Diagnosis of drug resistant TB

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1 Diagnosis of drug resistant TB Megan Murray, MD, ScD Harvard School of Public Health Brigham and Women s Hospital Harvard Medical School Broad Institute

2 Global burden of TB 9 million new cases year 2 million deaths year 2 billion people infected 80% cases in top 22 burden countries 25% case fatality Leading cause of death in HIV infected patients Dye, C et al. Global burden of tuberculosis: estimated incidence, prevalence and mortality by country. JAMA 1999; 282:

3 Dye, C. Tuberculosis, Lancet 2006

4

5

6 XDR = MDR plus resistance to a floroquinolone and an injectable second line drug (usually an aminoglycoside)

7 Latest global TB Estimates All forms of TB Greatest number of cases in Asia; greatest rates per capita in Africa Multidrug-resistant TB (MDR-TB) Estimated number of cases 9.15 million 489,000 Estimated number of deaths 1.65 million 120,000 Extensively drugresistant TB (XDR-TB) 40,000 20,000 HIV-associated TB 700, ,000 Adapted from a slide provided by Dr. Paul Nunn, WHO Geneva

8 Notified cases of MDR-TB ( ) and projected patients to be treated ( ) compared to estimated burden of MDR-TB Estimated 489,000 new MDR-TB cases each year non-glc GLC Proportion of the MDR case load being diagnosed and treated

9 Consequence of undiagnosed, untreated DR TB not only poor outcomes but also spread of DR TB

10 Reproductive number R0: the average number of secondary infectious cases directly infected by a single infectious case during the entire infectious period when s/he enters a totally susceptible population R0>1 R0<1 Disease can spread Disease cannot spread

11 Epidemic potential R 0 = bkd Where b = transmission probability k = contact rate D = average duration of infectiousness Epidemic potential of MDR/XDR depends on: Transmissibility of MDR/XDR Average number of contacts made between infectious cases and susceptible hosts Duration of infectious state in those with disease

12 New survey finds highest rates of drug-resistant TB to date

13 DOTS Sustained political commitment Access to quality-assured TB sputum microscopy Standardized short-course chemotherapy to all cases of TB under proper case-management conditions Uninterrupted supply of quality-assured drugs Recording and reporting system enabling outcome assessment

14 2 routes to drug resistant TB Inadequate treatment Acquired resistance Drug-sensitive Drug-resistant DOTS X Transmission Primary resistance Uninfected Drug-resistant

15 Limitations of Sputum Smear Microscopy Does not diagnose extrapulmonary TB

16 Limitations of Sputum Smear Microscopy Does not diagnose smear negative TB

17 Multivariate Predictors of Smear- Negative TB Predictors Multivariate OR (95% CI) P- value Expectoration TST result positive 0.3 ( ) 4.8 ( ) Infiltrate not typical of TB interaction HIV-positive status 0.3 ( ) 7.2 ( )

18 Alternatives to SSM Solid Culture Radiography

19 Culture Limitations Requires 3-8 weeks for diagnosis Needs lab infrastructure Biosafety issues Strengths Distinguishes between M. tuberculosis and atypical mycobacteria Can support phenotypic drug suceptibility testing

20 New methods: Growth based approaches that Liquid culture support DST Faster than solid media Direct solid media innoculation with innovative detection methods Faster than macroscopic colony detection Bacteriophage based tests Faster than culture methods

21 Liquid cultures: MGIT (mycobacterial growth indicator tube) Can reduce time to drug susceptibility test to 1-3 weeks Widely used in well resourced settings Costly

22 Liquid Culture: MODS Microscopic observation drug susceptibility culture Characteristic cording morphology of Mycobacterium tuberculosis seen at x20 magnification by inverted light microscopy. Image courtesy of David AJ Moore and Luz Caviedes.

23 TK TK medium rapid culture system for tuberculosis. Mycobacterial growth changes the colour of the medium from red to yellow. Common contaminants, such as fungi or Gram-negative bacteria, change the colour from red to green.

24 Griess Method = Nitrate reductase assay (NRA)

25 Sensitivity Griess method

26 Bacteriophage: Fast plaque

27 Molecular methods NAAT = nucleic acid amplification test Amplifies target DNA Species identification Identified mutations associated with DR

28 First line drug resistance: Rifampicin Single amino acid substitutions in the 81 bp core-region of the rpob gene responsible for conferring rifampicin (RIF) resistance. rpob codes for RNA polymerase.

29 Resistance: Isoniazid INH mono-resistant MDR MDR -plus Distributions of mutations in specific genes. Hazbon M et al. Antimcrobial agents and chemotherapy, 2006

30 Examples Cephead system Closed system with automated sputum processing, DNA extraction, gene amplification, and target detection into a single, handsfree test. Hain test GenoType MTBDR (Hain) and INNO-LiPA.Rif TB (Innogenetics) use PCR followed by amplicon hybridization onto a series of oligonucleotide probes to detect rifampin resistance with a 1-day turnaround.

31 What about second line drugs? Floroquinolones Ciprofloxacin Gatifolxacin Moxifloxacin Aminoglycosides Kanamycin Amikacin Streptomycin Capreomycin PAS Ethionoamide Cycloserine PZA Ethambutol

32 Rising rates of second line drug resistance

33 Current projects TB drug resistance mutation database Whole genome sequencing DR strains Gates Foundation study: TB archive

34

35

36 Mechanisms of Rifampin Resistance Rifampin Rifampin Binding Site rpob homology model target of Rifampin rpob homolog from Thermus aquaticus with rifampicin bound (crystal structure)

37 Evolution of the Extensively Drug-Resistant F15/LAM4/KZN Strain of Mycobacterium tuberculosis in KwaZulu-Natal, South Africa Manormoney Pillay and A. Willem Sturm Clinical Infectious Diseases 45(11): Development of drug resistance in the KwaZulu-Natal family of strains of Mycobacterium tuberculosis during the period Ca, capreomycin; E, ethambutol; Et, ethionamide; F, fluoroquinolones; I, isoniazid; K, kanamycin/amikacin; R, rifampicin; S, streptomycin; T, thiacetazone.

38 Drug Old Genes, New Mutations Mutations enriched in genes associated with drug resistance Strain MDR XDR inh katg S315T katg S315T inha -8TA inha -8TA rif rpob D435Y rpob N487S rpob D435G rpob L452P rpob I1106T streptomycin gidb gidb pyrazinamide pnca A132G pnca ethambutol ofloxacin gyra A90V kanamycin rrs A1401G Previously unreported mutations in these genes Compared to a closely related reference strain, F

39 Gates project Identify 2000 TB strains representing drug sensitive, mono-resistant, MDR to XDR Diverse lineage and geography Sequence 26 genes implicated in DR in TB Identify mutations in DR strains Publish all data in user friendly database

40 Ideal diagnostic for DR TB Can be used on sputum samples without the need for elaborate processing or culture. Can detect smear negative TB. Detects M. tuberculosis as well as presence of mutations that confer first and second line resistance. Does not require reference lab. Is easy to use, does not require specialized training Is cheap and readily obtained.

41 Thank you for your attention.

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