Comparison between Sequence Analysis and a Line Probe Assay for Testing Genotypic Resistance of Human Immunodeficiency Virus Type 1 to Antir
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微生物临床杂志 2005年第8期
Department of Microbiology and Parasitology
Department of Internal Medicine, La Paz University Hospital, Paseo de la Castellana 261, 28046 Madrid, Spain
ABSTRACT
The purpose of this study was to compare a line probe assay (LiPA) with sequence analysis for the detection of mutations conferring resistance to nucleoside and non-nucleoside inhibitors in human immunodeficiency reverse transcriptase and protease inhibitors. The limitations for interpreting LiPA make it unacceptable for routine clinical practice.
TEXT
The accumulation of retrospective and prospective data has led some expert panels to recommend the use of resistance testing in the management of human immunodeficiency virus type 1 (HIV-1)-infected patients as a part of routine clinical care (4, 5). Two different methods of genotypic resistance testing are used for assessing HIV-1 drug resistance: a line probe assay (LiPA), based on the principle of reverse hybridization (12), and sequence analysis by automated cycle sequencing. The LiPA (version 2.0) allows the study of wild-type and mutations M41L, T/A/N69D, K70R, L74V, V75T, M184V/I, T215D/S/A/Y/F, Q151M, K/R103N, V/I106A, and Y181C/I (the asterisk indicates a probe that was not included in version 1.0) of the reverse transcriptase (RT) gene and D30N, M46I, G48V, I50V, I/A54V, V/I82F/T/A, I84V, and L90M of the protease (PR) gene. Plasma samples from 54 previously treated (n = 49) or untreated (n = 5) HIV-1-infected patients were analyzed for drug genotypic resistance by both methods. Patients were monitored at the La Paz University Hospital, Madrid, Spain. Baseline characteristics are shown in Table 1. Samples were collected between April 2002 and October 2003. For the 54 patients, the RT gene was analyzed in 54 plasma samples, and the PR gene was analyzed in 43 plasma samples using sequencing analysis and the LiPA HIV-1 assays. The conventional EDTA tubes were used for blood collection. The RNA extraction method for LiPA and for sequencing analysis was the same. Viral load analysis and RNA extraction was performed from 500 μl of plasma using isopropanol and ethanol at 70% using the Cobas Amplicor HIV-1 Monitor 1.5 version (Roche Diagnostic Systems) according to the manufacturer's instructions. Amplification of the extracted RNA and sequencing analysis of the PR and the RT genes was performed by using the TruGene HIV-1 assay (Visible Genetics, Toronto, Ontario, Canada) on an automated DNA sequencer according to the manufacturer's recommendations. Testing involved clip sequencing of protease and codons 37 to 247 of RT from amplified cDNA in both the 3' and 5' directions. For LiPA, amplification of the extracted RNA and resistance testing was done with the new version 2.0 LiPA assay (VERSANT HIV-1 RT Resistance Assay and VERSANT HIV-1 Protease Resistance Assay; Bayer HealthCare LLC, Tarrytown, NY). All strips were read visually. A hybridization failure (indeterminate reaction) occurs in a LiPA assay when there are no signals for the specific probes for either the wild-type or the mutant codon. It is an uninterpretable result. Concordance was defined as the same interpretable result being obtained by both assays. Two types of discrepancies were studied—minor and major discrepancies—in which a wild-type codon was detected by one method and a mixed or mutant codon was detected by the other method, respectively.
The frequencies of antiretroviral resistance mutations by LiPA are shown in Table 2. LiPA-RT gave uninterpretable results in 5.95% (36 of 605 analyzed codons) of the RT gene (Table 3). The mutations detecting by sequencing that gave invalid results by LiPA were K103N (5.5%), M41L (7.4%), K70R M184V (3.7%) and T215Y V75T (1.8%). LiPA-PR gave uninterpretable results for 3.77% (13 of 344 analyzed codons), affecting the mutations by sequencing I54V L90M (4.6%). Comparison of genotypic results is shown in Table 4. The concordance between LiPA and sequence analysis was 97.6% for the RT (range, 93.9 to 100) and 95.7% for the PR codons (range, 87.2 to 100). Minor discrepancies reached 1.2% in the RT and 2.7% in the PR codons. Of 16 cases of codons with minor discrepancies, 15 cases were due to detection of mixed viral populations (mutant-wild type viruses) by LiPA and only wild-type virus by sequencing. Major discrepancies occurred in 1% of the RT and in 1.5% of the PR codons. These were due to detection of drug resistance mutations by sequencing and were not detected by LiPA. All sequences were submitted to the GenBank database and are available under accession number AY872282 to AY872335 (PR gene) and AY872336 to AY872389 (RT gene).
In our study, the rate of uninterpretable results is lower than for those reported in other studies performed with the previous assay version, which detected LiPA HIV-1 RT at rates of 18% (9) 15% (8), and 9.4% (11). In another study (1), the authors determined rates of hybridization failure of 8.44% for the RT gene and of 6.85% PR gene. For them, the performance of LiPA version 2.0 was significantly worse in strains belonging to the non-B subtypes. However, in our study, all strains belong to B subtypes. Uninterpretable results in LiPA correspond to HIV-1 genomes that have a nucleotide sequence that is different from the available LiPA probes. These failures limit the interpretation of the test and give incomplete information for the analysis of resistance. Overall, there was a good concordance between the results obtained with the two methods. The concordance rate is less for the PR codons than for the RT codons because there were more minor discrepancies in the former, mostly at codon 82 (10.3%). The reference method for detecting antiretroviral drug resistance-associated mutations is the sequencing of the RT and PR genes (5). However, several studies have shown that, in some circumstances, LiPA may be more sensitive than other techniques, including sequencing methods (7, 13). Drug-resistant variants, persisting as minority species, might not be detected with standard genotypic assays by population sequencing but would rapidly become apparent once antiretroviral therapy is initiated (5). Clonal resistance genotyping analysis by molecular cloning of HIV-1 PCR products (6) and single-genome sequencing are methods more sensitive for detecting minority populations (3), but it also requires intensive labor because many more clones must be sequenced. Proviral DNA sequencing from peripheral blood mononuclear cells has been studied to assess the presence of resistance mutations in archival virus populations (10), but systematic studies are needed. The minor discrepancies in data from our study suggest that LiPA has a greater sensitivity for detecting mutations present in only a small proportion of circulating viruses in clinical samples, as previously described in other reports (2, 7, 14). However, we consider that, as a consequence of limitations of LiPA and the number of uninterpretable results, it is not a method that would reliably detect mutated minority population in routine clinical care. Even with defined improvement in the number of uninterpretable results, LiPA version 2.0 would not sufficiently improve on the previous version. We find the number of indeterminate reactions to be unacceptably high.
ACKNOWLEDGMENTS
This work was supported by a grant from the La Paz University Hospital. It is a post-residence fellowship.
We thank R. Madero and N. Sastre for assistance with data analysis. We thank Bayer Diagnostic for the kind donation of LiPA kits for use in this study.
REFERENCES
Derdelinckx, I., K. Van Laethem, B. Maes, Y. Schrooten, K. De Schouwe, S. De Wit, K. Fransen, S. García-Ribas, M. Moutschen, D. Vaira, G. Zissis, M. Van Ranst, E. Van Wijngaerden, and A. M. Vandamme. 2003. Performance of the VERSANT HIV-1 Resistant Assays (LiPA) for detecting drug resistance in therapy-naive patients infected with different HIV-1 subtypes. FEMS Immunol. Med. Microbiol. 39:119-124.
Descamps, D., V. Calvez, G. Collin, A. Cecille, C. Apetrei, F. Damond, C. Katlama, S. Matheron, J. M. Huraux, and F. Brun-Vezinet. 1998. Line probe assay for detection of human immunodeficiency virus type 1 mutations conferring resistance to nucleoside inhibitors of reverse transcriptase: comparison with sequence analysis J. Clin. Microbiol. 36:2143-2145.
Devereux, H. L., C. Loveday, and M. Youle. 2000. Substantial correlation between HIV-1 drug-associated resistance mutations in plasma and peripheral blood mononuclear cells in heavily treated patients. AIDS 16:1025-1030.
EuroGuidelines Group for HIV Resistance. 2001. Clinical and laboratory guidelines for the use of HIV-1 drug resistance testing as part of treatment management: recommendations for the European setting. AIDS 15:309-320.
Hirsch, M. S., F. Brun-Vezinet, B. Clotet, B. Conway, D. R. Kuritzkes, R. T. D'Aquila, L. M. Demeter, S. M. Hammer, V. A. Johnson, C. Loveday, J. W. Mellors, D. M. Jacobsen, and D. D. Richman. 2003. Antiretroviral drug resistance testing in adults infected with human immunodeficiency virus type 1: recommendations of an International AIDS Society-USA Panel. Clin. Infect. Dis. 37:113-128.
Kearney, M., S. Palmer, F. Maldarelli, C. Bixby, H. Bazmi, D. Rock, J. Falloon, R. Davey, R. Dewar, J. Metcalf, J. Mellors, and J. Coffin. 2004. Single-genome sequencing is more sensitive than standard genotype analysis for detection of HIV-1 drug resistance mutations. Conference on Retroviruses and Opportunistic Infections, San Francisco, Calif.
Martinez-Picado, J., L. Sutton, M. Pia de Pasquale, A. V. Savara, and R. T. D'Aquila. 1999. Human immunodeficiency virus type 1 cloning vectors for antiretroviral resistance testing. J. Clin. Microbiol. 37:2943-2951.
Rusconi, S., S. L. Catamancio, F. Sheridan, and D. Parker. 2000. A genotypic analysis of patients receiving zidovudine with either lamivudine, didanosine, or zalcitabine dual therapy using the LiPA point mutation assay to detect genotypic variation at codons 41, 69, 70, 74, 184, and 215. J. Clin. Virol. 19:135-142.
Schmit, J. C., L. Ruiz, L. Stuyver, K. Van Laethem, I. Vanderlinden, T. Puig, R. Rossau, J. Desmyter, E. De Clercq, B. Clotet, and A. M. Vandamme. 1998. Comparison of the LiPA HIV-1 RT test, selective PCR and direct solid phase sequencing for the detection of HIV-1 drug resistance mutations J. Virol. Methods 73:77-82.
Servais, J., C. Lambert, E. Fontaine, J. M. Plesseria, I. Robert, V. Arendt, T. Staub, F. Schneider, R. Hemmer, G. Burtonboy, and J. C. Schmit. 2001. Comparison of DNA sequencing and a line probe assay for detection of human immunodeficiency virus type 1 drug resistance mutations in patients failing highly active antiretroviral therapy J. Clin. Microbiol. 39:454-459.
Stümer, M., B. Morgenstern, S. Staszewski, and H. W. Doerr. 2002. Evaluation of the LiPA HIV-1 RT assay version 1: comparison of sequence and hybridization based genotyping systems. J. Clin. Virol. 25:65-72.
Stuyver, L., A. Wyseur, A. Rombout, J. Louwagie, T. Scarcez, C. Verhofstede, D. Rimland, R. F. Schinazi, and R. Rossau. 1997. Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene. Antimicrob. Agents Chemother. 41:284-291.
Van Laethem, K., K. Van Vaerenbergh, J. C. Schmit, S. Sprecher, P. Hermans, V. De Vrocy, R. Schuurman, T. Harrer, M. Witvrouw, F. Van Wijngaerden, L. Stuyver, M. Van Ranstt, J. Desmyter, E. De Clercq, and A. M. Vandamme. 1997. Phenotypic assays and sequencing are less sensitive than point mutation assays for detection of resistance in mixed HIV-1 genotypic populations. J. Acquir. Immune Defic. Syndr. 22:107-118.
Villahermosa, M. L., G. Contreras, L. Perez-Alvarez, F. Bru, L. Medrano, E. Delgado, C. Colomo, M. Thomson, and R. Najera. 1998. Evaluation of mixtures of wild-type HIV-1 and HIV-1 with resistance point mutations against reverse transcriptase inhibitors. Antiviral Ther. 3:221-227.(S. García-Bujalance, C. L)
Department of Internal Medicine, La Paz University Hospital, Paseo de la Castellana 261, 28046 Madrid, Spain
ABSTRACT
The purpose of this study was to compare a line probe assay (LiPA) with sequence analysis for the detection of mutations conferring resistance to nucleoside and non-nucleoside inhibitors in human immunodeficiency reverse transcriptase and protease inhibitors. The limitations for interpreting LiPA make it unacceptable for routine clinical practice.
TEXT
The accumulation of retrospective and prospective data has led some expert panels to recommend the use of resistance testing in the management of human immunodeficiency virus type 1 (HIV-1)-infected patients as a part of routine clinical care (4, 5). Two different methods of genotypic resistance testing are used for assessing HIV-1 drug resistance: a line probe assay (LiPA), based on the principle of reverse hybridization (12), and sequence analysis by automated cycle sequencing. The LiPA (version 2.0) allows the study of wild-type and mutations M41L, T/A/N69D, K70R, L74V, V75T, M184V/I, T215D/S/A/Y/F, Q151M, K/R103N, V/I106A, and Y181C/I (the asterisk indicates a probe that was not included in version 1.0) of the reverse transcriptase (RT) gene and D30N, M46I, G48V, I50V, I/A54V, V/I82F/T/A, I84V, and L90M of the protease (PR) gene. Plasma samples from 54 previously treated (n = 49) or untreated (n = 5) HIV-1-infected patients were analyzed for drug genotypic resistance by both methods. Patients were monitored at the La Paz University Hospital, Madrid, Spain. Baseline characteristics are shown in Table 1. Samples were collected between April 2002 and October 2003. For the 54 patients, the RT gene was analyzed in 54 plasma samples, and the PR gene was analyzed in 43 plasma samples using sequencing analysis and the LiPA HIV-1 assays. The conventional EDTA tubes were used for blood collection. The RNA extraction method for LiPA and for sequencing analysis was the same. Viral load analysis and RNA extraction was performed from 500 μl of plasma using isopropanol and ethanol at 70% using the Cobas Amplicor HIV-1 Monitor 1.5 version (Roche Diagnostic Systems) according to the manufacturer's instructions. Amplification of the extracted RNA and sequencing analysis of the PR and the RT genes was performed by using the TruGene HIV-1 assay (Visible Genetics, Toronto, Ontario, Canada) on an automated DNA sequencer according to the manufacturer's recommendations. Testing involved clip sequencing of protease and codons 37 to 247 of RT from amplified cDNA in both the 3' and 5' directions. For LiPA, amplification of the extracted RNA and resistance testing was done with the new version 2.0 LiPA assay (VERSANT HIV-1 RT Resistance Assay and VERSANT HIV-1 Protease Resistance Assay; Bayer HealthCare LLC, Tarrytown, NY). All strips were read visually. A hybridization failure (indeterminate reaction) occurs in a LiPA assay when there are no signals for the specific probes for either the wild-type or the mutant codon. It is an uninterpretable result. Concordance was defined as the same interpretable result being obtained by both assays. Two types of discrepancies were studied—minor and major discrepancies—in which a wild-type codon was detected by one method and a mixed or mutant codon was detected by the other method, respectively.
The frequencies of antiretroviral resistance mutations by LiPA are shown in Table 2. LiPA-RT gave uninterpretable results in 5.95% (36 of 605 analyzed codons) of the RT gene (Table 3). The mutations detecting by sequencing that gave invalid results by LiPA were K103N (5.5%), M41L (7.4%), K70R M184V (3.7%) and T215Y V75T (1.8%). LiPA-PR gave uninterpretable results for 3.77% (13 of 344 analyzed codons), affecting the mutations by sequencing I54V L90M (4.6%). Comparison of genotypic results is shown in Table 4. The concordance between LiPA and sequence analysis was 97.6% for the RT (range, 93.9 to 100) and 95.7% for the PR codons (range, 87.2 to 100). Minor discrepancies reached 1.2% in the RT and 2.7% in the PR codons. Of 16 cases of codons with minor discrepancies, 15 cases were due to detection of mixed viral populations (mutant-wild type viruses) by LiPA and only wild-type virus by sequencing. Major discrepancies occurred in 1% of the RT and in 1.5% of the PR codons. These were due to detection of drug resistance mutations by sequencing and were not detected by LiPA. All sequences were submitted to the GenBank database and are available under accession number AY872282 to AY872335 (PR gene) and AY872336 to AY872389 (RT gene).
In our study, the rate of uninterpretable results is lower than for those reported in other studies performed with the previous assay version, which detected LiPA HIV-1 RT at rates of 18% (9) 15% (8), and 9.4% (11). In another study (1), the authors determined rates of hybridization failure of 8.44% for the RT gene and of 6.85% PR gene. For them, the performance of LiPA version 2.0 was significantly worse in strains belonging to the non-B subtypes. However, in our study, all strains belong to B subtypes. Uninterpretable results in LiPA correspond to HIV-1 genomes that have a nucleotide sequence that is different from the available LiPA probes. These failures limit the interpretation of the test and give incomplete information for the analysis of resistance. Overall, there was a good concordance between the results obtained with the two methods. The concordance rate is less for the PR codons than for the RT codons because there were more minor discrepancies in the former, mostly at codon 82 (10.3%). The reference method for detecting antiretroviral drug resistance-associated mutations is the sequencing of the RT and PR genes (5). However, several studies have shown that, in some circumstances, LiPA may be more sensitive than other techniques, including sequencing methods (7, 13). Drug-resistant variants, persisting as minority species, might not be detected with standard genotypic assays by population sequencing but would rapidly become apparent once antiretroviral therapy is initiated (5). Clonal resistance genotyping analysis by molecular cloning of HIV-1 PCR products (6) and single-genome sequencing are methods more sensitive for detecting minority populations (3), but it also requires intensive labor because many more clones must be sequenced. Proviral DNA sequencing from peripheral blood mononuclear cells has been studied to assess the presence of resistance mutations in archival virus populations (10), but systematic studies are needed. The minor discrepancies in data from our study suggest that LiPA has a greater sensitivity for detecting mutations present in only a small proportion of circulating viruses in clinical samples, as previously described in other reports (2, 7, 14). However, we consider that, as a consequence of limitations of LiPA and the number of uninterpretable results, it is not a method that would reliably detect mutated minority population in routine clinical care. Even with defined improvement in the number of uninterpretable results, LiPA version 2.0 would not sufficiently improve on the previous version. We find the number of indeterminate reactions to be unacceptably high.
ACKNOWLEDGMENTS
This work was supported by a grant from the La Paz University Hospital. It is a post-residence fellowship.
We thank R. Madero and N. Sastre for assistance with data analysis. We thank Bayer Diagnostic for the kind donation of LiPA kits for use in this study.
REFERENCES
Derdelinckx, I., K. Van Laethem, B. Maes, Y. Schrooten, K. De Schouwe, S. De Wit, K. Fransen, S. García-Ribas, M. Moutschen, D. Vaira, G. Zissis, M. Van Ranst, E. Van Wijngaerden, and A. M. Vandamme. 2003. Performance of the VERSANT HIV-1 Resistant Assays (LiPA) for detecting drug resistance in therapy-naive patients infected with different HIV-1 subtypes. FEMS Immunol. Med. Microbiol. 39:119-124.
Descamps, D., V. Calvez, G. Collin, A. Cecille, C. Apetrei, F. Damond, C. Katlama, S. Matheron, J. M. Huraux, and F. Brun-Vezinet. 1998. Line probe assay for detection of human immunodeficiency virus type 1 mutations conferring resistance to nucleoside inhibitors of reverse transcriptase: comparison with sequence analysis J. Clin. Microbiol. 36:2143-2145.
Devereux, H. L., C. Loveday, and M. Youle. 2000. Substantial correlation between HIV-1 drug-associated resistance mutations in plasma and peripheral blood mononuclear cells in heavily treated patients. AIDS 16:1025-1030.
EuroGuidelines Group for HIV Resistance. 2001. Clinical and laboratory guidelines for the use of HIV-1 drug resistance testing as part of treatment management: recommendations for the European setting. AIDS 15:309-320.
Hirsch, M. S., F. Brun-Vezinet, B. Clotet, B. Conway, D. R. Kuritzkes, R. T. D'Aquila, L. M. Demeter, S. M. Hammer, V. A. Johnson, C. Loveday, J. W. Mellors, D. M. Jacobsen, and D. D. Richman. 2003. Antiretroviral drug resistance testing in adults infected with human immunodeficiency virus type 1: recommendations of an International AIDS Society-USA Panel. Clin. Infect. Dis. 37:113-128.
Kearney, M., S. Palmer, F. Maldarelli, C. Bixby, H. Bazmi, D. Rock, J. Falloon, R. Davey, R. Dewar, J. Metcalf, J. Mellors, and J. Coffin. 2004. Single-genome sequencing is more sensitive than standard genotype analysis for detection of HIV-1 drug resistance mutations. Conference on Retroviruses and Opportunistic Infections, San Francisco, Calif.
Martinez-Picado, J., L. Sutton, M. Pia de Pasquale, A. V. Savara, and R. T. D'Aquila. 1999. Human immunodeficiency virus type 1 cloning vectors for antiretroviral resistance testing. J. Clin. Microbiol. 37:2943-2951.
Rusconi, S., S. L. Catamancio, F. Sheridan, and D. Parker. 2000. A genotypic analysis of patients receiving zidovudine with either lamivudine, didanosine, or zalcitabine dual therapy using the LiPA point mutation assay to detect genotypic variation at codons 41, 69, 70, 74, 184, and 215. J. Clin. Virol. 19:135-142.
Schmit, J. C., L. Ruiz, L. Stuyver, K. Van Laethem, I. Vanderlinden, T. Puig, R. Rossau, J. Desmyter, E. De Clercq, B. Clotet, and A. M. Vandamme. 1998. Comparison of the LiPA HIV-1 RT test, selective PCR and direct solid phase sequencing for the detection of HIV-1 drug resistance mutations J. Virol. Methods 73:77-82.
Servais, J., C. Lambert, E. Fontaine, J. M. Plesseria, I. Robert, V. Arendt, T. Staub, F. Schneider, R. Hemmer, G. Burtonboy, and J. C. Schmit. 2001. Comparison of DNA sequencing and a line probe assay for detection of human immunodeficiency virus type 1 drug resistance mutations in patients failing highly active antiretroviral therapy J. Clin. Microbiol. 39:454-459.
Stümer, M., B. Morgenstern, S. Staszewski, and H. W. Doerr. 2002. Evaluation of the LiPA HIV-1 RT assay version 1: comparison of sequence and hybridization based genotyping systems. J. Clin. Virol. 25:65-72.
Stuyver, L., A. Wyseur, A. Rombout, J. Louwagie, T. Scarcez, C. Verhofstede, D. Rimland, R. F. Schinazi, and R. Rossau. 1997. Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene. Antimicrob. Agents Chemother. 41:284-291.
Van Laethem, K., K. Van Vaerenbergh, J. C. Schmit, S. Sprecher, P. Hermans, V. De Vrocy, R. Schuurman, T. Harrer, M. Witvrouw, F. Van Wijngaerden, L. Stuyver, M. Van Ranstt, J. Desmyter, E. De Clercq, and A. M. Vandamme. 1997. Phenotypic assays and sequencing are less sensitive than point mutation assays for detection of resistance in mixed HIV-1 genotypic populations. J. Acquir. Immune Defic. Syndr. 22:107-118.
Villahermosa, M. L., G. Contreras, L. Perez-Alvarez, F. Bru, L. Medrano, E. Delgado, C. Colomo, M. Thomson, and R. Najera. 1998. Evaluation of mixtures of wild-type HIV-1 and HIV-1 with resistance point mutations against reverse transcriptase inhibitors. Antiviral Ther. 3:221-227.(S. García-Bujalance, C. L)