1. TB Diagnostics: Update on Policies and Pipeline
Thomas M. Shinnick, Ph.D.
Chair, Global Laboratory Initiative
Associate Director for Global Laboratory Activities
Division of TB Eliminations, NCHHSTP
9th WPRO Meeting of National TB Program Managers
Manila Philippines
December 9, 2014
National Center for HIV/AIDS, Viral Hepatitis, STD & TB Prevention
Division of Tuberculosis Elimination

2. Current Laboratory Diagnostic Gaps
Only about 53% of all new cases and 63% of new smear-positive cases are detected
AFB smear-based testing is inadequate in high HIV settings
Only 28% of MDR TB cases lab confirmed
Many XDR TB cases are not detected due to the lack of second-line DST

3. TB Laboratory Services
Key needs:
Quality assured testing for patient care
rapid, reliable detection and DST
Access to diagnostic tests for all patients
2007 WHA call for universal access to culture and drug susceptibility testing
Lab systems and tests appropriate for the setting
Safe lab working environment (Biosafety)

4. Courtesy WHO and FIND

5. WHO-Endorsed Technologies
LED Microscopy
Peripheral laboratory and higher
Culture (liquid and solid)
Regional or national level laboratory
Phenotypic DST
Molecular Line Probe Assay (LPA)
Xpert MTB/RIF
Sub-district and district level laboratory

6. Culture Culture in liquid media Culture positives – 2-3 weeks
Culture negative – 6 weeks
Culture on solid media
Culture positives – 3-4 weeks
Culture negative – 8 weeks
Culture is most sensitive tool for detection
Diagnostic delay limits the usefulness of culture as a diagnostic tool
Culture needed for phenotypic DST and monitoring of the response to therapy

7. Role of the Laboratory in DST
Detect drug resistance to enable clinician to design effective multidrug regimen
Initial M. tuberculosis isolate should be tested against primary drugs
INH, RIF, PZA, EMB
For Rif-R isolates, test secondary drugs as needed
FQ, AMI, KAN, CAP

8. Drug Susceptibility Testing
Culture-based methods
Proportion method
solid media
liquid media
Absolute concentration method
Relative ratio method
Test critical concentrations — the lowest concentration that inhibits growth of all susceptible strains and allows growth of all resistant strains
Molecular methods

9. Updated Critical Concentrations
World Health Organization
16 April, 2017
Updated Critical Concentrations
MGIT 960 LJ
7H10
7H11
Ciprofloxacin
removed
Ofloxacin
4 μg/ml
Levofloxacin
1.5 μg/ml
1μg/ml
Moxifloxacin
0.5, 2.0 μg/ml
Amikacin
30 μg/ml
Capreomycin
Kanamycin
2.5 μg/ml
Cycloserine

10. WHO-Endorsed Molecular Tests for TB
Molecular Line Probe Assays (LPA)
Xpert MTB/RIF Assays

11. Line-Probe Assays
LPA can be used to detect rifampicin and isoniazid resistance conferring mutations
AFB-smear positive specimens
Positive TB cultures
Results can be available in 24 hrs
Second-line LPA NOT recommended
Needs sophisticated laboratory
Needs good referral mechanisms
Suitable for high throughput testing at Central or Regional laboratory level

12. Xpert MTB/RIF Assay
Can detect rifampicin-resistance conferring mutations in
AFB-smear positive specimens
AFB-smear negative specimens
Positive TB cultures
Results can be available in 2 hrs
Minimal infrastructure and facility needs
Suitable for testing at sub-district/district hospital level laboratory

13. WHO Recommendations – Xpert
Xpert should be used rather than conventional microscopy, culture and DST as the initial diagnostic test in persons presumed to have pulmonary MDR TB or HIV-associated TB
Xpert may be used as a follow-on test to microscopy in persons, where MDR TB or HIV is of lesser concern, especially in further testing of smear-negative specimens
Xpert may be used rather than conventional microscopy, culture and DST as the initial diagnostic test in all persons presumed to have TB

14. WHO Recommendations – Xpert Paediatric TB
Xpert should be used rather than conventional microscopy, culture, and DST in children presumed to have MDR TB or HIV-associated TB
Xpert may be used rather than conventional microscopy, culture, and DST in all other children presumed to have pulmonary TB

15. WHO Recommendations - Xpert Extrapulmonary TB
Xpert should be used in preference to conventional microscopy, culture and DST as the initial diagnostic test in testing CSF from patients presumed to have TB meningitis
Xpert may be used as a replacement test for usual practice including conventional microscopy and culture in testing lymph nodes and tissues from patients presumed to have extrapulmonary TB

16. Scale-up PMDT Program in Parallel with Xpert MTB/RIF Scale-up
Laboratory capacity
conventional culture and DST
other molecular methods – e.g. LPA
specimen referral and reporting results
Treatment capacity
hospital based and ambulatory care
patient support and palliative care
infection control
Second-line drug management
Forecasting and ordering

17. DST Questions What is gold standard DST method? genotypic? phenotypic?
Correlation with treatment outcomes?
“Borderline” rifampicin resistance
prevalence
geographic distribution
Genotypic DST
not all mutations known for all drugs
silent mutations, polymorphisms
mutations of unknown significance
What is role of next generation DNA sequencing in determining DST

18. WHO-Endorsed Technologies
LED Microscopy
Peripheral laboratory and higher
Culture (liquid and solid)
Regional or national level laboratory
Phenotypic DST
Molecular Line Probe Assay (LPA)
Xpert MTB/RIF
Sub-district and district level laboratory

19. An overview of progress in the development and evaluation of TB diagnostics
Technologies in developmenta
Technologies scheduled for evaluation by WHO in 2015
Evaluated by WHO and not recommended
Volatile organic compounds
BreathLink, Menssana Research, USA
Prototype breathanalyzer device, Next Dimensions Technology, USA
Molecular technologies
TB LAMP, Eiken, Japan
Molecular technologies for genotypic DST (including sequencing technologies)
Line probe assays
Non-molecular technologies
Alere Determine TB-LAM, Alere, USA
Commercial serodiagnostics (all manufacturers)
Interferon-gamma release assays for the detection of active TB (all settings)
Molecular technologies
Alere Q, Alere, USA
B-SMART, LabCorp, USA
Gendrive MTB/RIF ID, Epistem, UK
LATE-PCR, Brandeis University, USA
GeneXpert XDR cartridge, Cepheid, USA
TruArray MDR-TB, Akkoni, USA
INFINITIMTB Assay, AutoGenomics, USA
FluoroType MTB / FluoroType MTB RNA, Hain Lifesciences, Germany
Culture-based technologies
BNP Middlebrook, NanoLogix, USA
TREK Sensititre MYCOTB MIC plate, Trek Diagnostic Systems/ Thermo Fisher Scientific, USA
Other technologies
TB Rapid Screen, Global BioDiagnostics, USA
TBDx, Signature Mapping Medical Sciences, USA
Technologies endorsed by WHO
On the market but evidence for use not yet submitted to WHO for evaluation
Molecular technologies
Xpert MTB/RIF (pulmonary , extrapulmonary and paediatric samples)
Lineprobe assays for the detection of MTB and rifampicin resistance conferring mutations in AFB smear positive sputum or MTB cultures
Microscopy
Light and LED Microscopy
Same-day -diagnosis
Culture-based technologies
Commercial liquid culture systems and rapid speciation
Non-commercial culture and DST (MODS, NRA, CRI)
Molecular technologies
iCubate System, iCubate, USA
TB drug resistance array, Capital Bio, China
EasyNAT TB Diagnostic kit, Ustar Biotechnologies, China
Truelab/Truenat MTB, Molbio/bigtec Diagnostics, India
a This is not an exhaustive list of technologies in development. Those listed are the ones documented in publications by UNITAID and TAG.

20. Evidence required for WHO review
Phase 5 Scale-up and policy refinement
Phase 1
Research and Development
Phase 4
Phased uptake and collection of evidence for scale-up
Phase 2 Evaluation and Demonstration
Phase 3
WHO evidence assessment
GRADE

21. WHO recommended diagnostics
Upstream research and development to define and validate a prototype
Laboratory validation under international standards, design-locked product
WHO interact with developers to discuss end-user requirements
Phase 1: Research and Development
Controlled trials at 3-5 trial sites in high-burden TB and HIV countries
Product registration with global and/or national regulatory authorities
Specifications, performance validated in trials at 5-10 sites of intended use
Phase 2: Evaluation and Demonstration
New technologies/new indications for use: Dossier with Phase 1 and 2 data
Generic technologies: ISO13:485 standards; equivalence shown in 2-3 SRLs
WHO is not a regulatory authority
WHO does not recommend technologies for individual country use
Phase 3: WHO evidence assessment using GRADE
Implementation in routine TB services in high-burden TB and HIV countries
Systematic evaluation: algorithms, workload, operational issues, CE
Lessons learnt by early implementers used for country adaptation
Phase 4: Phased uptake & evidence for scale-up
Scale-up, with subsequent data to inform and refine WHO policy guidance
Phase 5: Scale-up & policy refinement
WHO recommended diagnostics

23. Challenges for Adopting a New Test
Incorporate new test into national TB strategic plans and testing algorithms
coordination of implementing partners
education of clinicians, program, patients
coordination of lab and treatment scale-up
sustainability and affordability
Which services are needed for patient care?
access to DST for all relevant drugs
Specimen or patient referral system
when using standardized and individualized regimens

24. Implementation of New Technologies and Diagnostic Algorithms
Decided by Ministries of Health
Coordinate partners and funding to avoid duplication and maximize resources
Phased approach as part of national strategic plans for laboratory, program, PMDT, TB/HIV
Linked to drug access and program capacity
Based on local epidemiology

25. Improve Entire Path of Work
A new lab test, e.g., Xpert®, is just one tool
A systems approach is needed
Must strengthen all processed from specimen collection to reporting results

26. A Systems Approach Emphasizes access to services
Ensures timely flow of specimens and information
Uses appropriate methods to provide prompt, high quality results
Relies on a well-trained workforce
Involves a network of quality-assured public and private laboratories

27. New tests and diagnostic algorithms decided by Ministries of Health
Summary
New tests and diagnostic algorithms decided by Ministries of Health
Favor integrated technology and broad laboratory network development
Phased implementation as part of national strategic plan for lab and program
Use a systems approach to develop a network of laboratories that provide reliable, quality-assured testing
Build the system on quality principles and emphasize access to services and timely flow of specimens and information

28. Thank You
National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention
Division of TB Elimination

29. Target Product Profiles
Point-of-care, non-sputum based test
Point-of-care, triage test
Point-of-care sputum based test as a replacement for microscopy
Point-of-care drug susceptibility testing (at microscopy center level)
The clinical purpose of the test (e.g. triage, detection, DST, other)
Goal to be met (e.g. start TB treatment on that day; refer for confirmatory testing)
Target population (children, adults, community or HIV-clinic)
Intended level of implementation in the health care system (home, community, clinic, peripheral microscopy center, hospital)