multicenter evaluation of the entericbio gi panel · medical college of wisconsin froedtert...

MULTICENTER EVALUATION OF THE

ENTERICBIO GI PANELBy: Matthew Faron

Assistant Professor of Pathology

Medical College of Wisconsin

Milwaukee, WI

MEDICAL COLLEGE OF WISCONSIN

Froedtert hospital

650 beds -> growing 750

Clinical Laboratory

Cary Blair – 4,000/yr

Raw stools – 17,000/yr

EcoFix – 2,100/yr

Diagnostic Research – Clinical trials >50 studies

EntericBio GI panel

Verigene 1 enteric pathogen test

Developmental/Outcome

AI – Image analysis

NGS- Ion Torrent database validation

Comparison studies

OBJECTIVES

Discuss GI infections and diagnostics: what we do at MCW

Measure ways to look at value for stool panels

Examine the 510K data for the EntericBio GI assay

GI INFECTIONS -COMMUNITY VS NOSOCOMIAL

Community

Campylobact

er

34%

Salmonella

40%

Shigella

13%

STEC O157

3%

STEC non-

O157

2%

Vibrio

1%

Yersinia

1%

Cryptosporidiu

m

6% Cyclospora

0%

CDC FoodNet 2004-2015

92% of non-viral infections

Differs on patient population

Pediatrics

Rotavirus

Norovirus

Adult

C. difficile

Norovirus

Nosocomial

Viral ~60-70% infections

(Noro, Adeno, Sapo, Rota)

CAUSES OF COMMUNITY ACQUIRED

ILLNESSSeverity: Bacteria, viruses, parasites

Salmonella, Campylobacter, Shigella, STEC common, most severe → High impact!

Bacteria Inc/100k % hosp. total

Salmonella spp. 15.8 27%

Campylobacter

spp.

12.9 17%

Shigella spp. 5.5 23%

STEC non-O157 1.6 16%

STEC O157 0.9 39%

Vibrio spp. 0.4 24%

Yersinia spp. 0.3 27%

Viruses Inc/100k % hosp. total

Norovirus 75-150 1.5%

Parasites Inc/100k % hosp. total

Cryptosporidium 3.3 17%

Giardia 1.5 3%

Cyclospora 0.1 6%

Data from CDC FoodNet

LABORATORY DIAGNOSES - CULTURE Labor intensive

Screen (Non-specific) → Selective/differential media

BAP, MAC, XLD, HE, Campy, SMAC, MAC broth enrichment

“False positives” → Citro, Proteus, Pseudomonas, Serratia, VRE

Confirm

API, Phoenix, Vitek2, RapID NF, MALDI-TOF

How are we doing?

TAT – 48-72h

Clinically actionable? Infection Control?

95% of specimens are negative for target pathogens

Why so poor?

Culture is only 50-75% sensitive

High LoD, Fastidious bugs, Preanalytical phase (preservation/transport)

Culture is narrow spectrum

Focus on only 4 pathogens (Campylobacter, STEC, Salmonella, and Shigella)

SENSITIVITY OF CULTURE VS NAAT

Anderson, N. et al. JCM. 2014

Limit of defection?

NAAT is 1-2 log10 more sensitive than culture

Culture ~105 CFU/mL vs. NAAT ~104-105 CFU/mL

NAAT not subject to culture limitations

Low concentration pathogens not “hidden” by flora

Viability not needed

MOLECULAR DIAGNOSTICS

Can a multiplex molecular test solve these problems?

Sensitivity, speed, cost, value

► Benefits?

► Improved sensitivity – 1-2 logs

► Rapid TAT – 1-3 hours or 6-24 hours if batched

► Simplify ordering for clinicians

► Test for multiple pathogens (bacterial, viral, protozoa) associated with similar symptomology

► Challenges?

► Higher Laboratory Cost

► Instrumentation + Consumables

► Interpretation of results

► Results not consistent with clinical condition, history

► Pre test probability

► Multiple potential pathogens present

► EPEC treatment?

COST/BENEFIT – CAN THE LAB/PATIENTS

AFFORD THIS? Cost to the patient/healthcare insurer

Culture CPT code(s)

87045 – Salmonella + Shigella: $12.97

87046 – Added plates (Campy, SMAC): $9.69 ea.

87427 – stx EIA: $16.49

87077 – Workup of FP (API20): $16.63

Total: $50.00-$65.00

– Molecular Dx code(s)

– 87505 – multiplex 3-5 targets → $174.58

– 87506 - multiplex 6-11 targets → $290.45


Downward pressure on reimbursement, decreases likely coming → value-based?

– Less expensive Molec Dx test is better insulated against reduced payment

http://burnsmcdmedia.com/careersblog/wp-content/uploads/2012/01/GirlSolvingMathProblem.jpg

CURRENT USA RECOMMENDATIONS

Can we optimize the benefits of Molecular Dx?

Selective use of broad panels

CA-diarrhea

No sign of severe/systemic

illness

Immuno-competent

Culture

CA-diarrhea

Persistent (>7 days)

Travel Hx

Signs of severe/systemic illness

immunocompromised

Broad NAAT Panel

HA-diarrhea

>3 days admit

Hx of Abx

Cdiff NAAT

CAP Today, Jan 2016

But still missing important pathogens in “mild” CA-enteritis due to low culture sensitivity.

This could be an area of value for small panel tests.

WHAT DO WE DO IN WISCONSIN?

While easier to offer a single, broad panel,

it may not be in the best interest of the

patient and healthcare $$$s. Through

selective utilization the lab can provide

both better care and real value to the patient, provider, and healthcare system.

COMMUNITY ACQUIRED BACTERIAL

INFECTIONS

Culture

Blood agar (BAP), MacConkey agar (MAC)

Xylose Lysine Deoxycholate agar (XLD) – Salmonella and Shigella

MacConkey agar with Sorbitol (sMAC) – STEC

Campylobacter agar (CAMPY)

Cefsulodin Irgasan Novobiocin agar (CIN) – Yersinia and Aeromonas

Hektoen Enteric agar (HE) – Salmonella and Shigella

Thiosulfate Citrate Bile Salts Sucrose agar – vibrio on request

STEC – Validated the EntericBio STEC target

Poor sensitivity of culture and EIA

High impact – 40% hospitalized

Removed enrichement – TAT is now <24 hours (batch once a day)

Dont want to treat

OTHER BACTERIAL

C. difficile

Unformed raw stool

Cannot be from previously patient that was positive 14 days

Or negative last 7 days

Molecular only – Xpert assay – colonization screen

Molecular, reflex antigen – QUIK CHEK

Differentiate colonization vs active infection

PARASITES

Ecofix specimens or raw stool

Raw stool – Microsporidia, Isospora or Cyclospora

Ova and Parasite examination

Direct wet prep

Formalin Ethyl Acetate concentration

Merifluor DFA

Cryptosporidium and Giardia

VIRAL

Norovirus

Molecular LDT

Detects GI and GII

Rotavirus

Antigen test – Immunocard STAT

VALUE TESTING

Cost to the patient/healthcare insurer

Culture CPT code(s)

87045 – Salmonella + Shigella: $12.97

87046 – Added plates (Campy, SMAC): $9.69 ea.

87427 – stx EIA: $16.49

87077 – Workup of FP (API20): $16.63

Total: $50.00-$65.00

– Molecular Dx code(s)

– 87505 – multiplex 3-5 targets → $174.58



How else can we find value for molecular dx?

Possible unnecessary workup

O&P = $15.00

Crypto/Giardia DFA = $15.00

Rotavirus = $16.49

C. difficile = $48.00

Norovirus = $48.00

If all tests were ordered:

$198.27

VALUE TECHNOLOGIST TIME

Culture

Technologist for processing

Technologist for interpretation of cultures

O&P – highly technical

15-17 minutes, 35-40 additional workup

Molecular

Processing technologist

Biofire, Verigene

Molecular technologist

EntericBio, BD Max

Batch testing

BD max measured at ~2 minutes tech time

Reflex culture when positive for AST when requested

Mortensen et al. BMC Clinical Pathology 2015

COST PER POSITIVE

4 months data (April-July)

926 Stools

716 (77.32%)

Neg., no workup

926 (100%)

STX EIA

$8,004.88$12,837.60

167 (22.68%)

Pos., 1 workup

$1,279.61

43 (4.64%)

Pos., Mult. workup

$710.52

210 cultures require work-up172/210 = 81.9% Pure Waste

$1629.91

2/926 (0.2%) Positive

$22,832.61 total

$24.65/stool

$600.84/positive result

Is this efficient?

38/926 (4.1%) positive

cultures

TAT → 48-72 h

COST ANALYSIS OF STEC ALONE

EIA STEC – Premier EHEC

Previous method

~$13.90/test

April-July 2018 – 926 stools – 2 positives (0.2%)

$6,418.80 per positive result

EntericBio STEC – LDT

New Assay

~$21.55/test

April-July 2019 -1225 stools – 10 STEC positive specimens (0.8%)

$2,639.88 per positive result

WHERE CAN THE SCALES TIP TOWARDS VALUE –

FOR US

Small targeted approach

Broad panels test for unnecessary targets

C. difficile – No reason to order unless hospitalized and on abx.

~20% positive – based on research studies using molecular testing

Norovirus – symptoms usually distinguished due to vomiting (outbreak)

Rotavirus/Adenovirus/Sapovirus – Treating symptoms

Have a separate children’s hospital

Low cost

Limited budgets – is stool more impactful than other specimen updates

Final cost to the patient/healthcare system

ENTERICBIO PANEL - TARGETS

9 targets

6 bacterial

3 Parasites

Internal controls in each well

Bacterial

Salmonella

Campylobacter

Shigella/EIEC

STEC

Yersinia enterocolitica

Vibro spp.

Parasites

Cryptosporidium

Giardia lamblia

Entamoeba histolytica

CAUSES OF COMMUNITY ACQUIRED

ILLNESSSeverity: Bacteria, viruses, parasites

In addition – Entamoeba target

Bacteria Inc/100k % hosp. total

Salmonella spp. 15.8 27%

Campylobacter

spp.

12.9 17%

Shigella spp. 5.5 23%

STEC non-O157 1.6 16%

STEC O157 0.9 39%

Vibrio spp. 0.4 24%

Yersinia spp. 0.3 27%

Viruses Inc/100k % hosp. total

Norovirus 75-150 1.5%

Parasites Inc/100k % hosp. total

Cryptosporidium 3.3 17%

Giardia 1.5 3%

Cyclospora 0.1 6%

Data from CDC FoodNet

EVALUATION OF THE ENTERICBIO ASSAY

Data to support a 510K FDA submission

3 Geographical sites

Laboratory Alliance of Central New York

Regional Virus Laboratory, Royal Victoria Hospital- Belfast

The Medical College of Wisconsin

Fresh, frozen, and contrived testing

Cary-Blair stool specimens

Compared to FilmArray GI panel (Biofire Diagnostics, Salt Lake UT)

Discrepant testing

XTAG GI panel (Luminex, Austin TX)

Verigene GI panel (Luminex, Austin TX)

BD MAX enteric panel (BD Diagnostics, Sparks MD)

Compare results

EVALUATION – FRESH SPECIMENS

PPA = Positive Percent Agreement

NPA = Negative Percent Agreement

Organism TP TN FP FN TotalPPA%

(95% CI)

NPA%

(95% CI)

Salmonella 33 1466 0 3 1502 91.7 (78-97) 100 (99-100)

Campylobacter 62 1431 0 9 1502 87.3 (78-93) 100 (99-100)

Shigella/EIEC 17 1485 0 0 1502 100 (82-100) 100 (99-100)

STEC 11 1485 2 4 1502 73.3 (48-89) 99.9 (99-100)

Y. enterocolitica 5 1494 0 3 1502 62.5 (31-86) 100 (99-100)

Vibrio 0 1499 0 3 1502 0 (0-56) 100 (99-100)

Parasites

Cryptosporidium 10 1485 1 6 1502 62.5 (39-82) 100 (99-100)

Giardia lamblia 15 1485 1 2 1502 88.2 (66-97) 99.9 (99-100)

E. histolytica 0 1502 0 0 1502 N/A 100 (99-100)

EVALUATION – FROZEN SPECIMENS

Frozen STEC and Crypto was significantly different between fresh and frozen 73 vs 94 (confidence intervals)

Low number of fresh positives

Frozen specimens were selected based on culture – positive specimens at high concentrations

Biofire did release some user notices issuing possible increased FP rates for Campy, Crypto, Yersinia and Vibro


(95% CI)

NPA%

(95% CI)

Salmonella 12 195 0 2 209 85.7 (60-98) 100 (98-100)

Campylobacter 15 193 0 1 209 93.8 (72-99) 100 (98-100)

Shigella/EIEC 10 198 0 1 209 90.9 (62-98) 100 (98-100)

STEC 70 135 0 4 209 94.6 (87-98) 100 (98-100)


Vibrio 0 209 0 0 209 N/A 100 (98-100)

Parasites

Cryptosporidium 75 133 0 1 209 98.7 (93-100) 100 (98-100)

Giardia lamblia 29 180 0 0 209 100 (88-100) 100 (98-100)

E. histolytica 0 207 0 2 209 0- (0-66) 100 (98-100)

EVALUATION – COMBINED

Still had low numbers for Yersinia, Vibrio, and E. histolytica


(95% CI)

NPA%

(95% CI)

Salmonella 45 1661 0 5 1711 90.0 (77-96) 100 (99-100)

Campylobacter 77 1624 0 10 1711 88.5 (79-94) 100 (99-100)

Shigella/EIEC 27 1683 0 1 1711 96.4 (80-100) 100 (99-100)

STEC 81 1620 2 8 1711 91.0 (82-96) 99.9 (99-100)


Vibrio 0 1708 0 3 1711 0 (0-69) 100 (99-100)

Parasites

Cryptosporidium 85 1618 1 7 1711 92.4 (84-97) 99.9 (99-100)

Giardia lamblia 44 1664 1 2 1711 95.6 (84-99) 99.9 (99-100)

E. histolytica 0 1709 0 2 1711 0 (0-80) 100 (99-100)

CONTRIVED TESTING OF RARE TARGETS

Yersinia sensitivity goes to 92.2% with contrived testing

Vibrio sensitivity goes to 94.3% with contrived testing

Entamoeba sensitivity goes to 94.3% with contrived testing

The 1 FP was likely caused by high background setting in FastFinder

software

Organism TP TN FP FN PPA NPA

Yersinia 50 147 1 0 100 99.3

Vibrio 50 148 0 0 100 100

E. histolytica 49 148 0 1 98 100

DISCORDANT RESOLUTION

Bacterial targets were tested with Verigene GI panel (Luminex, Austin TX)

Parasites were tested using xTAG GI panel (Luminex, Austin TX)

FP results were not tested

a Some specimens were not available for testing

Organism FN Consensus agrees with EB Consensus agrees with BF

Salmonella 5 3 2

Campylobacter 10 9 1

Shigella/EIEC 1 1 0

STEC 8 8 0

Yersinia 5a 2 1

Vibrio 3 3 0

Crypto 7 4 3

Giardia 2 2 0

E. histolytica 2a 0 1

Total 43 32 8

DATA SUMMARY

Highly specific (NPA) assay with all targets ranging from 99-100%

Sensitivity ranged between targets

>90% for Shigella, Salmonella, STEC, Giardia, and Cryptococcus

All targets were >90% agreement with discrepant and contrived testing

Organism TP TN FP FN PPA NPA

Total Fresh 153 13332 4 30 83.6 (77-88) 99.9 (99-100)

Total Frozen 215 1653 0 13 94.3 (90-67) 100 (99-100)

Total 368 14985 4 43 89.5 (86-92) 99.9 (99-100)

Post Discrepant

and Contrived516 15164 4 13 97.5 (96-99) 99.9 (99-100)

CHALLENGES AND LIMITATIONS -COMPARISONS

Molecular vs culture

Gold standard – positive when you have an organism

Culture is less sensitive

Reconciling FP

Viruses and Parasites?

Molecular vs Molecular

Not a true gold standard and is a developed assay

No assay is perfect

During testing Biofire came out with a recall for elevated FP rates of Campylobacter and Cryptosporidium (None of our testing lot numbers)

Low FP rate of Yersinia and Vibrio due to production of media and non-viable organisms.

Extractions

Chemistry – primers and probes, amplification conditions.

COMPARING PLATFORMS

BD MAX EBP EntericBio Verigene EP xTAG GPP FilmArray GI

Targets 4 9 9 11 22

Automation Sample to Result

Heat extraction,

Automated pipettor

Sample to Result

Off-line Extraction,

Manual PCR setup

Sample to result

Technology RT-PCR RT-PCR PCR+Array PCR+xTAG Nested PCR

Throughput Batch, up to 24 Batch, up to 32 1 sample/run

Batch, limited by extractor

capacity1 sample/run

TAT 1.5 h 2.5 h 2.5 h 4 h 1 h

PANEL COMPARISONS

Bacterial/toxins

Salmonella

Campylobacter

Shigella/EIEC

STEC


Vibro spp.

Parasites

Cryptosporidium

Giardia lamblia


EntericBio Verigene/BD MAX*,extended+

Biofire

Bacterial/toxins

Salmonella*

Campylobacter*

Shigella/EIEC*

STEC*

Yersinia enterocolitica+

Vibro spp.+

ETEC+ only

P. shigelloides+only

Viruses

Norovirus

Rotavirus

Bacterial/toxins

Salmonella

Campylobacter

Shigella/EIEC

STEC


Vibro spp. (differentiates cholerae)

EAEC,EPEC,ETEC

E. coli 0157

Plesiomonas shigelloides

Parasites

Cryptosporidium

Giardia lamblia


Cyclospora cayetanensis

Viruses

Norovirus GI/GII

Rotavirus A

Adenovirus F40/41

Astrovirus

Saprovirus (I, II, IV, and V)

QUESTIONS?

Thank you collaborators

- LANCY

- Royal Victoria

- Serosep

- MDC associates

• New software for the Serosep EntericBio - Fast Finder

• Upload run files into the software

• Confirm template for control wells and specimens

• Report file of the results will populate

• Easy view of controls: QC and internal controls

• Color coded for easy review

• Can export amplification curves along with other auditing information

ENTERICBIO PANEL - TESTING

Set up for Cary-Blair specimens

Transfer with a flocked swab into buffer

Heat at 102 C for 30 minutes

Add tubes, strips, and QC to workstation

Execute run

Seal strips

Quick spin

Load 7500 dx

Run results through assay software – Post-trial addition

VALUE – IMPROVED TURNAROUND TIME

Rapid rule out for GI pathogens

Highly sensitive, High NPV for on-panel targets

Positive stools may not require further work-up

Negative result → focus further workup (Noro, TCBS, OX+, O&P)

Antibiotic stewardship

Hold empiric therapy?

Salmonella, STEC, Vibrio, Noro → No Abx

Shigella, Campylobacter, protozoa → AST/treat

Infection control

Identify outbreak or potential outbreak 48-72 h sooner! → Contain!!

Family members, school/daycare, LTC → Shigella, Norovirus, source STEC

HOWEVER! – THINGS TO CONSIDER

Complex interpretation

Carriage vs. causation

Most common targets detected by “broad panels” are C. diff and EPEC/EAEC

Any real utility in outpatient population? If not, better left unsaid?

Multiple potential pathogens in 15-30% of diarrheal stools

Antibiotic stewardship

Potential for over-prescription

EPEC/EAEC → Cipro? Bactrim? Any guarantee these work w/o AST?

Multiple pathogens → unnecessary therapy for “colonizer”, overly broad spectrum to cover all?

Viruses & C. difficile

Large proportion of positive are Noro/Sapo → important

Could this be done more cost effectively with cheaper LDT or single target test?

Noro (and Cdiff) need to remain a single analyte orderable - specific risk factors/population

multicenter evaluation of the entericbio gi panel · medical college of wisconsin froedtert...

Documents