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

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

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)

Courtesy WHO and FIND

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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

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

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

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

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

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

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

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)