Highly active antiretroviral therapy (HAART) is a combinatorial therapy for HIV-1 infection, using three or more anti-HIV-1 drugs. It has greatly contributed to HIV-1 prevention and treatment since its discovery. However, HIV-1 variants with drug-resistant mutations and decreased drug susceptibility are sometimes found in patients due to frustrated treatment regimens and poor adherence . These variants may set off wider drug resistance transmission, create serious setbacks for HIV-1 therapy . Detection of drug resistance trends, and mutations that facilitate drug resistance, in HIV-1infected patients is therefore critical to the selection of appropriate regimens and efficacious HIV-1 prevention and treatment.
HIV-1 drug resistance detection methods consist of genotypic and phenotypic drug resistance assays. The genotypic drug-resistance assay involves sequencing the HIV-1 viral pol gene in a sample and comparing the sequence to an HIV-1 drug resistance database, such as the Stanford HIV Drug Resistance Database (http://hivdb.stanford.edu/[HIV db]) [4, 12]. Phenotypic drug resistance assays measure the ability of HIV to grow in the presence of different drugs; they are often performed using methods based on peripheral blood monoclonal cells (PBMCs) or polymerase chain reaction (PCR) [5, 6]. Phenotypic drug resistance assays require cells to be cultured, and so are costly and time-consuming. However, phenotypic drug resistance can also be predicted from genotypic data according to the HIV db .
Monitoring HIV-1 drug resistance is rarely performed in developing countries because of the cost, but it is essential for effective selection of drugs. The main objective of this study was to evaluate trends of genotypic and predicted phenotypic drug resistance among drug-treated, HIV-1-infected patients in Hubei, China from 2004 to 2006.
A total of 290 drug-treated HIV-1-infected patients in former blood donors in Hubei, China between January 2004 and December 2006 were included in this study. After obtaining patients' consent, pol gene sequencing was performed for genotypic drug-resis tance tests. For each patient, CD4+ cell counts and HIV RNA loads were determined within 3 months of drug-resistance analysis. Characteristics of the 290 patients are shown in Table 1.
Table 1. Characteristics of drug-treated HIV-1-infected patients from 2004 to 2006
Nucleotide sequences encoding HIV-1 protease and reverse transcriptase genes (2.1 kb) were amplified from PBMC DNA using a nested PCR method. After TA cloning, DNA sequencing of PCR products was done with an automated ABI 3730 DNA sequencer (Applied Biosystems Inc., USA). Nucleotide sequences thus obtained were screened using the BLAST (National Center for Biotechnology Information, USA) and BioEdit programs (www.mbio.ncsu.edu/BioEdit/bioedit.html) to eliminate potential laboratory errors; some sequences had previously undergone HIV-1 epidemic analysis . Each sample's 2.1 kb sequence was submitted to the HIVdb for genotypic drug resistance analysis.
All drug-resistance mutations of each sequence were submitted to the HIVdb for drug resistance predictions. Samples submitted to the HIVdb were identified as having one of five levels of drug resistance: high, intermediate, low, potential low and susceptible. In this study, we labeled high resistance as H, intermediate resistance as I, and low resistance and potential low level resistance as L.
Changes in percentages of drug resistance mutations from 2004 to 2006 were analyzed using a Χ2 test. Results in which P > 0.05 were considered insignificant; P < 0.05 was considered significant; and P < 0.01 was considered highly significant. Analyses were performed using the SPSS for Windows, version 11.5 (SPSS Inc., Chicago, IL).
Genotypic drug resistance analysis
Interpretation of genotypic drug resistance
As shown in Table 2, from 2004 to 2006, we found highly significant increases in percentages of drug-treated patients carrying HIV-1 with either an nucleoside reverse-transcriptase inhibitor (NRTI) or nonnucleoside reverse-transcriptase inhibitor (NNRTI), both an NRTI and an NNRTI, any NRTI, ≥ 2 NRTIs, any NNRTI, ≥ 2 NNRTIs, any thymidine analogue mutation (TAM), or ≥ 2 TAMs.
Table 2. Drug resistance profiles
Over the study period (2004–2006), we found highly significant increases in percentages of drug-treated patients carrying HIV-1 variants with M41L, T215Y/F, D67N, K103N, G190A/S or Y181C/F mutations, significant increase in the percentage of drug-treated patients carrying HIV-1 with L210W, but no significant increases in percentages of drug-treated patients carrying HIV-1 with F116Y, M184V, K219Q/E, Q151M, K70R, K101E/P, V179D, Y188L/C, A98G or V106A mutation (Fig. 1).
Figure 1. Emerging trends of HIV-1 nucleoside reverse transcriptase inhibitors-resistance and non-nucleoside reverse transcriptase inhibitors-resistance mutations. The y-axis shows the percentages of HIV-infected patients carrying each drug-resistant mutation in each year. The x-axis shows names of mutations. Trends in changes in the percentage of each mutation in 2004, 2005 and 2006 were analyzed by Χ2test.
Of the NRTI-resistance mutations, patients with the M41L mutation increased from 1/108 (0.9%) in 2004 to 2/134 (1.5%) in 2005, and to 12/48 (25%) in 2006., Variants with T215Y/F increased fastest, from 1/108 (0.9%) in 2004, to 9/134 (6.7%) in 2005, and to 18/48 (37.5%) in 2006 (P < 0.01). Patients with the L210W mutation increased from 1/108 (0.9%) in 2004, to 3/134 (2.2%) in 2005, and to 4/48 (8.3%) in 2006 (0.01 < P < 0.05). Those with the D67N mutation were first noticed in 2005, 5/134 (3.7%); they increased to 7/48 (14.6%) in 2006 (P < 0.01).
Of the NNRTI-resistance mutations, K103N was the most abundant; it increased from 7/108 (6.5%) in 2004, to 17/134 (12.7%) in 2005, and to 16/48 (33.3%) in 2006 (P < 0.01). The G190A/S mutation increased from 1/108 (0.9%) in 2004, to 7/134 (5.2%) in 2005, and to 7/48 (14.6%) in 2006 (P < 0.01). The Y181C/F variant initially emerged in 2005 at 3/134 (2.2%), and increased to 8/48 (16.7%) in 2006 (P < 0.01).
To further analyze phenotypic drug resistance of each HIV-1infected drug-treated patient to NNRTIs and NRTIs, we predicted the phenotypic drug resistance of each patient using the HIVdb. As shown in Fig. 2 and Fig. 3, we found highly significant increases in percentages of HIV-1-infected drug-treated patients carrying high resistance to zidovudine (AZT) or stavudine (D4T) in NRTIs, and to delavirdine (DLV), efavirenz (EFV) or nevirapine (NVP) in NNRTIs; highly significant increases in percentages of HIV-1-infected drug-treated patients carrying intermediate resistance to abacavir (ABC), AZT, D4T, didanosine (DDI) or tenofovir disoproxil fumarate (TDF) in NRTIs, and to etravirine (ETR) in NNRTIs; and highly significant increases in the percentages of HIV-1-infected drug-treated patients carrying low and potentially low resistance to lamivudine (3TC), ABC, emtricitabine (FTC) or TDF in NRTIs, and to ETR in NNRTIs.
Figure 2. Emerging trends of predicted phenotypic drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs). The y-axis shows the percentages of HIV-infected patients carrying each drug-resistance mutation in each year. The x-axis shows the names of NRTIs and the levels of resistance to the NRTIs: high resistance (H), intermediate resistance (I), or low and potentially low resistance (L). Trends in changes in the percentage of each mutation in 2004, 2005 and 2006 were analyzed by Χ2test. 3TC: lamivudine; ABC: abacavir; AZT: zidovudine; D4T: stavudine; DDI: didanosine; FTC: emtricitabine; TDF: tenofovir disoproxil fumarate.
Figure 3. Emerging trends of predicted phenotypic drug resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs). The y-axis shows the percentages of patients harboring the predicted levels of resistance to the NNRTIs in each year. The x-axis shows the name of each NNRTIs and the levels of resistance to the NNRTIs. The levels of resistance are defined as high-level resistance (H), intermediate-level resistance (I), low-level resistance and potentially low-level resistance (L). Trends in changes in the percentage of each mutation in 2004, 2005 and 2006 were analyzed by Χ2test. DLV: delavirdine; EFV: efaviren; ETR: etravirine; NVP: nevirapine.
For predicted NRTI-related phenotypic drug resistance, we only found highly significant increases in patients carrying HIV-1 variants with high resistance to AZT or D4T, which increased from 1/108 (0.9%) or 1/108 (0.9%) in 2004, to 5/134 (3.7%) or 3/134 (2.2%) in 2005, and to 8/48 (16.7%) or 8/48 (16.7%) in 2006, respectively (P < 0.01). For predicted NNRTI-related drug resistance, we found highly significant increases in patients carrying HIV-1 variants with high resistance to DLV, EFV or NVP—from 7/108 (6.5%), 9/108 (8.3%) or 10/108 (9.3%) in 2004, to 19/134 (14.2%), 22/134 (16.4%) or 28/134 (20.9%) in 2005, and to 20/48 (41.7%), 23/48 (47.9%) or 27/48 (56.3%) in 2006, respectively (P < 0.01).
Among treated patients, no significant differences in age, gender or drug regimens were seen between those with drug-resistant variants and those with wild-type HIV-1 viruses (data not shown). However, we found a significant difference in HIV-1 viral RNA loads between those with drug -resistant variants and those with wild-type HIV-1 viruses; patients with high HIV-1 RNA loads are prone to carry drug-resistant variants (data not shown).