Video在线视频

大家好
-Hello.

我是Philip Butcher
My name is Philip Butcher.

我在英国伦敦大学圣乔治学院感染
I work at the Institute for Infection and Immunity

与免疫研究所工作
at St George's University of London in the UK.

今天 我将谈谈全基因组测序
Today, I am going to talk about the clinical usefulness

在改善多重耐药
of whole-genome sequencing as a way

和高度耐药结核病治疗方面的临床实用性
to improve the management of multiple-drug and extremely drug-resistant tuberculosis.

我讲述的主要信息内容
The key message and the content of my talk

包括耐药结核病的定义
will include a definition of drug-resistant tuberculosis,

用全基因组测序进行快速基因型抗生素
a touch on the potential for the rapid genotypic antibiotic susceptibility

易感性检测的可能性
to testing that we can do with whole-genome sequencing,

举例说明助于临床上解释突变的表型
try to consider a few examples of the phenotype-genotype correlation

与基因型相关性的例子
that is important for the clinical interpretation of mutations,

进一步展望下研究方向
and then consider where we are going.

结核病的短期治疗方案由4种药物组成
The short-term treatment for TB is made up of 4 drugs:

异烟肼 利福平 吡嗪酰胺和乙胺丁醇
isoniazid, rifampicin, pyrazinamide and ethambutol.

前2个月这四种药物都使用
All four drugs are given at the beginning for 2 months

之后为期4个月的疗程
followed by a continuation phase

只使用利福平和异烟肼
of rifampicin and isoniazid for 4 months.

这是一个非常有效的治疗方案
This is a very effective regimen.

但基本原理是
But the fundamental principle is

需要多种抗生素联合使用
that you need multiple combinations of antibiotics in order to

以预防结核病患病期可能出现的高频率突变
prevent the emergence of high-frequency mutations that can occur during TB disease.

结核抗生素耐药性的主要原因
In TB, the main source of antibiotic resistance

是靶基因突变
is due to mutations either in the target genes,

例如耐利福平相关的rpoB
such as rpoB for rifampicin,

耐异烟肼相关的inhA
inhA for isoniazid,

耐喹诺酮相关的gyrA
gyrA for quinolones

或是药物活化基因
or in drug activation genes

如活化异烟肼的katG
such as katG for the activation of isoniazid,

或活化吡嗪酰胺的pncA
or pncA for the activation of pyrazinamide.

在结核病中没有水平基因转移
There is no horizontal gene transfer,

质粒 噬菌体或转座子
plasmids, phages or transpositions in TB.

这一切都归因于突变
It is all down to mutations.

耐药结核病可以定义为
Drug-resistant TB can be defined as TB

对一线抗生素耐药的结核病
that is resistant to two of the front line antibiotics:

通常是利福平和异烟肼
normally rifampicin and isoniazid.

广泛耐药结核病（Extensive Drug Resistant TB, 简称“XDR-TB”）
XDR-TB is MDR-TB

是在耐多药结核病（Multidrug Resistant TB, 简称“MDR-TB”）的基础上
that is additionally resistant to an injectable

还对注射剂药物（诸如卡那霉素、阿米卡星和氟喹诺酮类药物）具有耐药性
such as kanamycin or amikacin, and one of the fluoroquinolones.

XDR-TB必须使用
XDR-TB then has to be treated

其他的三线抑菌抗生素治疗
with some of the other third-line and bacteriostatic antibiotics

但治疗效果不好
which do not work so well

并且可能有很强的毒性
and can be quite toxic.

XDR-TB是可以治疗的
XDR-TB is treatable.

但是治疗更加困难
But it is just more difficult

而且需要对这些抗生素的联合使用
and requires these combinations of antibiotics

有丰富的经验
that are often used empirically

这是关键
and that is the key point.

这是对抗生素的经验性选择
It is an empirical selection of antibiotics

全基因组测序的应用
nd whole-genome sequencing attempts to

试图为该选择提供一些遗传学依据
try to provide some genetic basis for that choice.

XDR-TB的治疗也非常昂贵
Treatment for XDR-TB is also very costly.

它有很多副作用
It has a lot of side effects

而且疗程长达2年
and can take up to 2 years.

治愈率通常只有50％到70％
The cure rates are often only between 50 and 70%.

通常 抗生素治疗
Normally, antibiotic treatment

是以表型药物敏感性试验为依据的
is guided by a phenotypic drug susceptibility testing.

但对于这些二线和三线抗生素
But in the case of these second-

是非常困难的
and third-line antibiotics,

而且通常需要数周的时间才能完成
that is very difficult to do and often takes weeks to perform.

从XDR-TB临床治疗的实际情况来看
In practical terms of clinical management of XDR-TB,

这太迟太久了
that is too little and too late.

全基因组测序实际上可以预测
Whole-genome sequencing can actually predict

与任何特定结核菌株相关的所有突变
all of the mutations associated with any particular strain of TB.

如果我们能尽快做到这一点
If we could do that as quickly as we can do

无论是GeneXpert还是涂片试验
either the GeneXpert or the smear testing,

都将真正加强全基因组测序
that would really enhance the ability of whole-genome sequencing to

对治疗决策的影响
impact on treatment decisions.

让我们看看结核病遗传耐药性的决定因素
If we look at the determinants of genetic resistance for TB,

这张幻灯片显示了
this slide shows just some of the mutations

一些可能与某些药物耐药性相关的突变
that you might find associated with resistance to certain drugs.

请看这张幻灯片的上部
If you look at the top of this slide,

我们看到katG或inhA的突变
you will see that mutations in katG or inhA

是异烟肼耐药性的原因
are responsible for resistance to isoniazid.

rpoB的突变是利福平耐药性的原因
Mutations in rpoB are responsible for resistance to rifampicin.

类似的
Similarly,

pncA基因的突变
mutations in the pncA gene

也是导致吡嗪酰胺耐药的原因之一
are responsible for pyrazinamide resistance

另有一些类似的发现
and so on.

在此我不赘述细节内容
I do not want to go through all of the other ones on there.

您可以自己查阅
You can read them for yourselves.

我们如何一次性识别和定位所有突变
How do we identify and map all of those mutations in one go?

在这方面 全基因组测序实际上
This is where whole-genome sequencing

比其它只能寻找一些选择性突变的检测
actually has the advantage over any of the other tests

都有优势
which only look for a few selective mutations.

我们一直使用这种方法
We have been using this

诊断圣乔治医院的XDR-TB临床病例
to look at clinical cases of XDR-TB at St George's.

在患者被诊断和菌培养阳性之后
After the patient is diagnosed and the culture becomes positive,

我们可以接触到结核菌株
we can then access the strain of TB.

可以从中提取DNA
We can extract the DNA from that

并在这里展示的测序仪上进行测序
and sequence it on one of the sequencing machines shown here.

然后 我们将获得的序列数据
Then we align the sequence data we get back

与参考基因组进行比对
with a reference genome

以确定在特定结核菌株中发现的突变
in order to identify the mutations that we see in that particular strain of TB.

然后用那些突变推断
Those mutations then can be used to infer

对于这种特定的菌株什么样的治疗是可用的
what treatment would be possible for this particular strain.

不是所有的突变都相同
Not all mutations are the same

它们对最小抑菌浓度（minimum inhibitory concentration; 简称“MIC”）的影响不同
and they all have different effects on the minimum inhibitory concentration.

以异烟肼为例
If you take isoniazid for instance

在katG基因中发现突变
and you find a mutation in the katG gene,

则通常是异烟肼高度耐药
this always confers a high level of resistance to isoniazid.

所以异烟肼不能用于治疗
So, isoniazid cannot be used in the therapy.

但是inhA启动子区域的突变实际上
But a mutation in the inhA promoter region

仅导致异烟肼低耐药性
actually only confers low level of resistance to isoniazid

如果异烟肼使用较高的浓度
and isoniazid can be still included in the regimen

则异烟肼仍是该治疗方案的可用药物之一
if it is given at a higher concentration.

莫西沙星耐药性更令人吃惊
This is even more startling with the moxifloxacin resistance.

gyrA基因发生A90V突变
There is a mutation in the gyrA gene called A90V

则使莫西沙星MIC低至1μg/ mL
which confers a low MIC value of 1 microgram/mL for moxifloxacin

但是其他喹诺酮类药物的MIC高很多
but much higher MIC levels for the other quinolones.

如果在TB突变体发现gyrA的A90V突变
If you find an A90V mutation in the gyrA of a TB mutant,

仍然可以在治疗中使用莫西沙星
you can still use moxifloxacin in the therapy.

这是已明确突变的另一个例子
This is another example of knowing the precise mutation

可为正确的治疗方案提供依据
that can provide evidence for the right type of treatment.

最后一个例子是利福平
My final example is rifampicin.

rpoB基因特定密码子
Mutations in the rpoB gene

507-533之间的突变与利福平耐药相关
between particular codons, 507 to 533, confer resistance to rifampicin.

但并非基因组该区域的所有突变
But not all mutations in that area of the genome

都可致利福平耐药
confer resistance.

其中一些仅是系统发育多态性
Some of them are just phylogenetic polymorphisms

而另一些只是导致利福平低耐药性
and some just confer low-level resistance.

同样 确定确切的突变实际上
Again, defining the exact mutation

可以提供利福平
can actually provide evidence for whether or not rifampicin

或其相关药物利福布汀是否可以使用的依据
or its related drug, rifabutin, can actually be used.

存在的突变与临床结果的关联性
This phenotype-genotype correlation has been difficult

缺乏充分数据的情况下
in the absence of substantial data

这种表型-基因型相关性是难以解释的
that links the presence of mutations with clinical outcome.

最近发表的论文达成了期待已久的共识
The recent papers being published long awaited consensus

实际上为我们如何解释
that actually gives evidence

这些分子药物敏感性试验提供了依据
as to how we can interpret these molecular drug susceptibility tests.

所提出的共识总结是
The summary of the consensus opinion presented in this paper

我们需要大量的研究来建立非常准确的
is that we need large studies to establish very accurately

表型与基因型相关性和相关的临床结果
this phenotype-genotype correlation and that linked clinical outcome

这样我们就能看到一个基于全基因组
so that we can see whether or not a modified regimen

测序数据的改良方案是否有效
based upon evidence from whole-genome sequencing actually works.

为了做到这一点
In order for this to happen,

我们也需要耐药突变体的国际数据库
we also need international databases of drug-resistant mutants.

汇总正在进行中
This also is being put together.

最后 有人说 我们应该需要开展
Lastly, they say that we should need clinical trials

临床试验研究来探讨基于分子检测的
to show the usefulness of individualized treatment

个体化治疗的有用性
based upon molecular assays.

全基因组测序将在临床上发挥巨大的作用
Whole-genome sequencing will have a very big clinical impact.

它可以给临床医生提供最好的证据
It can give the best possible evidence to clinicians to be able to

使他们决定能否使用某种抗生素
make those decisions about what antibiotic can and cannot be used.

它可以显示突变结核菌株
It can give a likely susceptibility of a mutant TB strain

对特定抗生素的可能易感
to a particular antibiotic.

它可以提供MIC水平的数据
It can give evidence about the MIC levels

以指导药物用量
so that drug levels can be monitored.

它也能确定不同类型的抗生素之间的
It will also identify cross resistance

交叉耐药性
between different types of antibiotics.

如果使用得当
If it is used correctly,

可以更快速 更有效地治疗病人
it will allow faster, more effective treatment delivered to the patient.

使得您的患者预后得到改善
Therefore, you get an improved patient outcome.

得益于此
As a result of that,

患者传染期缩短
you have a reduced time of infectivity,

进而减少了医院中
therefore reducing any possibility of onward transmission

出现后续传播或感染控制的可能性
or infection-control problems in a hospital.

通过尽早给予正确的抗生素治疗方案
By giving the right antibiotic regimen at the earliest point,

也可以阻止发展为耐药
we also prevent further resistance from developing.

而且 与许多其他方法相比
Also, whole-genome sequencing will be very cost-effective

全基因组测序的性价比很高
compared to many other methods.

有待解决的一些问题是
Some of the challenges that still remain to be addressed is

我们需要良好的生物信息学技术来分析数据
that we will need good bioinformatics skills to analyze the data

而且需要很多新的分析工具
and a range of new analysis tools are being made available.

基因型和表型相关性研究需要加强
This genotype-phenotype correlation needs to be strengthened

最近的共识报告是一个重要的方向
and this recent consensus report is an important way forward.

也需要可更新的优质数据库
Good databases that are updatable are also awaited.

这些始于一种叫ReSeq-TB的东西
These are beginning with something called ReSeq-TB

是一种国际分类
which is an international sorting for doing that.

最后
Last thing is

我们都需要了解结核病基因组的遗传学语言
that we all need to learn the genetics of the language of the TB genome.

在不了解结核病基因组的情况下
Without understanding the TB genome,

我们将无法应用全基因组测序
we will not be able to apply whole-genome sequencing to have any impact

对XDR-TB患者的临床护理发挥作用
on the clinical care of XDR-TB patients.

感谢您的聆听
Thanks for listening.