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Background: The association of the human herpesvirus-8/Kaposi's sarcoma (KS)-associated herpesvirus (HHV-8/KSHV) serology with various malignancies in Tanzania is not currently well established while previous studies were based on either PCR or immunofluorescence assays [IFA] but not with a sensitive enzyme-linked immunosorbent assay (ELISA). Selected archival diagnostic biopsies (n = 184) and sera from indigenous patients with KS (n = 120), non-KS tumors (n = 24) and non-neoplastic lesions (n = 40) at Muhimbili National Hospital (MNH), Tanzania, were evaluated by diagnostic histopathology, immunohistology [anti-HHV-8 latency-associated nuclear antigen (LANA)] and serology for HIV (ELISA) and HHV-8 (IFA and ELISA). Results: About 66.3% (n = 122) cases including AIDS-associated Kaposi's sarcoma (AKS) (n = 93), reactive conditions (n = 28) and only one non-KS tumour were HIV positive. Endemic KS (EKS) patients were mostly males (96.3%, 26/27) who were less (69.9%, 65/93) predominant in AIDS-associated (AKS). A high (89%) percentage of patients with anti-HHV-8 antibodies was found in the cohort including the HIV positive (92%) cases, males (81.2%), KS patients (93%), non-KS tumors (92%), and reactive conditions (75%). All HHV-8 seronegative KS cases were nodular stage whereas both sera and corresponding biopsies from early stage KS were HHV-8+. Assay sensitivity, positive predictive value (PPV) and specificity were 98.6%, 93.5% and 16.7% for IFA and 93.5%, 98.6% and 50.0% for ELISA respectively. Conclusion: HHV-8 seroprevalence at MNH appears high as expected among AKS cases and males but also in non-KS patients. ELISA showed a combination of high HHV-8 sensitivity as well as higher PPV and specificity than IFA which however, showed higher sensitivity. The apparent stage-dependent, inverted serum HHV-8 immunoreactivity supports a notion of viral immune-segregation during KS development. Routine HHV-8 screening should be considered particularly in patients at risk of KS and for selection of blood/organ donations. Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:10 http://www.infectagentscancer.com/content/3/1/10 examined and is therefore evaluated in the present study Background The HIV and AIDS epidemic has dramatically increased . the frequency of different malignancies particularly Kaposi's sarcoma (KS) and certain malignant lymphoma Results (ML) which are associated with the novel human herpes- Demography virus type 8 (HHV-8)/Kaposi's sarcoma-associated herpes A total of 184 selected biopsies and corresponding sera virus (KSHV) and have become a major health concern in available at MNH/MUHAS between 1990 and 2001 from sub-Saharan Africa including Tanzania [1-3]. The preva- indigenous African patients were included in the study. Of lence of HHV-8 varies from high-endemicity (30–70%) these patients, 120(65%) had KS, 24(13%) non-KS areas (sub-Saharan Africa), intermediate (5–20%) [Medi- tumors and 40(22%) non-malignant (reactive) condi- terranean] and low (≤ 5%) (Northern Europe, USA, tions. The male:female ratio was 2.75:1 for KS, 3:1 for south-east Asia and Japan) reflecting the KS and HIV epi- non-KS tumors and almost 1:1 for reactive, non-malig- demiology [4,5]. HHV-8 has a high (≈50%) prevalence in nant conditions [Figure 1(a)] and (Table 1). The mean age the healthy Tanzanian population (blood donors) for males was 36.6 years ranging from 12–70 and for although the prevalence in Tanzanian patients with and females 32.7 years ranging from 17–50 [Table 1]. without tumors is poorly documented . Seroconver- sion to HHV-8 precedes and therefore is predictive of KS Histopathology development  and serodetection may also help to con- Of the KS cases, most (68.7%, 82/120) were nodular stage firm KS diagnosis in suspect and/or borderline tumour [Figure 1(b)] with cutaneous (87.8%, 72/82) or lymphad- lesions. HHV-8 transmission is known to occur both hor- enopathic (12.2%, 10/82) localization [Table 2(a)]. The izontally (orally, sexually and parenterally) and vertically non-KS tumors included malignant lymphomas (45.8%), (mother-to-child) particularly in endemic areas [7,8]. epithelial cancers (25.0%), soft tissue tumors (25.0%) Obviously, large-scale HHV-8 screening would be benefi- and one neuroendocrine tumor (4.2%) [Table 2(b)]. Most cial and allow preventive/therapeutic interventions of non-neoplastic biopsies came from patients with reac- including possible anti-HHV-8 vaccination in at-risk pop- tive lymphadenitis and other inflammatory conditions. ulations, but this is not yet well documented in Africa, HIV serology particularly Tanzania. Previous studies on HHV-8 in Tan- zania were based on polymerase chain reaction (PCR) Most (66.3%, 122/184) studied sera were HIV positive assay which is not readily and widely available in (Table 1). Females were only 3.7% (1/27) among EKS resource-constrained developing countries [5,6,9]. Fur- patients and 30.1% (28/93) in AKS whereas males were thermore, the tested non-KS Tanzanian sera in the previ- predominant in both KS types which differences were sta- ous study by Massambu et al., (2003)  were very few tistically significant (χ = 7.96, p = 0.0048) [Table 1]. calling for the current larger study of hospital patients (KS, non-KS neoplasia and non-malignant clinical condi- Most KS cases 77.5%, (93/120) were HIV+ (AKS) and tions), for comparison with our previous reports includ- 22.5% (27/120) HIV- (EKS) [χ = 28.02, p < 0.001]. How- ing that on healthy blood donors, which was based on ever, most sera from patients with reactive conditions immunofluorescence assay (IFA) and real-time PCR , (70.0%, 28/40) were also HIV+ but most (95.8%, 23/24) The use of IFA necessitates culturing and subsequent non-KS tumor patients were HIV negative. The difference processing of a HHV-8+ body cavity-based lymphoma between HIV infection amongst KS and non-KS tumor (BCBL-1) cell line or another B-cell line (BCP-1) which patients was statistically significant (two-tailed p-value = may not be readily available and affordable in Tanzania 0.046) [Table 1]. particularly on a routine/large scale screening basis. HHV-8 serology Enzyme-linked immunoassays (ELISA) do not involve the use of cell cultures and thus provide an easy and more The HHV-8 serology results are summarized in table 3 and affordable screening method for HHV-8 infection. HHV-8 4. Most (89.0%, 164/184) screened cohort sera were sero-detection technology is still a developing area and HHV-8 seropositive based on either IFA (Figure 2) or new assays like ELISAs may not yet have been tested in the ELISA indicating a high prevalence among this cohort of high-endemicity African sera as compared to IFA [4,10]. MNH patients [Table 3 and 4]. HHV-8 seroprevalence was Our present study therefore, compares IFA and ELISA for highest (93.3%, n = 112/120) for KS cases, followed by HHV-8 serology of indigenous Tanzanians thus establish- non-KS tumors (91.7%, n = 22/24) and lowest (75.0%, n ing an assay for possible routine use. Furthermore, = 30/40) in non-malignant conditions. This difference in although it is known that HIV transactivates HHV-8/ HHV-8 seroprevalence between the three disease groups KSHV infection mostly by its Tat protein, the association was statistically highly significant (χ = 8.35, p = 0.0039) of HHV-8 and HIV with non-KS neoplasia and non-malig- [Table 3]. nant (reactive) conditions in Tanzania has not yet been Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:10 http://www.infectagentscancer.com/content/3/1/10 Cutaneous nodular AKS: gross p p Figure 1 laque) lesions [a] and a microgra icture of ph showing pr the right leg edomina of an A nce of spindle cells (SC) in frican patient showing mu a H & E section [b] × 400 ltiple nodules and macular (patch/ Cutaneous nodular AKS: gross picture of the right leg of an African patient showing multiple nodules and mac- ular (patch/plaque) lesions [a] and a micrograph showing predominance of spindle cells (SC) in a H & E section [b] × 400. Sex data was available for 154 HHV-8 seropositive cases 91.6–99.9) and specificity (50.0%, CI = 2.7–97.3). How- and most (81.2%, 125/154) of them were males and only ever, it appears [Figure 2(a–d)] that IFA can clearly visual- 18.8% (29/154) females. Male HHV-8 seroprevalence was ize latent [latency-associated nuclear antigen (LANA)] particularly high (90.2%, 92/102) among KS patients and [Figure (2a &2d)] as well as lytic [Figure 2b &2c] antibody less among non-KS tumors (77.3%, 17/22) and non-neo- reactivity allowing quantification of both latent and lytic plastic conditions (53.3%, 16/30) which difference HHV-8 infection within one individual [Figure 2(d)]. appeared statistically highly significant (χ = 20.86, p = 0.00003) [Figure 3]. HIV and HHV-8 correlations Evidently, most (70.3%, 114/162) HHV-8 seropositive Of all HHV-8 serology tests, 79 specimens were assayed by patients appeared also, co-infected with HIV and con- both IFA and ELISA and 92.4% (73/79) showed concord- versely, most (80.0%, 12/20) of those HHV-8 seronega- ance and most (91.1%, 72/79) were ELISA+/IFA+ as well tive were also non-reactive for anti-HIV antibodies which as one case (1.3%, 1/79) double negative [Figure 4]. How- difference appeared statistically highly significant (P = ever, 7.6% (6/79) the specimens were ELISA/IFA discord- 0.003 Fisher Exact Test) [Table 4]. ant including those ELISA-/IFA+ (6.3%, 5/79), and one ELISA+/IFA- case (1.3%, 1/79) [Figure 4]. Thus, although Immunohistochemistry (IHC) IFA appeared to have a higher (98.6%) assay sensitivity All early stage KS including patch (17.5%, 21/120) and [confidence interval (CI) = 91.6–99.9%], it also showed plaque (14.2%, 17/120) were cutaneous and HHV-8 lower specificity (16.7%, CI = 0.9–63.5) and positive pre- LANA+ (Figure 5) whereas two apparently HHV-8 seron- dictive value (PPV) [93.5%, CI = 84.8–97.6], compared to egative KS were nodular stage. Similarly, although all the ELISA which showed a reasonably high (93.5%, CI = HHV-8 positive KS sera came from patients with LANA+ 84.8–97.6) sensitivity, as well as higher PPV (98.6%, CI = biopsies (Figure 5), about 6.7% (8/120) of corresponding Page 3 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:10 http://www.infectagentscancer.com/content/3/1/10 Table 1: Demographic characteristics of studied MNH patients with KS, non-KS tumors and reactive lesions in association with HIV serostatus Histological Age Age-group (years) Sex HIV serostatus Overall cohort diagnosis characteristics (Years) (years) Adults (≥15) Children (<15) Male Female Positive Negative Kaposi's Mean age 35.8 na* 36.6 32.7 35.5 37.2 35.6 sarcoma (KS) [120 cases] Age-Range 17–70 na* 12–70 17–65 12–70 19–65 12–70 Total (%) 119 (99.2) 1 (0.8) 88 (73.3) 32 (26.7) 93 (77.5) 27 (22.5) 120 (100.0) Non-KS Mean age 36.5 8.5 31.1 22.4 na * 29.6 29.2 malignancies [24 cases] Age-Range 15–63 5–14 5–63 7–27 na * 5–63 5–63 Total (%) 18 (75.0) 6 (25.0) 18 (75.0) 6 (25.0) 1 (4.2) 23 (95.8) 24 (100.0) Non-malignant Mean age 34.9 8.0 37.5 26.1 31.8 31.2 31.6 cases [40 cases] Age-Range 18–52 2–11 9–52 2–47 2–52 9–44 2–52 Total (%) 36 (90.0) 4 (10.0) 21 (52.5) 19 (47.59 28 (70.0) 12 (30.0) 40 (100.0) Grand total (%) 173 (94.0) 11 (6.0) 127 (69.0) 57 (31.0) 122 (66.3) 62 (33.7) 184 (100.0) Key: na = not applicable, * = only one case available. KS sera were HHV-8 negative indicating a tissue-serum ble with a relatively high endemic incidence of HHV-8 discrepancy. infection [5,6]. The rather high prevalence of HIV infection in two-thirds Discussion All the three studied Tanzanian disease groups showed of the studied cohort patients at MNH is alarming but also comparable demographical features and the male pre- reflects a KS diagnostic selection bias. The dramatic (8- dominance in all KS types is concordant with previous fold) difference in the male:female ratio of EKS (1:27) and findings by us and others [5,6,8]. The relatively high sero- AKS (1:3) confirms previous reports from Tanzania, east- prevalence for HHV-8/KSHV in patients with non-KS ern Africa and elsewhere [8,11-15]. This sex ratio decline tumors and non-neoplastic (reactive) conditions has not in AKS apparently, reflects an increased frequency of HIV been previously documented in Tanzania and is compati- infected sexually active females, compared to males of the same age group and a suggested biological gender resist- ance for females to non-HIV forms of KS [EKS, classic Table 2: Histological diagnosis of (a) KS patients and (b) non-KS malignancies (CKS) and iatrogenic (IKS)] which clearly show a male predominance particularly in homosexuals (a) KS cases Number Percentage [2,8,11,14,16,17]. The finding that most non-KS tumors in our selected cohort were HIV negative emphasizes the Lymphadenopathic nodular KS 10 8.7 notion that essentially, KS today is an AIDS-defining Nodular KS 72 60 malignancy. Patch KS 21 17.4 Plaque KS 17 13.9 As expected by their KS diagnosis, the HHV-8 seropreva- Total 120 100 lence (90%) in the studied cohort sera was higher than that in the healthy non-hospitalized population of about (b) Non-KS malignancies Number Percentage 50% in Tanzania [5,6] and of 70% in sub-Saharan Africa Lymphomas 11 45.8 . The slightly lower seroprevalence observed in our pre- Epithelial tumors 6 25 vious PCR study could be attributed to the small KS sam- Neuroendocrine tumors 1 4.2 ple size compared to that in our present study  but Soft tissue tumors 6 25 differences in assay sensitivity/specificity should also be considered . Obviously, this high HHV-8 frequency in Total 24 100.0 patients at MNH implies a potential high risk of Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:10 http://www.infectagentscancer.com/content/3/1/10 Table 3: Summary of HIV and HHV-8 screening among MNH patients by either IFA or ELISA between 1990 and 2001 Assay IFA ELISA Disease group Serostatus HHV8+ HHV8- HHV8+ HHV8- Total KS HIV+ 42 (45.2) 3 (3.2) 46 (49.5) 2 (2.2) 93 (77.5) HIV- 10 (37.0) 2 (7.4) 15 (55.6) 0 27 (22.5) All cases 52 (43.3) 5 (4.2) 61 (50.8) 2 (1.7) 120 (100.0) Non-KS tumors HIV+ 1 (100.0) 0 0 0 1 (4.2) HIV- 8 (34.8) 1 (4.3) 13 (56.5) 1 (4.3) 23 (95.8) All cases 9 (37.5) 1 (4.2) 13 (54.2) 1 (4.2) 24 (100.0) Non-neoplastic HIV+ 11 (39.3) 3 (10.7) 14 (50.0) 0 28 (70.0) HIV- 0 10 (83.3) 2 (16.7) 0 12 (30.0) All cases 11 (27.5) 13 (32.5) 16 (40.0) 0 40 (100.0) Total 72 (39.1) 19 (10.3) 90 (49.0) 3 (1.6) 184 (100.0) parenteral/iatrogenic transmission for both patients and are yet not fully clarified although various biological and staff, particularly during blood transfusion and other socio-behavioral factors were suggested [6,8]. Recently in invasive procedures, as well as in intravenous (IV) drug a comparative genomic hybridization (CGH) and inter- users [4,5,18]. phase-fluorescence in-situ hybridization (interphase FISH) study, we have reported cytogenetic differences The higher (93%) HHV-8 prevalence among KS cases between male and female KS patients including the loss of compared to non-KS tumors and reactive lesions supports chromosome Y observed in all early and the majority of a causal relationship between the virus and primarily KS late male AKS and EKS representing a clonal genomic but other lymphoid tumors as primary effusion lym- change . Moreover, in early stage disease loss of Y phoma (PEL) or body cavity-based lymphoma (BCBL) chromosome was the only recurrent change found . and multicentric Castleman's disease (MCD) which may These features are apparently, mainly related to early male also be associated with HHV-8 [2,5,19] were surprisingly KS pathogenesis and may therefore indicate cytogenetic not evident in our study. reasons for the sex differences in KS . Furthermore, the higher seroprevalence of the oncogenic HHV-8 among Evidently, the finding that HHV-8 was more prevalent in male KS patients may also relate to the higher frequency males in all disease groups is concordant to our previous of KS in males [6,8,20,21]. reports and others [5,6,8,11,20] on HHV-8 seropreva- lence in KS and that KS is predominantly a male disease in Interestingly, the male predominance for HHV-8 sero- cutaneous AIDS-associated (AKS), African endemic (EKS), prevalence in our current study was also seen in non-KS classical (CKS) and iatrogenic [IKS]/post-transplantation and non-neoplastic patients suggesting a possible higher Kaposi's sarcoma [6,8,11,20] although, we have recently susceptibility/permissiveness of males to KSHV/HHV-8 reported an increased frequency of females with oral AKS infection and/or a high viral transmission among male (OAKS) . Furthermore, we have shown previously that homosexuals and intravenous (IV) drug users usually, HHV-8 is not only more frequent in males, but that they mostly males [8,18,22,23]. However, the association of appear to have higher viral (HHV-8) loads than females in sex with KS development and HHV-8 infection is yet to be both sera and tissue lesions by both immuhistochemistry fully elucidated. and real-time PCR [6,8]. Reasons for this sex differences Table 4: Association between HIV and combined (IFA and ELISA) HHV-8 sero-reactivity of studied patients at MNH (1990–2001) HHV8 [no. (%)] Total [no. (%)] Antibody Serostatus Positive Negative HIV Positive 114 (70.3) 8 (40.0) 122 (66.3) Negative 48 (29.7) 14 (80.0) 62 (33.7) Total 162 (88.0) 22 (12.0) 184 (100.0) Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:10 http://www.infectagentscancer.com/content/3/1/10 IFA m (green diffu Figure 2 icrophotographs sh se cytoplasmic owin staining) antibodies K8 g BCBL-1 cytospin cells reac .1 tivity to anti-HHV-8 LANA (pink intranuclear dots) [a] and to lytic IFA microphotographs showing BCBL-1 cytospin cells reactivity to anti-HHV-8 LANA (pink intranuclear dots) [a] and to lytic (green diffuse cytoplasmic staining) antibodies K8.1[b] and ORF 59 [c] 72 hrs after treatment with tetra-phobol acetate (TPA) to induce the lytic phase. KS patient's serum was added to BCBL-1 TPA treated cytospin cells producing, the lytic green diffuse cytoplasmic staining and latent white intranuclear dots. Note that majority of BCBL-1 cells reacted to latent (brown arrow) compared lytic (white arrow) antibodies in the index KS patient in [d], all sections × 400. Our finding that HHV-8 antibody seroprevalent patients therapeutic strategies among high-risk groups including at MNH were 3-times more often HIV co-prevalent than HIV-infected and AIDS patients. the HHV-8 seronegative cases is consistent with other reports [4,18,24] indicating corresponding seroepidemi- The finding that IFA showed greater sensitivity than ELISA ology for these viruses, partly explaining the much is probably due to that IFA methods shows antibodies to increased risk of KS development among those HIV and both lytic and latent antigens and that lytic are reportedly HHV-8 co-infected as compared to those infected with more sensitive . However, our finding that ELISA had either or neither of the viruses [2,4,8,18,24]. Furthermore, apparently, higher positive predictive value as well as spe- the findings support also the notion of HIV and HHV-8 cificity and still had a high sensitivity was unexpected and cross-talk, partly achieved through the transactivation of particularly favourable as it makes HHV-8 screening more KSHV by HIV-Tat as reported previously by us and others affordable in a resource-constrained country like Tanza- [2,6,9,25]. This HIV-HHV8 cross-talk, calls for exploration nia, mostly lacking the cell culture and fluorescence for the development and application of possible com- microscopy facilities needed in IFA. Consequently, ELISA bined anti-HIV, anti-HHV-8 and anti-KS prevention and can allow larger-scale screening of HIV high-risk groups Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2008, 3:10 http://www.infectagentscancer.com/content/3/1/10 ent stage-dependent tissue-serum discrepancy in HHV-8 Total HHV-8 antibody seroprevalence by sex and disease groups among MNH patients (1990-2001) antigen and antibody expression seems to support the notion of immune-segregation and/or selective virus clearance during the evolution KS. HHV-8 screening of patients at risk of KS and of blood and organ donors par- ticularly in high endemic areas will evidently help prevent development of KS. Methods Biopsies Diagnostic archival biopsies fixed in 10% neutral buffered KS Non-KS tumors Reactive lesions Female HHV8+ formalin and paraffin-embedded (FFPE) and correspond- Disease group Male HHV8+ ing, available sera and medical records of KS patients between 1990 and 2001 were retrieved and evaluated g Figure 3 Total HH roups am V-8 an ong MNH tibody seroprev patients (1990–200 alence by sex a 1) nd disease Total HHV-8 antibody seroprevalence by sex and dis- [Dept. Histopathology, Muhimbili National Hospital ease groups among MNH patients (1990–2001). (MNH)/Muhimbili University of Health and Allied Sci- ences (MUHAS), Dar es Salaam] and at the Immunopa- thology Lab [Karolinska University Hospital Solna, including blood/organ donors and thereby prevent KS Stockholm]. Furthermore, available biopsies and corre- development through early antiviral interventions sponding sera from non-KS tumors and reactive condi- (including vaccination) [4,5]. However, these findings are tions from the same period were also evaluated. The prone to observer errors, sampling bias (hospital data) biopsies of reactive conditions included all histologically including the fact that HHV-8 screening is a field that is non-malignant/inflammatory cases whose sera were still evolving methodologically . Nevertheless, the IFA available for analysis. None of the patients had received method also appears to afford visual evaluation of the per- any antiretroviral (ARV) or anti-tumour therapy before centage seroprevalence of lytic as well as latent anti-HHV- the biopsy was taken. 8 antibodies apparently, allowing categorization of patients as being in the productive and potentially infec- Histology tive (lytic) and non-productive but oncogenically more Routine hematoxylin and eosin (H & E) staining was done significant latent phase  and may therefore be useful as previously described [8,26] and microscopic evaluation when such characterization is desired for clinical or public was done independently by three pathologists as previ- health interventions. ously described . Our finding that all sera from early stage (patch-plaque) Serology KS were positive for anti-HHV-8 antibodies while all neg- Available sera from patients with histologically diagnosed ative sera were from LANA+ biopsies of nodular KS KS, non-KS malignancies as well as reactive conditions patients, further supports our previous notion of stage- were comparatively evaluated for HIV and HHV-8 serol- dependent tissue-serum discrepancy in viral antigen and ogy. Serology for HIV-1 evaluation (ELISA) was per- antibody expression probably due to virus tissue reten- formed at Microbiology/Immunology MUHAS as tion, immune-segregation and/or selective clearance dur- previously described [8,27,28]. ing KS evolution [6,19]. HHV-8 serology by IFA was performed [Swedish Institute Again, the clear relationship between HHV-8 and HIV for Infectious Diseases Control (SMI)] on cytospins of infections and KS prevalence underlines the interaction BCBL-1* cells  using patients sera as well as control between the two viruses being possibly facilitated in part, lytic (K8.1 and ORF 59) and latent (ORF 73 or LANA) by similar transmission patterns and also, their transacti- antibodies [Advanced Biotechnologies Inc. (Columbia, vation capacity [2,4,6,19]. MD)] as previously described [5,8]. Results were evalu- ated and documented by microphotography (Immunopa- thology Lab). Conclusion HHV-8 seroprevalence in patients at MNH Tanzania appears to be high as expected, in HIV+ and HIV- KS cases The HHV-8 infected body cavity-based lymphoma (BCBL- and males but also in the HIV seronegative non-KS 1) cells (kindly provided by G. Gaidano) were derived tumors. ELISA showed a combination of high HHV-8 sen- from a primary effusion lymphoma (PEL) [30,31], and sitivity as well as higher PPV and specificity compared to cultured [Immune and Gene Therapy Lab., Cancer Center IFA which however showed higher sensitivity. An appar- Karolinska (CCK)] in RPMI 1640 medium (Gibco, BRL, Page 7 of 9 (page number not for citation purposes) Percentage frequency Infectious Agents and Cancer 2008, 3:10 http://www.infectagentscancer.com/content/3/1/10 ELISA is a direct EIA based on the binding of HHV-8-spe- cific antibodies to lytic peptide antigens coupled to micro- 1; 1.3% 5; 6.3% 1; 1.3% titre test strips. Bound antibodies are detected by an anti- human IgG peroxidase conjugate and a 3,3',5,5'-tetrame- thylbenzidine (TMB) dark blue substrate reaction (BioFX Laboratories, Inc. Owings Mills, MD). The use of lytic pep- tide epitopes derived from various HHV-8 viral proteins ensures both a high sensitivity and specificity. The Kit has no detectable cross-reactivity with HIV/EBV antibodies. Control sera included those from known HHV-8+ KS 72; 91.1% patients while the HHV-8 infected BCBL-1 cells provided ELISA+/IFA+ ELISA-/IFA- ELISA-/IFA+ ELISA+/IFA- an internal positive control for IFA studies. The negative controls for both IFA and ELISA included sera from E b Figure 4 L etween ISA/IFA 1990 a percentage ser nd 2001 o-reactivity among MNH patients known HIV and HHV-8 negative non-KS patients and a ELISA/IFA percentage sero-reactivity among MNH buffer (PBS). patients between 1990 and 2001. Immunohistochemistry (IHC) Immunostaining with the avidin-biotin complex (ABC) UK) containing 10% heat-inactivated FCS serum, 2 mM L- immunoperoxidase technique was used on KS sections (5 glutamine, 100 IU/ml penicillin and 100 μg/ml strepto- μm) for detection of HHV-8 latency-associated nuclear mycin at 37°C and 5% CO . To induce lytic gene tran- antigen (LANA) to compliment histopathology and serol- scription, cells were cultured with 20 ng/ml of 12-O- ogy, as previously described [8,19,26,33]. Negative con- tetradecanoylphorbol-13-acetate (TPA) [Sigma Chemical trols included sections from non-KS tissues as well as Co., St. Louis, Mo.] as previously described, [5,28,32] and incubation with PBS instead of the primary antibody. Pos- harvested after 72 hours, washed with PBS and fastened itive controls included previously tested KS sections and by cytospin on SuperFrost slides (Menzel GmbH & Co FFPE sections of HHV-8 LANA+ cells from a body cavity- KG, Braunschweigh, Germany). The slides were fixed for based lymphoma (BCBL-1). The number of LANA+ cells/ 10 minutes in 4% paraformaldehyde (PFA) and washed HPF (× 400 magnification) was evaluated on micrographs before immunostaining as previously described [5,8]. as previously described [8,19,33]. Microscopy For HHV-8 antibody evaluation an IgG enzyme immu- noassay (EIA) Kit (96 Wells, HHV-317-02) Biotrin Inter- For microscopy and microphotography an Olympus national (Dublin, Ireland) was used. The Biotrin HHV-8 BX60, microscope with a digital camera (Sony DKC-5000) was used as previously described [8,19,33]. Statistical Analysis The EPI INFO 6 statistical software programme (CDC, Atlanta, GA) was used. P-values of ≤0.05 were considered statistically significant. The Fisher exact test was used to test the significance level where the sample size was small. Competing interests The authors declare that they have no competing interests. Acknowledgements We are greatly indebted to the MNH patients who provided their biopsies and sera as well as to Profs. L. Lema and P. Carneiro and the department of surgery for collecting biopsies and sera, Jafari Sufi and Onjection Byoban- gamba (MUHAS) for technological assistance and Anita Östborn (SMI, Swe- den) for shipments and Ms. Rose Mpembeni (MUHAS) for statistical review. Professors Gunnel Biberfeld and Anika Linde from SMI kindly facilitated A ous AK reactivity in SC (b Figure 5 section of an immunoperox S lesion shla owi ck a ng HHV-8 L rrows) × 60 idase stained n AN 0 A+ granul odular cuta ar nuclear ne- HHV-8 IFA studies. Ms. Susan Reid and Elaine Cameron of Dublin, Ireland A section of an immunoperoxidase stained nodular and Biotrin International are gratefully acknowledged for HHV-8 ELISA. cutaneous AKS lesion showing HHV-8 LANA+ granu- These studies were approved by the MUHAS Research Ethics Committee lar nuclear reactivity in SC (black arrows) × 600. and the Ethical Committee, Karolinska University Hospital Solna (Dnr 01- 096). 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Infectious Agents and Cancer – Springer Journals
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