Low prevalence of HCV, HIV, and HTLV-I/II infection markers in northwestern Greece: results of a 3-year prospective donor study (1995-1997).

Background: The risk of infection with transfusion-transmitted viruses has been reduced remarkably. A zero-risk blood supply, however, remains a popular goal. A 3-year prospective donor study was conducted in the Epirus region of Greece to determine the prevalence of human immunodeficiency virus (HIV), human T-lymphotropic virus (HTLV), hepatitis B virus, and hepatitis C virus (HCV). Herein, we report the prevalence of HIV, HTLV, and HCV infection markers in this area. Methodology: Between January 1, 1995 and December 31, 1997, 6696 donors were investigated for the presence of anti-HIV, anti-HTLV, and anti-HCV antibodies using standard enzyme immunoassays (EIA). Every sample with anti-HCV reactivity by third-generation EIA was further investigated using a third-generation recombinant immunoblot assay (RIBA 3.0) and HCV-RNA by a combination of polymerase chain reaction (PCR) and DNA EIA. Results: None of the donors tested positive for anti-HIV or anti-HTLV antibodies. In contrast, anti-HCV was detected in 41 donors (0.61%). Using a RIBA 3.0 test, eight donors tested positive and eight had indeterminate results, while 25 tested negative. Seven of the eight donors with both EIA and RIBA 3.0 reactivity had increased levels of aminotransferases and detectable serum HCV-RNA. The remaining 34 donors had repeatedly normal aminotransferases and three times negative HCV-RNA. Liver biopsy was performed in anti-HCV/HCV-RNA-positive donors (7/41). The lesions were compatible with chronic hepatitis C in all of them. Conclusion: A zero prevalence of HIV and HTLV infection markers was found. Although the number of annual donations in this study was relatively low, the negative data for HIV and HTLV clearly indicate that rates of these infections are low in our region and that infected donors will be seen infrequently. HCV infection in blood donors remains very low in our region and is similar to the data reported in other industrialized countries. In fact, the prevalence of definite HCV infection seems to be very low (7/6696; 0.1%). However, a significant proportion of anti-HCV-reactive donors by third-generation EIA (33/41) had indeterminate or negative results by the RIBA 3.0. The latter donors were repeatedly negative for HCV-RNA. This finding may indicate that some donors tested false-positive for anti-HCV, although the possibility of true HCV infection contracted in the distant past cannot be excluded. In our opinion, close attention to mandatory principles of transfusion medicine, along with the screening of plasma donors using nucleic acid amplification technology, are the only methods that can further ensure the safety of our blood supply.

Value of anti-HBc screening of blood donors for prevention of HBV infection: results of a 3-year prospective study in Northwestern Greece.

BACKGROUND: The risk of infection with transfusion-transmitted viruses has been reduced remarkably. However, a zero-risk blood supply remains a popular goal. Some authorities have introduced the screening for antibody to HBc (anti-HBc) as a surrogate test for the presence of several infectious agents. A 3-year prospective study was conducted in the Epirus region of Greece to determine the prevalence of several blood-borne viruses. One component of the study was the prevalence of HBV infection markers and the potential value of anti-HBc testing of donors in this area. STUDY DESIGN AND METHODS: Between January 1, 1995, and December 31, 1997, some 6696 donors were investigated for the presence of HBV infection markers by standard EIAS: Every sample that tested HBsAg-negative but anti-HBc-reactive alone or in combination with either or both antibodies to HBV e antigen (anti-HBe) and low-titered antibodies to HBsAg (anti-HBs <20 mIU/mL) was further investigated for the presence of HBV DNA by a combination of PCR and DNA EIA. RESULTS: Of these 6696 donors, 15.8 percent tested positive for at least one serologic marker of HBV infection (HBsAg prevalence, 0.85%). Anti-HBc reactivity alone or in combination with either or both anti-HBe and low-titered anti-HBs was found in 282 donors (4.2%). None tested HBV-DNA positive. No transfusion-associated HBV infections were recorded in the recipients of the above 282 blood units. CONCLUSION: A moderate prevalence of HBV infection markers was found. However, taking into account previous studies from this region, it appears that the HBsAg prevalence has declined. In addition, the present study cannot recommend the introduction of anti-HBc screening as a surrogate marker of occult HBV infection. The adoption of this exclusion criterion in this region would result in unacceptably high rejection rates among otherwise healthy donors. The absence of any case of transfusion-associated HBV infection after the transfusion of all HBsAg-negative, anti-HBc-positive units appears to provide further support for the negative HBV DNA results. Before a consideration of screening donors, efforts must be focused on reducing the number of false-positive anti-HBc results.

BRCA1 mutation analysis in breast/ovarian cancer families from Greece.

Germline mutations in BRCA1 gene account for varying proportions of breast/ovarian cancer families, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations the entire coding sequence of BRCA1 in 30 breast/ovarian cancer women with family history of two or more cases of breast cancer under age 50 and/or ovarian cancer at any age. Genomic DNA from patient was initially analyzed for truncating mutations in exon 11 with PTT followed by DNA sequencing. In the cases where no frameshift mutation was observed in exon 11, all other exons were screened with direct sequencing. Two novel (3099delT, 3277insG) and three already described (3741insA, 1623del5-TTAAA, 5382insC-twice) truncating mutations were identified. In addition, 6 point mutations (L771L, P871L, E1038G, K1183R, S1436S, S1613G) which are already classified as polymorphisms were identified. Three unclassified intronic variants (IVS16-68 G>A, IVS16-92 G>A, IVS18+65G>A) were also detected. These results show that BRCA1 deleterious mutations are present in a fraction (20%) of Greek breast/ovarian cancer families similar to other European countries. Mutations were detected in high- (>/=3 members) as well as in moderate-risk (2 members) families. This is the first report of BRCA1 mutation analysis in Greece.

Increased incidence of anti-cardiolipin antibodies in patients with hepatitis C is not associated with aetiopathogenetic link to anti-phospholipid syndrome.

OBJECTIVE: Chronic infection with hepatitis C virus (HCV) has been found to be associated with various diseases known as extra-hepatic manifestations of HCV. Recently, HCV has been implicated as a cause of the antiphospholipid syndrome (APLS). We conducted a study in a well-characterized area for epidemiological and prospective studies in the north-western part of Greece in order to address whether an aetiopathogenesis exists between HCV and APLS. DESIGN: Seventy-five patients with chronic hepatitis C were investigated for the presence of anti-cardiolipin antibodies (anti-CL) and for a past medical history supportive to the diagnosis of APLS. In addition, 24 patients with well-defined APLS (primary or secondary) and 12 patients with systemic lupus erythematosus (SLE) were tested for the presence of markers of HCV infection (anti-HCV and HCV RNA). The SLE patients were anti-CL-positive but none of them had developed any of the known clinical features of APLS. In addition, 267 healthy subjects were investigated for the presence of anti-CL. METHODS: IgG and IgM anti-CL were determined by a quantitative isotype-specific solid phase enzyme-linked immunosorbent assay set up in our laboratory. Anti-HCV was determined using a third-generation enzyme immunoassay and a confirmatory third-generation recombinant immunoblot assay. Active virus replication was defined by the detection of HCV RNA using a combination assay based on a reverse transcriptase polymerase chain reaction and a DNA enzyme immunoassay. RESULTS: Of the HCV patients, 37.3% had IgG and/or IgM anti-CL (P<0.00005 compared to healthy controls (2.25%)). However, the mean titres of each specific isotype were significantly lower in HCV patients compared with those found in the APLS patients (P<0.05 for IgM and P<0.001 for IgG isotypes). The mean titres of IgG anti-CL were also significantly lower in HCV patients compared with those found in the SLE patients (P<0.01). All patients with APLS or SLE (n = 36) tested negative for HCV infection markers. In addition, neither thrombotic events nor thrombocytopenia were associated with a positive anti-CL test in HCV patients. CONCLUSIONS: A significant proportion of HCV patients (37.3%) had detectable anti-CL of low titre. However, this finding was not associated with the development of APLS. On the other hand, none of the APLS patients was positive for HCV. Taken together, our data rather failed to reveal an aetiopathogenetic link between HCV and APLS. For this reason, testing for HCV in patients with APLS or follow-up for the possibility of the development of APLS in HCV patients cannot be suggested, at least in Greek patients. More prospective studies of longer duration are required in order to address whether HCV is involved or not in the aetiopathogenesis of APLS.

Absence of HCV viraemia in anti-HCV-negative haemodialysis patients.

BACKGROUND: Immunologic alterations have been reported in chronic haemodialysis (HD) patients. Some HD patients may have, therefore, an inability to produce detectable amounts of serum antibodies to hepatitis C virus (anti-HCV). Previous studies have shown the presence of HCV viraemia in anti-HCV-negative HD patients (ranging from 1 to 15%). However, the universal epidemiologic impact of these cases remains uncertain since there are conflicting results. In this context, we conducted a study in an attempt to investigate the presence of HCV viraemia among anti-HCV-negative HD patients in a well-defined geographic area of the northwestern part of Greece. METHODS: During a 6 month period, 81 anti-HCV-negative HD patients were tested twice for the presence of HCV RNA, using the reverse transcriptase polymerase chain reaction (RT-PCR) combined with a DNA enzyme immunoassay (DEIA). At the same time, periodic testing for anti-HCV by two commercially available third generation assays was done. In addition, 15 anti-HCV-positive HD patients and 20 non-HD patients with well established chronic HCV infection used as internal controls were tested for the presence of HCV RNA and anti-HCV. RESULTS: None of the anti-HCV-negative HD patients were shown to be viraemic by the combined RT-PCR and DEIA method. During the same time period, all remained anti-HCV negative by the third generation assays. By contrast, all the patients with known HCV-infection were positive by the two enzyme immunoassays, whereas 13 anti-HCV-positive HD patients (86.7%) and 18 non-HD patients (90%) were viraemic by RT-PCR. CONCLUSIONS: This study demonstrated that routine HCV RNA testing in anti-HCV-negative HD patients appears not to be necessary particularly when third generation assays are used for the detection of anti-HCV.

Association of mast cells with fibrosis and fatty infiltration in the minor salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: To determine the distribution and density of mast cells in the minor salivary glands of patients with primary Sjögren's syndrome (pSS) and of normal controls. METHODS: Minor salivary gland biopsies were obtained from 19 patients with pSS, 9 with systemic lupus erythematosus, one each with rheumatoid arthritis, sarcoidosis, and Hodgkin's disease, and from 10 individuals who had subjective xerostomia with normal salivary gland biopsies. Biopsy specimens were evaluated for the degree of inflammation according to Tarpley's classification. Sections were analysed for staining with Toluidine blue and with the mast cell specific marker c-kit. The data obtained were correlated with the histological findings of fatty infiltration, fibrosis and lymphocytic infiltration. RESULTS: There was a significant correlation between the number of mast cells identified and the degrees of fibrosis and fatty infiltrates. There was no correlation between the intensity of lymphoid infiltration and the number of mast cells. c-kit staining showed a high correlation when compared to Toluidine blue staining. CONCLUSION: Mast cells in the minor salivary glands of patients with pSS are strongly associated with fibrosis and cell acid infiltration. However, there is no correlation with parameters of disease activity such as lymphoid infiltration.

Mixed monoclonal cryoglobulinemia and monoclonal rheumatoid factor cross-reactive idiotypes as predictive factors for the development of lymphoma in primary Sjögren's syndrome.

OBJECTIVE: To prospectively investigate whether mixed monoclonal cryoglobulinemia (MMC) and monoclonal rheumatoid factor (mRF)-associated cross-reactive idiotypes (CRI) serve as predictive factors for the development of lymphoma in patients with primary Sjögren's syndrome (SS). METHODS: One hundred three consecutive patients with primary SS were evaluated from 1986 to 1991. In all patients, the amount of cryoglobulin was measured by ultraviolet absorption at 280 nm and 260 nm. The type of cryoglobulinemia was identified by agarose gel electrophoresis, combined with immunofixation. Sera from all patients were evaluated by enzyme-linked immunosorbent assay, using the corresponding monoclonal or polyclonal antibodies, for the presence of immunoglobulins bearing the idiotypes 17109 (V kappa IIIb associated), G-6 (VH1 associated), and 3rd SS (a rabbit polyclonal antibody raised against the Fab fragment of an IgM kappa mRF from a patient with primary SS). Data analysis was performed by logistic regression. RESULTS: Eighteen of the patients with primary SS (17.4%) had MMC during the first evaluation. There was a statistically significant correlation between the presence of MMC and a higher prevalence of autoantibodies to Ro/SS-A and La/SS-B, as well as extraglandular manifestations. During a 5-year period, 7 patients developed lymphoma. Six of the 7 (86%) had MMC before the appearance of lymphoma, compared with 12 of 96 (12.4%) of the remainder (r = 0.421, P < 0.0009). Patients who developed lymphoma had higher amounts of cryoglobulin than those who did not (mean +/- SD 53.4 +/- 44.7 mg/dl versus 26.8 +/- 20.6 mg/dl). CRIs 17109 and G-6 were also correlated with lymphoma development (r = 0.321, P < 0.006 and r = 0.22, P < 0.03, respectively). For both CRIs, this correlation was dependent on the presence of MMC, since a stepwise multiple comparison analysis revealed that their individual significance was abolished when their correlation with lymphoma in association with MMC was assessed. CONCLUSION: The determination of MMC can be used as a laboratory predictive factor for lymphoma development in primary SS. CRIs 17109 and G-6 may also be used to predict lymphoma development, especially when the monoclonal component is absent.

Cytokine mRNA expression in the labial salivary gland tissues from patients with primary Sjögren's syndrome.

The pattern of cytokine mRNA expression in frozen minor salivary gland tissues from patients with primary Sjögren's syndrome (pSS) (n = 12) and controls (n = 8) using an in situ hybridization technique and oligonucleotide probes of interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha and beta (TNF-alpha and TNF-beta), interleukin-6 (IL-6), interleukin-2 (IL-2) and its receptor (IL-2R), interleukin-4 (IL-4), interleukin-10 (IL-10), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta) was examined. In addition to in situ hybridization, immunohistochemistry was used to identify the subset of cells expressing IL-2 and IL-4 mRNA. Mononuclear cells involved in the minor salivary gland lesions of pSS patients were found to express mRNA for pro-inflammatory cytokines such as TNF-alpha and IL-1 beta, and cytokines involved in the regulation of B- and T-cell function (IL-2 and IL-6). In contrast, only three biopsies from patients with pSS express mRNA of inhibitory cytokines such as IFN-gamma and TGF-beta. Furthermore mRNA for IL-6 and IL-1 beta was also detected in the glandular epithelial cells suggesting that these cells may play a role in the pathogenesis of autoimmune lesion in Sjögren's syndrome. IL-10 mRNA was not detected while IL-4 mRNA was primarily detected in naïve T-lymphocytes of patients with a mild and early lesion. These results suggest that local production of cytokines by both mononuclear and epithelial cells may be involved in the immune-mediated destruction of exocrine glands in patients with pSS.

Inheritance of monoclonal rheumatoid factor cross-reactive idiotypes in primary Sjögren's syndrome (pSS). Comparative studies of a rabbit polyclonal anti-idiotype and 17109 monoclonal anti-idiotype.

A comparison of the presence of two idiotypes, one identified by a rabbit polyclonal antiidiotypic antibody, first found on a cryoprecipitable IgM chi rheumatoid factor (RF) from an SS patient (3rd SS) and the 17109 idiotype, identified by a monoclonal antibody was performed in 106 sera and eight minor salivary gland biopsies of Sjögren's syndrome (SS) patients and 125 sera from age-sex matched normal controls. Of 106 of SS patients' sera 36 had immunoglobulins positive for the 3rd SS idiotype. 17109 activity was more prevalent in SS patients positive for the polyclonal anti-idiotype 3rd SS, than those with negative idiotype (9/36 VS 2/70 chi 2 = 12.53 P less than 0.005). Cross inhibition studies, however, revealed that the polyclonal anti-idiotype binding was not inhibited by the 17109 moAb. 3rd SS and 17109 anti-idiotypes were reacted with immunoglobulins in the serum of 3.5% and 1.7% of normal human sera respectively. Immunohistologic studies demonstrated that 4/8 and 2/6 minor salivary gland biopsies had infiltrating plasma cells containing immunoglobulins bearing the 3rd SS and the 17109 idiotypes, respectively. The inheritance of both idiotypes was investigated in sera of 4 SS kindreds. In two kindreds with 3rd SS positive probands, the idiotype was detected in 3 first degree relatives of the same generation. 17109 activity was detected in the serum of a sister of the positive proband who had a high RF titer. These results suggest that the 17109 moAb recognizes a different epitope of that of the 3rd SS. The idiotypes of monoclonal RFs are not inherited and probably are produced by plasma cells infiltrating the labial minor salivary glands of SS patients.