ABSTRACT
ObjectiveTo evaluate the sensitivity and accuracy of an in-house detecting method of HIV-1 genotypic drug resistance system. MethodsTotally 130 serum specimens from Henan and Guangxi province were collected from April 2004 to October 2008 and tested in the Military HIV Testing Center of China. ViroSeqTM v2.0 (Abbott, Switzerland), a US FDA approved HIV genotypic drug resistance detecting system was utilized as the reference method. All the specimens were detected by the novel in-house method and the reference method to validate the difference in amplifying efficiency, drug resistance mutation detection and drug resistance report. ResultsConcerning the 14 850 known drug resistance mutation sites,14 752 (99. 3％ ) mutations can be detected by both of the two methods. Rates of concordance of detection in the regions of protease inhibitors-, reverse transcriptase inhibitors- and both two classes inhibitors-resistance were99.7％ ( Kappa =0. 909 9 , P ＜0. 01 ) , 99. 0％ (Kappa=0.952 1, P＜0. 01) and99.3％ (Kappa=0. 948 8, P ＜ 0. 01 ) respectively. Drug resistance reports from these two systems showed similar results (Kappa = 0. 637 4, P ＜ 0. 01 ). The in-house detecting system identified 34 novel mutations besides the ViroSeqTM drug resistance mutation database ( ViroSeqTM software v2. 7). Two mutations, V179F and K238T,had significant effect on HIV drug resistance. ConclusionsThe in-house genotyping system is an accurate,cost-effective method and has a high concordance with commercial ViroSeqTM genotyping system. Database from the in-house assay was superior to this of the ViroSeqTM assay.
ABSTRACT
Human Immunodeficiency Virus Type 1 exists in vivo as quasispecies, and one of the genome's characteristics is its diversity. During the antiretroviral therapy, drug resistance is the main obstacle to effective viral prevention. Understanding the molecular evolution process is fundamental to analyze the mechanism of drug resistance and develop a strategy to minimize resistance. Objective: The molecular evolution of drug resistance of one patient who had received reverse transcriptase inhibitors for a long time and had treatment which replaced Nevirapine with Indinavir was analyzed, with the aim of observing the drug resistance evolution pathway. Methods: The patient, XLF, was followed-up for six successive times. The viral populations were amplified and sequenced by single-genome amplification. All the sequences were submitted to the Stanford HIV Drug Resistance Database 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 the 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 maintained 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.
ABSTRACT
Objective To isolate stable passage primary HIV-1 drug resistant strains and observe replication dynamics of the drug resistant isolates and evolvement tendency of the drug resistant mutations in vitro.Methods Peripheral blood mononuclear cells(PBMCs)from 15 AIDS patients receiving highly active antiretroviral therapy(HAART)were collected,and the primary HIV-1 stains were separated utilizing co-cultivated with PBMCs from normal people.HIV-1 pol genes from those strains were obtained by RT-PCR and sequenced.The drug resistant mutations were analyzed in the Stanford HIV Drug Resistance Database.Results Eight strong positive strains were isolated from 15 AIDS patients with viral loads higher than 1000 copies/ml,and two of them were drug resistant.Drug resistant mutations of the two strains were respectively K103N/K238T and M184V/K103N/Y181C/H221Y which show high-level resistance to NVP and 3TC/NVP,respectively.K103N,M184V,Y181C and H221Y exist stably in the environment without drug pressure,however,RT K238T reverted to K238.Conclusion Two drug resistant strains were successfully isolated in vitro without drug pressure.Strains with K103N shows superior fitness and can exist steadily.Strains with M184V and K103N/Y181C/H221Y can also replicate stably in vitro without drug pressure.NNRTI mutation K238T reproduces astatically,which suggests that RT 238 codon might revert gradually to wild genotype.