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. EBBP3A, EBSPSA-V2

240

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Serosep Ltd., Annacotty Business Park, Annacotty, Limerick, Ireland. www.serosep.com

Technical support Email: entericbiosupport@serosep.com Tel: +353 61 358190

Bacterial Panel 3 User Manual

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Intended Use

For in vitro diagnostic use. The EntericBio realtime® Bacterial Panel 3 assay is a molecular diagnostic test for the direct

qualitative detection of Salmonella enterica spp., Shigella spp., Campylobacter jejuni/coli/lari, Yersinia enterocolitica

spp., Vibrio cholerae/parahaemolyticus and, Shiga-like toxin-producing E. coli (STEC, also known as Verotoxin-

producing E. coli, VTEC) stx1/stx2 in human stool samples. The assay is intended for use with stool samples from

symptomatic individuals to aid in the diagnosis of bacterial gastroenteritis.

Summary and Explanation

Infectious gastroenteritis is a significant cause of morbidity and mortality worldwide. The primary causes of bacterial

gastroenteritis include Salmonella enterica spp., Shigella spp. and shiga toxin-producing Escherichia coli and

Campylobacter jejuni.

The genus Salmonella comprises two species, namely S. enterica and S. bongori, with more than 2,500 serovars,

differentiated on the basis of their somatic (O) and flagellar (H) antigens by the White-Kauffmann-Le Minor Scheme1.

Salmonella isolates from humans are serotypes of Salmonella enterica. Gastroenteritis is the most common condition

caused by Salmonella species. Symptoms include abdominal pain, diarrhea, nausea and vomiting, often accompanied

by fever2.

The genus Shigella consists of four species; S. sonnei (1 serotype), S. flexneri (14 serotypes), S. boydi (18 serotypes)

and S. dysenteriae (13 serotypes), all of which are characterized serologically on the basis of the O antigen only, as

Shigella spp. lack the H antigen3. Infection with Shigella spp. manifests as a range of symptoms from watery diarrhea to

dysentery with frequent small volume feces containing blood, mucus and pus. The diarrhea may be accompanied by

fever and abdominal cramps4.

Strains of E. coli that produce the toxins Stx1 and Stx2 are termed Shiga-like toxin-producing E. coli (STEC, also known

as Verotoxin-producing E. coli, VTEC). The toxins are termed “shiga-like” due to their similarity to the toxin of Shigella

dysenteriae5. Infections vary in severity, from mild to bloody diarrhea, and may occur in any age group, although it is

more common in children. STEC are capable of causing two types of disease, namely hemorrhagic colitis6 and

hemolytic uraemic syndrome (HUS)7. HUS is a life-threatening disease characterized by thrombocytopenia, hemolytic

anemia, and acute renal dysfunction. Serogroup O157 is the most common cause of these illnesses, but at least 150

non-O157 STEC serotypes have been reported as agents of both sporadic and outbreak-associated disease8-10.

There are 11 species in the genus Yersinia, however only three of them are regarded as human pathogens: Y. pestis, Y.

enterocolitica and Y. pseudotuberculosis. Yersinia enterocolitica survives at low temperatures and can grow well at

refrigeration temperatures which contributes to its potential as foodborne pathogen11. Pigs are recognized as a source

of Y. enterocolitica strains and contaminated or undercooked pork is considered the main vector for infection with Y.

enterocolitica12, 13. However, this species has also been detected in other animal products12.

Yersinia enterocolitica species are divided into biotypes, depending on their genomic characteristics. Within the 6

biotypes currently recognized, only biotypes 1B and 2-5 are considered pathogenic to humans14. Main symptoms of

yersiniosis include abdominal pain with fever and diarrhea and often mimic other diseases such as appendicitis or

Crohn’s disease, therefore accurate and fast detection of Y. enterocolitica infection is important for patient management

decisions and avoidance of unnecessary surgical procedures.

Members of the genus Vibrio are Gram-negative, straight or curved, motile rods.

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Several Vibrio spp. are considered human pathogens and have been implicated in gastroenteritis. This includes species

such as V. cholerae (toxigenic and non-toxigenic serogroups), V. parahaemolyticus15. Most cases of Vibrio-caused

gastroenteritis can be linked to consumption of raw or undercooked seafood16.

The genus Campylobacter contains 25 species, however, Campylobacter jejuni accounts for about 90% of reported

infections and most of the remainder are caused by Campylobacter coli and lari17, 18. In human hosts diarrhea is usually

brief and sequelae are uncommon. Initial symptoms may be severe with fever and abdominal pain suggesting

appendicitis. Rarely, Campylobacter species infection may become invasive, with consequences ranging from transient,

self-limiting bacteremia, to fulminant Gram negative sepsis. Occasionally infection may produce sequelae such as

reactive arthritis, bursitis, endocarditis and neonatal sepsis. Acute post-infective demyelination may develop, affecting

the peripheral nervous system (Guillain-Barré Syndrome), and/or the central nervous system and cranial nerves (e.g.

the Miller-Fisher Syndrome)19.

Salmonella, Shigella, Yersinia enterocolitica and shiga toxin-producing E. coli are members of the family

Enterobacteriaceae. These organsims are Gram negative, oxidase negative and grow well aerobically at 37⁰C.

Campylobacters are curved, S-shaped or spiral Gram negative rods. They are motile, microaerophilic (optimum 5-10%

oxygen) and oxidase positive. Campylobacter species do not ferment or oxidize carbohydrates. A well-recognized

problem associated with identification of Campylobacter species is the unreliability of biochemical tests for this

purpose20. Several studies have shown that molecular-based detection methods result in increased detection of

Campylobacter spp. when compared to culture18, 21.

Traditional laboratory methods used to detect and identify these pathogen groups combine selective culture broths

and/or selective culture plates with a range of biochemical tests and serology. This approach is relatively labor intensive

with a typical time to result of between 48 and 96 hours. In addition, routine laboratory detection of STEC is generally

limited to non-sorbitol fermenting E.coli belonging to the O157 serogroup21, 22.

Test Principle The EntericBio realtime® Bacterial Panel 3 assay is a molecular diagnostic test for the simultaneous detection of

Salmonella enterica spp., Shigella spp., Campylobacter jejuni/coli/lari, Yersinia enterocolitica, Vibrio cholerae/

parahaemolyticus and Shiga-like toxin-producing E. coli (STEC) stx1/stx2 from human stool samples. The assay works

directly from the stool sample and does not require nucleic acid extraction/purification. The entire mastermix required to

perform each test is lyophilised into individual reaction wells. Each reaction well contains an Internal Amplification

Control (IAC) to monitor for PCR inhibition. A positive amplification control is also provided with each kit.

Briefly, a swab is coated with the stool sample, the swab is re-suspended in a tube of EntericBio Stool Preparation

Solution (SPS) and then placed on the EntericBio heatstation and heated at 103°C for 30 min. The heat-treated samples

are placed on the EntericBio workstation for fully automated transfer of the processed samples directly to the lyophilised

reaction wells using the EntericBio Gastro Panel programme. The wells are capped and transferred to the real-time

PCR instrument for automated amplification, detection and analysis with the EntericBio Bacterial Panel 3 programme.

An analytical run of 48 samples has a turnaround time to result of less than 3 hours with typically 30 minutes hands-on

time.

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Reagents Provided

EntericBio realtime® Bacterial Panel 3 (EBBP3A) 240 tests

o Assay A+: Salmonella/Shigella/Campylobacter (x5 pouches/48 tests) o Assay E: Yersinia/ Vibrio/ stx1/stx2 (x5 pouches/48 tests) o Reconstitution solution (500ul/green cap) o Positive control (red cap) o Re-suspension Buffer (50ml/green cap)

EntericBio realtime® SPS Kit (EBSPSA-V2) 240 tubes

o Stool Preparation Solution (240 x 4 ml/white pierceable cap)

Storage

The EntericBio realtime® Bacterial Panel 3 assay PCR kit should be stored 2-8oC and EntericBio realtime® SPS kit

should be stored at 2-25oC on receipt in the laboratory. Opened pouches should be used within 7 days.

Additional Equipment and Material Requirements

o Roche LightCycler 480 II (real-time PCR instrument option 1) (Product code: 05015278001) o ABI 7500 FAST Cycler (real-time PCR instrument option 2) (Product code: 4357362) o EntericBio workstation (Product code: 5070000450) o Eppendorf 5070 50µl filter tips (Product code: 0030014430) o Eppendorf 5430 Plate centrifuge (Product code: 5427000666) o Eppendorf MixMate (Product code: 5353000030) o EntericBio heatstation with block inserts for EntericBio racks (Product code: EBQBD4 SEROSEP) o Bioplastics LC480 plate adaptor (Product code: B79482-2) o EntericBio FLOQSwabs (Product code: 2E014N10.SER)

Warnings and Precautions

o The EntericBio Bacterial Panel 3 assay is for in vitro diagnostic use only.

o The EntericBio Bacterial Panel 3 assay should only be performed by suitably trained laboratory personnel.

o All clinical specimens and disposables/reagents in contact with clinical specimens should be treated as

potentially infectious and should be handled according to the relevant local/national safety guidelines and

regulations.

o Good laboratory practice for working with nucleic acid amplification technologies should be followed at all times.

o Strictly follow the test kit instructions and do not use the kit after the expiration date.

o The EntericBio PCR strips contain lyophilised PCR reagents in the form of a pellet inside each well. Ensure that

the lyophilised pellets are at the bottom of each well prior to use. If necessary, gently tap the PCR strips until

the pellets are on the bottom of the wells.

o Securely re-seal the PCR pouches promptly after use (with the desiccant inside) and remove any excess air

prior to sealing.

o The PCR strips and caps may be cut. It is extremely important to cut the strips/cap cleanly along the guide line

indent on the cap. It is important not to compromise the integrity of the cap or the well.

o Optical caps should always be handled with powder-free gloves.

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o Following amplification, the optical caps should never be removed from the PCR strips. Used PCR strips

should be disposed of in accordance with good laboratory practice for working with nucleic acid amplification

technologies.

Specimen Transport and Storage

Specimens should be transported to the lab as promptly as possible after collection. Samples not tested within 24 hours

of receipt in the laboratory should be stored at 2-8oC and tested within 5 days. For extended long-term storage samples

should be heat-treated in SPS solution as per the test procedure and stored at -20oC. Where culture/microscopic

confirmation of PCR positive samples is required, laboratories should refer to local/national criteria for acceptance of

samples.

Test Procedure

Positive Control Preparation

o Re-suspend the positive control vial (red Cap) with 200µl of reconstitution solution (500ul, green cap).

o Add the entire 200µl of the re-suspended positive control to a clearly labelled SPS tube. Mix well and store at

2-8⁰C.

o Prepare a fresh positive control for each new test kit (or after 10 weeks, whichever is sooner) and discard when

the test kit has been fully used.

Sample Preparation

o Label the required number of SPS tubes.

o Lightly coat the entire EntericBio FLOQswab with the stool sample then re-suspend the swab in the EntericBio

SPS solution.

o Securely cap the SPS tube and place in the EntericBio SPS racks A and B in the numerical order indicated on

the racks starting from numbered position 1 on rack A and ending in numbered position 46 on rack B (Figure 1).

o Close the lid(s) of the EntericBio SPS rack(s).

o Place the EntericBio SPS rack(s) with SPS tubes into its corresponding heat block adaptor on the EntericBio

heatstation set at 103°C for 30 min.

PCR Set Up

o Put on gloves. Fill an empty SPS tube with a minimum of 4 ml of the Re-suspension Buffer and place in

position 2 of EntericBio base in location A1 on the EntericBio workstation (Figure 1). This tube is also used as

the negative control.

o Insert the re-suspended Positive Control SPS tube in position 1 of the EntericBio base in location A1 on the

EntericBio workstation (Figure 1).

o Place the EntericBio SPS Rack A in position A1 and rack B in position B1 on the EntericBio workstation (Figure

1). Change gloves.

o Select the required number of EntericBio assay A+ and E PCR strips (including controls). Re-seal the pouches

correctly promptly after use.

o Load the strips onto the thermorack starting with Assay A+ in the first row and Assay E in the second row, then

load subsequent rows alternately. Align the strips with the square and rectangular symbols orientated to the left

hand side of the thermorack/instrument (Figure 1).

o De-cap the strips carefully and label them in numerical order immediately before starting the run (Figure 1).

o Turn on the EntericBio workstation and select the EntericBio realtime Gastro Panel protocol.

o Enter the required number of tests as per on-screen instructions.

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o Ensure the stage is loaded with EntericBio SPS rack A in position A1, EntericBio SPS rack B in position B1,

pipette filter tips in position A2 and PCR strips in position B2 (Figure 1). Ensure a clean discard waste bag is in

place on the discard bucket.

o When the stage is loaded start the run as per the on-screen instructions.

Figure 1. EntericBio workstation layout and PCR strip orientation.

o When the run is finished, immediately remove the thermorack and strips from the platform. Securely fasten the

optical caps onto the PCR strips.

o Place the strips securely (do not over tighten) onto the Eppendorf MixMate and mix for 60 sec at 2,000 rpm.

o Place the strips into the centrifuge and centrifuge for 60 sec at 1,500 rpm.

o Option 1 for LC480: Place strips into the LC480 adaptor in the numerical order labelled on the strips. Start

loading in position A1 of the adaptor, then fill the adaptor from top to bottom and left to right with the labelled

square and circle symbols aligned in alternate rows to the left hand side of the adaptor. Place the loaded

adaptor onto the LC480 instrument.

o Option 2 for ABI 7500 FAST: Place strips into the ABI 7500 FAST instrument in the numerical order labelled

on the strips. Start loading the strips from the top, then fill from top to bottom and left to right with the labelled

square and rectangular symbols aligned in alternate rows to the left hand side of the plate drawer.

o When the run is complete, remove used PCR strips from the instrument. Do not remove the optical caps from

the PCR strips following the amplification. Place in a sealable plastic bag, seal and dispose in accordance

with local / national guidelines.

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Amplification and Detection with Roche LightCycler 480 II instrument (option 1):

Amplification and Detection

o Select New Experiment from Macro in the LC480 front screen. In the pop up

screen select EntericBio realtime Bacterial Panel 3. Save the experiment in the required folder and the run will

then start automatically.

o Select the Sample Editor tab in the module bar.

o Select the Salmonella Subset from the Select Samples menu

o Enter the sample numbers in order starting at position A3 (Positive control= A1,Negative control=A2)

o When the experiment is finished a pop up for the traceable database appears. Type ‘ok’ to approve the run and

select ok to create the analysis.

o The Analysis screen will open automatically. Select each target to be analysed from the Open Existing

Analysis drop menu

o Select the Analysis Options tab to toggle between target analysis.

o From the module bar select the Report tab and select Generate to create the report, then save

the report or Print the report as required.

o If interfaced, select the navigator screen and select the run file to be exported. Click the Export tab

and change the file type to .xml.

o Select the appropriate location on the drive to export the run file and save it.

Results Analysis

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Positive

A sample is assigned as positive for the target where the sample produces a typical amplification curve for the

designated well.

Negative

A sample is assigned as negative for the target where there is no evidence of amplification. A sample must not be considered negative unless it is positive for the internal amplification control in the corresponding well.

Uncertain Occasionally, the amplification curve may be highlighted by the LC480 software as uncertain and the sample well is blue. Internal Amplification Control

A true negative sample must have a positive result for the internal amplification control. Negative samples that have a

failed internal control result should be retested from the original sample. If the sample is positive for any of the target

organisms in the corresponding well, the internal control result is dispensable due to competition in the reaction.

Positive Control

The respective positive control wells must be positive for each target in order to validate the results of the experimental

run.

Negative Control

The negative control wells must be negative for each target in order to validate the results of the experimental run.

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Amplification and Detection with ABI 7500 FAST instrument (option 2):

ABI 7500 Software:

o Select Template in the ABI 7500 software home screen. In the pop up screen select EntericBio realtime

Extended Bacterial Panel 3. Save the experiment in the required folder and start the run.

o Select the Plate Setup tab in the Setup menu.

o Enter the sample numbers in correct order in the Define Samples tab starting from Sample 3 position (Positive

control= Sample 1, Negative control=Sample 2)

o When the run is finished, the analysis is performed by selecting the Analysis tab from the

Experiment Menu. In the Plot Settings select Linear from the drop down menu in the Graph Type section.

o Change the View Plate Layout to View Well Table.

o Group by targets by selecting Target from the Group By drop down menu.

o Individual targets are analysed by selecting the target from the Target drop down menu in the Options tab.

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ABI 7500 Fast System SDS Software:

o Select New from the File menu. Click Browse and select EntericBio realtime Bacterial Panel 3 template from the

appropriate location. Click Finish to apply the template.

o Save the experiment in the required folder and start the run by clicking into the Instrument tab and selecting the

Start button.

o Select the Setup tab and enter the sample numbers in the correct order, starting from Positive Control in

position A1 and B1, Negative Control in position A2 and B2, followed by samples starting from position A3 and

B3.

o When the run is finished, the analysis is performed by selecting the Amplification plot tab from the Results tab.

Graph settings can be changed by right-clicking on the graph and selecting Linear/ Log option from the Post-run

setting section.

o Individual targets are analysed by selecting the target from the Detector drop down menu.

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o Ct values for the targets can be analysed by selecting the Report tab within the Results tab and grouping the

results by Detector or Sample name.

Results Analysis

Positive

A sample is assigned as positive for the target where the sample produces a typical amplification curve for the designated well

Negative

A sample is assigned as negative for the target where there is no evidence of amplification. A sample must not be considered negative unless it is positive for the internal amplification control in the corresponding well

Internal Amplification Control

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A true negative sample must have a positive result for the internal amplification control. Negative samples that have a

failed internal control result must be retested from the original sample. If the sample is positive for any of the target

organisms in the corresponding well, the internal control result is dispensable due to competition in the reaction.

Positive Control

The respective positive control wells must be positive for each target in order to validate the results of the experimental

run.

Negative Control

The negative control wells must be negative for each target in order to validate the results of the experimental run.

Results Interpretation

Salmonella

A positive result for Salmonella indicates the presence of the gene target specific for Salmonella enterica spp. in the

sample.

Shigella

A positive result for Shigella indicates the presence of the nucleic acid target for the Invasion Plasmid Antigen H (ipaH)

gene. This virulence factor can be carried by Shigella spp. and Enteroinvasive E. coli (EIEC).

Campylobacter

A positive result for Campylobacter indicates the presence of the gene target specific for Campylobacter jejuni,

Campylobacter coli or Campylobacter lari in the sample.

STEC/ VTEC

A positive result for STEC/ VTEC indicates the presence of the stx1 and/ or stx2 gene target in the sample.

A positive result with Cp >35 indicates a low level of target DNA in the sample, close to the limit of detection of the

assay. These results may not be reproducible and should be interpreted in conjunction with clinical and epidemiological

information

Yersinia

A positive result for Yersinia indicates the presence of the gene target specific for Yersinia enterocolitica in the sample.

Vibrio

A positive result for Vibrio indicates the presence of the gene target specific for Vibrio cholerae or Vibrio

parahaemolyticus in the sample.

Uncertain An uncertain result indicates that the test is ‘indeterminate’ for the presence of the nucleic acid target in the corresponding sample. The sample can be repeated from original faeces or tested with an alternative method.

Laboratories should consider local/ national guidelines when reporting positive and negative results.

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Analytical Performance Characteristics

Analytical Specificity (Inclusivity)

NCTC National collection of type cultures. NSRL National Salmonella Reference Laboratory (University College Hospital Galway, Ireland).

SARB Salmonella reference collection B (University College Cork, Ireland). CHO National E.coli Reference Laboratory (Cherry Orchard, Dublin, Ireland)

ATCC American type culture collection. ULjub University of Ljubljana, Slovenia.

DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen BEI BEI Resources

IHB Imelda Hospital Belgium

Strain Source Strain Source Strain Source Strain Source

C. coli DSMZ 4689 S. anatum SARB 2 S. flexneri NCTC 9950 Y. enterocolitica 4;3 Clinical, IHB

C. coli NCTC 12143 S.braenderup NCTC 05750 S. sonnei NCTC 7924 Y. enterocolitica 9;2 Clinical, IHB

C. jejuni NCTC 43442 S. bredeney NCTC 05731 S. sonnei NCTC 8220 Y. enterocolitica 36;1A Clinical, IHB

C. jejuni NCTC 12106 S.derby SARB 11 S. sonnei NCTC 8574 Y. enterocolitica 3;4 Clinical, IHB

C. jejuni NCTC 12109 S. dublin NCTC 09676 S. sonnei NCTC 9773 Y. enterocolitica 6;1A Clinical, IHB

C. jejuni NCTC 11828 S. enteritidis ATCC 13076 S. dysenteriae NCTC 5109 Y. enterocolitica 63;1A Clinical, IHB

C. jejuni subsp. doylei NCTC 11951 S. enteritidis PT4 NCTC 13349 S. dysenteriae NCTC 6340 E.coli O111 (VT1+VT2) Clinical (CHO)

C. lari NCTC 12144 S. gallinarum NCTC 423,287/91 S. dysenteriae NCTC 8571 E.coli O111 (VT1) Clinical (CHO)

C. lari NCTC 11458 S. gallinarum NCTC 13346 S. dysenteriae NCTC 4837 E.coli O182 (VT1) Clinical (CHO)

S. gaminara NCTC 5797 S. goldcoast NSRLd S. dysenteriae NCTC 8217 E.coli O26 (VT1) Clinical (CHO)

S. Virchow NCTC 05742 S. hadar ULjub MI 2 Y. enterocolitica BEI NR-204 E.coli O76 (VT1) Clinical (CHO)

S. manhattan NCTC 06245 S. heidelberg NCTC 5717 Y. enterocolitica BEI NR-209 E.coli O103 (VT1) Clinical (CHO)

S. newport SARB 36 S. infantis Uljub VF 35/94 Y. enterocolitica BEI NR-212 E.coli O26 (VT1+VT2) Clinical (CHO)

S. nottingham NCTC 07832 S. kentucky NCTC 05799 Y. enterocolitica BEI NR-205 E.coli O26 (VT1+VT2) Clinical (CHO)

S. panama SARB 40 S. livingstone NCTC 09125 Y. enterocolitica BEI NR-210 E.coli O157 (VT1+VT2) Clinical (CHO)

S. saint-Paul Uljub VF S-

13/95 S. london NCTC 05777 Y. enterocolitica BEI NR-213 E. coli O157 (VT2) Clinical (CHO)

S. senftenberg SARB 59 S. boydii NCTC 8576 Y. enterocolitica BEI NR-206 E. coli O157 (VT2) Clinical (CHO)

S. Stanley SARB 60 S. boydii NCTC 9328 Y. enterocolitica BEI NR-204 E. coli O157 (VT2) Clinical (CHO)

S. typhimurium ATCC 14028 S. boydii NCTC 9357 V. parahaemolyticus DSM 10027 E. coli O157 (VT2) Clinical (CHO)

S. typhimurium LT 2 NCTC 12416 S. boydii NCTC 9850 Y. enterocolitica BEI NR-207 E. coli O157 (VT2) Clinical (CHO)

S. typhimurium DT 104 NCTC 13348 S. flexneri NCTC 4 Y. enterocolitica BEI NR-2011 E. coli O157 (VT2) Clinical (CHO)

S. uganda NCTC 06015 S. flexneri NCTC 7885 V. cholerae Vircell MBC118

S.agona NCTC 11377 S. flexneri NCTC 8523 Y. enterocolitica 7,8;1A Clinical, IHB

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Analytical Specificity (Exclusivity)

Strain Source Strain Source Strain Source Strain Source

Yersinia aldovae DSM 18303 V. furnissii* DSM 19622 Aeromonas hydrophila DSM 17695 Clostridium difficile DSM 1296

Yersinia aleksicae DSM 114987 V. mimicus* DSM 19130 Alcaligenes faecalis DSM 30030

Clostridium perfringens DSM 756

Yersinia bercovieri DSM 118528 V. harvei DSM 19623 Arcobacter butzleri DSM 8739 Clostridium sordelli DSM 2141

Yersinia entomophaga DSM 122339 V. fischeri DSM 507 Bacillus cereus DSM 31

Cronobacter sakazaki DSM 4485

Yersinia

frederinsksenii

DSM 118490 P. damsela DSM 7482

Bacillus subtilis DSM 10 Cryptosporidium parvum ATCC PRA-67D

Yersinia intermedia DSM 118517 G. hollisae DSM 15132 Bacteroides fragilis DSM 2151 E. coli VTEC# Vircell MBC022

Yersinia kristensenii DSM 118543 V. diazotrophicus* DSM 2604 C.ampylobacter coli# DSM 4689 Edwardsiella tarda DSM 30052

Yersinia massiliensis DSM 121859 V. proteolyticus* DSM 30189 Campylobacter jejuni# DSM 4688

Encephaitozoon hellum BEI NR 9701

Yersinia mollaretti DSM 118520 V. natrigiens* DSM 759 Ewingella americana DSM 4580

Encephalitozoon cuniculi BEI NR 9703

Yersinia nurmii DSM 122296 V. pelagius DSM 21205 Giardia intestinalis ATCC 30888D

Encephalitozoon intestinalis BEI NR9702

Yersinia pekkanenii DSM 122769 V. campbellii DSM 19270 Hafnia alvei DSM 30163 Entamoeba dispar Clinical sample VS

Yersinia

pseudotuberculosis

DSM 18992 V. alginolyticus* DSM 2171

Klebsiella oxytoca DSM 5175 Entamoeba histolytica ATCC 30459D

Yersinia rhodei DSM 118270 Campylobacter lari# DSM 11375

Klebsiella pneumoniae DSM 30104 ETEC E. coli DSM 1103

Yersinia ruckeri DSM 118506 Candida albicans DSM 1577

Listeria monocytogenes DSM 20600

Enterobacter aerogenes DSM 30053

Yersinia similis DSM 118211 Citrobacter amalonaticus DSM 4593T Morganella morganii DSM 30164

Enterobacter cloacae DSM 30054

V. alginolyticus DSM 2171 Citrobacter freundi DSM 30039

Neisseria gonorrhoea DSM 9188

Enterococcus faecalis DSM 20478

V. fluvialis* DSM 19283 Citrobacter koseri DSM 4595

Plesiomonas shigelloides DSM 8224

Enterococcus faecium DSM 20477

Proteus vulgaris DSM 13387 Pseudomonas aeruginosa DSM 50071 Proteus mirabilis DSM 4479 EPEC E. coli DSM 8695

Providencia stuartii DSM 4539 Pseudomonas putida DSMZ 291 Rahnella aquatilis DSM 4594 Escherichia coli DSM 30083

Serratia marcescens DSM 1608 Saccharomyces cerevisiae DSM 1848 Shigella flexneri# DSM 7532

Stenotrophomonas maltophilia DSM 50170

Serratia odoriferae DSM 4582 Salmonella enteritidis# DSM 17420 Shigella sonnei# DSM 5570 Streptococcus agalactiae DSM 2134

Serratia rubidaea DSM 4480 Salmonella typhimurium# DSM 554

Staphylococcus aureus DSM 20231

Streptococcus bovis DSM 20480

Shigella boydii# DSM 7532 Serratia liquifaciens DSM 4487 Staphylococcus epidermidis DSM 20044

Streptococcus equinus DSM 20558

Escherichia hermannii DSM 4560

*See limitations of method # These were included in the exclusivity panel to confirm no cross-reaction occurs with other targets detected by the assay.

ATCC American type culture collection.

DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen

BEI BEI Resources Repository

VS St Vincent’s Hospital Sydney

Analytical Specificity (Interfering substances) A range of exogenous and endogenous substances were tested for potential PCR interference. No interference was

seen due to the presence of any of the substances tested.

Substance Result Substance Result

Amoxicillin NI Human DNA NI

Antacid NI Hydrocortisone NI

Anti-diarrhoeal NI Metronidazole NI

Cholesterol NI Mucin NI

Ciprofloxican NI Non-Steroidal Anti-Inflammatory (NSAID) NI

Erythromycin NI Trimethoprim NI

Haemorrhoidal cream NI Whole human blood NI

NI No interference

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External Quality Assessment

Distributions from two Independent Quality Assessment (EQA) Schemes – Quality Control for Molecular Diagnostics

(QCMD) and United Kingdom National External Quality Assessment Service (UK NEQAS) were tested with the

EntericBio realtime® Bacterial Panel 3 assay.

QCMD distribution panels

QCMD Bacterial (2012-2016) EQA distribution panels were tested and EntericBio realtime Bacterial Panel 3 assay

results were 98% concordant with expected results, with one observed discordant result (B16-04).

QCMD Reference

Sample EntericBio Result

QCMD Reference

Sample EntericBio Result

QCMD Reference

Sample EntericBio Result

B12-01 S. enteritidis Positive B12-02 C. lari Positive B12-03 C. jejuni Positive

B12-04 Y. enterocolitica Positive B12-05 S. enteritidis Positive B12-06 Negative Negative

B12-07 C. jejuni Positive B12-08 C. ureolyticus Negative B12-09 C. jejuni Positive

B12-10 S. flexneri Positive B12-11 C. lari Positive B12-12 S. enteritidis Positive

B12-13 C. jejuni Positive B12-14 S. flexneri Positive B12-15 Negative Negative

B12-16 C. ureolyticus Negative B12-17 C. jejuni Positive B12-18 S. enteritidis Positive

B12-19 C. jejuni Positive B12-20 Y. enterocolitica Positive B13-01 S. enteritidis Positive

B13-02 C. lari Positive B13-03 Y. enterocolitica Positive B13-04 C. jejuni Positive

B13-05 Negative Negative B13-06 S. enteritidis Positive B13-07 C. jejuni Positive

B13-08 S. flexneri Positive B13-09 C. jejuni Positive B13-10 C. ureolyticus Negative

B14-01 S. flexneri Positive B14-02 C. jejuni Positive B14-03 C. lari Positive

B14-04 C. jejuni Positive B14-05 Negative Negative B14-06 C. jejuni Positive

B14-07 Y. enterocolitica Positive B14-08 S. enteritidis Positive B14-09 C. coli Positive

B14-10 P. shigelloides Negative B15-01 P. shigelloides Negative B15-02 C. coli Positive

B15-03 S. enteritidis Positive B15-04 C. lari Positive B15-05 C. jejuni Positive

B15-06 Y. enterocolitica Positive B15-07 S. flexneri Positive B15-08 C. jejuni Positive

B15-09 Negative Negative B16-01 C. coli Positive B16-02 C. coli Positive

B16-03 C. lari Positive B16-04 C. coli/ P. shigelloides Negative B16-05 C. jejuni Positive

B16-06 S. enteritidis Positive B16-07 Negative Negative B16-08 S. flexneri Positive

B16-09 Y. enterocolitica Positive

UK NEQAS distribution panels 2013-2017

UK NEQAS Faecal Pathogens and General Bacteriology EQA distribution panels (2013-2017) were tested and

EntericBio realtime® Bacterial Panel 3 results were 100% concordant with expected results. Specimen Number

Sample EntericBio Result

Specimen Number

Sample EntericBio Result

Specimen Number

Sample EntericBio Result

1542 S. Enteritidis Positive 1543 S. Kentucky Positive 1709 Commensal flora Negative

1710 S. Typhimurium Positive 1840 Y. pseudotuberculosis Negative 1852 C. jejuni Positive

1853 S. Agona Positive 1885 S. Kentucky Positive 1968 S. Braenderup Positive

2012 S. flexneri Positive 2049 C. jejuni Positive 2086 S. Typhimurium Positive

2094 S. Braenderup Positive 2095 Commensal flora Negative 2096 C. jejuni Positive

2097 S. Typhimurium Positive 2174 V. parahaemolyticus Positive 2212 C. jejuni/coli Positive

2252 P. shigelloides Negative 2295 S. sonnei Positive 2340 S. Enteritidis Positive

2390 K. pneumoniae Negative 2391 N. meningitidis Negative 2392 Commensal flora Negative

2430 A. hydrophila Negative 2431 B. parapertussis Negative 2432 Y. enterocolitica Positive

2548 Commensal flora Negative 2549 L. monocytogenes Negative 2550 S. Oranienburg Positive

2597 S. aureus Negative 2598 B. fragilis Negative 2599 C. coli Positive

2628 β-haem Strep Negative 2629 N. gonhorrhoeae Negative 2630 S. Typhimurium Positive

2666 E. coli Negative 2667 C. ulcerans Negative 2668 S. sonnei Positive

2673 S. Oranienburg Positive 2674 C. coli Positive 2675 S. Typhimurium Positive

2676 S. sonnei Positive 2710 A. aphrophilus Negative 2711 L. pneumophila Negative

2712 Commensal flora Negative 2753 Commensal flora Negative 2754 P. mirabilis Negative

2755 V. cholerae Positive 2789 S. pneumoniae Negative 2790 C. septicum Negative

2791 Commensal flora Negative 2829 B. parapertussis Negative 2830 β-haem Strep. Negative

EBRT 59 rev 02 Page 16 of 20

Analytical sensitivity

Analytical sensitivity was determined by testing a standard dilution series for each target. Each dilution series consisted

of 8 replicates of 6 serial dilutions. Statistical results were generated using Minitab. The limit of detection of the assay

was established as <15 cell equivalents for Salmonella, Shigella, VTEC, Yersinia, Vibrio and Campylobacter with a

probability of greater than or equal to 95% by Probit analysis.

2831 S. boydii Positive 2870 C. macginleyi Negative 2871 S. milleri group Negative

2872 C. jejuni Positive 2920 S. aureus Negative 2921 A. fumigatus sp. Negative

2922 S. Enteritidis Positive 2969 S. gallolyticus Negative 2970 H. influenzae Negative

2971 Y. pseudotuberculosis Negative 3010 N. meningitidis Negative 3011 B. cepacia complex Negative

3012 C. difficile Negative 3128 S. aureus/ β-haem Strep

Negative 3129 B. pertussis Negative

3130 S. sonnei Positive 3173 A. haemolyticum Negative 3174 P. putida Negative

3175 S. Enteritidis Positive 3209 M. catarrhalis Negative 3210 C. jejuni Positive

3250 E. gallinarum Negative 3251 S. maltophilia Negative 3252 S. Montevideo Positive

3257 V. parahaemolyticus Positive 3258 S. Montevideo Positive 3259 C. jejuni Positive

3260 S. Enteritidis Positive 3294 S. pneumoniae Negative 3295 N. cyriacigeorgica Negative

3296 S. flexneri Positive 3338 S. marcescens Negative 3339 C. perfringens Negative

3340 V. parahaemolyticus Positive 3378 P. anaerobius Negative 3379 N. gonorrhoeae Negative

3380 Commensal flora Negative 3415 P. stutzeri Negative 3416 E. rhusiopathiae Negative

3417 Non-toxigenic C. difficile Negative 3456 A. niger species complex

Negative 3457 β-haem Strep. Negative

3458 C. jejuni Positive 3510 S. mutans Negative 3511 B. fragilis Negative

3512 S. Stanley Positive 3556 V. alginolyticus Negative 3557 C. albicans Negative

3558 Y. enterocolitica Positive 3598 C. noyvi Negative 3599 N. meningitidis Negative

3600 V. cholerae non-O1 Positive 3735 P. mirabilis Negative 3736 S. aureus Negative

3737 S. Typhimurium Positive 3780 S. lugdenensis Negative 3781 N. cyriacigeorgica Negative

3782 S. sonnei Positive

EBRT 59 rev 02 Page 17 of 20

Clinical Performance Characteristics

Assay A+

A total of 1,469 clinical samples were tested prospectively by participating clinical laboratories. Reference method used was culture, in accordance with HPA UK guidelines for the investigation of faecal specimens for bacterial pathogens. Discrepant results were confirmed using an alternative, FDA-approved molecular method.

EntericBio

Pos Neg

Reference

Method

Pos 104 3a

Neg 22 1332

o Sensitivity 0.97 (0.91 – 0.99, 95% CI)

o Specificity 0.98 (0.98 – 0.99, 95% CI)

o PPV 0.83 (0.75 – 0.89, 95% CI)

o NPV 0.99 (0.99 – 1.00, 95% CI)

EntericBio Routine Method

Salmonella 17(16) 19

Shigella 9 (4) 4

Campylobacter 100 (84) 84

Numbers in parentheses denote samples in agreement between the two methods. a 3/3 Salmonella positive on culture only post-enrichment and negative with the alternative molecular assay.

One additional Salmonella detected by the EntericBio assay was confirmed by the alternative assay. b Only 4/5 available for confirmation. 4/4 confirmed by the alternative molecular assay.

c Only 13/16 specimens were available for confirmation. 13/13 confirmed by the alternative molecular assay.

Assay E

A total of 245 clinical samples were tested by a participating clinical laboratory. Reference method included a combination of alternative CE-IVD molecular assay (Shiga-like toxin-producing E. coli) and culture (Yersinia, Vibrio). Additionally a total of 518 stored frozen, clinical specimens were tested with assay E of the Bacterial Panel 3 assay. Samples were previously screened by clinical laboratories using routine methods (PCR, culture, reference laboratory). All samples were processed accordingly to the EntericBio Bacterial Panel 3 instructions for use.

EntericBio

Pos Neg

Reference

Method

Pos 71 1

Neg 5 686

o Sensitivity 0.98 (0.91 – 1.00, 95% CI)

o Specificity 0.99 (0.98 – 0.99, 95% CI)

o PPV 0.93 (0.85 – 0.98, 95% CI)

o NPV 0.99 (0.99 – 1.00, 95% CI)

EntericBio Routine Method

STEC 45 (41) 42

Yersinia 29 (28) 28

Vibrio 2 (2) 2

EBRT 59 rev 02 Page 18 of 20

Limitations of Method

o Results from the EntericBio realtime® Bacterial Panel 3 assay should be interpreted in conjunction with patient’s clinical

signs and symptoms, medical history and other laboratory data available to the physician.

o The test is qualitative and does not provide a quantitative measurement of the detected organism nor indicate the amount

of the pathogen present in the sample.

o A positive PCR result does not indicate the presence of a viable organism in the sample at the time of testing. Samples

submitted for test of clearance/cure should be tested by routine culture methods.

o EntericBio realtime® Bacterial Panel 3 assay does not differentiate which Campylobacter species (coli or lari or jejuni) is

present in the sample.

o EntericBio realtime® Bacterial Panel 3 assay does not differentiate which Vibrio species (cholerae or parahaemolyticus) is

present in the sample23-25.

o EntericBio realtime® Bacterial Panel 3 assay does not differentiate which stx gene (stx1 or stx2) is present in the sample.

o The detection of the target is dependent upon appropriate specimen collection, transport, storage, handling and

preparation. Inappropriate procedure at any of these stages may result in incorrect results.

o The Invasion Plasmid Antigen H (ipaH) gene target is present in Shigella spp. and Enteroinvasive E. coli (EIEC). A

positive result for Shigella spp. that is not confirmed by culture may be due to the presence of nonviable organism,

differences in LOD between the methods or the presence of EIEC in the sample.

o stx1 gene can also be present in some Shigella spp. and is genetically indistinguishable from the stx1 gene of STEC. The

stx genes are mobile genetic elements and their presence has been reported in other members of Enterobacteriaceae

[15].

o The stx2 genotype stx2f is not detected with the EntericBio realtime® Bacterial Panel 3 assay. Reported detections of the

stx2f genotype in clinical samples from humans are rare.

o Mutations or polymorphisms in either primer or probe binding regions may have an impact on the detection of the targets,

resulting in sub-optimal detection or a false negative result with the EntericBio realtime® Bacterial Panel 3.

o In case of extremely low levels of target, below the analytical sensitivity of the assay, the target may be detected, but

results may not be reproducible.

o Strongly positive targets present in the sample may interfere with the detection of other targets present at low

concentrations (near the assay detection limit) within the reaction resulting in sub-optimal detection or a false negative

result due to PCR competition between targets in the multiplex reaction.

o Fluorescence leakage (cross-talk) may occur on the real-time PCR instruments between detection channels with

overlapping wavelengths. This can result in a weak false positive signal being detected due to crosstalk from a strongly

positive signal in the same reaction well.

o Laboratories that refer stx1/stx2 positive results to national STEC reference labs for confirmation should note that

anomalies may arise due to inherent differences between the EntericBio realtime® method and the method employed by

the reference laboratory.

o C. insulaenigrae when present at very high copy numbers may be detected in addition to C. jejuni/coli/lari due to

incomplete but significant sequence homology in the probe binding region. Reported cases of C. insulaenigrae from

suspected cases of enteritis in humans are rare.

o Vibrio furnissii, mimicus, fluvialis will be detected in the Vibrio assay. These organisms have been associated with

gastroenteritis in humans

o Vibrio diazotrophicus, proteolyticus, natrigiens, alginolytics, when present at very high copy numbers, may be detected in

the Vibrio assay, however reported cases from suspected cases of enteritis in humans are rare.

o Tests of clearance or status of asymptomatic carriage of Salmonella should include testing from a routine 24 hr

enrichment broth with the EntericBio realtime® assay and/or routine enriched culture. This should also be considered

where testing is requested on non-liquid stool samples submitted as part of a clinical follow up on patients with a recent

clinical history of foreign travel associated gastroenteritis.

o Cross-reactivity with organisms other than those listed in the Exclusivity table has not been evaluated.

EBRT 59 rev 02 Page 19 of 20

o Faecal samples that have been stored outside of the specimen transport and storage requirements of the EntericBio

realtime® Bacterial Panel 3 assay may lead to false negative results. Optimum performance is achieved when testing

fresh samples.

o The performance of the test has been evaluated on human stool specimens only.

o The performance of the EntericBio realtime® Bacterial Panel 3 has not been evaluated on immunocompromised

individuals and patients without symptoms of gastroenteritis.

o Positive and Negative Predictive values depend greatly on the disease prevalence and therefore the performance of the

EntericBio realtime® Bacterial Panel 3 assay may vary depending on the prevalence and population evaluated.

References

1. Grimont PAD, Weill F-X. Antigenic formulae of the salmonellae serovars (9th ed.) 2007; http://www.pasteur.fr/sante/clre/cadrecnr/salmoms/WKLM_2007.pdf

2. Gal-Mor O., Voyle E. C., Grassl G. A. 2014 Same species, different diseases: how and why typhoidal and non-typhoidal Salmonella enterica serovars differ. Front Microbiol Aug 4;5: 391

3. Li Y., Cao B., Liu B., Liu D., Gao Q., Peng X., Wu J., Bastin D. A., Feng L., Wand L. 2009. Molecular detection of all 34 distinct O-antigen forms of Shigella. J Med Microbiol: 58: 69-81.

4. Khan W. A., Griffiths J. K., Bennish M. L. 2013. Gastrointestinal and extra-intestinal manifestations of childhood shigellosis in a region where all four species of Shigella are endemic. PLoS One; May 17;8(5)

5. Gould L. H. et al. 2009. Recommendations for diagnosis of shiga toxin-producing Escherichia coli infections by clinical laboratories. MMWR Recomm Rep; 58 (RR12): 1-14.

6. Morandi E., Grassi C., Cellerino P., Massara P. P., Corsi F., Trabucchi E. 2003. Verocytotoxin-producing Escherichia coli EH 0157:H7 colitis. J Clin Gastroenterol; 36:44-6.

7. Siegler R., Oakes R. 2005. Hemolytic uremic syndrome; pathogenesis, treatment, and outcome. Curr Opin Pediatr; 5;17:200-4.

8. Gavin, P. J., et al. 2004. Evaluation of performance and potential clinical impact of ProSpecT Shiga toxin Escherichia coli microplate assay for detection of Shiga toxin-producing E. coli in stool samples. J. Clin. Microbiol.42:1652–1656.

9. Nielsen, E. M., Scheutz F., Torpdahl M. 2006. Continuous surveillance of Shiga toxin-producing Escherichia coli infections by pulsed-field gel electrophoresis shows that most infections are sporadic. Food Pathog. Dis. 3:81–87.

10. Safdar, N., Said A., Gangnon R. E., Maki D. G. 2002. Risk of haemolytic uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 enteritis. J. Am. Med. Assoc. 288:996–1001.

11. Bari M. L., Hossain M. A., Isshiki K., Ukuku D. 2011. Behavior of Yersinia enterocolitica in Foods. J Pathog 2011:420732. doi: 10.4061/2011/420732.

12. Fredriksson-Ahomaa M., Stolle A., Stephan R. 2007. Prevalence of pathogenic Yersinia enterocolitica in pigs slaughtered at a Swiss abattoir. Int J Food Microbiol Nov 1:119(3): 207-12.

13. Laukkanen-Ninios R., Fredriksson-Ahomaa M., Maijala R., Korkeala H. 2014. High prevalence of pathogenic Yersinia enterocolitica in pig cheeks. Food Microbiol Oct:43:50-2.

14. Karhukorpi J., Paivanurmi M. 2014. Differentiation of Yersinia enterocolitica biotype 1A from pathogenic Yersinia enterocolitica biotypes by detection of β-glucosidase activity: comparison of two chromogenic culture media and Vitek2. J Med Microbiol Jan: 63(Pt 1):34-7.

15. Kaysner C. A., DePaola A. Bacteriological Analytical Manual. Chapter 9: Vibrio, Food and Drug Administration.

16. Vibrio parahaemolyticus infections associated with eating raw oysters and clams harvested from Long Island Sound – Connecticut, New Jersey and New York. 1998. MWWR: 47:457-62

17. Snelling W. J, Matsuda M., Moore J. E., Dooley J. S. 2005. Campylobacter jejuni. Lett Appl Microbiol; 41:297-302

18. Bullman S., O’Leary J., Corcoran D., Sleator R. D., Lucey B. 2012. Molecular-based detection of non-culturable and emerging campylobacteria in patients presenting with gastroenteritis. Epidemiol Infect. Apr; 140(4): 684-8.

19. Thomas P. K. Peripheral neuropathy. In: Ledingham JGG, Warrell DA, editors. Concise Oxford Textbook of Medicine.Vol 1. Oxford: Oxford University Press; 2000. p. 1375.

20. On S. L. 1996. Identification methods for campylobacters, helicobacters and related organisms. Clin Microbiol Rev; 9(3): 405-22.

21. O’Leary J., Corcoran D., Lucey B. 2009. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens. J Clin Microbiol. 47(11): 3449-53.

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22. Investigation of Faecal Specimens for Enteric Pathogens. UK Standards for Microbiology Investigations. Standards Unit, Microbiology Services, Public Health England: Bacteriology | B 30 | Issue no: 8.1 | Issue date: 24.04.14 | Page: 1 of 41 https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/343955/B_30i8.1.pdf

23. Derber C., Coudron P., Tarr C., Gladney L., Turnsek M., Shankaran S., Wong E. 2011. Vibrio furnissii: an unusual cause of bacteremia and skin lesions afteringestion of seafood. J Clin Micro 49(6):2348-9.

24. Igbinosa E. O., Okoh A. I. 2010. Vibrio fluvialis: an unusual enteric pathogen of increasing public health concern. Int J Environ Res Public Health, 7(10):3628-43.

25. Chitov T, Kirikaew P, Yungyune P, Ruengprapan N, Sontikun K. An incidence oflarge foodborne outbreak associated with Vibrio mimicus. Eur J Clin Microbiol Infect Dis. 2009 Apr;28(4):421-4.

Glossary

Manufacturer

Use by Date

In vitro diagnostic medical device

Catalogue Number

Limit of Temperature

Consult instructions for use

Batch Code

Contains sufficient for <n> tests

CE conformity

Do not reuse