Multiplex Molecular Panels for Diagnosis of Gastrointestinal Infection: Performance, Result Interpretation, and Cost-Effectiveness

Abstract

Gastrointestinal disease is a major cause of morbidity and mortality worldwide, especially among young children and immunocompromised patients. Diarrhea may result from infection with a variety of microbial pathogens, including bacteria, viruses, or parasites. Historically, the diagnosis of infectious diarrhea has been made using microscopy, antigen tests, culture, and real-time PCR. A combination of these traditional tests is often required due to the inability to distinguish between infectious etiologies based on the clinical presentation alone. Recently, several multiplex molecular assays have been developed for the detection of gastrointestinal pathogens directly from clinical stool samples. These panels allow for the detection and identification of up to 20 pathogens in as little as 1 h. This review will focus on the multiplex molecular panels that have received clearance from the FDA for the diagnosis of diarrheal disease and will highlight issues related to test performance, result interpretation, and cost-effectiveness of these new molecular diagnostic tools.

FIG 1

Comparison of workflow, throughput, and turnaround time among three commercial, FDA-cleared multiplex GI platforms. (A) The Luminex xTag GPP assay requires an initial sample preparation step (45 to 60 min), followed by nucleic acid extraction (∼45 min) and multiplex PCR using a standard thermocycler (2.5 h). Subsequently, the amplified product is incubated with tagged beads to allow for hybridization (1 h), and, finally, data are acquired and analyzed on either the Magpix or Luminex 100/200 analyzers (∼10 min). (B) The Nanosphere Verigene enteric pathogens (EP) test involves pipetting 200 μl of sample into a supplied extraction tray. The extraction tray, reagents, and test cartridge are then loaded into the Verigene Processor SP (∼2 min). The Processor SP automates nucleic acid extraction, amplification, and hybridization with gold nanoparticles. Finally, the test cartridge is removed from the Processor and inserted into the Verigene Reader for data analysis, with a total turnaround time of ∼2 h per test. (C) The BioFire FilmArray GI panel uses a loading station to facilitate the addition of hydration solution and patient sample into the test pouch via a vacuum-controlled syringe (∼2 min). Once the sample has been added, the test pouch is inserted into the FilmArray instrument, which then automates nucleic acid extraction, nested PCR, and data acquisition using melting curve analysis (∼60 min).