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

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

5

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

6

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

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

9

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

10

Alere LAM assay performance in HIV+ population

11

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.

12

Shah, 2016

Cross-reactivity concerns

Specificity issues reported: False Positive (Alere antibody is a polyclonal)

13

Alere Determine TB LAM Ag assay

14

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

15

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!

16

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

17

“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)

18

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

19

Is LAM present in HIV(-) patients urine ?

20

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 !

21

Pre-analytical LAM concentration

22

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

23

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)

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

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)

27

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 !

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