), sequential use of screening and confirmatory tests for HHV-8 are likely to be required to address sensitivity and specificity issues; accordingly, an testing algorithm has been reported 235. These findings supported the need for critical investigation of the parameters that could influence the performance of these tests.

Although there is some variability in prevalence among similar populations with the same test, most data show that there is agreement within a defined range. The lack of concordance in HHV-8 diagnostic assays occurs primarily because not all HHV-8 infected persons exhibit all antibodies against all HHV-8 antigens at the same time 259. This phenomenon of single antibody reactivity is much more apparent in populations who are at low risk of infection, such as blood donors 259. Because of this, specificities are more variable than sensitivities among different laboratories 259.

Although refinement of the diagnostics assays is still possible, the greatest chances of success are in developing algorithms that make use of multiple assays for screening and then confirmation or alternatively, the use of assays that incorporate multiple antigens which have been shown to be highly immunogenic, perhaps during different stages of infection 259. It is possible to use a combination of latent and lytic antigen tests to determine a true positive as has been employed by several laboratories 112; however, recent data indicate that the humoral response to HHV-8 does not always produce both latent and lytic responses at the same time 260. In addition, antibodies directed against lytic antigens seem to be more prevalent than those for latent antigens.

IHC is a powerful serologic tool, but like Western blots can be tedious to perform. In the field of HHV-8 research and diagnostics, IHC has been used to locate HHV-8 proteins, assess involvement of HHV-8 in malignancies, detect specific HHV-8 gene expression, and to provide diagnosis of KS. The ability to identify which specific cell types or structures within a cell are expressing HHV-8 proteins can assist in the understanding of HHV-8 pathogenesis 261262 and determine the possible etiology of malignancies 263264265266267. Detection of specific HHV-8 gene expression, in particular LANA1 140211, has led to possible clinical applications for the diagnosis of KS in tissue samples 268269. This allows the exclusion of other neoplasms that can mimic KS 268269270. The ability of monoclonal and polyclonal antibodies to localize specific HHV-8 antigens should continue to improve HHV-8 diagnosis and our understanding of HHV-8 pathogenesis.