| Mutations associated with resistance in viral genome result in decrease of drug sensitivity to human immunodeficiency virus (HIV). Many literatures show that there is a significant correlation between drug resistance and the effect of anti-retroviral therapy (ART). Drug resistance is an independent factor of the failure of ART besides plasma viral load (VL), the CD4 cell-count number and the history of ART. The aim of this research is to learn about the occurrence and prevalence of drug-resistant strains in some regions accepting ART at different times by cross-sectional monitoring, to investigate the possible time of drug resistance occurrence, patterns of drug resistance and evolution of these patterns through 176 HIV-1 infected persons in Henan province through consecutive 5 years monitoring, to find out the information about novel drug-resistant mutations in main prevalent HIV-1 isolates and to obtain the information of polymorphism of pol gene through this study.In conclusion, this study can provide information of the rule of occurrence and prevalence about resistance and evolutionary character of HIV strains, enrich recognization about resistance mutations, and lay a foundation to detect resistance strain accurately and improve ART. Part 1: Prevalence of HIV-1 drug resistance stains by cross-sectional monitoring in ART regions in ChinaPatients infected HIV-1 in eight provinces, including Henan, Hebei, Shandong, Beijing, Guangdong, Gansu, Ningxia, Guangxi, were investigated by cross-sectional monitoring from March, 2004 to September, 2008. Monitor durations in different regions were as following: 5 years in Henan, 3 years in Hebei, Shandong and Guangxi provinces, and 2 years in Beijing, Guangdong and Gansu provinces, only 1 year in Ningxia autonomous region.Ratio of viral inhibit was used to evaluate ART effect. The partial pol gene was obtained by RT-PCR to determine drug resistance, then the information of drug resistance was acquired by submitting the sequence to the web http://hivdb.stanford.edu. 1113 AIDS patients were investigated during course of 5 years, which added up 2263 person-time. General information about viral inhibit and occurrence of resistance was acquired. Results are as follows:1. ART achieved better performance (inhibit ratio reaching 94.34% in Guangdong ), which could detain AIDS progression effectively, in monitored provinces except Henan.2. Resistant strains existed in most monitored provinces, but it was more serious in Henan because occurrence of resistant-strain showed high prevalence.3. The direct inducement of high prevalence about resistance is non-adherence in monitoring group of Henan, whose adherence only kept in 75%.4.For there existed high relevancy between HAART failure and resistance(OR=0.942), so HAART failure was mainly caused by occurrence of resistance.5. Such measures as intensifying the therapy surveillance, strengthening education about anti-retroviral curative effect, propagandizing AIDS prevention, could enhance ART and detain occurrences of resistance strains and epidemic outbreak effectively:Part 2: Cohort study on the development and epidemic of drug resistance in Henan provinceAlthough some studies like cross-sectional monitoring have been made in China, cohort study which was very important in the investigation of drug resistance was rare even though in other countries of the world. A five-year open cohort study about HIV-1 infected persons was focused on the development of drug resistance in one rural area of Henan province. Study about appearance order, pattern and stability of resistance mutations had been done through 5 years consecutive follow-up, whose aim was to master the rule of occurrence and development of resistance so that effective measures could be empolied to decrease and detain resistance occurrence.173 patients were recruited and followed up 10 times in 5 years, adding up to investigate 1085 person-time and 6510 person-month. Results are as follows:1. The resistance to nucleoside reverse transcriptase inhibitors (NRTIs) occured about 10 months (median test) after ART in the patients with non-adherence (~75%), whose therapeutic regimen was AZT/DDI/NVP. Peak of resistance occurrence appeared from 6 to 12 months after therapy.2. The resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) occured about 6.5 months (median test), and peak of ratio of resistance occurrence appeared during course of 4 to 6 months after therapy.3. 60% mutations associated with NRTIs resistance were single mutations in the early stage of ART (within 1 year), then multiple mutations gradually rose beyond 1 year. More late single mutation occurred, more stable it was and more possible it developed multiple mutations, whose contributation to resistance was greater. In addition, many mutations could result in resistance if multiple mutations were detected at first detection. Probability of occurrence about multiple mutations and high resistance showed rise with extending of therapy time.4. Mutation K103N was most common, Y181C(14.20%) and G190A(13.40%) took second place among all mutations associated with NNRTIs. All of them were stabe and could result in high resistance to NNRTIs. With longer therapy duration the amounts of single mutations and multiple mutations showed rise gradually, and the probability of showing high resistance also rose.5.Single mutations associated with resistance to NRTIs, such as T215Y/E, D67N, M41L and K219Q/E/T, were relatively stable, which could exist longer than six months in the body. The single mutations of NNRTIs resistance, K103N, Y181C and G190A were very stable, besides the multiple mutations M41L/L210LW/T215Y, M41L/E44DE/T215Y, M41L/E44D/T69D/V118I/L210W/T215Y, D67N/K70R/T215Y/K219E and L210W/T215Y.6. There was a significant correlation between the appearance of drug resistance and CD4 cell-count number, plasma viral load, that was when drug resistance occurred, the counted number of CD4 cells decreased and VL increased remarkably. Part 3: Screening novel resistance mutations among main HIV strains in ChinaRoutine resistance detection of HIV focuses on regions binding drugs, namely only including full protease and former 300 codons of reverse transcriptase. However, recent researchs show that mutations beyond enzyme binding regions can bring important effect to resiatance. There exists unknown mutations associating with resistance possibly resulting from many factors in China, and no people have been engaged in this field about resistance mutations beyond reverse transcriptase 300 codons. 971 sequences , including 354 sequences of full RT, firstly were sorted to identify their subtypes, then sequences were contrasted with corresponding consensus sequence accrording to subtype to find different amino acid mutations. After the above work was finished, whether mutations are novel or not would be identified. Results are as follows:1. There was no difference about polymorphism of protease in subtype B between the patients accepted ART and the naive ( F=0.875, P=0.579 > 0.05 ) .Comparing mutations of B subtype with of CRF01_AE in pol gene,result showed there was also no difference between them(F=0.058,P=0.576>0.05),which was related to that protease inhibitors didn,t be emploied among ART patients.2. Whether drugs were used or not had no effect on variance of RT gene in subtype B between ART patiens and the naive(F=0.003,P=0.756>0.05),but resistance mutations could be screened rapidly in the presence of drugs(F=19.008,P=0.007<0.05).Mutations in RT regions between subtype B and CRF01_AE were of no difference among ART patients(F=2.497,P=0.057>0.05).3. Frequency of 21 mutations which can bring into resistance to NRTIs was significantly higher among the ART patients in subtype B than the naive(F=19.008,P=0.007<0.05).TAMs (48.7%)and M184V(12.10%) were dominant in all mutation patterns of NRTIs, and Q151M mutation combination (5.77%)was inferior to them. K103N and Y181C were primary mutations in mutations associating with NNRTIs.4. 2 mutations ,I13V and R57K, have significant difference in subtype B between the ART patients and the naive through comparing 99 amino acids, difference with consensus sequence in protease. Frequency of R57K was higher in the na?ve than in the treated, but frequency of I13V was on the contrary. It was possible that all of them were polymorphism mutations.5. 560 amino acids of whole reverse transcriptase were compared order by order among the consensus B sequence between the treated and the navie to find significative difference mutations. Result showed 21 mutations had significant difference between two groups. Frequency of 3 mutations, I135V,S162C and V245E was remarkablely higher in the treated than the na?ve, other 18 mutations were on the contrary. 14 mutations among 21 were rarely reported and their relatives with resistance was undefined.6. 14 mutations were contrasted one by one with sequences registered in HIV Databse of Stanford University, results showed 5 potential new mutations, L238I, K366R, T369V, A371V and I375V, were possibly associated with ART and resiatance.7. Frequency of 5 potential new mutations, L238I, K366R, T369V, A371V and I375V, was as following: 26.33%, 21.74%, 8.30%, 7.51% and .7.20%.8. Pattern of new circulating recombination form (CRF) possibly existeds in injecting drug users and commercial sex workers.9. There was a significant correlation between distribution of subtypes and routes of infection. The patients who infected by blood donation/transfusion mostly were subtype B, but the patients who infected by drug abuse and sex transmission varied. The distribution of HIV-1 subtypes were regionally obviously. The HIV-1 in Henan, Hebei and Shandong province was B subtype, and CRF01_AE in Guangxi province. |
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