| Part One:Prevalence of HIV-1 drug resistance and factors related to drug resistance.Objective To investigate the prevalence of HIV-1 drug resistance in Guangxi, and explore the factors that may be related to HIV-1 drug resistance. Methods A survey was performed among 304 HIV-positive and AIDS subjects through questionnaires to collect informations on HIV-1 drug resistance. Peripheral venous blood samples were collected from all subjects. RNA were extracted from plasma, and nested RT-PCR were employed to amplify nucleic acid fragments encoding protease ( amino acids1-99) and reverse transcriptase (amino acids 1-285), and the amplified products were subjected to DNA sequencing. Sequences obtained were then analyzed with Stanford HIV drug resistance algorithm for informations about mutations and levels of drug resistance. Viral subtypes were determined by phylogenetic analysis with MEGA 4 software. ART-treated subjects were devided into groups with different characteristics, and then resistance rates were compared among these groups with Chi-Square test or Fisher's Exact test to observe the correlations between these characteristics and drug resistance. Results 219 pol sequences were obtained from 86 ART-treated subjects and 133 ART-naive subjects. Phylogenetic analysis indicated that subtypes could be determined for 218 samples, in which 176 were designated as subtype CRF01_AE, 26 as CRF07_BC, 13 as CRF08_BC, 2 as B, and 1 as C. One sample was undetermined with phylogenetic analysis. Mutations associated with resistance to PIs, NRTIs, and NNRTIs were present in 6.9% (14/219), 30.14% (66/219), 14.16% (31/219) of samples. CRF07_BC strains had a significantly higher prevalence of PI-related mutations than CRF01_AE strains, however, CRF08_BC had a higher prevalence of NRTI-related mutations than CRF01_AE and CRF07_BC. PI minor mutation A71V/T was more common in CRF08_BC than in CRF01_AE, and NRTI minor mutation T69S occured mainly in CRF08_BC, but rare in CRF01_AE and CRF07_BC. 18 out of 219 samples (8.22%) were found to harboured mutations that confered low to high levels of resistance to at least one antiretroviral drug (ARV), Of which 15 were from ART-treated subjects and 3 from ART-naive subjects, giving a resistance rate of 17.44% (15/86) for ART-treated subjects and 2.66% (3/133) for ART-naive subjects, respectively. Among the subjects harboured resistance-related mutations, the percentage of high-level resistance to NRTIs or NNRTIs was 77.78% and 72.22%, respectively, and the percentages of cross-resistance were both 100% within each drug class and 77.78% between two drug classes. Time to initial ART, Adherence to ART, and employment were related to resistance rate, other factors such as age, sex, education, nationality, viral subtype, transmission route, and regimen were not related to resistance risk. Resistance risk was not significant different between the group experienced 1 to 2 year ART treatment and the group experienced≥2 year ART treatment, however, a significant higher resistance risk was observed among subjects exposed to ART treatment exceeding 1 year compared to those exposed to treatment less than 1 year(OR=8.089,P<0.030). Resistance rate was higher in the group with 50-90% adherence compared to the group with≥90% adherence(OR=8.591, P=0.030). Farmer has a higher resistance risk than other employment workers and those unemployed (OR=4.223, P=0.017), and dose adherence was lower in farmer than in other employment workers and those unemployed (P=0.007). Conclusions The prevalence of HIV-1 drug resistance in Guangxi is at a relatively low level compared to many other provinces of China, however, high level resistance, cross resistance within or between drug classes are common in patients harboured mutations related to NRTIs and NNRTIs resistance. Therapy time, adherence to ART, and employment is related to resistance. Over 1 year of ART treatment, with low than 90% adherence, and farm employment may related to higher rate of drug resistance. Higher rate of drug resistance of farmers is related to lower adherence to ART regimens. It may be possible to reduce rate of drug resistance of farmers by providing a more convenient access to ART and education to promote adherence to ART regimens. The rates of drug resistance are not different among patients infected with different subtypes of HIV-1, but patterns of drug resistance may vary according to subtypes of the virus. A71V/T occur more frequently in CRF07_BC strains, however, T69S mainly in CRF08_BC strains.Part Two: Study on Genetic barrier to primary resistance mutations in subtypes CRF01_AE, CRF07_BC, and CRF08_BC.Objective To compare the genetic barriers to development of primary mutations among CRF01_AE, CRF07_BC, and CRF08_BC isolates, and discuss theoretically the genetic reasons underlying varying patterns and probabilities of resistance related mutations in different subtypes. Methods Selected pol sequences from all 133 ART naive subjects according to the result of Part One of the present study. Quality of sequences were checked and then subjected to recombinant analysis before inclusion to assure the quality of sequences and simgle subtype in pol region. Sequences with recombinant breakpoints that were not obviously different from reference sequences and codons with less than two ambiguities per codons were included. 21 primary PIs mutations, 16 primary NRTIs mutations, and 18 primary NNRTIs mutions were included according to Staford HIV drug resistance database. Drug resistance codons and their corresponding wild-type codons were determined using codon table, and then nucleotide transitions (ts) and transversions (tv) were count for each primary mutation. According to the phenomena that transitions occurr on average 2.5 more frequent than transversions, each transition was scored as 1, and each transversion scored as 2.5, and then, genetic barrier wich was taken as the sum of the scores was calculated for a particular drug resistance substitution. The mean genetic barriers for particular substitutions were compared among the three subtypes by Kruskal-Wallis test. When significance was found, the Nemenyi test was used to compare each pair of subtypes. Results Most wild-type codons were conserved among subtypes at positions where major PIs mutations may occur, only at position L10 and N88 codon usages varied between subtypes, but did not make an impact on genetic barriers because the types and quantities of nucleotide substitutions for particular resistance related mutations were the same in all three subtypes. At positions related to NRTIs resistance, different extent of variation in wild-type codon preferences were found among subtypes at position T/S69, K70, L74, V75, V118, L210, T215, and K219, and codon usage preferences at position T/S69, V118, and L210 had an impact on genetic barriers to resistance (P<0.001). CRF01_AE and CRF07_BC had higher genetic barriers for T/S69D substitution than CRF08_BC (P<0.01), and had lower genetic barriers for V118I and L210W substitution than CRF08_BC (P<0.01). At NNRTIs resistance-related positions, wild-type codon usage varied at position K103, V106, V108, and V/I179, and none but the synonymous differences at position V106 made an impact on genetic barrier for the V106M mutation (P<0.001). CRF07_BC had a lower genetic barrier than CRF01_AE and CRF08_BC (P<0.001), and no significance was seen between CRF01_AE and CRF08_BC. Conclusions The genetic barriers for evolution to most primary mutations are similar among CRF01_AE, CRF07_BC, and CRF08_BC, however, the genetic barriers vary among subtypes for L210W, V118I, V106M, and T/S69D mutations. These various genetic barriers for different subtypes suggest that under the same ARV selection, development of V118I and L210W may be easier for subtypes CRF01_AE and CRF07_BC than for CRF08_BC, but contrarily, CRF08_BC may be easier to develop T/S69D substitution than CRF01_AE and CRF07_BC. The V106M substitution may be more common for CRF07_BC strains that for CRF08_BC or CRF01_AE strains. Development of V118I, L210W and V106M may be more common for CRF07_BC strains that for CRF08_BC strains.Part Three: Development of a rapid subtyping assay for HIV-1 strains circulating in Guangxi.Objective To develop a nested multiplex PCR assay for easy determination of HIV-1 subtype CRF01_AE, CRF07_BC, CRF08_BC, B, and C circulating in Guangxi. Method Subtype-specific primers (inner primers) were designed for subtypes CRF01_AE, CRF07_BC, CRF08_BC, B, and C based on their gag sequences. Primer Gag F2 (forward) and Gag e2 (reverse) universal for HIV-1 M group strains were used as outer primers. 72 samples including 58 HIV-positive samples of known subtype, 14 HIV-positive samples of unknown subtype were included in this study. In addition, 5 positive control samples were used as posittive control, and subtype CRF01_AE, CRF07_BC, CRF08_BC, B and C had 1 sample each, and 10 samples from HIV-uninfected subjects were used as negative control. DNAs were extracted from whole blood and used as templates for first-round PCR with the primer pair Gag F2/Gag e2. Second-round PCR was performed by using the first-round PCR products as templates with subtype-specific primers for subtypes CRF01_AE, CRF_BC, B, and C. All these subtype-specific primers were simultaneously contained in one reaction mixture. PCR products were analyzed by electrophoresis on a 2.5% agarose gel, and subtype determination was made based on PCR product sizes. When CRF_BC were determined, in order to distinguish between CRF07_BC and CRF08_BC, an additional PCR reaction was performed to amplified the first-round PCR products, and followed by an analysis of electrophoresis on a 2.5% agarose gel to determine if the subtype was CRF07_BC or CRF08_BC. To check the accuracy of the subtyping results by nested multiplex PCR, all 72 samples to be subtyped were amplified by using the first-round PCR products as templates with primer pair 306/Cn-gag, and the 670-bp products were purified and analyzed by DNA sequenceing and phylogenetic analysis. The subtyping results by nested multiplex PCR were compared with that by sequence-based phylogenetic analysis, and the sensitivity and specificity of nested multiplex PCR were evaluated. 10 samples belonging to CRF01_AE, 10 samples belonging to CRF07_BC, and 5 samples belonging to CRF08_BC were subtyped three times by nested multiplex PCR, and repeatabilities were computed for detection of the three subtypes. Results 66 out of the 72 HIV-positive samples could be amplified by nested multiplex PCR, and all the 10 HIV-negative samples were negative. Of the 66 samples amplified, 40 were designated as CRF01_AE, 14 as CRF07_BC, 7 as CRF08_BC, 2 as subtype B, and 1 as subtype C. 306/Cn-gag could also amplified the same 66 samples amplified by nested multiplex PCR, and 64 sequences were obtained by sequencing, the remaining 2 samples failed sequencing. The sensitivity was 91.7% (66/72) for nested multiplex PCR and 88.9% (64/72) for sequence-based phylogenetic analysis. There were no significance between two methods with respect to the sensitivity (Fisher exact test, P=0.492). Since the same results were given to the 64 samples by the two methods, the specificity of nested multiplex PCR was 100% compared to sequence-based phylogenetic analysis. All samples belonging to CRF01_AE, CRF07_BC, and CRF08_BC in the repeatability assay could be correctly determined in three detection, and repeatabilities were computed to be 100% for detection of these three subtypes by nested multiplex PCR. Conclusions We have developed a nested multiplex PCR assay for accurate determination of HIV-1 subtypes CRF01_AE, CRF07_BC, CRF08_BC,and B circulating in Guangxi, China. This assay has high sensitivity, specificity, and repeatability, and is a simple, rapid, and low-cost alternative to sequence-based phylogenetic analysis for subtyping of HIV-1 strains circulating in Guangxi and the surrounding areas. |
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