1. Diagnosis of TB: what is new from the laboratory
Daniela Maria Cirillo
San Raffaele Scientific Institute
Milano, Italy

2. Topics Introduction: role of the laboratory in TB diagnosis
Integrated laboratory network
Biosafety guidelines
Quality Assurance in the diagnosis of tuberculosis
Laboratory diagnosis of tuberculosis
Diagnostic algorithms
Conclusion

3. Introduction Care of TB patients starts with a QA diagnosis
A robust network of Tb laboratories is required:
Adaquate biosafety
Modern diagnostics
SOPs
QAs
Integrated laboratory network

4. Integrated laboratory network
An integrated laboratory network is:
able to provide all primary diagnostic services without requiring patients to go to different laboratory facilities for specific tests
ONE STOP SHOP

5. The past Weakest component of health systems for:
slow policy change and constant underfunding
Inappropriate technology transfer
Inadequate infrastructure and biosafety
Staff skills and number
Inadequate lab strengthening plans
Insufficient technical assistance

6. The present
Joint effort to improve and expand TB lab capacity integrated into a global health system strengthening
Research on new diagnostics tools accelerated and diagnostic pipeline rapidly growing ( expected point of care by 2015)

7. Quality Assurance Programme
System designed to continuously improve the reliability and efficiency of laboratory services
Three main components:
Quality control (QC - IQA)
External Quality Assurance (EQA)
On site evaluation and review
Proficiency Testing / Panel testing (PT)
Blind rechecking
Quality improvement (QI)
QC: Also called Internal Quality Assurance, includes all means by which the laboratory controls operation, including instrument checks and checking new lots of laboratory reagents.
EQA: A system of objectively checking the laboratory’s results by means of an external agency, such as reference laboratory.
A process which allows participant laboratories to assess their capabilities by comparing their results with those in other laboratories in the network (intermediate and central laboratory) through panel testing and blinded rechecking. EQA also includes on-site evaluation of the
OSE: Periodic visits to the laboratory to assess laboratory practices. Use of standardized CHECKLIST and forms for reporting of results
Why? Obtain a realistic picture of laboratory practices Provide assistance with problem areas. Very poor performance may warrant an expanded visit to perform a comprehensive evaluation of laboratory procedures, assess quality practices, and assist with implementing corrective actions including providing training.
PT: “A program in which multiple samples are periodically sent to members of a group of laboratories for analysis and/or identification; laboratory’s results are compared with an assigned value (and/or with those of other laboratories in the group), and reported to the participating laboratories and others.” (NCCLS)
Rechecking: Random statistical sampling of specimens or slides for rechecking by a reference laboratory (intermediate or central laboratory) - Rechecking is always blinded , ensuring that the controller does not know the results from the peripheral laboratory. In other documents, this may also be referred to as rereading.
QI: A process by which the components of smear microscopy diagnostic services are analyzed with the aim of looking for ways to permanently remove obstacles to success. Data collection, data analysis, and creative problem solving are the key components of this process. It involves continued monitoring, identifying defects, followed by remedial action including retraining when needed, to prevent recurrence of problems. QI often relies on effective on-site evaluation visits.

8. EQA Benefits Allows comparison of performance and results
Serves as an early warning-system for problems
Identifies systematic problems
Provides objective evidence of laboratory quality
Serves as an indicator of where to direct improvement efforts
Identifies training needs
Results may be compared between laboratories offering not only an opportunity for performances to be compared but also for systematic kit problems to be identified.
Training needs may be identified and evaluated.
The problems encountered in a panel may then be addressed in the next panel, creating a circular process.

9. Laboratory biosafety Mtb is a class 3 risk pathogen
All biosafety strategies ( minimum requirements) should be based on procedures risk assessment
Based on:
Bacillary load of samples and workload
Viability of bacilli
Aeroosol generation
Typology of the patients
Fitness of the staff

10. Fluorescence Ziehl-Neelsen staining
MICROSCOPY
Rapid test
Inexpensive
Does not allow species identification
Not applicable to all samples
Specificity: >95%
Sensitivity: 25-65% (90 % of infectious cases)
Positive Predictive Value for TB depends on epidemiological situation
Fluorescence Ziehl-Neelsen staining
1st AFB smear
80-82 %
2nd AFB smear
10-14 %
3rd AFB smear
5-8 %
Excellent diagnostic test for TB control in high-incidence settings 10

11. LED Fluorescence Microscopy
Advantages:
increase in performance
increase in lamp lifetime
reduces initial, operating and maintenance costs (adaptable to existing microscopes)
No need for dark room
Batteries operated
Domanda 1

12. New Policy and Smear microscopy definition of a TB case
New definition in 2007*:
“person with al least one smear-positive sample (1 AFB is sufficient) out of a total of two examined”
2 samples regardless the collection time
*The definition/policy can be applied to countries performing microscopy under satisfactory quality assurance programmes 12

13. Same day approach
same-day-diagnosis approach (microscopy of two consecutive spot-spot sputum specimens) is equivalent, in terms of diagnostic accuracy, to conventional case-finding strategies by microscopy

14. WHO recommends that:
Countries that have successfully implemented the two- specimen case-finding strategy consider a switch to the same-day-diagnosis approach, especially in settings where patients are likely to default from the diagnostic process
Countries that are still using the three-specimen case-finding strategy consider a gradual change to the same-day-diagnosis approach.
Changes to a same-day-diagnosis strategy be preceded by a detailed situation assessment

15. WHO recommendations on sputum smear microscopy (2010)
ZN light microscopy performed on UNCONCENTRATED sputum is suitable for all laboratory service levels
Concentration of sputum is NOT recommended in programmatic settings
Fluorescence microscopy is recommended for increased sensitivity (add 10%)
LED microscopy is recommended over conventional fluorescence

16. TB Culture* Advantages Definitive diagnosis of TB
Increases case finding of 30-50%
Early detection of cases
Provide strains for DST and epidemiological studies
Disadvantages
Complex and expensive compared to microscopy
Requires complex handling of specimens
Skilled technicians
Appropriate infrastructure and biosafety levels
LIMITATIONS: need for decontamination and identification
*coverage /

17. Culture: solid/liquid
Low cost for reagents, not automated
Culture level infrastructure
Low contamination rate
Long time to positivity
Colony morphology
ID required
liquid
Complex and expensive can be automated (MGIT)
Highest infrastructure and biosafety levels
Case finding increased 10% over solid
Diagnostic delay reduced to days
ID required
Strip speciation tests needed for fast ID of Tbcomplex
Molecular test for speciation of most common mycobacteria

18. TB Culture: solid/liquid

19. Standardization of procedures still difficult to set up
Drug Susceptibility Testing (DST): the technical challenge still persists
Standardization of procedures still difficult to set up
The correct performance of DST requires the understanding of several steps such as:
Origin of resistance and interpretative criteria
Dosage and stability of the incorporated compounds
Anti-mycobacterial activity of the incorporate drug
Interpretation of results and data reporting 19

20. DST Definitive diagnosis of DRTB 3 main methods
Absolute concentration method
Proportion methods
Resistant Ratio method

21. DST (2) Indirect methods suitable for regional/ national laboratories
Adequate lab infrastructure and biosafety
Accuracy varies with the drugs tested:
R, H most accurate
Second line :
automated liquid DST are the gold standard
Not recommended in the absence of QA capacity (200 high risk specimen per year)
Formal link with Supranational Reference laboratories requested

22. Second line DST Recommended in appropriate structures for:
Aminoglycosides, Fluoroquinolones, Polypeptides
NOT recommended on routine for ethionamide, prothionamide, cycloserine,clofazimine, amox-clav, clarithromycin, linezolid)

23. WHO laboratory policies for culture and DST
Automated liquid culture (2007) Use of liquid culture in the contest of comprehensive plan for strengthening lab capacity starting with national/central level
Rapid speciation in combination with culture Second line DST (2008) to be conducted for selected drugs a central level
Available at

24. The effective treatment of MDR-TB is a life-saving intervention
Early diagnosis of both TB and DR-TB are the key for an effective Tb control
Novel technologies for rapid screening of anti-TB drug resistance have become a priority in Tuberculosis research

25. WHO policy on new diagnostics is evidence-driven
Following new investments in research new diagnostics are finally moving
Policy formulation needs to be an ongoing process at global and country level
WHO asses policies by a systematic, structured process

26. Drug-Resistant TB: Definitions
ISTC Training Modules 2008
Drug-Resistant TB: Definitions
Mono-resistant: resistance to a single drug
Poly-resistant: resistance to more than one drug, but not to the combination of isoniazid and rifampicin
Multidrug-resistant (MDR): resistance to at least isoniazid and rifampicin
Extensively drug-resistant (XDR): MDR plus resistance to fluoroquinolones and at least 1 of the 3 injectable drugs (amikacin, kanamycin, capreomycin)
Begin with the definitions for drug resistance:
Mono-resistant: The most common single drug-resistance pattern is mono-resistance to isoniazid. In general, this pattern of resistance is not usually associated with a worse outcome and does not require modification of the treatment regimen (as long as there are 4 drugs in the initial phase and rifampicin is included throughout the full duration of treatment). Rifampicin mono-resistance occurs, but is uncommon and is seen mainly in patients with HIV infection. The reasons for this association are not known.
Poly-resistant: A general term used when the organism is resistant to more than one drug, but not the combination of isoniazid and rifampicin.
MDR-TB: Resistance to at least isoniazid and rifampicin (the two most effective anti-tuberculosis drugs). MDR has a major adverse effect on the outcome of treatment. Patients with TB caused by MDR organisms generally require treatment with second line drug regimens.
XDR-TB: MDR-TB plus resistance to the 2 most important classes of 2nd-line agents used in MDR-TB treatment: the fluoroquinolones and at least 1 of 3 injectable agents (amikacin, kanamycin, capreomycin). In addition to meeting the defining criteria, XDR-TB cases are often resistant to all four 1st-line agents. Consequently, patients with XDR-TB are significantly more difficult to treat and require specialized care.
[Note: Material also covered (duplicate slide) in Management of Drug-Resistant TB module.]

27. Drug-Resistant TB: Definitions
ISTC Training Modules 2008
Drug-Resistant TB: Definitions
MONORESISTANT
Begin with the definitions for drug resistance:
Mono-resistant: The most common single drug-resistance pattern is mono-resistance to isoniazid. In general, this pattern of resistance is not usually associated with a worse outcome and does not require modification of the treatment regimen (as long as there are 4 drugs in the initial phase and rifampicin is included throughout the full duration of treatment). Rifampicin mono-resistance occurs, but is uncommon and is seen mainly in patients with HIV infection. The reasons for this association are not known.
Poly-resistant: A general term used when the organism is resistant to more than one drug, but not the combination of isoniazid and rifampicin.
MDR-TB: Resistance to at least isoniazid and rifampicin (the two most effective anti-tuberculosis drugs). MDR has a major adverse effect on the outcome of treatment. Patients with TB caused by MDR organisms generally require treatment with second line drug regimens.
XDR-TB: MDR-TB plus resistance to the 2 most important classes of 2nd-line agents used in MDR-TB treatment: the fluoroquinolones and at least 1 of 3 injectable agents (amikacin, kanamycin, capreomycin). In addition to meeting the defining criteria, XDR-TB cases are often resistant to all four 1st-line agents. Consequently, patients with XDR-TB are significantly more difficult to treat and require specialized care.
[Note: Material also covered (duplicate slide) in Management of Drug-Resistant TB module.]

28. Drug-Resistant TB: Definitions
ISTC Training Modules 2008
Drug-Resistant TB: Definitions
POLIRESISTANT
Begin with the definitions for drug resistance:
Mono-resistant: The most common single drug-resistance pattern is mono-resistance to isoniazid. In general, this pattern of resistance is not usually associated with a worse outcome and does not require modification of the treatment regimen (as long as there are 4 drugs in the initial phase and rifampicin is included throughout the full duration of treatment). Rifampicin mono-resistance occurs, but is uncommon and is seen mainly in patients with HIV infection. The reasons for this association are not known.
Poly-resistant: A general term used when the organism is resistant to more than one drug, but not the combination of isoniazid and rifampicin.
MDR-TB: Resistance to at least isoniazid and rifampicin (the two most effective anti-tuberculosis drugs). MDR has a major adverse effect on the outcome of treatment. Patients with TB caused by MDR organisms generally require treatment with second line drug regimens.
XDR-TB: MDR-TB plus resistance to the 2 most important classes of 2nd-line agents used in MDR-TB treatment: the fluoroquinolones and at least 1 of 3 injectable agents (amikacin, kanamycin, capreomycin). In addition to meeting the defining criteria, XDR-TB cases are often resistant to all four 1st-line agents. Consequently, patients with XDR-TB are significantly more difficult to treat and require specialized care.
[Note: Material also covered (duplicate slide) in Management of Drug-Resistant TB module.]

29. Drug-Resistant TB: Definitions
ISTC Training Modules 2008
Drug-Resistant TB: Definitions
MULTI DRUG RESISTANT
Begin with the definitions for drug resistance:
Mono-resistant: The most common single drug-resistance pattern is mono-resistance to isoniazid. In general, this pattern of resistance is not usually associated with a worse outcome and does not require modification of the treatment regimen (as long as there are 4 drugs in the initial phase and rifampicin is included throughout the full duration of treatment). Rifampicin mono-resistance occurs, but is uncommon and is seen mainly in patients with HIV infection. The reasons for this association are not known.
Poly-resistant: A general term used when the organism is resistant to more than one drug, but not the combination of isoniazid and rifampicin.
MDR-TB: Resistance to at least isoniazid and rifampicin (the two most effective anti-tuberculosis drugs). MDR has a major adverse effect on the outcome of treatment. Patients with TB caused by MDR organisms generally require treatment with second line drug regimens.
XDR-TB: MDR-TB plus resistance to the 2 most important classes of 2nd-line agents used in MDR-TB treatment: the fluoroquinolones and at least 1 of 3 injectable agents (amikacin, kanamycin, capreomycin). In addition to meeting the defining criteria, XDR-TB cases are often resistant to all four 1st-line agents. Consequently, patients with XDR-TB are significantly more difficult to treat and require specialized care.
[Note: Material also covered (duplicate slide) in Management of Drug-Resistant TB module.]

30. Drug-Resistant TB: Definitions
ISTC Training Modules 2008
Drug-Resistant TB: Definitions
MULTI DRUG RESISTANT
???
Begin with the definitions for drug resistance:
Mono-resistant: The most common single drug-resistance pattern is mono-resistance to isoniazid. In general, this pattern of resistance is not usually associated with a worse outcome and does not require modification of the treatment regimen (as long as there are 4 drugs in the initial phase and rifampicin is included throughout the full duration of treatment). Rifampicin mono-resistance occurs, but is uncommon and is seen mainly in patients with HIV infection. The reasons for this association are not known.
Poly-resistant: A general term used when the organism is resistant to more than one drug, but not the combination of isoniazid and rifampicin.
MDR-TB: Resistance to at least isoniazid and rifampicin (the two most effective anti-tuberculosis drugs). MDR has a major adverse effect on the outcome of treatment. Patients with TB caused by MDR organisms generally require treatment with second line drug regimens.
XDR-TB: MDR-TB plus resistance to the 2 most important classes of 2nd-line agents used in MDR-TB treatment: the fluoroquinolones and at least 1 of 3 injectable agents (amikacin, kanamycin, capreomycin). In addition to meeting the defining criteria, XDR-TB cases are often resistant to all four 1st-line agents. Consequently, patients with XDR-TB are significantly more difficult to treat and require specialized care.
[Note: Material also covered (duplicate slide) in Management of Drug-Resistant TB module.]

31. Drug-Resistant TB: Definitions
ISTC Training Modules 2008
Drug-Resistant TB: Definitions
EXTENSIVELY DRUG RESISTANT
Begin with the definitions for drug resistance:
Mono-resistant: The most common single drug-resistance pattern is mono-resistance to isoniazid. In general, this pattern of resistance is not usually associated with a worse outcome and does not require modification of the treatment regimen (as long as there are 4 drugs in the initial phase and rifampicin is included throughout the full duration of treatment). Rifampicin mono-resistance occurs, but is uncommon and is seen mainly in patients with HIV infection. The reasons for this association are not known.
Poly-resistant: A general term used when the organism is resistant to more than one drug, but not the combination of isoniazid and rifampicin.
MDR-TB: Resistance to at least isoniazid and rifampicin (the two most effective anti-tuberculosis drugs). MDR has a major adverse effect on the outcome of treatment. Patients with TB caused by MDR organisms generally require treatment with second line drug regimens.
XDR-TB: MDR-TB plus resistance to the 2 most important classes of 2nd-line agents used in MDR-TB treatment: the fluoroquinolones and at least 1 of 3 injectable agents (amikacin, kanamycin, capreomycin). In addition to meeting the defining criteria, XDR-TB cases are often resistant to all four 1st-line agents. Consequently, patients with XDR-TB are significantly more difficult to treat and require specialized care.
[Note: Material also covered (duplicate slide) in Management of Drug-Resistant TB module.]

32. Impact of Resistance on Outcome
ISTC Training Modules 2008
% of cases with failure or death, standard 4-drug regimen
Resistance pattern
New Cases (%)
Retreatment (%)
Pan-susceptible 4 10
Any Resistance 5 21
MDR 30 45
INH (not MDR) 6 23
RIF (not MDR) 13 29
Other 15
This table describes the outcome of treatment using a standard first line regimen in patients with various patterns of drug resistance.
Investigators conducted a retrospective cohort study of patients with TB in the Dominican Republic, Hong Kong, Italy, Ivanovo Oblast (Russian Federation), the Republic of Korea, and Peru.
The outcome was determined for new (previously untreated) and retreatment TB cases who received short-course chemotherapy with isoniazid, rifampicin, pyrazinamide, and either ethambutol or streptomycin between 1994 and Treatment outcome was defined according to WHO criteria.
In this table, death during treatment and treatment failure were combined to provide a measure of unfavorable outcome.
As the table shows, both MDR and rifampicin resistance were associated with a greater percentage of unfavorable outcomes than any of the other categories. Retreatment cases in all categories did less well than new cases. Again MDR cases had the highest percentage of unfavorable outcomes.
The success rate of retreated cases drops substantially in all categories. Successful treatment outcomes for both new and retreated cases are significantly lower for RIF-resistant and MDR cases.
[Reference: Espinal MA, et al. Standard Short-course Chemotherapy for Drug-Resistant Tuberculosis: Treatment Outcomes in 6 Countries. JAMA. 2000;283(19): ]
Espinal MA, et al. JAMA. 2000;283(19):

33. Mechanisms of resistance
Production of enzymes that modify or destroy the antibiotic molecule
Alteration of the target binding site
Active escretion of the molecule (efflux pump)
Membrane permeability reduction

34. Frequency of Resistance Mutations
ISTC Training Modules 2008
INH = 1 in 106
RIF = 1 in 108
EMB = 1 in 106
Str = 1 in 106
I + R = 1 in 1014
In any given population of TB bacilli, naturally occurring resistant organisms occur at the rates as shown.
[Review above]
The greater the burden of disease, the larger the bacillary population and the greater the risk for harboring drug-resistant mutants and acquiring drug resistance.
[Note: Year drug was discovered is listed under each drug.]
[Image Credit: CDC/Dr. Ray Butler; Janice Carr. Illustration Credit: National Institute of Allergy and Infectious Diseases (NIAID). Illustrator: Krista Townsend]

35. Genes involved in drug-resistance for major anti-tubercular drugs

36. RIF resistance as surrogate marker for MDR TB
RIF resistance as a mono resistance not very frequent (5-15% of them)
80-95% of RIF resistant strains are also resistant to INH
Key-drug in the anti-TB regimen
Low rate of spontaneous mutations
Mutations affect an hot-spot region in the rpoB gene
Khue et al. A 10-year prospective surveillance of Mycobacterium tuberculosis drug resistance in France 1995–2004. Eur Respiratory J. 2007
Ideal candidate for the molecular detection of resistance to the drug
PAGE | 36 36

37. Commercial Line Probe Assays
Hain Lifescience
Innogenetics
INNO-LiPA-Rif.TB 37

38. Yes (modified version)
Comparison GenoType® MTBDRplus and INNO-LiPA Rif.TB
GenoType® MTBDR
INNO-LiPA Rif.TB
Company
Hain Lifescience
Innogenetics
M. tuberculosis detection
Yes
Detection of RMP Resistance in M. tb Complex
Detection INH Resistance in M. tb Complex No
Strip Assay
PCR based
From liquid or solid culture
Direct assay
Yes (modified version)
TBCDetection: 23S-rRNA/16S-rRNA
RMP-Resistance: rpoB gene
INH-Resistance: katG gene/inhA gene
Universalcontrol
rpoB unicontrol
kat G/inHA unicontrol

39. New generation of LiPA performs better in both Sm+ and Sm- samples
Miotto al. JCM 2008

40. Policy statement by WHO and Partners June 27, 2008
Molecular line probe assays for rapid screening of patients at risk of MDR-TB
Policy statement by WHO and Partners
June 27, 2008
Endorsement of the two commercial line probe assays for rifampicin resistance detection:
Tests are CE marked and meet predefined performance targets in controlled evaluation studies
Both tests are highly sensitive and specific for rifampicin resistance detection from TB strains

41. Sputum samples Smear positive Smear negative Report as negative OR
if TB is highly suspected (HIV+,child..)
perform (liquid) culture
Rif/ Rif INH LiPA
Culture AFB positive,
TB morphology
MTB detected,
INH(*) and RMP susceptible
MTB detected,
INH and/or RMP resistant (MDR-TB)
Unsuccessful amplification
Standard MDR treatment
Culture (liquid/ solid)
DST ( first and second line)
Perform (liquid) culture
MTBC, INH(*) and RMP mutation
non detected NON MDR
No culture
Individualized
treatment
Culture AFB positive
monitoring
Start standard 1st line treatment
Treatment with 2nd-3rd
Drugs available/isolation ID
Monitoring sputum conversion

42. Possible automation on LiPA
LiPAs require:
Level II biosafety areas
Skilled laboratory staff
Amplicon Contamination control

43. XPERT TB-MDR •time-to-result < 2 h
•simple 1-step external sample prep.
•time-to-result < 2 h
•throughput: >16 tests / day / module
•no need for biosafety cabinet
•integrated controls
•true random access
•specific for MTB
•sensitivity similar to culture
•detection of rif-resistance via rpoB gene
•test cartridges for GeneXpert System
•several GeneXpert modules can be combined in 1 workstation
•~1 day technician training required

44. Simple Sample Processing – Direct Sputum
4. Transfer 2ml
to cartridge
2. Shake then stand 10 minutes
Begin Test…
3. Shake then stand
further 5 minutes
Add 2:1 Sample Buffer to sample

45. Moving to
“district” :
Dharamsala,
Delek Hospital

46. Sensitivity (cfu/ml) of pulmonary TB diagnostics
Solid
culture

47. Advantages Simple to perform Minimal training
Virtually cross contamination free
Minimal biosafety requirement
Higher sensitivity in paucibacillary samples (HIV+)

48. Proportion of TB cases detected

49. Time to detection

50. Time to RFP resistance detection

51. Potential limits of Xpert MTB/RIF technology
Unknown the performance at a district level
If RFP resistance is diagnosed at a low level MDR prevalence environment, the assay needs to be confirmed
Need to perform a culture for DST to evaluate other drug resistance
Need to perform a culture for monitoring issue (culture conversion)
It requires uninterrupted and stable electronic power supplies and yearly calibration
Size for storage issues

52. New WHO recommendations
WHO RECOMMENDATION, December 8th, 2010

53. New WHO recommendations
WHO RECOMMENDATION, December 8th, 2010

54. New WHO recommendations
WHO RECOMMENDATION, December 8th, 2010

55. Comparison GenoType® MTBDRplus and XpertTB/MDR
XpertTB-MDRplus
Company
Hain Lifescience
Cepheid
M. tuberculosis detection
Yes
Detection of RMP Resistance in M. tb Complex
Detection INH Resistance in M. tb Complex No
Fully automated /training
No/Yes
Yes/No
DNA tech
PCR
Mol Beacon
From liquid or solid culture NA
Direct assay
Level of biosafety II
microscopy
Time to results
Same day 2h
Cost per test
Low/Mod
Mod/High
Universal control
Extraction control
Cost of Maintenance
Contamination control
Low
High
Intermediate Reference labs
Patients testing from Sm/ C+ (Rif)
Fast tool Surveillance purposes
Potential to District level as fast
patients diagnostic tool, needs evaluation at district level

56. Sputum samples Xpert-TB/MDR-TB Contact tracing? Smear positive
Smear negative
Contact tracing?
Report as negative OR
if TB is highly suspected (HIV+,child..)
perform (liquid) culture
Rif/ Rif INH LiPA
Culture AFB positive,
TB morphology
MTB detected,
INH(*) and RMP susceptible
MTB detected,
INH and/or RMP resistant (MDR-TB)
Unsuccessful amplification
Standard MDR treatment
Culture (liquid/ solid)
DST ( first and second line)
Perform (liquid) culture
MTBC, INH(*) and RMP mutation
non detected NON MDR
No culture
Individualized
treatment
Culture AFB positive
monitoring
Start standard 1st line treatment
Treatment with 2nd-3rd
Drugs available/isolation ID
Monitoring sputum conversion

57. Genes involved in drug-resistance for major anti-tubercular drugs
40-80%

58. XDR molecular diagnosis: GenoType MTBDRsl (Hain Lifescience)

59. Technologies and laboratory appropriateness
Introducing new technology requires addressing of core elements:
Infrustructure, biosafety measures and maintenance
Equipment validation and maintenance
Specimen transport and referral mechanisms
Management of laboratory commodities and supplies
Laboratory information data and management system
Laboratory quality management
Strategies for HR development and retention
GLI road map at :

60. “Point of care assay” Closer if
Large scale innovation and delivery will be fully supported by:
Scientists and industry
Large operational research trials
Large view policy makers and regulators
Retooling of existing resources