Study The New Types Of HIV-1 Drug Resistance Genotypic Testing And The Dynamic Changes Of Drug Resistance Viral Quasispecies

Object:It has been proved that the most effective method to contral and prevention AIDS is the highly active antiretroviral therapy(HAART).But the HIV virus is highly variable,with the poor medication adherence of treatment,and many other factors.Immediately,the drug-resistance of HIV has become a severe problem.It is very important to detect the drug resistance mutations in treatment experience patients and treatment naive patients.The genotypic methodof detection HIV drug resistance mutationsis better than phenotypic detection method for widely application.But there are some deficiencies in the genotypic method of detection HIV drug resistance mutations.For example,the plasma specimens are hard to transport for long distance and long time.Sequencing and sequence assemble are inevtable in the process of detection,Drug-resistance mutaions are routinely detected using standard Sanger Sequencing,which does not detect nior variants with a frequency below 20%.It is necessary to development new types of HIV-1 genotypic resistance testing.Analyze the changes of drug resistance viral quasispecies under long-term HAART treatment.Low-frequencyvariants have been correlated to treatment failures and HIV transmission,and detection of these variants is helping to inform strategies for treatment and vaccinedevelopment.Mothods:1.Improvement the HIV-1 Drug Resistance Genotypic Testing for using Dried Blood Spot Specimens,and evaluation this testing through amplication sensitivity,precision and reproducibility.2.Detection of Major Drug Resistance mutation for HIV-1 by use of a multiplex Allele-specific assay,and evalution this assay by clinical specimens3.Detection of HIV-1 drug resistance mutations usingillumine high-throughput sequencing and analyze the changes of drug resistance viral quasispecies under long-term HAART treatment.Results:1.The amplification rate of DBS specimens with plasma viral load of 1,000-6,000 copies/mL was 96.2%(76/79).The nucleotide sequence identity was 99.710.34%and 99.6±0.25%within and between test runs,respectively.Moreover,there was a near perfect agreement of detecting drug resistance mutations intra-and inter-test runs with kappa value of 0.972 and 0.963,respectively.Between 64 pairs of plasma and DBS specimens,the nucleotide identity was excellented with 99.5±0.34%.As compared to the results of plasma specimens,the sensitivity and specificity for detecting drug resistance mutations in DBS specimens were 99.4%(95%CI,97.4-99.8%)and 99.8%(95%CI,99.7-99.9%),respectively.Totally 15 discordant drug resistance mutations were found.Among them,53.3%(8/15)were caused by mixture base.2.Using suspension array technology,established a multiplex allele-specific(MAS)assay that can simultaneously detect major drug resistance mutations of HIV-1 subtype B’ at 7 loci.Including M41L,M184V,K103N,V106A,K70R,K219Q and Q151M,and the limits of detection were 25%,25%,12.5%,12.5%,12.5%,12.5%and 12.5%,respectively.For clinical specimens,the detection rate were 100%(16/16),85.7%(30/35),89.2(25/28),83.3%(10/11),100%(14/14),92.5%(25/27)and 87.5%(7/8),respectively.3.Based on the deep sequencing technology of illumina.Established an assay which can detect more than 1%of the minor resistant mutations in HIV-1 B’ Subtype.Set up a systemdata flow analysis process based on Geneious(?)software.The NGS method detected all drug resistance mutations which can be detected by Sanger sequencing method.If this drug mutation is caused by mixed base,the deep sequencing method can show the mixed base proportion of this mutation.By analyzing the changes of drug resistance viral quasispecies under long-term HAART treatment.1.Many NNRTIs related minor drug resistence mutations(1.8-17.4%)have been detected in patients who due to poor adherence of HAART.But those minor drug resistance mutations can not be detected by Sanger sequencing method.2.Under the long-termpresure of the same drug formula,thefrequency of drug resistance mutations show increasing form 1.1%to 97.1%by duration of treatment,significantly.3.Replace single drug3TC(Lamivudine)in treatment formula,that can be found the frequency of M184V mutations increase form 3.4%to 97.4%dramatically.4.In some final death patients,a large number of minor drug resistence mutations can be detected after 3 to 12 monthsbeginning of HAART treatment,but those minor drug resistance mutations can not be detected by Sanger sequencing method.5.Did not detect out Pls related minor drug resistance mutations in treatment failure patients who has been treated byPIs.Conclusion:This study foucs on the genotypic method of detection HIV drug resistance mutations.Based on the different choice of sample types,convenience of detection method and the demand for detection minor drug resistance mutations.Development three different types of HIV-1 genotypic resistance mutations detection methods.Those methods have their own characteristics which can besuited for different region and different purpose.we can get the prefect similar results in detection drug resistance mutations using the DBS specimens with viral load below 6,000copies/ml and plasma specimens.Using liquid phase chip technology can quickly to detect major drug resistance mutations.Set up a system data flow analysis process based on Geneious(?)software.Many NNRTIs related minor drug resistence mutations have been detected in patients who due to poor adherence of HAART.In some final death patients,a large number of minor drug resistence mutations can be detected shortly after HAART treatment.