New Advances in Diagnosis and New Advances in Diagnosis and Treatment of TuberculosisTreatment of Tuberculosis

蘇維鈞蘇維鈞

臺北榮民總醫院臺北榮民總醫院

胸腔部胸腔部 呼吸感染免疫科呼吸感染免疫科

結核病是一個重要的公共衛生問題結核病是一個重要的公共衛生問題

••1/3 world’s population carry M. tuberculosis: not infectious‧8 million cases/year: contagious‧3 million deaths/year‧台灣地區每年約有一萬五千個新發生的結核病人，其中五千多人是有傳染性的結核病人。

臺灣結核病流行病學資料

臺臺 灣灣 地地 區區 主主 要要 死死 亡亡 原原 因因民國八十九年民國八十九年

順 合 計 男 性 女 性

死亡 每十萬 死亡 死亡 每十萬 死亡 死亡 每十萬 死亡

死 亡 原 因 人口 百分比 死 亡 原 因 男性人口 百分比 死 亡 原 因 女性人口 百分比

位 人數 死亡率 % 人數 死亡率 % 人數 死亡率 %

所有死亡原因 124,481 561.12 100.00 所有死亡原因 77,280 680.74 100.00 所有死亡原因 47,201 435.75 100.00

1 惡性腫瘤 31,554 142.23 25.35 惡性腫瘤 20,357 179.32 26.34 惡性腫瘤 11,197 103.37 23.72

2 腦血管疾病 13,332 60.10 10.71 腦血管疾病 7,822 68.90 10.12 腦血管疾病 5,510 50.87 11.67

3 心臟疾病 10,552 47.56 8.48 事故傷害 7,784 68.57 10.07 糖尿病 5,026 46.40 10.65

4 事故傷害 10,515 47.40 8.45 心臟疾病 6,341 55.86 8.21 心臟疾病 4,211 38.88 8.92

5 糖尿病 9,450 42.60 7.59 糖尿病 4,424 38.97 5.72 事故傷害 2,731 25.21 5.79

6 慢性肝病及肝硬化 5,174 23.32 4.16 慢性肝病及肝硬化 3,793 33.41 4.91 腎炎、腎徵候群及腎變性病 1,826 16.86 3.87

7 腎炎、腎徵候群及腎變性病 3,872 17.45 3.11 肺炎 2,104 18.53 2.72 慢性肝病及肝硬化 1,381 12.75 2.93

8 肺炎 3,302 14.88 2.65 腎炎、腎徵候群及腎變性病 2,046 18.02 2.65 肺炎 1,198 11.06 2.54

9 自殺 2,471 11.14 1.99 自殺 1,645 14.49 2.13 自殺 826 7.63 1.75

10 支氣管炎、肺氣腫及氣喘 1,604 7.23 1.29結核病 1,215 10.70 1.57 高血壓性疾病 765 7.06 1.62

其他 32,655 147.20 26.23 其他 19,749 173.96 25.56 其他 12,530 115.67 26.55

11 高血壓性疾病 1,602 7.22 1.29 支氣管炎、肺氣腫及氣喘 1,040 9.16 1.35 支氣管炎、肺氣腫及氣喘 564 5.21 1.19

12 結核病 1,534 6.91 1.23 高血壓性疾病 837 7.37 1.08 敗血症 417 3.85 0.88

13 胃及十二指腸之潰瘍 1,035 4.67 0.83 胃及十二指腸之潰瘍 706 6.22 0.91 先天性畸形 367 3.39 0.78

14 敗血症 856 3.86 0.69 源於周產期之病態 486 4.28 0.63 源於周產期之病態 352 3.25 0.75

15 源於周產期之病態 838 3.78 0.67 敗血症 439 3.87 0.57 胃及十二指腸之潰瘍 329 3.04 0.70

附 註：1.臺灣地區年中人口數 計22,184,530人, 男性11,352,389人, 女性10,832,140人。

2.死因分類號碼中* 符號表示其病名僅佔該號碼中之一部份疾病，28* 係包括國際詳細分類號碼 420-429 之全部疾病。

Rober koch (1843-1910)Discoverer of the tubercle bacillus in 1882Discoverer of the tubercle bacillus in 1882

痰液塗片耐酸性染色痰液塗片耐酸性染色●●不敏感不敏感 (Sensitivity 30(Sensitivity 30--50%, Specificity >99%)50%, Specificity >99%)

•• 101044--10105 5 bacilli/bacilli/mLmL●●不是直接證據不是直接證據

•• NTMNTM•• 其他其他Acid Fast stain (+) Acid Fast stain (+) 的細菌的細菌 ((NorcadiaNorcadia))

●● 最重要的肺結核診斷工具最重要的肺結核診斷工具

•• 快速報告快速報告

•• 需要配合的設備最少需要配合的設備最少

•• 找到最具感染性的病人找到最具感染性的病人

分枝桿菌培養分枝桿菌培養

J Respir Crit Care Med Vol 161. pp 1376–1395, 2000

Advantages and Disadvantages of Diagnostic MethodsAdvantages and Disadvantages of Diagnostic Methods

Costly; requires scanner which Costly; requires scanner which is not readily available in many is not readily available in many settingssettings

Enhanced visualization of Enhanced visualization of small lesions not seen on small lesions not seen on chest radiograph; can be chest radiograph; can be used for followused for follow--upup

Thorax CT scanThorax CT scan

Costly; requires qualified Costly; requires qualified laboratory; false positiveslaboratory; false positivesRapidRapidPCRPCR

False positives and false False positives and false negatives occur; does not negatives occur; does not distinguish between disease distinguish between disease and infectionand infection

Good for screening; can aid Good for screening; can aid in diagnosis if positivein diagnosis if positivePPDPPD

Indicative findings (Indicative findings (adenopathyadenopathy) ) not detected in many TB casesnot detected in many TB cases

Can help detect/differentiate Can help detect/differentiate lung pathologieslung pathologiesChest radiographChest radiograph

Low sensitivity, very slowLow sensitivity, very slowDefinitive diagnosis of Definitive diagnosis of M. M. tuberculosistuberculosisCultureCulture

Very low sensitivityVery low sensitivityRapidRapidSmear stainSmear stain

DisadvantagesDisadvantagesAdvantagesAdvantagesDiagnostic Diagnostic methodmethod

Impetus for Faster Diagnostic TechniquesImpetus for Faster Diagnostic Techniques

Molecular Techniques for Molecular Techniques for MycobacteriosisMycobacteriosisDiagnosisDiagnosis

Nucleic acid amplification Nucleic acid amplification techniquestechniques

Isothermal methods Isothermal methods (MTD)(MTD)Polymerase chain Polymerase chain reaction (PCR)reaction (PCR)Strand Displacement Strand Displacement AmplificationAmplification

Identification of isolatesIdentification of isolates

Nucleic acid probesNucleic acid probesRibotypingRibotyping (16S (16S rRNArRNA, 23S , 23S rRNArRNA)

Detection of drug resistanceDetection of drug resistance

Genomic analysis for drug Genomic analysis for drug susceptibility testingsusceptibility testing

Epidemiology and surveillanceEpidemiology and surveillance

Restriction fragment length Restriction fragment length polymorphism (RFLP)polymorphism (RFLP)

SpoligotypingSpoligotyping

Molecular epidemiologyMolecular epidemiology

Detection of laboratory Detection of laboratory errorserrors

)

Overview of in vitro Nucleic Acid Amplification Overview of in vitro Nucleic Acid Amplification Techniques for the Detection of Techniques for the Detection of M. tuberculosisM. tuberculosis

Target amplificationTarget amplificationPCR (IS6110, 65 kDa protein gene, 16S rDNA gene, MPB64 gene, 35 kDa protein gene, etc.)TMA (16S rRNA)SDA (IS6110)NASBA (16S rRNA)

Probe/primer amplificationProbe/primer amplificationLCR (Protein antigen b gene)Q-Beta (23S rRNA)

Signal amplificationSignal amplificationbDNA (as for PCR)

Infection with Infection with mycobacteriophagesmycobacteriophagesLRM (injected phage gene for luciferase)

Sensitivity of Various Assays Used to Sensitivity of Various Assays Used to Detect Mycobacteria in Clinical SamplesDetect Mycobacteria in Clinical Samples

Am J Clin Pathol 2000;114:940-950

Comparison of inComparison of in--house nested PCR, smear and house nested PCR, smear and culture results for the clinical diagnosis of TBculture results for the clinical diagnosis of TB

Diagnostic Yields of Single Tube Nested PCR Diagnostic Yields of Single Tube Nested PCR in Different Clinical Specimensin Different Clinical Specimens

0

10

20

30

40

50

60

70

80

Per

cent

pos

itive

(%)

Specimens

sputumpleural effusionascitesCSFbloodurinelung aspirateBAL/brusinggastric aspiratejoint fluidpericardial fluidLN aspiratetissue sectionnasopharyngeal aspiratespus

Comparison of Two FDAComparison of Two FDA--Approved Approved Nucleic Acid Amplification MethodsNucleic Acid Amplification Methods

Comparison of Different Amplification Methods Comparison of Different Amplification Methods in Direct Detection of Mycobacteriain Direct Detection of Mycobacteria

The Role of PCR Diagnostic TestingThe Role of PCR Diagnostic Testing

Improve the diagnosis of tuberculosis in:

patients with atypical presentation

patients treated with antibiotics

the immunodeficient patients

patients with extrapulmonary TB

Clinical Utility of Polymerase Chain Reaction Clinical Utility of Polymerase Chain Reaction (PCR) in Diagnosis of Tuberculosis(PCR) in Diagnosis of Tuberculosis

Identification by Nucleic Acid ProbeIdentification by Nucleic Acid Probe

Commercially available probes (Commercially available probes (AccuProbeAccuProbe))

Offer the specificity of DNA probes, with the simplicity and speOffer the specificity of DNA probes, with the simplicity and speed ed of Hybridization Protection Assay (HPA) technology.of Hybridization Protection Assay (HPA) technology.

M. tuberculosis complexM. tuberculosis complex

M. M. kansasiikansasii

M. M. aviumavium--intracellulareintracellulare

M. M. gordonaegordonae

Sensitivity and specificity of 100%Sensitivity and specificity of 100%

Relatively inexperienced lab personnel can perform Relatively inexperienced lab personnel can perform AccuProbeAccuProbeassaysassays

Results are obtained by utilizing GENResults are obtained by utilizing GEN--PROBEPROBE®® luminometersluminometers

False positive: False positive: MAC and MAC and M. M. terraeterrae--like organismslike organisms

Gene Targets For AntiGene Targets For Anti--tuberculosis Drugs And tuberculosis Drugs And

Mutations Associated With Drug ResistanceMutations Associated With Drug Resistance

Direct approach: Direct approach: molecular analysis of changes in gene sequencesmolecular analysis of changes in gene sequences

Probes for Reverse Dot Blot HybridizationProbes for Reverse Dot Blot Hybridization

1 cm

Representative Hybridization Patterns Obtained Representative Hybridization Patterns Obtained with RDB Assaywith RDB Assay

NTMNTM MDRTBMDRTBRDB Patterns of RDB Patterns of

MOTT and MDRTBMOTT and MDRTB

Sensitivity and Specificity of RDB Sensitivity and Specificity of RDB Hybridization in Detection of Rifampicin Hybridization in Detection of Rifampicin

Resistance from Clinical IsolatesResistance from Clinical Isolates

RDB results

Results of bioassays

Sensitivity SpecificityResistant

strainsSensitive

strains

Mutant type

33 0 100% 100%

Wild type 0 72

An Integrated Model For Reducing An Integrated Model For Reducing Transmission Of TuberculosisTransmission Of Tuberculosis

BMJ 1998;317:1263–4

Genotyping of Genotyping of M. tuberculosisM. tuberculosisRFLPRFLP

RFLP Patterns of RFLP Patterns of M. tuberculosisM. tuberculosis complex and NTMcomplex and NTM

M. tuberculosis complexM. tuberculosis complex NTMNTM

SpoligotypingSpoligotyping of of M. tuberculosis complexM. tuberculosis complexReversed Line BlottingReversed Line Blotting

Polymorphism of the chromosomal direct repeat (DR) locusPolymorphism of the chromosomal direct repeat (DR) locus

Clinical Applications of GenotypingClinical Applications of Genotyping

Molecular epidemiologyMolecular epidemiology: IS6110, TBN12: IS6110, TBN12Transmission patterns (TB outbreaks)Transmission patterns (TB outbreaks)

The natural histories of primary and The natural histories of primary and reinfectionreinfection tuberculosis (exogenous tuberculosis (exogenous reinfectionreinfection and reactivation)and reactivation)

Detection of laboratory errorsDetection of laboratory errorsCrossCross--contamination of specimens (false contamination of specimens (false positive)positive)

初次短程治療初次短程治療

藥藥 物物 INH+EMB+RMP+PZA INH+EMB+RMPINH+EMB+RMP+PZA INH+EMB+RMP

Limitations of the AntiLimitations of the Anti--tuberculosis tuberculosis ChemotherapyChemotherapy

The duration of treatment required for curing

patients cannot be reduced below 6 months.

High rates of patient nonadherencenonadherence

Increased mortality and drug resistancedrug resistance

All drugs must be taken togethertaken together.

Unpleasant side-effects

Second-line drugs: expensive, toxicexpensive, toxic.

The Need for New Strategies for the The Need for New Strategies for the Treatment of TuberculosisTreatment of Tuberculosis

To improve current treatmentTo improve current treatment

by by shortening the total duration of shortening the total duration of TxTx and/or and/or

by providing for more widely spaced by providing for more widely spaced intermittent intermittent TxTx

To improve the treatment of To improve the treatment of MDR TBMDR TB

To provide for more effective treatment of latent To provide for more effective treatment of latent

tuberculosis infection tuberculosis infection (LTBI)(LTBI)

DOTSDOTS (短程直接觀察治療法短程直接觀察治療法)(Direct Observed Treatment, Short Course)

送送 藥藥 到到 手手

服服 藥藥 入入 口口

吞吞 了了 再再 走走

New Drugs Therapies (I)New Drugs Therapies (I)Existing drugs:Existing drugs:

Rifabutin (spiropiperidyl derivative of rifampicin S)Less susceptible to the known mechanisms of resistance (rpoB mutation position; type of amino acid change)Wider spectrum of activity encompasses the NTM (prophylaxis against MAC infections in AIDS patients)

KRM-1648 (benzoxazinorifamycin)Very short course regimens of therapy: 4-months or less (combines with INH or PZA)

New Drugs Therapies (II)New Drugs Therapies (II)New drugsNew drugs: developed from existing lead molecules: developed from existing lead molecules

FluoroquinolonesFluoroquinolones ((MICsMICs: 0.06: 0.06--0.5 0.5 µµg/ml)g/ml)SparfloxacinSparfloxacinMoxifloxacinMoxifloxacinLevofloxacinLevofloxacinCiprofloxacinCiprofloxacinOfloxacinOfloxacin

Advantages:Advantages:Concentrate well within macrophages and distribute into the Concentrate well within macrophages and distribute into the lungs during therapylungs during therapy

Drawback:Drawback:Rapidly emerging resistanceRapidly emerging resistanceConsiderable cross resistance to the entire class of Considerable cross resistance to the entire class of quinolonesquinolonesDamage to cartilage growthDamage to cartilage growth

Children, adolescents, pregnant womenChildren, adolescents, pregnant women

New Drugs Therapies (III)New Drugs Therapies (III)Dormant bacilliDormant bacilli

Completely new lead moleculesCompletely new lead moleculesOxazolidinonesOxazolidinones: : DuPDuP 721721NitroimidazolesNitroimidazoles: CGI 17341 : CGI 17341

Nitro radical anion → nitrite + imidazole radical → DNA damageAt 0.1 to 0.3 mmg/ml, CGI 17341 inhibited the drug-susceptible and multi-drug-resistant strains of Mycobacterium tuberculosis.

No cross-resistance with isoniazid, rifampin, streptomycin, or ethambutol.

Comparable to those of isoniazid and rifampin; superior to those of streptomycin, ciprofloxacin or norfloxacin, and oxazolidinone DuP 721

The MIC was not affected when the pH of the medium.

Antimicrob Agents Chemother. 1993;37(2):183-6

防疫危機，肺結核院內感染防疫危機，肺結核院內感染台北市振興醫院集體爆發肺結核院內感染，六十七人疑似台北市振興醫院集體爆發肺結核院內感染，六十七人疑似

有肺結核症狀，總計共有七人感染。有肺結核症狀，總計共有七人感染。

關渡醫院疑爆發肺結核院內感染。關渡醫院疑爆發肺結核院內感染。

台北榮總傳肺結核病例！疾管局：排除群聚與院內感染台北榮總傳肺結核病例！疾管局：排除群聚與院內感染......

根據調查台灣醫師結核病發生率為每十萬人口六十四

人，護士結核病發生率為每十萬人八十六人，一般統

計，醫護人員結核病發生率為一般人口的一點五倍，比

較落後國家甚至可以高到到十倍，台灣醫護人員染結核

病並未出現偏高現象。

醫院內爆發肺結核流行之原因醫院內爆發肺結核流行之原因

延遲診斷

未接受有效的治療

未辨識出的抗藥性

通風不良 –隔離室正壓、廢氣再循環呼吸器 –使結核菌霧化支氣管鏡檢查

換藥 (dressing change)沖洗髖關節結核 –激起水霧屍體解剖 (autopsy)

肺結核院內感染控制方法肺結核院內感染控制方法

「盡早找出傳染病例並有效治療」最為重要「盡早找出傳染病例並有效治療」最為重要

改善通氣設備改善通氣設備

足夠通氣量；減少廢氣再循環足夠通氣量；減少廢氣再循環

廢氣消毒廢氣消毒 –– HEPA filters, UVGIHEPA filters, UVGI

負壓隔離室負壓隔離室 ––門窗常保持關閉、門窗常保持關閉、

注意氣流方向、換氣量注意氣流方向、換氣量 >6 AC/h>6 AC/h

紫外線殺菌照射紫外線殺菌照射 (UVGI) (UVGI)

維護費用較「改善通氣設備」、「負壓隔離室維護費用較「改善通氣設備」、「負壓隔離室 」低廉」低廉

安全性高安全性高 –– 不會導致皮膚癌、白內障不會導致皮膚癌、白內障

適用於高危險場所適用於高危險場所 –– 隔離室、急診處隔離室、急診處

保護性口罩保護性口罩(N95)(N95)

病人在隔離病房內不須戴用，教導病人在隔離病房內不須戴用，教導

病人咳嗽時以手掩口。病人咳嗽時以手掩口。

病患離開隔離病房時使用。病患離開隔離病房時使用。

醫護人員在下列狀況須穿戴高效能醫護人員在下列狀況須穿戴高效能

口罩口罩

結核病隔離室內結核病隔離室內

高危險操作高危險操作 –– 如：支氣管鏡檢、屍體如：支氣管鏡檢、屍體

解剖解剖

運送結核病病人運送結核病病人

攜手共抗結核病攜手共抗結核病