Detection of HCV salivary antibodies by a simple and rapid test.

Hepatitis C (HCV) is common in developing countries, where blood sampling and expensive sophisticated methods for detection are less available. Hemodialysis patients have high prevalence of HCV and may resemble sick populations in developing countries in relation to immunosuppression and antibodies production. For these reasons anti-HCV antibodies were assayed in saliva of hemodialysis patients by ImmunoComb II assay that is less laborious, relatively inexpensive and easy to perform If the findings are confirmed by larger studies this method may be useful especially in developing countries. Serum and saliva samples were obtained from 37 hemodialysis patients and assayed by ImmunoComb II kit. In positive PCR patients the saliva test had 100% sensitivity, which was as good as serum anti-HCV Axsym testing. Saliva testing had a similar or better specificity than the serum method.

Optical fiber immunosensor based on a poly(pyrrole-benzophenone) film for the detection of antibodies to viral antigen.

We describe herein a newly developed optical microbiosensor for the diagnosis of hepatitis C virus (HCV) by using a novel photoimmobilization methodology based on a photoactivable electrogenerated polymer film deposited upon surface-conductive fiber optics, which are then used to link a biological receptor to the fiber tip through light mediation. This fiber-optic electroconductive surface modification is done by the deposition of a thin layer of indium tin oxide on the silica surface of the fiber optics. Monomers are then electropolymerized onto the conductive metal oxide surface; thereafter, the fibers are immersed in a solution containing HCV-E2 envelope protein antigen and illuminated with UV light (wavelength approximately 345 nm). As a result of the photochemical reaction, a thin layer of the antigen becomes covalently bound to the benzophenone-modified surface. The photochemically modified fiber optics were tested as immunosensors for the detection of anti-E2 protein antibody analyte that was measured through chemiluminescence reaction. The biosensor was tested for sensitivity, specificity, and overall practicality. Our results suggest that the detection of anti-E2 antibodies with this microbiosensor may enhance significantly HCV serological standard testing especially among patients during dialysis, which were diagnosed as HCV negative, by standard immunological tests, but were known to carry the virus. If transformed into an easy to use procedure, this assay might be used in the future as an important clinical tool for HCV screening in blood banks.

Hepatitis C virus infection in renal failure patients in the absence of anti-hepatitis C virus antibodies.

The magnitude and clinical significance of Hepatitis C virus (HCV) infection in dialysis patients is controversial and underestimated. This study was conducted in order to evaluate the correlation between HCV replication and antibody response to HCV in dialysis patients. HCV infection in dialysis patients was evaluated over a period of 3 years and compared to HCV infection in Liver Clinic patients. Sera were collected from 310 dialysis patients and tested for anti-HCV and HCV-RNA. In addition, HCV genotype and HCV viral load were determined in HCV-RNA-positive sera. Anti-HCV was detected in 43 (14%) of the dialysis patients. Of these, 37 (86%) were HCV-RNA-positive. Among the 267 HCV-seronegative dialysis patients, 25 (9%) were found to be HCV-RNA-positive in more than one sample during the study. These patients were characterized by low viral load; at least two orders of magnitude lower than in the group of HCV-seropositives. In contrast, in the Liver Clinic patients, HCV-RNA was found exclusively in HCV-seropositive patients. Comparison of the genotype pattern in the two groups did not reveal a difference. Our results suggest that HCV infection in dialysis units may be underestimated due to cases of low viral load, depending on the method of RNA extraction and sensitivity of the test used. Low viral load might contribute to the lack of humoral immune response seen in some dialysis patients.

Hepatitis C virus infection and genotypes in Southern Israel and the Gaza Strip.

The Gaza Strip borders the southern part of Israel and Egypt. There is a remarkable difference in the prevalence of antibodies to hepatitis C virus (HCV) between Israel (0.5%) and Egypt (10%). A few thousand inhabitants cross the borders daily from the Gaza Strip to both countries. The objectives of this study were to investigate the prevalence of HCV infection in the Gaza Strip, an area that was not studied before, and to study HCV transmission in the Gaza Strip by characterizing the genotypes of HCV in Southern Israel and the Gaza Strip and comparing them with those found in Egypt. HCV prevalence in the Gaza Strip was found to be 2.2%, relatively higher than in Israel but lower than in Egypt. The most common genotypes found were type 1 b in Southern Israel and type 4 in the Gaza Strip, corresponding to the most prevalent genotype in Egypt. Similarity between type 4 isolates from the Gaza Strip and Egypt was illustrated further by sequence analysis of the HCV 5' noncoding region (NCR).

Chlamydia trachomatis and HIV infection.

To study the interactions between Chlamydia trachomatis (CT) and human immunodeficiency virus (HIV) infections, we examined CT serologies in sequential serum samples of male homosexuals (MHS), followed over a mean period of 4 years. Of the MHS studied, 77 were HIV(-), 18 were HIV(+) and 10 patients seroconverted during the study period. Seventy matched heterosexual controls were tested concomitantly. CT-specific antibodies of both IgG and IgA isotypes were determined by an immunoperoxidase assay, indicating past and active infection respectively. Anti-CT IgG was frequently observed in both HIV(-) and HIV(+) MHS (40-50% vs. 23% of controls) and IgA antibodies were also common in both MHS groups (15-20% vs. 1.5% of controls). After HIV infection, no increase in CT antibodies occurred. We found serological data suggestive of active CT infection preceding seroconversion in 3 of 10 seroconverters vs. 5% of matched MHS controls who remained HIV(-) (P < 0.025), indicating a possible effect of CT infection on the acquisition of HIV should be further studied.

Serum antibodies in rickettsia patients as determined by immunoblotting technique.

The Western-blot technique (WB) was used to determine which polypeptides of Israeli spotted fever (ISF) isolates and other spotted fever group rickettssia (SFGR) reference isolates (G212, S484, A828) and two reference strains. R. Rickettsii (Sheila Smith strain) and R. conorii (Boutonneuse fever), were used as antigen sources for the WB. Immunoperoxidase assay (IPA) seropositive (titer greater than 80) and seronegative (titer less than) sera were examined with the separated polypeptides of the above strains. WB analysis of the rickettsial polypeptide-serum reactions showed that R. conorii and the three isolates of ISF reacted identically with the sera, except that in the three ISF strains a 175 kD protein was present. It was also observed that all of the IPA seropositive sera examined reacted with the following polypeptides: 18kD, 20kD, 22kD (28kD to 37 kD LPS group), while each seropositive and seronegative serum reacted differently with polypeptides 23kD, 42kD, 45kD, 46kD, 52kD, 55kD, 70kD, 82kD, 105kD, 125kD, 155kD and 175kD. Using this technique, no heat labile polypeptides (preelectrophoretic treatment: 100 degrees C for 2 min vs 37 degrees C for 20 min) were observed in SFGR strains used in this study. Our results indicate that the immunoblot technique shows no difference between R. conorii and ISF antigens except the existence of 175kD protein antigen in the latter.

Comparison of structural polypeptides, detected by immunoblotting technique, in the sera of spotted fever group rickettsia positive cases--symptomatic versus asymptomatic.

In an attempt to characterize the nature of symptomatic versus asymptomatic spotted fever group rickettsia (SFGR) infection, the immune response to R. conorii (boutonneuse fever) structural polypeptides was studied by Western-blot immunoassay. Sera from immunoperoxidase assay (IPA), SFGR seropositive (titre greater than or equal to 80) individuals, symptomatic and asymptomatic and from SFGR seronegative (IPA titre less than 80) individuals living in a kibbutz community in the desert region of Southern Israel were examined by immunoblot. This community suffered from a very high morbidity rate due to SFGR (21-fold higher than the national reported average). The entire community (n-326) has been followed-up since 1985, with serial serum samples being examined for specific IgG antibodies by IPA. The intensity of the immunoblot reaction correlated with specific IgG antibody titres as determined by IPA. This correlation was also observed between the decrease in the IgG titre and the strength of the antibody-antigen reaction by immunoblot over time for a given individual. IPA seropositive sera from asymptomatic as well as symptomatic spotted fever cases reacted to 8 individual polypeptides. In both cases antibodies to 22 kD, 24 kD, 26 kD, 28 kD, 30 kD, 32 kD, 34 kD, and 37 kD were found. In the IPA seronegative sera, antibodies to polypeptides in the range of 24 kD to 32 kD were not detected. The lack of detectable differences by immunoblotting between SFGR symptomatic vs, asymptomatic cases might be explained by other aspects of the immune response of each infected individual, and/or it is possible that virulent and non-virulent antigenically closely related SFGR strains infected symptomatic vs. asymptomatic individuals.

Sequential serological studies of homosexual men with and without HIV infection. Epstein-Barr virus activation preceding and following HIV seroconversion.

Viral cofactors may be important in the pathogenesis of HIV infection and the development of AIDS, but their role is still imperfectly understood. Sequential serological studies were performed in a cohort of 100 homosexual men and 70 matched healthy controls over a mean period of 4 years. Of the patients, 18 were found to be HIV+ on admission to the study and 15 seroconverted to HIV+ during the follow up (seroconversion group). Serum antibodies of both IgG and IgA isotypes against Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were determined. IgG antibodies indicate past infection, while a marked increase in IgG titre or a positive IgA titre were taken to indicate active infection or reactivated latent infection. EBV and CMV infections were about two to four times more prevalent in the homosexual men both HIV- and HIV+, compared with controls. Active infections were increased in the homosexual men and particularly in the HIV+ patients. The seroconversion group revealed activation of both EBV and CMV following HIV infection. When the antibody profile of seroconverting patients at the time preceding seroconversion was compared with a matched group of 39 homosexual men who remained HIV-, no change was found in CMV antibodies, but four out of 15 (26.6%) of the patients had high titres of anti-EBV IgA preceding seroconversion, as compared with only one out of 39 (2.6%) of HIV- homosexual men (P less than 0.05). This suggests a role for EBV reactivation in the pathogenesis of HIV infection in some patients.

Chlamydia specific IgG and IgA antibodies in women with obstructive infertility as determined by immunoblotting and immunoperoxidase assays.

The prevalence rate of IgG and IgA antibodies to Chlamydia was analyzed in 50 women with laparoscopy-verified tubal infertility and in 50 age-matched control women by single serovar (L2) inclusion immunoperoxidase assay (IPA) and by immunoblotting technique (IB). Women with tubal infertility had significantly (p less than 0.001) elevated IPA Chlamydia IgG antibody titer greater than or equal to 128 and greater than or equal to 256 than controls (64% vs 16%. Odds ratios = 9.3 and 50% vs 10%, Odds ratio = 9 respectively). The prevalence rate of IPA IgA antibody titer (greater than or equal to 16) to Chlamydia was also significantly higher (p less than 0.001) in women with tubal infertility than controls (48% vs 8%, Odds ratio = 10.6). Antibodies to at least 19 chlamydial structural polypeptides ranging in molecular weight from 30 kD to 204 kD, were detected by the IB technique in the IPA seropositive sera. Antibodies to 57-60 kD were detectable in almost all the IPA IgG and IgA seropositive sera. The prevalence rate of IgG antibody to 57 kD-60 kD was significantly higher in women with obstructive infertility than healthy woman (84% vs. 56% p less than 0.01; Odds ratio = 3.8). More significantly, higher differences to 57-60 kD polypeptide were found in the case of IgA between the infertile women and controls (52% vs. 10%, p less than 0.001; Odds ratio = 9.7). The significance of IPA and IB technique for screening of infertile women is discussed.

Detection of IgG and IgA antibodies to Chlamydia trachomatis in sera of patients with chlamydial infections: use of immunoblotting and immunoperoxidase assays.

The immune response to individual structural polypeptides of Chlamydia trachomatis was studied in 75 sera from symptomatic and asymptomatic women with culture-proved genital infections and from apparently healthy women who were culture-negative for C. trachomatis. The immunoblotting technique and the single serovar (L2) inclusion immunoperoxidase assays were used for measurement of the various antibodies. Antibodies to 18 structural polypeptides, ranging in molecular weight from 29 to 204 Kdaltons, were detected by the immunoblotting technique in sera from seropositive women. The immunoperoxidase assay showed that sera with high titers of IgG and IgA antibodies to C. trachomatis reacted with more polypeptides than did sera with low titers in this test. Antibodies to the 60- and 62-Kdalton polypeptides were detected in almost all sera positive for IgG and IgA antibodies, irrespective of chlamydial shedding. About 40% of sera with high IgG and IgA titers reacted with 39-, 57-, 64-, 72-, 86-, 105-, 155-, and 204-Kdalton polypeptides. The prevalence of IgA antibodies to C. trachomatis was higher among women with culture-proved chlamydial infections than among apparently healthy controls.

Virus-specific serum IgG, IgM, and IgA antibodies in cytomegalovirus mononucleosis patients as determined by immunoblotting technique.

The immune response to individual human cytomegalovirus (CMV) structural polypeptides was studied in paired sera from 15 adult CMV mononucleosis (CMV-MN) patients and healthy controls by immunoblotting technique (IB). IgM and IgG antibodies to at least 11 structural polypeptides with molecular weights of 28K, 49K, 55K, 57K, 66-70K, 82K, 87K, 110K, 150K, 205K, and 235K were detected in the patients' sera in the serum sample obtained in the acute phase of the disease. IgA antibodies to polypeptides with molecular weights of 66-70K, 82K, 110K, and 150K were also detected in these sera. In healthy seropositive adults, IgG antibodies with the same molecular weight polypeptides, excluding the 205K and 235K polypeptides, were detected as in convalescent CMV-MN patients. A prominent reactivity of IgM and IgA antibodies to the 66-70K and 150K polypeptides was noted in the acute sera from all the CMV-MN patients examined, but not in a number of late convalescent sera. The potential implications of these findings in the development of specific serological tests are discussed.

Persistence of a positive test for IgM antibodies to hepatitis A virus in late convalescent sera.

We had the opportunity to carry out a long-term follow up of 15 patients with viral hepatitis Type A. The serum IgM response to hepatitis A virus (HAV) was investigated with a commercially available kit (HAVAB-M). IgM antibodies were detected in all specimens collected during the acute phase of the disease and during early convalescence (first 3 months). However, there was a clear decrease in the "sample-to-cutoff" ratio (s/co ratio) after the first 2 months. Serum samples were available from six patients 30 to 32 months after onset of illness. Two of these were positive for IgM anti-HAV, but the s/co ratios were low. These results point to the need for careful evaluation of the diagnosis of acute or recent viral hepatitis Type A when low s/co ratios are obtained.

Detection and persistence of specific IgA antibodies in serum of patients with hepatitis A by capture radioimmunoassay.

The serum immunoglobulin A (IgA) response to hepatitis A virus (HAV) was investigated with a sensitive capture radioimmunoassay. In serial serum samples drawn from 15 patients with viral hepatitis A, IgA anti-HAV antibodies reached their highest titer between 1-2 weeks after onset and peak titers ranged from 10,000-20,000. Serum samples were available from six patients 30-32 months after onset of illness. These samples were all positive for IgA anti-HAV and some had titers similar to peak titers during illness. However, the height of the titration curves, expressed as the binding ratio (BR) at a dilution of 1/1000, was in all cases significantly lower at 30-32 months than during acute illness and early convalescence. The significance of the persistence of the IgA anti-HAV and possible reasons for the change in the BR are discussed.