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A quantitative nucleic acid sequence-based amplification assay (NASBA-QT) for detection of hepatitis C virus RNA (HCV-RNA) was evaluated and compared with the HCV branched-DNA (bDNA) assay (Chiron Corporation) and the HCV MONITOR assay (Roche Diagnostic Systems). For this evaluation five panels were designed: (1) serial dilutions of genotype 1b in-vitro HCV-RNA; (2) standards of in-vitro HCV-RNA genotypes 1a, 1b, 2, 3, 4, and 5; (3) a proficiency panel consisting of 12 HCV-RNA positive plasma samples of different genotypes and HCV-RNA concentrations and a genotype 1a and 1b 3-fold dilution series; (4) a panel of 67 HCV-RNA positive plasma samples obtained from patients with HCV infection and (5) an HCV-RNA positive control sample, diluted 50-fold in 25 different HCV-RNA negative plasma samples. The quantitative detection limit was found to be 10(3) copies per 100 microl and the qualitative detection limit 10(2.3) per 100 microl. The amplification efficiency was independent of the plasma matrix, but dependent on the HCV genotype. The HCV NASBA-QT assay was more than 10 times as sensitive as the bDNA assay while the quantitative results of both assays were highly concordant. The HCV NASBA-QT assay was comparable in sensitivity with the HCV MONITOR assay, but the HCV MONITOR assay yielded consistently lower values. It is concluded that the HCV NASBA-QT assay is a reliable assay for quantitative HCV-RNA detection in various settings.

Using an experimental assay for isothermal amplification of HIV RNA in plasma (NASBA, Organon Teknika, Boxtel, The Netherlands), 70 samples from 59 HIV-1-infected persons and 29 samples from 28 uninfected volunteer blood donors were tested for the presence of HIV-1 RNA. The HIV-1-positive plasma samples were drawn from patients at various stages of infection: two samples from persons with signs of acute HIV infection (CDC stage I); 29 samples from 25 symptom-free persons (CDC stage II) and 39 samples from 32 persons with AIDS-related symptoms (CDC stage IV). All samples from HIV-1-infected persons had HIV-1 RNA, irrespective of the stage of infection (100% sensitivity). Testing the donor samples in duplicate, initially 54 58 samples (93%) tested negative for HIV-1 RNA. Repeated testing of the 4 samples with inconsistent test results showed all samples to be negative (specificity 100%). Detection of HIV-1 RNA in plasma by isothermal amplification (NASBA) promises to be a valuable alternative to the detection of HIV-1 RNA or DNA by the polymerase chain reaction.

The influence of different storage temperatures and anticoagulation conditions on the HIV-1 RNA load as measured by NASBA-QT was examined. Blood specimens from 14 HIV-1 infected individuals were processed within 2 h after collection. The HIV-1 RNA load remained stable for at least 6 months when samples were frozen directly at -70 degrees C in lysis buffer. This lysis buffer fully inactivated the virus. When whole EDTA blood was stored, the HIV-1 RNA load was stable for 72 h at 25 degrees C, but it declined within 24 h at 4 degrees C. The HIV-1 RNA load in whole heparinized blood declined significantly after 24 h at both 4 and 25 degrees C. It was slightly lower (average of 0.18 log ml-1) than in whole EDTA blood. At 4 degrees C, the HIV-RNA load in serum and EDTA-plasma stored with lysis buffer did not decline up to 14 days. At + 30 degrees C, however, the load declined significantly after 2 days. Of clinical significance, the mean load in EDTA plasma was 0.5 log ml-1 higher than in serum. This difference was patient dependent (range 0.1-0.7 log ml-1). We thus recommend, for quantifying HIV-1 RNA by NASBA, to use preferably EDTA blood which is kept at room temperature until plasma separation. When using heparinized blood, the plasma should be stored frozen within 8 h.

Eighty-six laboratories participated in a collaborative study and tested the second EUROHEP HCV-RNA reference panel. The coded panel comprised 4 HCV-RNA positive plasma samples (one weak positive), 6 HCV-RNA negative plasma samples and two dilution series of HCV-RNA genotype 1 and 3 plasma standards. The 86 laboratories submitted 136 coded data forms for evaluation. Of these data sets 99 were tested using a PCR assay developed in-house, 28 using a commercially available HCV-PCR test (AMPLICOR, Roche Diagnostic Systems) and 9 using other amplification methods. Twenty-two data forms (16%) had faultless results, 39 (29%) missed the weak positive sample only and 75 data sets (55%) had false positive and/or false negative results. Participants using the commercial HCV-PCR test tended to reach a sufficient quality score more often than investigators using assays developed in-house (64% versus 45%, P = 0.11). The UNG system in the commercial HCV-PCR test did not prevent five laboratories generating false-positive results in the 6 HCV-RNA negative samples. Among the laboratories with satisfactory results, up to 10000-fold differences in sensitivity were observed in the dilution series. The 50% and 90% laboratories detection endpoints in the dilution series of the HCV genotype 1 plasma standard were approximately 600 genome equivalents per ml (geq/ml) and 7750 geq/ml according to a standard applied in a signal amplification assay (bDNA, Chiron). Our results suggest that the detection efficiency for genotype 3 by commercial HCV-RNA assays is lower than by the in-house assays. Internationally characterized HCV-RNA plasma standards should be made available for validation and standardization of HCV-RNA assays for HCV diagnosis and virological safety testing of blood products.

The extent of hepatitis C virus (HCV) RNA loss during increasing periods of fixation of liver tissue in formalin was examined. For this purpose human liver tissue, known to be HCV RNA positive and stored at − 70°C until use, was cut into small slices (n = 9), which were fixed in phosphate-buffered formalin for increasing periods of time before embedding in paraffin. Nucleic acids were extracted from each slice of formalin-fixed, paraffin-embedded liver tissue and HCV RNA loss during fixation was semi-quantified by testing 10-fold dilutions of each extract in an HCV cDNA-PCR assay. The endpoint dilution for HCV RNA detection by cDNA-PCR in liver slices fixed in buffered formalin for 8–24 h was comparable to the endpoint dilutions found for ‘fresh’, non-fixed liver slices. After fixation for 2–3 days the endpoint dilution for HCV RNA detection was 102 to 103-fold less. After 2–4 weeks of formalin-fixation, HCV RNA was detectable from undiluted nucleic acid extracts only. It is concluded that formalin-fixed, paraffin-embedded liver biopsies can be used for HCV RNA detection by cDNA-PCR, on condition that the liver tissue has been embedded in paraffin within 24 h of formalin-fixation.