Paul Neuwald

Significance of serum hepatitis C virus RNA levels in chronic hepatitis C.

Hepatitis C virus (HCV) is the main cause of parenteral non-A, non-B hepatitis and serum can be tested for the virus itself by reverse-transcription polymerase chain amplification. What of the level of this viraemia? To find out if quantitative study of HCV RNA might be useful clinically we took advantage of participation in trials of interferon-alpha in patients with chronic HCV infection and applied a new assay, branched DNA (bDNA) signal amplification. Paired serum and liver biopsy specimens from 47 patients with confirmed chronic HCV infection and evidence of HCV RNA in their serum were studied. The quantitative bDNA assay (detection limit 350,000 equivalents/mL [eq/mL]) was positive in 34 sera (sensitivity 72%). Patients who acquired HCV infection by blood transfusion had a higher viraemia (median 2,701,000 eq/mL, n = 29) than health workers and intravenous drug users (635,000 eq/mL, n = 13; p < 0.01). Patients with a sustained complete response to interferon-alpha therapy had lower pre-treatment viraemia levels (median at bDNA cut-off, n = 11) than complete responders who relapsed after the drug was stopped (1,613,000 eq/mL, n = 15; p < 0.01) and non-responders (3,066,000 eq/mL, n = 20; p < 0.01). High viraemia levels were not related to the histological diagnosis but were associated with lobular inflammation, lymphoid aggregates, and bile-duct lesions. These findings indicate that mode of acquisition is an important determinant of HCV viraemia and that patients with low HCV viraemia levels are more likely to respond to interferon in a sustained fashion.

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Direct and quantitative detection of HIV-1 RNA in human plasma with a branched DNA signal amplification assay.

AimTo determine the relative effect of sample matrix on the quantitation of HIV RNA in plasma. MethodTwo HIV-positive specimens were diluted into five and 10 different HIV-negative plasma samples, respectively. Branched DNA signal amplification technology and reverse-transcriptase polymerase chain reaction were used to measure the viral load. ResultsIn one sample the viral load by polymerase chain reaction ranged from undetectable to 1.9 x 105 copies/ml, and the branched DNA results ranged from 2.6 x 104 to 4.2 x 104 HIV RNA equivalents/ml. In the other sample the corresponding figures were 6.3 x 104 to 5.5 x 105 copies/ml and 5.7 x 104 to 7.5 x 104 HIV RNA equivalents/ml. ConclusionIn contrast to reverse-transcriptase polymerase chain reaction the branched DNA signal amplification assay does not require a separate extraction step or enzymatic amplification of the target. Therefore this measurement is less affected by the sample matrix and the signal generated is directly proportional to the viral load.

Ribavirin or Alpha Interferon Treatment of Chronic Hepatitis C: Assessment of Antiviral Efficacy

The efficacy of interferon (IFN) in the treatment of chronic hepatitis C is limited, and there is therefore a need for other antiviral drugs. We studied 28 patients treated with oral ribavirin, 8 patients treated with IFN-α, and 7 untreated patients. Normalization or a 50% decline of serum ALT was observed in 19 (68%) of the 28 ribavirin-treated patients. There was no significant change in HCV-RNA in those in whom the levels of virus were measured. The gradual decline of ALT without rapid decrease in virus load differed from the effects seen in αIFN-responsive patients. The ALT decline during ribavirin treatment was observed even in the presence of cirrhosis and also in patients who had not responded to previous treatment with αIFN. These results suggest a difference in antiviral mechanisms between ribavirin and IFN.