Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The polymerase chain reaction with prior reverse transcription of RNA into cDNA was applied to hepatitis C virus RNA detection in human serum samples of different origin. In order to eliminate false negative results, the following steps were optimized: RNA extraction, reverse transcription, and oligonucleotide primer selection. We compared different RNA extraction methods using guanidinium salt/detergent and proteinase K digestion/phenol extraction, and tested virus particle enrichment with polyethylene glycol precipitation and ultracentrifugation. RNA extraction with guanidinium salt/detergent was the most efficient method. Ultracentrifugation of single samples did not improve hepatitis C virus RNA detection. Polyethylene glycol precipitation performed poorly. Recombinant thermostable reverse transcriptase produced cDNA from fewer samples than did Moloney murine leukaemia virus reverse transcriptase. Nested oligonucleotide primers from the 5'-terminal non-coding region of the hepatitis C virus genome amplified cDNA from more samples than did primers from the coding regions. Thirty six anti-hepatitis C virus antibody positive samples were tested; nested primers (nucleotides 6 to 327 and 15 to 288) yielded 21 amplificates, whereas primers from the coding region produced 16 amplificates (nucleotides 4684-5276) and 5 amplificates (nucleotides 5166-5270), respectively. The most efficient combination of steps was RNA extraction with guanidinium salt solution, reverse transcription with Moloney murine leukaemia virus reverse transcriptase and nested polymerase chain reaction primed with primers from the 5'-terminal non-coding region of the hepatitis C virus genome. Other combinations produced more false negative results. Three different groups of anti-hepatitis C virus antibody positive individuals had markedly different viraemia patterns: Hepatitis C virus RNA was detected in the sera of only 10% of anti-hepatitis C virus antibody positive blood donors, but in 90% of anti-hepatitis C virus antibody positive patients with clinically manifest hepatitis C, and 90% of anti-hepatitis C virus antibody positive haemophiliacs who had received plasma products in the past which had not been virus-inactivated. No hepatitis C virus RNA could be detected in the sera of 450 anti-hepatitis C virus antibody negative blood donors with elevated serum alanine aminotransferase catalytic concentrations.
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PMID:Improved detection of hepatitis C virus RNA by reverse transcription and polymerase chain reaction. 128 41

The prevalence of hepatitis C virus (HCV) infection among the patients of a hemodialysis unit in Taiwan was determined by an immunoblot and reverse transcriptase-polymerase chain reaction algorithm to be 58.8% (67 of 114 patients) after serological surveys with two advanced-generation enzyme-linked immunosorbent assays (ELISAs) for anti-HCV and a C 100-3 single-antigen test. The results of the second-generation ELISAs, the supplementary immunoblot test, and the test for HCV RNA were in good agreement with each other, from 86.0 to 98.2%. The first-generation C 100-3 test lacked the sensitivity of the four other systems. The two advanced-generation screening ELISAs for anti-HCV, a multiple-recombinant-antigen test, the Abbott second-generation ELISA, and a synthetic peptide multiple-antigen test, the UBI HCV EIA, provided reliable and virtually equivalent detection of potentially infected blood. Antibodies to capsid 1 and capsid 2 determinants of the Liatek immunoblot system were the most frequently detected reactivities to HCV in the HCV-infected hemodialysis patients. The percentage of HCV-infected patients with abnormal liver function (alanine aminotransferase level, greater than 100 IU/liter) was higher than that of the uninfected patients. The prevalence of HCV infection was correlated to the duration of hemodialysis treatment and the amount of blood transfused, and the most common transmission mode was thought to be patient-to-patient transmission through the dialysis equipment. Several means of reducing the frequency of transmission between hemodialysis patients are suggested.
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PMID:Hepatitis C markers in hemodialysis patients. 768 54

Hepatitis C virus (HCV) infection is common in hemodialysis patients, as determined by antibody assays and qualitative polymerase chain reaction (PCR) analysis of serum HCV RNA. To further characterize HCV infection in this population, we measured the viral load in infected hemodialysis patients by a quantitative, competitive PCR assay (QC-PCR) for HCV RNA. Hepatitis C virus RNA levels were correlated with serologic, biochemical, and demographic features of a cohort of hemodialysis patients. Sera from 208 hemodialysis patients were screened for HCV RNA (5' conserved region) by reverse transcriptase PCR (RT-PCR) and HCV-specific antibody. Forty-four patients were antibody positive (21%); among these patients, 34 (77%) were HCV RNA positive. No viremic, seronegative patients were identified. Hepatitis C virus RNA levels quantitated by QC-PCR ranged from 3 x 10(5) to 10(8) molecules of HCV RNA/mL. Male patients had significantly higher mean and median HCV RNA levels (10(7) molecules/mL) compared with female patients (3.6 x 10(6) molecules/mL and 3 x 10(6) molecules/mL, respectfully; P = 0.02). No other demographic or clinical feature of this cohort correlated with HCV RNA levels. Intravenous drug abuse was the most frequently identified risk factor (29% of seropositive patients) for infection with HCV in this population. No association between HCV RNA levels and hepatic enzyme levels (alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase, alkaline phosphatase) was apparent. Hepatitis C virus infection is highly prevalent in our hemodialysis population, and hemodialysis patients, particularly males, have high levels of HCV in serum.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Quantitation of hepatitis C viral RNA in sera of hemodialysis patients: gender-related differences in viral load. 797 21

To analyze the serological, clinical and histological significance of hepatitis B virus (HBV) replication among a group of patients with chronic delta hepatitis (CDH), we have studied the clinical and the histological activity in 49 patients with CDH. The HBV-DNA was analyzed by dot-blot and polymerase chain reaction (PCR). Concomitant infection with hepatitis C virus (HCV) was analyzed by reverse transcriptase (RT)-PCR, HDV replication by dot-blot, and human immunodeficiency virus (HIV) infection by enzyme-linked immunosorbent assay. The subjects were divided into three groups according to HBV-DNA status: group I: 14 patients HBV-DNA dot-blot positive; group II: 29 patients HBV-DNA positive only by PCR, and group III: 6 patients HBV-DNA negative by dot-blot and PCR. We have found HBV-DNA by dot-blot in 28.5% of patients, and by PCR in 87.7%. Also 22 patients were anti-HCV positive (86.3% had HCV-RNA by RT-PCR). The first group (HBV-DNA dot-blot positive) had significantly higher serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) than those in the second and third groups. Likewise, serum ALT and AST were significantly higher in the second group (HBV-DNA positive by PCR) than in those of the third group. Histological inflammatory activity was significantly higher in the group of patients with HBV-DNA detectable by dot-blot. The prevalence of serum HDV-RNA and IgM anti-HDV were similar in the three groups. These results were similar in the anti-HCV-positive and -negative patients. In conclusion, these data suggest that: (1) persistence of HBV replication is a major determinant of severe liver damage in chronic delta hepatitis, and (2) HCV and HIV infections do not influence the natural history of CDH.
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PMID:Correlation between hepatitis B viremia and the clinical and histological activity of chronic delta hepatitis. 799 89

The infectivity titer of a standard stock of the SAR-55 strain of hepatitis E virus (HEV) was determined in cynomolgus macaques (Macaca fascicularis) and the effect of dose on the course of the infection was examined by weekly monitoring of alanine aminotransferase (ALT) and anti-HEV levels. Antibody to HEV (anti-HEV) was measured with ELISAs based on ORF-2 recombinant antigens consisting of either a 55 kDa region expressed in insect cells or shorter regions expressed as fusion proteins in bacteria. The ELISA based on the 55 kDa antigen was generally more sensitive. The infectivity titer of SAR-55 was 10(6) cynomolgus 50% infectious doses per gram of feces. The infectivity titer corresponded to the HEV genome titer of the inoculum as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Anti-HEV IgM was detected in only a portion of the animals that had an anti-HEV IgG response. Biochemical evidence of hepatitis was most prominent in animals that were inoculated with the higher concentrations of virus and the incubation period to seroconversion was prolonged in animals that received the lower doses.
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PMID:Infectivity titration of a prototype strain of hepatitis E virus in cynomolgus monkeys. 808 60

The hepatitis C virus (HCV) genome was sought in the saliva of 76 chronic HCV carriers (mean age nearly 60 years) in a rural Japanese town, who had high serum titers of c-100 and anti-core second generation antibodies. In 27 samples (27 cases, 36%), the HCV-RNA genome was detected by the reverse transcriptase - polymerase chain reaction with either of two sets of primers covering two regions of the HCV genome: the 5'noncoding region and the region encompassing the putative envelope (E1). Transaminase values at the time of sampling were higher in the patients with than in those without detectable HCV RNA in saliva (p = 0.04 for alanine aminotransferase, p = 0.04 for aspartate aminotransferase; Wilcoxon test). The prevalence of the positivity was higher by 5'noncoding primers (14/59 vs. 15/68). Our data show that the severity and duration of hepatic dysfunction influence the detectability of the HCV genome in the saliva. This has been a controversial point among investigators.
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PMID:Correlation of detectability of hepatitis C virus genome in saliva of elderly Japanese symptomatic HCV carriers with their hepatic function. 855 81

Response to interferon-alpha (IFN-alpha) treatment in hepatitis C is poorer when cirrhosis is present. In the third Australian multicentre hepatitis C trial, Aushep-3, we examined the efficacy and tolerability of an intensive 24-week course of interferon-alpha 2a in Child-Pugh grade A patients with chronic hepatitis C and cirrhosis. This was an open uncontrolled trial of 4.5 million units (MU) of IFN-alpha 2a daily for 24 weeks; follow-up was 48 weeks. Chronic hepatitis C and cirrhosis were confirmed histologically. HCV RNA was determined in serum by reverse transcriptase polymerase chain reaction (PCR), and viral genotyping was by line-probe assay. Treatment response was defined as a reduction of alanine aminotransferase (ALT) to less than 1.5 times the upper limit of normal (and by at least 50% of pretreatment values) at weeks 20 and 24. Sustained response was defined as normal serum ALT after treatment from trial week 28 until week 48. Among the 56 patients, a treatment response occurred in 18 (32% by intention-to-treat; 42% of those who completed treatment) and eight (14%) had a sustained response. At 24 weeks, HCV RNA was not detectable in 12 of 17 treatment responders, and remained negative at 48 weeks in six of eight sustained responders. Treatment response by genotype occurred in 75% of patients with HCV type 2, in 38% with HCV type 3a and in 12% with HCV genotype 1. Sustained response occurred in only one (4%) patient with HCV genotype 1 but in five (20%) with genotypes 2 or 3a. Among 13 patients withdrawn, nine were for adverse effects, most often haematological; 10 others underwent dose reduction for adverse effects. It is concluded that a sustained biochemical and viral response to treatment with IFN-alpha 2a can be obtained in some patients with hepatitis C and cirrhosis, particularly those with genotypes 2 or 3a. Therefore, patients with cirrhosis should be considered for interferon treatment on an individual basis. Genotyping may improve case selection, but vigilance is required for haematological complications.
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PMID:Efficacy and tolerance of a 6-month treatment course of daily interferon-alpha 2a for chronic hepatitis C with cirrhosis. The Australian Hepatitis C Study Group. 931 Sep 30

Hepatitis C virus (HCV) infection often persists in association with chronic hepatitis. Different factors have been proposed to determine the clinical outcome of HCV infection. The aim of this study was to examine three different factors of HCV infection among injecting drug users. Nineteen untreated HCV seroconverters were tested longitudinally for the presence of HCV RNA by reverse transcriptase (RT) PCR, and results were quantified by the branched-DNA (bDNA) assay. HCV genotypes were determined with the first sample taken after HCV seroconversion. To assess the natural course of infection, serum alanine aminotransferase (ALT) levels were measured at three stages in every individual. The concordance between bDNA and RT-PCR was 98.9%. Three distinct patterns were found, according to the HCV RNA load after seroconversion during a mean follow-up period of 5 years (range, 1 to 8 years). HCV genotype 1a was predominant (52.6%). There was a significant increase in serum ALT levels (mean 55.5 U/liter) in the early phase of HCV infection, compared with basal serum ALT levels before HCV seroconversion and at the end of the follow-up period. Three distinct HCV RNA load profiles were found, without apparent relationship to genotype and serum ALT levels in the first 5 years of HCV infection.
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PMID:Different hepatitis C virus (HCV) RNA load profiles following seroconversion among injecting drug users without correlation with HCV genotype and serum alanine aminotransferase levels. 954 1

The prevalence of hepatitis G virus (HGV) RNA was compared in a cohort of 89 thalassemic children (age range, 1-16 years) with a history of multiple blood transfusions, recruited from the hematology outpatient clinic at Thailand's Chulalongkorn Hospital and in specimens from 200 blood donors at the Red Cross in Bangkok. 29 specimens (32.6%) from thalassemic children, compared with 10 (5%) from blood donors, demonstrated detectable HGV RNA by reverse transcriptase analysis. 48% of the HGV-RNA-positive thalassemic children had elevated alanine aminotransferase levels, compared with 51.9% of the cohort without detectable HGV RNA; a finding that supports the assumption HGV infection does not cause detectable hepatitis. HGV RNA prevalence was 11.8% among children with 2-10 transfusions, 48.8% with 11-50 transfusions, 21.7% in those with 51-100 transfusions, and 16.7% among those with over 100 transfusions. This pattern suggests that at least some of the children recovered from HGV infection and may have developed immunity to reinfection. The clinical significance of HGV, as well as the apparent immunity acquired against reinfection, merit further investigation.
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PMID:High prevalence of hepatitis G virus infection in multiply transfused children with thalassaemia. 957 Feb 37

The development of policies to prevent nosocomial transmission of hepatitis C virus (HCV) infection in hemodialysis units is critically dependent on the understanding of the relationship between tests for anti-HCV, HCV RNA, and HCV genotype and the patients' clinical characteristics. We tested sera from all patients on the renal transplant waiting list at the New England Organ Bank between November 1986 and June 1990 for anti-HCV by a third-generation enzyme-linked immunosorbent assay (ELISA3) and a third-generation recombinant immunoblot assay (RIBA3). All ELISA3-positive sera were tested for HCV RNA by reverse transcriptase "nested" polymerase chain reaction, and the genotype was characterized by restriction fragment length polymorphism. Sera were available in 1,544 of 3,243 (48%) patients on the waiting list, of whom 287 (19%) tested positive for anti-HCV by ELISA3. Two hundred eighty-six randomly selected, anti-HCV-negative patients served as controls. Compared with anti-HCV-negative controls, anti-HCV-positive patients had a longer duration since initiation of renal replacement therapy, higher number of previous kidney transplants and blood transfusions, higher proportion of patients with anti-HBc, history of liver disease, history of non-A, non-B hepatitis, and elevated serum alanine aminotransferase, and lower serum albumin concentrations. Of the 287 anti-HCV-positive sera, 261 (91%) were reactive by RIBA3, 21 (7%) were indeterminate, and five (2%) were nonreactive. HCV RNA was detected in 224 of 275 (81%) ELISA3-positive patients, in whom additional sera were available. There were no significant differences in clinical or laboratory characteristics between ELISA3-positive patients with and without HCV RNA. Genotypes 1a, 1b, 2a, 2b, 3a, and 4 were present in 53%, 23%, 8%, 10%, 4%, and 2% of patients, respectively. Infection with one, two, or three different HCV genotypes was present in 92%, 7%, and 1%, respectively. There was no significant association between the type or number of HCV genotypes and RIBA3 reactivity. There were no major differences in clinical or laboratory characteristics between genotypes or between single and mixed infection. In summary, this study provides detailed information regarding the relationship between tests for anti-HCV, HCV RNA, and HCV genotypes and the clinical and laboratory characteristics of a large, well-characterized cohort of patients referred for renal transplant.
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PMID:Serologic and virologic profiles of hepatitis C infection in renal transplant candidates. New England Organ Bank Hepatitis C Study Group. 963 34


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