Environment Health Techniques 1120
Jing Yang et al.
Research Paper Discordance between genotypic resistance and pseudovirus phenotypic resistance in AIDS patients after long-term antiretroviral therapy and virological failure Jing Yang1,2, Wenqing Geng1, Min Zhang1, Xiaoxu Han1 and Hong Shang1 1
2
Key Laboratory of AIDS Immunology of Ministry of Health, Department of Laboratory Medicine, the First Affiliated Hospital of China Medical University, Shenyang, China Infection Control Department, General Hospital of Shenyang military Area Command, Shenyang, China
Sixteen original recombinant pseudoviruses were generated by cloning the reverse transcriptase and protease genes of human immunodeficiency virus (HIV)-1 from patients into a plasmid vector (pNL4-3-DE-EGFP). By site-directed mutagenesis two restriction endonuclease sites, ApaI and AgeI, were inserted into pNL4-3-DE-EGFP. Phenotypic susceptibility of recombinant pseudoviruses to five different classes of antiretroviral drugs was determined using a luciferase reporter assay system. The results were subjected to comparative analyses to detect genotype–phenotype associations. Among 16 strains tested, 12 strains had a discordant genotype–phenotype resistance pattern to at least one drug. In five strains resistance to two, in two strains to three, and in one strain resistance to four drugs was detected. HIV resistance genotyping could predict the phenotype for nevirapine and azidothymidine. For lamivudine, 20 -30 -didehydro-20 30 dideoxythymidine and didanosine, phenotypic resistance testing was necessary. The study showed that in patients who experienced long-term highly active antiretroviral therapy and virological failure, there is some discordance between genotypic and phenotypic HIV drug resistance. To address the issue of limited resources in China, genotypic and phenotypic resistance testing should be done for different drugs in order to guide clinical therapy more effectively. Abbreviations: HIV – human immunodeficiency virus; AIDS – acquired immunodeficiency syndrome; HAART – highly active anti-retroviral therapy; NRTI – nucleoside reverse transcriptase inhibitor; NNRTI – non-nucleoside reverse transcriptase inhibitor; RT-PCR – reverse transcriptase polymerase chain reaction; IC50 – 50% inhibitory concentration; TCID50 – 50% tissue culture infective dose; AZT – azidothymidine; ddI – didanosine; d4T – 20 -30 -didehydro-20 -30 -dideoxythymidine; 3TC – lamivudine; NVP – nevirapine Keywords: HIV-1 / Drug resistance / Genotype–phenotype resistance / Highly Active Antiretroviral Therapy (HAART) Received: June 16, 2013; accepted: October 11, 2013 DOI 10.1002/jobm.201300415
Introduction Acquired immune deficiency syndrome (AIDS) is a disease of the human immune system caused by infection with human immunodeficiency virus (HIV) that presents a significant threat to human health. An estimated 780 thousand people in China were infected with HIV-1 in 2011, among that 154 thousand were AIDS patients [1].
Correspondence: Professor Hong Shang, Key Laboratory of Immunology of Ministry of Health, the First Affiliated Hospital of China Medical University, No.155 Nanjing North Street, Shenyang 110001, China E-mail: [email protected] Phone: þ86 24 83282634 Fax: þ86 24 83282634 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Currently, there is no cure or vaccine for AIDS. Highly active antiretroviral therapy (HAART) has been proven to be the most effective therapy to treat AIDS and control HIV-1 transmission [2]. However, the extensive use of HAART has led to high mutation rates of the HIV virus and drug resistance. HIV-1 drug resistance has become the most important cause of antiretroviral activity impairment [3]. Studies in South Africa, Uganda, and Brazil found that drug resistance mutation rates in patients experiencing HAART and virological failure were over 86%. Beyond that drug resistance mutation sites continued to increase after long-term HAART and treatment failure [4–6]. The pilot study for the National Free ART Program in China
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J. Basic Microbiol. 2014, 54, 1120–1125
Discordance between genotypic and phenotypic drug resistance in AIDS patients
started 2002 [7]. Along with the promotion of free antiretroviral therapy, nearly one hundred thousand patients had received free ART. The drug resistance rate, however, is growing year by year as patients are undergoing treatment for longer periods of time. Previous studies concentrated on patients receiving therapy for 2–3 years [8–12], but there were no reports about patients who received therapy for more than 3 years and failed HAART. We have found that reverse transcriptase (RT) resistance-associated mutation rate was 100% (27/27) (data not shown). This rate is higher than the 86.7% found by Brazilian authors [6], and is also significantly higher than those reported in short-term treatment studies (32.4–70.5%) conducted in China [8, 9]. Resistance may be assessed either by genotype or phenotype-based assays. Whether the two assays had concordance is controversial. In the study presented here, we focused on the relationship between the genotypic and phenotypic HIV drug resistance profiles of patients treated with two nucleoside RT inhibitors (NRTIs) plus one nonnucleoside RT inhibitor (NNRTI) regimen for more than 3 years. This data will support clinicians rationally select a drug resistance assay and help guide future treatment of HIV/AIDS patients in China.
Materials and methods Unless otherwise stated, all commercial molecular biological kits/assays were used according to manufacture’s protocols. Patients Sixteen AIDS patients who received HAART over 3 years and experienced one of virological failure which was defined as HIV RNA >400 copies ml1 after 24 weeks in a treatment-naïve patient initiating therapy or after HAART, HIV viral load has reduced to “undetectable (625) R(H:138) R(H:421) R(H:30.5) R(H:10.9) R(I:5.4) S(0.8) R(H:55.5) R(I:6) S(2.4) R(H:181) R(H:33.1) R(H:54.6) R(H:66.5) R(H:58.5)
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
H, high-level resistance; I, intermediate-level resistance; L, low-level resistance; p, potential resistance; s, sensitive.
R(I:6.7) R(H:15.2) R(I:6.7) S(0.6) R(I:5.5) R(I:4.1) S(2.8) S(3.1) R(H:14) S(3.7) R(I:6.8) S(1.1) S(0.3) S(3.3) S(0.5) S(1.2) M41L D67N T69D K70R L210W T215Y M41L D67N K70R V118I M184V L210W T215F K219Q M41L D67N L74V V75T L210W T215Y K219N – D67N K70R L74V Q151KLM T215I K219Q M41L D67N L74V L210WT215Y M41L E44D L74I V118I M184V L210W T215Y M41L V75I V118I T215S D67H T69N K70R M184V T215F K219E – M41L E44D D67N K70R L210S T215Y D67N K70R T215F K219Q M41LM E44DE M184V M41L E44D L74V V118I L210W T215Y M184V M184V T215F 1006 1008 1014 1019 1022 1033 1060 1074 1081 1082 1085 1086 1103 1110 130 494
K101E G190S K103R V106I V179DE Y188L V106I V179D Y181C H221C K103N G190A K238T V90I K103S G190A K101AET K103N G190A K238T Y188L V106A F227L V106M G190A F227L A98G V179D Y181C G190A H221Y K103K G190A K238T K103N G190S K103N P225H V90I K101P K103S
R(H) R(H) S(P) S R(L) S(P) R(H) S(P) R(H) S S(P) S R(H) R(L) R(H) R(H)
R(H) R(H) R(H) S R(H) R(H) R(H) R(I) R(H) S R(H) R(H) S R(H) S R(I)
R(H) R(H) R(H) S R(H) R(H) R(I) R(I) R(H) S R(H) R(H) S(P) R(H) S R(I)
R(L) R(I) R(H) S R(H) R(H) R(H) R(I) R(I) S R(I) R(L) R(L) R(H) S R(L)
R(H) R(H) R(I) R(H) R(H) R(H) R(H) R(I) R(H) S R(H) R(H) R(H) R(I) R(H) R(H)
R(H:>19.2) R(H:>19.2) R(H:>19.2) S(1.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2)
R(H: > 1250) S(3.3) R(H:>1250) S(2.3) R(H:70) R(H:93) R(H:136) R(H:231) R(H:119) R(H:852) R(H:>1250) S(1) S(1.5) R(H:126) S(1.3) R(I:5.5)
R(I:7.9) S(1.1) R(I:7.9) S(0.9) R(I:>8.7) S(2.5) R(I:4.5) R(I:5.3) R(I:>8.7) R(I:5.8) R(I:>8.7) S(0.9) S(0.5) S(2.5) R(I:6) S(1.1)
NVP ddI d4T AZT NRTI ID
NNRTI
3TC
AZT
d4T
ddI
NVP
3TC
Phenotypic resistance (level: fold increase of IC50) Genotypic resistance (level) Genotypic resistance mutations
Table 1. Susceptibility to 3TC, d4T, ddI, AZT, and NVP of pseudovirus containing mutations associated with drug resistance.
Discordance between genotypic and phenotypic drug resistance in AIDS patients
1123
resistance assays, we detected for ddI, 3TC, d4T, AZT, and NVP a discordance of 50% (8/16), 37.5%(6/16), 31.3%(5/ 16), 18.8% (3/16), and 18.8% (3/16). These values are congruent with those reported by Hirsch et al. [17]. The authors conducted genotypic and phenotypic HIV drug resistance testing as well, showing that the results by two methods in 79% of the drugs is the same, coincidence rate highest in NVP (92%), lowest in ddI (57%). And the result is also concordant with that of Dunne et al. [18], in this study the concordance between phenotypic and genotypic susceptibility testing was 81% for nucleoside RT inhibitors, and 91% for non-nucleoside RT inhibitors. Statistical analysis showed that though there was a difference between genotypic and phenotypic resistance patterns, the difference was not significant. A genotypic resistance pattern with a phenotypic sensitivity profile was detected for AZT (2 cases), d4T (5 cases), ddI (6 cases), and NVP (2 cases). On the other hand, a genotypic pattern of sensitivity with a phenotypic resistance profile was detected for 3TC (6 cases), AZT (1 case), ddI (2 cases), and NVP (1 case). For genotypic resistance patterns with phenotypic sensitivity cases, we found that these cases contained multiple drug-resistant mutations so genotypic resistance tests were positive. Previous studies showed that M184V resistance mutation can lead to both 3TC resistance and increased AZT sensitivity [19]. For patients experiencing long-term antiretroviral therapy and virological failure, a variety of mutations may have complex interactions, leading to the discordance of genotypic and phenotypic resistance. For genotypic sensitivity and phenotypic resistance cases, we speculate that because the HIV-1 subtype was B0 in this study, there may be some mutations not yet included in the Stanford HIV drug database based on subtype B HIV-1 strains. Thus, the genotypic resistance interpretation is sensitive and drug resistance can be detected by phenotypic resistance testing. In these cases, we may draw the wrong conclusions simply by genotypic resistance testing to predict phenotype. Phenotypic resistance testing can make up for the errors associated with genotypic resistance testing and detect the net effect of the interactions among resistance mutations. The phenotypic resistance testing can provide more reliable, intuitive resistance results, and thus guide clinical treatment more clearly. Study conducted by Sarmati et al. [15] also indicated that discordance between the genotypic and phenotypic drug resistance profiles in 64% of HIV-1 strains isolated from 25 patients treated with two NRTIs was detected. They thought that predicting resistance phenotype from genotypic data had important limitations and a phenotypic analysis could be performed in spite of an HIV
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Table 2. Analysis of the genotypic and phenotypic resistance to RT inhibitors drugs.
Resistance pattern Genotype
Phenotype
Resistance Resistance Sensitivity Sensitivity Resistance Sensitivity Sensitivity Resistance p-value for x2 testing
Antiretroviral drug 3TC
AZT
d4T
ddI
NVP
9 1 0 6 >0.05
10 3 2 1 >0.75
7 4 5 0 >0.25
7 1 6 2 >0.25
13 0 2 1 >0.5
genotypic sensitivity pattern. Differently from our study, the phenotypic analyses in their report were performed with native virus isolates. In China, neither virus isolate nor recombinant virus phenotypic resistance testing can be performed frequently due to limited resources. Therefore, it would be most efficient to use the genotypic resistance results to predict the phenotype for NVP and AZT. The results indicate that for 3TC sensitive cases in genotypic resistance testing, we should consider the possibility of phenotypic resistance; the drug should be selected carefully. In addition, for d4T and ddI, even if some cases showed resistance by genotypic resistance testing, phenotypic resistance testing should still be conducted to determine whether or not the drugs can be used for treatment. In a word, for 3TC, d4T, and ddI, phenotypic resistance testing is necessary. In summary, there was discordance between genotypic resistance and pseudovirus phenotypic resistance in patients who experienced long-term antiretroviral therapy and virological failure in China. Genotypic assays are rapid to perform but complex to interpret, mainly in patients who were extensively treated with antiretroviral drugs and carried a complex virus population showing multi-drug resistance. The results of pseudovirus phenotypic resistance testing can truly reflect the resistance condition and guide clinical treatment more directly. To address the issue of limited resources in China, genotypic and phenotypic resistance testing should be performed for different drugs in order to guide clinical therapy more effectively.
References [1] Ministry of Health and UNAIDS and the World Health Organization, 2011. AIDS epidemic Assessment Report in China in 2011. [2] Hammer, S.M., Eron, J.J. Jr., Reiss, P., Schooley, R.T. et al., 2008. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA, 300, 555–570. [3] Sabin, C.A., Hill, T., Lampe, F., Matthias, R. et al., 2005. Treatment exhaustion of highly active antiretroviral therapy (HAART) among individuals infected with HIV in the United Kingdom: multicentre cohort study. BMJ, 330, 695–699. [4] Pillay, V., Pillay, C., Kantor, R., Venter, F. et al., 2008. HIV type 1 subtype C drug resistance among pediatric and adult South African patients failing antiretroviral therapy. AIDS. Res. Hum. Retroviruses, 24, 1449–1454. [5] Lyagoba, F., Dunn, D.T., Pillay, D., Kityo, C. et al., 2010. Evolution of drug resistance during 48 weeks of zidovudine/lamivudine/tenofovir in the absence of real-time viral load monitoring. J. Acquir. Immune. Defic. Syndr., 55, 277–283. [6] Toledo, P.V., Carvalho, D.S., Romagnoli, L., Marcinko, G. et al., 2010. HIV-1 genotypic resistance profile of patients failing antiretroviral therapy in Parana, Brazil. Braz. J. Infect. Dis., 14, 360–371. [7] Zhang, F.J., Pan, J., Yu, L., Wen, Y. et al., 2005. Current progress of China’s free ART program. Cell Res., 15, 877– 882. [8] Li, J., Li, H., Liu, H., Li, H. et al., 2006. A cohort study of evolving of HIV-1 drug resistance in Henan Province, China. Wei. Sheng. Wu. Yu. Gan. Ran (Article in Chinese), 1, 211–216. [9] Yin, C., Lu, H., Lou, G., Li, X. et al., 2006. Prevalence of drugresistant human immunodeficiency virus type 1 in antiretroviral-treated patients of China (article in Chinese). Chin. J. Infect. Dis., 24, 164–167.
Acknowledgment This work was supported by Mega-Projects of National Science Research for the 12th Five-Year Plan (2012ZX10001-006).
[10] Han, X., Zhang, M., Cui, W., Liu, B. et al., 2005. Efficacy of anti-HIV treatment and drug-resistance mutations in some parts of China (article in Chinese). Zhonghua. Yi. Xue. Za. Zhi, 85, 760–764.
Conflict of interest statement
[11] Cai, F., Wei, F., Sun, H., Tan, Y. et al., 2009. Analysis of genotypes and subtypes of drug-resistance genes in patients with HIV-1 infection in Shanghai (article in Chinese). Lab. Med., 24, 788–791.
There are no financial conflicts of interest. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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[12] Wei, F., Wu, H., Zhang, T., Li, Q. et al., 2009. Analysis of drug resistance results in 20 cases of HIV/AIDS patients (article in Chinese). Shoudu. Yi. Ke. Da. Xue. Xue. Bao, 30, 626–630. [13] Zhang, H., Zhou, Y., Alcock, C., Kiefer, T. et al., 2004. Novel single-cell-level phenotypic assay for residual drug susceptibility and reduced replication capacity of drug-resistant human immunodeficiency virus type 1. J. Virol., 78, 1718– 1729. [14] Wei, X., Decker, J.M., Liu, H., Zhang, Z. et al., 2002. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother., 46, 1896– 1905. [15] Sarmati, L., Nicastri, E., Parisi, S.G., d’Ettore, G. et al., 2002. Discordance between genotypic and phenotypic drug resistance profiles in human immunodeficiency virus type
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1 strains isolated from peripheral blood mononuclear cells. J. Clin. Microbiol., 40, 335–340. [16] Hanna, G.J., D’Aquila, R.T., 2001. Clinical use of genotypic and phenotypic drug resistance testing to monitor antiretroviral chemotherapy. Clin. Infect. Dis., 32, 774–782. [17] Hirsch, H.H., Drechsler, H., Holbro, A., Hamy, F. et al., 2005. Genotypic and phenotypic resistance testing of HIV-1 in routine clinical care. Eur. J. Clin. Microbiol. Infect. Dis., 24, 733–738. [18] Dunne, A.L., Mitchell, F.M., Coberly, S.K., Hellmann, N.S. et al., 2001. Comparison of genotyping and phenotyping methods for determining susceptibility of HIV-1 to antiretroviral drugs. AIDS, 15, 1471–1475. [19] Whitcomb, J.M., Parkin, N.T., Chappey, C., Hellmann, N.S. et al., 2003. Broad nucleoside reverse-transcriptase inhibitor cross-resistance in human immunodeficiency virus type 1 clinical isolates. J. Infect. Dis., 188, 992–1000.
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Jing Yang et al.
Research Paper Discordance between genotypic resistance and pseudovirus phenotypic resistance in AIDS patients after long-term antiretroviral therapy and virological failure Jing Yang1,2, Wenqing Geng1, Min Zhang1, Xiaoxu Han1 and Hong Shang1 1
2
Key Laboratory of AIDS Immunology of Ministry of Health, Department of Laboratory Medicine, the First Affiliated Hospital of China Medical University, Shenyang, China Infection Control Department, General Hospital of Shenyang military Area Command, Shenyang, China
Sixteen original recombinant pseudoviruses were generated by cloning the reverse transcriptase and protease genes of human immunodeficiency virus (HIV)-1 from patients into a plasmid vector (pNL4-3-DE-EGFP). By site-directed mutagenesis two restriction endonuclease sites, ApaI and AgeI, were inserted into pNL4-3-DE-EGFP. Phenotypic susceptibility of recombinant pseudoviruses to five different classes of antiretroviral drugs was determined using a luciferase reporter assay system. The results were subjected to comparative analyses to detect genotype–phenotype associations. Among 16 strains tested, 12 strains had a discordant genotype–phenotype resistance pattern to at least one drug. In five strains resistance to two, in two strains to three, and in one strain resistance to four drugs was detected. HIV resistance genotyping could predict the phenotype for nevirapine and azidothymidine. For lamivudine, 20 -30 -didehydro-20 30 dideoxythymidine and didanosine, phenotypic resistance testing was necessary. The study showed that in patients who experienced long-term highly active antiretroviral therapy and virological failure, there is some discordance between genotypic and phenotypic HIV drug resistance. To address the issue of limited resources in China, genotypic and phenotypic resistance testing should be done for different drugs in order to guide clinical therapy more effectively. Abbreviations: HIV – human immunodeficiency virus; AIDS – acquired immunodeficiency syndrome; HAART – highly active anti-retroviral therapy; NRTI – nucleoside reverse transcriptase inhibitor; NNRTI – non-nucleoside reverse transcriptase inhibitor; RT-PCR – reverse transcriptase polymerase chain reaction; IC50 – 50% inhibitory concentration; TCID50 – 50% tissue culture infective dose; AZT – azidothymidine; ddI – didanosine; d4T – 20 -30 -didehydro-20 -30 -dideoxythymidine; 3TC – lamivudine; NVP – nevirapine Keywords: HIV-1 / Drug resistance / Genotype–phenotype resistance / Highly Active Antiretroviral Therapy (HAART) Received: June 16, 2013; accepted: October 11, 2013 DOI 10.1002/jobm.201300415
Introduction Acquired immune deficiency syndrome (AIDS) is a disease of the human immune system caused by infection with human immunodeficiency virus (HIV) that presents a significant threat to human health. An estimated 780 thousand people in China were infected with HIV-1 in 2011, among that 154 thousand were AIDS patients [1].
Correspondence: Professor Hong Shang, Key Laboratory of Immunology of Ministry of Health, the First Affiliated Hospital of China Medical University, No.155 Nanjing North Street, Shenyang 110001, China E-mail: [email protected] Phone: þ86 24 83282634 Fax: þ86 24 83282634 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Currently, there is no cure or vaccine for AIDS. Highly active antiretroviral therapy (HAART) has been proven to be the most effective therapy to treat AIDS and control HIV-1 transmission [2]. However, the extensive use of HAART has led to high mutation rates of the HIV virus and drug resistance. HIV-1 drug resistance has become the most important cause of antiretroviral activity impairment [3]. Studies in South Africa, Uganda, and Brazil found that drug resistance mutation rates in patients experiencing HAART and virological failure were over 86%. Beyond that drug resistance mutation sites continued to increase after long-term HAART and treatment failure [4–6]. The pilot study for the National Free ART Program in China
www.jbm-journal.com
J. Basic Microbiol. 2014, 54, 1120–1125
Discordance between genotypic and phenotypic drug resistance in AIDS patients
started 2002 [7]. Along with the promotion of free antiretroviral therapy, nearly one hundred thousand patients had received free ART. The drug resistance rate, however, is growing year by year as patients are undergoing treatment for longer periods of time. Previous studies concentrated on patients receiving therapy for 2–3 years [8–12], but there were no reports about patients who received therapy for more than 3 years and failed HAART. We have found that reverse transcriptase (RT) resistance-associated mutation rate was 100% (27/27) (data not shown). This rate is higher than the 86.7% found by Brazilian authors [6], and is also significantly higher than those reported in short-term treatment studies (32.4–70.5%) conducted in China [8, 9]. Resistance may be assessed either by genotype or phenotype-based assays. Whether the two assays had concordance is controversial. In the study presented here, we focused on the relationship between the genotypic and phenotypic HIV drug resistance profiles of patients treated with two nucleoside RT inhibitors (NRTIs) plus one nonnucleoside RT inhibitor (NNRTI) regimen for more than 3 years. This data will support clinicians rationally select a drug resistance assay and help guide future treatment of HIV/AIDS patients in China.
Materials and methods Unless otherwise stated, all commercial molecular biological kits/assays were used according to manufacture’s protocols. Patients Sixteen AIDS patients who received HAART over 3 years and experienced one of virological failure which was defined as HIV RNA >400 copies ml1 after 24 weeks in a treatment-naïve patient initiating therapy or after HAART, HIV viral load has reduced to “undetectable (625) R(H:138) R(H:421) R(H:30.5) R(H:10.9) R(I:5.4) S(0.8) R(H:55.5) R(I:6) S(2.4) R(H:181) R(H:33.1) R(H:54.6) R(H:66.5) R(H:58.5)
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
H, high-level resistance; I, intermediate-level resistance; L, low-level resistance; p, potential resistance; s, sensitive.
R(I:6.7) R(H:15.2) R(I:6.7) S(0.6) R(I:5.5) R(I:4.1) S(2.8) S(3.1) R(H:14) S(3.7) R(I:6.8) S(1.1) S(0.3) S(3.3) S(0.5) S(1.2) M41L D67N T69D K70R L210W T215Y M41L D67N K70R V118I M184V L210W T215F K219Q M41L D67N L74V V75T L210W T215Y K219N – D67N K70R L74V Q151KLM T215I K219Q M41L D67N L74V L210WT215Y M41L E44D L74I V118I M184V L210W T215Y M41L V75I V118I T215S D67H T69N K70R M184V T215F K219E – M41L E44D D67N K70R L210S T215Y D67N K70R T215F K219Q M41LM E44DE M184V M41L E44D L74V V118I L210W T215Y M184V M184V T215F 1006 1008 1014 1019 1022 1033 1060 1074 1081 1082 1085 1086 1103 1110 130 494
K101E G190S K103R V106I V179DE Y188L V106I V179D Y181C H221C K103N G190A K238T V90I K103S G190A K101AET K103N G190A K238T Y188L V106A F227L V106M G190A F227L A98G V179D Y181C G190A H221Y K103K G190A K238T K103N G190S K103N P225H V90I K101P K103S
R(H) R(H) S(P) S R(L) S(P) R(H) S(P) R(H) S S(P) S R(H) R(L) R(H) R(H)
R(H) R(H) R(H) S R(H) R(H) R(H) R(I) R(H) S R(H) R(H) S R(H) S R(I)
R(H) R(H) R(H) S R(H) R(H) R(I) R(I) R(H) S R(H) R(H) S(P) R(H) S R(I)
R(L) R(I) R(H) S R(H) R(H) R(H) R(I) R(I) S R(I) R(L) R(L) R(H) S R(L)
R(H) R(H) R(I) R(H) R(H) R(H) R(H) R(I) R(H) S R(H) R(H) R(H) R(I) R(H) R(H)
R(H:>19.2) R(H:>19.2) R(H:>19.2) S(1.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2) R(H:>19.2)
R(H: > 1250) S(3.3) R(H:>1250) S(2.3) R(H:70) R(H:93) R(H:136) R(H:231) R(H:119) R(H:852) R(H:>1250) S(1) S(1.5) R(H:126) S(1.3) R(I:5.5)
R(I:7.9) S(1.1) R(I:7.9) S(0.9) R(I:>8.7) S(2.5) R(I:4.5) R(I:5.3) R(I:>8.7) R(I:5.8) R(I:>8.7) S(0.9) S(0.5) S(2.5) R(I:6) S(1.1)
NVP ddI d4T AZT NRTI ID
NNRTI
3TC
AZT
d4T
ddI
NVP
3TC
Phenotypic resistance (level: fold increase of IC50) Genotypic resistance (level) Genotypic resistance mutations
Table 1. Susceptibility to 3TC, d4T, ddI, AZT, and NVP of pseudovirus containing mutations associated with drug resistance.
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resistance assays, we detected for ddI, 3TC, d4T, AZT, and NVP a discordance of 50% (8/16), 37.5%(6/16), 31.3%(5/ 16), 18.8% (3/16), and 18.8% (3/16). These values are congruent with those reported by Hirsch et al. [17]. The authors conducted genotypic and phenotypic HIV drug resistance testing as well, showing that the results by two methods in 79% of the drugs is the same, coincidence rate highest in NVP (92%), lowest in ddI (57%). And the result is also concordant with that of Dunne et al. [18], in this study the concordance between phenotypic and genotypic susceptibility testing was 81% for nucleoside RT inhibitors, and 91% for non-nucleoside RT inhibitors. Statistical analysis showed that though there was a difference between genotypic and phenotypic resistance patterns, the difference was not significant. A genotypic resistance pattern with a phenotypic sensitivity profile was detected for AZT (2 cases), d4T (5 cases), ddI (6 cases), and NVP (2 cases). On the other hand, a genotypic pattern of sensitivity with a phenotypic resistance profile was detected for 3TC (6 cases), AZT (1 case), ddI (2 cases), and NVP (1 case). For genotypic resistance patterns with phenotypic sensitivity cases, we found that these cases contained multiple drug-resistant mutations so genotypic resistance tests were positive. Previous studies showed that M184V resistance mutation can lead to both 3TC resistance and increased AZT sensitivity [19]. For patients experiencing long-term antiretroviral therapy and virological failure, a variety of mutations may have complex interactions, leading to the discordance of genotypic and phenotypic resistance. For genotypic sensitivity and phenotypic resistance cases, we speculate that because the HIV-1 subtype was B0 in this study, there may be some mutations not yet included in the Stanford HIV drug database based on subtype B HIV-1 strains. Thus, the genotypic resistance interpretation is sensitive and drug resistance can be detected by phenotypic resistance testing. In these cases, we may draw the wrong conclusions simply by genotypic resistance testing to predict phenotype. Phenotypic resistance testing can make up for the errors associated with genotypic resistance testing and detect the net effect of the interactions among resistance mutations. The phenotypic resistance testing can provide more reliable, intuitive resistance results, and thus guide clinical treatment more clearly. Study conducted by Sarmati et al. [15] also indicated that discordance between the genotypic and phenotypic drug resistance profiles in 64% of HIV-1 strains isolated from 25 patients treated with two NRTIs was detected. They thought that predicting resistance phenotype from genotypic data had important limitations and a phenotypic analysis could be performed in spite of an HIV
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Table 2. Analysis of the genotypic and phenotypic resistance to RT inhibitors drugs.
Resistance pattern Genotype
Phenotype
Resistance Resistance Sensitivity Sensitivity Resistance Sensitivity Sensitivity Resistance p-value for x2 testing
Antiretroviral drug 3TC
AZT
d4T
ddI
NVP
9 1 0 6 >0.05
10 3 2 1 >0.75
7 4 5 0 >0.25
7 1 6 2 >0.25
13 0 2 1 >0.5
genotypic sensitivity pattern. Differently from our study, the phenotypic analyses in their report were performed with native virus isolates. In China, neither virus isolate nor recombinant virus phenotypic resistance testing can be performed frequently due to limited resources. Therefore, it would be most efficient to use the genotypic resistance results to predict the phenotype for NVP and AZT. The results indicate that for 3TC sensitive cases in genotypic resistance testing, we should consider the possibility of phenotypic resistance; the drug should be selected carefully. In addition, for d4T and ddI, even if some cases showed resistance by genotypic resistance testing, phenotypic resistance testing should still be conducted to determine whether or not the drugs can be used for treatment. In a word, for 3TC, d4T, and ddI, phenotypic resistance testing is necessary. In summary, there was discordance between genotypic resistance and pseudovirus phenotypic resistance in patients who experienced long-term antiretroviral therapy and virological failure in China. Genotypic assays are rapid to perform but complex to interpret, mainly in patients who were extensively treated with antiretroviral drugs and carried a complex virus population showing multi-drug resistance. The results of pseudovirus phenotypic resistance testing can truly reflect the resistance condition and guide clinical treatment more directly. To address the issue of limited resources in China, genotypic and phenotypic resistance testing should be performed for different drugs in order to guide clinical therapy more effectively.
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Acknowledgment This work was supported by Mega-Projects of National Science Research for the 12th Five-Year Plan (2012ZX10001-006).
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Conflict of interest statement
[11] Cai, F., Wei, F., Sun, H., Tan, Y. et al., 2009. Analysis of genotypes and subtypes of drug-resistance genes in patients with HIV-1 infection in Shanghai (article in Chinese). Lab. Med., 24, 788–791.
There are no financial conflicts of interest. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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