核苷（酸）类似物耐药的检测—Keeffe教授 vs. 张欣欣教授

Emmet Keeffe  美国斯坦福大学医学院

张欣欣  上海交通大学医学院附属瑞金医院

Hepatology Digest: With nucleoside/nucleotide analogues treatment， what are the clinical criteria for diagnosis of antiviral resistance and how can we detect the antiviral resistant mutations， Professor Keeffe?

《国际肝病》：核苷(酸)类抗病毒药物产生耐药的诊断标准是什么？我们如何检测耐药的相关变异呢？Keeffe教授？

Prof. Keeffe: Well I think the traditional approach has been to have a baseline serum HBV DNA level and then you have to monitor the patient at some regular interval， probably initially every three months with repeated serum HBV DNA levels. They will typically fall with all of the oral antiviral agents and then will either become undetectable or will reach some low nadir level. As the patient is then monitored every three or six months， resistance is defined by so-called “breakthrough” when serum HBV DNA level will increase by more than one log. Now today in some countries although not everywhere around the world， we have access to actually measure for the resistant viral species through resistance testing. So that is often performed as a confirmatory test to see if there is a specific mutation to whichever agent is being utilized. There’s always a differential diagnosis when the serum HBV DNA level rises which is the fact that the patient may not be taking their medication， so in fact there is not a resistance virus they’ve simply become non-compliant for economic or personal reasons and are no longer taking their medication.

Keeffe教授：传统方法是首先确定一个血清学HBV DNA水平基线，再对患者进行定期监测，开始时可能每隔3个月就要监测一次血清HBV DNA水平。口服抗病毒药物后，病毒水平会有明显的下降，随后或是检测不到或是达到一个最低水平。然后如前所述，每隔3~6个月再对患者进行检测，耐药的定义是所谓的病毒学“突破”——此时血清中HBV DNA水平会升高超过1 log。现在，在一些国家——并不是世界上所有的地方，我们可以通过耐药检测测量实际耐药的病毒株。因此，这种检测通常用作确证试验，用以探查是否有特异变异及这种变异针对何种药物。当血清HBV DNA水平升高的时候，需要进行鉴别诊断，因为有些时候患者并没有服用药物，因而此时实际上并没有产生病毒耐药，只是因为患者的个人原因或经济原因而未依从治疗。

Hepatology Digest: Professor Zhang， Professor Keeffe mentioned access to tests. Can you talk about the situation in China and testing and access in general. Can you speak about that?

《国际肝病》：张欣欣教授，Keeffe教授提到了现在已经可以进行的这种检测，您能不能大体介绍一下这种检测方法在中国的应用？

Prof. Zhang: Yes， in China we also have the detection of HBV DNA done regularly about two or three months during the antiviral treatment especially in the big cities. But in some small cities or small hospitals， DNA commercial detection kits may not be available. Moreover， detection of genotypic mutation is not very popular in China because of the reagent. It is too expensive and not very popular but DNA detection it is quite popular. So we based it on the breakthrough of DNA to monitor the antiviral therapy and monitor the appearance of mutation.

张欣欣教授：是的，在中国，特别是在一些大城市，我们也有检测HBV DNA水平的方法，一般是在抗病毒治疗过程中，每隔2~3个月检测一次。但在一些小城市或小医院，还没有这种商业化的DNA水平检测设备。而且由于试剂的原因，基因变异的检测在中国也不很广泛。由于试剂太昂贵，所以应用不广泛。但DNA检测在中国应用还是比较广泛的。因此，我们通过HBV DNA水平突破来监测抗病毒治疗效果和是否有变异的出现。

Prof. Keeffe: And I think it’s perfectly adequate to do it that way. There’s a sequence of events that occurs when resistance develops and that is first of all the development of a resistant species that becomes the dominant virus within the patient. That’s followed by a raise in the serum HBV levels and then later a raise in the ALT level and potential clinical deterioration. So if one intervenes at the time there’s a rise in the HBV DNA level there is plenty of time to map out a new strategy on how to best manage the patient.  Would you agree? I think it is perfectly adequate.

Keeffe教授：我认为这样做足够了。耐药的发生包括了一系列事件：首先耐药株出现并成为患者体内的优势株，随后导致血清HBV DNA水平的升高，接下来是ALT的升高及潜在的临床症状恶化。因此，如果在HBV DNA水平升高的同时给予干预，也有足够的时间来规划对患者最有利的新治疗方案。您同意我的观点么？我认为这足够了。

Prof. Zhang: Yes， and also I would like to ask Professor Keeffe， in some patients， the patient has the breakthrough of the HBV DNA levels but the genotypic mutation is negative so in that case what do you think about this?

张欣欣教授：是的，我也想问Keeffe教授，有些患者出现了HBV DNA突破，但基因变异检测是阴性的。那么您如何看待这种病例？

Prof. Keeffe: Well that may be non-compliance or it may be that the fact that the assay that is used is simply not sensitive enough to detect that there is a beginning emergence of a resistant viral species. It just may not be dominant enough depending on which assay system is utilized. So I think it is quite reliable， particular it has been recommended that the HBV DNA level be repeated twice so that there is confirmation that it is rising. And typically it is well more than one log over the lowest levels， typically two or three logs. So if that is confirmed I think that’s very good evidence that there is fact failure of the drug - provided that the patient says ‘Doctor， I’m taking my pill every day’.

Keeffe教授：那可能是患者依从性不好，或不过是因为所应用的检测方法，对早期出现的耐药病毒株不够敏感。或许仅仅是对你所使用的检测系统而言，耐药病毒株不是优势株。因此我认为HBV DNA检测很可靠，特别是我们推荐进行两次HBV DNA水平的重复检测来确定它是否升高。一般超过最低水平1 log以上，通常是2~3 log。因此我认为，假如那些患者说“医生，我每天都吃药”，一旦确定有HBV DNA水平的升高，就能充分证明药物治疗失败。

Prof.Zhang: But really， we actually meet some - not many -but some patients who have good compliance and we have repeated the HBV DNA assays and we’re sure they have breakthrough but no genotypic mutations. I think even if there is some mutant strain as a minor strain in the serum of the patients， if it is not the dominant strain it could not lead to breakthrough.
 
张欣欣教授：但实际上，我们的确遇见了这样一些患者——不是很多，这些患者依从性很好，我们对其做了两次HBV DNA检测，并肯定他们有病毒学突破而没有基因变异。我认为，即使在患者血清里存在一些未成为优势株的突变株，也不能导致病毒学突破。

Prof. Zhang: I just wondered what do you think about why for some patients after antiviral therapy with nucleos-(t)ide analogues， at the beginning almost all the patients have a decrease in HBV DNA. For example， from ten to seven or eight， or ten to five or ten to four. And it stops at that stage. What do you think about the reason for this phenomenon?

张欣欣教授：我很想知道，为什么有些接受核苷（酸）类似物抗病毒治疗的患者，在开始时几乎所有的患者都有HBV DNA水平的降低，例如从10 log降到7 log或8 log，或者从10 log降到5 log或4 log，但却停在这个阶段不再下降。您认为这种现象的原因是什么？

Prof. Keeffe: Yeah I’ve thought about that long and hard as well. I really don’t know. I have no idea. And we often use the term “a sub-optimal response” to apply to a patient like that. Now in the early days when antiviral therapies first became available， we were quite excited if we could use a drug like lamividine， which was the first drug we had access to， and drive the viral load down from 109， or 105 or 104. We though that was terrific， we thought we’d done a very good job for our patient. Well we learned later that it was not really fully adequate because though there was benefit initially that resistance had a high likelihood of occurring with some loss of the benefit. But why that occurs that’s a long answer to your short question， I really don’t know.

Keeffe教授：是的，我也被这个问题困扰了很久。我确实不太清楚原因。我们常用术语“次理想应答”来描述这种患者。在早些日子，当抗病毒治疗刚刚出现时，如果我们能使用拉米夫定——这是当时能用的第一种药物，我们会很兴奋，因为我们有办法使病毒载量从9 log降到5 log或4 log。我们认为这非常好，我们对患者治疗非常成功。然而，随后我们看到治疗不是很充分，虽然刚开始有益处，但随后发生耐药的概率很高。为什么会发生这种情况，这个问题的答案很复杂，我确实不太清楚。

Hepatology Digest: When it comes to quasispecies and HBV there seems to be an agreement that HBV quasispecies are the same as in HCV. Professor Zhang， how do these quasispecies influence the progress of the disease in HBV， can you talk a little about that subject?

《国际肝病》：谈到准种，我听说对HBV有种共识：HBV准种和HCV准种是类似的。张欣欣教授，这些准种是如何影响疾病进展的，您能不能谈谈这个话题？

Prof. Zhang: Well the HBV quasispecies is just like the HCV quasispecies. It’s not clear what the function of the quasispecies is. According to some previous studies， maybe the quasispecies of HBV also play some important roll in the pathogenesis， or it may influence the outcome of the treatment， or even the efficacy of treatment. For example， it has been reported that the evolution of HBV quasispecies during the e-antigen seroconversion and the interferon treatment the evolution of quasispecies may influence the efficacy of treatment. Recently in our study we have looked at the quasispecies of HBV in patients before treatment and 4 weeks after lamivudine treatment. And what we found very interesting is that is for these treatment patients actually baseline， the complexity and the diversity of the quasispecies for responders and non-responders is the same. No significant difference. But after 4 weeks of treatment with lamivudine， the complexity and diversity of the respondents become much lower than those of non-respondents. Even after the HBV DNA is decreased rapidly during the early stage of antiviral treatment that means the patients may be respondent. I mean he has become HBV DNA negative during the first three months of treatment， but the quasispecies complexity continues to increase， not decrease. Six months after that the patient has a relapse and the mutation appears. So it is a very interesting phenomenon. Maybe the quasispecies， the interaction between the different strains in the serum of the patient influence the antiviral efficacy. But I still do not know the mechanisms.

张欣欣教授：HBV准种类似于HCV准种。这些准种的功能还不是很清楚。根据以前的一些研究，可能HBV准种在病理过程中也起重要作用，或者说它可能影响治疗预后，甚至是疗效。例如，据报道，在HBeAg发生血清转换过程和干扰素治疗期间，HBV准种存在进化，这种进化会影响治疗效果。最近，我们观察了HBV准种在治疗前和拉米夫定治疗4周后的情况。我们发现，不论对治疗是否有应答，治疗前患者的准种实际基线的复杂性和多样性都是一致的，无统计学差异。但在拉米夫定治疗4周后，有应答患者准种的复杂性和多样性明显低于无应答者，即使在抗病毒治疗的早期阶段HBV DNA水平迅速降低意味着可能出现应答。我的意思是，即使在最初3个月治疗中患者已经出现HBV DNA转阴，但准种的复杂性可能还在持续增加而没有降低。6个月后，病情会复发并有变异产生。这是个非常有趣的现象。或许患者血清中准种之间的相互作用对抗病毒效果造成了影响，但其确切机制并未明确。

Hepatology Digest: What about as far as hepatitis B and C and comparing the progression of the disease and the natural history， and between those two?

《国际肝病》：在疾病的进展以及自然史方面，HBV和HCV有什么区别呢？

Prof. Zhang: Yes， it’s different between hepatitis B and C.  Because after the HBV infection， only 5% patients when infected during the adult age will evolved into a chronic disease or even cirrhosis.  But for hepatitis C， it’s about more than 50% or even 70% will evolve to chronic. Actually in Chinese clinics we meet many many hepatitis B not hepatitis C. So my impression is we talk a lot about hepatitis C but in clinical， in china， we don’t meet so many patients， but for hepatitis B yes. This very reason maybe because in hepatitis B there’s vertical transmission in China. If the infection occurs during the natal age， many many immunotolerances can lead to the chronicity. 

张欣欣教授：是的，HBV和HCV是有区别的。因为在HBV感染后，如果是在成年感染的，只有5%的患者会进展为慢性肝病甚至是肝硬化。但如果是HCV感染，会有超过50%甚至70%的患者会发展成慢性肝病。实际上，在中国临床上，我们遇见很多很多乙肝患者而不是丙肝患者。因此，我的感觉是我们进行了许多关于丙肝的讨论，但在中国，临床上我们见不到那么多丙肝患者，而乙肝患者却很多。可能由于在中国，HBV多数是通过垂直传播的。如果感染发生在婴儿期，大量的免疫耐受会导致肝病慢性化。

Prof. Keeffe: Hepatitis C when it comes chronic after infection， tends to have a relatively linear progression that’s slow over time， like hepatitis B. It may accelerate a little bit with older age pass the age of fifty-five or sixty.  On the other hand Hepatitis B may have a more variable natural history because there is immunotolerance phase when there’s no active disease and then there’s a period of disease activity during the immune clearance phase sometimes around the age of thirty or forty or so that may be brief or may be prolonged then there is a period of quiescence， the inactive carrier， but then there may be reactivation.  So Hepatitis B is considerably more complex in natural history for those reasons compared to Hepatitis C.

Keeffe教授：当感染HCV并发展为慢性疾病时，会趋向于形成近似线性回归方程，随着时间的进展，疾病的发展会慢下来，类似于HBV。在老年或者年龄超过55岁、60岁的病例，疾病发展可能会稍微加速。另外，HBV的自然史要更多变一些。HBV有免疫耐受期，这时疾病处于非活动期；随后在免疫清除期会有明显的疾病活动。免疫清除期一般在30或40岁，也有提前的或延后的，之后就是静止期，这时期非活动性的携带者有可能被重新激活。所以，与丙肝相比，乙肝的自然史更复杂些。

Hepatology Digest: Professor Zhang you just mentioned the vertical transmission.  How are the efforts progressing in China to prevent and what阵 being done to help prevent that?

《国际肝病》：张教授，您刚才提过垂直传播。现在中国有什么措施可以预防病毒感染，有何成效？

Prof. Zhang: I think it’s the vaccination. Yes， we vaccinate all the babies since 1992. The prevalence of HBV infection has decreased remarkably during the past ten years， more than 10 years.

张欣欣教授：我认为是疫苗。是的，我们从1992年起对所有的婴儿进行免疫接种。在过去的10年中，甚至超过10年，HBV感染的流行已经有了明显的下降。

Prof. Keeffe: We’ll be out of a job in a generation.

Keeffe教授：那再过一代人，我们就要失业了。