Recent emergence of XDR TB have brought home the fact that reliable laboratory work up is essential if we are going to control tuberculosis.
- 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
- 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
- 1. Aminoglycosides Kanamycin
- 2. Thioamides Ethionamide
- 3. Polypeptide Capreomycin
- 5. Cycloserine/ Terizidone
12. 13. 14. Resistance due to mutation in target gene
-
- Resistance to two drugs=sum of mutational frequency
-
- 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
-
-
- Management of drug supply
-
-
- Process of drug delivery to the patient
-
-
- Compliance of the patient
-
-
- Distance of healthcare centre
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
-
- Poor adherence to medication
-
- Insufficient monitoring of therapy
-
- Drug interactions in co-morbidities
-
- Poorly functioning TB control program
-
- 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
-
- Relapse or recurrence after previous successful treatment
-
- Hospitalization within the past two years
-
- 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 :
-
-
-
- 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)
-
-
- + all 3 injectables (not just to one)
-
-
-
- These cases will have < 50% chances of success & so
would behave differently from other XDR TB strains
-
-
- 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
-
-
- 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.
- Identification by Niacin &
- Treatment duration from 9 M to 18 M
Slow diagnostic methods 39. Progress made inlaboratory diagnosis
of tuberculosis
- Fluorochrome stain for AFB smear Mid1970s
- 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
-
-
- First line oral drugs: INH (H); rifampicin (R), ethambutol
(E),pyrazinamide (Z), rifabutin (Rib)
-
-
- Injectable agents Kanamycin (Km), amikacin (Am),
capreomycin(CM),viomycin (Vm), streptomycin (S)
-
-
- Fluoroquinolones Moxifloxacin (Mfx), levo (Lfx), Oflox
(Ofx)
-
-
- Oral bacteriostaticEthionamide (Eto), protionamide (Pto),
-
-
- secondline drugs cycloserine (Cs), terizidone (Trd), PAS
-
-
- 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
-
- 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
-
-
- High power rather than oil immersion
-
-
- Black background, yellow bacilli
-
-
- Increased sensitivity by 10%
62. Improved diagnostics: Specimen processing
-
-
-
- long contact with bleach will lyse bacilli
63. Culture based detection Gold standard Slow : weeks Low
sensitivity 64. Improving detection
65. Improving detection
- Microscopic observation of micro colonies (MODSA)
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
-
-
- 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
- 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
-
- Detection & Identification
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