Infection with GB virus C and reduced mortality among HIV-infected patients.

BACKGROUND: The flavivirus GB virus C (GBV-C, also designated hepatitis G virus) was identified in a search for hepatitis viruses, but no disease is currently known to be associated with it. We investigated the relation between coinfection with GBV-C and the long-term outcome in patients infected with the human immunodeficiency virus (HIV). METHODS: A total of 197 HIV-positive patients were followed prospectively beginning in 1993 or 1994. Of these patients, 33 (16.8 percent) tested positive for GBV-C RNA, 112 (56.9 percent) had detectable antibodies against the GBV-C envelope protein E2, and 52 (26.4 percent) had no marker of GBV-C infection and were considered unexposed. We assessed the relation between GBV-C infection and the progression of HIV disease. We also tested 169 GBV-C-positive plasma samples with a quantitative branched-chain DNA (bDNA) assay in order to investigate possible correlations between GBV-C viral load and both the CD4+ cell count and the HIV load. RESULTS: Among the patients who tested positive for GBV-C RNA, survival was significantly longer, and there was a slower progression to the acquired immunodeficiency syndrome (AIDS) (P<0.001 for both comparisons). Survival after the development of AIDS was also better among the GBV-C-positive patients. The association of GBV-C viremia with reduced mortality remained significant in analyses stratified according to age and CD4+ cell count. In an analysis restricted to the years after highly active antiretroviral therapy became available, the presence of GBV-C RNA remained predictive of longer survival (P=0.02). The HIV load was lower in the GBV-C-positive patients than in the GBV-C-negative patients. The GBV-C load correlated inversely with the HIV load (r=-0.33, P<0.001) but did not correlate with the CD4+ cell count. CONCLUSIONS: Coinfection with GBV-C is associated with a reduced mortality rate in HIV-infected patients. GBV-C is not known to cause any disease, but it is possible that its presence leads to an inhibition of HIV replication. However, GBV-C infection could also be a marker for the presence of other factors that lead to a favorable HIV response.

Prevalence of primary resistance to zidovudine and lamivudine in drug-naive human immunodeficiency virus type-1 infected patients: high proportion of reverse transcriptase codon 215 mutant in circulating lymphocytes and free virus.

The presence of primary zidovudine (AZT)-resistance (mutation T215Y/F) or lamivudine (3TC)-resistance (mutation M184V) was evaluated in 90 drug-naive patients infected with human immunodeficiency virus type-1 (HIV-1) between 1987 and 1997. The proportion of mutant strains in proviral samples or plasma viral samples was determined using a differential hybridization assay. Mutation T215Y/F was found in five (5.6%) patients infected between 1994 and 1997, whereas none of these patients harbored the mutation M184V. The T215Y/F mutation was present in the virus and/or provirus and persisted for at least two years. In one patient, the mutant provirus was associated with only wild-type free virus. Four of these patients were followed, and two were treated subsequently to a regimen containing AZT but with low response. The persistence of primary resistance mutations might depend on the proportion of these mutations at the time of infection, although mutant provirus might not give rise to replicating variants.

Hepatitis G virus and human immunodeficiency virus coinfection: response to interferon-alpha therapy.

The prevalence and consequences of hepatitis G virus (HGV) infection were determined in 180 patients with human immunodeficiency virus (HIV) infection (predominantly male homosexuals) who participated in a trial that compared treatment with zidovudine versus interferon (IFN)-alpha versus the combination. HGV RNA levels were measured by branched DNA signal amplification assay. Initially, 66 (37%) had HGV RNA. Sexual transmission was the sole risk factor for infection in all but 4 subjects. Pretreatment clinical features were similar between HGV RNA-positive and -negative patients. After 6 months, only 5% treated with zidovudine became HGV RNA negative, compared with 95% who received IFN-alpha alone and 66% on combination therapy with low-dose IFN-alpha. After therapy, HGV RNA levels returned to baseline in most subjects. Thus, HGV infection is common among HIV-infected homosexual males but does not appear to influence clinical features in early HIV infection. HGV RNA levels are suppressed by IFN but not by zidovudine.

The clinical significance of simultaneous infection with hepatitis G virus in patients with chronic hepatitis C.

OBJECTIVES: Hepatitis G virus (HGV) is a recently discovered member of the flavivirus family that has been associated with acute and chronic hepatitis. HGV infection has been reported to coexist in 10-20% of patients with chronic hepatitis C. The significance of simultaneous infection with HGV and hepatitis C virus (HCV) remains to be clarified, as do the effects on HGV of therapeutic interventions such as interferon treatment or liver transplantation. THE AIMS OF OUR STUDY WERE: 1) to examine the frequency of HGV infection in the settings of liver transplantation and interferon therapy for hepatitis C; and 2) to compare HGV RNA levels before and after liver transplantation or interferon treatment. METHODS: Pre-treatment sera were available in 65 patients with chronic hepatitis C treated with interferon; pretransplant sera were available in 49 patients transplanted for end stage liver disease associated with chronic hepatitis C. Information collected included age, sex, risk factors for hepatitis, concurrent liver disease, patient and allograft survival, biochemical response to interferon, histological activity index, and degree of fibrosis/cirrhosis. HCV genotyping was performed by sequencing the NS-5 region. HGV quantitation was performed using a research-based branched DNA (bDNA) assay with a set of probes directed at the 5' untranslated region. RESULTS: HGV was detected in 10 of 49 patients (20%) before transplant and in 13 of 65 patients (20%) treated with interferon. There was a female predominance among HGV-positive compared with HGV-negative transplant patients (80% vs 20%; p < 0.01), but such a difference was not observed in the interferon-treated group. Hepatic iron concentration was lower in hepatic explants from patients who were HGV-positive than in those who were HGV-negative (318 +/- 145 microg/g dry weight vs 1497 +/- 2202 microg/g dry weight; p = 0.02). HCV exposure after 1980 was more common in the HGV-positive patients than in those who were HGV-negative for the entire study population (10 of 20 [50%] vs 16 of 66 [24%]; p = 0.03), as well as for the nontransplant subgroup (8 of 12 [67%] vs 12 of 39 [31%]; p = 0.03). HGV RNA levels declined at 1 yr after transplant in seven of eight patients. Among nine patients tested during or after interferon treatment, HGV RNA levels declined from pretreatment levels in all and disappeared in three. CONCLUSIONS: Among patients with chronic hepatitis C treated with either interferon or liver transplantation, the frequency of coinfection with HGV is about 20%. HGV may be a more recent virus in the US than HCV. Coinfection with HGV does not appear to affect the likelihood of response to interferon in patients with hepatitis C. Finally, HGV RNA levels appear to decline after both liver transplantation and interferon therapy, suggesting possible suppression by increased HCV replication in the former case, and a possible drug treatment effect in the latter.

Preparation of genotype-specific HCV RNA transcripts for assessing HCV detection and quantification assays.

Hepatitis C virus (HCV), the etiological agent responsible for the majority of cases of parenterally acquired liver disease, is found throughout the world. HCV is an enveloped virus with a small, single-stranded RNA genome. Because it uses an error-prone, RNA-dependent RNA polymerase, HCV has a high spontaneous mutation rate, and isolates of HCV display significant genetic heterogeneity. Isolates of HCV have been classified into at least six major genotypes and multiple subtypes based on sequencing and phylogenetic analysis (1). These genetic variants of HCV show a diverse geographical distribution. HCV types 1a, 1b, 2b, and 3a are the most prevalent in the US and western Europe (2,3), although all six major genotypes have been noted.

Hepatitis after liver transplantation: the role of the known and unknown viruses.

This study was designed to determine the cause of posttransplantation hepatitis in patients undergoing transplantation for liver disease of nonviral cause; the role of acquired hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis G virus (HGV) in posttransplantation hepatitis; and the course of posttransplantation hepatitis of unknown cause. Two hundred forty-three patients underwent transplantation for nonviral liver diseases (mean age, 48 years; 103 men, 140 women). Serological and virological assays for HBV and HCV were performed pretransplantation to exclude preexisting infection and posttransplantation to investigate the cause of posttransplantation hepatitis. Histology was graded on all available biopsy specimens; posttransplantation hepatitis was assessable in 150 patients. Posttransplantation hepatitis was present in 29% (44 of 150) of the patients after a median follow-up of 47 months (range, 1 to 101 months). Actuarial survival was significantly lower in patients with posttransplantation hepatitis compared with patients without (71% v 89% at 5-year follow-up; P = .03). HCV and HBV were identified posttransplantation in 14% and 9% of patients with hepatitis, respectively. After the exclusion of HCV and HBV infection, 22% (33 of 150) of the patients had posttransplantation hepatitis of unknown cause. HGV was present in 58% of these patients, but HGV was equally prevalent in patients without posttransplantation hepatitis. When patients with HBV and HCV were excluded, there was no difference in survival between patients with posttransplantation hepatitis compared with patients without (P = .08, log-rank test). Posttransplantation hepatitis was present in approximately 30% of the patients undergoing transplantation for nonviral diseases, with a median follow-up of 47 months. Known hepatitis viruses (HBV, HCV) were present in one fourth of the patients with posttransplantation hepatitis; 22% (33 of 150) of the patients had hepatitis of unknown cause, suggesting that other, as yet undiscovered, hepatitis viruses may exist.

Activity of a ritonavir plus saquinavir-containing regimen in patients with virologic evidence of indinavir or ritonavir failure.

OBJECTIVE: To evaluate the virologic activity of a ritonavir plus saquinavir-containing regimen in patients who have failed an indinavir or ritonavir-containing regimen. DESIGN: Patients were identified through a retrospective study evaluating the incidence of indinavir or ritonavir failure in our clinic. PATIENTS: Eighteen patients failing indinavir or ritonavir therapy and who switched to a ritonavir-saquinavir-containing regimen were evaluated. Indinavir or ritonavir failure was defined as a plasma viral load > 1500 copies/ml (branched DNA) after 16 weeks of continuous therapy. INTERVENTIONS: All patients switched to ritonavir (400 mg twice daily) plus saquinavir (400 mg twice daily) and received concurrent therapy with two nucleoside reverse transcriptase inhibitors (NRTI). Twelve of the 18 patients modified their NRTI regimen at the time ritonavir-saquinavir was initiated. OUTCOME MEASURES: Plasma viral load was monitored using a branched DNA assay. Genotypic analysis was performed using a point mutation differential hybridization technique, and was confirmed with direct sequencing. RESULTS: Fourteen out of 18 patients completed at least 24 weeks of therapy; the remaining four patients discontinued therapy after week 12 due to a lack of virologic response or intolerance. Plasma viral load decreased a median 1.4 log10 after 4 weeks of treatment with ritonavir-saquinavir. Only four patients had a greater than 0.5 log10 decrease in viral load after 24 weeks of therapy. In eight out of 10 patients evaluated, the V82A mutation was present at the time of the switch to ritonavir-saquinavir. Viral rebound on ritonavir-saquinavir was associated with the emergence of mutations at amino acids 46, 48, 54 and 90. CONCLUSION: The combination of ritonavir, saquinavir and two NRTI resulted in a moderate but transient suppression of viral replication in patients who have failed indinavir or ritonavir therapy. Failure of ritonavir-saquinavir may be associated with the emergence of mutations associated with resistance to ritonavir/saquinavir monotherapy, particularly the L90M mutation.

Quantitation of hepatitis G and C viruses in the liver: evidence that hepatitis G virus is not hepatotropic.

Hepatitis G virus (HGV) is prevalent in patients with chronic liver disease and has been previously detected in liver specimens. However, it is unknown whether the virus is replicating in the liver or is simply a contaminant from serum. We sought to determine whether HGV was hepatotropic and to determine whether coinfection with HGV and hepatitis C virus (HCV) influenced the level of either virus. Virus was quantitated using branched DNA (bDNA) assay for both HGV and HCV in the liver explants and pretransplant serum samples from 30 transplant recipients: Group I, HGV/HCV coinfection (n = 10); group II, HCV infection alone, (n = 8); group III, HGV alone (n = 12). In patients with coinfection HCV (RNA) titers in liver were consistently higher than those for HGV RNA (median 1.13 x 10(8) and 360,000 Eq/g respectively, P < .01). The ratio of liver/serum viral RNA was significantly higher for HCV than for HGV (median 129 and 0.3 respectively, P < .01). Levels of HCV RNA were similar in patients with HCV infection alone versus those with HGV/HCV coinfection (median; liver = 1.15 x 10(7) vs. 1.13 x 10(8) Eq/g, serum = 500,000 vs. 200,000 Eq/mL) and levels of HGV RNA in liver and serum were similar in patients with HGV infection alone compared to those with HGV/HCV coinfection (median; liver = 1.2 x 10(6) vs. 4.0 x 10(5) Eq/g, serum = 4.5 x 106 vs. 2.6 x 10(6) Eq/mL). Levels of either virus appeared unaffected by the presence of an additional virus. The high ratio of HCV RNA levels in liver compared to serum is consistent with its known hepatotropism, but this pattern was not observed for HGV. The median liver/serum ratio of HGV RNA was less than unity, a finding consistent with serum contamination of liver tissue. Thus we conclude that the liver is not the main site of HGV replication.

Hepatitis G virus infection in patients transplanted for cryptogenic cirrhosis: red flag or red herring?

BACKGROUND: The significance of hepatitis G (HGV) infection in liver transplant recipients is not known. We set out to determine the pre-orthotopic liver transplantation (OLT) prevalence, the pre- and postoperative viral titers of HGV, and the allograft histology in patients infected with HGV who underwent OLT for cryptogenic cirrhosis. METHODS: HGV RNA was measured using a research-based branched DNA assay. The assay used a target-specific probe set that was based on the 5'-untranslated region of the HGV genome. Allograft histology was assessed with protocol liver biopsies in all patients who survived longer than 6 months. RESULTS: The preoperative prevalence of HGV infection in recipients transplanted for cryptogenic cirrhosis was 26%. Thirty-seven percent (12 of 33) of recipients who had serum available in the first postoperative month had HGV infection. Mean HGV RNA levels were 9.8 (+/-4.2) (viral molecular equivalents/ml x 10[6]) before OLT and 37.5 (+/-10.7) at 1 year after OLT. In 4 of the 11 cryptogenic recipients in whom HGV RNA was detectable in the first postoperative month, HGV RNA fell to undetectable levels at the most recent follow-up (mean 70 months). Of the five cryptogenic recipients who continue to have measurable HGV RNA, three have unexplained hepatitis histologically. CONCLUSIONS: These findings suggest the following: 1) The prevalence of HGV infection in patients undergoing OLT for cryptogenic cirrhosis is about 25%. 2) In recipients persistently infected with HGV, mean HGV RNA titers increase after OLT. 3) HGV RNA becomes undetectable in about one third of recipients who had detectable HGV RNA in the first month after OLT. 4) Hepatitis of uncertain etiology occurs in 60% (3 of 5) of persistently HGV-infected cryptogenic recipients.

Hepatitis C and G co-infection: response to interferon therapy and quantitative changes in serum HGV-RNA.

Hepatitis G virus (HGV), a positive sense RNA virus, is distantly related to hepatitis C virus (HCV): its genetic organization and identity are consistent with the Flaviviridae family. Coinfection with HGV occurs in 10% to 20% of HCV-infected subjects. These similarities raise two theoretical questions. First, could HGV coinfection play any role in the response of HCV to antiviral therapy and second, would this coinfected population have changes in serum HGV-RNA induced by interferon. To address these questions, 98 patients with documented chronic HCV underwent interferon therapy (3 million units three times a week) for 6 months. Response to therapy was categorized using standard biochemical criteria. Changes in HGV-RNA levels were evaluated before, during, and after interferon therapy by a quantitative branched DNA amplification research-based assay. Eleven of 98 (11%) patients with HCV infection had detectable serum HGV-RNA. There was no difference between the groups (HGV+ vs. HGV-) when baseline alanine aminotransferase (ALT) values, HCV-RNA levels, HCV genotype, histological severity, or other demographic features were analyzed. Interferon response was similar in both groups and HGV was not associated with outcome following therapy. Antiviral therapy appeared to induce a reduction in HGV-RNA load in five of nine patients coinfected with HCV serially tested. In two patients, the fall in serum HGV-RNA correlated with biochemical response, independent of changes in HCV-RNA. These observations indicate that a larger study of an HGV population is required to more clearly define the relationship between HCV and HGV coinfection and their response to antiviral therapy.

Effect of interferon-alpha and ribavirin therapy on serum GB virus C/hepatitis G virus (GBV-C/HGV) RNA levels in patients chronically infected with hepatitis C virus and GBV-C/HGV.

GB virus C/hepatitis G virus (GBV-C/HGV) is a newly described virus associated with hepatitis in humans, and GBV-C/HGV coinfection is common in patients chronically infected with hepatitis C virus (HCV). To determine the clinical impact of GBV-C/HGV infection in such patients and the effect of interferon-alpha and ribavirin therapy on serum GBV-C/HGV RNA levels, GBV-C/HGV RNA was detected and quantitated in serum samples from 62 chronically infected HCV patients by a combination of a qualitative nested reverse transcription-polymerase chain reaction and a newly developed quantitative branched DNA assay: 10 patients were positive for serum GBV-C/HGV RNA. There were no differences in the clinical, biochemical, and histologic features of the patients with GBV-C/HGV-HCV coinfection compared with those with HCV infection alone. Interferon-alpha treatment caused a marked but usually transient reduction in serum GBV-C/HGV RNA, and ribavirin had, at most, a modest antiviral effect.

A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml.

The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification. Addition of a 14 site preamplifier was found to increase the signal/noise ratio 8-fold. A set of 74 oligonucleotide probes was designed to the consensus HIV POL sequence. The detection limit of this new HIV branched DNA amplifier assay was approximately 50 molecules/ml. The assay was used to measure viral load in 87 plasma samples of HIV- infected patients on triple drug therapy whose RNA titers were <500 molecules/ml. In all 11 patients viral load eventually declined to below the detection limit with the new assay.

Influence of hepatitis G virus infection on the severity of liver disease and response to interferon-alpha in patients with chronic hepatitis C.

BACKGROUND: Dual infection with hepatitis G virus (HGV) and hepatitis C virus (HCV) is common. The effect of HGV infection on chronic hepatitis C is not well known. OBJECTIVE: To assess the prevalence of HGV infection; the effect of HGV infection on the clinical, virologic and histologic features of patients with chronic hepatitis C treated with interferon-alpha; and the influence of HGV infection on response to interferon-alpha therapy. DESIGN: Retrospective study. SETTING: A university hospital in France. PATIENTS: 228 patients with chronic hepatitis C treated with interferon-alpha (3 million U or 5 million U subcutaneously 3 times a week for 3, 6, or 12 months). MEASUREMENTS: Before initiation of treatment, serum HGV RNA and serum HCV RNA were detected with branched-DNA assays and HCV genotype was determined with a line probe assay. Serum HGV RNA and serum HCV RNA were detected by polymerase chain reaction at the end of treatment and 6 months after treatment. RESULTS: Infection with HGV was detected in 21% of patients and 32% of intravenous drug users. The median serum HGV RNA level was 33 x 10(6) genome equivalents/mL. Infection with HGV was more frequently found in men with a history of intravenous drug use and was associated with HCV genotype 3a (P = 0.02) independent of the source of infection. Serum HCV RNA levels, liver histologic findings, and response to interferon-alpha therapy did not differ between patients with and those without HGV infection. The loss of serum HGV RNA was not correlated with the biochemical response contrarily to the loss of serum HCV RNA. CONCLUSIONS: Infection with HGV occurred frequently in this sample of patients with chronic hepatitis C, especially in patients infected with HCV genotype 3a. The level of HGV viremia was high relative to the level of HCV viremia. Infection with HGV did not influence the severity of liver disease or response to interferon-alpha therapy.

Detection of GB virus-C/hepatitis G virus RNA in serum by reverse transcription polymerase chain reaction.

Three PCR methods based on the GB virus-C/hepatitis G virus (GBV-C/HGV) 5'UTR and NS3 genomic region were used for the detection of GBV-C/HGV RNA in serum of 62 patients with chronic hepatitis C virus (HCV) infection. Ten of 62 (16%) patients were found to have GBV-C/HGV RNA, which was confirmed by sequence analysis of the 5'UTR PCR amplicon. All methods appear to be specific, but methods based on the 5'UTR appear to be more sensitive.

Hepatitis G virus in patients with cryptogenic liver disease undergoing liver transplantation.

To examine the prevalence of hepatitis G virus (HGV) in end-stage liver disease of unknown cause and the role of HGV infection in posttransplantation hepatitis, we studied 46 patients undergoing liver transplantation (mean age, 50 years; M:F, 18:28) with cryptogenic cirrhosis. HGV RNA was detected by polymerase chain reaction (PCR) and was quantified by a branched DNA (bDNA) assay. The prevalence of HGV RNA was determined in samples collected before and after liver transplantation and was found to be 22% and 67%, respectively. We evaluated the prevalence of posttransplantation hepatitis in 25 patients, 16 of whom were HGV-positive and 9 were HGV-negative. The proportion of patients with hepatitis was not significantly different in the two groups (38% in HGV-positive and 22% in HGV-negative patients). The median histological scores were significantly higher in liver biopsies from patients with HGV infection than in those without HGV infection (2 [range, 0-14] and 1 [range, 0-3]; P = .01), but the histological scores were low overall. The duration of follow-up was similar in the two groups. HGV RNA levels were not correlated with the severity of liver disease based on histological score (r = -.08). Graft survival and patient survival were not significantly different. We concluded that liver disease was frequent (32%) after transplantation in patients with a pretransplantation diagnosis of cryptogenic cirrhosis, although the disease was generally mild. Although HGV RNA was demonstrable in the majority (67%) of patients after transplantation, there was no relationship between the presence of HGV RNA and the presence of posttransplantation liver disease. The finding of posttransplantation hepatitis in the absence of known viruses (A-G), suggests that other, as-yet-unidentified viruses may be important.

An enhanced-sensitivity branched-DNA assay for quantification of human immunodeficiency virus type 1 RNA in plasma.

The quantification of human immunodeficiency virus type 1 (HIV-1) RNA has facilitated clinical research and expedited the development of antiretroviral drugs. The branched-DNA (bDNA) assay provides a reliable method for the quantification of HIV-1 RNA in human plasma and is considered one of the most reproducible assays ready for use in clinical trials. A series of oligonucleotide probe design and solution changes have been developed to enhance the sensitivity of the bDNA assay while maintaining its performance characteristics. Among the changes incorporated into the enhanced-sensitivity bDNA (ES bDNA) assay to reduce the background level and enhance the signal are the use of shorter overhang sequences of target probes for capture, the cruciform design of target probes for amplification, and the addition of preamplifier molecules. The ES bDNA assay is at least 20-fold more sensitive than the first-generation bDNA assay, yet it maintains a high level of accuracy, linearity, and reproducibility. Further, quantification values obtained with the ES bDNA assay and the first-generation bDNA assay are highly correlated, thus allowing for meaningful comparisons of HIV-1 RNA levels in specimens tested with either assay. The ES bDNA assay may be useful in determining the prognostic value of HIV-1 RNA levels of below 10,000 copies per ml and in assessing the clinical benefit of antiretroviral therapy-induced decreases in plasma HIV-1 RNA sustained at levels of below 10,000 copies per ml.

Detection of Trypanosoma brucei spp. in human blood by a nonradioactive branched DNA-based technique.

We have developed a nonradioactive branched DNA (bDNA)-based assay for the diagnosis of the African trypanosomiases in simple buffy coat preparations of human blood. Two repetitive DNA sequences specific to the Trypanosoma brucei complex were chosen as targets of the bDNA assay, a technique which amplifies the signal from a target molecule rather than the target itself. Comparable sensitivities were observed with cloned target sequences, purified T. brucei DNA, procyclic trypanosomes, and bloodstream trypomastigotes. The results of bDNA analysis of human blood samples from Côte d'Ivoire (n = 50) showed excellent agreement with those of buffy coat microscopy. The bDNA technology offers certain advantages over alternative molecular biological techniques, including the simplicity of sample preparation and of the procedure itself, the stability of the reagents, the ability to process large numbers of samples simultaneously, and freedom from crosscontamination artifacts. We have successfully applied the bDNA technique to the detection of T. brucei in clinical samples from regions where T. brucei infection is endemic; to our knowledge, this is the first report of the molecular detection of T. brucei in human blood.

Accurate quantification of hepatitis C virus (HCV) RNA from all HCV genotypes by using branched-DNA technology.

In studies monitoring disease progression and therapeutic response, it is essential that the method used for hepatitis C virus (HCV) quantification not be influenced by genotypic variability. The branched DNA assay provides a reliable method for the quantification of HCV RNA. A modified set of oligonucleotide probes for the branched DNA assay was developed to enhance the efficiency of binding to genotypic variants of HCV. The improved branched DNA assay (HCV RNA 2.0) yielded highly reproducible quantification of hepatitis C virus RNA and displayed a nearly 600-fold dynamic range in quantification up to 120 Meq of HCV RNA per ml. The quantification limit was set at 0.2 Meg of HCV RNA per ml to ensure a specificity of > or = 95%. With this lowered quantification limit and the enhanced hybridization of the probes, the HCV RNA 2.0 assay exhibited a high level of sensitivity (96%) and was virtually unaffected by the genotypic variability of HCV. The HCV RNA 2.0 assay may be a useful tool for following HCV RNA levels throughout the course of disease, selecting patients for therapy, and evaluating therapeutic response.

Levels of hepatitis C virus in blood donors infected with different viral genotypes. International HCV Collaborative Study Group.

The level of hepatitis C virus (HCV) in the serum of 337 blood donors infected with different viral genotypes was investigated by branched DNA assay. Viral genotype was deduced by restriction analysis of the virus 5'-noncoding region. Samples included genotypes 1a, 1b, 2a, 2b, 3,4,5, and 6. Multivariate analysis revealed that the ranges of HCV levels were similar for all viral genotypes and subtypes (P=.18), with the possible exception of genotype 4. Virus levels were significantly lower in female than in male subjects (P<.001) but did not correlate with donor age (P=.06) or genotype or with donor age, sex, or country. These results indicate a similar replicative capacity in vivo for different HCV genotypes and clarify the influence of host and virus factors on disease severity and responsiveness to interferon treatment.

Preparation and characterization of RNA standards for use in quantitative branched DNA hybridization assays.

RNA standards were developed for use in quantitative hybridization assays such as the Quantiplex HCV RNA Assay and Quantiplex HIV RNA Assay, which are based on branched DNA signal amplification. In vitro transcripts ranging in size from 0.5 to 9.4 kb were prepared and purified by phenol extraction following gel electrophoresis or column chromatography. Aliquots of the transcripts were digested to nucleosides and phosphate and then quantified by phosphate analysis against the U.S. National Institute of Standards and Technology phosphate standard. The quantitation was checked by OD260 and by either hyperchromicity or isotopic tracer analysis. The quantitation of each lot of RNA agreed within 20% by the three methods. The reproducibility of the methods was tested by preparing a total of 13 lots of standard RNAs. The average percentage full-length RNA of the 13 lots was 82%, with a range of 59 to 97%. The standard RNAs were used to test the ability of the branched DNA hybridization assay to quantify all target RNAs accurately regardless of size or slight variations in sequence. Standard Hepatitis C virus (HCV) RNAs of 1.3, 2.2, and 3.2 kb showed that size has no detectable effect on quantitation in the branched DNA hybridization assay. Three different lots of standard 3.2-kb HCV RNA were serially diluted and quantified over a thousand-fold range in the branched DNA hybridization assay. The average signal per attomole of target varied by less than 20% among the 3 lots. Standard HCV RNA transcripts were also prepared from clones of HCV subtypes 1b and 3a to study the effects of target sequence diversity and probe design on quantitation by hybridization.(ABSTRACT TRUNCATED AT 250 WORDS)