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Enteric PCR: test evaluations and application of PCR in clinical microbiology Kate Templeton NHS Enteric PCR: test evaluations and application of PCR in clinical microbiology Kate Templeton NHS Lothian 1

WHO data 2 WHO data 2

Workload in Edinburgh, UK 3 Workload in Edinburgh, UK 3

Positives in Scotland Pathogen 2013 2014 Norovirus 1915 1306 Rotavirus 1301 346 Shigella sonnei Positives in Scotland Pathogen 2013 2014 Norovirus 1915 1306 Rotavirus 1301 346 Shigella sonnei 62 49 Shigella flexneri 19 34 Yersinia entercolitica 6 4 Campylobacter 6163 5293* Listeria 15 15 C. difficile 1731 1710 Salmonella 813 543* Cryptosporidium 429 VTEC 219 Data HPS 282* 4 * Reported at week 40

Service challenges we face Increased workload Less staff Skilled staff retiring Less money improve Service challenges we face Increased workload Less staff Skilled staff retiring Less money improve clinical service Equity of access Patient centred care Fast and accurate results 5

Current enteric pathogen processing 6 Current enteric pathogen processing 6

Overlapping process Norovirus • Hospitalised/outbreaks in community C. diffiicle • 90% of above plus Overlapping process Norovirus • Hospitalised/outbreaks in community C. diffiicle • 90% of above plus any person >15 with diarrhoea Enteric culture Crypto • All tested for C. diff plus others with signs of gastroenteritis Microscopy A subset of those getting culture, e. g travel, immunocompromsied 7

Patient presents in Hospital with acute diarrhoea – what most likely Norovirus Campylobacter C. Patient presents in Hospital with acute diarrhoea – what most likely Norovirus Campylobacter C. difficle VTEC Shigella Salmonella Combine No. V and C. diff Could be any of them ! Stool only requested for both C. diff and Norovirus – 30% of time. 8

C. Difficle Challenges Screen with GDH or PCR ( toxin b) Confirm with toxin C. Difficle Challenges Screen with GDH or PCR ( toxin b) Confirm with toxin test Screening with PCR is an option 9

1 9 9 9 0 7 8 8 5 6 6 6 4 5 1 9 9 9 0 7 8 8 5 6 6 6 4 5 4 4 5 3 3 1 1 2 9 0 0 9 0 4 90 0 4 94 0 4 96 0 4 00 1 4 03 1 4 06 1 4 09 1 4 12 1 4 15 1 4 18 1 4 21 1 4 24 1 4 27 1 4 30 1 4 33 1 4 36 1 4 39 1 41 42 4 45 1 4 48 1 4 51 1 4 54 1 4 57 1 4 60 1 4 64 1 4 67 1 4 69 1 C. Difficile Challenges 10. 0% 9. 0% 8. 0% Equivocals 48/95 - PCR neg 7. 0% 1. 75 visits/ case for IPCN 6. 0% 45% moved to side room 5. 0% % equiv 4. 0% % Pos 3. 0% 2. 0% 1. 0% 0. 0% 10

Parasites – what is best method for diagnosis? Cyst of Giardia lamblia Cyst of Parasites – what is best method for diagnosis? Cyst of Giardia lamblia Cyst of Entamoeba histolytica Cyst of Cryptosporidium parvum Reference: Resources in Medical Microbiology. Scion Publishing Ltd. Viewed 24 th September 2013 at

Parasite PCR is gold standard Microscopy 30 -50% sensitive 1, 2 12 1 van Parasite PCR is gold standard Microscopy 30 -50% sensitive 1, 2 12 1 van Lint 2014 et al 2 Stark 2011 et al

PCR as an option Norovirus • Hospitalised/outbreaks in community PCR C. Difficile PCR • PCR as an option Norovirus • Hospitalised/outbreaks in community PCR C. Difficile PCR • 90% of above plus any person >15 with diarrhoea Enteric culture VTEC Crypto PCR • All tested for C. diff plus others with signs of gastroenteritis Parasites PCR 13

Can we do this with less staff and same money? 14 Can we do this with less staff and same money? 14

Norovirus on BDMax BDMAX is an Integrated extraction and PCR platform Evaluated in 2012 Norovirus on BDMax BDMAX is an Integrated extraction and PCR platform Evaluated in 2012 for Norovirus testing Introduced in 2013

The BDMAX Extraction on URS PCR on cartridge The BDMAX Extraction on URS PCR on cartridge

Workflow 1 m. L H 2 O + 25 MG SAMPLE PREP EXTRACT 1 Workflow 1 m. L H 2 O + 25 MG SAMPLE PREP EXTRACT 1 m. L H 2 O + 25 MG SAMPLE *SBT SAMPLE PREP PCR **URS EXTRACT ANALYSE PCR CARTRIDGE PCR ANALYSE *Sample Buffer Tube **Universal Reagent Strip

Validation Results RESULTS Platform TP TN FP FN Sensitivity Specificity ABi IC 99 233 Validation Results RESULTS Platform TP TN FP FN Sensitivity Specificity ABi IC 99 233 2 3 97% (91. 0 -99. 2) BDMAX IC 96 233 2 6 94% (87. 1 -97. 6) PPV (%) NPV (%) 99% (96. 6 -99. 8) 98 (92. 3 -99. 7) 99 (96. 0 -99. 7) 99% (96. 6 -99. 8) 98 (92. 1 -99. 6) 98 (94. 4 -98. 9) ASSAY COMPARISON P Value Cohen’s Κ easy. MAG/ABi NO IC vs. easy. MAG/ABi 7500 IC 1. 00 0. 96 BDMAX IC vs. easy. MAG/ABi NO IC 0. 67 0. 89 easy. MAG/ABi 7500 IC vs. BDMAX IC 0. 80 0. 94

Outcomes for the service BDMAX requires less technical skill and hands on time to Outcomes for the service BDMAX requires less technical skill and hands on time to perform the test and analyse results BDMAX currently being run by band 2 s and 4 s Lab TAT – 4 hours 17 mins Compared to Cepheid -3 hours 50 mins 70 -80% of results received by IPCN by 15. 00 Decrease in lost beds 2007 -9 3678 beds closed due to Norovirus – Daniel et al 2011 20013 -15 253 beds closed due to Norovirus 7 days testing Weekend testing informs ward closures at weekend

Multiplex with C. difficile for same staff Cost of GDH - £ 6 per Multiplex with C. difficile for same staff Cost of GDH - £ 6 per test Current consumable cost =£ 108, 000 If combined PCR – get 7000 test( no additional charge – as already doing No. V) Additional PCR cost =£ 110, 000 Advantages – No missed diagnosis Staff time overall saving 20

Now for the rest!! 21 Now for the rest!! 21

22 22

Validation Sample set: • 412 Stool samples • 189 – from viral lab • Validation Sample set: • 412 Stool samples • 189 – from viral lab • 233 – from bacteria enterics lab and E. coli ref lab • Process with GPP panel and compare to existing routine (and new) in-house real-time PCR as well as culture

189 Virology Samples Noro Rota Adeno Neg PCR 53 13 13 110 GPP 50 189 Virology Samples Noro Rota Adeno Neg PCR 53 13 13 110 GPP 50 11 2 67 Missed by GPP 10 (ct>35) 2 (ct>38) 11 (ct 14 -40) Sensitivity 90% 94% 20% Additional positives • 6 C. Diff • 3 Salmonella • 32 Camplyobacter • 2 Crypto Numerous samples with > 1 pathogen 25 had Bacteria and virus 1 Noro, Rota, Camplyobacter and C. diff

Bacteria Enterics Samples GPP Culture Missed by GPP Salmonella 24 32 12 Shigella 9 Bacteria Enterics Samples GPP Culture Missed by GPP Salmonella 24 32 12 Shigella 9 4 0 Camplyobacter 45 39 1 C. difficile 24 23 1 ETEC 8 5 0 STEC 7 8 3 O 157 7 10 (Inc IMS) 3 Yersinia enterocolitica 0 0 0 Vibrio chloera 0 1 1

Parasite Enteric Samples Microscopy GPP PCR Missed By GPP Giardia 4 6 6 0 Parasite Enteric Samples Microscopy GPP PCR Missed By GPP Giardia 4 6 6 0 Cryptosporidium 12 12 13 1 Entamoeba histolytica 0 0

Additional Positives • • 10 Norovirus positives – in Enteric samples 29 Dual Infections Additional Positives • • 10 Norovirus positives – in Enteric samples 29 Dual Infections 5 Triple infections 1 with 4.

Got Local grant to assess PCR for bacteria/parasites Awarded £ 39 K from the Got Local grant to assess PCR for bacteria/parasites Awarded £ 39 K from the ELHF http: //www. elhf. co. uk/ 28

Phase One: Retrospective Testing BDMAX ENTERIC PANELS ENTERICBIO GASTRO PANEL 2 CULTURE AND MICROSCOPY Phase One: Retrospective Testing BDMAX ENTERIC PANELS ENTERICBIO GASTRO PANEL 2 CULTURE AND MICROSCOPY LUMINEX XTAG GPP INHOUSE ASSAYS 29

Quick Assay Comparison BDMAX Enteric Bacterial Panel Plus Enteric Parasite Panel Salmonella Spp. Campylobacter Quick Assay Comparison BDMAX Enteric Bacterial Panel Plus Enteric Parasite Panel Salmonella Spp. Campylobacter jejuni/coli Shigella spp/EIEC VTEC Stx 1/Stx 2 Shigella dysenteriae. Giardia lamblia C. hominis/parvum Entamboeba histolytica 24 Samples per run Luminex x. TAG® GPP Ad. V 40/41, No. V GI/II, Rota A C. diff toxin A/B, Campylobacter spp. , E. coli O 157, ETEC HL/HS ET, VTEC(Stx)1/2, Shigella spp. , Salmonella spp. , V. cholerae, Y. enterocolitica, Cryptosporidium spp. , Giardia spp. E. histolytica 96 Samples per run* Serosep Enteric. Bio realtime Gastro Panel 2 Salmonella Spp. Campylobacter jejuni/coli/lari Shigella spp/EIEC VTEC (stx 1/stx 2) Shigella dysenteriae C. parvum/hominis Giardia lamblia 46 Samples per run Sample to Answer: ~3 Hrs Sample to Answer: 4. 5 Hrs Sample to Answer: <3 Hrs Hands-on-time: 30 mins Hands-on-time: 1. 75 Hrs Hands-on-time: 30 mins Low Complexity High Complexity Low Complexity £ 20. 82 per assay Plus external controls £ 75 plus £ 4 extraction Via easy. Mag 30 ~ £ 12. 50

PANEL Salmonella Shigella VTEC Camplyobacter Giardia Crytosproridium 50 negatives Positives collected over 1 year PANEL Salmonella Shigella VTEC Camplyobacter Giardia Crytosproridium 50 negatives Positives collected over 1 year period Approx 20 -30 of each 31

POSITIVES BDMAX SEROSEP 0 0 108 22 2 19 CULTURE/MICROSCOPY THIS EXCLUDES AND UNRESOLVED, POSITIVES BDMAX SEROSEP 0 0 108 22 2 19 CULTURE/MICROSCOPY THIS EXCLUDES AND UNRESOLVED, INDETERMINATE OR DUAL INFEC

SALMONELLA BDMAX SEROSEP 1 0 0 8 1 0 3 CULTURE/MICROSCOPY SALMONELLA BDMAX SEROSEP 1 0 0 8 1 0 3 CULTURE/MICROSCOPY

CAMPYLOBACTER BDMAX SEROSEP 3 0 0 36 11 4 10 CULTURE/MICROSCOPY CAMPYLOBACTER BDMAX SEROSEP 3 0 0 36 11 4 10 CULTURE/MICROSCOPY

CRYPTOSPORIDIUM BDMAX SEROSEP 0 0 0 9 2 2 1 CULTURE/MICROSCOPY CRYPTOSPORIDIUM BDMAX SEROSEP 0 0 0 9 2 2 1 CULTURE/MICROSCOPY

VTEC BDMAX SEROSEP 1 0 2 35 3 7 13 CULTURE/MICROSCOPY VTEC BDMAX SEROSEP 1 0 2 35 3 7 13 CULTURE/MICROSCOPY

Dual Infections ENT # C & M BDMAX SEROSEP COMMENT 10 Salmonell a POS Dual Infections ENT # C & M BDMAX SEROSEP COMMENT 10 Salmonell a POS Salmonella and VTEC POS. Looks genuine. 69 Salmonell a POS Salmonella POS This could be a possible contamination and Campy POS. on the Serosep plate as there a lot of campys on the run. 107 Campy POS Shigella POS This does look genuine but there is and Campy POS. another strong Shigella positive close in the run. 201 Salmonell a POS Salmonella POS and VTEC POS. 208 Campy POS Shigella POS I think this looks genuine for the and Campy POS. additional Shigella POS. 230 Campy POS Shigella and Campy POS. VTEC POS. NEGATIVE. The VTEC result looks genuine but is at a much lower level than the Salmonella POS. There are two strong Shigella positives either side of this sample so this could be likely contamination.

Hands on time STEP SEROSEP PROTOCOL TIME PER RUN Innoculate SPS 00: 20 Boil Hands on time STEP SEROSEP PROTOCOL TIME PER RUN Innoculate SPS 00: 20 Boil SPS 00: 30 Set up workstation 00: 03 Robotic dispensing 00: 40 Mix 00: 01 Centrifuge 00: 01 Real Time PCR 01: 22 Analysis 00: 10 HANDS ON TIME 00: 35 TIME TO RESULT 03: 07 38

Overall Xtag BDMax GPP Serso. Sep Inhouse Set-up time 01: 35 00: 55 03: Overall Xtag BDMax GPP Serso. Sep Inhouse Set-up time 01: 35 00: 55 03: 30 02: 30 PCR 01: 22 02: 42 04: 38 01: 40 Hands on time 00: 35 00: 30 03: 38 00: 50 Time to result 03: 32 04: 07 08: 09 05: 00 46 24 96 46 batch size 39

Next phase - Test in parallel one system for 2 months with culture methods Next phase - Test in parallel one system for 2 months with culture methods Report out in parallel in LIMS system Act on results Support from IPCN Public health Protocols developed 40

Summary In evaluations – need to address clinical problem Use of automation – it Summary In evaluations – need to address clinical problem Use of automation – it is only solution All systems look to perform satisfactory in retrospective testing Cost Making case within own resources are main reasons for chance of success Be imaginative !! 41

Acknowledgments Microbiology – molecular Microbiology Dr. Juliet Kenicer Dr. Lesley Allison and SERL Dr. Acknowledgments Microbiology – molecular Microbiology Dr. Juliet Kenicer Dr. Lesley Allison and SERL Dr. Mary Hanson Dr. Richard Othieno Laura Mc. Kenzie Julie White ELHF for providing funding for project NES for funding clinical science traineeship 42

Any Questions? Any Questions?