FAPIC Workshop Point of Care Device CommercializationFAPIC Workshop Point of Care Device Commercialization

Michael G. LorenzMolzym GmbH & Co. KGBremen, Germany, y

Development of a fast method for culture‐independent ID and characterisation p pof pathogens

Automated system development: DNA extraction – highly multiplexed amplification – array detection (species, resistance, virulence) – read out

Decentralised and laboratory devices Clinical evaluation

POC d i   P h D L b d i   P h R bPOC device « PathoDoc » 1ml extraction protocol: EDTA blood Pathogen detection integrated

(amplification  arraying  imaging)

Lab device « PathoRobot » 5ml extraction protocol:

EDTA blood, CSF  Separate amplification  detection units(amplification, arraying, imaging)

1 sample Separate amplification, detection units 1‐8 samples

SelectNA™plus – 1ml ReleasedApril 2016

Liquid handling & vacuumfiltration robot

Micro‐Dx™E i

Specimens Micro‐Dx™A l iExtraction

Liquid biopsies (1ml)•Joint aspirates

Analysis

•EDTA blood•Ascites•CSF•Swabs

Real‐Time PCR:•16S BacteriaEluate•Swabs

•...Tissue & other biopsies•Heart valves

•16S Bacteria•18S FungiSequencingSepsiTest™‐BLAST

Eluate

•Bone•Soft tissue•Abscesses•...

24.11.2016 5

...

l d ( l ) Interior validation (at Molzym)▪ Analytical parameters and workflow (LOD, reagent stability, cross‐contamination, decontamination, risk assessment), , )

▪ Analysis of clinical samples (stored, fresh); comparator: manual system (CE‐IVD SepsiTest™‐UMD)

E i   lid i  (b   i ) Exterior validation (beta testing) Reconsideration of the system for improvementE t i   l ti  t ti   t  ti  l b   i t i Exterior evaluation: testing at routine labs against in‐house tests, culture

Documentation  CE‐IVD marking Documentation, CE‐IVD marking

Limit of detection (LOD)  Gram-negative bacteria Strain100% 0 %

LOD( )expressed as colony forming units per millilitre pass when comparable to 

100% ≥50 %Escherichia coli ECO1 100 75Klebsiella pneumoniae KLPN4 100 n.d.Gram-positive bacteriaEnterococcus faecalis EK1 25 n.d.Staphylococcus aureus STA1 50 25Streptococcus agalactiae STGB2 10 n.d. pass when comparable to 

previously evaluated systems

f

YeastsCandida albicans CA1 10 5Candida glabrata CAGL 10 5Candida krusei CAKR 50 10Candida parapsilosis CAPA 10 5Candida tropicalis CATR 5 3

Cross contamination of samples: side‐by‐side extraction of positive (spiking) p ( p g)and negative samples pass when not detectable

Micro‐Dx™ vs  manual system (CE‐IVD SepsiTest™‐UMD)Micro Dx  vs. manual system (CE IVD SepsiTest UMD) pass when greatly comparable: 82% identical positives

Species / genus Micro-Dx™ UMD (manual) Both SampleAbiotrophia defectiva 1 1 1 heart valveBartonella washoensis 0 1 0 EDTA blood

Tissue biopsies No.Sum 37

heart valves 21aortic valve prothesis 1

pleura tissue 1aortic occluder 1necrotic tissue 3

muscle 1hip tissue 2

Bartonella washoensis 0 1 0 EDTA bloodCandida albicans 1 1 1 unspecified tissue

Candida glabrata 1 1 1 EDTA blood

Cladosporium spp. or Davidiella spp. (fungus) 1 1 1 hip tissue

Enterococcus faecalis 1 1 1 heart valve

Mesorhizobium loti 0 1 0 aortic valve

Microbacterium testaceum/resistens/paraoxydans 1 1 1 blood culture

infected hematoma 3pericard biopsy 2

other tissue 2Liquid samples & swabsSum 29

EDTA-blood 18pleural aspirate 3

knee joint aspirate 2blood culture 2

Moraxella osloensis 1 0 0 aortic valve

Neisseria meningitidis 1 0 0 blood culture

Rhodococcus qingshengii/erythropolis 0 1 0 joint aspirate

Staphylococcus aureus 4 4 4 heart valves (3x), joint aspirate

Staphylococcus spp. (CoNS) 4 3 3 heart valves (3x), valve prothesis

Streptococcus spp. 9 10 7 pleura tissue, heart valves (6x), hip tissue, CSF 1

bone marrow 1e-Swabs 2

Total 66

UMD pos UMD neg SumMicro-Dx™ pos 23 4 27Micro-Dx™ neg 7 32 39S 30 36 66

p pp p , ( ), p ,hematomas (3x)

Streptococcus pseudopneumoniae/pneumoniae 1 1 1 CSF

Mixed sequences (UMD) neg Streptococcus spp. Staphylococcus spp. Propionibacterium

acnes

0 pericard biopsy

Staphylococcus hominis

Staphylococcus spp. Streptococcus spp.

1 pericard biopsy

Sum 30 36 66 Corynebacterium spp.Clostridium spp. Clostridium spp.

Bacillus firmansEnterococcus faecalis

1 hip tissue

Micro‐Dx™ vs. in‐house PCR vs. cultureMicro Dx  vs. in house PCR vs. culture pass: 88% identical positives – Micro‐Dx™‐positive, in‐house test‐negative results were regarded 100% relevant

Mi D ™ i i  I h  PCR i   l

Tissue biopsies No.Sum 28

heart valve 22spinal disc 2th ti 4

Sample Micro-Dx™ CultureAspirate Staphylococcus aureus negHeart valve Streptococcus mutans negCSF Staphylococcus aureus negHeart valce Streptococcus spp. Staphylococcus epidermidis *

St t

Micro‐Dx™‐positive, In‐house PCR‐negative samples

other tissue 4Liquid samples & SwabsSum 36

CSF 5joint aspirates 19

ascites 1

Abscess tissue Streptococcus pyogenes neg

Aortic valve Streptococcus dysgalactiae neg

Pleura aspirate Streptococcus pyogenes neg

Tissue proximal Streptococcus sanguinis neg

Heart valve Streptococcus mitis group neg

Aortal swab Streptococcus mitis group negwound swab 9

endotracheal exudate 2Total 64

Aortal swab p group neg

CSF Streptococcus spp. (mixed) neg

* regarded as contaminant

In-house PCR pos In-house PCR neg SumMicro-Dx™ pos 14 12 26Micro Dx posMicro-Dx™ neg 2 36 38Summe 16 48 64

Mi D ™   i h   i l l l ID  Micro‐Dx™ vs. in‐house commercial molecular ID system pass: 100%  identical positives – 100% true Micro‐Dx™ results on top of in‐house method

Micro-Dx™(41 samples)

Stavnsbjerg et al. 2016

h l l bl Do as much external evaluations as possible Do testings at routine laboratories  for at least 

6 weeks to gather 50‐100 sample results6 weeks to gather 50 100 sample results Do clinical studies focussed on particular diseases Promote publishing of results Constantly consider system improvements as a 

consequence of customers’ feed back

FAPIC !

V lid ti  b   ti Validation by consortium▪ Analytical parameters and workflow (LOD, reagent stability, decontamination, risk assessment)

▪ Analysis of limited number of clinical and environmental samples; comparator: culture, other results, patient history

Reconsideration of the protocol for improvementeco s de at o o t e p otoco o p o e e t Evaluation: prospective clinical studies in Belgium and Croatia including 4,000 samples from 500 sepsis 

ti tpatients.>>> Diagnostic performance vs. culture, time‐to‐result, patient outcome, cost analysisp , y