Title Urine LAM detection: what is the future ? Author Emmanuel - PowerPoint PPT Presentation

Title Urine LAM detection: what is the future ? Author Emmanuel Moreau Event, location and date Advanced TB Diagnostics; Jun 19th 2018

Presentation outline LAM antigen= a perfect candidate ? The one IVD-grade urine LAM test: Alere lateral flow assay Current LAM testing limitations Next generation of LAM assays 2

Unmet Need in TB diagnostics 3

LAM: origin, role and structure What is LAM ? Why LAM as a target for diagnosis ? How to detect LAM ? 4

LAM is a bacterial cell wall component Lawn, 2102 LAM a ~17 kDa transmembrane glycolipid with LAM = ~60% of bacterial cell wall distinctive mannose capping = 15% of bacterial dry weight  maintains cell wall integrity (Hamasur, 2015)  Immunomodulation of host response  Interacts with macrophage mannose receptors for bacterial uptake 5

LAM structure is very similar among mycobacterial species ManLAM from M. tuberculosis culture AraLAM fro M. chelonae culture PILAM from M. smegmatis culture 6

Pathogen antigen for stand-alone diagnostics Direct proof of pathogen presence  High clinical specificity: if detected = the pathogen’s here ! Not a surrogate or indirect marker (≠ host marker protein) Not a long lasting marker ≠ antibodies (serology) ≠ memory T-cells (IGRA) Potential marker of disease course and pathogen dynamics 7

Immunodetection of LAM Free circulating LAM : detectable in serum, sputum and urine of active TB patients 8

Assays for detection of LAM in urine Research/”in development” assays IVD IVD Inverness Alere /Alere Los Alamos National Labs Karolinska Institutet Chemogen ? National University of Singapore /Abbott “Clearview” Colorado State University ELISA ELISA LFA ELISA Karolinska Institutet Uri TB direct 2001 2004 2007 2012 2015 2018… WHO Policy Guidance for Alere LFA LAM assay 9

Alere Determine TB LAM Ag 10

Alere LAM assay performance in HIV+ population  Pooled sensitivity: 44% [31-60%] and specificity: 92% [83-96%] WHO LAM Policy Guidance, 2015 11

Alere Determine TB Alere LAM assay shows limited sub-optimal sensitivity in TB+ HIV+ patients Sensitivity is better in TB+ HIV+ patients with low CD4 count Shah, 2016 WHO Conditional recommendation: Alere Determine TB may be used for TB diagnosis in HIV+ patients critically ill and/or CD4 count < 100/uL Specificity performance is unclear. Low quality of evidence led the WHO to strongly recommend against screening with Alere LAM assay. 12

Cross-reactivity concerns Specificity issues reported: False Positive (Alere antibody is a polyclonal) 13

Alere Determine TB LAM Ag assay Alere LAM test Urine LAM detection partly met in HIV+ patients not met 25 min 3.5$/test Visual reading LFA None (60 uL urine) Level 0/1 14

LAM testing is complementary to Smear and Xpert Diagnostic yield x2 Diagnostic yield x3 LAM + Smear : 19.4% LAM + Smear : 18.9% to 46.8% to 60.8% LAM + Xpert : 26.6% LAM + Xpert : 24.3% to 52.5% to 60.5% Lawn, 2017 15

Even at existing suboptimal performance, LAM saves lives! The reduction of relative risk of dying was 17% with LAM testing Peter, 2016 16

Alere Determine TB Alere LAM assay is a valuable diagnostic tool in a niche population of HIV+ TB+ patients “It saves lives !” Alere Determine TB assay is in the WHO’s Model List of Essential In Vitro Diagnostics No more discussions about discontinuation of the assay by Abbott  “We need a better, more sensitive LAM assay”  how much more sensitive ?  what’s LAM concentration in urine ? 17

Biological origin of LAM in urine Proposed model ( Wood, 2012) The more advanced HIV infection is (CD4 ⇓ ), the more urine LAM ⇑ Decreased immune response: • Increased free LAM (not immunocomplexed) • Extrapulmonary TB (renal tract infection) 18

Is LAM present in HIV(-) patients urine ? Using current methods, LAM is hardly detected in TB+ HIV- patients If LAM concentration is likely to be low  concentrate the sample ! Paris, 2018: • Capture of urine LAM by hydrogel nano-cages • Detection by immuno-macroarray 19

Is LAM present in HIV(-) patients urine ? 1000 Alere cut-off LAM can be detected in HIV(-) urine ! For “High Sensitivity”, cut-off should be <20 pg/mL !!! (vs 500-1000 pg/mL for Alere LFA) 20

The future: High-Sensitive LAM assay HS-Troponin central lab tests can achieve analytical sensitivity (LOQ) of ~5 pg/mL in blood; HS-Troponin POC tests (LOQ) of ~50 pg/mL Requirement for HS LAM= 100-fold improvement of analytical sensitivity of Alere assay Leads for improvement: • Pre-concentration of LAM in urine samples • Better antibodies • Advanced assay design and signal amplification  but still PoC and easy to use ! 21

Pre-analytical LAM concentration Paris, 2018 immunoarray, chemotraps 100-fold, HIV- 22

Pre-concentration of LAM Ultrafiltration: (+) easy, standard (-) interfering cross-reagents (-) transferable to POC LFA assay ? Capture (+) specific to LAM Magnetic beads (-) cost ? Chemical traps (-) additional steps for POC 23

Improved LAM antibodies Monoclonal antibodies with sharp specificity to ManLAM epitopes (Choudhary, 2018) High affinity MAbs to pick up lower LAM concentrations in samples (Amin, 2018) 24

Signal amplification Use of a portable reader instrument • Luminescence decay-time sensors (Weigl’s group / Global Good) Enhance signal output of LFA • Enzyme-amplified colorimetric detection (Mologic) • Silver amplification (Fujifilm) 25

Next generation of urine LAM assays Improved LAM assay TPP Alere LF-LAM • FIND and Fujifilm ✖ ฀ Only in HIV • Stage-gates and 7 milestones completed within budget Intended Use & Target • «Product Design» input completed Population patients with low CD4 counts • Evaluated 989 antibody pairs using patient urine on high ✖ ฀ ~40% sensitivity platform Sensitivity in all HIV + • «Product Development» completed ✖ ฀ ~1000pg Analytical sensitivity • Initial Evaluation completed LAM/ml • NEXT STEPS: Evaluation on frozen samples from >1200 ✔ ฀ Specificity mostly HIV+ patients will be completed soon ✔ ฀ Sample Type ✔ ฀ Time to Result Global Good/ Intellectual Ventures ✔ ฀ Ease of use • Data later this year – so far little information ✔ ฀ Manufacturing cost Others India Institute of Science paper-based PoC in pre-development •

Detection of LAM in sputum Otsuka Pharmaceutical • Drug development company • Need real-time assessment of efficacy in TB drug development trials • Sputum pre-treatment (20’ @100 ° C) then classic ELISA test • LoQ = 15 pg/mL of LAM • Sensitivity (banked samples): 100% in S+/C+; 51% in S-/C+ (vs 79% by Xpert) Sputum LAM correlates with changes in sputum cfu, and with MGIT TTD  direct relation between LAM concentration and pulmonary bacilli load. 27

Conclusion Currently, urine LAM is a valuable antigen target for diagnosis of TB patients with advanced HIV infection Alere Detect TB LAM lateral flow assay is a true POC test beneficial to HIV critically ill patients but has strong limitations Second generation of High Sensitive test are likely to reach LAM’s full clinical potential and extend its use to HIV negative patients. Recent developments are promising ! 28

Thank you Questions?

LAM stability Is LAM stable at RT ? Is LAM stable when stored frozen ? And after freeze/thaw cycle(s) ? Current knowledge: conflicting message • Heat-stable (Clearview, Otsuka IFUs) • Can sustain 3 Freeze/Thaw cycles (Alere’s Determine TB IFU) • Sensitive to Freeze/Thaw cycles (personal communications) One and unique answer ? It is method dependent !  : targeted epitopes Each assay needs to be validated for sample stability individually 30

The reality of molecular interactions 31

TPPs for TB detection and triage tests 32