3. A key driver of this process is overprescription of
antibiotics, and we are running out of options:
1. Feasibility: why do it, can we do it?
Antibiotic resistance is a major issue.
http://www.who.int/drugresistance/publications/infographic-antimicrobial-resistance-20140430.pdf
https://www.cdc.gov/drugresistance/about.html
4. Just search online for “antibiotic resistance timeline”!
http://www.nature.com/nchembio/journal/v3/n9/fig_tab/nchembio.2007.24_F1.html
5. The financial burden is considerable = an opportunity exists to generate revenue
while saving public and private funds (and lives).
http://www.who.int/drugresistance/documents/AMR_report_Web_slide_set.pdf
6. Antibiotic stewardship initiatives mean that there might be funding available.
£10M for a
POC test!
https://www.whitehouse.gov/sites/default/files/docs/national_action_plan_for_combating_antibotic-resistant_bacteria.pdf
http://www.safetyandquality.gov.au/
our-work/healthcare-associated
-infection/antimicrobial-steward
ship/
A US national action plan means funding
can be sought from
NIH, BARDA, DoD, etc.
7. OK, OK, it's a great idea. Can we do it?
1. RPS focuses on point-of-care testing (lateral flow); good angle vs overprescription
2. RPS has a relationship with Kyowa Medex, who hold patents relating to MxA,
which is a biomarker of viral infection (named for association with myxovirus infection,
downstream of IL-6 and IFN-α/β/λ)
This led to a search for additional biomarkers of infection:
C-reactive protein (CRP): widely used as marker of inflammation, agnostic as to
cause thereof, not especially well regarded in terms of sensitivity/specificity
Procalcitonin (PCT): also more of an inflammatory marker but somewhat specific for
bacterial infection
8. 2. Optimization: from prototype to initial design freeze
Reference ranges:
● MxA: a cutoff of ~35 ng/ml distinguishes normal (~<5-10 ng/ml) from elevated
● CRP: opinions vary in non-cardiovascular context; normal ~ <10 µg/ml
These are numbers we can work with on a lateral flow test.
● PCT: < 0.15 ng/ml healthy, 0.15-2.0 ng/ml likely infection, >2.0 ng/ml serious risk
These, not so much: requires high sensitivity and narrow range distinguishing
positive from negative.
9. Basic anatomy of a lateral flow test:
Wong &Tse, Lateral Flow Immunoassay
13. Lifting the conjugate out of the plane of
fluid flow in order to avoid infringing patents
14. sample type
sample buffer
conjugate:
antibody pairs?
gold, latex, fluoro?
control:
internal to test or sample?
materials: backing,
sample pad, waste
pad, conjugate pad,
membrane limit of detection
predicate test?
gold standard/comparator?
fit to cassette +
“favorites” =
smaller testing
matrix
15. sample type
sample buffer
conjugate:
antibody pairs?
gold, latex, fluoro?
control:
internal to test or sample?
screen by ELISA
visual read, multiple
colors: latex
materials: backing,
sample pad, waste
pad, conjugate pad,
membrane limit of detection
predicate test?
gold standard/comparator?
fit to cassette +
“favorites” =
smaller testing
matrix
16. sample type
sample buffer
limit of detection
predicate test?
gold standard/comparator?
conjugate:
antibody pairs?
gold, latex, fluoro?
control:
internal to test or sample?
screen by ELISA
visual read, multiple
colors: latex
blood
pH
Detergent/s
Salt
Blocking
- BSA
- PEG
- PVA
Viscosity (sugars)
etc.
17. sample type
sample buffer
limit of detection
predicate test?
gold standard/comparator?
conjugate:
antibody pairs?
gold, latex, fluoro?
control:
internal to test or sample?
screen by ELISA
visual read, multiple
colors: latex
blood
pH
Detergent/s
Salt
Blocking
- BSA
- PEG
- PVA
Viscosity (sugars)
etc.
e.g. ck IgY +
rb anti-IgY
usu. anti-hu.IgG
18. sample type
sample buffer
limit of detection
predicate test?
gold standard/comparator?
conjugate:
antibody pairs?
gold, latex, fluoro?
control:
internal to test or sample?
screen by ELISA
visual read, multiple
colors: latex
blood
pH
Detergent/s
Salt
Blocking
- BSA
- PEG
- PVA
Viscosity (sugars)
etc.
e.g. ck IgY +
rb anti-IgY
usu. anti-hu.IgG
none
Biofire, culture,
PCR, bloodwork
cutoff not pres/abs
define sample volume
19. 3. Verification: did we build the device right?
Analytical
● spike-and-recovery in blood
● small scale testing in human subjects
Interfering substances/conditions
● globinopathies, RF, ...
Robustness
● guard-bands for buffer volume
● guard-bands for timing
● run upside-down, on angle, drop, etc
Stability
● accelerated-aging studies provides scope for real-time
20. 4. Design Transfer
Typical:
● write SOPs
● oversee pilot batch/es, amend SOPs
● mfg makes 3 batches at scale (process validation)
● those three batches used for clinical trial and final evaluation of
performance parameters (analytical, stability)
22. A brief word about a molecular approach to the same problem:
● microarray analysis; one patient cohort, n = 317
● validated vs existing data sets
● bacteria 71 probes, virus 33 probes, noninfectious 26 probes
● 87% vs clinical adjudication
23. ● microarray analysis, one patient cohort n = 58
● sets of 18-33 probes
● “better than white cell count”
24. ● 7 genes whose expression patterns distinguish viral from bacterial
● in silico only, yet to be tested on patients
● potential to combine with previously identified 11 gene set for sepsis dx
