【摘要】： HIV耐藥性是指由于發(fā)生特定的病毒基因突變,導(dǎo)致病毒對藥物敏感性下降的現(xiàn)象。大量研究顯示,耐藥性與抗病毒治療效果之間存在顯著的相關(guān)性,在控制了血漿病毒載量、CD4細(xì)胞計(jì)數(shù)和抗病毒治療歷史等因素以后,耐藥性是獨(dú)立的導(dǎo)致抗病毒治療失敗的因素。本研究通過對我國部分省份艾滋病病人的多次橫斷面研究,闡明在接受抗病毒治療后,HIV-1耐藥性毒株在不同時(shí)間點(diǎn)的發(fā)生和流行水平;以河南省某地的176名艾滋病患者為對象,通過5年的隊(duì)列研究,分析針對不同藥物的耐藥發(fā)生的可能時(shí)間、耐藥突變發(fā)生模式及其隨時(shí)間的演變;了解在我國特有治療模式下主要流行毒株中存在的新型耐藥突變發(fā)生情況及pol區(qū)基因的多態(tài)性,最終為認(rèn)識我國HIV-1耐藥毒株的發(fā)生和流行規(guī)律、了解耐藥性HIV-1毒株的分子進(jìn)化特征、豐富對耐藥相關(guān)突變的認(rèn)識提供依據(jù)、為準(zhǔn)確檢測耐藥毒株、提高艾滋病抗病毒治療效果奠定基礎(chǔ)。 第一部分我國部分地區(qū)HIV-1耐藥性毒株流行的橫斷面研究 2004年3月至2008年9月,以河南、河北、山東、北京、廣東、甘肅、寧夏及廣西8省市的艾滋病病人為研究對象,進(jìn)行多次橫斷面研究,其中河南連續(xù)監(jiān)測5年,河北、山東、廣西連續(xù)監(jiān)測3年,北京、廣東、甘肅連續(xù)監(jiān)測2年,寧夏監(jiān)測1年。通過病毒抑制率分析抗病毒治療的效果;使用RT-PCR方法獲得pol區(qū)目的基因,提交序列到斯坦福大學(xué)HIV耐藥數(shù)據(jù)庫獲得耐藥信息。共調(diào)查艾滋病患者1113名,累積研究2263人次,了解和掌握了抗病毒治療地區(qū)治療后病毒抑制和耐藥發(fā)生情況。主要結(jié)果如下: 1.在監(jiān)測省份,除河南省外,艾滋病抗病毒治療均取得了良好的效果(病毒抑制率最高可達(dá)94.34%),延緩了艾滋病的進(jìn)程; 2.耐藥毒株在多個(gè)省份都有發(fā)生,但在河南呈高度流行趨勢(約65%); 3.河南耐藥監(jiān)測人群中,服藥依從性僅能維持75%左右,依從性差是導(dǎo)致該地區(qū)耐藥毒株高發(fā)的直接原因; 4.耐藥發(fā)生與抗病毒治療失敗之間呈高度相關(guān)(OR=0.942),耐藥是導(dǎo)致抗病毒治療失敗的關(guān)鍵因素; 5.應(yīng)加強(qiáng)治療監(jiān)管和抗病毒治療教育、提高服藥依從性,以延緩HIV-1耐藥的發(fā)生與流行,提高治療成功率。 第二部分河南省HIV-1耐藥性毒株發(fā)生和流行的隊(duì)列研究 當(dāng)前我國已經(jīng)開展了多次HIV耐藥的橫斷面研究,但是隊(duì)列研究還不多,在世界范圍內(nèi)也比較少見,而這樣的研究對于揭示耐藥的發(fā)生發(fā)展規(guī)律是非常重要的。我們對河南省某鄉(xiāng)接受抗病毒治療的艾滋病病人進(jìn)行了5年開放式隊(duì)列研究,通過分析不同時(shí)間點(diǎn)耐藥突變出現(xiàn)的先后順序、耐藥突變發(fā)生和演變的模式等,分析在當(dāng)?shù)靥厥庵委熌Ｊ较?耐藥性突變發(fā)生發(fā)展的規(guī)律。隊(duì)列共納入173例艾滋病患者,隨訪了1085人次、6510人月,最多的隨訪10次。主要結(jié)果如下: 1.服藥依從性保持在75%左右的、使用AZT/DDI/NVP治療方案的艾滋病患者,針對NRTIs發(fā)生耐藥的時(shí)間中位數(shù)是抗病毒治療后10個(gè)月左右,耐藥發(fā)生高峰在治療后6-12個(gè)月; 2.該監(jiān)測人群針對NNRTIs耐藥的發(fā)生時(shí)間中位數(shù)為治療后6.5個(gè)月,在治療后4-6個(gè)月耐藥突變發(fā)生率最高。 3.首次發(fā)生的NRTIs耐藥突變,早期(治療1年內(nèi))發(fā)生的60%是單一位點(diǎn)突變,1年后以突變型形式出現(xiàn)的增多。NRTIs單一突變發(fā)生越晚,保持穩(wěn)定或發(fā)展為突變型的可能性越大,對耐藥的貢獻(xiàn)也越大。首次發(fā)生的NRTIs耐藥突變,如果是突變型,則多數(shù)導(dǎo)致中高度耐藥。隨治療時(shí)間的延長,突變型出現(xiàn)并導(dǎo)致中高度耐藥的概率呈上升趨勢。 4.以單一位點(diǎn)形式出現(xiàn)的NNRTIs耐藥突變,K103N最多(32.3%),Y181C(14.20%)和G190A(13.40%)次之,它們均可穩(wěn)定存在并導(dǎo)致對NNRTIs的高度耐藥。隨治療時(shí)間延長,以單一位點(diǎn)突變和突變型形式出現(xiàn)的NNRTIs耐藥突變逐漸增加,導(dǎo)致高度耐藥的可能性亦增加。 5. NRTIs單突變位點(diǎn)M41L、D67N、T215Y/F與K219Q/E/T相對穩(wěn)定,可在體內(nèi)穩(wěn)定存在6個(gè)月以上。突變型M41L/L210LW/T215Y、M41L/E44DE/T215Y、M41L/E44D/T69D/V118I/L210W/T215Y、D67N/K70R/T215Y/K219E、L210W/T215Y都比較穩(wěn)定。NNRTIs穩(wěn)定的單位點(diǎn)突變?yōu)镵103N、Y181C與G190A, 6. HIV-1耐藥的發(fā)生與CD4、病毒載量具有高度相關(guān)性,即耐藥發(fā)生時(shí)CD4下降、病毒載量明顯上升。 第三部分新型HIV-1耐藥相關(guān)突變位點(diǎn)的篩選 以往HIV-1耐藥檢測多數(shù)只針對藥物的結(jié)合區(qū),即只局限于蛋白酶與逆轉(zhuǎn)錄酶氨基酸密碼子300位以前。最新的研究提示,酶結(jié)合以外區(qū)域的基因突變也有可能對耐藥產(chǎn)生重要影響。在我國,多種因素導(dǎo)致可能存在未被認(rèn)識的HIV-1耐藥相關(guān)突變位點(diǎn),且我國還沒有針對RT區(qū)300位以外密碼子耐藥突變的研究。我們針對前期研究獲得的接受和未接受抗病毒治療的的971條HIV-1 pol基因序列(其中354條是包含300位密碼子以外的RT全長序列),首先進(jìn)行基因分型,然后分別與相應(yīng)亞型的共享序列進(jìn)行逐個(gè)密碼子比對,篩選在接受與未接受抗病毒治療的序列中存在顯著差異的突變位點(diǎn),然后再檢索該突變位點(diǎn)在數(shù)據(jù)庫中的收錄情況,確定該位點(diǎn)是否可能為新型耐藥相關(guān)突變位點(diǎn)。主要結(jié)果如下: 1.HIV-1 B′亞型治療與未治療人群序列中,蛋白酶抑制劑耐藥相關(guān)突變的發(fā)生率無顯著差異(F=0.875,P=0.5790.05),B′亞型與CRF01_AE亞型治療人群中蛋白酶抑制劑耐藥突變的發(fā)生率亦無顯著性差異(F=0.058,P=0.5760.05),這與多數(shù)病人未使用蛋白酶抑制劑治療有關(guān); 2.HIV-1 B′亞型治療與未治療序列中,藥物的使用與否對pol區(qū)基因序列的基因變異影響不大(F=0.003,P=0.7560.05),但藥物存在加快了對耐藥突變位點(diǎn)的選擇(F=19.008,P=0.0070.05)。HIV-1 B′亞型與CRF_01AE亞型pol區(qū)基因序列突變發(fā)生沒有顯著差異(F=2.497,P=0.0570.05)。 3.HIV-1 B′亞型治療序列中,21個(gè)逆轉(zhuǎn)錄酶抑制劑耐藥相關(guān)突變位點(diǎn)的發(fā)生率顯著高于未治療序列(F=19.008,P=0.0070.05)。治療序列中NRTIs的耐藥突變模式以TAMs和M184V為主,分別為48.7%和12.1%,其次為Q151M突變復(fù)合體(5.77%);NNRTIs耐藥突變以K103N和Y181C為主。 4.分析HIV-1 B′亞型治療與未治療序列中蛋白酶區(qū)密碼子突變,通過與蛋白酶共享序列99個(gè)密碼子逐一核對,發(fā)現(xiàn)2個(gè)突變(I13V和R57K)在兩組序列中有差異,R57K在未治療序列中出現(xiàn)的頻率顯著高于治療序列,I13V則相反。這2個(gè)位點(diǎn)可能都是多態(tài)性突變位點(diǎn)。 5.將B′亞型共享序列與相應(yīng)亞型治療序列和未治療序列逆轉(zhuǎn)錄酶全長560個(gè)密碼子逐一比對,發(fā)現(xiàn)21個(gè)突變位點(diǎn)在兩組人群中具有顯著性差異,3個(gè)突變位點(diǎn)(I135V、S162C、V245E)的發(fā)生率在未治療序列顯著高于治療序列,18個(gè)突變位點(diǎn)的發(fā)生率在治療序列中顯著高于未治療序列,其中有14個(gè)是報(bào)道較少且與耐藥相關(guān)性不明確的位點(diǎn)。 6.將初步篩選的14個(gè)突變位點(diǎn)逐個(gè)與美國斯坦福大學(xué)HIV耐藥數(shù)據(jù)庫中收錄序列進(jìn)行比較,確定L238I、K366R、T369V、A371V、I375V可能是與抗病毒治療及耐藥相關(guān)的突變。 7.逆轉(zhuǎn)錄酶區(qū)300位密碼子以后可能與耐藥相關(guān)的突變位點(diǎn)的發(fā)生率分別為:L238I為26.33%、K366R為21.74%、T369V為8.30%、A371V為7.51%、I375V為7.20%。 8.在我國以吸毒、性接觸為主要感染途徑的人群中,可能存在HIV-1毒株的新型重組模式,即CRF_BC重組毒株與B′亞型的二次重組。 9.HIV-1亞型分布與感染途徑密切相關(guān),以獻(xiàn)/輸血為主要感染途徑的人群主要以B′亞型為主,而吸毒、性傳播途徑的人群亞型則較為豐富。HIV-1分布地域特征明顯,在河南、河北、山東等地區(qū)地區(qū)以B′亞型為主,而在廣西則以CRF01_AE為主。
[Abstract]:HIV resistance refers to the decline in drug susceptibility due to specific mutations in viral genes. A large number of studies have shown that there is a significant correlation between drug resistance and antiviral efficacy. Drug resistance is independently guided by factors such as plasma viral load, CD4 cell count and antiviral treatment history, etc. Factors contributing to the failure of antiretroviral therapy. Through a series of cross-sectional studies on AIDS patients in some provinces of China, this study clarified the occurrence and prevalence of HIV-1 drug-resistant strains at different time points after antiretroviral treatment; and 176 AIDS patients in a certain area of Henan Province were selected as subjects and analyzed through a five-year cohort study. The possible time of drug resistance, the pattern of drug resistance mutation and its evolution with time; the occurrence of new drug resistance mutation and the polymorphism of pol gene in the main epidemic strains under the unique treatment mode in China; and finally to understand the occurrence and epidemic regularity of drug-resistant HIV-1 strains in China, and to understand the drug-resistant HIV-1 strains. The molecular evolutionary characteristics of the virus strains enrich the knowledge of drug resistance-related mutations, and lay a foundation for accurate detection of drug-resistant strains and improving the efficacy of anti-HIV treatment.
Part one cross-sectional study on the prevalence of HIV-1 drug-resistant strains in some areas of China
From March 2004 to September 2008, cross-sectional studies were carried out on AIDS patients in Henan, Hebei, Shandong, Beijing, Guangdong, Gansu, Ningxia and Guangxi provinces and cities. Among them, 5 years of continuous surveillance in Henan, 3 years of continuous surveillance in Hebei, Shandong and Guangxi, 2 years of continuous surveillance in Beijing, Guangdong and Gansu, and 1 year of continuous surveillance in Ningxia. To analyze the effect of antiviral therapy, the target gene of Pol region was obtained by RT-PCR, and the sequence was submitted to Stanford University HIV resistance database to obtain drug resistance information.
1. Except Henan Province, the anti-HIV treatment in the monitoring province has achieved good results (the highest inhibition rate of the virus can reach 94.34%) and delayed the progress of AIDS.
2. drug-resistant strains occurred in many provinces, but showed a high prevalence in Henan (about 65%).
3. In Henan drug resistance surveillance population, drug compliance can only maintain about 75%, poor compliance is the direct cause of the high incidence of drug-resistant strains in this area;
4. The occurrence of drug resistance was highly correlated with the failure of antiviral treatment (OR = 0.942). Drug resistance was the key factor leading to the failure of antiviral treatment.
5. We should strengthen treatment supervision and antiviral treatment education, improve drug compliance, to delay the occurrence and epidemic of HIV-1 resistance, improve the success rate of treatment.
The second part is a cohort study on the occurrence and prevalence of HIV-1 drug-resistant strains in Henan province.
At present, China has carried out a number of cross-sectional studies on HIV resistance, but the cohort study is still rare in the world, and such research is very important to reveal the occurrence and development of drug resistance. By analyzing the sequence of drug resistance mutations at different time points, the pattern of drug resistance mutation occurrence and evolution, and the regularity of drug resistance mutation occurrence and development under the local special treatment mode, 173 AIDS patients were enrolled in the cohort, followed up 1 085 people, 6510 people a month, the most 10 times.
1. Drug compliance was maintained at about 75%. The median time of drug resistance to NRTIs was about 10 months after antiviral treatment in AIDS patients treated with AZT/DDI/NVP regimen. The peak of drug resistance was 6-12 months after treatment.
2. The median duration of NNRTIs resistance was 6.5 months after treatment, and the highest incidence of NNRTIs resistance mutation was 4-6 months after treatment.
3. The first NRTIs resistant mutation, 60% of which occurred in the early stage (within one year of treatment) was single site mutation, and increased in the form of mutation after one year. The later the single NRTIs mutation occurred, the greater the possibility of stability or development of mutation, the greater the contribution to drug resistance. The number of mutants leads to moderate to high drug resistance, and the probability of mutants leading to moderate to high drug resistance increases with the duration of treatment.
4. The NNRTIs resistance mutations in the form of single locus were most common in K103N (32.3%), followed by Y181C (14.20%) and G190A (13.40%). Both of them could be stable and lead to high resistance to NNRTIs. Increase.
5. NRTIs single mutation sites M41L, D67N, T215Y/F and K219Q/E/T are relatively stable, and can be stable in vivo for more than 6 months. Mutant M41L/L210LW/T215Y, M41L/E44DE/T215Y, M41L/E44D/T69D/V118I/L210W/T215Y, D67N/K70R/T215Y/K219E, L210W/T215Y are relatively stable. The mutant M41L/L210L210LW/T215Y, M41L/E44D/T69D/T69D/V118I/L210W/T215Y, and L210W/T215Y are stable.
6. The occurrence of HIV-1 resistance is highly correlated with CD4 and viral load, that is, CD4 decreases and viral load increases significantly when drug resistance occurs.
The third part is the screening of new HIV-1 resistance related mutation sites.
In the past, most HIV-1 drug resistance tests only focused on the drug-binding region, that is, only limited to the protease and reverse transcriptase amino acid codon before 300. Recent studies suggest that mutations in the region outside the enzyme-binding may also have an important impact on drug resistance. In our country, 971 HIV-1 pol gene sequences (354 of which are full-length RT sequences containing codons other than 300) received or not received antiviral therapy were studied. Genotyping was performed first, and then the corresponding subtypes were identified. After codon-by-codon alignment, the mutant sites with significant differences between those receiving antiviral treatment and those not receiving antiviral treatment were screened, and then the data of the mutant sites in the database were retrieved to determine whether the mutant site might be a novel drug resistance-related mutant.
1. There was no significant difference in the incidence of protease inhibitor resistance-related mutations between the HIV-1 B'subtype and untreated population (F = 0.875, P = 0.5790.05). There was no significant difference in the incidence of protease inhibitor resistance mutations between the HIV-1 B' subtype and CRF01_AE subtype (F = 0.058, P = 0.5760.05). Enzyme inhibitors are related to treatment.
2. In the treatment and untreated sequences of HIV-1 B'subtype, the use of drugs had little effect on gene mutation in Pol region (F = 0.003, P = 0.7560.05), but the presence of drugs accelerated the selection of resistant mutation sites (F = 19.008, P = 0.0070.05). There was no significant difference between HIV-1 B' subtype and CRF_01AE subtype in Pol region (F = 2.003, P = 0.7560.05). .497, P=0.0570.05).
3. In the treatment sequence of HIV-1 subtype B', the incidence of resistance-related mutations of 21 retroviral transcriptase inhibitors was significantly higher than that of untreated sequences (F = 19.008, P = 0.0070.05). In the treatment sequence, the resistance mutation patterns of NRTIs were mainly TAMs and M184V, 48.7% and 12.1% respectively, followed by Q151M mutation complex (5.77%). 181C is the main.
4. The mutation of protease codon in the untreated and untreated HIV-1 B'subtypes was analyzed. Two mutations (I13V and R57K) were found to be different in the two groups of sequences by checking 99 codons of the shared sequence with the protease. The frequency of R57K in untreated sequences was significantly higher than that in untreated sequences, whereas that of I13V was opposite. Polymorphic mutation sites.
5. By comparing the shared sequence of B'subtype with the treatment sequence and untreated sequence, we found that 21 mutation sites were significantly different between the two groups. The incidence of 3 mutation sites (I135V, S162C, V245E) in untreated sequence was significantly higher than that in untreated sequence, and 18 mutation sites occurred. The rate in the treatment sequence was significantly higher than that in the untreated sequence, 14 of which were less reported sites with unclear correlation with drug resistance.
6. Comparing the 14 mutation sites with the HIV drug resistance database of Stanford University, we found that L238I, K366R, T369V, A371V, I375V may be related to antiviral therapy and drug resistance.
7. The incidence of resistance-related mutations after 300 codons in the reverse transcriptase region were 26.33% in L238I, 21.74% in K366R, 8.30% in T369V, 7.51% in A371V and 7.20% in I375V, respectively.
8. There may be a new recombinant pattern of HIV-1 strain in the population with drug abuse and sexual contact as the main route of infection in China, that is, the recombinant strain of CRF_BC and the recombinant strain of B'.
9. The distribution of HIV-1 subtypes is closely related to the route of infection. The main route of infection is blood donation/transfusion, while drug abuse and sexual transmission are abundant. The distribution of HIV-1 subtypes is obvious in Henan, Hebei, Shandong and other regions, while CRF01_AE is dominant in Guangxi.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2009
【分類號】：R181.3

【引證文獻(xiàn)】

相關(guān)期刊論文 前2條

1 吳守麗;嚴(yán)延生;;HIV耐藥性及耐藥檢測研究進(jìn)展[J];中國病毒病雜志;2012年02期

2 鄒雯;劉穎;王健;高國建;董繼鵬;咸慶飛;;HIV耐藥的研究現(xiàn)狀[J];中國中藥雜志;2013年15期

相關(guān)碩士學(xué)位論文 前2條

1 宋丹;鄭州地區(qū)男男同性戀人群HIV-1感染者基因亞型及耐藥分析[D];鄭州大學(xué);2012年

2 邱麗君;福建省HIV-1耐藥性檢測及新耐藥相關(guān)突變位點(diǎn)的研究[D];福建醫(yī)科大學(xué);2013年

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本文編號：2251092