An international collaborative study to establish the 1st international standard for HIV-1 RNA for use in nucleic acid-based techniques

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Abstract

Twenty-six laboratories from 10 different countries participated in a collaborative study to establish the 1st International Standard for HIV-1 RNA for use in nucleic acid-based techniques (NAT). Three candidate preparations were tested all based on genotype B viruses. The candidates were tested by each laboratory at a range of dilutions in four independent assays and the results collated and analysed statistically. All three candidates gave results that were tightly grouped, with little difference between the results from different laboratories or from the use of different assays. Studies of relative potency showed good agreement between laboratories. There were no significant differences between five commercial assay types, except that candidate XX showed a slightly lower potency compared to YY and ZZ with a single commercial assay. The reason for this was not established. Degradation studies showed that the freeze-dried preparations were stable at −20, 4 and 20°C for 26 weeks, the longest period studied, but that they became difficult to reconstitute after 3 weeks at 45°C and 9 weeks at 37°C. As a result of the study, the World Health Organisation (WHO) Expert Committee on Biological Standardisation (ECBS) established the preparation referred to as candidate YY (NIBSC Code No. 97/656) as the 1st International Standard for HIV-1 RNA for use with NAT with an assigned potency of 100 000 International Units per vial.

Introduction

Over the past decade, PCR and other nucleic acid-based techniques (NAT), have improved dramatically our ability to detect viruses such as the human immunodeficiency virus (HIV) and hepatitis C virus (HCV) at increasingly earlier stages of infection as well as facilitating an accurate measurement of virus load (Busch et al., 1995). Their application in the field of HIV patient management has provided clinicians with a powerful prognostic indicator. It has enabled clinicians to monitor the benefits of combined drug therapy such as HAART, with the result that viral load can be reduced to very low or even undetectable levels (Carpenter et al., 1996, Mellors et al., 1996, Saag et al., 1996). In the area of blood and blood product safety, the use of NAT can reduce the likelihood of donations given during the ‘window period’, that period between infection and demonstrable seroconversion, from being administered or used in the manufacture of blood products and thereby resulting in transmissions of blood-borne viruses. As a result of the transmission of HCV by intravenous immunoglobulin in 1994 (Schneider and Geha, 1994, James and Mosley, 1995) the US FDA introduced a requirement for the testing for HCV RNA in all immunoglobulin preparations that had not been inactivated virally (Scribner, 1994, Zoon, 1995). From mid-1999, the European Union has required that plasma pools used for manufacturing plasma derivatives must be tested and found non-reactive for HCV RNA (Mueller-Breitkreutz and Allain, 1999). Although the testing of plasma pools for HIV-1 RNA by NAT is not yet a mandatory requirement, with the improvement in assay sensitivity that has been achieved recently and the desire to reduce the risk of virus transmission by blood transfusion and blood products as much as is practically possible, a number of organisations have already implemented voluntary screening for HIV-1 by NAT and more are sure to follow.

The WHO International Working Group on Standardisation of Gene Amplification Techniques for the Virological Safety Testing of Blood and Blood Products (SoGAT) recommended that the establishment of a WHO International Standard for HIV-1 RNA based on a genotype B virus be given a high priority (Rogers et al., 1997). Such a standard would be given a defined unitage and could be used to calibrate the range of working reagents currently in use. This would facilitate inter-laboratory comparisons and help reduce variations between different laboratories and between different commercial and ‘in-house’ assays (Coste et al., 1996, Revets et al., 1996, Schuurman et al., 1996, Fransen et al., 1998, Bootman et al., 1999). It would also provide an independent quantitative standard that could be used to monitor the sensitivity of assays used in the manufacture of blood products.

In response to this need, we identified three candidate standards for evaluation in an international collaborative study, with the aim of establishing an International Standard for HIV-1 RNA.

Section snippets

Preparation of the candidate standards

Three candidate standards were evaluated in the collaborative study, two of which (XX and YY) were freeze-dried and the third (ZZ) was frozen at −70°C. Details of each preparation is given below:

  • 1.

    Candidate XX was based on a genotype B (env V3, gag) field isolate of HIV-1 provided by Dr P Simmonds, University of Edinburgh, UK. It was isolated post-mortem from a patient who had died from an AIDS-defining illness (patient 4 in Donaldson et al., 1994). The virus was supplied as a low-passage PBMC

Results

From a total of 26 laboratories which participated in the study, 27 data sets were reported, 20 (74%) of which were in a quantitative format and 7 (26%) qualitative. Of these, 18 and 3, respectively were from commercially available kits. The quantitative assays included the Abbott LCx HIV Quantitative RNA Assay (LCx), the Bayer (formerly Chiron) Quantiplex HIV RNA bDNA Assay version 3.0 (bDNA), the Organon Teknika NASBA NucliSens HIV-1 QT Assay (Nuclisens) and the Roche Amplicor HIV-1 Monitor

Discussion

The measurement of HIV-1 RNA in the plasma is being used extensively in HIV patient management as a prognostic marker and in monitoring the effects of antiviral chemotherapy (Carpenter et al., 1996, Mellors et al., 1996, Saag et al., 1996), and increasingly by manufacturers of blood products. A number of studies have performed comparative evaluations of a variety of clinical samples with different NAT assays and differences in sensitivity as well as discordant results have been identified (

Acknowledgements

We wish to thank the EU Programme EVA/MRC Centralised Facility for AIDS Reagents, NIBSC, UK, for distributing the panels and Janet Bootman for her help and support in setting up the study. We are especially grateful to all the participants in the collaborative study (see Appendix A) without whom it could not have been undertaken. This work was supported in part by a grant from the World Health Organisation.

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