Comparative Diagnostic Accuracy Study of Three Multiplex Molecular Assays for Bacterial Gastroenteritis

Comparative diagnostic accuracy study of three multiplex molecular assays for bacterial gastroenteritis

Simon Goldenberg Consultant Microbiologist and Infection Control Doctor Guy’s & St Thomas’ NHS Foundation Trust Honorary Senior Lecturer King’s College London 1000 Top 10 most frequent reasons for emergency 900

admission, Guy’s & St Thomas’ Hospital, 800 2014/15 700

600

500

emergency admissions emergency 400

300

200 Number Number of 100

Primary diagnosis code Clinical features are not helpful for predicting the etiology of diarrhoea

• Duration of diarrhoea • Abdominal pain • Vomiting • Blood stools • Number of bowel movements • Fever • Requirement for IV hydration

This emphasizes the need for more rapid, sensitive, and simple methods to improve the diagnostic yield

Chan et al. Hong Kong J Emerg Med. 2003;10:162-168 Svenungusson et al. Clin Infect Dis. 2000 30 (5): 770-778 Munk Petersen et al. Scand J Gastroenterol 1996;31:906-11 Svanteson et al. Scand J Infect Dis 1988;20:303-14 aripiprazole, acarbose, acamprosate, acebutolol, aceclofenac, acemetacin, acetazolamide, acyclovir, acitretin, activated charcoal, agalsidase beta, alendronate, alfuzosin, allopurinol, almotriptan, amphotericin, amiloride, aminophylline, amitriptyline, amlodipine, amoxicillin, anastrozole, anidulafungin, allantoin, aspirin, atenolol, atorvastatin, aztreonam, azathioprine, azithromycin, barium sulphate, brinzolamide, baclofen, bendroflumethiazide, belimumab, betaxolol, bezafibrate, bisoprolol, bleomycin, bisacodyl, bosutinib, bromocriptine, buprenorphine, bumetanide, busulfan, buspirone, calcium carbonate, calcium folinate, calcium glubionate, calcium lactobionate, caffeine, colestilan, caspofungin, capecitabine, captopril, carglumic acid, carbamazepine, carboplatin, cetirizine, carvedilol, cefadroxil, cephalexin, cefotaxime, cefradine, ceftazidime, ceftriaxone, cefuroxime, celecoxib, celiprolol, chlorambucil, chlorphenamine, colesevelam, ciprofloxacin, cimetidine, citalopram, cisplatin, clarithromycin, clindamycin, clofarabine, clopidogrel, clozapine, codeine phosphate, colchicine, colestipol, colistimethate, corifollitropin alfa, crisantaspase, cytarabine, dacarbazine, daptomycin, darifenacin, decitabine, dantrolene, daunorubicin, deferasirox, deferiprone, desferrioxamine, desloratadine, dihydrocodeine, diazepam, diclofenac, digoxin, diltiazem, docusate sodium, dipyridamole, disodium pamidronate, docetaxel, domperidone, donepezil, dorzolamide hydrochloride, doxazosin, doxorubicin, doxycycline, ethosuximide, esomeprazole, emtricitabine, enalapril, etanercept, entacapone, epirubicin, epoprostenol, etoposide, epoetin alfa, erdosteine, erythromycin, estradiol, ethambutol, ethosuximide, etodolac, ethinylestradiol, exemestane, fentanyl, fenofibrate, ferrous sulphate, ferrous fumarate, flecainide

Electronic Medicines Compendium www.medicines.org.uk aripiprazole, acarbose, acamprosate, acebutolol, aceclofenac, acemetacin, acetazolamide, acyclovir, acitretin, activated charcoal, agalsidase beta, alendronate, alfuzosin, allopurinol, almotriptan, amphotericin, amiloride, aminophylline, amitriptyline, amlodipine, amoxicillin, anastrozole, anidulafungin, allantoin, aspirin, atenolol, atorvastatin, aztreonam, azathioprine, azithromycin, barium sulphate, brinzolamide, baclofen, bendroflumethiazide, belimumab, betaxolol, bezafibrate, bisoprolol, bleomycin, bisacodyl, bosutinib, bromocriptine, buprenorphine, bumetanide, busulfan, buspirone, calcium carbonate, calcium folinate, calcium glubionate, calcium lactobionate, caffeine, colestilan, caspofungin3141, capecitabine drug, captopril, preparationscarglumic acid, carbamazepine, carboplatin, cetirizine, carvedilol, cefadroxil, cephalexin, cefotaxime, cefradine, ceftazidime, ceftriaxone, cefuroxime, celecoxib, celiprolol, chlorambucil, chlorphenamine, colesevelam, ciprofloxacin, cimetidine, citalopram, cisplatin, clarithromycin, clindamycin,with clofarabine, clopidogreldiarrhoea, clozapine, codeine asphosphate, a colchicine, colestipol, colistimethate, corifollitropin alfa, crisantaspase, cytarabine, dacarbazine, daptomycin, darifenacin, decitabine, dantrolene, daunorubicin, deferasirox, deferiprone, desferrioxamine, desloratadine, dihydrocodeine, diazepam,known diclofenac, digoxin, unwanted diltiazem, docusate sodium, effect dipyridamole , disodium pamidronate, docetaxel, domperidone, donepezil, dorzolamide hydrochloride, doxazosin, doxorubicin, doxycycline, ethosuximide, esomeprazole, emtricitabine, enalapril, etanercept, entacapone, epirubicin, epoprostenol, etoposide, epoetin alfa, erdosteine, erythromycin, estradiol, ethambutol, ethosuximide, etodolac, ethinylestradiol, exemestane, fentanyl, fenofibrate, ferrous sulphate, ferrous fumarate, flecainide

Electronic Medicines Compendium www.medicines.org.uk Managing diarrhoea in hospital is time consuming and inefficient

• 20% of infection control team time is taken is spent on management of diarrhoea • Almost all patients (99%) with suspected infectious diarrhoea are isolated in a side room • Mean number of wards closed annually due to infectious diarrhoea = 12 per hospital • 25% of the time causative pathogens are identified after patient discharge

Buchanan et al J Hosp Infect 2015 doi: 10.1016/j.hin.2014.12.021. ID 1 CCDA1 24h Antimicrobial susceptibility testing 2-10h3 CCDA 18-24h 2

6 4 Biochemical ID and agglutination tests Selenite-F 18- ABC5 18-24h ID 24h 2 Antimicrobial susceptibility testing 2-10h3 3.7h

ID Biochemical ID and agglutination tests CT-SMAC7 18-24h 2 Antimicrobial susceptibility testing 2-10h

ID Biochemical ID and agglutination tests XLD8 18-24h 2 Antimicrobial susceptibility testing 2-10h

Gram stain, basic Selective culture Species level identification using Antimicrobial Sample biochemistry transportation +/- enrichment MALDI-TOF MS and biochemical susceptibility testing (Oxidase etc) identification (API 20E)

Total time = 66.5 hours, range - 64 hours (negative) to 118 hours (positive)

Conventional culture based detection of bacterial enteric pathogens Poor sensitivity of conventional and culture based tests compared with molecular assays

Study Method n % positive % positive conventional molecular Amar. Eur J Clin Microbiol Infect Dis. In house 2205 19 42 2007 de Boer. J Clin Microbiol. 2010 In house 13,974 6.4 19.2

Wessels. Clin Microbiol Infect. 2014 Luminex xTAG GPP 393 11.2 22.1

Mengelle. Clin Microbiol Infect. 2013 Luminex xTAG GPP 440 32.7 40

McAuliffe. J Infect. 2013 Fast Track Diagnostics 530 18 30

Onori. Diagn Microbiol Infect Dis. SeeGene Seeplex ACE 245 66.5 76.3 2014 Cunningham. J Clin Microbiol. 2010 In house 392 26.5 96.7

Biswas. Eur J Clin Microbiol Infect BD Max Enteric Panel 434 86.1 94.4 Dis. 2014 Problems with current culture dependent diagnostic methods:

• Inability to differentiate clinically between different cause of IG • Fragmented test request procedure • Inefficient laboratory workflow • Laborious workflow – slow, long TAT • Poor sensitivity – lack of clinical confidence Enterics panel evaluation:

• RBioPharm RIDA Gene Bacterial Stool Panel and EHEC/EPEC Manual two-tube real time PCR on ABI7500 Separate automated extraction using Promega Maxwell instrument (magnetic beads) Salmonella, Camylobacter, Y. enterocolitica*, Shigella, E. coli

• Fast Track Diagnostics Bacterial Gastroenteritis panel Manual two-tube real time PCR on ABI7500 Separate automated extraction using same extract as above Salmonella, Campylobacter, Y. enterocolitica*, Shigella, E. coli, C. difficile*

• BD Max Gastroenteritis panel Fully automated reat time PCR with built in extraction Salmonella, Campylobacter, Shigella, E. coli

• Conventional selective culture methods: CCDA – Campylobacter, XLD – Salmonella/Shigella CT-SMAC EC0157 Selenite F broth & ABC Harlequin – Salmonella

*C. difficile and Y. enterocolitia not assessed in this evaluation

Enterics panel evaluation:

• 434 non-duplicate samples tested by all 4 methods:

• 116 retrospective samples selected on the basis of culture positivity • Remaining 318 consecutive samples tested semi- prospectively

Gold standard / True positive:

• Either culture positive OR • 2/3 positive molecular tests Results 18

2 Campylobacter

0 Shigella

Culture Salmonella Fast Track Diagnostics RIDA-Gene BD Max Relative performance characteristics of molecular panels and conventional culture combined samples (n=434)

RIDA®GENE Bacterial Stool FTD® Bacterial BD MAX™ Enteric Bacterial Conventional culture Panel and EHEC/EPEC Gastroenteritis Panel Panel Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity Sensitivity Specificity (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI) Campylobacter 91.1 98.8 80.2 99.4 92.1 100 91.1 100 (83.8 – 95.8) (97 – 99.7) (71.1 – 87.5) (97.8 – 99.9) (85 – 96.5) (98.9 – 100) (83.8 – 95.8) (98.9 – 100)

Shigella 94.4 99.7 88.9 100 94.4 100 86.1 100 (81.3 – 100) (98.6 – 100) (73.9 – 96.9) (99.1 – 100) (81.3 – 99.3) (99.1 – 100) (70.5 – 95.3) (99.1 – 100)

Salmonella 55 99 45 99.8 75 100 85 100 (31.5 – 76.9) (97.5 – 99.7) (23.1 – 68.5) (98.7 – 100) (50.9 – 91.3) (99.1 – 100) (62.1 – 96.8) (98.9 – 100)

Shiga-toxin 0 99.5 100 99.8 100 99.5 100 100 producing E. (0 – 97.5) (98.3 – 99.9) (25 – 100) (98.7 – 100) 25 – 100) (98.3 – 99.9) (25 – 100) (99.2 – 100) coli RIDA®GENE bacterial Fast Track Diagnostics BD Max™ Enteric Conventional culture stool panel and bacterial bacterial panel Relative performance EHEC/EPEC gastroenteritis panel characteristics of Campylobacter, prevalence = 5.3% molecular panels and Sensitivity 100 88.2 100 52.9 conventional culture for (95% CI) (80.5 – 100) (63.6 – 98.5) (80.5 – 100) (27.8 – 77) Specificity 99.7 100 100 100 the prospective sample (95% CI) (98.2 – 100) (98.8 – 100) (98.8 – 100) (98.8 – 100) only (n=318). PPV 94.4 100 100 100 (95% CI) (72.7 – 99.9) (78.2 – 100) (80.5 – 100) (66.4 – 100) NPV 100 99.3 100 97.4 (95% CI) (98.8 – 100) (97.6 – 99.9) (98.8 – 100) (95 – 98.9) Shigella, prevalence = 4.4% Sensitivity 85.7 92.9 100 71.4 (95% CI) 57.2 – 98.2) (66.1 – 99.8) (76.8 – 100) (41.9 – 91.6) Specificity 100 100 100 100 (95% CI) (98.8 – 100) (98.8 – 100) (98.8 – 100) (98.8 – 100) PPV 100 100 100 100 (95% CI) (73.5 – 100) (75.3 – 100) (76.8 – 100) (69.2 – 100) NPV 99.3 99.7 100 98.7 (95% CI) (97.7 – 99.9) (98.2 – 100) (98.8 – 100) (96.7 – 99.6) Salmonella, prevalence = 1.3% Sensitivity 25 50 100 100 (95% CI) (0.6 – 80.6) (6.8 – 93.2) (39.8 – 100) (39.8 – 100) Specificity 99.7 99.7 99.7 100 (95% CI) (98.2 – 100) (98.2 – 100) (98.2 – 100) (98.8 – 100) PPV 50 66.7 80 100 (95% CI) (13 – 99.7) (9.4 – 99.8) (28.4 – 99.5) (39.8 – 100) NPV 99.1 99.4 100 100 (95% CI) (97.3 – 99.8) (97.7 – 99.9) (98.8 – 100) (98.8 – 100) RIDA®Gene Bacterial and FTD® Bacterial BD MAX™ Enteric Bacterial EHEC/EPEC panels Gastroenteritis panel Panel 16s-rDNA (Campylobacter) glyA and mapA (Campylobacter tuf (Campylobacter) Targets detected ipaH (Shigella, Enteroinvasive E. jejuni and Campylobacter coli) ipaH (Shigella, Enteroinvasive E. coli) ipaH (Shigella) coli) eae (EHEC/EPEC) ttr (Salmonella) spaO (Salmonella) ttr (Salmonella) vtx 1A, 1B, 2A and 2B stx 1a and stx 2a (Shiga toxin stx 1 and stx 2 (Shiga toxin (Enterohaemorrhagic E. coli) producing E. coli and Shigella producing E. coli and Shigella ail (Yersinia enterocolitica) dysenteriae) dysenteriae) tcdB (Clostridium difficile) ail (Yersinia enterocolitica) Total hands on time 50 mins (including separate 50 mins (including separate 20 mins (based on 24 samples) extraction) extraction) Total laboratory turnaround 176 mins 171 mins 163 mins time (based on 24 samples) 40 mins Maxwell, 86 mins PCR 40 mins Maxwell, 81 mins PCR Minimum run size 1 (plus 2 controls) 4, 6 or 12 depending on kit 1 (controls built in) (plus 2 controls) Maximum run size 47 (plus 2 controls)1 47 (plus 2 controls)1 24 (controls built in)

Fully automated? No No Yes

Reagent storage -20°C -20°C Room temperature

Result interpretation Requires interpretation of PCR Requires interpretation of PCR No interpretation required curves curves (detected/not detected) Consumables cost per sample2 £22.42 £23.48 £22.50

1 Based on testing with ABI 7500 real-time PCR thermocycler 2 Includes all additional consumables (including extraction) based on a reagent rental contract for 3000 samples per year over x years, excludes tax. Cost for RIDA® Gene and FTD® panels does not include cost of purchase / rental or maintenance of ABI7500 instrument. Most positives from community patients 28 / 36 (78%) (prospective) 88 / 116 (76%) (retrospective)

33 samples from hospitalised patients: all except one sample sent within D3 of admission

One sample had Campylobacter and was submitted on D4

Despite advising clinicians of the limited usefulness ~25% of requests for stool culture are from patients with onset of diarrhoea within 3 days of admission Testing strategy at GSTT:

Continue current testing methods for Norovirus (in house PCR) and C. difficile (GDH plus Toxin EIA/PCR)

Adopt BD Max Bacterial Stool Panel, culture positive Salmonella, Shigella and E. coli O:157 for AST and typing.

Campylobacter positives will not be routinely cultured (recommend treatment with macrolide)

Demand management savings of ~25% partially offsets the additional cost of PCR

Culture: £19.18 x 8300 = £159,194 BD Max with demand management: £30 x 6800 = £204,000

Vote Now What proportion of your hospitals beds are in single rooms?

1. < 10% 3.2% 2. 10- 19% 19.4% 3. 20 - 29% 29.0% 4. 30 - 39% 3.2% 5. 40 - 49% 0.0% 6. 50 - 59% 0.0% 7. > 60% 0.0% 8. Don't know 45.2%

Vote Trigger Vote Now What proportion of patients with clinically significant diarrhoea are presumptively isolated?

1. > 20% 6.1% 2. 21 - 40% 3.0% 3. 41 - 60% 0.0% 4. 61 - 80% 21.2% 5. > 80 % 24.2% 6. Don't know 45.5%

Vote Trigger Vote Now What method(s) does your laboratory use for the detection of Campylobacter, Salmonella, Shigella and E. coli O:157?

1. Culture with identification based on biochemical methods 29% 2. Culture with identification based on MALDI-TOF MS 15% 3. Molecular panel 29% 4. Combination of 1 and 2 6% 5. Combination of 1, 2 and 3 21% 6. Other 0%

Vote Trigger Vote Now What is the average turnaround time for detection (and AST if done) of Campylobacter, Salmonella, Shigella and E. coli O:157?

1. < 10 hours 8.8% 2. 11 - 24 hours 17.6% 3. 25 - 48 hours 20.6% 4. 49 - 72 hours 44.1% 5. > 72 hours 8.8% 6. Don't know 0.0%

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