xxdr tb

xxdr tb

1. XXDR-TB XXDR TB Presenter - Dr. Nisha Singh

2. sir John Crofton First to study and recognize the importance of drug resistance in TB.

3. History Drug resistance type Mechanism of drug resistance Detection methods for drug resistance TB and HIV PMDT New Anti TB Drug Vaccines

4. Problem of TB in India (WHO SEARO REPORT 2015-2016) Estimated incidence - 2.8 MILLION CASES 167 (156-179) per 1 lac population annually 75 new smear positive PTB cases/1lakh population per year Estimated prevalence of TB disease 195 (131-271) per 1 lac population annually Estimated mortality 4 8 0,000 deaths due to TB each year Over 1000 deaths a day 2 deaths every 3 minutes Gujarat and Maharashtra has indicated multi drug resistance levels of 3% among new TB cases and 12-17% among previously treated TB patients.

5. HISTORY MDR-TB was first seen in the early 90’s First case of XDR – TB in India was diagnosed in P. D. Hinduja National Hospital and Medical Research Centre, Mumbai in 2006 In 2007 (May) – First report of two documented cases of extremely drug resistant (XXDR) TB) in Italy

6. XXDR TB in India In January 2012 it was reported that twelve cases of TB had been diagnosed in Mumbai which were referred to as totally drug resistant (TDR) TB/XXDR NTI Bangalore confirms 8 strains to be resistance to all known first and second line TB drugs and 2 from Mumbai were sensitive to second line drug There have also been six cases in Bangalore and a further two in New Delhi⁵ “ We have little to offer these patients except for drastic surgery and medication for some relief,” Dr Zarir Udwadia 4

7. Categories of Antituberculosis Drugs: WHO Group 1 – First-line drugs : Isoniazid, rifampicin, ethambutol , pyrazinamide Group 2 - Injectable agents: Kanamycin, amikacin , capreomycin , streptomycin Group 3 - Fluoroquinolones : Levofloxacin, moxifloxacin , ofloxacin Group 4 - Oral bacteriostatic agents: Ethionamide , cycloserine , para- aminosalicylic acid (PAS), prothionamide , terizadone Group 5 – Unclear role: Clofazamine , linezolid, amoxicillin/ clavulanate , Imipenem / cilastatin , thioacetazone , high-dose isoniazid, clarithromycin,

8. Drug resistance - types

9. 9 Definitions of drug resistant TB TB condition Definition Monoresistant TB: resistant to a single drug Polyresistant TB: resistant to at least two drugs, but not involving Isoniazid and Rifampicin simultaneously. Multidrug resistant (MDR) Resistance to: Isoniazid Rifampicin Extensively drug resistant (XDR) Resistance to: Isoniazid Rifampicin Any fluoroquinolone At least one of the three injectable drugs: capreomycin , kanamycin , amikacin Extremely drug resistant (XXDR) Resistance to: All first-line anti-TB drugs All second-line anti-TB drugs

10. 10 Random spontaneous mutations creating wild TB bacteria drug-resistant Selection of drug-resistant TB bacteria by inadequate treatment (resulting in monotherapy) due to: inadequate prescription , supply and intake of drugs NATURAL DRUG RESISTANCE ACQUIRED DRUG RESISTANCE MDR /XDR/XXDR MDR-TB can be amplified into XDR-TB OR XXDR-TB by: Inadequate/interrupted treatment with second line anti-TB drugs Indiscriminate use of second-line drugs Non-adherence to national and/or international guidelines Development of anti-tuberculosis drug resistance PRIMARY DRUG RESISTANCE

11. Antimycobacterial agents Genes involved in resistance Mechanism of resistance Isoniazid i ) katG (catalase peroxidase) i) mutations in KatG result in failure to generate an active intermediate of INH. ii) inh A ( enoyl –ACP reductase synthesis) ii) overexpression of inhA allows continuation of mycolic acid iii) ahp C (alkyl hydroperoxide reductase) iii) ahpC mutations as a marker for lesions in KatG Rifampicin rpoB (β subunit of RNA polymerase) Mutations in rpoB prevent interaction with rifampicin Streptomycin rpsl (ribosomal protein S12) Mutation prevents interaction with Streptomycin Ethambutol EmbAB ( arabinosyl transferase ) Overexpression or mutation of embAB allows continuation of arabinan biosynthesis. Pyrazinamide PncA Loss of pyrazinamidase activity results in decreased conversion of pyrazinamide to pyrazinoic acid, the putative active moiety Fluoroquinolones GYra (DNA gyrase subunit A) Mutations in gyrA prevent interaction with fluoroquinolones . Mechanism of resistance of Anti-tubercular drugs

12. Spontaneous mutations develop as bacilli proliferate to >10 8 Drug Mutation Rate Rifampisin 10 -8 Isoniazid 10 -6 Pyrazinamide 10 -6

13. Drug resistance in XXDR TB ³ A tomic force microscopes confirmed morphological variation in XXDR-TB isolates . bacilli were round , oval or even multiple branching forms In addition, various type of cell division i.e., symmetrical, asymmetrical and budding were found. The cell wall thicker MDR-TB isolates Pilli like structure that protruded from the head, tail or side poles of the bacilli were also detected

14. Does HIV infection favor TB drug resistance? Documented evidence: Nosocomial transmission (HIV facilities, prisons, etc.) Poor adherence to TB treatment by HIV-positive patients Malabsorption of TB drugs (advanced immunosuppression, chronic diarrhea) Acquired rifampicin resistance (diarrhea, antifungal treatment, antiretroviral treatment) Poor TB programme performance (overload of TB cases due to HIV epidemic, unknown association of HIV) 14

15. P henotypic methods Genotypic methods Proportion method Resistance ratio method Absolute concentration method 4.Colorimetric methods 5. Nitrate reductase assay 6. BACTEC TB-460 7. Mycobacterial Growth Indicator Tube (MGIT) 8. Phage-based methods 9. E test 10. Microscopic observation broth-drug susceptibility assay 11. Thin-layer agar method Automated DNA sequencing 2.PCR SSCP 3.Solid-phase hybridization techniques 4.Line Probe Assay - INNO- LiPA Rif TB - GenoType MTBDR assay 5.Rifoligotyping 6.Real Time PCR (RIF) 7.DNA Microarrays 8.Cleavage fragment length polymorphism (CFLP) 9.Dideox fingerprinting ( ddF ) 10.Hybridization protection assays (HPA) Detection methods for resistance in TB

16. AUTOMATED LIQUID BASED CULTURE BACTEC 460-TB Based on microbacterial metabolism Faster than solid culture MB/ Bact T® system Based on detection of CO2 DST 8 to 12 days BACTEC Mycobacteria Growth indicator Tube (MGIT)960 Consumption of oxygen DST 8 to 12 days

17. ESP culture system II Based on the detection of pressure changes within the headspace,due to gas production or gas consumption by microbial growth. . Phage Assay Methodology Expose strains to drug (RMP) Infect with D29 phage Grow with M.smegmatis Resistance correlated with plaque formation

18. Microscopic Observations Drug Susceptibility (MODS) Assay It is rapid drug susceptibility testing method for M. tuberculosis ( Rapid detection of MDR-TB ) Due to faster growth of M. tuberculosis in liquid culture characteristic cord formation observed early Incorporation of drugs permit concomitant DST

19. MOLECULAR METHODS DNA sequencing The Line Probe assay ( LiPA ) WHO recommended GenoType MTDBRplus Strips ( Hain Lifescience Most accurate and reliable method Detect both previously recognized and unrecognized mutations For rifampicin only Detect rpoB mutations of rifampicin Can be used directly on sputum specimens, Results within 1-2 days

20. GeneXpert MTB/RIF

21. PCR SSCP Based on property of single stranded DNA to fold into a tertiary structure whose shape depends on it’s sequence Single strands of DNA differing by only one or few bases will fold into different conformations with different mobility's on a gel ,producing single strand conformation polymorphism Detect - RIF , INH ,SM and ciprofloxacin resistance.

22. Microarrays Gryadunov et al : developed a biochip for detection of rifampicin-resistant and isoniazid-resistant strains of M. tuberculosis . The newest generation of TB-biochips identifies mutations responsible for the emerging resistance of M. tuberculosis so the highly effective second-line fluoroquinolone antibiotics can be administered

23. Characteristics of several drug susceptibility testing methods

24. Secondary Drugs testing: [lack of standardized methods!] XXDR Diagnostic technology CHOICE Molecular DST (LPA DST) FIRST Liquid culture isolation and LPA DST SECOND Solid culture isolation and LPA DST THIRD Liquid culture isolation and Liquid DST FOURTH Solid culture isolation and DST FIFTH

25. New Anti TB drugs Bedaquiline Delamanid Pretomanid NC-002, NC-003 Sutezolid SQ 109 Benzothiazinones Repurposed Anti TB drugs Linezolid Clofazamine Imipenem / Meropenem Amoxicillin – Clavulanate Thioacetazone Clarithromycin ( Mendell , et al.Principle and Practice of INFECTIOUS DISEASES;2005:2852-2886 .) Bedaquiline 2016 – RNTCP has introducing BDQ through conditional access programme at 6 sites in India

26. Programmatic Management of Drug Resistant TB (PMDT) in India Efficient and timely identification of patients who require DST Quality-assured laboratory capacity (Smear, Culture-DST, rapid molecular test ) Efficient drug procurement and supply chain management Adherence to difficult-to-take regimens for long periods Prompt identification and management of side-effects Recording and reporting and Human and financial resources

27. Vaccine Vaccine Description Development stage BCG30 rBCG with plasmid over expressing M. tuberculosis Ag85A Phase I VPM-X rBCG with chromosomal expression of listeriolysin (for endosome escape) Phase I Crucell Ad35 Recombinant adenovirus 35 expressing M. tuberculosis antigens 85A, 85B boosts BCG or rBCG Phase II MVA85A Oxford Modified vaccinia Ankara 85A vector expressing M. tuberculosis antigen 85A, boosts BCG Phase II GSK M72 M.tuberculosis protein (Rv1196), combined with an inactive serine protease Rv0125, boosts BCG Phase II