Multiplex PCR to Diagnose Gastrointestinal Infections: Key Limitations in Interpretation

ABSTRACT

Multiplex polymerase chain reaction (PCR) panels are increasingly used for the evaluation of acute diarrhea, providing rapid and sensitive detection of a broad range of enteric pathogens. However, their widespread adoption has created important interpretive challenges for clinicians. Positive results may reflect colonization, prolonged shedding, or organisms of uncertain pathogenicity rather than active infection, and current panels do not provide antimicrobial susceptibility or quantitative information. Incomplete pathogen coverage and false‐positive detections may further complicate result interpretation, particularly in patients with mild or self‐limited disease. Inappropriate test ordering or overinterpretation of results may lead to unnecessary antimicrobial therapy, delayed evaluation of non‐infectious causes of diarrhea, and increased healthcare utilization. This review summarizes the key limitations of multiplex GI PCR panels and provides a practical framework for integrating molecular results with clinical presentation, disease severity, and patient risk factors to support informed management decisions in everyday clinical practice.

1. Introduction

Acute gastroenteritis remains one of the most common reasons for presentation to urgent care centers, outpatient clinics, and emergency departments [1, 2, 3]. Because clinical features are often nonspecific and overlap across infectious and non‐infectious etiologies, laboratory testing plays a central role in diagnostic evaluation, particularly in patients with severe disease, prolonged symptoms, or risk factors for complications [4, 5, 6].

The widespread adoption of syndromic multiplex polymerase chain reaction (PCR) panels has fundamentally altered the diagnostic approach to gastrointestinal (GI) infections [7]. By enabling the simultaneous molecular detection of multiple enteric pathogens from a single stool specimen, these assays have largely replaced conventional diagnostic strategies in many clinical settings. Their increasing use has been driven not only by technical advances in molecular diagnostics but also by operational pressures favoring rapid, comprehensive testing. A range of commercially available nucleic acid amplification test (NAAT) platforms are currently used in clinical laboratories for the diagnosis of gastrointestinal infections. These systems vary in target composition and include, among others, the BioFire FilmArray system [8], QIAstat‐Dx Gastrointestinal Panel [9], xTag Gastrointestinal Pathogen Panel [10], Verigene Enteric Pathogens Panel [11], BioCode Gastrointestinal Pathogen Panel [12], Seegene Allplex Gastrointestinal Panel [13], RIDAGENE Bacterial Stool Panel [14], EasyScreen Enteric Parasite Detection Kit [15], and enteric panels available for the BD MAX system [16, 17, 18, 19]; several of these assays are FDA‐cleared for clinical use in the United States, including the BioFire FilmArray GI Panel, xTAG GPP, and BD MAX enteric panels. The specific molecular targets and primer designs used by these assays are not always fully disclosed by manufacturers, which may limit independent assessment of assay performance and contribute to uncertainty when unexpected or discordant results occur.

However, the transition from traditional methods to highly sensitive (94.5%–100%) [8, 20, 21, 22] molecular panels has shifted the diagnostic challenge from pathogen detection to result interpretation. Molecular identification of enteric organisms does not necessarily imply causation, and positive results may reflect colonization, prolonged shedding, or organisms of uncertain pathogenicity rather than active infection [23]. In addition, multiplex PCR panels do not capture the full spectrum of infectious or non‐infectious causes of diarrhea nor provide ancillary information, such as antimicrobial susceptibility data or clonal typing.

As a result, the clinical impact of multiplex GI PCR testing is determined by how and when these assays are used and how their results are interpreted in the context of the patient's presentation and risk profile. Misinterpretation of multiplex GI PCR results may lead to unnecessary antimicrobial therapy, additional diagnostic testing, delayed evaluation of non‐infectious causes of diarrhea, and increased healthcare utilization. This review examines the key limitations in the interpretation of multiplex GI PCR panel results and discusses their implications for clinical decision‐making (Table 1).

TABLE 1.

Selected interpretive limitations common to multiplex GI PCR panels.

Limitation	Example
Incomplete pathogen coverage	Toxin‐mediated foodborne illness, less common pathogens
No antimicrobial susceptibility	Shigella, Campylobacter
Lack of quantitative data	Viral shedding versus infection
Colonization versus infection	C. difficile, EPEC, EAEC
Detection of low‐pathogenicity organisms	Y. enterocolitica 1A, E. dispar
Assay‐specific false positives	Norovirus (selected platforms), Vibrio

2. Strengths of Multiplex GI PCR Panels

Multiplex GI PCR panels offer several well‐established advantages that have driven their widespread adoption in clinical practice. Compared with conventional stool culture, microscopy, and antigen‐based testing, these assays provide markedly higher analytic sensitivity, enabling more reliable detection of enteric pathogens, including organisms present at low abundance or that are difficult to recover by traditional methods [8, 16, 18, 24]. In addition, multiplex panels deliver rapid diagnostic results, with multiple studies showing that multiplex panels reduce diagnostic turnaround times from days to hours when compared with conventional stool culture, microscopy, and antigen‐based testing [25], as well as reduced utilization of other stool tests per patient [26]. The availability of rapid, organism‐specific results has also been associated with earlier initiation of antimicrobial therapy and a higher likelihood of targeted treatment rather than empiric therapy, highlighting an additional clinical benefit of multiplex testing [27]. In patients with inflammatory bowel disease (IBD), multiplex GI PCR panels are particularly valuable, as identification of an enteric pathogen has been shown to significantly influence management decisions; patients with detected infections are less likely to have IBD therapies initiated or escalated [28], and individuals with IBD are more likely than patients without IBD to have at least one enteric pathogen identified [3]. In this population, multiplex testing may be particularly helpful when patients present with acute worsening of diarrhea, as distinguishing infection from an inflammatory flare can be clinically challenging. Identification of an enteric pathogen may prompt a trial of targeted antimicrobial therapy prior to modification of immunosuppressive treatment, although results should be interpreted cautiously since detection of some organisms may represent colonization rather than the cause of symptoms. Another important strength of multiplex PCR testing is broad pathogen coverage [29] and improved detection of fastidious, slow‐growing, or historically underrecognized pathogens, such as diarrheagenic Escherichia coli and Plesiomonas shigelloides [8, 30, 31]. As a result, multiplex panels have expanded the recognized spectrum of causes of infectious diarrhea and reduced the number of cases previously labeled as idiopathic or gastroenteritis of unknown etiology.

Several studies have also highlighted the utility of multiplex panels in identifying enteric pathogens among populations at increased risk for sexually transmitted enteric infections, such as men who have sex with men (MSM) [27, 32, 33, 34]. In high‐income countries, multiplex PCR testing has additionally contributed to the identification of shigellosis outbreaks among individuals with limited access to hygiene, including persons experiencing homelessness (PEH), enabling faster implementation of treatment, infection control measures and community‐level interventions [35, 36]. Beyond individual patient care, multiplex GI PCR panels have also played an important role in outbreak detection and public health surveillance, including the unexpected recognition of outbreaks caused by Cyclospora cayetanensis and Cryptosporidium spp., which were previously identified only through acid‐fast staining of stool specimens [37, 38], as well as improved detection of Shigella outbreaks, facilitating earlier public health notification and intervention [34, 35, 39]. Finally, the ability of multiplex panels to simultaneously detect multiple pathogens has provided new insights into the epidemiology of mixed infections. In reported studies, coinfections have been detected in 16.4%–27.5% of tested specimens [20, 27, 29]. In a multicenter study by Spina et al., the most frequently observed combination was Campylobacter and enteropathogenic E. coli (EPEC), but triple infections with enteroaggregative E. coli (EAEC), EPEC, and enterotoxigenic E. coli (ETEC) were also reported. EPEC or EAEC was present in 84% of samples with multiple pathogens detected [29]. In clinical practice, detection of mixed bacterial or parasitic infections may influence management decisions and may require combination antimicrobial therapy [27]. Coinfections are now recognized to occur disproportionately in specific at‐risk groups, including MSM, travelers returning from low‐ and middle‐income countries, and PEH [27, 34, 40].

3. Self‐Limited Disease and Implications for Testing

Most episodes of acute infectious diarrhea are self‐limited and resolve with supportive care alone, particularly in otherwise healthy individuals. Consequently, multiplex GI PCR testing is not routinely indicated for all patients with acute diarrhea and is best reserved for those with severe or complicated illness (e.g., bloody diarrhea, systemic symptoms, or persistent disease) or those at increased risk for adverse outcomes, including immunocompromised patients and older adults [4, 6]. Inappropriate use of multiplex PCR panels in low‐risk patients increases the likelihood of detecting incidental or clinically irrelevant organisms, potentially leading to unnecessary treatment, additional testing, or patient concern.

4. Incomplete Pathogen Coverage

Despite their broad design, multiplex GI PCR panels do not capture the full spectrum of infectious causes of acute diarrhea. Important gaps in panel content remain, and awareness of these limitations is essential to avoid misinterpretation of negative results or missed diagnoses in selected clinical scenarios.

Several emerging or less commonly recognized bacterial pathogens are not included in most commercially available panels. These include some species of Aeromonas, non‐jejuni Campylobacter species, Providencia alcalifaciens, Escherichia albertii, Klebsiella oxytoca, and Laribacter hongkongensis [41, 42]. The strength of evidence supporting enteropathogenicity varies among these organisms. While E. albertii has been increasingly recognized as a diarrheal pathogen, the pathogenic potential of others, including certain Aeromonas species and K. oxytoca, may be context‐dependent. Yersinia pseudotuberculosis is an established enteric pathogen that can present with diarrhea, mesenteric lymphadenitis and pseudo‐appendicitis, but is not detected by most multiplex panels [43]. Although individually uncommon, these organisms have been increasingly implicated in diarrheal illness and may be clinically relevant in patients with compatible exposures or epidemiologic risk factors [41, 42]. Their detection continues to rely on targeted culture‐based methods or specialized molecular assays [41].

Coverage limitations also extend to region‐specific pathogens, particularly parasitic organisms endemic in certain geographic areas. With the exception of Giardia, Entamoeba histolytica, Cryptosporidium, and Cyclospora, most parasitic causes of diarrhea—including microsporidia, Strongyloides, and Ascaris—are not detected by multiplex PCR panels [44]. In patients with a relevant travel history, chronic symptoms, or eosinophilia, conventional ova and parasite (O&P) examination remains an essential diagnostic tool [44, 45].

Multiplex PCR panels also fail to identify several important causes of foodborne illness. Pathogens such as Listeria monocytogenes and toxin‐mediated illnesses caused by Staphylococcus aureus, Bacillus cereus, and Clostridium perfringens are not detected by these assays and account for a substantial proportion of acute gastrointestinal disease [46, 47]. Diagnosis of these conditions should be guided by clinical and exposure history and typically requires specialized testing performed in public health or reference laboratories, often involving demonstration of toxin production or detection of the toxin itself rather than identification of the organism alone.

A related interpretive challenge is the potential over‐reliance on a negative multiplex PCR result in evidence against an infectious etiology. Given the pathogens not included in current panels, a negative result should not be equated with the absence of infection when clinical suspicion remains high, and additional diagnostic evaluation may be warranted.

5. Lack of Antimicrobial Susceptibility Information

A major limitation of multiplex GI PCR panels is the absence of phenotypic antimicrobial susceptibility information. While these assays provide rapid organism identification, they do not offer data on antimicrobial resistance, which remains critical for guiding therapy in selected cases of bacterial gastroenteritis and for supporting public health surveillance.

In contrast to multiplex PCR panels used for bloodstream infections or pneumonia, GI panels do not include targets for antimicrobial resistance genes. The lack of susceptibility information may limit their role in definitive treatment selection, particularly in infections where resistance to commonly used agents is prevalent. Rising resistance to macrolides and fluoroquinolones among Shigella and Campylobacter isolates has been reported in several geographic regions, particularly in travelers or in men who have sex with men, making empiric therapy increasingly unreliable in some settings [33, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61]; in such cases, clinicians may be forced to base treatment decisions on local epidemiology in the absence of susceptibility results.

The lack of resistance data has additional implications in the context of sexually transmitted enteric infections, such as shigellosis. In these cases, administration of ineffective antimicrobial therapy may prolong bacterial shedding and facilitate ongoing transmission, with consequences extending beyond the individual patient to the community level. This concern is amplified when organisms detected by PCR fail to grow in culture, precluding subsequent susceptibility testing and limiting opportunities for public health intervention and strain typing to support outbreak investigation.

Together, these considerations underscore the continued importance of reflexive culture and targeted susceptibility testing following positive multiplex PCR results. While molecular panels have streamlined pathogen detection, they cannot replace culture‐based methods for resistance determination, surveillance, and epidemiologic studies. Integrating multiplex PCR testing with complementary diagnostic approaches remains essential for informed clinical management and public health response.

6. Lack of Quantitative Information

Most multiplex GI PCR panels generate qualitative results that report the presence or absence of specific pathogens without providing information on organism burden. The absence of quantitative data makes it difficult to distinguish low‐level carriage from clinically meaningful infection. This challenge is particularly relevant for organisms that may be present in asymptomatic individuals or shed for prolonged periods following symptom resolution.

In other areas of infectious diseases, quantitative measures—such as viral load in HIV, hepatitis B, or cytomegalovirus infection—play a central role in diagnosis, risk stratification, and treatment monitoring. In contrast, multiplex GI PCR panels do not currently provide analogous metrics that could help to contextualize positive results. Cycle threshold (Ct) or crossing point (Cp) values, which might serve as a surrogate measure of organism burden, are not reported to clinicians and lack standardized interpretive thresholds across platforms, although some assays generate such parameters as part of their analytical output, which may occasionally assist in the evaluation of results that do not correspond to the clinical presentation [62]. A previous study showed that higher Ct values on real‐time PCR were significantly lower in cases than in controls for some enteric bacterial pathogens [63], while another study found that the incidence of some bacterial causes of diarrhea was higher when tested by qPCR compared to other non‐quantitative assays [64].

The lack of quantitative information therefore limits the ability of multiplex GI PCR testing to inform clinical decision‐making beyond pathogen identification. Future incorporation of standardized quantitative or semi‐quantitative reporting may help improve discrimination between colonization and active infection, at least for certain pathogens; however, until such approaches are validated, quantitative results must be interpreted cautiously and in conjunction with the clinical presentation.

7. Distinguishing Colonization From Infection

The high analytic sensitivity of multiplex GI PCR panels is a major strength but necessitates a clinical context to distinguish colonization from active infection. Because PCR assays detect microbial nucleic acids rather than viable organisms or toxin production, they cannot reliably differentiate between true infection and colonization.

This challenge is well illustrated by Clostridioides difficile, a well‐recognized example of the inability of laboratory testing to distinguish colonization from disease in which asymptomatic colonization is common, particularly among hospitalized patients and long‐term care residents [65, 66]. Molecular assays, including those used in multiplex PCR panels, detect genes encoding toxins A and B (e.g., tcdA and tcdB), rather than the toxin itself; a positive result may reflect either C. difficile infection (CDI) or colonization with toxigenic C. difficile. PCR positivity alone therefore does not establish disease in the absence of appropriate clinical correlation. Although NAAT is the most sensitive laboratory method for detecting toxigenic C. difficile [67], accurate diagnosis of CDI requires integration of laboratory results with the clinical presentation [68]. Current diagnostic guidelines differ somewhat in their emphasis on toxin detection: European guidance (ESCMID/ECDC) recommends multistep algorithms incorporating toxin assays [69], whereas North American guidance (IDSA/SHEA) allows NAAT‐based strategies in settings with appropriate diagnostic stewardship [70]. Similar interpretive challenges arise in immunocompromised populations, such as hematopoietic stem cell transplant recipients, in whom diarrhea frequently results from non‐infectious causes including conditioning regimens, graft‐versus‐host disease, or medication effects [71]. In these patients, the differential diagnosis is broad and may also include opportunistic infections not detected by standard multiplex panels, such as cytomegalovirus colitis, microsporidiosis, or other parasitic infections, further underscoring the importance of integrating molecular results with clinical context.

Interpretive uncertainty also applies to several diarrheagenic E. coli pathotypes detected by some multiplex PCR panels. EPEC and EAEC are commonly identified in both symptomatic and asymptomatic individuals, particularly in otherwise healthy adults, complicating attribution of causality when detected in isolation [72, 73]. However, published evidence and anecdotal reports suggest that EPEC and EAEC can cause persistent diarrhea in both children and adults, including in high‐income countries [72, 74]. In these cases, careful integration of clinical features (i.e., duration of symptoms), host factors, and epidemiologic context can help to avoid overdiagnosis and unnecessary treatment.

8. Organisms of Uncertain or Low Pathogenicity

Multiplex GI PCR panels may detect organisms whose clinical relevance is uncertain or inherently low, creating additional challenges for result interpretation. Examples include Yersinia enterocolitica biotype 1A and Entamoeba dispar. Y. enterocolitica biotype 1A lacks key virulence determinants found in pathogenic Y. enterocolitica strains, including the virulence plasmid (pYV) and associated genes required for epithelial invasion and toxin production [75]. Because molecular assays detect targets that are not directly linked to these virulence determinants, positive PCR results cannot distinguish Y. enterocolitica biotypes. Biotype 1 A is generally regarded as nonpathogenic or of minimal pathogenic potential despite its inclusion as a reportable target on some multiplex PCR panels. Detection of this organism may therefore represent transient colonization or incidental carriage rather than the cause of clinical disease.

Similarly, although multiplex PCR panels are designed to detect Entamoeba histolytica, assay cross‐reactivity with the closely related nonpathogenic species Entamoeba dispar has been reported, particularly when E. dispar is present at higher concentrations [76]. E. dispar has been described as an intestinal colonizer, including among MSM in which enteric protozoa may be detected in the absence of invasive disease [77]. In such patients, molecular detection of E. histolytica without a compatible clinical presentation may reflect E. dispar colonization or non‐invasive infection rather than true amebiasis [77]. These findings underscore the importance of distinguishing pathogenic E. histolytica from non‐pathogenic Entamoeba species when interpreting molecular test results. More broadly, the detection of organisms with low or uncertain pathogenic potential should be interpreted cautiously and correlated with the clinical presentation and epidemiologic context before attributing symptoms to the identified organism.

9. Hybrid and Overlapping Pathotypes in Diarrheagenic E. coli

Diarrheagenic Escherichia coli are traditionally classified into six major pathotypes based on the presence of specific virulence determinants: enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shiga toxin–producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), and diffusely adherent E. coli (DAEC) [78]. Commercial multiplex GI PCR panels detect selected virulence genes associated with several diarrheagenic E. coli pathotypes (e.g., STEC, ETEC, EPEC, EAEC, and EIEC), but no currently available panel targets all recognized pathotypes, and DAEC is generally not included. Although STEC, ETEC, and EIEC are well‐established causes of diarrheal disease, the clinical significance of other pathotypes—particularly EAEC, DAEC, and some EPEC strains—remains less certain, as these organisms are frequently detected in asymptomatic individuals.

The increasing recognition of hybrid diarrheagenic E. coli strains highlights an important interpretive limitation of multiplex GI PCR panels. These assays detect individual virulence genes used to define traditional E. coli pathotypes rather than complete strains or functional pathogenicity. Consequently, organisms harboring virulence determinants from multiple pathotypes, such as EAEC and EPEC, may be reported as overlapping detections though the clinical relevance of such hybrids remains incompletely understood.

Hybrid strains driven by combinations involving Shiga toxin (e.g., EPEC/STEC or EAEC/STEC hybrids carrying stx) have been implicated in severe cases of diarrhea, including E. coli O157:H7 and the German outbreak serotype E. coli O104:H4 that are frequently complicated by hemolytic‐uremic syndrome [79, 80]. Furthermore, hybrid EPEC/EAEC strains causing acute diarrhea have been reported in Brazil, but information regarding the impact on illness severity is not available [81]. Because multiplex PCR testing does not assess virulence gene expression, toxin production, invasiveness, or organism burden, distinguishing clinically meaningful infection from incidental detection remains challenging.

10. False‐Positive Detections

In addition to the general interpretive challenges discussed above, several assay‐ or target‐specific limitations have been reported for particular multiplex GI PCR platforms. False‐positive detections have been reported for selected targets on multiplex GI PCR panels, most notably for the norovirus assay on the BioFire FilmArray GI Panel [82], prompting both institutional mitigation strategies, through confirmatory testing using alternative singleplex molecular assays, and regulatory review. Manufacturer‐initiated post‐marketing performance follow‐up studies demonstrated preserved clinical sensitivity but reduced specificity compared to the original studies (negative percent agreement 96.5% vs. 98.8%) [83], reinforcing concerns regarding analytic false positivity and supporting recommendations for confirmatory testing when results are discordant with the clinical presentation or outside the usual seasonal incidence.

In parallel, it is important to recognize that not all apparent false‐positive norovirus results reflect assay failure. Prolonged viral shedding represents a major contributor to clinically misleading PCR positivity. Norovirus RNA may remain detectable in stool for weeks after symptom resolution, particularly in young children, older adults, and immunocompromised individuals [84, 85, 86]. In addition, norovirus can be detected in asymptomatic individuals [87], further complicating interpretation in patients with alternative explanations for diarrhea. In these settings, a positive PCR result may reflect prior or incidental infection rather than the cause of current symptoms.

Similar interpretive challenges have been described for Campylobacter. In a recent study, the implementation of the BioFire FilmArray GI Panel was associated with an increase in Campylobacter detections that were not confirmed by stool culture (culture confirmation occurred in 62% of BioFire detections, compared with 78% of detections using other culture‐independent methods, p < 0.05), prompting analysis of melt curve data [88]. Specimens with PCR‐positive/culture‐negative results frequently exhibited atypical melt curve patterns compared with PCR‐positive/culture‐positive samples, leading to the suggestion that some detections may represent false positives. However, it is also possible that some atypical melt curves reflect amplification of variant Campylobacter sequences that differ from the reference strains used to define standard melt curve signatures, and some culture‐negative cases may represent fastidious strains not culturable on selective media. Of note, a recent study found irregular amplification curves on the BD MAX Extended Enteric Bacterial Panel that were not confirmed by culture for Vibrio spp. and Yersinia enterocolitica [89], while another study on the BD MAX Enteric Bacterial Panel showed that Campylobacter spp. detections that were not confirmed by culture had higher Ct values than confirmed ones [90]. Some false‐positive Vibrio detections may result from DNA contamination of holding media, which includes agar as a component [91]. Because the multiplex assay detects Vibrio spp. at the genus level, these detections cannot be attributed to specific pathogenic Vibrio species. Further research is needed to distinguish which atypical curves represent analytic false‐positive detections and which reflect true molecular identification of genetically divergent or fastidious bacterial strains.

11. Strategies to Improve Interpretation and Clinical Use

Optimizing the clinical impact of multiplex GI PCR panels requires a diagnostic stewardship approach that addresses both appropriate test utilization and thoughtful result interpretation. A proposed algorithm for the interpretation of multiplex GI PCR panel results is shown in Figure 1. Table 2 summarizes practical considerations for clinicians interpreting multiplex GI PCR results, including guidance on appropriate test ordering, interpretation of positive and negative findings, use of complementary diagnostic testing, and the role of diagnostic stewardship. Testing should be targeted to clinical scenarios in which results are likely to influence management and interpreted in the context of patient presentation and epidemiologic risk. At the same time, syndrome‐based multiplex testing strategies may identify unexpected pathogens that would not otherwise be considered on clinical grounds alone, including community‐acquired Clostridioides difficile infection. These approaches may therefore contribute not only to individual patient management but also to improved detection of atypical or emerging pathogens and to a better understanding of the epidemiology of enteric infections. Figure 2 highlights patient populations and clinical scenarios in which multiplex GI PCR testing is most likely to affect clinical decision‐making.

FIGURE 1.

Proposed algorithm for the interpretation of multiplex GI PCR panels. Created with BioRender.com.

TABLE 2.

Practical tips for gastroenterologists: Interpreting multiplex GI PCR panel results.

Order testing selectively Reserve multiplex GI PCR panels for patients with severe, persistent, or bloody diarrhea, systemic illness, immunocompromised status, or public health concerns; avoid routine testing in mild, self‐limited diarrhea.

Interpret positive results in clinical context PCR detects microbial nucleic acids rather than viable organisms or toxin; positive results may reflect colonization, prolonged shedding, or organisms of uncertain pathogenicity.

Do not equate a negative panel with absence of infection Multiplex panels do not detect all enteric pathogens (e.g., toxin‐mediated foodborne illness, some parasites); further evaluation may be warranted when clinical suspicion remains high.

Use complementary testing when indicated Consider reflex stool culture for susceptibility testing and public health surveillance in patients with bacterial gastroenteritis, and ova and parasite examination in patients with relevant travel or exposure history.

Collaborate and steward diagnostic resources Engage the microbiology laboratory or infectious diseases specialists for discordant or complex results and apply diagnostic stewardship principles to minimize overdiagnosis and unnecessary treatment.

FIGURE 2.

When to use multiplex GI PCR testing. Created with BioRender.com.

Reflex testing algorithms remain an important adjunct to molecular diagnostics. For selected bacterial pathogens, follow‐up stool culture enables antimicrobial susceptibility testing, strain characterization, and public health surveillance, helping to offset key limitations of PCR‐based assays. Close collaboration among infectious diseases specialists, gastroenterologists, and the microbiology laboratory further supports consistent test ordering practices, meaningful result reporting, and interpretation of complex or ambiguous findings.

12. Conclusions

Multiplex PCR panels have become a cornerstone of gastrointestinal infection diagnostics, providing rapid and sensitive detection of a broad range of enteric pathogens. However, their high analytic sensitivity and wide scope introduce interpretive challenges, including incomplete pathogen coverage, absence of antimicrobial susceptibility data, detection of colonization or low‐pathogenicity organisms, and lack of quantitative information. Some of these challenges are inherent to multiplex molecular testing in general, whereas others reflect assay‐ or target‐specific limitations observed with particular diagnostic platforms.

Clinical judgment remains central to the effective use of these assays. Thoughtful test selection, integration of molecular results with a clinical context, and application of diagnostic stewardship principles are essential to maximizing benefit. Recognizing these limitations allows clinicians to use multiplex GI PCR results more effectively to guide management and avoid unnecessary interventions across clinical settings.

Conflicts of Interest

GST and FCF did not receive payment for their work on this manuscript. GST (AI182462) and FCF (AI150041, AI160130) are recipients of support from the National Institutes of Health. FCF has been a paid consultant for bioMérieux and Abbott Diagnostics.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.