Title Author
Event, location and date
Urine LAM detection: what is the future ?
Emmanuel Moreau 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
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Unmet Need in TB diagnostics
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LAM: origin, role and structure
What is LAM ?
Why LAM as a target for diagnosis ?
How to detect LAM ?
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LAM is a bacterial cell wall component
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LAM = ~60% of bacterial cell wall = 15% of bacterial dry weight (Hamasur, 2015)
LAM a ~17 kDa transmembrane glycolipid with distinctive mannose capping
maintains cell wall integrity Immunomodulation of host response Interacts with macrophage mannose receptors for bacterial
uptake
Lawn, 2102
LAM structure is very similar among mycobacterial species
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AraLAM fro M. chelonae culture
PILAM from M. smegmatis culture
ManLAM from M. tuberculosis culture
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
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Immunodetection of LAM
Free circulating LAM : detectable in serum, sputum and urine of active TB patients
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Assays for detection of LAM in urine
Research/”in development” assays
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Karolinska Institutet ELISA
2001
Chemogen ELISA
2012 2007 2004
Inverness /Alere “Clearview” ELISA
Alere /Abbott LFA
2015
Los Alamos National Labs National University of Singapore Colorado State University Karolinska Institutet Uri TB direct
2018…
?
IVD IVD
WHO Policy Guidance for Alere LFA LAM assay
Alere Determine TB LAM Ag
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Alere LAM assay performance in HIV+ population
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Pooled sensitivity: 44% [31-60%] and specificity: 92% [83-96%]
WHO LAM Policy Guidance, 2015
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
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.
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Shah, 2016
Cross-reactivity concerns
Specificity issues reported: False Positive (Alere antibody is a polyclonal)
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Alere Determine TB LAM Ag assay
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25 min 3.5$/test
Visual reading LFA
None (60 uL urine)
Level 0/1
Urine LAM detection
Alere LAM test
not met
partly met in HIV+ patients
LAM testing is complementary to Smear and Xpert
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Diagnostic yield x2 LAM + Smear : 19.4% to 46.8% LAM + Xpert : 26.6% to 52.5%
Diagnostic yield x3 LAM + Smear : 18.9% to 60.8% LAM + Xpert : 24.3% to 60.5%
Lawn, 2017
Even at existing suboptimal performance, LAM saves lives!
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Peter, 2016
The reduction of relative risk of dying was 17% with LAM testing
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
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“We need a better, more sensitive LAM assay” how much more sensitive ? what’s LAM concentration in urine ?
Biological origin of LAM in urine
Proposed model (Wood, 2012)
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The more advanced HIV infection is (CD4 ⇓), the more urine LAM ⇑
Decreased immune response: • Increased free LAM (not immunocomplexed) • Extrapulmonary TB (renal tract infection)
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
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Is LAM present in HIV(-) patients urine ?
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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)
1000 Alere cut-off
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 !
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Pre-analytical LAM concentration
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Paris, 2018 immunoarray, chemotraps 100-fold, HIV-
Pre-concentration of LAM
Ultrafiltration: (+) easy, standard (-) interfering cross-reagents (-) transferable to POC LFA assay ?
Capture (+) specific to LAM (-) cost ? (-) additional steps for POC
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Magnetic beads Chemical traps
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)
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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)
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TPP Alere LF-LAM
Intended Use & Target Population
✖️Only in HIV patients with low CD4 counts
Sensitivity in all HIV + ✖️~40%
Analytical sensitivity ✖️~1000pg LAM/ml
Specificity ✔️ Sample Type ✔️ Time to Result ✔️ Ease of use ✔️ Manufacturing cost ✔️
Improved LAM assay • FIND and Fujifilm • Stage-gates and 7 milestones completed within budget • «Product Design» input completed • Evaluated 989 antibody pairs using patient urine on high
sensitivity platform • «Product Development» completed • Initial Evaluation completed • NEXT STEPS: Evaluation on frozen samples from >1200
mostly HIV+ patients will be completed soon
Global Good/ Intellectual Ventures • Data later this year – so far little information
Others
• India Institute of Science paper-based PoC in pre-development
Next generation of urine LAM assays
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)
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Sputum LAM correlates with changes in sputum cfu, and with MGIT TTD direct relation between LAM concentration and pulmonary bacilli load.
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 !
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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
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The reality of molecular interactions
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TPPs for TB detection and triage tests
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