LETTER TO THE EDITOR
Issues on Results of the External Quality Assessment for Proviral DNA Testing of HIV-1 Tropism in the Maraviroc Switch Collaborative Study Jörg Berg,a Christian Paar,a Heribert Stoiber,b Thomas Klimkaitc
W
ith great interest we read the article by Tu et al. on the external quality assessment for the genotypic HIV-1 coreceptor tropism testing from proviral DNA (1). This large multicenter, multinational study conducted by opinion leaders in the field will undoubtedly have implications for the tropism testing for the routine clinical laboratory. We feel, however, that several points need clarification prior to a generalized translation into routine clinical tropism testing. 1. A number of protocols have been published for the genotypic HIV-1 coreceptor tropism testing from virus or provirus (2–5), but no optimal primers and protocol have been defined for a most reliable amplification of the highly variable V3 loop region. On this basis it is rather surprising that the study did not at first standardize amplification and sequencing methods for all laboratories. For example, in Table 1 of the article by Tu et al. we noticed that laboratory B reported false-positive rates (FPRs) regarded as representing X4 replicates (i.e., replicates with an FPR of less than 10% according to the geno2pheno bioinformatic algorithm) in 4 of the 5 samples, which were preclassified as R5. Interestingly and in contrast, the comparable but standardized study by Svicher et al. showed a high concordance of proviral genotypic tropism testing of 96.4% across all participating laboratories by using one standardized laboratory protocol (3). 2. In phase 2 of the trial it appears absolutely critical to have a standardized panel of samples in sufficient amounts used. This would have been clonal samples, e.g., DNA prepared from cultured cells infected with a spectrum of phenotypically defined HIV-1 strains. The authors did not specify in detail, how their standardization was obtained (Tu et al., page 2065, 2nd paragraph). In particular, we wonder about reasons for obtaining the excessively high FPRs in three X4 samples (no. 4, 8, and 19; Fig. 2 in that article), ranging from 0% to 90% within one sample. Similar results were presented in Fig. 3 of the article for 7 samples sequenced up to 32 times. Even the inexperienced reader will notice that ranges covering up to 90% of the scale will be completely useless for diagnostics. Additionally, the results of 10 of 19 samples show an FPR variability of ⬎40%. This is in contradiction to findings by others, who compared clinical samples by populationbased sequencing and ultradeep pyrosequencing (5): X4 sequences were not detectable by ultradeep sequencing, when an FPR ⬎ 60% was obtained by population-based sequencing. This implies that the samples used by Tu et al. were not suitable for the intentions of the study.
December 2013 Volume 51 Number 12
3. A further principal issue in genotypic testing of HIV-1 tropism is the choice of cutoff in geno2pheno to discriminate between R5 and X4. Use of clonal samples in phase 2 of the study could have tackled the problem at least to some meaningful extent. 4. Finally, in clinical genotypic testing of HIV-1 tropism, from proviral or plasma samples, the importance of testing in triplicates remains elusive and is not as clear-cut as the authors maintain (4, 6). As testing in triplicates is tedious and costly and does not readily fit into the work flow of a routine clinical laboratory, it is paramount to scientifically justify this. Regrettably, the setup of the study by Tu et al. has not addressed this issue. In conclusion, we feel that extrapolations from the clinical routine testing of HIV-1 coreceptor tropism from HIV-DNA cannot convincingly be made from the results of this study unless the issues raised have been addressed conclusively. ACKNOWLEDGMENT We declare that we have no conflicts of interest.
REFERENCES 1. Tu E, Swenson LC, Land S, Pett S, Emery S, Marks K, Kelleher AD, Kaye S, Kaiser R, Schuelter E, Harrigan R; MARCH Laboratory Group and the MARCH Study Group. 2013. Results of external quality assessment for proviral DNA testing of HIV tropism in the Maraviroc Switch Collaborative Study. J. Clin. Microbiol. 51:2063–2071. 2. McGovern RA, Thielen A, Portsmouth S, Mo T, Dong W, Woods CK, Zhong X, Brumme CJ, Chapman D, Lewis M, James I, Heera J, Valdez H, Harrigan PR. 2012. Population-based sequencing of the V3-loop can predict the virological response to Maraviroc in treatment-naïve patients of the MERIT Trial. J. Acquir. Immune Defic. Syndr. 61:279 –286. 3. Svicher V, Alteri C, Montano M, D’Arrigo R, Andreoni M, Angarano G, Antinori A, Antonelli G, Allice T, Bagnarelli P, Baldanti F, Bertoli A, Borderi M, Boeri E, Bon I, Bruzzone B, Callegaro AP, Capobianchi MR, Carosi G, Cauda R, Ceccherini-Silberstein F, Clementi M, Chirianni A, Colafigli M, D’Arminio Monforte A, De Luca A, Di Biagio A, Di Nicuolo G, Di Perri G, Di Pietro M, Di Santo F, Fabeni L, Fadda G, Galli M, Gennari W, Ghisetti V, Giacometti A, Gori C, Gori A, Gulminetti R, Leoncini F, Maffongelli G, Maggiolo F, Manca G, Gargiulo F, Martinelli C, Maserati R, Mazzotta F, Meini G, Micheli V, et al. 2012. Performance testing of genotypic tropism testing on proviral DNA in clinical practice: results from the DIVA study group. New Microbiol. 35:17–25.
Address correspondence to Jörg Berg, [email protected]. For the author reply, see doi:10.1128/JCM.02207-13. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02124-13
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Institute of Laboratory Medicine, General Hospital Linz, Linz, Austriaa; Section of Virology, Department of Hygiene, Microbiology and Social Medicine, Medical University of Innsbruck, Innsbruck, Austriab; Institute of Medical Microbiology, Department Biomedicine, Petersplatz Building, University of Basel, Basel, Switzerlandc
Letter to the Editor
4. Paar C, Geit M, Stekel H, Berg J. 2011. Genotypic prediction of human immunodeficiency virus type 1 tropism by use of plasma and peripheral blood mononuclear cells in the routine clinical laboratory. J. Clin. Microbiol. 49:2697–2699. 5. Svicher V, Cento V, Rozera G, Abbate I, Santoro MM, Armenia D, Fabeni L, Bruselles A, Latini A, Palamara G, Micheli V, Rizzardini G, Gori C, Forbici F, Ippolito G, Andreoni M, Antinori A, CeccheriniSilberstein F, Capobianchi MR, Perno CF. 2013. The genotypic false
positive rate determined by V3 population sequencing can predict the burden of HIV-1 CXCR4-using species detected by pyrosequencing. PLoS One 8:e53603. doi:10.1371/journal.pone.0053603. 6. Symons J, Vanderkerkhove L, Paredes R, Verhofstede C, Bellidido R, Demecheleer E, van Ham PM, van Lelyveld SF, Stam AJ, Van Versendaal D, Nijhuis M, Wensing AM. 2012. Impact on triplicate testing on genotypic HIV-1 tropism prediction in routine clinical practice. Clin. Micobiol. Infect. 18:606 – 612.
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Issues on Results of the External Quality Assessment for Proviral DNA Testing of HIV-1 Tropism in the Maraviroc Switch Collaborative Study Jörg Berg,a Christian Paar,a Heribert Stoiber,b Thomas Klimkaitc
W
ith great interest we read the article by Tu et al. on the external quality assessment for the genotypic HIV-1 coreceptor tropism testing from proviral DNA (1). This large multicenter, multinational study conducted by opinion leaders in the field will undoubtedly have implications for the tropism testing for the routine clinical laboratory. We feel, however, that several points need clarification prior to a generalized translation into routine clinical tropism testing. 1. A number of protocols have been published for the genotypic HIV-1 coreceptor tropism testing from virus or provirus (2–5), but no optimal primers and protocol have been defined for a most reliable amplification of the highly variable V3 loop region. On this basis it is rather surprising that the study did not at first standardize amplification and sequencing methods for all laboratories. For example, in Table 1 of the article by Tu et al. we noticed that laboratory B reported false-positive rates (FPRs) regarded as representing X4 replicates (i.e., replicates with an FPR of less than 10% according to the geno2pheno bioinformatic algorithm) in 4 of the 5 samples, which were preclassified as R5. Interestingly and in contrast, the comparable but standardized study by Svicher et al. showed a high concordance of proviral genotypic tropism testing of 96.4% across all participating laboratories by using one standardized laboratory protocol (3). 2. In phase 2 of the trial it appears absolutely critical to have a standardized panel of samples in sufficient amounts used. This would have been clonal samples, e.g., DNA prepared from cultured cells infected with a spectrum of phenotypically defined HIV-1 strains. The authors did not specify in detail, how their standardization was obtained (Tu et al., page 2065, 2nd paragraph). In particular, we wonder about reasons for obtaining the excessively high FPRs in three X4 samples (no. 4, 8, and 19; Fig. 2 in that article), ranging from 0% to 90% within one sample. Similar results were presented in Fig. 3 of the article for 7 samples sequenced up to 32 times. Even the inexperienced reader will notice that ranges covering up to 90% of the scale will be completely useless for diagnostics. Additionally, the results of 10 of 19 samples show an FPR variability of ⬎40%. This is in contradiction to findings by others, who compared clinical samples by populationbased sequencing and ultradeep pyrosequencing (5): X4 sequences were not detectable by ultradeep sequencing, when an FPR ⬎ 60% was obtained by population-based sequencing. This implies that the samples used by Tu et al. were not suitable for the intentions of the study.
December 2013 Volume 51 Number 12
3. A further principal issue in genotypic testing of HIV-1 tropism is the choice of cutoff in geno2pheno to discriminate between R5 and X4. Use of clonal samples in phase 2 of the study could have tackled the problem at least to some meaningful extent. 4. Finally, in clinical genotypic testing of HIV-1 tropism, from proviral or plasma samples, the importance of testing in triplicates remains elusive and is not as clear-cut as the authors maintain (4, 6). As testing in triplicates is tedious and costly and does not readily fit into the work flow of a routine clinical laboratory, it is paramount to scientifically justify this. Regrettably, the setup of the study by Tu et al. has not addressed this issue. In conclusion, we feel that extrapolations from the clinical routine testing of HIV-1 coreceptor tropism from HIV-DNA cannot convincingly be made from the results of this study unless the issues raised have been addressed conclusively. ACKNOWLEDGMENT We declare that we have no conflicts of interest.
REFERENCES 1. Tu E, Swenson LC, Land S, Pett S, Emery S, Marks K, Kelleher AD, Kaye S, Kaiser R, Schuelter E, Harrigan R; MARCH Laboratory Group and the MARCH Study Group. 2013. Results of external quality assessment for proviral DNA testing of HIV tropism in the Maraviroc Switch Collaborative Study. J. Clin. Microbiol. 51:2063–2071. 2. McGovern RA, Thielen A, Portsmouth S, Mo T, Dong W, Woods CK, Zhong X, Brumme CJ, Chapman D, Lewis M, James I, Heera J, Valdez H, Harrigan PR. 2012. Population-based sequencing of the V3-loop can predict the virological response to Maraviroc in treatment-naïve patients of the MERIT Trial. J. Acquir. Immune Defic. Syndr. 61:279 –286. 3. Svicher V, Alteri C, Montano M, D’Arrigo R, Andreoni M, Angarano G, Antinori A, Antonelli G, Allice T, Bagnarelli P, Baldanti F, Bertoli A, Borderi M, Boeri E, Bon I, Bruzzone B, Callegaro AP, Capobianchi MR, Carosi G, Cauda R, Ceccherini-Silberstein F, Clementi M, Chirianni A, Colafigli M, D’Arminio Monforte A, De Luca A, Di Biagio A, Di Nicuolo G, Di Perri G, Di Pietro M, Di Santo F, Fabeni L, Fadda G, Galli M, Gennari W, Ghisetti V, Giacometti A, Gori C, Gori A, Gulminetti R, Leoncini F, Maffongelli G, Maggiolo F, Manca G, Gargiulo F, Martinelli C, Maserati R, Mazzotta F, Meini G, Micheli V, et al. 2012. Performance testing of genotypic tropism testing on proviral DNA in clinical practice: results from the DIVA study group. New Microbiol. 35:17–25.
Address correspondence to Jörg Berg, [email protected]. For the author reply, see doi:10.1128/JCM.02207-13. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02124-13
Journal of Clinical Microbiology
p. 4285– 4286
jcm.asm.org
4285
Downloaded from http://jcm.asm.org/ on March 18, 2015 by University of Saskatchewan
Institute of Laboratory Medicine, General Hospital Linz, Linz, Austriaa; Section of Virology, Department of Hygiene, Microbiology and Social Medicine, Medical University of Innsbruck, Innsbruck, Austriab; Institute of Medical Microbiology, Department Biomedicine, Petersplatz Building, University of Basel, Basel, Switzerlandc
Letter to the Editor
4. Paar C, Geit M, Stekel H, Berg J. 2011. Genotypic prediction of human immunodeficiency virus type 1 tropism by use of plasma and peripheral blood mononuclear cells in the routine clinical laboratory. J. Clin. Microbiol. 49:2697–2699. 5. Svicher V, Cento V, Rozera G, Abbate I, Santoro MM, Armenia D, Fabeni L, Bruselles A, Latini A, Palamara G, Micheli V, Rizzardini G, Gori C, Forbici F, Ippolito G, Andreoni M, Antinori A, CeccheriniSilberstein F, Capobianchi MR, Perno CF. 2013. The genotypic false
positive rate determined by V3 population sequencing can predict the burden of HIV-1 CXCR4-using species detected by pyrosequencing. PLoS One 8:e53603. doi:10.1371/journal.pone.0053603. 6. Symons J, Vanderkerkhove L, Paredes R, Verhofstede C, Bellidido R, Demecheleer E, van Ham PM, van Lelyveld SF, Stam AJ, Van Versendaal D, Nijhuis M, Wensing AM. 2012. Impact on triplicate testing on genotypic HIV-1 tropism prediction in routine clinical practice. Clin. Micobiol. Infect. 18:606 – 612.
Downloaded from http://jcm.asm.org/ on March 18, 2015 by University of Saskatchewan
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