New Approaches to the Diagnosis and Management of Tuberculosis

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1 New Advances in Diagnosis and Treatment of Tuberculosis

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5 Rober koch ( ) Discoverer of the tubercle bacillus in 1882

(Sensitivity 30-50%, Specificity >99%) 10 4-10

6 (Sensitivity 30-50%, Specificity >99%) bacilli/ml NTM Acid Fast stain (+) (Norcadia)

7 J Respir Crit Care Med Vol 161. pp , 2000

8 Advantages and Disadvantages of Diagnostic Methods Diagnostic method Advantages Disadvantages Smear stain Culture Chest radiograph PPD PCR Thorax CT scan Rapid Definitive diagnosis of M. tuberculosis Can help detect/differentiate lung pathologies Good for screening; can aid in diagnosis if positive Rapid Enhanced visualization of small lesions not seen on chest radiograph; can be used for follow-up Very low sensitivity Low sensitivity, very slow Indicative findings (adenopathy( adenopathy) not detected in many TB cases False positives and false negatives occur; does not distinguish between disease and infection Costly; requires qualified laboratory; false positives Costly; requires scanner which is not readily available in many settings

9 Impetus for Faster Diagnostic Techniques

10 Molecular Techniques for Mycobacteriosis Diagnosis Nucleic acid amplification techniques Isothermal methods (MTD) Polymerase chain reaction (PCR) Strand Displacement Amplification Identification of isolates Nucleic acid probes Ribotyping (16S rrna,, 23S rrna) ) Detection of drug resistance Genomic analysis for drug susceptibility testing Epidemiology and surveillance Restriction fragment length polymorphism (RFLP) Spoligotyping Molecular epidemiology Detection of laboratory errors

11 Overview of in vitro Nucleic Acid Amplification Techniques for the Detection of M. tuberculosis Target amplification PCR (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 amplification LCR (Protein antigen b gene) Q-Beta (23S rrna) Signal amplification bdna (as for PCR) Infection with mycobacteriophages LRM (injected phage gene for luciferase)

12 Sensitivity of Various Assays Used to Detect Mycobacteria in Clinical Samples Am J Clin Pathol 2000;114:

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

14 Diagnostic Yields of Single Tube Nested PCR in Different Clinical Specimens Percent positive (%) Specimens sputum pleural effusion ascites CSF blood urine lung aspirate BAL/brusing gastric aspirate joint fluid pericardial fluid LN aspirate tissue section nasopharyngeal aspirates pus

15 Comparison of Two FDA-Approved Approved Nucleic Acid Amplification Methods

16 Comparison of Different Amplification Methods in Direct Detection of Mycobacteria

17 The 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

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

19 Identification by Nucleic Acid Probe Commercially available probes (AccuProbe( AccuProbe) Offer the specificity of DNA probes, with the simplicity and speed ed of Hybridization Protection Assay (HPA) technology. M. tuberculosis complex M. kansasii M. avium-intracellulare intracellulare M. gordonae Sensitivity and specificity of 100% Relatively inexperienced lab personnel can perform AccuProbe assays Results are obtained by utilizing GEN-PROBE luminometers False positive: MAC and M. terrae-like organisms

20 Gene Targets For Anti-tuberculosis tuberculosis Drugs And Mutations Associated With Drug Resistance Direct approach: molecular analysis of changes in gene sequences

21 Probes for Reverse Dot Blot Hybridization 1 cm

22 Representative Hybridization Patterns Obtained with RDB Assay

23 RDB Patterns of MOTT and MDRTB NTM MDRTB

24 Sensitivity and Specificity of RDB Hybridization in Detection of Rifampicin Resistance from Clinical Isolates Results of bioassays RDB results Resistant strains Sensitive strains Sensitivity Specificity Mutant type % 100% Wild type 0 72

25 An Integrated Model For Reducing Transmission Of Tuberculosis BMJ 1998;317:1263 4

26 Genotyping of M. tuberculosis RFLP

27 RFLP Patterns of M. tuberculosis complex and NTM M. tuberculosis complex NTM

28 Spoligotyping of M. tuberculosis complex Reversed Line Blotting Polymorphism of the chromosomal direct repeat (DR) locus

29 Clinical Applications of Genotyping Molecular epidemiology: : IS6110, TBN12 Transmission patterns (TB outbreaks) The natural histories of primary and reinfection tuberculosis (exogenous reinfection and reactivation) Detection of laboratory errors Cross-contamination of specimens (false positive)

31 INH+EMB+RMP+PZA INH+EMB+RMP

32 Limitations of the Anti-tuberculosis tuberculosis Chemotherapy The duration of treatment required for curing patients cannot be reduced below 6 months. High rates of patient nonadherence Increased mortality and drug resistance All drugs must be taken together. Unpleasant side-effects Second-line drugs: expensive, toxic.

33 The Need for New Strategies for the Treatment of Tuberculosis To improve current treatment by shortening the total duration of Tx and/or by providing for more widely spaced intermittent Tx To improve the treatment of MDR TB To provide for more effective treatment of latent tuberculosis infection (LTBI)

35 DOTS (Direct Observed Treatment, Short Course)

36 New Drugs Therapies (I) 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)

37 New Drugs Therapies (II) New drugs: : developed from existing lead molecules Fluoroquinolones (MICs MICs: : µg/ml) Sparfloxacin Moxifloxacin Levofloxacin Ciprofloxacin Ofloxacin Advantages: Concentrate well within macrophages and distribute into the lungs during therapy Drawback: Rapidly emerging resistance Considerable cross resistance to the entire class of quinolones Damage to cartilage growth Children, adolescents, pregnant women

38 New Drugs Therapies (III) Dormant bacilli Completely new lead molecules Oxazolidinones: DuP 721 Nitroimidazoles: : CGI Nitro radical anion nitrite + imidazole radical DNA damage At 0.1 to 0.3 mg/ml, CGI 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

43 (dressing change) (autopsy)

44 HEPA filters, UVGI >6 AC/h (UVGI)

PowerPoint 簡報

PowerPoint 簡報 2016 1 2 960 150 120 48 HIV 40 19 3 2035 (SDGs) WHOTB 2020 2025 2030 2035 TB 2015 TB 2015 TB 35% 75% 90% 95% 20% 50% 80% 90% 0% 0% 0% 0% 4 Projected acceleration of TB incidence decline to target levels