entericbio realtime dx - serosep ltd. · the genus shigella consists of four species; s. sonnei (1...

EBRT 63 rev 03 of 22

. EBGPDX, EBSPSA-V2

240

EBRT 63 rev 03

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

Technical support Email: [email protected] Tel: +353 61 358190

EntericBio realtime Dx User Manual

http://www.serosep.com/

mailto:[email protected]


Intended Use

For in vitro diagnostic use. The EntericBio realtime® Dx 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, Shiga-like toxin-producing E. coli (STEC, also known as Verotoxin-producing E. coli,

VTEC) stx1/stx2 , Cryptosporidium parvum/ hominis, Giardia lamblia and Entamoeba histolytica 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., Shiga-like 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, diarrhoea, 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 diarrhoea to dysentery with frequent

small volume faeces containing blood, mucus and pus. The diarrhoea 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 diarrhoea, and may occur in any age group, although it is

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

uremic syndrome (HUS) 7. HUS is a life-threatening disease characterized by thrombocytopenia, haemolytic anaemia, 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 products11.

Yersinia enterocolitica species are divided into 6 biotypes (1A, 1B, 2-5), depending on their genomic characteristics. Main

symptoms of yersiniosis include abdominal pain with fever and diarrhoea and often mimic other diseases such as appendicitis

or Crohn’s disease11, 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.

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. parahaemolyticus14. Most cases of Vibrio-

caused gastroenteritis can be linked to consumption of raw or undercooked seafood15.

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 lari16, 17. 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 (e.g. the Miller-Fisher Syndrome)18.

Salmonella, Shigella, Yersinia enterocolitica and Shiga-like 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

purpose19. Several studies have shown that molecular-based detection methods result in increased detection of

Campylobacter spp. when compared to culture17, 20.

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 labour

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 serogroup20,21.

Cryptosporidium and Giardia are the two leading causes of parasite-associated diarrheal disease22. Symptoms can

include diarrhoea, abdominal cramps, fever, nausea, vomiting and loss of appetite in addition to greasy stools and

flatulence in cases of giardiasis.

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 ruminants22.

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 colonizes and reproduces in the small

intestines of several vertebrates, causing giardiasis, with G. lamblia assemblages A and B most frequently reported in

humans23.

Entamoeba is a genus of single-cell protozoan parasites within which at least six species can be found in

humans among which Entamoeba histolytica is the only pathogenic species24. The symptoms of amoebiasis include

diarrhoea (mild to severe), often with mucus and blood and may occur several months after the initial infection with E.

histolytica24. Furthermore, the infection may become chronic and spread extra-intestinally, mostly to the liver or to the

brain, where it is associated with higher mortality rates24. Microscopic examination of stools does not allow

differentiation between E. histolytica and non-pathogenic E. dispar species as these two are morphologically

indistinguishable25. Therefore, molecular-based detection methods offer increased specificity for the detection of E.

histolytica in patients in a diagnostic setting.

Traditional laboratory methods using light microscopy to identify these parasites tend to be labour intensive and

subjective26 and molecular methods have been shown to improve detection of those parasites in patients27-30.


Test Principle

The EntericBio realtime® Dx 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, Shiga-like

toxin-producing E. coli (STEC) stx1/stx2, Cryptosporidium parvum/ hominis, Giardia lamblia and Entamoeba

histolytica 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 Dx programme. The wells are capped and transferred to the real-time

PCR instrument for automated amplification, detection and analysis with the EntericBio Dx programme. An analytical

run of 32 samples has a turnaround time to result of less than 3 hours with typically 30 minutes hands-on time.

Reagents Provided

EntericBio realtime® Dx (EBGPDX) 240 tests

o PCR strips (4 tests/strip) (10 pouches/24 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® Dx 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 Dx assay is for in vitro diagnostic use only.

o The EntericBio Dx 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.

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. 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 30 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 location A1 and rack B in location B1 on the EntericBio workstation

(Figure 1). Change gloves.

Figure 1. EntericBio workstation layout and PCR strip orientation.

o Select the required number of EntericBio Dx PCR strips (including controls). One strip is sufficient for 4 tests.

Re-seal the pouches correctly promptly after use.

o Load the strips onto the thermorack one after another, without any gaps. Align the strips with the L and R

symbols orientated to the left and right hand side of the thermorack/instrument respectively. Ensure that the

arrows on both sides of the strip point upwards (Figure 2).

Figure 2 Orientation and Alignment of the EntericBio realtime Dx PCR strip on the thermorack.

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 Dx protocol.

o Enter the required number of tests to be processed as per on screen instructions

Orientation Orientation

Sample 1 Sample 2 Sample 3 Sample 4 Left Align Right

Align


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

pipette filter tips in location A2 and PCR strips in location 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.

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 L and R

symbols aligned to the left and right hand side of the adaptor respectively. Place the loaded adaptor onto the

LC480 instrument.

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

strips. Start loading in position A1 of the plate drawer, then fill the drawer from top to bottom and left to right

with L and R symbols aligned to the left and right hand side of the plate drawer respectively.

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.

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 Dx. 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

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.

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 Dx. 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.


ABI 7500 Fast System SDS Software:

o Select New from the File menu. Click Browse and select EntericBio realtime Dx 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.

1. To enter Sample ID, select the SETUP tab. Highlight the wells A7, A8, A9 (Sample 1 and scan the first Sample

ID barcode from the Sample Worklist. Continue scanning samples in the order they are listed in the EntericBio

Sample Worklist.

If it is not possible to scan the barcode, Sample ID can be manually entered and sign on the keyboard can be used to proceed to the next sample

o Positive Control are pre-assigned to position A1, A2 and A3, Negative Control in position A4, A5 and A6

respectively.


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.

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

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 (not differentiated)

STEC/ VTEC

A positive result for STEC/ VTEC indicates the presence of the stx1 and/ or stx2 gene target in the sample (not

differentiated).

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.

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.

Entamoeba

A positive result for Entamoeba indicates the presence of the gene target specific for Entamoeba histolytica in the

sample.

A positive result with Cp >35 may indicate 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

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.


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 Cryptosporidium parvum ATCC PRA-67D

S.agona NCTC 11377 S. flexneri NCTC 8523 Y. enterocolitica 7,8;1A Clinical, IHB Giardia intestinalis WB ATCC 30957D

Giardia intestinalis

Portland-1 ATCC 30888D

Entamoeba histolytica

Rahman BEI NR-179

Entamoeba histolytica

HM-1:IMSS ATCC 30459D


Analytical Specificity (Exclusivity)

Strain Source Strain Source Strain Source Strain Source

Aeromonas hydrophila DSM 17695 Entamoeba histolytica# ATCC 30459D

Pseudomonas

putida DSMZ 291

V. furnissii*B DSM 19622

Alcaligenes faecalis DSM 30030

Enterobacter

aerogenes DSM 30053 Rahnella aquatilis DSM 4594

V. harveiB DSM 19623

Arcobacter butzleri DSM 8739 Enterobacter cloacae DSM 30054

Saccharomyces

cerevisiae DSM 1848

V. mimicus*B DSM 19130

Bacillus cereus DSM 31 Enterococcus faecalis DSM 20478

Salmonella

enteritidis# DSM 17420

V. natrigiens*B DSM 759

Bacillus subtilis DSM 10 Enterococcus faecium DSM 20477

Salmonella

typhimurium# DSM 554

V. pelagiusB DSM 21205

Bacteroides fragilis DSM 2151 Escherichia coli DSM 30083 Serratia liquifaciens DSM 4487 V. proteolyticus*B DSM 30189

Campylobacter coli# DSM 4689 E. coli EPEC DSM 8695

Serratia

marcescens DSM 1608

Yersinia aldovaeB DSM 18303

Campylobacter jejuni# DSM 4688 E. coli ETEC DSM 1103 Serratia odoriferae DSM 4582 Yersinia aleksicaeB DSM 114987

Campylobacter lari# DSM 11375 E. coli STEC# Vircell MBC022 Serratia rubidaea DSM 4480 Yersinia bercovieriB DSM 118528

Candida albicans DSM 1577 Escherichia hermannii DSM 4560 Shigella boydii# DSM 7532 Yersinia entomophagaB DSM 122339

Citrobacter amalonaticus DSM 4593T Ewingella americana DSM 4580 Shigella flexneri# DSM 7532 Yersinia frederinskseniiB DSM 118490

Citrobacter freundi DSM 30039 Giardia intestinalis# ATCC 30888D Shigella sonnei# DSM 5570 Yersinia intermediaB DSM 118517

Citrobacter koseri DSM 4595 Hafnia alvei DSM 30163

Staphylococcus

aureus DSM 20231

Yersinia kristenseniiB DSM 118543

Clostridium difficile DSM 1296 Klebsiella oxytoca DSM 5175

Staphylococcus

epidermidis DSM 20044

Yersinia massiliensisB DSM 121859

Clostridium perfringens DSM 756 Klebsiella pneumoniae DSM 30104

Stenotrophomonas

maltophilia DSM 50170

Yersinia mollarettiB DSM 118520

Clostridium sordelli DSM 2141

Listeria

monocytogenes DSM 20600

Streptococcus

agalactiae DSM 2134

Yersinia nurmiiB DSM 122296

Cronobacter sakazaki DSM 4485 Morganella morganii DSM 30164 Streptococcus bovis DSM 20480 Yersinia pekkaneniiB DSM 122769

Cryptosporidium parvum#

ATCC PRA-

67D Neisseria gonorrhoea DSM 9188

Streptococcus

equinus DSM 20558

Yersinia

pseudotuberculosisB

DSM 18992

Edwardsiella tarda DSM 30052

Plesiomonas

shigelloides DSM 8224

V. alginolyticus*B DSM 2171 Yersinia rhodeiB DSM 118270

Encephaitozoon hellum BEI NR 9701 Proteus mirabilis DSM 4479 V. campbelliiB DSM 19270 Yersinia ruckeriB DSM 118506

Encephalitozoon cuniculi BEI NR 9703 Proteus vulgaris DSM 13387 V. diazotrophicus*B DSM 2604 Yersinia similisB DSM 118211

Encephalitozoon

intestinalis BEI NR9702 Providencia stuartii DSM 4539

V. fischeriB DSM 507

Entamoeba dispar

Clinical

sample VS

Pseudomonas

aeruginosa DSM 50071

V. fluvialis*B DSM 19283

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

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


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® Dx assay.

QCMD distribution panels

QCMD Bacterial (2012-2016) and Parasitic (2014-2016) EQA distribution panels were tested and EntericBio realtime

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

QCMD Bacterial Panel

QCMD Parasitic Panel

QCMD Reference

Sample EntericBio Result

QCMD Reference


QCMD Reference


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

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

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

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

P15-08 E. histolytica Positive P16-01 G. lamblia Positive P16-02 C. parvum Positive

P16-03 G. lamblia Positive P16-04 E. dispar/ B. hominis Negative P16-05 E. histolytica Positive

P16-06 C. parvum Positive P16-07 Negative Negative P16-08 E. histolytica Positive

P16-09 D. fragilis/ B. hominis Negative


UK NEQAS distribution panels 2013-2017

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

EntericBio realtime® Dx results were 100% concordant with expected results.

Specimen Number


Specimen Number


Specimen Number


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

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


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, STEC, Yersinia, Vibrio and Campylobacter

and <15 genome copies for Entamoeba histolytica, Cryptosporidium and Giardia lamblia with a probability of greater

than or equal to 95% by Probit analysis.

Clinical Performance Characteristics

A total of 245 clinical samples were tested by a participating clinical laboratory. Reference method included a

combination of alternative CE-IVD molecular assay (Salmonella, Shigella, Campylobacter, Shiga-like toxin-producing

E. coli, Cryptosporidium, Giardia), culture (Yersinia, Vibrio) and microscopy (Entamoeba).

EntericBio

Pos Neg

Reference

Method

Pos 103 0

Neg 2 149

Sensitivity 1.00 (0.96 – 1.00 95% CI)

Specificity 0.99 (0.95 – 0.99 95% CI)

PPV 0.98 (0.93 – 0.99 95% CI)

NPV 1.00 (0.97 – 1.00 95% CI)

EntericBio Dx Routine Method

Salmonella 9 (9) 9

Shigella 14 (14) 14

Campylobacter 17 (17) 17

STEC 12 (12) 12

Yersinia 20 (20) 20

Vibrio 2 (2) 2

Entamoeba 3 (1)a 1

Giardia 15 (15) 15

Cryptosporidium 13 (13) 13

a) 2 additional E. histolytica were confirmed by sequencing

Numbers in parentheses denote samples in agreement between the two methods.


Limitations of Method

o Results from the EntericBio realtime® Dx 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.

o The EntericBio realtime® Dx assay is not recommended for samples submitted for test of clearance/cure – these

samples should be tested by routine culture methods.

o EntericBio realtime® Dx assay does not differentiate which Campylobacter species (coli or lari or jejuni) is present in the

sample.

o EntericBio realtime® Dx assay does not differentiate which Vibrio species (cholerae or parahaemolyticus) is present in

the sample23-25.

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

o EntericBio realtime® Dx assay does not differentiate which Cryptosporidium species (parvum or hominis) 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® Dx 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® Dx.

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 32-34

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 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. 35


o 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.

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.

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

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

15. 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

http://www.pasteur.fr/sante/clre/cadrecnr/salmoms/WKLM_2007.pdf


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

17. 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.

18. 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.

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

20. 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.

21. 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

22. 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

23. 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.

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

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

25. Entamoeba histolytica and Entamoeba dispar. Bench Aids. DPDx Laboratory diagnosis of amebiasis. Center for Disease Control and

Prevention. Online resources: http://www.cdc.gov/dpdx/resources/pdf/benchAids/Entamoeba_benchaid.pdf

26. Investigation of Specimens other than Blood for Parasites. UK Standards for Microbiology Investigations. Standards Unit, Microbiology

Services, Public Health England: Bacteriology | B 31 | Issue no: 4.1 | Issue date: 09.05.14 | Page: 2 of 48


27. Chiu C. H. and Ou J. T. 1996. Rapid identification of Salmonella serovars in feces by specific detection of virulence genes, invA and

spvC, by an enrichment broth culture-multiplex PCR combination assay. J Clin Microbiol. Oct; 34(10): 2619–2622.

28. Stensvold Cr. R., Nielsen H. V. 2012. Comparison of microscopy and PCR for detection of intestinal parasites in Danish patients supports

an incentive for molecular screening platforms. J Clin Microbiol 50(2): 540-1.

29. Chalmers R. M., Campbell B. M., Crouch N., Charlett a., Davies A. P. 2011. Comparison of diagnostic sensitivity and specificity of seven

Cryptosporidium assays used in the UK. J Med Microbiol 60(Pt 11): 1598-604.

30. ten Hove R., Schuurman T., Kooistra M., Möller L., van Lieshout L., Verweij J. J. 2007. Detection of diarrhoea-causing protozoa in

general practice patients in The Netherlands by multiplex real-time PCR. Clin Microbiol Infect 13(10):1001-7

31. 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 after ingestion of seafood. J Clin Micro 49(6):2348-9.

32. 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.

33. Chitov T, Kirikaew P, Yungyune P, Ruengprapan N, Sontikun K. An incidence of large foodborne outbreak associated with Vibrio

mimicus. Eur J Clin Microbiol Infect Dis. 2009 Apr;28(4):421-4.

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

14(11):1800-2


http://www.cdc.gov/dpdx/resources/pdf/benchAids/Entamoeba_benchaid.pdf



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