Drugresistanttuberculosis 100227020029 Phpapp01
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  • 1. Drug Resistant Tuberculosis A Great Human Concern Dr.T.V.Rao MD
  • 2. MDR TB –Great Human Concern
  • 3. HISTORY of Tuberculosis
    • Tuberculosis Is an Ancient Disease Identified as Spinal Tuberculosis in Egyptian Mummies History dates to 1550 – 1080 BC Identified by PCR
  • 4. A Tribute to Robert Koch Discoverer of Mycobacterium Tuberculosis
  • 5. Global Status
    • Nine million people suffer from tuberculosis
    • Two million people die each year.
    • Tuberculosis accounts for one-third of AIDS deaths world wide every year.
  • 6. USAID Report on Tuberculosis in India
    • India has more new tuberculosis (TB) cases annually than any other country, ranking first among the 22 high-burden TB countries worldwide, according to the World Health Organization’s (WHO’s) Global TB Report 2009. TB remains one of the leading infectious causes of mortality in India, causing more than 331,000 deaths in 2007. There were approximately 1.96 million new TB cases in India in 2007, representing more than 21 percent of all TB cases worldwide
  • 7. Nobody is absolutely Immune to Tuberculosis
  • 8.
    • 1908-1920 (Calmette and Guerin)
      • Vaccine (BCG)
        • Attenuated strain Mycobacterium Bovis
    • 1943
      • Streptomycin developed
    • 20th November 1944
      • Critically ill TB patient injected dramatically recovered
    Pharmacological discoveries
  • 9. Selman Abraham Waksman Nobel Prize for his discovery in 1952 .   
  • 10. Pharmacological discoveries
    • 1956-1960
      • Combination therapy of INH and PZA cures TB
    • 1955 Cycloserine
    • 1962 Ethambutol
    • 1963 Rifampicin
    • 1970-1977
      • Combination of Rifampicin and I soniazid adopted as International regime for treatment of TB
  • 11. Introduction
    • Tuberculosis is an ancient disease & it remains the leading cause of death of human being.
    • It is mainly caused by Mycobacterium tuberculosis
  • 12. Typical tubercle bacilli
      • Human type M.tuberculosis.
      • Bovine type M.bovis.
      • Vole type M.microti.
      • Human type M.africanum.
  • 13. Multi Drug Resistant Tuberculosis MDR-TB
  • 14. Definition
    • MDR-TB caused by strains of Mycobacterium Tuberculosis resistant both Rifampicin and Isoniazid with or without resistance to other drugs.
    • Single Isoniazid or Rifampicin resistance is not MDR - TB
    • MDR TB is a laboratory diagnosis
  • 15. MDR-TB & XDR-TB THE 2008 REPORT % of MDR-TB among new TB cases 1994-2007
  • 16. Classification of Drugs
    • 3 Groups depending upon the degree of effectiveness and potential side effects
      • First Line: (Primary agents)
        • are the most effective and have lowest toxicity. Isoniazid Rifampin
      • Second Line:
        • Less effective and more toxic effects
        • include (in no particular order): p-amino salicylic acid, Streptomycin, Ethambutol
      • Third Line
        • are least effective and most toxic. Amikacin, Kanamycin, Capreomycin, Viomycin, Kanamycin, Cycloserine
  • 17. Several Drugs becoming resistant
  • 18. Basic concepts – Keep facts
    • Primary (Initial) resistance
    • TB patient’s initial Mycobacterium tuberculosis population resistant to drugs
    • Secondary (Acquired) resistance
    • Drug-resistant M. tuberculosis in initial population selected by inappropriate drug use (inadequate treatment or non-adherence )
  • 19. What is multidrug-resistant tuberculosis (MDR TB)?
    • Multidrug-resistant TB (MDR TB) is TB that is resistant to at least two of the best anti-TB drugs, isoniazid and rifampicin. These drugs are considered first-line drugs and are used to treat all persons with TB disease
  • 20. When to suspect MDR TB
    • Re-treatment patients who’s sputum smear remains positive after three months’ of intensive therapy
    • Treatment failure and interruption cases
    • Close contacts of MDR tuberculosis cases
    • Positive diagnoses with;
      • TB culture and susceptibility testing
  • 21. What is extensively drug resistant tuberculosis (XDR TB)?
    • Extensively drug resistant TB (XDR TB) is a relatively rare type of MDR TB. XDR TB is defined as TB which is resistant to isoniazid and rifampin, plus resistant to any fluoroquinolone and at least one of three injectable second-line drugs (i.e., amikacin, kanamycin, or capreomycin).
  • 22. Why XDR - TB a grave concern
    • Because XDR TB is resistant to first-line and secondline drugs, patients are left with treatment options that are much less effective.
    • XDR TB is of special concern for persons with HIV infection or other conditions that can weaken the immune system. These persons are more likely to develop TB disease once they are infected, and also have a higher risk of death once they develop TB.
  • 23. Global Estimates 16,000 27,000 XDR TB 1,16,000, 4,24,000 MDR TB 1.6 million 8.8 million All forms TB Estimated Number of Deaths Estimated Number of Cases Classification
  • 24. Extensively Drug-Resistant Mycobacterium tuberculosis , India
    • The first XDR TB cases in India and the emergence of XDR TB is reported by Rajesh Mondal* and Amita Jain* *King George's Medical University, Lucknow, India Volume 13, Number 9–September 2007 in Emerging Infectious Diseases.
  • 25. Global incidence of tuberculosis Still rising as a result of the growing epidemic in Africa 0 100 200 300 400 500 600 1990 1995 2000 2005 2010 2015 Incidence per 100,000 per year World Cent. Euro, East. Europe Est Market East. Medit. Lat. America West. Pacific Sth East Asia AFR high HIV AFR low HIV
  • 26. Are we Returning to a Pre-antibiotic Era Drug susceptible TB*§ MDR-TB 1990§ XDR-TB 2006§ Total DR ? Resistance to H&R – Treatable with 2 nd line drugs Resistance to 2 nd line drugs – Treatment options seriously restricted Resistance to all available drugs – No treatment options *or limited resistance manageable with 4 drug regimen - DOTS
  • 27. WHO Surveillance and Incidence of MDR TB Dye et al. Global Burden of Multidrug-Resistant TB. JID 185(8), 2002 5.3 Ivory Cost 6.6 Dominican 5.8 Iran 3.4 India 6.0 Russia 2.8 China (DOTS) 7.7 China (non-DOTS) 9.0 Latvia 14.1 Estonia % MDR TB of all new cases Country
  • 28. Genesis of MDR TB
    • Resistance is a man-made amplification of a natural phenomenon .
    • Inadequate drug delivery is main cause of secondary drug resistance .
    • Secondary drug resistance is the main cause of primary drug resistance due to transmission of resistant strains.
    • MDR due to spontaneous mutations is not possible as the genes encoding resistance for anti TB are unlinked .
  • 29. Strains with genetic drug resistance Wild M. TB strain Acquired drug resistance Primary drug resistance Spontaneous mutation Selection: inadequate treatment Transmission Development of anti-tuberculosis drug resistance Pablos-Mendez et al. WHO, 1997
  • 30. Factors Contributing to Development and Spread of MDR and XDR TB
    • Weak TB programs (DOTS)
      • Low completion/cure rates
      • Lack of treatment follow up and patient support
      • Unreliable drug supply
      • Diagnostic delay
    • Absent or inadequate infection control measures
    • Uncontrolled use of 2 nd line drugs
  • 31.
    • INH
      • Chromosomally mediated
      • Loss of catalase/peroxidase
      • Mutation in mycolic acid synthesis
      • Regulators of peroxide response
    Mechanism of resistance
  • 32.
    • Rifampin
      • Reduced binding to RNA polymerase
        • Clusters of mutations at “Rifampin Resistance Determining Region” (RRDR)
      • Reduced Cell wall permeability
    Mechanism of resistance
  • 33. Gene location associated Drug-Resistant M.tuberculosis
    • Drug Gene
    • Isoniazid Kat G, Inh A, Kas A
    • Rifampicin rpo B
    • Ethambutol emb B
    • Streptomycin rps L
    • Pyrazinamide pnc A
    • Fluoroquinolones gyr A
    • Dubaniewicz A, et al. Molecular sub-type of the HLA-DR antigens in pulmonary tuberculosis. Int J Infect Dis2000;4:129-33.
  • 34. Drug Susceptibility Testing
  • 35. Susceptibility Testing
    • 􀂄 Direct and indirect testing
    • 􀂄 Primary Drugs testing
    • 􀂄 Isoniazid
    • 􀂄 Rifampicin
    • 􀂄 Ethambutol (*)
    • 􀂄 Pyrizinamide (*)
  • 36. Drug susceptibility testing (DST)
    • DST is recommended for all new cases for all first line drugs with specimens taken before initiating treatment .?
    • Accuracy is more important than speed
    • DST results should come from a small number of well-equipped, experienced laboratories who participate and perform well in an international DST quality control scheme.
    • The WHO Supranational Laboratory Quality Control Network offers the greatest global coverage for this
  • 37. Drug susceptibility Testing
    • Assessment of grwoth inhibition on solid media containing various dilutions of the drug, in comparison with the test strains.
    • As the method depend observation of grwoth Results are not available until several weeks after isolation of the organism.
  • 38. Other accredited Methods
    • Radiometric and Non radiometric methods
    • Nucleicacid technology – effective upto 95% in mutations to rifampicin resistance to gene rpoB gene
  • 39. Drug susceptibility testing (DST )
    • As a minimum, laboratories supplying DST data, should correctly identify resistance to isoniazid and rifampicin in over 90% of quality control samples in two out of the last three quality control rounds.
  • 40. Detection of Rifampicin Drug susceptibility testing (DST) is more important .
    • Early identification of mycobacterial growth as M. tuberculosis complex and the identification of rifampicin resistance should be the first priority as rifampicin resistance invalidates standard 6 month short-course chemotherapy and is a useful marker in most countries for MDR-TB .
    • Laboratories should aim to identify isolates as M. tuberculosis complex and perform rifampicin resistance in 90% of isolates within 1-2 working days. This is technologically feasible.
  • 41. Drug susceptibility testing
    • For DST laboratories, modern molecular techniques permit the successful identification of isoniazid resistance in at least 75% of mycobacterial cultures within 1-2 working days and are useful preliminary screens for isoniazid resistance.
  • 42. Secondary Drugs testing:[lack of standardized methods!]
    • Ofloxacin, quinolones
    • Ethionamide
    • Kanamycin
    • Capreomycin
    • ! Ensure quality control and quality assurance ?
  • 43. MODS M icroscopic O bservation of D rug Susceptibility T esting
  • 44. MODS affordable Technically Feasible
    • MODS arose during experiments conducted by Luz Caviedes under the guidance of Professor Robert Gilman at Universidad Peruana Cayetano Heredia in Lima, Peru in the late 1990s in which a colorimetric test for TB growth was being investigated. The observation that microcolonies could be seen under the microscope long before a colour change occurred prompted the development of MODS.
  • 45. Review Article in Indian Journal of Medical Microbiology
    • Caviedes L, Moore DA. Introducing mods: A low-cost, low-tech tool for high-performance detection of tuberculosis and multidrug resistant tuberculosis. Indian J Med Microbial 2007;25:87-8
  • 46. Observation of Grwoth in liquid Media
    • MODS depends upon three key principles (which have been known for decades): (1) Mycobacterium tuberculosis grows faster in liquid (broth) than on solid media, (2) in liquid cultures M. tuberculosis grows in a visually characteristic manner (tangles, cording) which can be observed under the microscope long before the naked eye could visualize colonies on solid agar
  • 47. Least time required for detection of MDR
    • Incorporation of anti-TB drugs into broth cultures at the outset enables direct susceptibility testing from sputum samples
  • 48. MODS more streamlined
    • Recently completed operational field studies have served to refine and streamline the methodology further and importantly validate MODS as a test for TB detection and MDRTB detection directly from sputum.
  • 49. Inverted Microscope a minimal need
    • Characteristic “ tangles “ of M.tuberculosis can be visualised under microscope long before colonies to the naked eye
  • 50. MODS for detection of MDR - TB
    • The scientific observations have proved that a single MODS culture of sputum sample offers more rapid and sensitive detection of tuberculosis and Multidrug-resistant tuberculosis than the existing gold standard methods used.
  • 51. Advantages of MODS methodology in MDR detection
    • All the chemical ingredients are available locally, except few which can be acquired easily.
    • Existing infrastructure in District and Teaching hospital can be adopted for implementation of MODS
    • Risk to technician handling the specimens is minimal, there is no absolute need to obtain grade III safety cabinets,
    • Technology transfer is easier all the new technical manpower can be trained easily.
  • 52. Performing MODS Assay
    • The MODS assay was performed as described in standard protocols,
    • Broth cultures were prepared in 24 well tissue culture plates ( Becton Dickinson) each containing decontaminant, 7H9 broth (Becton Dickinson), oxalic acid, albumin, dextrose, and catlase (OADC) (Becton Dickinson) and Polymyxin, Amphotericin B, Nalidixic acid,trimethoprim and azlocillin (PANTA)
  • 53. MODS Assay ( Contd) ‏
    • For each sample, 12 wells were used;
    • Four in control wells, no drug was used and each of the remaining 8 wells, contained one of the four drugs at one of the two concentrations tested.
    • The cultures were examined under an inverted light microscope at magnification of 40x every day ( except weekends ) from 4 to day 15, on alternative days from 16 today 25 and twice weekly from 26 to 40day.
  • 54. Sample layout on MODS plate (2 samples per plate) No plate contained 2 samples from the same patient
  • 55. Drug susceptibility Testing In MODS
    • Drug susceptibility testing was performed with the use of MODS assay,
    • Growth in the drug free control wells but not in drug containing wells, indicates susceptibility
    • The drug concentration were as follows
    • Isoniazid , 0.1 and 0.4 µg/milliliter
    • Rifampicin 1 and 2 µg per millilitre
  • 56. Differentiation from Typical and Atypical Mycobacterium
    • Nontuberculous mycobacteria (NTM) were recognized by their lack of cording or (in the case of Mycobacterium chelonae that uniquely among NTM does form cords) rapid overgrowth of wells by day 5.
  • 57. In MODS growth is identified by cording on Microscopy
  • 58. MODS assay ( Contd) ‏
    • To minimize cross contamination and occupational exposure, plates were permanently sealed inside plastic zip lock bags after inoculation and were subsequently examined with in the bag
  • 59. Observation of growth in MODS
    • Positive cultures were identified by cord formation, characteristic of M.tuberculosis grwoth,in liquid medium in drug free control wells.
  • 60. MODS in Atypical Mycobacterium
    • Non tuberculous mycobacterium were recognised by their lack of cording or, for M,chelonae ( which forms cords) by rapid overgrwoth by day 5.
  • 61. Contamination in MODS Assay
    • Fungal or bacterial contamination was recognised by rapid overgrowth and clouding in wells.
    • If contamination was detected, the original samples was cultured again after being decontaminated once more
  • 62. Honduras study comparing the LJ medium
    • Per specimen, there was concordance between MODS and LJ culture in 94.2% MODS tests were also less prone to contamination than LJ cultures. 62 [3.8%] vs (95 [5.8%] of 1,639 samples, respectively (P ≤0.01).
  • 63. PCR: Molecular susceptibility testing RMP resistance INH resistance Hain Genotype MTBDR Hain Genotype MTBDR INNO-LiPA Rif.TB assay
  • 64. Confirming MODS results
    • Spacer Oligonucleotide typing Spoligotyping , polymerase chain reaction with multiple primers, or both were applied to all isolates from each of the three types of cultures in order to confirm the presence of M.tuberculosis.
  • 65. MODS and MDR detection
    • The drug sensitivity for Rifampicin and Isoniazid can be tested and established the presence of MDR.
    • In view of being chronic disease it is highly essential to establish MDR Tuberculosis at centers serving DOTS under WHO guidelines
    • Starting and establishing centers to identify MDR at every district and Teaching Medical centers leads to better control of Tuberculosis
  • 66. Why MODS is a better method for MDR TB detection
    • If a MODS culture was negative on day 15,there is 99.7% chance that the sample is truly culture negative.
    • The negative MODS cultures can be discarded after 3 weeks
  • 67. Biosafety concerns in MODS technology
    • Legitimate concerns about biosafety with other liquid culture systems do not really apply to MODS, indeed the converse is the case. After inoculation with decontaminated sample the MODS plates are permanently sealed in ziplock polythene bags through which the microscopic examination is made, thus spillage of the mycobacterial "soup" cannot occur.
  • 68. Lower Grade Biosafety is adequate
  • 69. N 95 mask protects from Biohazard
  • 70. No transfer of Materials needed in MODS
    • As no secondary sub-culture is needed (because this is direct and not indirect susceptibility testing) no further manipulation is required - this zero potential for aerosolisation or accident compares favourably with the hazard associated with preparation of a standardized inoculum for indirect DST.
  • 71. Computer pattern recognition of Mycobacterium tuberculosis in MODS culture
  • 72. Automation in MODS
  • 73. M . tuberculosis in MODS x10 objective (sputum sample inoculation ) Day 6 Day 16 Day 17 Day 7 Day 8 Day 9 Day 10 Day 11 Day 12 Day 13 Day 14 Day 15
  • 74. MODS can be used in Extra pulmonary Tuberculosis
  • 75. Draw backs of MODS
    • One possible drawback however, could be the inability of the laboratory technicians to distinguish between TB and some NTM. This could potentially have clinical impact in settings where NTM prevalence is high and not all mycobacteria respond to anti-TB treatment.
  • 76. Other WHO-Endorsed Tools
    • Liquid culture (e.g. MGIT, BacT/ALERT)
    • Capilia TB
      • Rapid strip test that detects a TB-specific antigen from culture
    • Molecular line probe assays (e.g. GenoType MTBDRplus, INNO-LiPA Rif.TB)
      • Strip test for detection of TB and drug-resistance conferring mutations
  • 77. WHO Controls the Tuberculosis related work
    • The laboratory methods for anti-tuberculosis drug susceptibility testing should be selected from among those that are WHO-recommended, and all laboratory processes should be quality-assured in cooperation with a partner Supranational Reference Laboratory (SRL)
  • 78. XDR-TB in South Africa August 2006
    • 53 of 544 patients defined as XDR-TB cases
    • • 52 of the 53 patients died on average within 25
    • days, including those on antiretroviral therapy
    • • Further investigations being carried out
    • • XDR-TB likely in bordering African countries
  • 79. Molecular Fingerprinting
    • 26 of 30 (87%) XDR TB isolates found to be genetically similar
    • Majority of patients had no previous history of TB treatment Suggestive of recent infection with drug-resistant strain
    • Additional cases identified in 28 of the 68 hospitals in KZN KwaZulu Natal
  • 80. CDC Updates Guidelines for Nucleic Acid Amplification Techniques to Diagnose Tuberculosis
    • NAAT results should be interpreted in conjunction with the AFB smear results.
    • NAAT and smear positive: start Rx despite pending culture results. PPV 95%
    • Smear negative, NAAT positive: use clinical judgment to either treat or await culture
  • 81. Selection from automated systems for molecular and bacteriological rapid diagnostics
    • PCR:
    • Roche/COBAS®: Amplicor® amplification kits
    • Roche/COBAS® : LightCycler® (real-time-PCR)
    • Roche/COBAS® : TaqMan 48®
    • (increases the specificity of real-time-PCR)
  • 82. Microscopy and Culturing still a top priority
  • 83. Is PCR methods a solution ?
    • PCR can't yet replace neither microscopy, culturing and competent clinical examination.
  • 84. No testing method replaces clinical assessment
  • 85. Extreme Drug resistant Tuberculosis (XDR-TB)
    • Resistant to all first line drugs namely; Isoniazid and Rifampin and
    • Three or more second line drugs (SLD’S) that are used to treat MDR-TB
      • Thequinalones like Ofloaxin
        • Or
      • Aminoglygocides like Capreomycin & Kanamycin
    • No third-line drugs available to treat XDR-TB since none has been developed in the last 40 years.
    • Dr.T.V.Rao MD
  • 86. XDR-TB: 8/23/06
    • “ Rapidly Fatal in South Africa” Tugela Ferry, KwaZulu-Natal
    • 10% isolates resistant to ALL 1 st and 2 nd line agents
    • 51/52 XDR dead in median 16 days after first positive sputum
    • 67% AIDS deaths w/ MDR TB
  • 87. Czech Republic The boundaries and names shown and the designations used on this map do not imply the expression of any opinion whatsoever on the part of the WHO concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.  WHO 2005. All rights reserved Ecuador Georgia Argentina Bangladesh Germany Republic of Korea Armenia Russian Federation South Africa Portugal Latvia Mexico Peru USA Brazil UK Sweden Thailand Chile Based on information provided to WHO Stop TB Department 13 September 2007 Spain Islamic Republic of Iran China, Hong Kong SAR France Japan Norway Canada Italy Netherlands Estonia Lithuania Ireland Romania Israel Azerbaijan Poland Slovenia India Australia Mozambique Vietnam Countries with confirmed XDR-TB cases as of September 2007
  • 88. Summary Drug resistant TB
    • Drug-resistant TB poses a grave public health threat especially in high HIV prevalence settings
    • XDR-TB strains have been found in all regions of the world
    • XDR-TB occurs as a result inadequate TB control programmes
    • XDR-TB, if identified early, can be treated and cured but experience limited to low HIV prevalence settings
    • Infection control measures must be strengthened
    • XDR-TB underlines the need for investment in basic TB control plus development of new TB diagnostics, treatments and vaccines
  • 89. Health Care Workers and MDR TB
    • Recognised risk for health care workers
    • Risk assessment
    • High risk – Prolonged closed contact with infectious patients
    • Smear positive MDR TB patients
    • Medium risk –Primary health care centres involved
    • Sputum collection on TB suspects
    • Low risk –Health care support staff e.g. cleaners
    • Porters and admin staff
    • Dr.T.V.Rao MD
  • 90. Koch failed to conquer tuberculosis, which still causes enormous health problems worldwide 100 years after his Nobel award.
    • The scientific academies noted that the triumphant discovery of 1882 was followed by a succession of failures: first of all, the failed attempt to present tuberculin as a remedy against tuberculosis in 1890-91, which severely damaged Koch's reputation
    • Medical History, 2001, 45: 1-32 CHRISTOPH GRADMANN *
  • 91. Are there any solutions for effective Diagnosis in TB ?
  • 92. Many more powerful hands needed to Control Tuberculosis
  • 93. Contribute your Knowledge, Wisdom, to prevent spread and control of Tuberculosis
  • 94. Created by Dr.T.V.Rao MD for ‘e’ learning Programme Email [email_address]