3 The diagnostic tests

3 The diagnostic tests

The assessment compared 3 intervention tests with 1 comparator.

The interventions

xTAG Gastrointestinal Pathogen Panel

3.1 The xTAG Gastrointestinal Pathogen Panel (Luminex) is a CE‑marked qualitative, highly multiplexed polymerase chain reaction (PCR) test that can simultaneously detect and identify nucleic acids from up to 15 gastroenteritis-causing viruses, parasites and bacteria (see table 1). It can analyse human stool samples that are fresh, frozen or in a holding medium, and the results should be used in conjunction with other clinical and laboratory findings. It is intended to be used in a laboratory setting.

Table 1 Pathogens detected and identified by the xTAG Gastrointestinal Pathogen Panel

Bacteria and bacterial toxins

Viruses

Parasites

Campylobacter

Adenovirus 40/41

Cryptosporidium

Clostridium difficile, toxin A/B

Norovirus GI/GII (genogroup)

Entamoeba histolytica

Escherichia coli O157

Rotavirus A

Giardia

Enterotoxigenic Escherichia coli LT/ST

Shiga-like toxin-producing Escherichia coli stx1/stx2

Salmonella

Shigella

Vibrio cholerae

Yersinia enterocolitica

3.2 The assay uses reverse transcription PCR and the procedure includes 5 distinct phases:

  • pre-treatment of the sample

  • nucleic acid extraction and purification using an automated nucleic acid extraction system

  • broad-range PCR reaction using a thermal cycler

  • bead hybridisation and detection using a thermal cycler

  • data acquisition and analysis (using Luminex 100/200 or MAGPIX analyser).

3.3 Ten microlitres of purified sample are needed for the first broad-range PCR reaction, which amplifies nucleic acids present in the sample. Five microlitres of the broad-range PCR products are then added to a hybridisation and detection reaction, in which target nucleic acids bind to species-specific tagged beads. If pathogen nucleic acid is present, fluorescence is emitted by a streptavidin and R‑Phycoerythin conjugate, which is included in the reaction. Fluorescence intensity is measured by either the Luminex 100/200 or MAGPIX analyser to determine which bacterial, viral or parasitic DNA is present in the sample. Positive and negative controls should be included in each test run. The company recommends that each run includes 3 negative controls (RNase-free water) and at least 1 positive control (known positive samples). The assay also contains an internal control that is added to each sample before extraction and shows whether the assay is functioning as intended.

3.4 The estimated turnaround time for the xTAG Gastrointestinal Pathogen Panel is 5 to 6 hours, including sample preparation time. Up to 96 samples (including controls) can be processed in 1 run, depending on the capacity of a laboratory's PCR thermal cyclers. The test does not give any information on antimicrobial resistance genes or antimicrobial susceptibility.

FilmArray GI Panel

3.5 The FilmArray Gastrointestinal Panel (BioFire Diagnostics) is a CE‑marked qualitative, highly multiplexed PCR test that can simultaneously detect and identify up to 22 pathogens (see table 2) from stool samples in Cary Blair transport media. It is intended for use in a clinical laboratory and the results should be used in conjunction with other clinical and laboratory findings.

Table 2 Pathogens detected and identified by the FilmArray GI Panel

Bacteria

Viruses

Parasites

Campylobacter (jejuni, coli and upsaliensis)

Adenovirus F 40/41

Cryptosporidium

Clostridium difficile (toxin A/B)

Astrovirus

Cyclospora cayetanensis

Plesiomonas shigelloides

Norovirus GI/GII

Entamoeba histolytica

Salmonella

Rotavirus A

Giardia lamblia

Yersinia enterocolitica

Sapovirus (I, II, IV and V)

Vibrio (parahaemolyticus, vulnificus and cholerae)

Vibrio cholerae

Enteroaggregative Escherichia coli

Enteropathogenic Escherichia coli

Enterotoxigenic Escherichia coli lt/st

Shiga-like toxin-producing Escherichia coli stx1/stx2

Escherichia coli O157

Shigella/Enteroinvasive Escherichia coli

3.6 The FilmArray GI Panel is intended for use with the FilmArray and the FilmArray 2.0 system. The FilmArray systems are integrated and include automated sample preparation. The FilmArray system can process 1 sample per hour, whereas the FilmArray 2.0 system allows several FilmArray systems to be linked to process up to 8 samples per hour depending on how many modules are available in a laboratory (1 sample per hour per module). All reagents needed for sample preparation, reverse transcription, PCR and detection are provided freeze-dried in a single-use pouch. Before inserting the reagent pouch into the analyser, the sample is combined with sample buffer and is injected into the pouch along with a hydration solution. After the pouch has been inserted, the system automatically processes a sample through the following stages:

  • nucleic acid purification

  • broad-range reverse transcription PCR

  • second-stage 'nested' PCR with species-specific primers

  • detection with melting curve analysis.

3.7 The system extracts and purifies nucleic acids, which then undergo reverse transcription and are amplified in the first broad-range PCR reaction. A second nested PCR reaction containing species-specific primers is run to detect and identify any pathogens in the sample by fluorescence. Each single-use pouch also contains 2 internal controls: 1 RNA process control assay and 1 control assay for the second-stage PCR. Both controls must be positive for the sample to be reported. Results are reported automatically using the FilmArray software. The test does not give any information on antimicrobial resistance genes or antimicrobial susceptibility.

Faecal Pathogens B assay

3.8 The Faecal Pathogens B assay (AusDiagnostics) is a CE‑marked highly multiplexed PCR test that can detect and identify up to 15 pathogens from nucleic acid extracted from fresh faecal samples. The assay is intended to be used in conjunction with other clinical and laboratory findings. The pathogens that can be identified by the assay are shown in table 3.

Table 3 Pathogens detected and identified by the Faecal Pathogens B assay

Bacteria

Viruses

Parasites

Salmonella species

Rotavirus A

Giardia lamblia (18s)

Shigella species and Shigella/Enteroinvasive Escherichia coli

Norovirus genogroup I

Cryptosporidium (parvus and hominis)

Campylobacter species

Norovirus genogroup II

Entamoeba histolytica (not dispar)

Clostridium difficile

Adenovirus group F and group G

Shiga toxin 1 and 2

Sapovirus

Escherichia coli O157

Astrovirus

3.9 The assay is intended to be used in conjunction with the High-Plex Multiplex Tandem PCR system and the Easy-Plex results software. The assay procedure includes the following processes:

  • nucleic acid extraction and purification

  • broad-range PCR (using the High-Plex MultiPlex Tandem PCR system)

  • real-time PCR with species-specific primers (using the High-Plex Multiplex Tandem PCR system)

  • detection with melting curve analysis.

3.10 In the first PCR step, broad-range primers are used and the product of this reaction is diluted and divided into several real-time PCR reactions, which use nested species-specific primers to detect and identify any pathogens in the sample by fluorescence. Results are reported using the Easy-Plex results software. When multiple pathogens are present in a sample, the software indicates the relative quantitation between the targets, which may allow the relative importance of each detected pathogen to be determined. Each tube used for the broad-range PCR reaction includes an internal positive control (SPIKE), and the company recommends that each run includes both positive and negative (water) controls. Up to 24 samples, including positive controls, can be processed in 1 run. The estimated test turnaround time is 3 to 4 hours. The test does not give any information on antimicrobial resistance genes or antimicrobial susceptibility.

The comparator

3.11 The comparator used in this assessment is the syndromic algorithm for routine testing in sporadic cases from Public Health England's UK standards for microbiology investigations for gastroenteritis and diarrhoea. The standard recommends that, except for requests for a single-organism screen, all samples should be screened for the following pathogens that are commonly associated with gastrointestinal infection:

  • Campylobacter species

  • Salmonella species

  • Shigella species

  • Verocytotoxic Escherichia coli including O157

  • Clostridium difficile (for antibiotic-associated diarrhoea)

  • Cryptosporidium

  • rotavirus (children younger than 5)

  • adenovirus (children younger than 5)

  • norovirus (children younger than 5 and people in hospital).

3.12 The testing pathway incorporates a range of tests including:

  • microbiological culture

  • nucleic acid amplification tests

  • immunoassays

  • microscopy.

3.13 People who have a history of recent travel (to areas other than western Europe, North America, Australia or New Zealand) have additional primary testing for Vibrio and Plesiomonas species by bacterial culture. A 2‑staged testing approach is currently recommended for Clostridium difficile, which involves first testing for glutamate hydrogenase using either a nucleic acid amplification test or enzyme immunoassay. If the test is positive, a sensitive toxin enzyme immunoassay should be done to detect the toxins that cause illness. The syndromic algorithm also notes that laboratories may decide to only test for rotavirus during cooler months when the incidence peaks (November to April). Blood cultures may also be done if a patient is systemically unwell.

3.14 In clinical practice, it is likely that the integrated multiplex PCR tests would be used in conjunction with a toxin assay when Clostridium difficile is detected and with culture when Escherichia coli, Salmonella, Shigella or Yersinia are detected to confirm toxin production or give additional identification or antimicrobial sensitivity information.

  • National Institute for Health and Care Excellence (NICE)