REMoxTB – a microbiological challenge Dr Anna Bateson Dr. Tim McHugh, Robert Hunt, Emma Cunningham (Emily Bongard, Holly Ciesielczuk)

Mexico Clinical site are sending sputum samples to Denver Cape Town and Stellenbosch Clinical sites are sending sputum samples to laboratory in Stellenbosch Westdene, Brits, Tembisa, Soweto 4 clinical sites are sending sputum samples to laboratory in Johannesburg India 28 clinical sites are sending sputum samples to central laboratory in Delhi Thailand 2 clinical sites in Bangkok sending sputum samples central laboratory in Bangkok Kuala Lumpar Malaysia China 2 clinical sites in Beijing and Tianjin sending samples to laboratory in Tianjin Nairobi, Kenya Moshi, Tanzania Mbeya, Tanzania Lusaka, Zambia REMoxTB – Clinical sites and TB laboratories Durban 2 clinical sites (DICTU and CAPRISA) sending samples to central laboratory

REMoxTB Laboratory and Quality Manual Implementation of a single REMoxTB Laboratory and Quality Manual across all sites Detailed standard operating protocols for all TB microbiology techniques to be carried out in the trial These protocols must be followed to ensure uniform laboratory practises across all the trial sites The REMoxTB Quality contains details of the required quality checks and associated reporting forms. Strict adherence to the laboratory and quality manual is essential for the strength and integrity of the trial data

Sputum decontamination (LM2) NaOH/NALC Sputum smear (LM3/LM4) ZN stain Liquid culture (LM5) MGIT Solid culture (LM5) L/J slope Sputum transfer (LM1) Overview of TB microbiology protocols - I Drug susceptibility testing MGIT (LM5) Molecular speciation Accuprobe (LM6) Positive LJ or MGIT M.tb complex confirmed BASELINE AND POTENTIAL RELAPSE ONLY (positive culture at or after week 17) Molecular detection of resistance from sputum - HAIN assay (LM8) Rifampicin/Isonazid/Fluroquinolones SCREENING ONLY – only at sites with high levels of MDR and/or FQ resistance

BASELINE AND SUSPECTED RELAPSE ONLY DNA extraction (LM7) IS6110 RFLP typing (LM9) MIRU typing (LM10) UCL, LONDON Whole genome sequencing SANGER CENTRE, CAMBRIDGE M.tb confirmed positive culture (LJ or MGIT) Overview of TB microbiology protocols - II

Site preparation and start up Preparing laboratories to join REMoxTB (UCL and CRA) – liaise with the site laboratory staff to ensure everything is in place to start the study – equipment, staff, documentation Pre-Site Initiation training (UCL) – Presentation and practical sessions, observation of procedures and competency – Review of logistics for sample transport and data reporting to meet trial requirements

Site follow up and monitoring 10-patient QC visit (UCL and CRA) – Detailed review of all processes, laboratory procedures and documentation – Follow up training as required Routine monitoring visits (CRA) – After 50 patients (or 3 months), every 6 months and at the end of the study – Similar to 10-patient QC, but restricted to detailed review of documentation UCL role after site initiation – Assisting with the resolution of issues and providing further training and/or a more detailed review – Long range QC review of data from the REMoxTB database

Capacity Development Advise in the design, rebuilding or remodeling of containment level 3 laboratory space to improve safety standards (in line with international standards) – e.g. India, China, Malaysia, Moshi Provision of equipment – e.g. MGIT, Accuprobe, Hain (most sites) Training and implementation of new assays such as Hain and MGIT based sensitivity testing (not part of routine service) Introduction of standard quality processes – provision of reference strains, enrolment in international EQA schemes Clinical trials experience – training and experience in documentation required for Phase III regulatory study

Contamination Rates - I

Contamination Rates- II Do contamination rates vary according to the time in treatment? Increase not seen with MGIT data Smaller sample numbers at later timepoints – analysis ongoing

Smear microscopy as predictor of culture positivity during treatment (Dr. Michael Murphy)

Smear grading and culture positivity during treatment - LJ Good correlation between smear grading and week growth seen on LJ slopes early in treatment Smear grading corresponds to decreasing viable bacteria as treatment progresses

Drug resistance - I Drug resistant isolates from all sites are being sent to UCL – Baseline (pre-treatment) isolates (~1900) – Late exclusions MDR, rifampicin monoresistance, moxifloxacin resistance – Post week 17 positive cultures – acquired resistance Further analysis of drug resistant isolates Repeat DST to confirm, MIC determination In moxi-resistant isolates, determine cross resistance to other fluoroquinolones (FQ) Characterise resistance conferring mutations (e.g. sequencing of GyrA, RpoB, KatG etc)

Drug resistance – II Surveillance data from geographically diverse populations – Patterns of resistance, Baseline levels of moxifloxacin resistance Acquired resistance during treatment - failure/relapse – Frequency of drug resistant allelles Hain assay data in some sites (isonaizid/rifampicin/FQ resistance mutations) – Comparison of Hain and Culture based DST Fitness – Fitness of acquired drug resistant isolates compared to pre- treatment isolate - likelihood of mutations to fix in the population

Current available data Baseline samples – 161/1211 resistant to any of the drugs tested (13.3%) – 1203 SIRE results available 4 rifampcin monoresistant 18 MDR – 1153 Moxifloxacin results available (excludes India) – 8 Moxi resistant (excludes data from Thailand/Lusaka – testing problems) Acquired resistance during treatment (at or after week 17) 117 results available – 4 isolates with new drug resistance – Includes 1 moxifloxacin resistant isolate (Stellenbosch) – to be confirmed