Prevalence Of HIV-1 Drug Resistance In Henan Province And Analysis Of New Resistance Associated Mutations

Drug resistance of HIV-1 is the main obstacle for antiretroviral therapy (ART). The information about the prevalence and molecular evolution of HIV-1 drug resistance is significant for understanding drug resistance mechanism and the supervision of ART. At the beginning, in-house genotyping method was evaluated. Then this method was used in the cross-sectional monitoring of drug resistance in Henan province. The prevalence of drug resistance and its impact on ART were analyzed and some advices about ART were provided. Combined with the information about the HIV-1 drug resistance epidemiology in Henan province, some new mutations associated with ART failure were screened and viruses with these mutations were constructed by site-directed mutagenesis and transfection strategies. And the impacts of these mutated viruses on antiretroviral drugs were measured by phenotypic resistance test. At last, a new method for the analysis of quasispecies drug resistance was successfully constructed. Single-Genome Amplification (SGA) was used to analyze the molecular evolution of Indinavir (IDV) drug resistance in one patient, and the new IDV resistance mutation pattern was identified.PartⅠEvaluation of an in-house method for HIV-1 genotyping resistance test used in ChinaObjective: Evaluating the sensitivity and accuracy characteristics of an in-house genotypic resistance system widely used in China. Methods: This in-house method was evaluated by comparing with an US FDA-approved genotyping system, ViroSeq? v2.0 (Abbott, Switzerland). The characteristics of the detection and interpretation of drug resistance mutations were validated through drawing a parallel between these two tests in 130 plasma samples. Results: Out of the 14850 mutations detected, 14752 (99.34%) mutations was concordant between these two methods (kappa value was 0.9488; P value was less than 0.00001). Through the comparison between these two methods, the rates of concordance in protease inhibitors-, reverse transcriptase inhibitors-resistance mutations were 99.70% and 99.01% respectively, and kappa values were 0.9099 and 0.9521 respectively (P values were all less than 0.00001). The comparison of drug resistance reports showed similar results (Kappa value was 0.6374, P value was less than 0.0001), suggesting there was high concordance between these two tests in drug resistance mutation interpretation. There were 34 mutations excluded from ViroSeq? drug resistance mutation database (ViroSeq? software v2.7) which were detected by one or both tests. Two mutations, V179F and K238T in RT region, had significance in drug resistance, indicating the database used by in-house assay was superior to ViroSeq?. Conclusions: The in-house genotyping system is an accurate, cost-effective method and had high concordance with commercial ViroSeq? genotyping system in the detection and interpretation of drug resistance mutations.PartⅡCross-sectional monitoring of the prevalence of drug resistance in Henan provinceObjective: The impact of current treatment regimens were investigated through this cross-sectional monitoring of drug resistance in Henan province, such as the treatment efficacy, immune system rebuilding and the prevalence of drug resistance variants, which could provide evidence to improve treatment regimen. Methods: This cross-sectional study included 120 HIV-1 infected patients who began to ART from 2003 or 2004. The information of patients'clinical manifestation and drug adherence was obtained through face to face review. All patients'CD4+ T cells counts and HIV-1 viral loads were measured to evalutate the ART efficacy and immune rebuilding, and in-house drug resistance test was performed in treatment-failure patients. Results: There were three ART regimens used in these patients, AZT/DDI/NVP group (n=102), AZT/3TC/NVP group (n=12), D4T/3TC/NVP group (n=6). The average of CD4+ T cell counts was 377±218 cells/ml and the median was 367 cells/ml, and 64 (53.3%) patients'CD4+ T cell counts were higher than 350 cells/ml. Out of 114 patients with viral load data, 33 patients'viral loads were less than 50 copies/ml, and the remaining patients'viral loads had an average of 4.09±1.10 lg copies/ml (the median was 3.87 lg copies/ml). Effective viral inhibition (viral load is less than 1000 copies/ml) was found in 47 patients (41.23%). No statistics significance was found among these three ART regimens'efficacies (P=0.53) through Fisher Exact test. In treatment failure patients, the ratio of reverse transcriptase inhibitors (RTIs) resistance was 69.0% (40/58), and there was no protease inhibitors (PIs) resistance variants. The ratio of nucleoside reverse transcriptase inhibitors (NRTIs) resistance and non-nucleoside reverse transcriptase inhibitors (NNRTIs) resistance were 53.4% (31/58) and 67.2% (39/58), respectively. NVP had the highest drug resistance ratio (67.24%, 39/58) in these NNRTIs used in these patients, and AZT was the highest in NRTIs (53.4%, 31/58). In the patients who had drug resistant variants, variants which showed high- and intermediate-level resistance were much more than those with low-level resistance, indicating variants existed in treatment failure patients were mostly high- and intermediate-level and had cross-drug resistance. M41L and T215Y in RT region were the most prevalence NRTIs mutations; K103N and Y181C were the most prevalence NNRTIs mutations. Conclusions: The treatment regimens currently used in these patients were not suitable, because these regimens did not gain realistic antiretroviral efficacy. Drug resistance variants existed in most treatment failure patients, suggesting protease inhibitors should be involved in new regimens. PartⅢIdentification of new drug resistance associated HIV-1 mutationsBecause of the prevalence of different HIV-1 subtypes, ART and the interaction among virus, host and drugs, more and more drug resistance mutations were identified. Reverse transcriptase (RT) is the most important target for ART, and many mutations which had significance in drug resistance had been reported and applied for drug resistance detection and monitoring. But almost all of mutations are located in the polymerase domain of RT; the mutations in ligation domain are rare. Objective: Some new selected RT mutations'impact on drug resistance was analyzed. Methods: The mutated pNL4.3 viruses were constructed by site-directed mutagenesis and transfection strategies. The 50% inhibitory concentration (IC50) change folds of each mutated virus were measured through the comparison of the wild and mutated viruses. Results: All 7 mutated pNL4.3 viruses were constructed successfully, and the mutations were D123E, V292I, K366R, T369A, T369V, A371V and I375V in RT region respectively. (1) Mutations A371V and T369V in RT could confer resistance to AZT, and the IC50 change folds of variants were 3.60 and 2.83 respectively. But the other 5 mutations decreased the IC50 of AZT, especially mutation T369A which decreased the IC50 by 35.67 times. (2) Mutation A371V and T369V could increase the EFV IC50, and the change folds were 4.55 and 2.87 respectively. The other 5 mutated viruses increased the susceptibility of virus to EFV, especially the I375V mutation. When I375V mutation occurred alone, the IC50 of EFV decreased to 1.23 nM while wild virus was 8.11 nM. (3) Mutation T369V conferred resistance to NVP by increasing IC50 by 11.55 times, while other 6 mutations had no impact on NVP susceptibility. Conclusions: Mutation A371V in RT region could cause low-level resistance to AZT (IC50 change fold was 3.60). T369V could cause low- and intermidiate-level resistance to EFV and NVP respectively (IC50 change fold were 4.55 and 11.55 respectively). The impact of mutations at codon 369 in RT ligation domain on AZT, EFV and NVP susceptibility was confirmed for the first time. T369A mutation could increase the susceptibility to AZT and EFV, while T369V resulted in the resistance to AZT, EFV and NVP.PartⅣAnalysis of HIV-1 quasispecies evolution of Indinavir resistance by Single-Genome AmplificationHIV-1 exists in vivo as quasispecies, and wild variants and resistanct variants coexist. And their proportion exhibit dynamic evolution and development during the selection pressure of drugs. Understanding the molecular evolution process is fundamental to analyzing the mechanism of drug resistance. Objective: The molecular evolution of drug resistance in one patient was analyzed. This patient had received reverse transcriptase inhibitors (RTIs) for a long time and recently replaced Nevirapine (NVP) with Indinavir (IDV). Methods: The patient, XLF, was followed-up six times successively. The viral populations were amplified and sequenced by single-genome amplification (SGA). All the sequences were submitted to Stanford HIV Drug Resistance Database (SHDB) for the analysis of genotypic drug resistance. Results: 149 entire protease and 171 entire reverse transcriptase sequences were obtained from these samples, and all sequences were identified as subtype B. Before the patient received Indinavir, the viral population only had some polymorphisms in the protease sequences. After the patient began Indinavir treatment, the variants carrying polymorphisms declined while variants carrying the secondary mutation G73S gained an advantage. As therapy was prolonged, G73S was combined with M46I/L90M to form a resistance pattern M46I/G73S/L90M, which then became the dominant population. 97.9% of variants had the M46I/G73S/L90M pattern at XLF6. During the emergence of protease inhibitors resistance, reverse transcriptase inhibitors resistance always maintained at high levels. Conclusion: Indinavir-resistance evolution was observed by single-genome amplification. During the course of changing the regimen to incorporate Indinavir, the G73S mutation occurred and was combined with M46I/L90M.