Lack of association between hepatitis C infection and development of AIDS-related lymphoma.

Hepatitis C virus (HCV) has been associated with various lymphoproliferative disorders, and a high prevalence (9%-32%) of chronic HCV infection has been demonstrated among patients with lymphoma. Dual coinfection by HIV and HCV has been demonstrated in approximately 40% of certain populations of HIV-infected individuals. Because of this high prevalence of coinfection by HIV and HCV, the known relations between HCV and lymphoproliferative disorders, and the association of HIV and B cell lymphoma, the potential association between chronic HCV and the development of AIDS-related lymphoma was examined. The prevalence of HCV infection in HIV-infected patients with lymphoma was compared with that in patients with AIDS, diagnosed on the basis of an illness other than lymphoma. Risk factors for HCV infection, overall, were also evaluated. Evidence of HCV infection was ascertained by assessing anti-HCV antibodies, and HCV RNA in serum. The study consisted of 99 homosexual/bisexual men with AIDS-related lymphoma, and 43 other AIDS patients. HCV infection was detected in 11 of 99 (11.1 %) men with lymphoma, and in 5 of 43 (11.6%) other AIDS patients. Further, in patients with AIDS-related lymphoma, no relation was found between HCV infection and lymphoma histology or site. History of use of injected illicit drugs was associated with a significantly elevated risk of HCV infection in the combined group of lymphoma and other AIDS patients. The current study demonstrates no relation between dual infection by HIV and HCV and subsequent increased risk of lymphoma.

Prevalence of hepatitis G virus RNA in the sera of patients with HIV infection.

OBJECTIVE: The routes of transmission of the hepatitis G virus (HGV) are similar to those responsible for infection with HIV. We sought to evaluate the prevalence of HGV RNA in the sera of HIV-infected patients. METHODS: The sera of 157 HIV-infected patients were assayed by reverse transcriptase-polymerase chain reaction (RT-PCR) using established primers for HGV. Patients were divided into group 1 (positive circulating hepatitis B surface antigen [HBsAg]), group 2 (positive anti-hepatitis C virus [HCV] antibody) and group 3 (without markers for HBV or HCV). RESULTS: The overall prevalence of HGV RNA was 22%; prevalence was higher in group 1 (49%) than in groups 2 (16%) or 3 (7%). Patients with positive HGV RNA had laboratory values similar to HGV RNA-negative patients except for higher CD4 counts. Patients with an estimated risk duration of < or = 14 years were more likely to be HGV RNA-positive than patients at risk for >15 years. HGV RNA was found as frequently in patients with a homosexual lifestyle as in injection drug users (IDU). Multivariable analysis showed that the presence of HBsAg was the strongest factor associated with the presence of HGV RNA in serum. CONCLUSIONS: Patients with HIV and HBV coinfection are significantly more likely to be HGV RNA-positive. Patients with a risk factor duration for >15 years were less likely to be HGV RNA-positive, pointing to a decrease in HGV RNA prevalence over time. This study supports the notion that homosexual lifestyle, in addition to injection drug usage and blood product transfusion, is a risk factor for HGV infection.

Clinical significance of hepatitis C viral RNA status and its correlation to antibodies to structural HCV antigens in anti-HCV reactive patients with normal liver tests.

Extensive serological testing and HCV RNA determination by RT-PCR was performed in serum, PBMCs, and liver tissue in thirteen anti-HCV reactive patients with persistently normal liver tests. Absolute concordance in the status of HCV RNA between serum, PBMCs, and liver was noted. Five patients were HCV RNA positive but only three had mild histological changes. Eight patients were HCV RNA negative in all three sites and had virtually normal liver histology. Patterns of reactivity in RIBA 2.0 strip immunoblot assay did not differentiate viremic from nonviremic patients. ELISA testing using multiple individual HCV recombinant antigens from the structural and non-structural regions of HCV demonstrated mean antibody titers to the structural antigens, in particular HCV E2 antibodies, to be significantly lower in HCV RNA negative patients. The status of HCV RNA in the serum appears to infer the status of HCV RNA in the liver and PBMCs in patients with persistently normal liver tests. Patients with persistently normal liver tests and undetectable HCV RNA have probably spontaneously cleared HCV infection.

A new restriction-modification system, KpnBI, recognized in Klebsiella pneumoniae.

A unique DNA restriction-modification (R-M) system has been identified in the GM236 strain of Klebsiella pneumoniae using the newly isolated phage, SBS. The system was designated KpnBI. The gene (hsdRKpnBI) complementing the restriction activity of the KpnBI system was cloned in pBR322. The nucleotide sequence of the cloned DNA revealed one open reading frame (ORF) of 3035 bp. Analysis of the deduced amino-acid sequence shows seven helicase motifs which are common to the restriction (R) subunit of both type-I and type-III R-M systems. Computer analysis (Dendrogram) of the R polypeptide of KpnBI suggests a closer relationship to EcoR124/3I, a member of type-IC family, than to other representative type-I and type-III systems.

Short-term prednisone therapy affects aminotransferase activity and hepatitis C virus RNA levels in chronic hepatitis C.

BACKGROUND/AIMS: The effects of corticosteroids on chronic hepatitis B have provided insight into the mechanism of liver cell injury caused by hepatitis B. In this study, this model was applied to investigate the effects of prednisone on alanine aminotransferase (ALT) and hepatitis C virus (HCV) RNA levels in chronic hepatitis C. METHODS: Ten patients with chronic hepatitis C who had increased levels of ALT and HCV RNA detectable in serum were given a 7-week course of a tapering dose of prednisone. Quantitation of serum HCV RNA was determined by polymerase chain reaction (PCR) and by branched-chain DNA amplification. RESULTS: ALT levels decreased in 8 of 10 patients during therapy. Mean ALT levels of all 10 patients decreased from 184 to 84 U/L (P = 0.002) and then rebounded in 7 of the 8 patients after discontinuation of prednisone. HCV RNA was detectable by the branched DNA technique in 9 of 10 patients. These values increased in all 9 patients during prednisone therapy. The mean serum HCV RNA levels increased from 40.9 before treatment to 414.3 Eq/mL x 10(5) during treatment (P = 0.043). Using PCR, HCV RNA titers increased one log-fold in 8 of 10 patients (geometric mean of 1:4420 to 1:23410). HCV RNA levels decreased to pretreatment values within a mean of 2.8 weeks (range, 1-5) after discontinuation of prednisone. CONCLUSIONS: These responses in ALT and HCV RNA suggest the participation of an immune-mediated mechanism in the liver cell injury in chronic hepatitis C.

Clinical significance of concomitant hepatitis C infection in patients with alcoholic liver disease.

The significance of antibodies to hepatitis C virus in patients with chronic alcoholic liver disease is unclear. Prior studies have utilized the first-generation enzyme-linked immunosorbent assay, which is limited by problems with sensitivity and specificity. Hepatitis C virus infection in 137 patients with biopsy-proven alcoholic liver disease was assessed with second-generation hepatitis C virus antibody assays and reverse transcription-polymerase chain reaction for detection of hepatitis C virus RNA in the serum. The patients were categorized into three groups according to results of serological testing. Discriminant-function analysis was used to determine which factors (risk, biochemical and histological) could best differentiate the three groups. Thirty-three patients were reactive on second-generation enzyme-linked immunosorbent assay/second-generation recombinant immunoblot assay and RNA positive (group 1). Twelve were reactive on second-generation enzyme-linked immunosorbent assay/second-generation recombinant immunoblot assay but RNA negative (group 2). Eighty-six were nonreactive on second-generation enzyme-linked immunosorbent assay, and six were reactive on second-generation enzyme-linked immunosorbent assay but negative on second-generation recombinant immunoblot assay and negative for hepatitis C virus RNA (group 3). Seventy-six percent of patients in group 1 and 58% in group 2 had parenteral risk factors, compared with only 1% in group 3 (p < 0.00001). The mean ALT level was higher in group 1 patients (p < 0.05). The mean histologic activity index was significantly higher in group 1 (p = 0.0007).(ABSTRACT TRUNCATED AT 250 WORDS)

The stability of serum hepatitis C viral RNA in various handling and storage conditions.

To investigate whether certain handling and storage conditions of serum samples could alter the sensitivity and specificity of the hepatitis C virus (HCV) RNA assay, we studied serum samples obtained from five patients known to be positive for HCV RNA and two patients with autoimmune chronic active hepatitis. Samples were subjected to one of the following conditions: (1) immediate storage at -20 degrees C, (2) five freeze-thaw cycles, (3) storage at 4 degrees C for 5 days, and (4) storage at room temperature for 5 days. Detection of HCV RNA was performed by polymerase chain reaction. Titers of HCV RNA were determined by serial end point dilutions. We found that the titer of HCV RNA was reduced by only one logfold in samples subjected to conditions 2 through 4 in two of the five patients. False-positive results were not seen with the serum samples that were subjected to similar conditions from the two negative control patients. We conclude that serum HCV RNA is resistant to degradation under routine laboratory handling and storage conditions.

Spontaneous exacerbation of disease activity in patients with chronic delta hepatitis infection: the role of hepatitis B, C or D?

Forty-six patients with chronic hepatitis delta virus infection were followed between 6 and 116 mo (mean = 32.8 mo; median = 24 mo). Nineteen patients (41%) demonstrated clinical courses with episodes of biochemical reactivation (ALT levels greater than or equal to 10 times baseline values [group A]). Twenty-seven patients (59%) had stable clinical courses without biochemical reactivation (group B). Patients in group A were younger than those in group B (30.5 vs. 35.3 yr; p = 0.03), were less likely to be intravenous drug abusers (16% vs. 52%; p = 0.01) and were more likely to be homosexual (58% vs. 22%; p = 0.01). Serum hepatitis B virus DNA, hepatitis delta virus RNA, IgM antibody to HBc, HBeAg, antibody to HBe and IgG and IgM antibody to hepatitis delta virus were measured in all patients. In group A, these markers were studied before and during reactivation and during remission. In group B, these parameters were studied in a random fashion at 7- to 10-mo intervals. The presence of antibodies to human immunodeficiency virus and hepatitis C virus was assessed in all patients. A total of 38 biochemical reactivation episodes was noted among the 19 patients in group A. Eleven had sequential changes in hepatitis delta virus markers, suggesting that the exacerbations were due to hepatitis delta virus. In three, the sequential changes of viral markers were consistent with the exacerbations due to hepatitis B virus. In five other patients, no sequential changes in viral markers could be demonstrated to correlate with the biochemical exacerbations.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved detection of hepatitis C virus antibodies in high-risk populations.

Sera from 483 patients at high (group 1, n = 313) and lower (group 2, n = 170) risk for exposure to hepatitis C were tested for antibodies to hepatitis C using first-generation (c100-3) and second-generation enzyme-linked immunosorbent assays and four-antigen recombinant immunoblot assay. The second-generation enzyme-linked immunosorbent assay and nitrocellulose-based immunoblot assay differ from c100-3-based systems in the addition of expression products from the NS3/NS4 (c33c, c200) and putative nucleocapsid (c22-3) region of the hepatitis C genome. In group 1, the sensitivity of detection of hepatitis C antibodies was 45%, 55% and 46% by the first- and second-generation enzyme-linked immunosorbent assays and recombinant immunoblot assay, respectively. In group 2, antibodies were detected by each test system in 26%, 32% and 7% of patients, respectively. Most sera (99%) reactive with the first-generation enzyme-linked immunosorbent assay were reactive with the second-generation enzyme-linked immunosorbent assay (in group 1, 89% of these specimens demonstrated reactivity to at least one antigen with the immunoblot assay, compared with only 31% in group 2). An additional 12% (group 1) and 6% (group 2) of specimens demonstrated reactivity with the second-generation enzyme-linked immunosorbent assay only (of these, 75% [group 1] and 9% [group 2] demonstrated reactivity to at least one antigen with the immunoblot assay). Ninety-eight percent of specimens not reactive with both enzyme-linked immunosorbent assay test systems were also nonreactive by recombinant immunoblot assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Host dependent modulation of hepatitis delta virus replication in chronic delta hepatitis.

To determine whether host dependent differences modulated hepatitis delta virus replication in chronic delta hepatitis, we tested HDV RNA in homosexual and intravenous drug abuser populations. Overall, the seroprevalence of HDV RNA in the two groups with matching clinical characteristics was 72% (76/106 patient visits). A trend for greater prevalence of HDV RNA was noted at initial presentation in homosexuals (82%) compared to intravenous drug abusers (60%, P less than 0.05) and this trend appeared to be maintained during two years of sequential follow-up. The seroprevalence of co-appearing IgM and IgG anti-HD antibodies was similar in the two groups of patients. However, in HDV RNA positive homosexuals IgG anti-HD antibody was more prevalent, and additionally, assumed concordance with HDV RNA of 92% although the significance of this observation is unclear. The difference in prevalence of HIV in the two groups did not reach statistical significance. Prospective studies are required to confirm differences in HDV replication in various patient groups and to define underlying mechanisms.

Acute delta hepatitis: serological diagnosis with particular reference to hepatitis delta virus RNA.

To evaluate serologic diagnosis of hepatitis delta virus, we tested HDV RNA in stored sera from 48 patients with acute delta hepatitis who were identified with anti-HD antibodies. Initial sera were positive for HDV RNA in 27 of 48 (56%) patients. In comparison, isolated IgM anti-HD was present in 18 (38%) patients, although IgM and IgG anti-HD were present concurrently in 16 (33%) additional patients. Overall, either HDV RNA or IgM anti-HD was present in 69% of the initial sera. The HDV infection was self-limiting in all except two patients who died of fulminant hepatitis and nine others in whom chronic delta hepatitis ensued. Patterns of HDV seropositivity during progression to chronicity induced variable persistence, disappearance or recrudescence of either HDV RNA or IgM and IgG anti-HD. Results of HDV RNA and IgM anti-HD tests were concordant in only 40-50% of instances. Our results indicate that serological testing for HDV RNA is direct and will demonstrate HDV replication in a large number of cases with acute delta hepatitis. Testing for IgM anti-HD could provide supplemental evidence for HDV infection. Sequential testing for these markers will facilitate assessment of the outcome of acute HDV infection.

Genomic hsd-Mu(lac) operon fusion mutants of Escherichia coli K-12.

Genomic (chromosomal)hsd-Mu(lac) operon fusions have been constructed in two strains of Escherichia coli K-12 for the three hsd genes, hsdRK, hsdMK and hsdSK, using MudX and lambda placMu53. Expression of hsdK mutants ranged from 16 to 74 units (u) (with a mean of 52 u) for fusions to promoter pres and ranged from 26-75 u (also with a mean of 52 u) for fusions to promoter pmod. The expression of the two hsdK promoters was measured in different stages of growth. The pres fusion mutant showed a lag in beta-galactosidase (beta Gal) production, as compared to the pmod fusion mutant. One r-Km-K mutant (JR205) showed more than ten times the beta Gal activity of other insertion mutants. The activity of this mutant decreased by 20-fold upon the transfer of F101-102, which includes the wild-type hsd region. Positive gene-dosage effect was observed using F' plasmids containing the hsd-lacZ region.

Evaluation of a commercial anti-delta EIA kit for detection of antibodies to hepatitis delta virus.

Anti-hepatitis delta virus (anti-HDV) antibodies were measured by solid phase IgG and IgM capture radioimmunoassays (RIA) as well as by a competitive binding enzyme immunoassay (EIA) in both acute and chronic HDV infections. EIA anti-delta test measures total delta antibody without discriminating IgM from IgG anti-delta. Low titer IgG antibodies were detected by both techniques with equal sensitivity. High titer IgG antibodies reached the end point sooner with EIA than with RIA (10(-3) versus greater than 10(-6)). When IgM anti-HDV was present without accompanying IgG anti-HDV, EIA failed to identify the antibody. Presence of high titer rheumatoid factor in the serum and lipemic samples produced false-positive results by EIA. Usage of undiluted serum samples for EIA probably exaggerates the factors contributing to false-positive reaction.

Interactions of HDV, HBV and HIV in chronic B and D infections and in reactivation of chronic D infection.

From this study, we can conclude that there is significant influence of HIV infection on the clinical course of chronic HDV as follows: In these patients, there is simultaneous replication of both HBV and HDV and the suppression of HBV by HDV is modified. There is decreased antibody response to HDV, however, the degree of liver injury is not altered. Although these patients tend to have "reactivation episodes" as frequently as the HIV negative group, no correlation of serum ALT to HDV-RNA could be found. The possibility of these episodes resulting from injury due to other viruses such as non-A, non-B cannot be excluded.

Study of preneoplastic changes of liver cells by immunohistochemical and molecular hybridization techniques.

The status of hepatitis B virus DNA was investigated by in situ hybridization in multifocal areas of a noncancerous hepatitis B virus-associated cirrhosis. This liver exhibited a marked degree of dysplasia and adenomatous hyperplasia. The results of these studies were correlated with the histopathology and immunohistochemical stains for hepatitis B core and surface antigens. There was clear evidence of a marked reduction to absence of hepatitis B viral DNA by in situ hybridization and absence of HBc and HBsAg in the foci of liver cell dysplasia and adenomatous hyperplasia. These results support the hypothesis that liver cell dysplasia and adenomatous hyperplasia are preneoplastic in nature.

Study of reactivation of chronic hepatitis delta infection.

Five patients with chronic hepatitis delta virus (HDV) infection suffered spontaneous episodes of liver enzyme elevation on a background of otherwise biochemically stable liver disease. In all five patients these episodes were accompanied by a rise in serum levels of anti-HDV IgM, HDV antigen and HDV RNA. These episodes of increased HDV replication accompanied by biochemical evidence of liver injury are reminiscent of reactivation in chronic hepatitis B. Surges of increased HDV replication may be important in the progression of liver disease in chronic HDV infection.

Detection of hepatitis delta virus in serum and liver tissue by molecular hybridization. Validation of a rapid spot-hybridization technique.

Serologic tests for detection of delta hepatitis virus (HDV) antigen and antibody have recently been supplemented with a Northern blot hybridization assay for HDV RNA. However, this technique is cumbersome for analysis of multiple samples. In order to simplify detection of HDV RNA, the authors have tested a spot-hybridization method with a new HDV cDNA probe. Their method has proved to be rapid, sensitive, and specific for HDV RNA even when less ultracentrifugation was used for recovering serum RNA. Results for HDV RNA were concordant by both spot and Northern blot hybridization in 12 serum samples from patients with known delta hepatitis, whereas in seven cases spot-hybridization was superior in detecting liver HDV RNA. The concordance between HDV RNA by spot hybridization and delta antigen was complete, whereas that between delta IgM (44% overall) or IgG (67% overall) was less strong. The authors' observations indicate that this new technology permits detection of HDV RNA with relative ease and could be applicable to the evaluation of large numbers of cases with delta hepatitis.

Correlation of IgM anti-hepatitis D virus (HDV) to HDV RNA in sera of chronic HDV.

One hundred forty-four serum samples from 52 patients with chronic hepatitis D virus infection were analyzed for hepatitis D virus RNA by dot-blot hybridization using hepatitis D virus cDNA probe labeled with 32P. The results were correlated with the presence of serum IgM anti-hepatitis D virus and hepatitis D antigen in liver biopsy specimens when available. Although there was a trend of positive correlation between serum hepatitis D virus RNA and IgM anti-hepatitis D virus, no statistical significance could be found. In the serum samples with hepatitis D virus RNA, 32% were found to be negative for IgM anti-hepatitis D virus. Therefore, in chronic hepatitis D virus, absence of IgM anti-hepatitis D virus does not rule out active viral infection, as suggested by previous studies. There was a strong correlation between serum hepatitis D virus RNA and hepatic hepatitis D virus antigen. These data indicate that detection of hepatitis D virus RNA in serum samples is a reliable noninvasive marker of active viral infection.