AUTHOR REPLY
Reply to “Issues on Results of the External Quality Assessment for Proviral DNA Testing of HIV-1 Tropism in the Maraviroc Switch Collaborative Study” Elise Tu,a Luke C. Swenson,b Sally Land,d Sarah Pett,a,c Sean Emery,a Kat Marks,e Anthony D. Kelleher,a,c,e Steve Kaye,f Rolf Kaiser,g Eugene Schuelter,g Richard Harrigan,b on behalf of the MARCH Laboratory Group and the MARCH Study Group
W
e appreciate the comments by Berg et al. regarding our article. First, the aim of the quality assessment program reported in this study was not to standardize the methodology across the network of laboratories involved but to assess the outcome of laboratories testing clinical material using their laboratory’s in-house standard protocol for determination of HIV-1 tropism using proviral DNA. As MARCH is an international study, the different HIV subtype distribution necessitated having nonstandardized methodologies and, hence, use of different primers required to amplify different clades. We agree that samples had a high degree of variability, with some samples having a very wide range in their false-positive rates (FPRs), and these were solid findings. Our external quality assessment (EQA) samples were derived from real patient samples and deliberately selected to be similar to those recruited to MARCH, i.e., long-term aviremic patients. The use of only clones or other homogeneous samples would therefore not be a true representation of the genetic diversity of real patient samples and would have led to a misrepresentation of the performance of laboratories testing such samples for diagnostics. One could have separately evaluated the two different issues, diversity of samples and diversity of laboratory performance. However, we presented the data in one single examination to assess the overall impact of such material as an international quality assessment program. Furthermore, it was interesting that Berg et al. compared our study to that of Svicher et al. This study used samples from viremic patients (⬎10,000 copies/ml) and tested HIV RNA, which is more likely to deliver uniform results because the circulating virus is likely to reflect the fittest clone/quasispecies circulating in that patient at that time. The DNA in long-term aviremic patients is likely to be far more heterogeneous, reflecting multiple quasispecies of archived virus
December 2013 Volume 51 Number 12
of varying fitness. We felt very strongly that a clonal panel would not be helpful for the purposes of our program. The observed variability validates the importance of performing this EQA, and these data will be critical for interpreting the results of the MARCH study itself. We acknowledge that triplicate testing is costly and not feasible in all laboratory settings. Nevertheless, we felt triplicate testing was essential, considering both the inherent variability of proviral DNA and the lack of information from rigorously conducted randomized clinical trials, to the clinical relevance of this test. Therefore, we consider it far more important to detect X4 viruses and be conservative in terms of patient safety. Overall, and for the reasons described both in the article and in this response, we do not believe that singlicate testing of a clonal standard would reliably ensure that a patient’s virus would likely respond to the protocol drug, maraviroc. In summary, we feel that Berg et al. have misinterpreted the intent of the EQA program established specifically for MARCH. Proviral DNA, obtained from real patient samples, was associated with a high degree of variability, and the most suitable approach for quality assessment for determination of viral tropism was developed for this study.
Address correspondence to Elise Tu, [email protected], or Luke C. Swenson, [email protected]. E.T. and L.C.S. are co-primary authors. This is a response to a letter by Berg et al. (doi:10.1128/JCM.02124-13). Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02207-13
Journal of Clinical Microbiology
p. 4287
jcm.asm.org
4287
Downloaded from http://jcm.asm.org/ on June 3, 2015 by FLORIDA INTL UNIV/GL810
The Kirby Institute, University of New South Wales, Sydney, NSW, Australiaa; BC Centre for Excellence in HIV/AIDS, Vancouver, British Columbia, Canadab; Immunology, HIV and Infectious Diseases Clinical Services Unit, St. Vincent’s Hospital, Sydney, Australiac; NRL, Fitzroy, Victoria, Australiad; Centre for Applied Medical Research (AMR), St. Vincent’s Hospital, Sydney, NSW, Australiae; Imperial College London, Norfolk Place, London, United Kingdomf; Institut für Virologie, der Universität zu Köln, Cologne, Germanyg
Reply to “Issues on Results of the External Quality Assessment for Proviral DNA Testing of HIV-1 Tropism in the Maraviroc Switch Collaborative Study” Elise Tu,a Luke C. Swenson,b Sally Land,d Sarah Pett,a,c Sean Emery,a Kat Marks,e Anthony D. Kelleher,a,c,e Steve Kaye,f Rolf Kaiser,g Eugene Schuelter,g Richard Harrigan,b on behalf of the MARCH Laboratory Group and the MARCH Study Group
W
e appreciate the comments by Berg et al. regarding our article. First, the aim of the quality assessment program reported in this study was not to standardize the methodology across the network of laboratories involved but to assess the outcome of laboratories testing clinical material using their laboratory’s in-house standard protocol for determination of HIV-1 tropism using proviral DNA. As MARCH is an international study, the different HIV subtype distribution necessitated having nonstandardized methodologies and, hence, use of different primers required to amplify different clades. We agree that samples had a high degree of variability, with some samples having a very wide range in their false-positive rates (FPRs), and these were solid findings. Our external quality assessment (EQA) samples were derived from real patient samples and deliberately selected to be similar to those recruited to MARCH, i.e., long-term aviremic patients. The use of only clones or other homogeneous samples would therefore not be a true representation of the genetic diversity of real patient samples and would have led to a misrepresentation of the performance of laboratories testing such samples for diagnostics. One could have separately evaluated the two different issues, diversity of samples and diversity of laboratory performance. However, we presented the data in one single examination to assess the overall impact of such material as an international quality assessment program. Furthermore, it was interesting that Berg et al. compared our study to that of Svicher et al. This study used samples from viremic patients (⬎10,000 copies/ml) and tested HIV RNA, which is more likely to deliver uniform results because the circulating virus is likely to reflect the fittest clone/quasispecies circulating in that patient at that time. The DNA in long-term aviremic patients is likely to be far more heterogeneous, reflecting multiple quasispecies of archived virus
December 2013 Volume 51 Number 12
of varying fitness. We felt very strongly that a clonal panel would not be helpful for the purposes of our program. The observed variability validates the importance of performing this EQA, and these data will be critical for interpreting the results of the MARCH study itself. We acknowledge that triplicate testing is costly and not feasible in all laboratory settings. Nevertheless, we felt triplicate testing was essential, considering both the inherent variability of proviral DNA and the lack of information from rigorously conducted randomized clinical trials, to the clinical relevance of this test. Therefore, we consider it far more important to detect X4 viruses and be conservative in terms of patient safety. Overall, and for the reasons described both in the article and in this response, we do not believe that singlicate testing of a clonal standard would reliably ensure that a patient’s virus would likely respond to the protocol drug, maraviroc. In summary, we feel that Berg et al. have misinterpreted the intent of the EQA program established specifically for MARCH. Proviral DNA, obtained from real patient samples, was associated with a high degree of variability, and the most suitable approach for quality assessment for determination of viral tropism was developed for this study.
Address correspondence to Elise Tu, [email protected], or Luke C. Swenson, [email protected]. E.T. and L.C.S. are co-primary authors. This is a response to a letter by Berg et al. (doi:10.1128/JCM.02124-13). Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02207-13
Journal of Clinical Microbiology
p. 4287
jcm.asm.org
4287
Downloaded from http://jcm.asm.org/ on June 3, 2015 by FLORIDA INTL UNIV/GL810
The Kirby Institute, University of New South Wales, Sydney, NSW, Australiaa; BC Centre for Excellence in HIV/AIDS, Vancouver, British Columbia, Canadab; Immunology, HIV and Infectious Diseases Clinical Services Unit, St. Vincent’s Hospital, Sydney, Australiac; NRL, Fitzroy, Victoria, Australiad; Centre for Applied Medical Research (AMR), St. Vincent’s Hospital, Sydney, NSW, Australiae; Imperial College London, Norfolk Place, London, United Kingdomf; Institut für Virologie, der Universität zu Köln, Cologne, Germanyg
