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. EBGP2A-V2, 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

Gastro Panel 2 User Manual

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Intended Use

For in vitro diagnostic use. The assay is intended for use with liquid/loose stool samples submitted from symptomatic

patients for routine investigation of gastroenteritis. The EntericBio realtime® Gastro Panel 2 assay is a molecular

diagnostic tests for the direct, qualitative detection of Salmonella enterica spp., Shigella spp./ Enteroinvasive E. coli,

Campylobacter jejuni/coli/lari, Cryptosporidium parvum/ hominis, Giardia lamblia and VTEC (stx1/stx2) in human stool

samples.

Summary and Explanation

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

gastroenteritis include Campylobacter spp., Salmonella enterica spp., Shigella spp. and Verotoxigenic Escherichia coli.

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

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

Scheme [1]. Salmonella isolates from humans are serotypes of Salmonella enterica. Gastroenteritis is the most common

condition caused by Salmonella species. Symptoms include abdominal pain, diarrhoea, nausea and vomiting, often

accompanied by fever.

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 characterised serologically on the basis of the O antigen

only, as Shigella spp. lack the H antigen [2]. Infection with Shigella spp. manifests as a range of symptoms from watery

diarrhoea to dysentery with frequent small volume faeces containing blood, mucus and pus. The diarrhoea may be

accompanied by fever and abdominal cramps.

Strains of E. coli that produce the toxins stx1 & stx2 are termed Verotoxigenic E. coli (VTEC). The toxins are

termed “shiga-like” due to their similarity to the toxin of Shigella dysenteriae [3]. Infections vary in severity from mild to

bloody diarrhoea and may occur in any age group, although it is more common in children. VTEC are capable of

causing two types of disease, namely haemorrhagic colitis [4] and haemolytic uraemic syndrome (HUS) [5]. 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 VTEC serotypes have been reported as

agents of both sporadic and outbreak-associated disease [6-8].

The genus Campylobacter contains 25 species but only some of those have been firmly established as a cause

of human gastroenteritis. Campylobacter jejuni accounts for about 90% of reported infections and most of the remainder

are caused by Campylobacter coli and lari [9]. In human hosts diarrhoea 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 bacteraemia, 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 [10] (e.g. the Miller-Fisher Syndrome).

Salmonella, Shigella and Verotoxigenic 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 oxidise carbohydrates. A well-recognised problem associated with identification of

Campylobacter species is the lack of effective discriminating tests. Traditional laboratory methods used to detect and

identify these four pathogen groups combine selective culture broths and/or selective culture plates with a range of

biochemical tests and serology. This approach is relatively labour intensive with a typical time to result of between 48

and 96 hours. In addition routine laboratory detection of VTEC is generally limited to non-sorbitol fermenting E.coli

belonging to the O157 serogroup [11-14].

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Cryptosporidium and Giardia are the two leading causes of parasite associated diarrhoeal disease [15].

Cryptosporidium is a genus of apicomplexan protozoans that contains at least seven species associated with human

diseases with the most frequently isolated species being C. hominis and C. parvum. C. hominis is mainly found in

humans while C. parvum can also be isolated from cattle and other ruminants [15].

Giardia is a genus of anaerobic flagellated protozoan parasites with a life cycle that alternates between an actively

swimming trophozoite and an infective resistant cyst. The organism colonises and reproduces in the small intestines of

several vertebrates, causing giardiasis with G. lamblia assemblages A & B most frequently reported in humans.

These organisms are usually transmitted through water and food contaminated with animal faeces. Animal

reservoirs, particularly cattle, are an important vector for transmission of Cryptosporidium and Giardia spp. [16] and

outbreaks are commonly reported [17]. Person-to-person transmissions (particularly in childcare settings) have also

been described [17]. Symptoms can include diarrhoea, abdominal cramps, fever, nausea, vomiting and loss of appetite

in addition to greasy stools and flatulence in cases of giardiasis. However asymptomatic infections have also been

reported [17]. Traditional laboratory methods using light microscopy to identify these parasites tend to be labour

intensive and subjective [18] therefore molecular-based methods are exploited as an alternative in a clinical

microbiology setting.

Test Principle

The EntericBio realtime® Gastro Panel 2 assay is a molecular diagnostic tests for the simultaneous detection of

Salmonella enterica spp., Shigella spp./Enteroinvasive E. coli, Campylobacter jejuni/coli/lari, Cryptosporidium parvum/

hominis, Giardia lamblia and VTEC 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 2 programme. The wells are capped and transferred to the real-time

PCR instrument for automated amplification, detection and analysis with the EntericBio Gastro Panel 2 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.

Reagents Provided

EntericBio realtime® Gastro Panel 2 (EBGP2A-V2) 240 tests

o Assay A+ Salmonella/Shigella/Campylobacter (x5 pouches/48 tests)

o Assay B+ VTEC/Cryptosporidium/Giardia (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 4ml/pierceable cap)

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Storage

The EntericBio realtime® Gastro Panel 2 assay and EntericBio realtime® SPS kit should be stored at 2-8⁰C on receipt

in the laboratory. Resealed 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 GastroPanel 2 assay is for in vitro diagnostic use only.

o The EntericBio GastroPanel 2 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.

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.

o If assay pouches or component vials are damaged or show any sign of deterioration prior to use, do not use.

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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 (<6

months) 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(s) 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 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 B+ 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 B+ in the second row, then

load subsequent rows alternately. Align the strips with the square and circle 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 clinical specimens followed by the number of controls + clinical specimens.

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

pipette filter tips to position A2 and PCR strips to 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 circle 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 Gastro Panel 2. 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.

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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.

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

Gastro Panel 2. 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 Gastro Panel 2 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.

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

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.

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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

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 corresoponding 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.

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.

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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.

VTEC (stx1/stx2)

A positive result for 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.

Cryptosporidium

A positive result for Cryptosporidium indicates the presence of the gene target specific for C. parvum and/ or C. hominis

in the sample.

Giardia

A positive result for Giardia indicates the presence of the gene target specific for G. lamblia 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 (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

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 W Cryptosporidium Reference Unit, Wales, UK

Strain Source Strain Source Strain Source Strain Source

C. coli DSMZ 4689 S. anatum SARB 2 S. boydii NCTC 8576 E. coli O157 (VT2) Clinical (CHO)

C. coli NCTC 12143 S.braenderup NCTC 05750 S. boydii NCTC 9328 E. coli O157 (VT2) Clinical (CHO)

C. jejuni NCTC 43442 S. bredeney NCTC 05731 S. boydii NCTC 9357 E. coli O157 (VT2) Clinical (CHO)

C. jejuni NCTC 12106 S.derby SARB 11 S. boydii NCTC 9850 E. coli O157 (VT2) Clinical (CHO)

C. jejuni NCTC 12109 S. dublin NCTC 09676 S. flexneri NCTC 4 E. coli O157 (VT2) Clinical (CHO)

C. jejuni NCTC 11828 S. enteritidis ATCC 13076 S. flexneri NCTC 7885 E. coli O157 (VT2) Clinical (CHO)

C. jejuni subsp. doylei NCTC 11951 S. enteritidis PT4 NCTC 13349 S. flexneri NCTC 8523 E. coli O157 (VT2) Clinical (CHO)

C. lari NCTC 12144 S. gallinarum NCTC 423,287/91 S. flexneri NCTC 9950 E. coli O157 (VT2) Clinical (CHO)

C. lari NCTC 11458 S. gallinarum NCTC 13346 S. sonnei NCTC 7924 E. coli O157 (VT2) Clinical (CHO)

S. gaminara NCTC 5797 S. goldcoast NSRLd S. sonnei NCTC 8220 E. coli O157 (VT2) Clinical (CHO)

S. Virchow NCTC 05742 S. hadar ULjub MI 2 S. sonnei NCTC 8574 E. coli O157 (VT2) Clinical (CHO)

S. manhattan NCTC 06245 S. heidelberg NCTC 5717 S. sonnei NCTC 9773 E. coli O145 (VT2) Clinical (CHO)

S. newport SARB 36 S. infantis Uljub VF 35/94 S. dysenteriae NCTC 5109 E. coli O157 (VT2) Clinical (CHO)

S. nottingham NCTC 07832 S. kentucky NCTC 05799 S. dysenteriae NCTC 6340 E. coli O157 (VT2) Clinical (CHO)

S. panama SARB 40 S. livingstone NCTC 09125 S. dysenteriae NCTC 8571 E. coli O157 (VT2) Clinical (CHO)

S. saint-Paul Uljub VF S-13/95 S. london NCTC 05777 S. dysenteriae NCTC 4837 E. coli O146 (VT2) Clinical (CHO)

S. senftenberg SARB 59 E.coli O182 (VT1) Clinical (CHO) S. dysenteriae NCTC 8217 E.coli O26 (VT1+VT2) Clinical (CHO)

S. Stanley SARB 60 E.coli O111 (VT1) Clinical (CHO) E.coli O103 (VT1) Clinical (CHO) E.coli O111 (VT1+VT2) Clinical (CHO)

S. typhimurium ATCC 14028 E.coli O26 (VT1) Clinical (CHO) E.coli O103 (VT1) Clinical (CHO) E.coli O157 (VT1+VT2) Clinical (CHO)

S. typhimurium LT 2 NCTC 12416 E.coli O76 (VT1) Clinical (CHO) E.coli O26 (VT1) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO)

S. typhimurium DT 104 NCTC 13348 E.coli O103 (VT1) Clinical (CHO) E.coli O36 (VT1) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO)

S. uganda NCTC 06015 E. coli O26 (VT1) Clinical (CHO) E.coli O26 (VT1) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO)

S.agona NCTC 11377 E. coli O103 (VT1) Clinical (CHO) E. coli O157 (VT1) Clinical (CHO) E.coli O103 (VT1+VT2) Clinical (CHO)

E.coli O26 (VT1+VT2) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO)

E.coli O26 (VT1+VT2) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO)

E.coli O157 (VT1+VT2) Clinical (CHO) E.coli O26 (VT1+VT2) Clinical (CHO) E.coli O91 (VT1+VT2) Clinical (CHO) E.coli O91 (VT1+VT2) Clinical (CHO)

C. parvum Wild type (W) C. hominis Wild type (W) G. lamblia assemblage A Wild type (W) G. lamblia assemblage B Wild type (W)

C. parvum ATCC PRA-67D G. lamblia ATCC - 50580

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Exclusivity

Strain Source Strain Source Strain Source Strain Source

Arcobacter butzleri DSMZ 8739 Aeromonas hydrophila DSMZ 17695 Bacillus cereus DSMZ 31 Bacteroides fragilis DSMZ 2151

Candida albicans DSMZ 1577 Campylobacter coli # DSMZ 4689 Campylobacter jejuni # DSMZ 4688 Citrobacter freundi DSMZ 30039

Enterobacter cloacae DSMZ 30054 Enterococcus faecalis DSMZ 20478 Escherichia coli DSMZ 30083 Escherichia coli

(EPEC) DSMZ 8695

Escherichia coli (ETEC) DSMZ 1103 Klebsiella pneumonia DSMZ 30104 Klebsiella oxytoca DSMZ 5175 Pseudomonas

aeruginosa DSMZ 50071

Proteus mirabilis DSMZ 4479 Proteus vulgaris DSMZ 13387 Staphylococcus aureus DSMZ 20231 Saccharomyces

cervisiae DSMZ 1848

Salmonella Enteritidis # DSMZ 17420 Staphylococcus

epidermidis DSMZ 20044 Shigella flexneri # DSMZ 7532 Serratia liquifaciens DSMZ 4487

Salmonella Typhimurium # DSMZ 554 Vibrio parahaemolyticus DSMZ 10027 Yersinia enterocolitica DSMZ 4780 Entamoeba

histolytica ATCC 30459

Encephalitozoon cuniculi BEI NR 9703 Encephalitozoon

intestinalis BEI NR 9702

Encephalitozoon

hellum BEI NR 9701 Toxoplasma gondii BEI NR 15430

Cryptosporidium canis W23645 Cryptosporidium

cuniculus* W29592 Cryptosporidium felis W30949

Cryptosporidium

Horse Type* W25500

Cryptosporidium

meleagridis* W25535

Cryptosporidium Skunk

type W23573

Cryptosporidium

ubiquitum* W26353

Cryptosporidium

viatorum W25244

Cryptosporidium xiaoi W20202 Cryptosporidium

andersoni A823 Cryptosporidium baylei W15502

Campylobacter

insulaenigrae* DSMZ 17739

*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

W Cryptosporidium Reference Unit, Wales

BEI BEI Resources Repository

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® Gastro Panel 2 assay.

QCMD distribution panels

QCMD Bacterial (2012-2015) and Parasitic (2014-2015) EQA distribution panels were tested and EntericBio realtime

Gastro Panel 2 assay results were 100% concordant with expected results.

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 Negative 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 Negative B13-01 S. enteritidis Positive

B13-02 C. lari Positive B13-03 Y. enterocolitica Negative 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 Negative 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 Negative B15-07 S. flexneri Positive B15-08 C. jejuni Positive

B15-09 Negative Negative

P14-01 E. histolytica Negative P14-02 G. lamblia Positive P14-03 G. lamblia Positive

P14-04 E. dispar Negative P14-05 Negative Negative P14-06 C. parvum/hominis Positive

P14-07 E. histolytica Negative P14-08 C. parvum/hominis Positive P15-01 G. lamblia Positive

P15-02 C.parvum/hominis Positive P15-03 E. histolytica Negative P15-04 C. parvum/hominis Positive

P15-05 Negative Negative P15-06 G. lamblia Positive P15-07 G. lamblia Positive

P15-08 E. histolytica Negative

EBRT 52 rev 04

UK NEQAS distribution panels 2013-2016

UK NEQAS Faecal Pathogens and General Bacteriology EQA distribution panels (2013-2016) were tested and

EntericBio realtime® Gastro Panel 2 results were 100% concordant with expected results.

Analytical sensitivity

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

performed in triplicate and consisted of 12 replicates of 8 dilutions. Statistical results were generated using

Minitab. The limit of detection of the assay was established as <15 cell equivalents for Salmonella, Shigella, VTEC,

Campylobacter, Cryptosporidium <5 genome copies, and <1 genome copies for Giardia lamblia; with a probability of

greater than or equal to 95% by Probit analysis.

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 Negative 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 Negative

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 Negative 2789 S. pneumoniae Negative 2790 C. septicum Negative

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

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

EBRT 52 rev 04

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 3

Neg 22 1,332

EntericBio

Positive

Reference Method

Positive

EntericBio & Reference

Method Positive

Salmonella a 17 19 16

Shigella b 9 4 4

Campylobacter c 100 84 84 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.

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)

EBRT 52 rev 04

Assay B+

A total of 486 frozen, clinical specimens were tested with part B+ of the Gastro Panel 2 assay. Samples were previously screened by clinical laboratories using routine methods. All positive samples were confirmed by national reference laboratory testing. All samples were processed accordingly to the EntericBio Gastro Panel 2 instructions for use.

EntericBio

Pos Neg

Reference Method Pos 191 3

Neg 6 287

EntericBio

Positive

Reference Method

Positive

EntericBio & Reference

Method Positive

VTEC 73 74 73

Giardia d 60 59 58

Cryptosporidiume 64 61 60

d 2/2 confirmed by an alternative molecular assay.

e Only 3/4 specimens were available for confirmation 3/3 confirmed by an alternative molecular assay.

o Sensitivity 0.98 (0.95 – 0.99, 95% CI)

o Specificity 0.98 (0.95 – 0.99, 95% CI)

o PPV 0.97 (0.93 – 0.99, 95% CI)

o NPV 0.99 (0.97 – 0.99, 95% CI)

EBRT 52 rev 04

Analytical Performance

Limitations of Method

Results from the EntericBio realtime® Gastro Panel 2 assay should be interpreted in conjunction with patient’s clinical

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

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.

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.

EntericBio Gastro Panel 2 assay does not differentiate which shiga toxin gene (stx1 or stx2) is present in the sample.

EntericBio Gastro Panel 2 assay does not differentiate which Cryptosporidium species (parvum or hominis) is present in

the sample.

EntericBio Gastro Panel 2 assay does not differentiate which Campylobacter species (coli or lari or jejuni) is present in the

sample.

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.

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.

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

[19].

The stx2 genotype stx2f is not detected with the EntericBio realtime Gastro Panel 2 assay. Reported detection of the stx2f

genotype in clinical samples from humans is very rare.

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® Gastro Panel 2.

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.

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.

Fluorescence leakage (cross-talk) may occur in the real-time cycler 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.

Laboratories that refer stx1/stx2 positive results to national VTEC 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.

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.

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

enrichment broth with the EntericBio 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.

Cryptosporidium spp. cuniculus / meleagridis / ubiquitum and Cryptosporidium Horse genotype may be detected in

addition to C. parvum and C. hominis due to significant sequence homology in the probe binding region. Nevertheless,

reports of human infections with these species are rare [20].

EBRT 52 rev 04

In-silico sequence analysis of Giardia canis, Giardia bovis and Giardia cati (assemblage C, D, E, F) suggest that these

may be detected in addition to G. intestinalis due to significant sequence homology in the probe binding region, however

these have not been reported in humans.

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

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

realtime Gastro Panel 2 assay may lead to false negative results. Optimum performance is achieved when testing fresh

samples.

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

The performance of the EntericBio GastroPanel 2 has not been evaluated on immunocompromised individuals and

patients without symptoms of gastroenteritis.

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

EntericBio Gastro Panel 2 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. Li Y. et al. 2009. Molecular detection of all 34 distinct O-antigen forms of Shigella. J Med Microbiol : 58: 69-81.

3. 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.

4. 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.

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

6. 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.

7. 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.

8. 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.

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

10. 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.

11. Health Protection Agency 2008. Investigation of faecal specimens for bacterial pathogens. National Standard Method BSOP 30 Issue

6.1.

12. Koziel M., Corcoran D., O’Callaghan I. Sleator R. D., Lucey B. 2013. Validation of the EntericBio Panel II multiplex PCR system for the

detection of Campylobacter Spp., Salmonella spp., Shigella spp., and verotoxigenic E. coli for use in a clinical diagnostic setting.

Diagn Microbiol Infect Dis. 75: 46–49.

13. Koziel M., Kiely R., Blake L., O’Callaghan I., Corcoran G., Lucey B., Sleator R .D. 2013. Improved detection of bacterial pathogens in

patients presenting with gastroenteritis using the EntericBio Real-Time Gastro Panel I® assay J Clin Microbiol ;51(8):2679-85

14. 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. 47:3449–3453

15. Caccio, S.M., Thompson R. C. A, McLauchlin J., Smith H. V. 2005. Unravelling Cryptosporidium and Giardia epidemiology. Trends in

Parasitology: 21(9), 430-437. Version 1.02 18 19/7/10

16. Budu-Amoako E., Greenwood S. J., Dixon B. R., Barkema H. W., McClure J. T. 2012. Giardia and Cryptosporidium on dairy farms and

the role these farms may play in contaminating water sources in Prince Edward Island, Canada. J Vet Intern Med. 26(3): 668-73.

17. Centers for Disease Control and Prevention. 2012. Cryptosporidiosis Surveillance – United States, 2009-2010 and Giardiasis

Surveillance – United States, 2009-2010. MMWR 61(5).

18. Health Protection Agency 2014. Investigation of specimens other than blood for parasites. UK Standards for Microbiology

Investigation. Standards Unit, Microbiology Services, Public Health England. B31, issue 4.1. 09 May 2014.

19. Herols S., Karch H., Schmidt H. Shiga toxin-encoding bacteriophages – genomes in motion. 2004. Int J Med Microbiol. 294(2-3):115-

21.

20. Robinson G., Elwin K., Chalmers R.M. 2008. Unusual Cryptosporidium genotypes in human cases of diarrhea. Emerg Infect Dis.

14(11):1800-2.

EBRT 52 rev 04

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