Advantages for Using Multiplex PCR Assays for Certain Infectious Syndromes

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The page below is a sample from the LabCE course PCR Fundamentals: Focus on Multiplex PCR Assay and the Advantages over Singleplex Assays. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Advantages for Using Multiplex PCR Assays for Certain Infectious Syndromes

Multiplex PCR has been exploited in FDA-cleared diagnostic assays for infectious syndromes, such as sepsis/bacteremia (blood stream infections), respiratory (especially viral detection), and gastrointestinal (GI) infections. Appropriate samples are tested on the system and all three reactions - sample preparation (DNA/RNA extraction), amplification, and detection - are performed automatically by the instrument in a closed environment. The rational is for use of these multiplex PCR systems for these infectious syndromes is explained below.
Blood culture identification: The identification of organisms (and certain gene markers of antimicrobic resistance directly from blood culture broth) is both diagnostic and prognostic. The laboratory, working with infectious disease clinicians and the pharmacy (for antimicrobic stewardship), can rapidly modify and treat patients based on results generated within hours of positivity (cite references).
Typically, conventional culture and antimicrobial susceptibility testing (AST) can take up to 72 hours to produce results from a positive blood culture. The benefit of rapid multiplex PCR testing has shown positive patient outcomes due to a reduction in the time to place patients on the appropriate antimicrobics. This extrapolates to a reduction in antibiotic resistance (where judicious use of narrow spectrum drugs are used). Currently, there are several FDA approved systems on the market, with others to follow.
Respiratory panels: Virology testing has been dramatically transformed by molecular methodologies, providing the direct identification of respiratory viruses and select bacterial agents, delivering results in hours, versus days to weeks using conventional testing methods. Respiratory tract infections are one of the most common causes of morbidity and mortality in all age groups. The clinical presentation of different organisms can be similar, making it impossible to reliably predict the causative pathogen based solely on clinical symptoms. The 2020 COVID19 pandemic clearly demonstrated that the rapid and specific diagnosis of viral respiratory infections is important for patient and public health management. Clinicians are becoming more aware that viral respiratory infections other than influenza can be very severe, driving interest in a rapid and comprehensive multiplex PCR test that can detect the most common pathogens. This can benefit patient management, as well as surveillance monitoring of respiratory viral infections. Multiplex PCR for respiratory viral pathogens has replaced the inferior antigen detection assays.
Currently, there are many molecular testing options for influenza, including FDA-cleared assays as a point-of-care test. In addition to the widely available molecular assays designed to detect influenza A (with and without subtyping), influenza B, and/or respiratory syncytial virus (RSV) from nasopharyngeal swabs, multiplex PCR assays that detect multiple respiratory viruses are being used more frequently by clinical laboratories. Currently, there are several FDA-cleared multiplex molecular respiratory viral panels. The benefit to patients has been documented to decrease the duration of antibiotic use, the length of inpatient stay, and time spent in isolation.
GI Panels: Infectious diarrhea can be caused by bacterial, viral, or parasitic pathogens and remains a significant healthcare burden worldwide. Although most GI infections are self-limiting, they can be severe, and even fatal, in young children, the elderly, and other immunocompromised individuals. Because the pathogen cannot be ascertained by clinical presentation, clinicians often order a bacterial culture and also an ova and parasite (O&P) exam on stool samples from patients with diarrhea.
Conventional testing for bacteria and toxins (Shiga toxin in particular) is labor intensive and can take three to five or more days for results. In addition, the O&P exam lacks sensitivity, is time consuming, and requires highly trained personnel for meaningful interpretation and detection of parasites causing diarrhea. Finally, although viral infections are an important cause of GI illness outbreaks, the majority of laboratories do not perform viral culture or antigen testing, except for rotavirus, so the infection goes undetected and potentially spreads. GI panels offer the detection and differentiation of bacterial, viral, and parasitic agents of infection within hours.