抗HIV药物的耐药检测

抗HIV药物的耐药检测

Robert W. Shafer,M.D.

译者：邹宝华住院医师

北京协和医院内科

Email：hellenzou0105@gmail.com

张峣博士

北京协和医院内科

Email：zhangyao1@gmail.com

审阅者：李敬云教授

解放军军事医学科学院

The development of drug resistance depends on the size and heterogeneity of the HIV population within an individual, the extent to which virus replication continues during therapy, the ease of acquisition of a particular mutation (or set of mutations), and the effect of drug resistance mutations on drug susceptibility and virus fitness. HIV drug resistance can also be transmitted between individuals. HIV strains resistant to at least one of the three classes of antiretroviral drugs account for about 10% of new infections in the United States and Europe (5, 22, 36, 44, 52, 61).

耐药性的发生取决于患者体内病毒群体的大小及异质程度、治疗过程中病毒持续复制的程度、发生某一突变（或某一组突变）的难易程度以及耐药突变对药物敏感性和病毒适应性影响的程度。耐药病毒株可以在个体之间传播。在美国和欧洲，大约10%的HIV新发感染者对3类抗病毒药物中的至少1种耐药（5，22，36，44，52，61）。

A single mutation can confer high-level resistance to certain antiretroviral drugs, allowing detection of drug-resistant mutants within weeks of initiating an incompletely suppressive regimen. Other drugs require multiple mutations for high-level drug resistance. Additive or synergistic drug combinations that block HIV replication can slow or prevent the emergence of drug resistant virus.

对某些药物，单一的点突变就可能导致高度耐药，往往在未完全病毒抑制的抗病毒治疗后数周内就可检出耐药突变。而有些药物需要多个突变才能出现高度耐药。联合应用有相加和协同作用的药物，能够阻断病毒复制，从而减缓或阻止耐药病毒的产生。

Clinical Significance of HIV Drug Resistance

HIV药物耐药性的临床意义

The presence of drug resistance before starting a new drug regimen is a strong independent predictor of virologic response to that regimen, and several expert panels have recommended resistance testing to help select treatment in particular clinical situations (8, 14, 20, 23, 24, 28, 45, 57).

在新的药物治疗方案开始前是否存在耐药性是预测对治疗病毒学应答的一项主要的独立的指标。很多专家组推荐在某些情况下应进行耐药检测来帮助选择合适的治疗方案（8，14，20，23，24，28，45，57）。

Resistance testing is recommended for some previously untreated patients — including pregnant women, persons with acute HIV infection and recently infected persons in areas where transmitted resistance is common — to help select initial antiretroviral therapy. Resistance testing is also strongly recommended for patients failing one or more HAART regimens. Drug therapy should be continued during resistance testing, as wild-type virus emerges rapidly after treatment is stopped (16, 58). The usefulness of resistance testing is likely to progressively diminish in persons failing multiple treatment regimens because resistant virus from past failed regimens may no longer be reliably detected.

对某些未治疗过的患者也推荐进行耐药检测，以帮助选择初始抗病毒治疗方案，包括孕妇、HIV急性感染者和在耐药毒株广泛传播地区的感染者。强烈推荐对一种或多种的HAART治疗方案失败的患者进行耐药检测。在进行耐药检测期间应不间断药物治疗，因为在治疗停止以后野生型毒株会快速出现。（16，58）。耐药检测的作用在于减少多个治疗方案失败的危险，因为过去失败的治疗方案产生的耐药病毒不是总能够稳定地检测到。

Review Article: Hirsch MS, et al. Antiretroviral drug resistance testing in adult HIV-1 infection: 2008 recommendations of an International AIDS Society-USA Panel. Clin Infect Dis 2008;47:266-285.

Methods of Testing for HIV Drug Resistance

HIV耐药性的检测方法

Drug resistance can be measured using either genotypic or phenotypic assays. Genotypic assays involve the detection of mutations known to be associated with drug resistance. In phenotypic assays, a fixed inoculum of HIV is cultured in the presence of serial dilutions of an inhibitory drug. Most resistance assays can reliably detect viruses present in the plasma at a concentration of 1,000 copies/ml or higher. Because resistance is not an all-or-nothing phenomenon, the most informative resistance tests report three levels of resistance: susceptible, low-level resistance, and high-level resistance.

可以采用基因型或表型实验检测耐药性。基因型实验检测耐药相关的基因突变。表型实验将HIV于存在系列稀释药物的培养基中进行培养。多数耐药实验能可靠地检测出血浆中浓度为1000拷贝/ml及以上的病毒。由于耐药不是“全或无”的现象，多数耐药检测报告将耐药性报告为3个程度：敏感、低度耐药和高度耐药。

Phenotypic Susceptibility Testing

药物敏感性的表型检测

Two companies have developed standardized phenotypic susceptibility assays amenable to high-throughput performance (Virco, Mechelen, Belgium and ViroLogic, South San Francisco, CA, USA) (27, 43). Both assays amplify the entire protease gene, much of RT and some of gag from HIV RNA isolated from plasma. Amplified cDNA is incorporated into a pol-deleted recombinant virus construct and virus replication is measured in the presence and absence of a range of antiretroviral drug concentrations. Drug susceptibility is reported as the concentration of drug required to inhibit virus replication by 50% (IC₅₀). Fold resistance is calculated by comparing the measured IC₅₀ to the IC₅₀ of wild-type control isolates. Recombinant-based assays are easier, quicker and provide more uniform results than previous non-recombinant-based assays.

目前有两家公司（比利时的Virco公司，南旧金山的ViroLogic公司）已经建立了标准化、高通量的表型敏感性检测技术，（27，43）。两个公司的技术都是从患者血浆中分离的HIV RNA中扩增全长蛋白酶基因、绝大部分逆转录酶基因和部分gag基因，将扩增的cDNA整合到pol基因缺失的重组病毒上，然后在有和没有系列稀释药物的条件下检测病毒的复制。药物敏感性报告为抑制50%病毒复制时所需的药物浓度（IC₅₀ ），通过与野生型病毒株IC₅₀的比值来计算耐药的倍数。这种基于重组病毒的试验比以前使用的不是基于重组病毒的试验方法更为简单和快捷，能够提供更具可比性的结果。

Genotypic Susceptibility Testing

药物敏感性的基因型检测

Genotypic susceptibility testing relies primarily on dideoxynucleotide sequencing. HIV sequencing using this method is highly reproducible in experienced laboratories (46). There are two available kit, each of which has been approved by the FDA for use in clinical settings: TRUEGENE (Visible Genetics, Inc) and VIROSEQ (Applied Biosystems, Inc).

基因型药物敏感型检测主要基于双脱氧核酸的序列测定。在有经验的实验室里，用这种方法进行HIV测序的重复性很好（46）。目前有两种试剂盒被美国FDA批准用于临床，他们分别是：TRUEGENE (Visible Genetics公司) 和 VIROSEQ (Applied Biosystems公司)。

Genotypic tests are used more commonly than phenotypic tests in clinical settings because of their wider availability, lower cost, shorter turnaround time, and superior insight into the evolving potential for resistance. Several clinical trials have also shown genotypic assays to be more useful in predicting drug resistance than phenotypic assays. (3, 9, 10, 17, 25, 38, 40, 56). Genotypic tests detect mutations present as mixtures, even if the mutation is present at a level that is too low to affect drug susceptibility in a phenotypic assay. Genotypic tests also detect transitional mutations that do not cause drug resistance by themselves but indicate the presence of selective drug pressure and evolving drug-resistance.

在临床上，基因型检测比表型检测的应用更广泛，原因是基因型检测的试剂更易操作，费用较低，操作时间短，并且可以更好的了解耐药性的进化过程。有些临床试验显示在预测耐药性方面，基因型检测比表型检测更有用（3，9，10，17，25，38，40，56）。基因型检测的结果可能是一些突变基因的混合，即使是在表型测试方法中，某些突变基因型由于水平很低而未显示对药物敏感性的影响。基因型试验也可检测出一过性突变，这种突变本身不会导致耐药的产生，但提示药物选择压力的存在和随后可能出现的耐药。

Limitations of Susceptibility Testing

敏感性试验的局限性

Several factors limit the utility of both genotypic and phenotypic testing. First, it is often difficult to discern the contribution of drug resistance to virologic failure. Because many highly resistant viruses are less fit than wildtype viruses, patients often benefit from continuing therapy despite the presence of drug resistance (13, 19, 37, 42). Second, minor variants in an HIV population within an individual often go undetected. Third, resistance tests only detect the most recently circulating virus variants. Patients are unlikely to respond to a regimen they previously failed, even if a susceptibility test does not reveal drug resistance. Fourth, extensive cross-resistance within each drug class may thwart a clinician trying to select useful treatment options.

一些因素限制了基因型和表型检测的使用。首先，明确耐药在抗病毒治疗失败中的作用经常是困难的。原因是很多高度耐药的病毒的复制适应性较野生型病毒的明显降低，即使出现耐药患者仍可从继续的治疗中获益（13，19，37，42）。第二，体内HIV群体中的少量突变毒株通常无法检测出来。第三，耐药检测只能检出近期存在的病毒变异株。即使敏感性试验未发现耐药，患者对既往失败的治疗方案也往往是反应不好。第四，某类药物中广泛的交叉耐药影响了临床医师对治疗方案的选择。

Genotypic Resistance Reports

耐药基因型的检测报告

Mutations in HIV RT and protease are identified by listing the single letter code for the corresponding subtype B consensus sequence amino acid, the position number, and the single letter code for the mutation (e.g. T215Y). This shorthand may also be used to indicate more than one mutation at a specific position or to indicate the presence of a mixture in a particular sequence (e.g. V82AFST). Mutations known to be associated with drug resistance are called drug-resistance mutations. Mutations occurring in untreated patients are called polymorphisms. However, some polymorphisms occur with increased frequency in treated patients and have been shown to contribute to drug resistance.

HIV逆转录酶和蛋白酶突变的表示方式是：B亚型共享序列编码的氨基酸的字母、后接突变位置数字，再接突变序列编码的氨基酸的字母（如T215Y）。这种标记方式可以表示某一特定位点的多种突变，或存在这些突变的混合（如V82AFST）。与耐药性相关的突变叫做耐药性突变。发生在未经治疗患者中的突变称为多态性。然而，某些基因多态性在接受治疗的患者中逐渐增多，并与耐药相关。

In addition to providing a list of protease and RT mutations, most genotypic reports provide a prediction of the virologic response to treatment with each of the FDA-approved drugs. However, until a standard interpretation system is developed it will probably be necessary for physicians to have some familiarity with the clinical significance of mutations associated with drug resistance. It is also important to consider a patient's treatment history and past drug susceptibility test results rather than blindly following resistance reports.

除了蛋白酶和逆转录酶的突变列表以外，多数基因型测试报告还提供对每一种FDA批准的抗病毒药物治疗病毒学应答的预测。然而，由于目前没有标准的解释系统，临床医师需要熟悉和了解耐药性突变的临床意义。同时应参考患者的治疗史和过去的药物敏感性检测结果，而不仅仅盲目地依靠当前的耐药报告结果。

Phenotypic Resistance Reports

耐药表型的检测报告

Phenotypic resistance reports are increasingly accompanied by information on the clinical significance of different levels of reduced drug susceptibility, which varies among different antiretroviral drugs. The derivation of these "clinical cut-offs" is evolving rapidly and the cut-offs themselves differ between the Virco and ViroLogic assays. For the drugs tenofovir, d4T, ddI, and ddC, small reductions in susceptibility may be clinically significant, yet may not be possible to measure reproducibly.

随着对各种药物敏感性降低临床意义的认识不断深入，耐药性表型的检测报告也正在增加。“临床临界值”的含义正在快速进化，Virco和ViroLogic两个方法的临界值是不同的。对于tenofovir、d4T, ddI和ddC，敏感性轻度降低就可能对临床有不小的影响，但目前的技术还不能保证很好的重复性。

Protease Inhibitor (PI) Resistance

对蛋白酶抑制剂的耐药

Mutations at more than 20 residues are associated with PI resistance (reviewed in (41, 45)) (Table 1). The substrate cleft mutations and the mutation L90M reduce binding affinity between the inhibitor and the mutant protease, while others involve mechanisms such as alterations in enzyme catalysis, effects on dimer stability, alterations in inhibitor binding kinetics, and re-shaping of the active site (19, 41). Mutations at protease cleavage sites often develop to compensate for the decreased fitness of enzymes containing protease mutations but do not cause resistance by themselves (12, 62).

超过20种突变与对蛋白酶抑制剂耐药相关(文献41, 45为相关综述) (表１)。底物裂隙处的突变和L90M 突变降低了药物与突变蛋白酶的结合，还涉及其他耐药的机制：例如酶催化活性的改变、对二聚体稳定性的影响、对药物结合动力学的影响以及活性位点的重塑。蛋白酶裂解部位经常发生突变以补偿含蛋白酶突变毒株的复制适应性的下降，但这些突变本身并不导致耐药。

The dynamic susceptibility range between typical wildtype isolates and the most highly PI-resistant isolates is about 100-fold (27, 43). Although low levels of PI resistance (e.g. 2 to 3-fold) are clinically significant, they may be overcome by high serum PI concentrations. Higher levels of PI resistance result from the accumulation of multiple PI-resistance mutations. Most patients developing virologic failure during treatment with one PI have a diminished virologic response to a second PI (11, 32, 48). Successful cases of salvage therapy in patients failing a PI regimen usually include regimens with dual PIs or a change to a new PI in combination with an NNRTI (4, 18, 63).

典型的野生型毒株与多数对蛋白酶抑制剂高度耐药的毒株相比，药物敏感性差别的动态范围大约有100倍（27，43）。虽然对蛋白酶抑制剂低度耐药（如２-３倍）对临床也有影响，但可以通过提高血药浓度来克服。更高水平的蛋白酶抑制剂耐药是由多个蛋白酶抑制剂耐药突变的积累造成的。多数使用一种蛋白酶抑制剂治疗病毒学失败的患者对第二种蛋白酶抑制剂治疗的病毒学应答也会降低（11，32，48）。在蛋白酶抑制剂治疗失败的病人中，挽救治疗成功的通常使用包括两种蛋白酶抑制剂，或一种新蛋白酶抑制剂联合一种非核苷酸逆转录酶抑制剂的治疗方案（4，18，63）。

Nucleoside RT Inhibitor (NRTI) Resistance

核苷类逆转录酶抑制剂的耐药

There are two biochemical mechanisms of NRTI drug resistance mediated by mutations in the RT enzyme: discrimination against NRTIs during polymerization and more rapid hydrolytic removal or excision of chain-terminating NRTIs (2, 7, 39). The dynamic susceptibility range is >100-fold for ZDV and 3TC, and 15 to 20-fold for ddI, d4T, ddC, and abacavir in most drug susceptibility assays (27, 43).

逆转录酶的突变通过2种生化机制导致对苷类逆转录酶抑制剂耐药：在聚合反应中排斥核苷类逆转录酶抑制剂，和更快地水解或剪切掉使得链合成终止的核苷类逆转录酶抑制剂（2，7，39）。在多数药物敏感性检测中，ZDV和3TC的药物敏感性动态范围可大于100倍、对于 ddI、d4T、ddC和阿巴卡韦通常为15-20倍（27，43）。

The RT mutations M41L, D67N, K70R, L210W, T215F, and K219QE mediate hydrolytic removal of chain-terminating NRTIs (2, 7, 39) (Table 2). Although these mutations are often called thymidine analog mutations, or TAMs, because they arise after treatment with ZDV and d4T, they also confer cross-resistance to abacavir, ddI, ddC, and tenofovir.

逆转录酶的突变，如M41L, D67N, K70R, L210W, T215F和K219QE可以介导使得链末端终止的核苷类逆转录酶抑制剂的水解（2，7，39）（表2）。尽管这些突变通常被称为胸苷类似物突变，或TAMs，因为它们出现在ZDV和d4T治疗以后，它们也会导致对阿巴卡韦、ddI, ddC和泰诺福韦的交叉耐药。

K65R, L74V, Q151M, and M184VI prevent the addition of NRTIs to the growing DNA chain (Table 2). M184V, which is adjacent to two of the three catalytic aspartates in the conserved YMDD motif of RT, is the most common of these mutations. It confers high-level resistance to 3TC (>100-fold in most assays) and low levels of resistance to abacavir, ddI, and ddC. However, both M184V and L74V interfere with the activity of the TAMs (6, 21) and hypersensitize isolates to ZDV and possibly d4T (35, 54). These effects are clinically significant and probably explain the prolonged antiviral activity of many common dual NRTI combinations.

K65R, L74V, Q151M和 M184VI可以阻止核苷类逆转录酶抑制剂与正在延伸的DNA链相结合（表2）。M184V，邻近逆转录酶保守的YMDD基序中三个天门冬氨酸中的两个，是这类突变中最常见的，它可导致对3TC高度耐药（绝大部分试验结果为大于100倍）和对阿巴卡韦, ddI和ddC的低度耐药。然而，M184V和L74V均会对TAM的活性产生影响（6，21），因而增加对ZDV或d4T的敏感性（35，54）。上述效应是有临床意义的，它可能解释为什么多种常见的两种核苷类逆转录酶抑制剂方案能够长期有效。

Patients switching from one dual NRTI combination to another will generally have some response; however, if high-level resistance to one combination has emerged, it is unlikely that the second NRTI combination will retain significant antiviral activity. Multidrug-resistance within the NRTI class usually results from the accumulation of multiple TAMs in combination with M184V and one or more accessory mutations. About 5% to 10% of patients receiving ddI and ZDV or d4T develop the mutation Q151M (47), which is usually followed by mutations at positions 62, 75, 77, and 116. This mutation combination confers high-level resistance to each of the NRTIs except 3TC (29, 49). A two amino acid insertion at residue 69 also confers multidrug resistance when present with T215FY and other TAMs (60).

患者在两种含双核苷类逆转录酶抑制剂的方案间转换时，往往会有一些治疗反应。但是如果对其中一种组合产生了高度耐药，那么对第二种方案很难保持显著的抗病毒活性。对NRTI的多药耐药往往是由于多种TAMs突变的积累，加上M184V以及其他一种或多种附加突变。大概5%到10% 接受ddI、ZDV或d4T治疗的患者会发生Q151M突变（47），此后通常会发生62、75、77位点的突变。这种突变的组合导致对除3TC以外的各种核苷类逆转录酶抑制剂高度耐药（29，49）。存在T215FY和其他TAMs突变时，69位点两个氨基酸的插入也可导致多药耐药（60）。

Non-nucleoside RT Inhibitor (NNRTI) Resistance

非核苷酸逆转录酶抑制剂的耐药

The NNRTIs delavirdine, efavirenz, and nevirapine inhibit HIV RT allosterically by binding to a hydrophobic pocket close to the active site (33, 53). A single mutation in this NNRTI-binding pocket may result in high-level resistance to one or more NNRTIs. Resistance usually emerges rapidly when NNRTIs are administered as monotherapy or in the presence of incomplete virus suppression, perhaps through the selection of a pre-existing population of mutant viruses (26, 30, 59). The dynamic susceptibility range for each of the three available NNRTIs is often several hundred-fold.

非核苷酸逆转录酶抑制剂地拉韦啶、依发韦仑和奈韦拉平通过与紧邻活性位点的疏水性口袋的共价结合来抑制HIV逆转录酶的活性（33，53）。NNRTI结合口袋的单一突变就可能导致对一种或多种NNRTI的高度耐药。在单用1种NNRTI治疗时或对病毒抑制不充分时，这种耐药往往迅速出现，可能是通过对已存在的突变株的选择（26，30，59）。出现耐药后，对现有三NNRTI的动态敏感性范围经常为数百倍。

The NNRTI resistance mutations are clustered between residues 98-108, 179-190, and 225-236 (Table 3). K103N, which is the most common, confers >25-fold resistance to each of the NNRTIs and appears sufficient to cause virologic failure with each of the NNRTIs (15, 31, 51). Y181CI is the second-most commonly occurring NNRTI-resistance mutation. It causes >30-fold resistance to nevirapine and delavirdine and 2 to 3-fold resistance to efavirenz. Other clinically relevant NNRTI resistance mutations occur at residues 98, 100, 106, 108, 179, 188, 190, 225, 227, 230, and 236. Based on the low genetic barrier of resistance to most NNRTIs and the high levels of cross-resistance between available NNRTIs, treatment with a second NNRTI following virologic failure with a different NNRTI is usually unsuccessful (51).

NNRTI的耐药突变成簇地分布在98-108, 179-190, 225-236氨基酸残基间（见表３）。其中最常见的，K103N,可以导致对所有NNRTI大于25倍的耐药，这足以导致每一种NNRTI治疗的病毒学失败（15，31，51）。Y181CI是第二常见的NNRTI耐药突变，会导致对地拉韦啶和奈韦拉平大于30倍的耐药，对依发韦仑2-3倍的耐药。其他有临床意义的NNRTI耐药相关突变发生在98, 100, 106, 108, 179, 188, 190, 225, 227, 230, 和236位点上。由于多数NNRTI的遗传障碍较低和交叉耐药率很高，在治疗病毒学失败后换用另一种NNRTI往往不会成功。

There is no cross-resistance between NRTIs and NNRTIs. Indeed, several NNRTI-resistance mutations hypersensitize isolates to NRTIs (34) and combinations of certain NRTI-resistance mutations hypersensitize isolates to NNRTIs (50). These interactions help explain the clinical synergy observed when two NRTIs are combined with an NNRTI (1, 55).

NRTIs与NNRTIs之间没有交叉耐药，实际上一些NNRTIs的耐药突变会增加NRTIs的敏感性（34），而某些NRTI耐药突变的组合可增加对NNRTI的敏感性（50）。上述相互影响有助于解释临床上两种NRTIs联用一种NNRTIs时出现的协同效应（1，50）。

Tables and Figures

Table 1. Protease Inhibitor (PI) Resistance Mutations [Download PDF]

表1. 蛋白酶抑制剂的耐药突变

Table 2. HIV Nucleoside RT Inhibitor (NRTI) Resistance Mutations [Download PDF]

表2. NRTI的耐药突变

Table 3. HIV Non-Nucleoside RT Inhibitor (NNRTI) Resistance Mutations [Download PDF]

表3. 非核苷类逆转录酶抑制剂的耐药突变

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