Gastrointestinal Multiplex Real-time PCR Panel

The GI pathogen multiplex molecular assay is a multiplex real-time PCR test, able to detect 19 stool bacterial, viral and parasitic pathogens, including: Salmonella species, Shigella/ Enteroinvasive Escherichia coli, Shiga-toxin producing E.coli (STEC), Enteropathogenic E.coli,  Enterotoxigenic E. coli, Enteroaggregative E. coli, Campylobacter species, pathogenic Yersinia enterocolitica, toxin gene carrying Clostridiodes difficile, Rotavirus, Norovirus, Human Adenoviruses, Sapovirus, Astrovirus, Giardia sp., Cryptosporidium sp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica.


The assay is a laboratory developed test by PHML.  Its performance characteristics has been verified according to the accreditation requirements.


Useful For

Detection of common infectious diarrhea pathogens in inpatient and outpatient settings.


Indications for Testing

All patients with a history of non-formed stool that is assumed to be caused by the bacterial, viral, or parasitic agents; especially if the duration is > 7days, with certain warning signs and risk factors such as fever, bloody diarrhea, dysentery, severe abdominal pain, dehydration, and immunocompromised state.


Limits of the Testing

As with all laboratory test interpretation, it is important to interpret in the clinical context.

As this is an assay utilizing a molecular detection technique; the panel will only determine the presence of DNA/RNA of the pathogen. This test is NOT a test of cure, nor a test for viability of the pathogens.

The assay includes the pathogens limited as in the list above. It does not test for all potential infectious agents of diarrheal disease. A negative result will not completely rule out infection in patients with a high pretest probability for gastrointestinal infection.


Testing Algorithm

If toxigenic C. difficile DNA is detected, toxin testing will be performed automatically. 1


Special Instructions and Forms

Not Applicable


Method Name

Multiplex Real-time Polymerase Chain Reaction (RT-qPCR)


Reporting Name


Ordering Mnemonic



Stool testing

Enteric testing

Stool Culture









Diarrheagenic E. coli

E. coli

Traveler’s diarrhea




Cyclospora cayetanensis

E. histolytica

D. fragilis

Specimen Required


Specimen Minimum Quantity Room temperature Refrigerated Frozen
Non-formed Stool in Cary Blair or Enteric Transport media1 0.5 ml of stool No Yes2 Yes3
Non-formed stool in sterile cup (not recommended)4 0.5 ml of stool No Yes2 Yes3


  1. Transport media is available through the NL PHML, order through this website
  2. Refrigerate specimen if it WILL be delivered to the NL PHML within 72 hours
  3. Freeze specimen immediately if transport to the NL PHML is going to be greater or equal to 72 hours
  4. NOT RECOMMENDED. Submitting specimen in sterile cup will reduce viability if reflex bacterial cultures are required for antibiotic susceptibility and surveillance.


Reject Due To

Patient previously tested positive within 7 days

Patient previously tested negative twice within 7 days

Specimen arrived at room temperature

Specimen not labeled

Duplicate specimens collected on the same day

Specimen container over filled

Quantity not sufficient for assay

Specimen received in other transport media

Infectious Diarrhea pathogens can be transmitted in many ways including food and water consumption, contact with fomites, and even droplet ingestion (see chart below).  Additionally, toxigenic C. difficile may be present in a carrier state and the patient’s gastrointestinal flora was disrupted causing C. difficile infection (CDI).  Most commonly this is caused by antibiotics killing off ‘good flora’, however immunosuppression, gastrointestinal surgery and other disturbances can increase risk of CDI.


Chart of the Pathogens of the Gastrointestinal Multiplex Real-time PCR Panel

Shigella/EIEC Risks/Transmission: Direct person-person, contaminated food/water, oro-genital/oro-anal contact, travel to endemic area
Incubation Period: 1 to 7 days (Shigella) & 2-48 hours (EIEC)
Symptoms: Watery diarrhea, bloody/mucoid diarrhea, fever, abdominal pain/cramping, dehydration
EHEC/STEC Risks/Transmission: Undercooked beef, contaminated produce, drinking/recreational water, person-person contact, petting-zoos
Incubation Period: 1 to 9 days
Symptoms: Severe abdominal pain, bloody diarrhea, fever uncommon, may be complicated by Thrombotic Thrombocytopenic Purpura and Hemolytic-Uremic Syndrome (TTP-HUS)
EPEC Risks/Transmission: contaminated food/water, person-person contact
Neonates and young children at higher risk of infection
Incubation Period: 6-48 hours
Symptoms: watery/non-bloody diarrhea, vomiting, low-grade fever. Chronic/untreated infection may lead to severe malnutrition
ETEC Risks/Transmission: travel to endemic area, cruise ships, heavily contaminated/untreated water
Incubation Period: 2-48 hours
Symptoms: Watery diarrhea, severe dehydration
EAEC Risks/Transmission: travel to endemic area, HIV-positive, young children. Mode of transmission unknown
Incubation Period: 8-52 hours
Symptoms: acute, watery diarrhea in children/travellers. May cause chronic, persistent diarrhea in children and HIV-positive individuals
Salmonella Risks/Transmission (non-typhoidal salmonella): contaminated animal products (poultry, ground meat, eggs, dairy), exposure to animals/pets/pet foods, HIV-positive
Incubation Period: 12-48 hours
Symptoms: abdominal cramping, fever, non-bloody diarrhea, bacteremia
chronic carriage in asymptomatic patients may occur
Campylobacter Risks/Transmission: commercial poultry, unpasteurized milk, untreated water, exposure to pets/cattle/animals, direct person-person, oro-genital oro-anal contact
HIV-Positive/immunocompromised patients at higher risk
Incubation Period: 1-7 days
Symptoms: abdominal cramping, fever, myalgias, bloody or watery diarrhea. Bacteremia/endovascular infections
Y. enterocolitica Risks/Transmission: exposure to pigs, undercooked pork, contaminated produce/dairy, untreated water
Incubation Period: 4-7 days
Symptoms: fever, abdominal pain, bloody/watery diarrhea, exudative pharyngitis
May mimic acute appendicitis/mesenteric adenitis
Toxigenic C. difficile Risks/Transmission: antibiotic use/overuse, hospitalization, older age, gastric acid suppression, immunosuppression, inflammatory bowel disease.  May be community acquired without hospitalization
Fecal-oral transmission via ingestion of spores
Incubation Period: Unknown
Symptoms: loose diarrhea, abdominal cramping, leukocytosis. Can present with adynamic ileus, toxic megacolon, and bowel perforation




Reference Values

Not Detected




Clinical Reference

  1. Crobach MJ, Planche T, Eckert C, Barbut F, Terveer EM, Dekkers OM, Wilcox MH, Kuijper EJ. European Society of Clinical Microbiology and Infectious Diseases: update of the diagnostic guidance document for Clostridium difficile infection. Clin Microbiol Infect. 2016;22:S63–81. doi: 10.1016/j.cmi.2016.03.010.
  2. Versalovic, James and Carroll, Karen C. and Funke, Guido and Jorgensen, James H. and Landry, Marie Louise and Warnock, David W.(ed). 2011. Manual of Clinical Microbiology, 10th Edition
Status Days Analytic Time Maximum Laboratory Time Specimen Retention
Routine Daily 5h 2 days 14 days


The laboratory developed multiplex PCR gastrointestinal panel utilized nucleic acid amplification techniques making use of Taqman probes to detect the DNA/RNA of various pathogens.  To determine performance characteristics the test was validated by comparing the assay to a commercial assay, and investigating any discordant results with a secondary commercial PCR.  This modified non-reference standard is used below to determine positive and negative agreement for the assay.


Pathogen Positive Agreement

(95% Confidence Interval)

Negative Agreement

(95% Confidence Interval)

Shigella sp. and Enteroinvasive E. coli 100% (86.3-100%) 100% (98.3-100%)
Shiga-Toxin producing E. coli 100% (89.1-100%) 99.5% (96.9-99.9%)
Enteropathogenic E. coli 100% (90.2-100%) 100% (97.6-100%)
Giardia lamblia 100% (81.8-100%) 100% (97.8-100%)
Enteroaggregative E. coli 93.3% (76.5-98.8%) 100% (97.8-100%)
Enterotoxigenic E. coli 100% (86.3-100%) 100% (97.8-100%)
Dientamoeba fragilis 100% (83.4-100%) 100% (97.8-100%)
Cryptosporidium species 100% (82.2-100%) 100% (97.8-100%)
Cyclospora cayetanensis 100% (79.1-100%) 100% (98.0-100%)
Salmonella species 100% (90.0-100%) 99.2% (96.9-99.9%)
Campylobacter jejuni/coli/lari/ upsaliensis/ hyointestinalis 100% (90.2-100%) 99.6% (97.2-99.9%)
Entamoeba histolytica 100% (79.95-100%) 100% (97.8-100%)
Yersinia enterocolitica 96.8% (81.5-99.8%) 100% (98.2-100%)
Toxigenic Clostridium difficile 100% (94.2-100%) 99.6% (97.2-99.9%)
Rotavirus group A 100% (86.7-100%) 100.0 (98.1-100%)
Norovirus Genogroup I/II 97.6% (85.6-99.9%) 99.6% (97.3-99.9%)
Adenovirus species 100% (82.8-100%) 99.6% (97.5-99.9%)
Astrovirus serotypes 1-8 100% (84.0-100%) 100%  (98.1-100%)
Sapovirus genogroup I-V 100% (82.2-100%) 100%  (98.1-100%)



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