1.MDR / XDR TB What Microbiologists should know Dr. Ashok Rattan, Chief Executive, Fortis Clinical Research Ltd., Adviser, Religare SRL Diagnostics inFortis / Escorts Hospitals inDelhi & NCR

2. Milestones in Laboratory Diagnosis of Tuberculosis

Koch(1882) Alkaline methylene blue,vesuvin as counterstain

Ehrlich (1882) Discovered acid fastness,hot fuchsin in aniline oil

Ziehl(1883) Phenol as mordant

Neelsen(1884) Combined dye & mordant

Lehmann&

Neumann (1896)Myco bacteria

3.

Story of anti-tuberculosis chemotherapy is a miniature history of anti-infective chemotherapy

First half of 20 thcentury

• Thick lipid rich wall of mycobacteria postulated to make chemotherapy impossible

• Sulfonamides & Penicillin had no useful activity against MTb

4. Prof Selman A Waksman & Albert SchatzStreptomyces griseus Streptomycin The drug would lead the path in the elimination of The Great White Plague 5. British Medical Research Council (BMRC) trial 1948

Investigated efficacy of streptomycin monotherapy

Most patients who were treated with the drug developed resistant strains

6. Jorgen Lehman para aminosalicyclic acid (PAS) 1943 Lehman J. On the effect of isomers of para aminosalicyclic acid And related substances on the tuberculostatic effect of PAS. Experientia 1949; 5: 365 62. Turnbull FW et al. Streptomycin resistance after treatment withPAS alone. BMJ 1953; 1: 1244 - 46 7. PAS helps prevent emergence of resistance to SM 8. INH, a pro drug with anti TB activity (1952) first synthesized in 1912 as MAO inhibitor 9. 10. 11. Second line anti TB drugs six different classes of drugs

Category Drugs .

1. Aminoglycosides Kanamycin

Amikacin

2. Thioamides Ethionamide

Prothiomide

3. Polypeptide Capreomycin

4. FQ Ciprofloxacine

Ofloxacin

5. Cycloserine/ Terizidone

6. PAS

12. 13. 14. Resistance due to mutation in target gene

Rifampicin 3.32 x 10-9

INH 2.56 x 10-8

Streptomycin 2.29 x 10-8

Ethambutol 1.0x 10-7

• Risk of simultaneous

• Resistance to two drugs=sum of mutational frequency

• For Rif, SM, INH=1025

• P = 1 (1 - r)n

• P= probability of drug resistance, r = mutation rate

• N = number of bacilli in a lesion (usually 10-- 8 )

15. rpoB gene mutations 16. Mitchisen Actively dividing mycobacteriamimicked by in vitro tests(aerobic environment) Myobacteria withspurts of metabolism Microaerophilic conditions Intracellular mycobacteria Acidic pH Dormant Mycobacteria Metabolic activity Rate of division INH RMP RMP PZA 17. The 5 Components of theDOTS Strategy:

Sustained political commitment

Access to quality-assured sputum microscopy

Standardized short-course chemotherapy for all cases of TB under proper case management conditions, including Directly Observed Treatment (DOT)

Uninterrupted supply of quality-assured drugs

Recording and reporting system enabling outcome assessment of all patients and assessment of overall program performance

18. There is a gathering storm of drug resistant tuberculosis and thestrategy of WHO and other national & international agencies hasfailed to control the disease or prevent emergence ofMDR / XDR TB Doctors without borders MSF Alert Fall 2006 19. Tuberculosis now There is a gathering storm of drug resistant tuberculosis and the strategy ofWHO, other national & international agencies has failed to control the diseaseor prevent emergence of MDR/XDR TB-Doctors without borders MSF Alert Fall 2006 500,000 MDR TB 2 million death 10 million cases DOTS detects 70% Cures 85% 2 billion personsWith latent TB 20. MDR/XDR TB is Man Made

MDR TB is a laboratory Diagnosis

Simultaneousresistance to INH and Rifampicin

Generated as a consequence ofhuman error

• Patient, Prescriber or Health care system

• • Prescription of drugs

• • Management of drug supply

• • Case management

• • Process of drug delivery to the patient

• • Compliance of the patient

• • Distance of healthcare centre

• • Lack of transport

21. MDR-TB Why worry ?

• DOTS don't yield acceptable cure rates

• Treatment is more expensive

• • Treatment of susceptible strains$ 200

• • Treatment of MDR infections $ 20,000

• Without effective treatment, transmission will continue

• Private tragedy, Public Health Emergency

• Use of DOTS alone in MDR will lead to generation of XDR TB

22. Contributing factors toDevelopment of MDR/XDR TB

Patient

• Poor adherence to medication

Provider:

• Prescribing errors

• Suboptimal dosing

• Insufficient monitoring of therapy

• Drug interactions in co-morbidities

Healthcare system:

• Poorly functioning TB control program

• Medication stock outs

• Lack of staff

• Inadequate access for patients: distance, transportation

• Policies that rely on smear alone to diagnose TB

23. Risk factors for drug resistant TB

Residence in a region with known high MDR/XDR TB prevalence

Known exposure to MDR/XDR TB patients

HIV/AIDS

History of

• Previous TB therapy

• Relapse or recurrence after previous successful treatment

• Hospitalization within the past two years

• Nonadherence or default

• Incarceration

• Substance abuse

• Malaborsption

• Medications known to cause drug interaction with ATT

24. Predicting the future of XDR Tuberculosis Sally Blower & Virginie Supervie. Lancet 2007; 7: 443

WHO specified targets for reducing global tuberculosis epidemic

• Detecting & treating at least 70% of sputum smear positive cases (currently 53% are detected)

• Attaining 85% cure rate (currently 82% of drug sensitive cases are cured

• Epidemics that are driven mainly by drug sensitive strains my be controlled by achieving WHO targets (2005)

25. Predicting the future of XDR Tuberculosis Sally Blower & Virginie Supervie. Lancet 2007; 7: 443

In South Korea & Latvia: 15 19% MDR

Without effective control of MDR TB epidemics XDR TB would become uncontrollable 26. Pillay & Sturm. CID 2007; 45: 1407- 14

Possible Reason for conversion of MDR to XDR within a decade

Introduction of DOTS & DOTS + in TB Control

No susceptibility testing or Drug Susceptibility Surveillance

27. Venn diagram of SNP Jassal & Bishoi: Lancet Infect Dis 2009; 9: 10 - 30 28.

Basis of TB drug resistance is selection of bacterial mutants with innate resistance to chemotherapy

Generated by three inter related mechanisms

• Conversion of wild type pan sensitive strain to resistant strain during therapy (acquired resistance)

• Increased development of resistance in drug resistant strains because of inappropriate chemotherapy (amplified resistance)

• Transmission of drug resistant cases (transmitted resistance) [fuelled by HIV epidemic]

29. Generation of resistance withmonotherapySusceptible Resistant (SM) 30. Generation of resistance withmonotherapySusceptible Resistant (SM R) (ETH R) ETH 31. 50 countries 32. What is XDR TB Laboratory Based Diagnosis

March 2006: MMWR(2000 2004)

• INH+ RIF (MDR) + R to at least 3 of 6 classes of second line agents :

• • • Aminoglycosides;

• • • Polypeptides;

• • • FQ;

• • • Thioamides;

• • • Cycloserine;

• • • PAS

WHO Task Force Oct 2006

• • • MDR + FQ + any one of Amikacin/ Kanamycin/ Capreomycin,

Bactericidal & reliablein vitromethodsavailable 33. Is the current definition ofXDR TB adequate ?

• • Is XDR TB predictive of poorer clinical response ?

• • • Success rate of >90% achieved with pza+ethionamide+cycloserine

• Most accurate diagnosis of XDR TB would be:

• • R to all first line drugs (not only INH+Rif)

• • + FQ (Moxifloxacin)

• • + all 3 injectables (not just to one)

• • • These cases will have < 50% chances of success & so would behave differently from other XDR TB strains

• If not,

• • we would soon also be talking about XXDR (extensively extensively drug resistant TB) or

• • CDR TB (completely drug resistant)

34. M. tuberculosis Drug susceptibile XDR M. tuberculosis Drug susceptible INH + RIF Resistant =MDR FQResistant InjectableResistant 35. 1 stline drugs 2 nd line drugs INH INH RIF RIF PZA PZA Etham Etham FQ FQ Injectable Injectable Ethio Ethio Cyclo Cyclo PAS PAS MDR TB XDR TB 2 most important 2 most important 36.

Preventing transmission ofM tuberculosisrelies on

• Accurate&Rapiddiagnosis

• PT resultsTAT

• Key barrier

• • Current case detection rates are low

• • Methods insensitive & slow

• Delays enhance likelihood of transmission

• Potential to produce clusters of secondary infections

• • Transmission in nosocomial or congregate settings

37. Commonest method fordiagnosis of TB 1885 2005 38.

AFB smear

Culture on LJ

Identification by Niacin &

Nitrate test

Sensitivity by RR

Culture report in 8wk

DST report in 8 wks

Treatment duration from 9 M to 18 M

Slow diagnostic methods 39. Progress made inlaboratory diagnosis of tuberculosis

Fluorochrome stain for AFB smear Mid1970s

BACTEC 460TB Early 1980s

DNA probes for AFB +ve respiratory Late1980s

Automated broth systems for cultureEarly 1990s

HPLC on AFB +ve culturesEarly 1990s

Drug susceptibility by Bactec Early 1990s

Nucleic acid amplification on all smear +ve1996

Use of courier to transport specimen1997

Us of NAA TB testing on smear ve2000

Reporting of results by fax2000

Lipa for identification and Rif resistance1997

Genotype MTBDR for subspecies2003

40. Think Globally Actlocally 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. Sample & culture positive load for AFB Bactec at GGN (September 2007-October 2008) sample number = 1656 51. Total samples processed = 1656 (smear +ve=302, -ve=1354)Total culture positive =453 (Mtb=393, MOTT=60) 30.62 13.79 8 41 MOTT SmearNegative (n=1354) 34.28 17.15 66 133 M.tb 28 6.6 10 21 MOTT 26.12 9.64 191 258 M.tbSmearPositive (n=302)Speed of growth on LJ in days Speed of growth on MGIT in days LJ +ve MGIT +ve Mycobacteriaspp. SmearResult 52. Speed Of Growth Of M.tb (MGIT 960 vs LJ Media) 1 stwk 2 ndwk 3 rdwk 4 thwk 5 thwk 6 thwk 166 0 132 23 55 46 23 63 13 48 2 24 53. Policy guidance on DST of second line anti tuberculosis drugs : WHO 2008

Alternative method for grouping ATT drugs

Grouping Drugs

• Group 1

• • First line oral drugs: INH (H); rifampicin (R), ethambutol (E),pyrazinamide (Z), rifabutin (Rib)

• Group 2

• • Injectable agents Kanamycin (Km), amikacin (Am), capreomycin(CM),viomycin (Vm), streptomycin (S)

• Group 3

• • Fluoroquinolones Moxifloxacin (Mfx), levo (Lfx), Oflox (Ofx)

• Group 4

• • Oral bacteriostaticEthionamide (Eto), protionamide (Pto),

• • secondline drugs cycloserine (Cs), terizidone (Trd), PAS

• Group 5

• • Agents with unclear role Clofazimine (Cfz), linezolid (Lzd), Amox Clav,

54. Policy guidance on DST of second line anti tuberculosis drugs :WHO 2008 55. Mtb isolated = 393 Total sensitivity performed = 94 MDR TB= 50 isolates XDR TB=2 isolatesSensitive to all the drugs = 19 isolates 56. Samples for culture = 1656 Culture positive=455 MTb=393 MOTT=62 DST performed=94 MDR=50 XDR TB=2 R to atleast one drug=23 S to all drugs=19 2008: NCR & North India 57. 58. Reporting TAT for10 Drugs panel / both 1 st& 2 ndline drugs 10 6 thSeptember28 th August9HH0022857 12 19 thAugust6 thAugust9HH006343 13 9 thAugust25 thJuly9HG032563 12 17 thJuly5 thJuly9HG005659 14 15 thJuly1 stJuly9HG000914 6 26 thAugust20 thAugust9HH007858 19 24 thJune 5 thJune 9HF006055 11 2 ndJuly20 thJune9HF025442 14 26 thJune 11 thJune9HF013364 18 28 thJune 9 thJune9HF010873 17 25 thJune 7 thJune 9HF008253 24 25 27 th May 9HE034724 16 6 thJune22 NDMay9HE028517 TAT in days Reported date Accession date Accession No 59. 60. Smear examination

ZN method

Kinyoun cold stain

Auromine O

• Rapid, economical, simple

• • Adequate sensitivity in symptomatics

• • Overall Low sensitivity (22 78% of culture)

• • Limit of detection 1 x 10 4vs 10 2bacilli/ml

• • Species cannot be identified

61. How to improve microscopyLED Microscopy

Fluorescent microscope

• Pro:

• • High power rather than oil immersion

• • Black background, yellow bacilli

• • Increased sensitivity by 10%

• Con:

• • High cost, maintenance

FluoLED Microscopy

62. Improved diagnostics: Specimen processing

• Improve yield

• Safer for lab personnel

• • Bleach centrifugation

• • Flotation method

• • Bleach sedimentation

• • • long contact with bleach will lyse bacilli

63. Culture based detection Gold standard Slow : weeks Low sensitivity 64. Improving detection

CO 2production

O 2consumption

• Radiometric

• Fluorescent

• Colourimetric

• Pressure sensor

• Decrease TAT by half

14C Palmitate 14CO 2

65. Improving detection

Alamar Blue

Microscopic observation of micro colonies (MODSA)

Bacteriophage

66. 67. Genotype Mycobacterium test

DNA Strip technology: Three steps

• 1. DNA isolated from culture

• 2. Multiplex amplification with biotinylated primers 230 bp product

• 3. Reverse hybridization:

• • Chemical denaturation of amplified product

• • Hybridization of SS biotin labeled amplicon to membrane bound probes

• • Stringent washing

• • Addition of Steptavidin/alkaline phosphatase

• • AP mediated staining reaction

68. 69. Genotype Mycobacterium CM/AS 70. Genotype MTBDR assay 71. M icroscopicallyO bserved D rugS usceptibility 24 wells plate (BD),12 wells used per sample, 4 drug free control, 8 containing drugs Middlebrook 7H9 broth (BD)INH0.1&0.4 ug/ml OACDRMP1&2 PENTAEtham 2.5&5 720 ul inoculum usedSM2&6 Zip lock,examined from 4 to 15 days daily, A/D 40 72. MODS7 days (6to8) MBBacT13(10to 14) LJ26(21to 33) 73. 74. Potency of anti-TB drugs againstM. tuberculosis Gatifloxacin 75. What does the clinicians really want ? Is itM. tuberculosis Is it MDR ? What drugs can I use ? 76. Laboratory Quality standards Turn Around Time (TAT) benchmarks

Smear examination must be carried out and reported within 24 hours of receipt of specimen

Isolation & identification ofM. tuberculosisshould occur in 14 to 21 days

The whole process, including susceptibility to first line drugs should be completed in 30 days of specimen receipt.

77. Immediate goal of any diagnostic mycobacteriology laboratory

IdentifyM. tuberculosis

Determine Rifampicin resistance

At least in smear positive cases within 2 weeks.

Keep a look out for XDR TB

78. Evidence based Summary

Use of rapid identification tests in combination with rapid culture (liquid) has greatly reduced overall turnaround time.

Use a combination of tests rather than a single test for diagnosis

It is the system of choice where funding is available

79. Consequence of this strategy

Diagnosis of TB patients : 38% faster

Initiation of therapy : 4.6 days earlier

Reduction in mortality : 31%

Reduction in medical costs: 18%

80. Take home message

Key to reliable results is the

submission of high quality specimens

Appropriate infrastructure to handle samples

Staff training, using SOP & validated tests

Quality assurance procedures

Close interaction with clinician and program officers

81. SRL TB Diagnosis Report card Ideal situation Aim for the moon

Prompt delivery of specimen to the lab

Prompt use of rapid, state of the art methods

Reporting of smear results to specimen submitter within 1 day

Report of culture identification (Mtb or not) in 21 days

Report on susceptibility results within 30 days

82. Diagnosis of TB

Current strategy:

• Detection

• • Microscopy

• • Culture

• Identification

• • Biochemicals

• • HPLC

• • PCR, Probes

• Susceptibility

• • First line

• • Second line

Future strategy

• Detection & Identification

• Susceptibility

Cepheid is NOW

24 hrs, 6 wks, 6 months2 hrs, 24 hrs complete report 83. If problem is man made,solution too will have to bewo man made Thank you for your attention