Study On HIV-1Primary Drug Resistance,Subtype With Viral Tropism Of Drug-naive AIDS Patients And Evaluation Of Therapeutic Efficacy And Secondary Drug Resistance Of Special AIDS Patients In Some Parts Of China

It is well known that highly active antiretroviral therapy （HAART） is the most effective therapeutics for HIV/AIDS patients. However, Drug resistance of HIV-1is the main obstacle for it. Of not only the primary drug resistance in treat-free patients but also the secondary drug resistance in treat-experience patients, HIV-1mutation to drugs can induce the failure of antiviral therapy and accelerate the transmission to new population finally. Moreover, special viral subtype may play an important role in improving AIDS fast progression. Hereby, it will be very helpful to understand the epidemical of HIV primary drug resistance and to conduct the adjustment of treat strategy in AIDS patients via analyzing on prevalence of HIV-1primary drug resistance and subtype and viral tropism. Besides, due to unbalance healthy resource and relative shortage of antiviral medicine along with complicated situation, most special patients such as co-infected by HBV or first-line failure patients have not received comfortable evaluation and seasonable therapy yet.PART I. Prevalence of primary drug resistance and subtype with viral tropism of treatment-naive AIDS patientsSection I. Prevalence of genotypic primary drug resistance of HIV-1and viral subtype in ChinaObjective:To investigate the genotypic primary drug resistance and distribution of HIV-1subtype along with viral tropism in drug-naive AIDS patients in some parts of China.Methods:Total914adult drug-naive AIDS patients were collected from twenty areas of China from1991to2010. The entire protease gene and255codons of the reverse transcriptase of HIV-1were amplified and sequenced. Furthermore, genotypic drug resistance and their subtype were analyzed with Stanford database and software. Based on the bio-math system model, Ka/Ks ration was calculated with different virus pol sequences and identified the pol gene mutation patterns under different selection pressures.Results:（1） A total of82.5%（754/914） pol gene sequences were amplified successfully. In which were70.9%cases transmitted by sexual. The average prevalence of primary drug resistance was4.64%（35/754） during1990-2010including3.97%（30/35） and0.66%（5/754） mutation for one and two family, respectively. The primary resistance incidence of last five years was8.67%, significant higher than other prior time period:1.61%within<1995（p=0.01）,2.0%within1996-1999（p=0.004） and4.58%within2000-2004（p=0.013）.（2） The proportion of primary drug resistance genes for RT was68.57%, while42.86%for Pro gene.There were no significant difference of primary mutation among NRTI（1.99%）, NNRTI（1.72%）and PI（1.59%）, p=0.759. High level resistance were observed in NNRTIs （10/13,76.9%） rather than in NRTIs （1/15,6.7%） and PIs （3/12,25.0%）。The most common NRTI mutations in TAMs was K70NR （11.11%） and L210WM （8.33%）. Non-TAMs area was V75IML （13.89%） and V118I （13.89%）, the proportion of multi-drug resistance was T69Ins13.89%. Most common NNRTI mutation were K103N （13.89%）, V179AE （11.11%） and Y181C （11.11%）. M46I （11.11%） in PI was common mutation, followed by N88D （8.33%）, N83K （8.33%） and A71T （8.33%）. There were34.1%（11/35） cases of primary drug resistance patients from Beijing.（3） Ka/Ks ratio displayed that during2002-06years and2007-10years, HIV-1pol gene received the strong positive selection pressure with a significant increase in the mutation rate. Ka/Ks ratio higher areas were mainly distributed in the pol gene of the protease gene （36-99aa）, the RT gene RT-1（162-333aa） and RT-thumb （337-406aa） matching up national therapy time-window. Cumulative Ka/Ks ratio reflects the dynamic frequency of pol gene mutation may reflect national the changing treatment strategy and implementation of the program.（4） In the754cases of patients, the main subtypes were AE subtype227cases （30.13%）, B/B’subtype205cases （27.18%） and BC subtypes187cases （24.82%）. Prior to1995AIDS patients were infected by subtype B about60%, but by2010was reduced to about13%（p<0.05）; whereas AE subtype in the early1990s, less than1%, has now risen to47.3%（p<0.01）. But the prevalence of07/08BC was stabile20.8-25.9%within two decades.（5） Of754patients with subtype distribution were regional differences, subtype B/B’mainly in Henan （76.4%）, Beijing （26.8%）, Shaanxi （35.0%） and Shanghai （21.9%）. AE subtype mainly in Beijing （32.6%）, Shanghai （37.0%）, Guangdong （42.1%）, Fujian （40.4%） and Shaanxi （40.0%）.07/08_BC subtypes were mainly distributed in Yunnan （58.0%）, and Guangdong （23.8%）, Beijing （24.7%） and Fujian （21.7%）.（6） In the transmitted pathway study of754cases,520cases with sexual transmission infection （69.0%）, of which314cases of heterosexual contact （41.7%）,206cases of homosexual sexual contact （27.3%）; only152patients （20.2%） infection by blood. About38.8%percent were CRF01_AE subtype in full sexual infected patients （42.7%from homosexual and36.3%from heterosexual transmission of infected persons）. The incidence of8.78%（18/205） primary mutation in B subtype was significant higher than5.29%（12/227） and1.07%（2/178） in CRF₀1AE and in07BC/08BC, respectively （p=0.017）.Conclusion:It is the first report of primary drug resistance during the past20years in parts of China. We found that the average rate was4.64%of primary drug resistance. It was from the earliest1.61%gradually increased to the current8.67%, about increased five-fold within two decades mainly mutated resistance to RT area. Computational Biology Ka/Ks ratio reflects the dynamic pol gene mutation frequency in the types of medicines as well as treatment strategies and implementation impact from the programs by the government. Except IDU individuals, treated-free patients of B subtype in the early years were given main proportion to ones of AE subtype recently consisting with the change of transmission from blood to sexually route. The common subtype by sexual transmission was AE subtype with different prevalence area distributions.Section â…¡. HIV-1AE subtype is associated with X4tropism and fast HIV progression in patients infected through sexual transmissionObjective:To research the relationship between molecular epidemiology of the HIV-1CRF01_AE subtype as a possible risk factor and fast HIV-1progression in drug-naive patients.Methods:We analyzed HIV-1tropism by utilizing samples from201treatment-naive patients in our multicenter cohort （12research centers in different provinces of China）. Tropism was determined by V3loop sequencing. Data from235treatment-naive patients infected sexually （including aforementioned201patients） in this cohort with the estimated date of seroconversion （EDS） were retrospectively evaluated. Median time from EDS to AIDS was analyzed by Kaplan-Meier curves. Hazard ratios were determined by Cox proportional model.Results:CRF01_AE subtype was predominant （46.0%）, especially in the men having sex with men （MSM） group. Further analysis revealed that the proportion of X4tropism was higher in the CRF01_AE subtype （45.5%） than in others （C/CRF07_BC/CRF08_BC,4.3%; B,6.1%; p<0.001）. CRF01_AE subtype was associated with faster progression from EDS to AIDS （4.8vs.6.4years, p=0.018） compared with non-CRF01_AE subtypes. In a multivariate model, the adjusted hazard ratio （aHR） of CRF01_AE was1.42（95%CI,0.99-2.03, p=0.057）, independent of HIV-1viral load; it was also associated with fast progression to advanced immunodeficiency （aHR,1.81,95%CI,1.03-3.18, p=0.038）.Conclusion:CRF01AE, a predominant HIV-1subtype in Chinese HIV-1sexually infected patients, tends to be associated with fast progression to AIDS and advanced immunodeficiency, which might be ascribed to high proportion of X4tropism. Further investigation of these risk factors may have significant implications to clinical practice and policy-making.PART â…¡. Evaluation of Therapeutic Efficacy and Secondary Drug Resistance of Special AIDS patients in some parts of ChinaSection â… . Evaluation of efficacy and emergence of drug resistance in HIV-HBV co-infected patients in China:2-year multi-central pilot studyObjective:To study the efficacy of suppression of HIV and HBV viral load with HAART including3TC-or3TC+TDF regimens and the occurrence of secondary drug resistance in HIV-HBV co-infected patients during2years treatment in China.Methods:Total55AIDS patients with HBsAg positive for at least six months were selected from913enrolled subjects.3TC+TDF+EFV/NVP or3TC+AZT/d4T+EFV/NVP were for patients within96weeks. HIV-RNA and HBV-DNA viral load were detected at baseline,12weeks,48weeks and96weeks from plasma. All samples were detected HIV and HBV drug resistance against3TC using Luminex fluorescent beads and common Sanger sequencing. Using One-way ANOVA repeated measures test （Kruskal-Wallis nonparametric test） to compare CD4cell counts, HIV RNA and HBV DNA before and during3TC treatment at baseline and at each follow-up point.Results:After96weeks on HAART, CD4count at baseline with149/ul was up to281/ul at96weeks. Both HIV-1RNA and HBV-DNA loads were lower to the detection limit （<20copies/ml and <20IU/ml）. HIV-1RNA was significantly reduced in the treatment of12W. According to different groups, the rate of HIV VL decline in3TC+TDFgroup was slightly lower than in only3TC group which can play more powerful efficacy to control HBV-DNA load almost100%suppression at48weeks. No case appeared rebound of HBV-DNA in3TC+TDF group, while in3TC group,26.7%（12/45） appeared rebound of HBV-DNA. HBV resistance mutations rt204rt180against3TC was about60%and80%, respectively. Only one patient （2.2%） in the group3TC was found resistance mutations for HIV at Ml84V and K103N.Conclusion:This study is the first report of efficacy on HIV-HBV co-infected patients and drug resistance analysis during two years HAART.3TC or3TC+TDF-containing HAART regimen was able to control HIV and HBV viral replication. But it was a little bit weak effective anti HBV using only3TC-group. Moreover,3TC monotherapy to HBV can lead to its resistance mutations after long-term treatment.Section â…¡. Efficacy and drug resistance of TDF/3TC/LPVr in treated failure of HIV-infected Chinese adults:a2-year prospective multicenter pilot cohort studyObjective:To evaluate the efficacy of the second-line highly active antiretroviral therapy regimen TDF/3TC/LPVr in AIDS patients with resistance to first-line anti-retroviral drugs in China.Methods:84eligible AIDS patients were studied prospectively for2years. CD4count and HIV-1VL were measured at baseline and each subsequent visit. Genotype drug resistance testing was performed at baseline and on VL rebound in house. The plasma lopinavir concentration was determined by a validated HPLC coupled to UV detection. Additional, routine clinical laboratory including serum creatinine, eGFR and CRcl were detected.Results:At baseline,47.6%,19.0%and17.9%patients had high-or middle-level resistance to3TC or TDF or dual resistance, respectively. The common resistant mutation were TAMs、M184V、K103NS and Y181CI. After2years of therapy, the median CD4count increased from191to341cells/mm3（P<0.001）, and the median VL decreased from4.39to<1.60log10（cps/mL）（P<0.001） in ITT assay.66.7%patients had a VL<40cps/mL while VL failure or rebound occurred in11patients.In72.7%（8/11） patients the absence of valid drug levels indicated poor adherence.68drug-related adverse events were reported in26.6%of patients. Gastrointestinal disorders, hepatotoxicity and rash were common.94.1%adverse events （64/68） were grade â…  or â…¡. The levels of serum creatinine, eGFR and CrCl showed significant difference （P=0.000） compared to baseline at48and96weeks.Conclusions:This study indicates that TDF/3TC/LPVr （even LPVr monotherapy） can suppress HIV replication and improve renal function for2years in first-line drug resistance patients in China. Measuring drug plasma concentration can help to monitor adherence and to evaluate clinical efficacy.