| Background and objectiveAcquired immunodeficiency syndrome(AIDS)is a global public health problem that seriously affects human health.Antiretroviral therapy(ART)can inhibit HIV replication,reduce viral load,increase the life expectancy of infected persons,and reduce new infections.But ART cannot eradicate HIV,and patients need to take medicine for life.It may cause poor compliance due to adverse drug reactions,economic level,psychological factors and other reasons.ART stop is one of the extreme manifestations of poor compliance.At present,there are few studies on the characteristics of drug resistance during ART stop using second generation sequencing technology in China.And there are few reports that affect the effect of retreatment.In this study,the HIV genotype resistance detection method based on second generation sequencing will be established.Then study the changes of drug-resistant strains in patients with withdrawal and their impact on follow-up treatment to provide basic data for the sustainability of antiviral treatment.Methods1.In this study,the enzyme fragmentation method was used to construct the library.A detection method for HIV genotype resistance mutation sites based on the Illumina Miseq second generation sequencing platform was established.2.HIV-positive plasma samples with viral load?1000copies/mL were selected.Evaluate the feasibility of the method through library uniformity,sequencing depth and coverage.The accuracy,precision and repeatability of the method were evaluated based on amplification sensitivity,nucleotide consistency with first-generation sequencing,and intra-and inter-batch consistency.3.In order to further verify the genotype resistance detection method.A total of 332 samples were tested in parallel using genotype resistance testing methods based on first and second generation sequencing.Based on the results of first-generation sequencing,the sensitivity and specificity of second-generation sequencing to detect drug-resistant mutations under different detection thresholds were evaluated.4.Through a cross-sectional survey,211 HIV/AIDS patients who discontinued antiretroviral treatment were included in some areas of my country in 2016.Next generation sequencing was used to detect mutation sites at 5%and 20%thresholds.Analyze the investigation data of the research subjects and the changes of drug resistance mutation sites after drug withdrawal.5.Through a retrospective cohort survey,64 HIV/AIDS patients who had stopped ART from Queshan,Henan Province and Fuyang,Anhui Province were observed.The changes of viral load,CD4+T lymphocyte count and HIV drug resistance before and after drug withdrawal were analyzed.In addition,analyze the virus suppression situation and influencing factors of retreatment patients.Results1.Establish and evaluate HIV drug resistance detection methods based on second generation sequencing:The enzyme fragmentation method was used to construct the library and the HIV drug resistance detection method based on second generation sequencing was established.The average sequencing depth of the method is 109798 ×.And the minimum sequencing depth of each base position of the research object is above 10000×.The nucleotide sequence can cover the gene regions corresponding to the clinically significant drug resistance mutation sites in the PR and RT regions.In this study,NGS can detect all mutation sites detected by Sanger sequencing.When the site mutation frequency is higher than 10%,the detection results of the two sequencing methods are completely consistent.There was no difference between the results of 4 repeated trials of the same sample.The repeatability results of the wild-type pNL4-3 plasmid showed no mutations,indicating that the method is more stable.When the threshold is set to 1%or 5%,there is a statistical difference between the detection methods based on the first and second generation sequencing,and when the threshold is 1%,the consistency between the two methods is low(?=0.0941).As the threshold increased from 1%to 20%,the sensitivity of second generation sequencing decreased,while the specificity increased from 16.7%to 94.7%.In addition,the presence of non-specific mutation sites such as K65ER,D67GEN,F77L,L100VI was not detected above the 5%threshold,but the frequency was higher at the 1%threshold.The drug resistance results showed that NGS could detect 56.9%of mutation sites that were not detected by SS.But in 3.6%of patients,the mutation frequency of drug resistance sites was more than 20%only detected by NGS.In 0.9%of patients,drug resistance sites were not detected by NGS but by SS,mainly caused by mixed bases.2.Results of the 2016 cross-sectional survey of patients who discontinued ART in some areas:A total of 211 HIV/AIDS patients who discontinue ART were included in this cross-sectional survey.They received ART from 2010 to 2014,and the drugs they took before the withdrawal were mainly first-line drugs containing 3TC.There were 174 patients with discontinued virus whose viral load was?1000copies/mL.The results showed that 55(26.1%,55/211)patients had drug resistance mutation sites,and the drug resistance sites were mainly M46VI,K65R,K103N,E138AG and G190AE.80.7%of the patients with viral load?1000copies/mL in patients who were discontinued for 1-12 months.The proportion of patients with viral load?1000 copies/mL for 12 months and above was 84.3%.27.5%of the patients who stopped the drug for 1-12 months carried drug resistance sites.However,24.5%of patients who withdrew for 12 months and above carried drug resistance sites.Chi-square test results of drug withdrawal time,viral load,and drug resistance rate show that the longer the drug withdrawal time,the higher the proportion of viral load?1000 copies/mL(p<0.0001),and the lower the drug resistance rate(p<0.0001).In addition,the results show that HIV/AIDS patients can detect PIs mutation sites such as D30N,M46VI,Q58E,N88D,NRTIs mutation sites such as K65R,D67G,and NNRTIs mutation sites K103N,V106AM,after 1-12 months of drug withdrawal.E138AG,V179DE,G190AE,Y188CL,P225H.The sites that still existed when patients stopped taking drugs for 12 months and above were M46VI,K65R,K103N,E138AG,V179DE,G190AE,and Y188CL,.3.Analysis of drug resistance in patients with withdrawal after receiving early ART:The discontinued patients included in this part of the study started receiving ART in 2003-2004.The treatment regimen before drug withdrawal is based on the early first-line regimen(AZT/d4T+ddI+NVP).There were 64 people before drug withdrawal,and 52 and 40 people were followed up after 12 and 24 months of drug withdrawal,respectively.According to the World Health Organization(WHO)standards,the viral load after treatment is less than 1000copies/mL for viral suppression.The patients were divided into two groups according to the viral load before ART stop.The viral load was less than 1000copies/mL as the virus suppression group,and the viral load was higher than or equal to 1000copies/mL as the virus uninhibited group.The results showed that the proportion of viral load?1000 copies/mL of 30 patients in the viral suppression group at 12 and 24 months after drug withdrawal were 84.0%(21/25)and 100.0%(21/21).The viral load of patients in the virus uninhibited group remained above 1000copies/mL after drug withdrawal.The median of CD4+cells in patients with withdrawal dropped from 285(IQR 175-443)/?L before withdrawal to 223(IQR 111-345)and 282(IQR 177-348)pcs/?L.The results showed that the resistance sites in NRTIs were mainly K65RE,and it and V75TIA and Q151M sites still exist after 12 months of drug withdrawal.The drug resistance sites such as M41L,K70N,L74V,F116Y,K219E disappeared after 12 months of withdrawal.The most common NNRTIs resistance sites were K103ENST(95.2%),Y181C(38.1%),and G190AE(19.1%),which gradually decreased with the extension of drug withdrawal time,and could still be detected 24 months after drug withdrawal.The drug resistance sites such as K101E,H221Y,F227L,M230I,K238NT and Y188L have not been detected at 12 months or 24 months after drug withdrawal.The evolutionary pressure of single amino acid mutation and the results of co-mutation analysis showed that the disappearance of mutation sites L74V and V75T were related to sites K103N and Y188L,respectively.In addition,L74V,Q151M and T215Y are all affected by the site Y181C.Combined with the results of the disappearance of the mutation site of patients who stopped the drug,it was found that 9.5%of patients with the mutation site L74V and Y181C disappeared together.The mutation sites Q151M and Y181C disappeared together at a rate of 4.8%.Also in 4.8%of patients,the T215Y site and K103N and Y181C mutation sites disappeared together.The results of the COX regression model of virus suppression after re-treatment showed that the sex,duration of drug withdrawal,and ART regimen were the influencing factors of virus suppression after re-treatment.Although the mutation site of the patients before re-treatment has no effect on the virus suppression of the patients who retreated.However,at the 5%threshold,patients with drug resistance that are resistant to both NRTIs and NNRTIs tend to have a lower viral suppression rate after retreatment.Conclusions1.The library constructed by enzyme fragmentation method is evenly distributed in the pol gene region.It can meet the requirements of HIV drug resistance genotype detection based on the next generation sequencing method.2.The HIV genotype drug resistance detection method based on second generation sequencing is established.Its accuracy,repeatability and precision are better.Compared with the first generation sequencing results,the accuracy,sensitivity and specificity are higher at the 5%threshold,and low frequency drug resistance sites can be detected.3.After stopping ART,HIV/AIDS patients receiving ART have increased viral load and decreased CD4+cell count.Mutation sites such as K65R,L74V,V75T,Q151M,Y188L,H221Y gradually disappeared with the time of ART stop.And the mutation sites of L74V,Q151M and Y181C disappeared synergistically.The K103N,E138A,G190A and other NNRTIs-related mutations can still exist after 12-24 months of ART stop,but the frequency of mutations decreases as the time of ART stop increases.4.The gender,duration of drug withdrawal and ART regimen of patients with drug withdrawal are the influencing factors of viral suppression after re-treatment. |
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