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., Dientamoeba fragilis, Entamoeba histolytica.
The assay is a laboratory developed test by PHML. Its performance characteristics has been verified according to the accreditation requirements.
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.
If toxigenic C. difficile DNA is detected, toxin testing will be performed automatically. 1
Special Instructions and Forms
Multiplex Real-time Polymerase Chain Reaction (RT-qPCR)
Diarrheagenic E. coli
|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|
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.
(95% Confidence Interval)
(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%)|
|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%)|