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我是Howard Takiff
-I am Howard Takiff.

我在巴黎巴斯德研究院
I work at the Institut Pasteur in Paris,

中国深圳市南山区
at the Nanshan CCDC

慢性病防治院（NanshanCCDC）
in Shenzhen, China,

和委内瑞拉加拉加斯的
and at IVIC in Caracas,

国家科学研究院（Venezuelan Institute for Scientific Research, IVIC）工作
Venezuela.

我将介绍一下结核病耐药性的简要历史
I am going to present a brief history of drug resistance in TB.

数千年来 结核病一直困扰着人类
Tuberculosis has plagued humanity for thousands of years.

古埃及 古希腊和古印度的医生
Ancient Egyptian, Greek and Indian healers

推荐了许多治疗方法
recommended many therapies

包括动物血液 牛奶 放血
including animal blood, milk,bleeding,

休息和移动到高处
rest and moving to higher altitudes.

在欧洲
In Europe,

国王赐予的触摸被认为可以治愈淋巴结核
the King's touch was supposed to cure scrofula,

这是一种伴有颈部淋巴结肿大的结核病
a form of tuberculosis with enlarged cervical lymph nodes.

许多其他物质和奇怪的方法
Many other substances and strange measures

成为治疗肺痨的处方
were prescribed for consumption.

疗养院取得了一定的成功
Some success was achieved with sanatoriums

在那里
where high altitude,

高海拔 休息和良好的营养
rest and good nutrition cured or decreased symptoms

可以治愈或减轻一些有财力病人的症状
in some of the fortunate patients

这些富裕的病人可以负担得起这种治疗
who could afford this form of therapy.

但真正的革命发生在1943年
But the real revolution came in 1943

当时Selman Waxman
when streptomycin was purified

Elizabeth Bugie
from Streptomyces griseus,

和Albert Shatz
a bacteria isolated from the soil of the Andes

从安第斯山脉土壤中分离出来的
by Selman Waxman,

灰色链霉菌中提纯出链霉素
Elizabeth Bugie and Albert Shatz.

1944年11月20日
On November 20, 1944,

第一次在一位危重病人身上使用链霉素
for the first time it was given to a critically ill patient.

他痰中的细菌出乎意料地消失
The bacteria surprisingly disappeared

且病人迅速地恢复过来
from his sputum and he made a rapid recovery.

然而 链霉素存在问题
There were problems however with streptomycin.

它需要连续注射数月
It had to be injected for months

并且会引起听力问题甚至导致耳聋
and it caused hearing problems and even deafness.

在使用的第一年内
Within the first year of use,

由于细菌迅速对链霉素产生耐药性
some patients began to worsen on treatment

一些患者的治疗开始恶化
because the bacteria had rapidly developed streptomycin resistance.

1944年对氨基水杨酸（Para-aminosalicylic acid PAS）
Para-aminosalicylic acid

首度在一位患者上使用
was first given to a patient in 1944.

您可以看到它与阿司匹林非常相似
You can see it is very similar to aspirin.

病人很快康复
The patient made a dramatic recovery.

PAS是无毒的 可以口服
PAS is non toxic and can be taken orally.

到了20世纪40年代后期
So,by the late 1940s,

PAS联合链霉素治愈了
PAS together with streptomycin

约80％的肺结核患者
cured about 80% of patients with pulmonary TB.

但是20％没有治愈
But 20% were not cured,

尤其是那些有大面积病变或有空洞的患者
especially those with extensive disease or cavities.

1952年
In 1952,

三家制药公司研发了异烟肼（isoniazid INH）
isoniazid was found by three pharmaceutical firms.

它与烟酰胺很相似 并且非常有效
It is very similar to nicotinamide and it is very effective.

但由于两位布拉格化学家
But it could not be patented

Meyer和Mally
because two Prague chemists,

早在1912年已经从煤焦油中
Meyer and Mally,

分离出INH并作为博士论文的一部分
had isolated isoniazid from coal tar in 1912

因此无法获得专利
as part of their PhD thesis.

因为它不能获得专利
But because it could not be patented,

每个病人只需花费100美元使用INH
INH only cost 100 dollars per patient

而链霉素和PAS需要花费3500美元
as opposed to the 3 500 dollars for streptomycin and PAS.

但是当单独使用INH时
But when INH was given alone,

耐药性进化得很快
resistance developed rapidly.

到1955年
By 1955,

出现三联疗法
there was triple therapy: streptomycin,

链霉素 PAS和INH联合使用2年
PAS and INH together for two years

治愈率接近100％
achieved cure rates approaching 100%.

1948年 氨硫脲出现了
In 1948,there was thioacetazonetablets.

接下来 对烟酰胺进行修饰后得到吡嗪酰胺
Then, modifications of nicotinamide led to the development of pyrazinamide

它很有效 但毒性太大
which was very effective but much too toxic.

1956年
Then in 1956,

又有了乙硫异烟胺和丙硫异烟胺
there was ethionamide and prothionamide.

但真正的进展是在1959年
But the real advance came in 1959

利福平（rifampicin, RIF）非常有效
with rifampicin which was extremely effective.

但是单独使用时
But when given alone,

耐药性进化得很快
resistance developed rapidly.

所以 它和异烟肼一起使用
So, it was given with isoniazid.

两种药物联合使用9个月的疗效
When together they were given for 9 months,

与2年的三联疗法一样
it was as effective as 2 years of triple therapy.

然后在1961年
Then in 1961,

乙胺丁醇出现
there was ethambutol.

1962年
In 1962,

吡嗪酰胺再次用于治疗
pyrazinamide was given again

但剂量较低且不会产生毒性
but at lower and non-toxic doses.

当吡嗪酰胺加入INH
When pyrazinamide was added to isoniazid,

RIF和乙胺丁醇时
rifampicin and ethambutol,

治疗时间可缩短至6个月
the treatment could be shortened to just 6 months.

1942年
In 1942,

René Dubos推测
René Dubos hypothesized

是抗生素选择导致了耐药性
that antibiotics selected for resistance.

但在1943年
But in 1943,

Salvador Luria和Max Delbruck
Salvador Luria and Max Delbruck

说明即使没有药物选择也会发生随机突变
showed that random mutations occur even without selection

并可能引起耐药
and can cause resistance.

他们还观察到链霉素耐药菌株
It was also observed that streptomycin-resistant strains

可能传播给其他人并导致疾病
could be transmitted to others and cause disease.

1955年
In 1955,

在英国进行的
in the first national drug-resistance survey in Britain,

第一次全国耐药性调查中
it was found that there were TB strains

发现了已经对链霉素
that had already developed resistance to streptomycin,

PAS和INH产生耐药性的结核菌株
PAS and isoniazid.

在美国
In the USA,

二十世纪六十年代初期
in the early 1960s,

INH耐药率为6.3％
isoniazid resistance was 6.3%

仅仅几年后的六十年代后期
but just a few years later, in the late 1960s,

耐药率升至9.7％
it was up to 9.7%.

还发现即使没有药物选择
It was also found that resistant strains did not revert to sensitive

耐药菌株也不会恢复敏感性
even with no selection.

如果菌株对链霉素耐药
If a strain was resistant to streptomycin,

即使病人没有使用链霉素
even if the patient was not given streptomycin,

菌株仍然耐链霉素
the strain remained resistant to streptomycin.

大约1/10^6的细菌会随机突变
About 1 in 10 to the 6th bacteria will be spontaneously resistant

而自发地对任何抗生素产生耐药性
to any antibiotic by random mutations.

一个空洞可以有10^9个细菌
A cavity can have 10 to the 9th or to the 10th bacteria,

所以它会包含一些
so it will contain some bacteria

对所使用的抗生素耐药的细菌
that are resistant to any antibiotic given.

然而
However,

细菌不太可能对两种抗生素均产生耐药性
it is unlikely that any bacteria will have resistance to two antibiotics

因为这种情况发生的概率
because the frequency would be

大约是1/10^6乘以1/10^6
about 10 to the 6th x 10 to the 6th

也就是1 / 10^12
or about 1in 10 to the 12th.

但如果四种药物不规律使用
But if the four drugs are not taken routinely

或未完全吸收
or not absorbed completely,

其中三种药物的浓度可能较低
the concentration of three drugs could be low,

使得对第四种药物有抗性的杆菌存活
allowing bacilli resistant to the fourth drug to survive,

生长和繁殖
grow and reproduce.

然后
Then,

当药物浓度再次降低时
when drug concentrations are again low,

对第二种药物也具有抗性的杆菌
bacilli also resistant to a second drug

可以存活和繁殖
could survive and reproduce.

继发性耐药是在对以前药物敏感的菌株
Secondary resistance is resistance that develops while on therapy

进行治疗时发生的耐药
in a previously drug-sensitive strain.

这是继发性耐药的一个例子
Here is an example of secondary drug resistance.

来自里昂的糖尿病患者Luc
Luc,the diabetic patient in Lyon,

再次咳嗽 发烧
came in again with cough and fever.

他之前曾接受过两次结核病治疗
He was previously treated twice for TB

但依从性很差
but he was poorly compliant.

他没有服用所有的药物
He did not take all of his medicines.

分子流行病学研究表明
Molecular epidemiology studies

他体内有和刚开始治疗时
show that he has the same strain of TB

相同的结核菌株
that he had when he first started treatment.

他体内菌株最初是对药物敏感的
His strain was originally drug sensitive

但现在是耐多药的
but is now multi-drug resistant.

原发性耐药是指最初感染的即为耐药菌株
Primary resistance is initial infection with a strain that is already resistant.

早在20世纪70年代就出现了
Outbreaks of drug-resistant strains or primary resistance

耐药性菌株或者原发性耐药的暴发
were seen as early as the 1970s.

然而在20世纪90年代
In the 1990s however,

有许多耐多药病例的暴发
there were many multidrug-resistant outbreaks,

主要是在HIV阳性患者中
mostly in HIV-positive patients.

当时没有有效的HIV治疗方法
There was no effective treatment for HIV at that time.

所以这类病人很容易患结核病和其它传染病
So,patients were very susceptible to TB and other infections.

HIV患者经常出现在门诊和医院
HIV patients were frequently in clinics and hospitals.

有研究发现耐多药（Multidrug-resistant MDR）菌株
It was found that the MDR strains

正在医院和医疗机构内的
were being transmitted amongst the HIV-positive patients

HIV阳性患者之间传播
within the hospitals and health facilities.

起初 药敏试验不是常规进行的
Drug-sensitivity testing at that time was not routine at first.

传统的药敏试验
Traditional drug-sensitivity testing

需要2到3个月才能出结果
took 2 to 3 months to get results.

但到了3个月的时候
But by then,by 3 months,

超过一半的HIV阳性的
more than half of the HIV-positive MDR patients

MDR病人已经死亡
had died.

耐多药结核病（Multidrug-resistantTB, MDR-TB）的结核菌株
MDR-TB is tuberculosis strains

至少耐RIF和INH
that are resistant to at least rifampicin and isoniazid.

INH抑制合成霉菌酸
Isoniazid inhibits the enzyme InhA

所必需的InhA酶
which is essential for making mycolic acids.

RIF结合RNA聚合酶的rpoB亚基
Rifampicin binds the rpoB subunit of the RNA polymerase.

可能这些关键酶的突变
It might be thought that mutations in these critical enzymes

使结核杆菌变弱和适应性降低
make the bacillus weak and less fit.

事实上 一些耐药菌株的毒力较弱
Indeed, some resistant strains were less virulent

不易传播
and not readily transmitted

但是出现多次高度传播的
but there were many outbreaks

耐多药菌株导致的暴发
of highly-transmitted MDR strains.

INH是一种无活性的前体药物
Isoniazid is given as an inactive prodrug

必须由KatG活化成其活化形式
that must be activated by KatG into its active form.

KatG是一种细菌过氧化氢酶和过氧物酶
KatG is a bacterial catalase peroxidase

可以保护芽孢杆菌免受氧化应激
that protects the bacillus from oxidative stress.

如果KatG缺失或灭活
If KatG is missing or inactivated,

因为INH不能被活化
the bacteria is resistant to isoniazid,

细菌对耐药
it cannot be activated.

但细菌适应性降低
But maybe the bacteria is less fit and more easily killed

更容易被免疫细胞杀死
by cells of the immune system.

但是KatG有一个特殊的突变
However, there is a special mutation in KatG.

KatG酶的315位氨基酸
The KatG enzyme with mutations

从丝氨酸突变为苏氨酸
that change the amino acid 315 from serine to threonine

不会活化INH
will not activate INH,

因此产生高度耐药性
so produces high-level resistance.

高度耐药性在InhA靶标
A high-level resistance that is much higher than mutations in the InhA target

或启动子中具有更多的突变
or in the promoter to the InhA

因此产生了更多的InhA酶
that makes more of the InhA enzyme.

但这种315位氨基酸
But the KatG enzyme

发生置换的KatG酶
with this 315 substitution

几乎保留了全部的过氧化氢酶
retains nearly full catalase

和过氧物酶活性
and peroxidase activity,

因此细菌适应性未降低
so it has no loss of fitness.

而且可以预见的是
And predictably,

大多数暴发的MDR菌株
most outbreak MDR strains

具有KatG315位从丝氨酸到苏氨酸的置换
have the KatG serine-315-threonine substitution.

95％至98％的RIF耐药突变的发生
95 to 98% of rifampicin-resistant mutations

在位于RNA聚合酶5个亚基之一的
are in an 81 base per segment of RpoB

RpoB片段的第81碱基中
which is one of the five subunits of the RNA polymerase.

RIF耐药突变可能会影响聚合酶的功能
RIF-resistance mutations may compromise polymerase function

但大多数暴发菌株在RNA聚合酶的
but most outbreak strains also have additional mutations,

其他亚基中或RpoB基因的另一个位点中
either in other subunits of the RNA polymerase or in another place,

也有额外的突变
in the RpoB gene.

这些突变可能会恢复完整的RNA聚合酶功能
These may restore full RNA polymerase function

但也恢复全面适应性
but also restore full fitness

使菌株可以传播并导致严重的疾病
so that the strains can be transmitted and cause severe disease.

如果有许多MDR-TB
Where there are many patients who have MDR-TB,

这可能是由于原发性耐药
this is probably the result of primary resistance,

即高度传播性的MDR菌株在社区内流行
highly-transmitted MDR strains circulating within the community.

例如 突尼斯的一次暴发
For instance,here is an outbreak from Tunisia.

您可以看到
You can see

所有这些MDR菌株的限制性
all of these MDR strains

内切酶片段长度多态性 (Restriction Fragment Length Polymorphism RFLP)
look exactly the same

结果看起来完全相同
by RFLP.

对敏感性结核病有效的药物
MDR-TB is not cured

并不能治愈MDR-TB
by drugs for sensitive TB.

如果MDR-TB患者接受无效药物的治疗
If patients with MDR-TB receive ineffective drugs,

则可能对其它药物产生耐药性
they can develop resistance to additional drugs,

并发展广泛耐药结核病（extensively-drug-resistant TB,XDR-TB）
and develop XDR-TB or extensively-drug-resistant TB.

XDR-TB是指在MDR-TB的基础上
XDR-TB means MDR-TB plus resistance

对氟喹诺酮和任何一种注射药物耐药
to a fluoroquinolone and resistance to any of the injected drugs.

治疗MDR-TB和 XDR-TB的二线
Second- and third-line drugs for MDR-TB

和三线药物非常昂贵
and XDR-TB are very expensive.

许多低资源国家承担不起
Many low-resource countries

治疗MDR-TB和 XDR-TB的费用
cannot afford the cost of treating MDR- and XDR-TB.

他们需要外部资助的资金
They need funding from outside sources

如全球基金
such as the Global Fund.

下面是原发性耐药的例子
Here an example of primary drug resistance.

东欧女性HIV感染者Sylvia
We have Sylvia who is an HIV-positive woman in Eastern Europe.

她有发烧 咳嗽和体重下降的症状
She has fever,cough and weight loss.

MDR-TB治疗4个月后
After 4 months of treatment for MDR-TB,

她仍然是BK阳性
she is still BK-positive.

她的痰里有细菌
She has bacilli in her sputum.

这是因为她感染了北京基因型XDR菌株
It is because she is infected with an XDR-TB strain

该菌株在她的国家传播
of the Beijing family that is circulating in her country

导致许多XDR-TB病例
and causing many XDR-TB cases.

政府卫生系统难以找到足够的药物
The government health system has trouble finding adequate drugs

来治疗这些病人
to treat these patients

治疗这些病人的高额费用
and the high cost of treating them

给国家卫生系统造成了很大的负担
is a large burden on the National Health System.

综上所述
In summary.

多药物联合治疗对于治愈结核病是必要的
Multidrug therapy is necessary to cure TB

因为如果只使用一种或两种药物
because resistance develops rapidly

耐药性就会迅速发展
if only one or two drugs are given.

说到继发性耐药
This is secondary resistance.

在出现多例MDR-TB患者的地方
In places with many MDR-TB cases,

大多数可能是由于社区内
most are probably the result of transmission of MDR-TB strains circulating

流行的MDR-TB菌株的传播
within the community.

至于原发性耐药
This is primary resistance.

如果MDR菌株
If MDR strains are not treated with several drugs

没有被一些其敏感的药物治疗
to which they are sensitive,

则会产生额外的耐药性
they can develop additional resistance,

成为 XDR-TB
become XDR-TB,

那将需要不同的药物
will require different drugs

而且很难治愈
and are very difficult to cure.

然后 XDR-TB菌株可能在社区内传播
Then,the XDR-TB strains can be transmitted within the community.

感谢您的关注
Thank you for your attention.