Search PubMed⌕ Search

Biomedical subjects

D R Gretch

Publications and source records attributed to D R Gretch.

At least 55 records · Page 3Linked to original sources
PubMed1997

Diagnostic tests for hepatitis C.

Diagnostic tests for hepatitis C can be divided into the following two general categories: 1) serological assays that detect antibody to hepatitis C virus (anti-HCV); and 2) molecular assays that detect, quantify, and/or characterize HCV RNA genomes within an infected patient. Serological assays have been subdivided into screening tests for anti-HCV, such as the enzyme immunoassay (EIA), and supplemental tests such as the recombinant immunoblot assay (RIBA). Three generations of anti-HCV tests have been developed, and each generation has resulted in an improvement in the sensitivity of detecting anti-HCV. Supplemental anti-HCV tests are designed to resolve false-positive testing by EIA, and are appropriately used in low-prevalence settings in which false-positive anti-HCV tests remain a problem. Third-generation anti-HCV tests (EIA-3 and RIBA-3, respectively) contain antigens from the HCV core, nonstructural 3, nonstructural 4, and nonstructural 5 genes. Detection of HCV RNA in patient specimens by polymerase chain reaction (PCR) provides evidence of active HCV infection and is potentially useful for confirming the diagnosis and monitoring the antiviral response to therapy. Optimal HCV PCR assays at present have a sensitivity of less than 100 copies of HCV RNA per milliliter of plasma or serum. Standardization and proficiency testing of diagnostic laboratories performing HCV PCR remains an important problem for future study. Two main technologies exist for assessing HCV RNA levels or viral load. Quantitative PCR is the most sensitive test for determining hepatitis C viral load, whereas the branched-chain DNA test appears to be the most precise method. Major limitations of the current tests are inadequate dynamic range and high variability of PCR-based assays, and poor sensitivity of the branched-chain DNA test. Molecular tests have also been developed to classify HCV into distinct genotypes; the clinical importance of HCV genotype determination remains a subject for future investigation.

Genotype↗
PubMed1997

Use and interpretation of HCV diagnostic tests in the clinical setting.

The rapidly emerging and sometimes complicated field of HCV diagnostics can be simplified by classification of tests into two general categories: serologic tests which screen for anti-HCV antibodies, and molecular tests which are used to assess HCV viremia and characterize viral infection at the genetic level. Antibody tests include the highly sensitive screening enzyme immunoassays (current versions: EIA-2 and EIA-3), and supplemental tests such as the recombinant immunoblot assay (RIBA-2). Molecular assays such as HCV RNA polymerase chain reaction (PCR) may play an important role in confirming HCV infection in several clinical situations, such as immunosuppressed patients with chronic hepatitis C, patients with acute hepatitis who might be in the diagnostic "window" period prior to seroconversion, and seropositive patients with normal ALT values. Quantitative HCV-RNA tests, such as quantitative PCR (Q-PCR) and branched DNA (bDNA), provide valuable tools for assessing the level of HCV viremia prior to and during therapy. Genotype tests allow classification of HCV infection in one of six distinct HCV genotypes, although the clinical relevance of HCV genotype tests has not been established.

False Positive Reactions↗
PubMed1997

Assessment of hepatitis C virus quasispecies heterogeneity by gel shift analysis: correlation with response to interferon therapy.

Hepatitis C virus (HCV) quasispecies heterogeneity was assessed in pretreatment sera of 22 patients treated with interferon (IFN). Quasispecies heterogeneity was quantitated by analysis of 490 hypervariable region 1 (HVR1) clones using gel shift analysis (GSA), which allowed determination of two components of HCV quasispecies heterogeneity: genetic complexity (number of variants) and genetic diversity (mean genetic distance between variants). HCV genotype and pretreatment RNA titer were similar between responders (n = 12) and nonresponders (n = 10). GSA correlated well with nucleotide sequencing for estimating HCV genetic diversity (R = .952; P < .001). A positive correlation of .666 (P < .001) was observed between genetic diversity and complexity, suggesting that these components of quasispecies heterogeneity were related. However, while there were no significant differences in pretreatment complexity or genetic diversity among response groups, higher pretreatment genetic diversity was predictive of response failure (P = .041). Assessment of HVR1 heterogeneity by GSA revealed important relationships between quasispecies genetic diversity, complexity, and response to IFN therapy and suggested a need for analysis of other HCV genes.

Cloning, Molecular↗
PubMed1996

Tracking hepatitis C virus quasispecies major and minor variants in symptomatic and asymptomatic liver transplant recipients.

To evaluate the possibility that distinct viral quasispecies play a role in the pathogenesis of progressive hepatitis C virus (HCV) infection, we performed a detailed evaluation of HCV quasispecies before and after liver transplantation in five patients infected with HCV genotype 1, three of whom developed severe recurrent hepatitis C and two of whom developed asymptomatic posttransplant infections with high-titered viremia. HCV quasispecies were characterized by using a combination of nucleotide sequencing plus heteroduplex tracking assay of the second envelope gene hypervariable region (HVR). An average of 30 HVR clones were analyzed per specimen; an average of five specimens were analyzed per patient over a 6- to 24-month study period. The complexity of HCV quasispecies in pretransplant serum varied, ranging from one to nine genetically distinct variants for the five patients. However, in all five cases, relatively homogenous quasispecies variants emerged after liver transplantation. In the three patients who developed recurrent hepatitis, quasispecies major variants present in pretransplant serum were efficiently propagated immediately after liver transplantation and were propagated throughout the course of acute and chronic hepatitis. In contrast, in the two asymptomatic cases, we observed rapid depletion of pretransplant quasispecies major variants from posttransplant serum, followed by emergence of new quasispecies variants by posttransplant day 30. Genetic analysis suggested that in these cases, the new quasispecies variants were derived from minor variants present at relatively low clonal frequency (less than 5% of HVR clones) within the pretransplant quasispecies populations. These data demonstrate that quasispecies tracking patterns are associated with the rapidity and severity of HCV-associated liver disease after liver transplantation. Further characterization of HCV quasispecies in animal model systems is warranted.

Amino Acid Sequence↗
PubMed1996

Elimination of hepatitis C virus RNA in infected human hepatocytes by adenovirus-mediated expression of ribozymes.

Hepatitis C virus (HCV), a positive-strand RNA virus, is the major infectious agent responsible for causing chronic hepatitis. Currently, there is no vaccine for HCV infection, and the only therapy for chronic hepatitis C is largely ineffective. To investigate new genetic approaches to the management of HCV infection, six hammerhead ribozymes directed against a conserved region of the plus strand and minus strand of the HCV genome were isolated from a ribozyme library, characterized, and expressed from recombinant adenovirus vectors. The expressed ribozymes individually or in combination were efficient at reducing or eliminating the respective plus- or minus-strand HCV RNAs expressed in cultured cells and from primary human hepatocytes obtained from chronic HCV-infected patients. This study demonstrates the potential utility of ribozyme therapy as a strategy for the treatment of hepatitis C virus infection.

Adenoviridae↗
PubMed1995

Assessment of hepatitis C viremia using molecular amplification technologies: correlations and clinical implications.

OBJECTIVE: To compare two recently developed molecular techniques for quantitating the levels of hepatitis C virus (HCV) RNA in the serum of patients with a wide spectrum of chronic hepatitis C. DESIGN: Serum samples from 299 patients with HCV viremia, 101 control patients without HCV infection, and 19 consecutive patients receiving systemic interferon therapy were evaluated by a commercially available branched-chain DNA (bDNA) assay and a quantitative competitive polymerase chain reaction (PCR). SETTING: University-based hepatology clinics and reference virology laboratory. PATIENTS: Patients with HCV viremia as defined by results of qualitative RNA PCR, including 53 HCV-infected blood donors, 34 patients receiving renal dialysis, and 212 patients attending a hepatology clinic. RESULTS: Results of in vitro and in vivo experiments indicated that the sensitivity and dynamic range of the PCR assays were greater than those of the bDNA assay. Detection of HCV viremia by the bDNA assay was highly dependent on viral RNA titers, with a sensitivity of 5% at HCV RNA titers of 5.0 logs per mL or less and 94% at titers of 5.5 logs per mL or greater. The best correlation between assays was observed in specimens with HCV RNA titers between 6.0 and 7.5 logs per mL (r = 0.73). In patients with high-titer HCV viremia, including liver transplant recipients and patients with cirrhosis, quantitative PCR results were an average of 12-fold higher than bDNA assay results. Results of repetitive testing of discordant specimens showed that these discrepancies were caused by a high kit-to-kit coefficient of variation (112%) in the bDNA assay. Of 19 patients receiving interferon therapy, 9 (47%) became bDNA negative, but only 5 became quantitative PCR negative. The bDNA-negative, quantitative PCR-positive patients all had relapse when therapy was discontinued. CONCLUSIONS: The bDNA assay has a narrower linear range for quantitation of HCV viremia than quantitative PCR. Because persons with low HCV titers may respond well to therapy, seropositive persons with negative bDNA results should be retested with PCR-based assays. Similarly, the bDNA assay may underestimate the true degree of HCV viremia in persons with end-stage infection (> 10(7) RNA equivalents/mL of sera). Despite these limitations, the combination of bDNA- and PCR-based assays appears to be optimal for selecting and following patients during interferon therapy.

Case-Control Studies↗
PubMed1995

Hepatitis C--associated glomerular disease in liver transplant recipients.

Hepatitis C virus (HCV) infection may be associated with extrahepatic illness including renal disease. We investigated the clinical and virological characteristics of three patients who developed a mesangial proliferative and sclerosing glomerulopathy alone or in association with membranoproliferative glomerulonephritis after liver transplantation for end-stage liver disease secondary to HCV infection. Using polymerase chain reaction technology and the IgM RIBA assay, viral load, genotype and IgM antibody response to HCV in the setting of glomerulonephritis was evaluated. Within 1 year of transplantation, the patients showed decreased renal function, proteinuria and recurrent hepatitis C liver disease. Likewise, HCV viral load increased following transplantation, whereas the viral genotypes remained unchanged. Although the first patient presented with classic type II cryoglobulinemia in association with glomerulonephritis, the second patient developed an IgM directed specifically against the hepatitis C core antigen. The third patient developed a low-titered IgM directed against the hepatitis C core antigen with rheumatoid factor activity but without cryoglobulinemia. All of the patients show IgM in glomerular capillary walls by biopsy. One patient has shown a clinical response to interferon (IFN) alfa-2b therapy without evidence of hepatic allograft rejection. The second and third patients have not responded to IFN or developed hepatic rejection. This study suggests that HCV-associated glomerulonephritis may complicate liver transplantation in conjunction with the production of increased amounts of IgM of variable specificity. The posttransplant setting may provide a unique situation in which to investigate the specific requirements for the onset of renal disease.

Adult↗
PubMed1995

Successful cyclophosphamide treatment of cryoglobulinemic membranoproliferative glomerulonephritis associated with hepatitis C virus infection.

A 54-year-old man with cryoglobulinemia and chronic hepatitis C infection presented with progressive renal insufficiency caused by membranoproliferative glomerulonephritis. Because of a steady decline in renal function, cyclophosphamide therapy was instituted. Within 1 month of starting therapy, his cryoglobulins disappeared, and in 3 months, his creatinine clearance had improved from 56 mL/min to 89 mL/min. At no point in his course was there clinical evidence of liver disease. After 1 year, cyclophosphamide was successfully stopped. Fourteen months later, his creatinine clearance is 105 mL/min. These results suggest that cyclophosphamide may be useful therapy for patients with cryoglobulinemic membranoproliferative glomerulonephritis and hepatitis C virus infection who have progressive renal insufficiency.

Cryoglobulinemia↗
PubMed1995

Membranoproliferative glomerulonephritis associated with hepatitis C virus infection responsive to interferon-alpha.

A 42-year-old man with hepatitis C virus infection, cryoglobulinemia, hypocomplementemia, and nephrotic syndrome is reported. The kidney biopsy showed membranoproliferative glomerulonephritis. The patient was treated with interferon-alpha for 2 months. After the treatment, serum hepatitis C virus RNA became negative and nephrotic syndrome remitted. Repeat biopsy of the kidney revealed an improvement in renal histology. This case confirms an association between hepatitis C virus infection and membranoproliferative glomerulonephritis and the usefulness of interferon-alpha for treatment.

Adult↗
PubMed1995

Characterization of simple and complex hepatitis C virus quasispecies by heteroduplex gel shift analysis: correlation with nucleotide sequencing.

In infected humans, hepatitis C virus (HCV) exists as a quasispecies typically characterized by multiple nucleotide substitutions within the second envelope gene hypervariable region 1 (HVR1). In the current study, we used heteroduplex gel shift analysis (GSA) of HVR1 sequences amplified directly from patients' sera to define two patterns of HCV quasispecies: (i) simple quasispecies, which gave a mostly homogeneous gel shift profile with a single predominant band and (ii) complex quasispecies, which gave a gel shift profile with multiple bands. Recombinant HVR1 libraries were generated from two patients with complex HCV quasispecies (cases 1 and 2) and two patients with simple HCV quasispecies (cases 3 and 4), and 129 individual clones were analysed by either GSA, nucleotide sequencing or both techniques. In case 1 we identified a highly complex HCV quasispecies with 11 distinct HVR1 variants differing by 1-51 nucleotide changes. We found a general but not absolute correlation between GSA pattern and the number or position of nucleotide changes within HVR1. In case 2, the complex HCV quasispecies consisted of three distinct major variants; GSA of individual HVR1 clones allowed us to reconstruct the complex quasispecies pattern in vitro. In case 3, the simple quasispecies comprised 66% homogeneous clones and 33% unique minor variants differing by 1-3 nucleotides from the consensus sequence. In case 4, the simple quasispecies was 84% homogeneous, but six unique major shift variants were identified among 31 clones by GSA. In summary, HCV quasispecies can be characterized based on GSA profiles following direct PCR amplification of HVR1 sequences from patients' serum; the GSA profiles approximate the clonal population of HCV as determined by clonal analysis. GSA of HVR1 clones showed a strong correlation with nucleotide sequencing.

Base Sequence↗
PubMed1995

Hepatitis C virus infection and membranoproliferative glomerulonephritis in Japan.

The prevalence of hepatitis C virus (HCV) infection was determined in 146 adult patients with various types of glomerulonephritis and renal diseases monitored between 1990 and 1993. Serum HCV antibody (HCV Ab) was evaluated, and positive cases were tested for HCV RNA by polymerase chain reaction. HCV infection was present in 1 (1.7%) of 58 cases of immunoglobulin A nephropathy, 0 (0%) of 14 cases of lupus nephritis, 0 (0%) of 12 cases of minimal change nephrosis, and 0 (0%) of 28 cases of other renal diseases, which is similar to the 2% prevalence observed in healthy blood donors in Japan. In contrast, HCV Ab was observed in 2 (8.3%) of 24 cases of membranous nephropathy and 6 (60%) of 10 cases of membranoproliferative glomerulonephritis (MPGN) Type I. The prevalence of HCV infection in MPGN patients was significantly higher than the frequency of HCV infection observed in the other patients with renal diseases (P < 0.001). HCV RNA was present in all cases in which HCV Ab was present. The six patients with HCV-MPGN were similar to the four patients with idiopathic MPGN with respect to age, presence of nephrotic syndrome, and renal dysfunction, but had a higher incidence of liver dysfunction, cryoglobulinemia, rheumatoid factor, and hypocomplementemia (low C3). HCV infection is present in a large percentage of patients with MPGN in Japan and clinically may differ slightly from other cases of MPGN.

Adolescent↗
PubMed1995

Transmission of the hepatitis-C virus by tissue transplantation.

The hepatitis-C virus has been the most prevalent cause of chronic hepatitis in both blood and organ recipients. The introduction of a second-generation immunoassay for antibodies to the hepatitis-C virus (HCV 2.0) provided the opportunity to determine if the hepatitis-C virus can be transmitted through tissue transplantation. Banked sera from tissue donors that had previously been found to be non-reactive to the first-generation hepatitis-C virus antibody assay (HCV 1.0) and non-reactive for antibodies to hepatitis-B core antigen were retested with HCV 2.0. The sera from two donors were reactive; the transplant records of recipients of tissues from these donors were reviewed, and the surgeons or hospitals were contacted. The tissue recipients were tested with HCV 2.0, and positive sera were tested for hepatitis-C virus RNA by polymerase chain reaction. Viral nucleic acids isolated from viremic donors and recipients were analyzed for identity by sequencing of the hepatitis-C virus envelope gene (E2) hypervariable region. There were twenty-one grafts, which had been treated with gamma radiation, from one donor; thirteen had been transplanted to twelve recipients. Serum samples from six of the recipients were tested; one was reactive. This patient had other risk factors for infection with the hepatitis-C virus, and sequence analysis demonstrated non-identity between the donor and recipient hepatitis-C virus isolates. Nine of twelve grafts from a second donor had been transplanted in nine recipients. Serum samples from five patients were tested with HCV 2.0; four were reactive. In three of the four patients, the sera were determined to be positive for the hepatitis-C virus by polymerase chain reaction. E2 sequence analyses of hepatitis-C virus RNA isolates from two of these recipients demonstrated sequence identity with the donor isolate. The results of the present report demonstrate that the hepatitis-C virus can be transmitted by bone, ligament, and tendon allografts. They also support the need for testing of all tissue donors for antibodies to the hepatitis-C virus before the tissue is released for transplantation. The results also suggest that seventeen kilo-gray of gamma radiation may inactivate the hepatitis-C virus in tissue.

Adolescent↗
PubMed1995

Persistent hepatitis C virus infection after liver transplantation: clinical and virological features.

We report a prospective clinical and virological study of 18 patients undergoing orthotopic liver transplantation, selected because of hepatitis C virus (HCV) RNA positivity before transplantation. Nine of the 18 patients (50%) developed chronic active hepatitis (CAH) in liver allografts during the first year posttransplantation; hepatitis was first observed between 6 and 25 weeks posttransplantation. HCV viremia was measured for all patients before transplantation and on posttransplantation days 3, 7, and 14, and months 1, 6, 12, and 24 to 41, by quantitative competitive RNA polymerase chain reaction (QC-PCR). HCV RNA levels on posttransplantation days 3, 7, and 14 were significantly higher among patients who subsequently developed CAH versus those who did not (P < .02 by t-test and Mann-Whitney test on all three dates). However, HCV RNA levels in sera obtained at 1, 6, and 12 months posttransplantation did not correlate with CAH at 1 year or with HCV genotype determined in posttransplantation sera. At least two serial liver biopsy specimens from each patient were stained for HCV nonstructural 4 (NS4) antigen by immunohistochemistry. The intensity of cytoplasmic staining of NS4 antigen was significantly higher for specimens with CAH versus those without CAH (P = .028 by chi 2). Three patients developed bridging fibrosis in liver allografts during the first year after transplantation; all three patients had intense (3+) immunostaining for NS4 antigen, and the infecting genotypes were 1a, 1b, and 1a plus 1b, respectively. In summary, the 18 patients all developed high-titer viremia by 1 month after liver transplantation, whereas CAH developed in 50% of allografts during the first year after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic Disease↗
PubMed1995

Hepatitis B and transplantation.

Hepatitis B infection can add significantly to the morbidity and mortality of transplantation. Patients with hepatitis B can have severe recurrent disease following liver transplantation. Reactivation of occult disease can occur during chemotherapy or immunosuppression. Hepatitis B also can be acquired at the time of transplantation. Intensive study of transplant patients with hepatitis B has led to a new understanding of the immunobiology of hepatitis B virus.

Hepatitis B↗
PubMed1994

Hepatitis C virus-associated glomerulonephritis. Effect of alpha-interferon therapy.

Hepatitis C virus (HCV) infection may present as a primary glomerular disease. We report 34 adult patients who presented with proteinuria and had circulating anti-HCV antibodies. Primary risk factors included a history of intravenous drug abuse (56%) or blood transfusion (18%). Patients presented with nephrotic syndrome (71%) or with non-nephrotic proteinuria (29%) and had membranoproliferative or acute proliferative glomerulonephritis on renal biopsy. Signs of clinical liver disease were infrequent (18%), though elevated liver function tests were common (66%) and liver biopsy in 16 of 18 patients showed chronic active hepatitis. Cryoglobulinemia was frequent (59%), but only 44% had extrarenal manifestations. In 100% of cases tested, HCV RNA could be found in the serum or cryoprecipitates. Fourteen patients received interferon alpha for 6 to 12 months with a significant reduction in proteinuria but no improvement in renal function. A good clinical response correlated with disappearance of HCV RNA from the serum during treatment; however, relapse of viremia and renal disease was common after completing therapy. Evidence for HCV infection should be sought in all patients with primary glomerular disease. The optimal treatment strategy, however, remains to be defined.

Adult↗