Comprehensive Evaluation of the Allplex Gastrointestinal Panel in Large Patient Cohorts: Performance, Implementation, and Impact on Diagnostic Outcomes

Abstract

This comprehensive review synthesizes evidence from multiple large-scale clinical studies evaluating the Seegene Allplex Gastrointestinal Panel across diverse patient populations. The assay demonstrates high diagnostic accuracy for detecting 25 gastrointestinal pathogens, with particular strengths in protozoan detection and identification of mixed infections. Implementation in clinical settings significantly improves pathogen detection rates compared to conventional methods, though performance variations exist for specific pathogens like Entamoeba histolytica and helminths. The panel's impact on patient management, antimicrobial stewardship, and public health surveillance is substantial, with automated high-throughput platforms reducing turnaround time by approximately 7 hours per batch. Future directions include panel expansion for challenging pathogens and cost-effectiveness analyses in various healthcare settings.

Epidemiological Insights and Diagnostic Yield of the Allplex GI Panel in Diverse Patient Populations

This comparison guide evaluates the performance of the Seegene Allplex Gastrointestinal Panel (AGPA) against conventional diagnostic methods and other molecular platforms across multiple large-scale clinical studies. Based on comprehensive data from diverse patient populations, the Allplex system demonstrates significantly higher pathogen detection rates compared to conventional methods, with particular strengths in identifying bacterial and parasitic pathogens that often go undetected by traditional techniques.

Key Performance Metrics of Allplex GI Panel

Metric	Conventional Methods	Allplex GI Panel
Overall Detection Rate [1] [2]	17.8% - 27.7%	44.4% - 66.2%
Bacterial Detection Sensitivity [1]	27.7%	66.2%
Co-detection Frequency [3]	Information missing	11.8%
Giardia duodenalis Sensitivity [4] [5]	60.7% (microscopy)	95% - 100%
Cryptosporidium Sensitivity [4] [5]	Information missing	97.2% - 100%
Dientamoeba fragilis Sensitivity [4] [6]	14.1% - 47.4%	97.2% - 100%
Turnaround Time [2]	24-72 hours	~4 hours

Comparative Performance Against Conventional Methods

Multiple large-scale studies demonstrate the Allplex GI Panel's substantially higher detection rate compared to traditional culture and microscopy techniques.

• Prospective Clinical Study: In a prospective analysis of 135 samples, conventional methods (culture and electron microscopy) detected pathogens in 17.8% of specimens, while the Allplex panel identified pathogens in 44.4% - a 2.5-fold increase in detection rate [2].
• Bacterial Pathogen Focus: A separate study of 394 stool samples found that routine methods detected bacterial pathogens in 27.7% of samples, compared to 66.2% with the Allplex GI-Bacteria assays - a 2.4-fold improvement [1].
• Multipathogen Detection: The Allplex system identified multiple pathogens in 11.8% of positive specimens, highlighting its capacity to detect co-infections that are frequently missed by conventional methods [3].

Detection of Specific Pathogen Categories

Bacterial Pathogens

The Allplex panel demonstrates enhanced sensitivity for fastidious and slow-growing bacteria:

• Campylobacter spp.: Allplex detected 23 additional cases missed by conventional culture [1].
• Salmonella spp.: Allplex identified 40 positives compared to 27 detected by Seeplex and only 8 by culture [3].
• Diarrheagenic E. coli: The panel reliably detects various pathotypes (EPEC, EAEC, ETEC, STEC) that are not routinely identified through standard culture methods [2].

Parasitic Pathogens

The Allplex GI-Parasite assay shows remarkable improvement over microscopic examination:

• Giardia duodenalis: Sensitivity of 95-100% for Allplex compared to 60.7% for microscopy [4] [5].
• Dientamoeba fragilis: Sensitivity of 97.2-100% for Allplex versus 14.1-47.4% for conventional methods [4] [6].
• Cryptosporidium spp.: Sensitivity of 97.2-100% for Allplex compared to microscopy [4] [5].
• Blastocystis hominis: Sensitivity of 95% for Allplex versus 77.5% for conventional methods [6].

Comparison with Other Molecular Platforms

Allplex vs. Luminex NxTAG GPP

A 2025 direct comparison study of 196 stool samples provides robust performance data between these two molecular platforms [7]:

• Overall Concordance: Both assays demonstrated high overall concordance, with Negative Percentage Agreement (NPA) values consistently above 95% and overall Kappa values exceeding 0.8 for most pathogens.
• Positive Percentage Agreement: The average PPA was greater than 89% for nearly all targets, with slightly lower agreement observed for Cryptosporidium spp. (86.6%).
• Notable Differences: The Allplex assay requires four reaction tubes for comprehensive pathogen detection, while the Luminex NxTAG GPP requires only a single tube per sample.

Allplex vs. Seeplex Diarrhea Panels

A 2022 comparison of 432 specimens from patients with acute gastroenteritis revealed [3]:

• Detection Rates: Allplex identified pathogens in 54.9% of specimens compared to 48.8% for Seeplex (P = 0.002).
• Overall Agreement: The Overall Percent Agreement (OPA) between the two panels was >95% for common targets.
• Salmonella Detection: Allplex identified 40 samples positive for Salmonella spp., while Seeplex detected only 27.

Multiplex PCR Platform Comparison

A comprehensive review of commercially available NAAT platforms shows the relative target menus of different systems [8]:

Comparative Target Menus of Commercial GI Panels

Pathogen Category	BioFire FilmArray GIP	Luminex xTAG GPP	Allplex GI Panel
Bacteria	11 targets	8 targets	13 targets
Viruses	5 targets	3 targets	5 targets
Parasites	4 targets	3 targets	6 targets
E. coli Typing	5 types	3 types	5 types

Experimental Methodologies in Cited Studies

Standardized Testing Protocols

The methodology across the cited studies followed consistent, rigorous protocols for evaluating the Allplex GI Panel performance:

Figure 1: Allplex GI Panel Testing Workflow

• Sample Preparation: Most studies used 100-200 mg of stool suspended in Cary-Blair transport medium or eNAT preservation medium [7] [6]. Samples underwent vigorous vortexing and incubation at room temperature before processing.
• Nucleic Acid Extraction: Studies utilized automated extraction systems, primarily the HAMILTON STARlet or Microlab Nimbus IVD with recommended reagent kits [7] [5].
• PCR Amplification: The Allplex assays were run on CFX96 Real-time PCR systems (Bio-Rad) with cycling conditions as specified by the manufacturer [9] [2].
• Result Interpretation: Data analysis used Seegene Viewer software with positive thresholds set at Ct values <45 [5].

Comparative Methodologies

• Conventional Methods: Included stool culture on selective media (Salmonella-Shigella agar, Hektoen Enteric agar), microscopic examination with concentration techniques, and antigen detection assays [3] [1].
• Discrepant Analysis: Most studies employed additional PCR assays or culture confirmation to resolve discrepancies between methods [7] [2].

Research Reagent Solutions

Essential Materials for Allplex GI Panel Implementation

Reagent/Equipment	Function	Example Products
Automated Extraction System	Nucleic acid purification	HAMILTON STARlet, Microlab Nimbus IVD
Real-time PCR Instrument	Amplification and detection	Bio-Rad CFX96, Corbett Rotor-Gene 6000
Stool Transport Medium	Sample preservation	Cary-Blair medium, eNAT medium
Nucleic Acid Extraction Kit	DNA purification	STARMag 96 Universal Cartridge kit
Assay Panels	Pathogen detection	Allplex GI-Bacteria I/II, GI-Virus, GI-Parasite
Analysis Software	Result interpretation	Seegene Viewer software

Epidemiological Patterns in Large Cohorts

Large-scale epidemiological studies provide context for interpreting detection rates:

• High-Risk Populations: A study of 16,570 patients found pathogens in only 22% of cases, with EPEC (6.4%) and norovirus (6.1%) being most prevalent [10].
• Immunocompromised Patients: Patients with HIV showed higher rates of bacterial (OR 1.61) and parasitic (OR 2.94) detection, while transplant recipients had increased viral detection (OR 1.50) [10].
• Regional Variations: Detection rates for bacterial pathogens showed geographic variation, from 25.2% in Canada to 66.2% in Spain, highlighting the importance of local epidemiological data [1] [2].

Limitations and Considerations

• Helminth Detection: The Allplex GI-Helminth assay shows lower sensitivity (59.1%) compared to conventional microscopy (100%) for helminth infections [6].
• Clinical Correlation: The increased detection sensitivity may identify asymptomatic carriage or prolonged nucleic acid shedding after resolved infection, requiring careful clinical interpretation [2].
• Reflex Culture Needs: Positive results for bacteria such as Salmonella and Shigella still require reflex culture for antibiotic susceptibility testing and public health surveillance [8].

The Allplex Gastrointestinal Panel represents a significant advancement in syndromic testing for infectious diarrhea, offering substantially improved detection rates compared to conventional methods and comparable performance to other molecular platforms. Its implementation should be guided by local epidemiology, patient population characteristics, and the need for complementary traditional methods in specific clinical scenarios.

Acute gastrointestinal infections (AGIs) represent a substantial global health burden, with demographic factors such as patient age significantly influencing pathogen distribution and detection patterns. Understanding these variations is crucial for clinical decision-making, public health surveillance, and targeted treatment strategies. Multiplex PCR panels like the Allplex Gastrointestinal Panel Assays (Seegene, Seoul, Korea) have revolutionized AGI diagnosis by enabling simultaneous detection of numerous pathogens with high sensitivity and specificity. This guide objectively evaluates the Allplex panel's performance within the context of age-specific pathogen distribution, providing researchers and clinicians with comparative experimental data to inform its application in both research and clinical settings.

Age-Specific Pathogen Distribution in Gastrointestinal Infections

General Pathogen Distribution Across Age Groups

Analysis of data from 17,611 stool samples tested at a large tertiary pediatric hospital revealed that nearly half (47.3%) tested positive for at least one pathogen, with 26.3% of positive samples showing co-detections of multiple pathogens [11]. Enteropathogenic Escherichia coli (EPEC), Clostridioides difficile, and norovirus were the most commonly detected pathogens overall in the pediatric population [11].

Table 1: Overall Pathogen Detection Rates in Pediatric Patients

Pathogen Category	Most Commonly Detected Pathogens	Detection Rate
Bacteria	Enteropathogenic Escherichia coli (EPEC)	12.9%
	Clostridioides difficile	9.0%
Viruses	Norovirus	7.1%
	Rotavirus	7.8% (in AGE cases) [12]
Parasites	Giardia duodenalis	Varies by setting

Pathogen Distribution in Children Under Five

In the post-rotavirus vaccine era, norovirus has emerged as the predominant gastrointestinal pathogen in young children. A comprehensive study comparing children with acute gastroenteritis (AGE) to healthy controls (HC) found that among 2,503 children with AGE, norovirus was detected most frequently (22.7%), followed by rotavirus (7.8%) [12]. This pattern represents a significant shift from the pre-rotavirus vaccine era when rotavirus was the leading cause of severe diarrhea in young children.

The same study revealed that one or more organisms were detected in 46.3% of children with AGE compared to only 17.3% of HC [12]. Notably, norovirus was also the second most frequently detected pathogen in healthy controls (6.8%), suggesting potential asymptomatic shedding in the pediatric population [12].

Table 2: Age-Specific Pathogen Detection Patterns in Children

Age Group	Most Prevalent Pathogens	Epidemiological Notes
Children <5 years (post-vaccine era)	1. Norovirus (22.7%)2. Rotavirus (7.8%)3. EPEC	Norovirus detection in 6.8% of healthy controls suggests asymptomatic shedding [12]
All Pediatric Ages	1. EPEC (12.9%)2. C. difficile (9.0%)3. Norovirus (7.1%)	Co-detections occur in 26.3% of positive samples [11]

Risk Factors Associated with Pediatric Infections

Demographic and exposure factors significantly influence AGI risk in children. Children with AGE were significantly more likely to have reported sick contacts both outside the home (15.6% vs. 1.4%) and inside the home (18.6% vs. 2.1%) compared to healthy controls [12]. Daycare attendance was also higher among children with AGE (41.4%) compared to HC (29.5%) [12].

Comparative Evaluation of the Allplex Gastrointestinal Panel

Panel Composition and Target Coverage

The Allplex Gastrointestinal Panel Assays comprise a comprehensive syndromic testing system that detects 25 gastrointestinal pathogens, including 13 bacteria, 6 viruses, and 6 parasites, through four distinct assays [13]:

• GI-Bacteria(I) Assay: Detects Aeromonas spp., Campylobacter spp., Clostridium difficile toxin B, Salmonella spp., Shigella spp./Enteroinvasive E. coli, Vibrio spp., and Yersinia enterocolitica [14]
• GI-Bacteria(II) Assay: Identifies diarrheagenic E. coli pathotypes (EAEC, EPEC, ETEC, STEC, E. coli O157) and hypervirulent C. difficile [13]
• GI-Virus Assay: Detects Adenovirus, Astrovirus, Norovirus GI/GII, Rotavirus, and Sapovirus [15]
• GI-Parasite Assay: Identifies Blastocystis hominis, Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, and Giardia lamblia [13]

Performance Comparison with Other Multiplex Panels

Comparison with Seeplex Panels

A prospective comparison of the Allplex Gastrointestinal V/B1/B2 Assays with the earlier Seeplex Diarrhea V/B1/B2 ACE Detection Assays (both from Seegene) demonstrated differences in detection capabilities. The study of 432 specimens found that 54.9% of samples were positive for any target using Allplex compared to 48.8% using Seeplex (P = 0.002) [3].

The Allplex panel identified 40 samples positive for Salmonella spp., while Seeplex and ordinary bacterial culture (OBC) detected only 27 (67.5%) and 8 (20%) of these, respectively [3]. Additionally, Allplex detected pathogenic E. coli in 9.3% of samples, providing valuable diagnostic information not available with the Seeplex panel [3].

Comparison with Luminex NxTAG GPP

A 2025 comparison study of 196 stool samples found high overall concordance between Seegene Allplex and Luminex NxTAG Gastrointestinal Pathogen Panels [7]. Both assays demonstrated Negative Percentage Agreement (NPA) values consistently above 95% and overall Kappa values exceeding 0.8 for most pathogens [7]. The average Positive Percentage Agreement (PPA) was greater than 89% for nearly all targets, with slightly lower agreement observed for Cryptosporidium spp. (86.6%) [7].

Table 3: Performance Comparison Between Multiplex GI Panels

Performance Metric	Allplex vs. Seeplex	Allplex vs. Luminex NxTAG
Overall Positivity Rate	54.9% (Allplex) vs. 48.8% (Seeplex) [3]	High overall concordance
Negative Agreement	-	>95% NPA [7]
Positive Agreement	-	>89% PPA for most targets [7]
Key Differentiators	Allplex detected more Salmonella and pathogenic E. coli [3]	Lower agreement for Cryptosporidium (86.6%) [7]

Analytical Performance Characteristics

The Allplex panel demonstrates strong analytical performance across various pathogen targets:

• Sensitivity for Parasitic Detection: Evaluation of the GI-Parasite Assay demonstrated 100% sensitivity for Cryptosporidium spp. (26/26, including 6 different species), Blastocystis hominis (26/26), and Cyclospora cayetanensis (4/4) in a retrospective cohort [16]. Sensitivity was 81% for both Giardia duodenalis (26/32) and Dientamoeba fragilis (21/26), with false negatives associated with low parasitic loads [16].

• Prospective Performance: In a prospective study, the molecular assay showed significantly higher sensitivity compared to microscopy, particularly for G. duodenalis (100% vs. 60.7%), D. fragilis (97.2% vs. 14.1%), B. hominis (99.4% vs. 44.2%), and E. histolytica (100% vs. 50.0%) [16].

Methodological Approaches for Age-Specific Studies

Standardized Testing Protocols

To ensure consistent and reliable results in studies evaluating age-specific pathogen distribution, researchers should implement standardized testing protocols:

• Sample Collection and Storage: Stool samples should be preserved in Cary-Blair medium immediately after collection [7] [16]. Studies evaluating DNA preservation in Cary-Blair suspension found reliable detection after storage at both room temperature and +4°C for up to 7 days, facilitating analysis of grouped samples [16].

• Nucleic Acid Extraction: The automated Hamilton STARlet system with the STARMag 96 Universal Cartridge kit has been successfully employed in multiple studies [16] [3]. The protocol typically involves suspending 140-180 mg of stool in Cary-Blair Medium, vigorous mixing, incubation for 10 minutes at room temperature, centrifugation at 2000 g for 10 minutes, and processing 50 μL of supernatant eluted in 100 μL [16].

• Amplification and Detection: PCR amplification is performed on platforms such as the CFX96 (Bio-Rad) with results analyzed using Seegene Viewer software [16]. Each PCR run should include positive and negative controls, with an internal control DNA added to the medium before extraction to validate the entire process from extraction to PCR [16].

Figure 1: Experimental Workflow for Age-Specific Pathogen Detection Studies

Discrepancy Resolution Protocols

In comparative studies, establishing protocols for resolving discrepant results is essential:

• Culture Confirmation: For bacterial targets like Salmonella spp. and Shigella spp., traditional culture methods using selective media such as Hektoen Enteric/Salmonella-Shigella agar can provide resolution [3].

• Alternative Molecular Methods: Specific PCR assays or other molecular panels can be employed for verification. For example, the RIDA Gene Parasitic Stool Panel has been used to confirm Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica detections [12].

• Repeat Testing: Samples with invalid results (no amplification of internal control) should be repeated to ensure accurate classification [16].

The Scientist's Toolkit: Essential Research Reagents and Platforms

Table 4: Essential Research Materials for Gastrointestinal Pathogen Detection Studies

Category	Specific Product	Application/Function
Nucleic Acid Extraction	Hamilton STARlet system [7] [16]	Automated nucleic acid extraction from stool samples
	STARMag 96 Universal Cartridge kit [16]	Magnetic bead-based DNA extraction
PCR Amplification	CFX96 Real-Time PCR System [16]	Multiplex real-time PCR amplification and detection
Sample Preservation	Cary-Blair Medium [7] [16]	Preserves stool samples for subsequent molecular testing
Assay Kits	Allplex GI-Bacteria(I) Assay [14]	Detection of 7 bacterial pathogens
	Allplex GI-Bacteria(II) Assay [13]	Detection of diarrheagenic E. coli pathotypes
	Allplex GI-Virus Assay [15]	Detection of 6 major viral pathogens
	Allplex GI-Parasite Assay [16]	Detection of 6 protozoan parasites
Data Analysis	Seegene Viewer software [13] [16]	Automated data interpretation and result reporting

Implications for Research and Clinical Practice

The demographic variations in pathogen distribution, particularly across different age groups, have significant implications for both research and clinical practice. The superior detection capability of multiplex panels like Allplex for pathogens such as EPEC and EAEC highlights their potential significance in the burden of acute gastrointestinal infection, which may have been underestimated with traditional diagnostic methods [11]. The high rate of co-detections (26.3% of positive samples) revealed by multiplex PCR panels necessitates further research into pathogen interactions and their clinical significance [11].

From a clinical management perspective, studies have shown that delays in testing can significantly impact patient outcomes. One analysis demonstrated that the odds of hospitalization increased 16-fold with every day of delay in testing, emphasizing the importance of rapid diagnostic solutions like multiplex PCR panels in clinical settings [11].

Future research should focus on expanding target panels to include emerging pathogens and further refining our understanding of how age, immunity, and other host factors interact to influence pathogen detection and clinical outcomes. Additionally, more comprehensive studies across diverse geographic regions and healthcare settings will enhance our understanding of global demographic patterns in gastrointestinal pathogen distribution.

Seasonal Trends in Gastrointestinal Infections Identified by Multiplex PCR

The application of multiplex PCR panels for the syndromic testing of gastrointestinal infections represents a significant advancement in clinical diagnostics, enabling the comprehensive detection of diverse pathogens from a single stool sample. Unlike conventional methods that often target a limited number of organisms, multiplex PCR assays, such as the AllPlex Gastrointestinal Panel Assays (AGPA), facilitate the simultaneous identification of 25 gastrointestinal pathogens, including viruses, bacteria, and parasites, with high sensitivity and specificity [13]. This detailed analytical capability provides unprecedented opportunities to investigate the complex epidemiology and seasonal patterns of enteric infections. Framed within a broader thesis on the evaluation of the AllPlex Gastrointestinal Panel through large patient cohort research, this analysis leverages data from multiple surveillance studies to delineate seasonal trends, thereby informing public health strategies, diagnostic protocols, and therapeutic interventions aimed at mitigating the burden of gastroenteritis.

Performance of Multiplex PCR in Gastrointestinal Pathogen Detection

Comparative Detection Rates versus Conventional Methods

The transition from conventional diagnostic methods to multiplex PCR has markedly improved the detection of enteric pathogens. Traditional techniques, including stool culture for bacteria and microscopy for viruses and parasites, are often limited by low sensitivity, prolonged turnaround times, and an inability to detect certain fastidious or non-culturable organisms [17]. Evidence from a 2018 comparative study demonstrated that the AGPA detected over twofold more bacterial and viral pathogens than conventional methods in a prospective analysis of 135 samples, with 44.4% of samples positive by AGPA compared to 17.8% by conventional methods [17]. This enhanced detection is largely attributable to the assay's capacity to identify pathogens such as enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and non-O157 Shiga toxin-producing E. coli (STEC), which are frequently missed by routine culture [17]. Moreover, the AGPA demonstrated 96% sensitivity in detecting pathogens from archived culture-positive samples, confirming its robustness [17].

Table 1: Detection Rate Comparison Between AllPlex GI Panel and Conventional Methods

Study Reference	Sample Size	Positive by Conventional Methods	Positive by AllPlex GI Panel	Key Findings
Pagotto et al. (2018) [17]	135	24/135 (17.8%)	60/135 (44.4%)	AGPA detected significantly more pathogens, including diarrheagenic E. coli
Robert-Gangneux et al. (2025) [18]	3,495	286 samples (protozoa)	909 samples (protozoa)	Multiplex PCR was significantly more sensitive for detecting intestinal protozoa
Lebanese Surveillance Study (2024) [19]	271	Not Specified	192/271 (71%)	High detection rate, with 54% of positive cases being mixed infections

Enhanced Detection of Protozoan Parasites

The superior sensitivity of multiplex PCR is particularly evident in the diagnosis of intestinal protozoa. A large prospective study on 3,500 stool samples over three years found that multiplex PCR (AllPlex GI Panel) detected protozoa in 909 samples, compared to only 286 samples identified by classical microscopy [18]. The assay demonstrated exceptional sensitivity and specificity for key protozoa: 100% sensitivity and 100% specificity for Entamoeba histolytica; 100% sensitivity and 99.2% specificity for Giardia duodenalis; 97.2% sensitivity and 100% specificity for Dientamoeba fragilis; and 100% sensitivity and 99.7% specificity for Cryptosporidium spp. in a multicentric Italian study [5]. This heightened detection capability underscores the limitations of microscopy, which is labor-intensive, operator-dependent, and suffers from poor sensitivity, especially for parasites present in low numbers [18] [5].

Seasonal Trends in Gastrointestinal Infections

The etiology of gastrointestinal infections exhibits distinct temporal patterns, influenced by environmental factors, human behavior, and pathogen characteristics. Syndromic surveillance using multiplex PCR provides detailed insights into these seasonal fluctuations, which are often obscured by the limited scope of conventional testing.

• Viral Infections: Norovirus, a highly contagious viral pathogen, demonstrates clear seasonality, with outbreaks typically peaking in cooler winter months. Data from the BIOFIRE Syndromic Trends network indicate a peak in norovirus detection in March-April in the Northern Hemisphere [20]. In contrast, rotavirus, another major viral cause of gastroenteritis, has shown shifts in its epidemiological pattern following the introduction of universal vaccination programs [17].
• Bacterial Infections: The prevalence of bacterial pathogens also fluctuates seasonally. A one-year multicenter study in Lebanon utilizing the AllPlex assay found that bacterial infections, particularly those caused by pathogenic E. coli strains, were highly prevalent during the summer, fall, and winter months [19]. This contrasts with Clostridioides difficile, for which detection remains relatively constant throughout the year, reflecting its association with healthcare settings and antibiotic use rather than seasonal drivers [20].
• Parasitic Infections: The seasonality of parasitic infections is less well-defined, though some patterns emerge. For instance, the waterborne parasite Cryptosporidium is more frequently transmitted during summer months, linked to recreational water activities and the parasite's chlorine resistance [20].

Table 2: Seasonal Patterns of Common Gastrointestinal Pathogens

Pathogen	Category	Peak Season(s)	Notes on Transmission & Risk Factors
Norovirus	Virus	Winter (Peak: Mar-Apr) [20]	Highly contagious; person-to-person, contaminated food/water [20]
Rotavirus	Virus	Historically winter; pattern altered by vaccination [17]	Primarily affects young children [17]
Enterotoxigenic E. coli (ETEC)	Bacterium	Summer, Fall, Winter [19]	A leading cause of traveler's diarrhea
Cryptosporidium spp.	Parasite	Summer [20]	Waterborne transmission; chlorine-tolerant [20]
Clostridioides difficile	Bacterium	Year-round [20]	Linked to antibiotic use and healthcare exposure

Experimental Protocols and Methodologies

To ensure the reliability and comparability of data on seasonal trends, standardized protocols for sample processing and analysis are critical. The following methodology is compiled from several key studies evaluating the AllPlex GI Panel [17] [19] [16].

Sample Collection and Nucleic Acid Extraction

• Specimen Type: Fresh human stool samples are collected from patients presenting with symptoms of acute gastroenteritis (e.g., passage of three or more loose stools in 24 hours, often accompanied by fever or bloody diarrhea) [13] [19].
• Storage and Transport: Stool samples are frequently suspended in a transport medium, such as Cary-Blair medium or ASL Stool Lysis Buffer, and can be stored refrigerated or frozen until processing. Studies have validated that DNA remains stable in Cary-Blair suspension for up to 7 days at both room temperature and 4°C, facilitating batch testing [16].
• Nucleic Acid Extraction: Automated extraction systems, such as the MagNA Pure Compact System (Roche) or the Microlab Nimbus system (Hamilton), are commonly used. Typically, 300-500 µL of the stool suspension supernatant is used for extraction, with nucleic acids eluted in a volume of 50-100 µL [17] [5]. An internal control is added to each sample to monitor extraction efficiency and PCR inhibition.

Multiplex PCR Amplification and Analysis

• PCR Setup: The AllPlex Gastrointestinal Panel Assays are one-step real-time RT-PCR assays. For each sample, 5 µL of extracted nucleic acid is added to the PCR master mix [19].
• Cycling Conditions: Amplification is performed on a CFX96 Real-Time PCR Detection System (Bio-Rad) under the following conditions: 20 minutes at 50°C for reverse transcription, 15 minutes at 95°C for initial denaturation, followed by 45 cycles of 10 seconds at 95°C (denaturation), 1 minute at 60°C (annealing), and 30 seconds at 72°C (extension) [19].
• Result Interpretation: The assay utilizes Seegene's MuDT technology, which allows reporting of multiple Ct values for different analytes in a single channel. Results are automatically interpreted using Seegene Viewer software, with a Ct value below 40 (or 45, depending on the study) considered positive [13] [16] [5].

Figure 1: Workflow for Multiplex PCR Detection of Gastrointestinal Pathogens.

The Scientist's Toolkit: Key Research Reagents and Materials

The implementation and validation of multiplex PCR for gastrointestinal pathogen detection rely on a suite of specialized reagents and instruments. The following table details essential components used in the featured studies.

Table 3: Essential Research Reagents and Materials for Multiplex PCR Testing

Item Name	Manufacturer	Function in Experimental Protocol
AllPlex Gastrointestinal Panel Assays	Seegene	Multiplex one-step real-time RT-PCR kit for simultaneous detection of 25 GI pathogens [13]
ASL Stool Lysis Buffer	Qiagen	Buffer for stool sample suspension and initial lysis, facilitating nucleic acid release [17] [5]
MagNA Pure Compact / Microlab Nimbus	Roche / Hamilton	Automated nucleic acid extraction systems for standardized and high-throughput processing [17] [5]
CFX96 Real-Time PCR Detection System	Bio-Rad	Thermal cycler with fluorescence detection capabilities for real-time PCR amplification and data capture [17] [19]
Seegene Viewer Software	Seegene	Automated software for interpretation of multiplex PCR results, including Ct value reporting and target identification [13]

Syndromic surveillance using the AllPlex Gastrointestinal Panel and other multiplex PCR assays has fundamentally refined our understanding of the seasonal dynamics of gastrointestinal infections. The technology's high sensitivity and comprehensive pathogen coverage have consistently revealed a greater burden of illness and more frequent mixed infections than previously documented by conventional methods. The identification of distinct seasonal peaks for key pathogens like norovirus in winter and enterotoxigenic E. coli in summer provides critical data for directing public health interventions, such as targeted vaccination campaigns, food safety initiatives, and public awareness efforts during high-risk periods. Furthermore, the ability of multiplex PCR to rapidly and accurately identify the etiologic agent of gastroenteritis supports antimicrobial stewardship and improves patient management. Continued large-scale, multi-year surveillance using these advanced molecular tools is essential for monitoring shifting epidemiological trends, evaluating the impact of interventions, and ultimately reducing the global burden of infectious gastroenteritis.

The advent of syndromic multiplex PCR panels has revolutionized the detection of gastrointestinal pathogens, revealing a complex landscape of mixed infections that traditional culture-based methods largely overlooked. This comprehensive analysis evaluates the performance of the Allplex Gastrointestinal Panel in detecting co-infections across diverse patient populations. By synthesizing data from multiple clinical studies, we demonstrate that molecular testing significantly increases the detection of multiple pathogens simultaneously-present in stool specimens, with co-infection rates ranging from 16% to 59% in positive samples. The Allplex system demonstrates high sensitivity and specificity across most bacterial, viral, and parasitic targets, though performance varies for specific pathogens such as Cryptosporidium and Aeromonas species. Our findings underscore the clinical importance of recognizing mixed infection patterns, which are associated with increased healthcare utilization and may represent either true co-infections or colonization in vulnerable patient populations. These insights are crucial for researchers, clinical microbiologists, and public health professionals working to optimize the diagnosis and management of infectious gastroenteritis.

Infectious diarrheal diseases remain a leading cause of global morbidity and mortality, particularly affecting children in developing countries where they account for approximately 1.34 million deaths annually [21]. The conventional approach to diagnosing gastrointestinal infections has relied on culture-based methods, antigen detection, and single-pathogen PCR assays, which have limited sensitivity and prolonged turnaround times [1]. These technical limitations have historically obscured the true prevalence and clinical significance of mixed enteric infections, where multiple pathogens are detected simultaneously in a single patient.

The introduction of syndromic multiplex PCR panels has dramatically transformed gastrointestinal pathogen detection by enabling simultaneous identification of numerous bacteria, viruses, and parasites in a single test [7]. These comprehensive panels include the Allplex Gastrointestinal Panel (Seegene, Seoul, Korea), which detects 25 different gastrointestinal pathogens, and the BioFire FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT), which targets 22 pathogens [22] [23]. These advanced molecular diagnostics have uncovered surprisingly high rates of co-infections, challenging traditional concepts of gastroenteritis etiology and necessitating reevaluation of clinical interpretations when multiple pathogens are detected.

Understanding the frequency and patterns of co-detected pathogens is crucial for several reasons: First, mixed infections may potentially lead to more severe clinical presentations than single-pathogen infections [21]. Second, the detection of multiple pathogens complicates clinical decision-making regarding targeted antimicrobial therapy. Third, the public health implications of co-infections may differ from single-pathogen outbreaks. Finally, accurate detection of co-infections provides valuable insights into microbial interactions within the gut ecosystem and their collective impact on patient outcomes.

This review synthesizes current evidence on mixed infection rates detected by the Allplex Gastrointestinal Panel and comparable multiplex PCR systems, analyzing patterns of co-detection across different patient populations and geographic regions. We evaluate the technical performance of these assays, examine clinical correlations, and discuss implications for both patient management and public health surveillance.

Methodology of Multiplex PCR Testing for Gastrointestinal Pathogens

The Allplex Gastrointestinal Panel Assay System

The Allplex Gastrointestinal Panel (Seegene, Seoul, Korea) is a comprehensive multiplex real-time PCR system designed for the simultaneous detection of major pathogens responsible for infectious gastroenteritis. The assay employs proprietary technologies including Dual Priming Oligonucleotide (DPO) and TOCE (Three-Oligo Competitive Enzyme) to ensure high specificity and sensitivity while minimizing false-positive results [13] [14]. The system incorporates UDG (Uracil-DNA Glycosylase) treatment to prevent carry-over contamination and includes an internal control to validate the entire process from nucleic acid extraction to amplification.

The complete Allplex system comprises four distinct panels that collectively detect 25 gastrointestinal pathogens:

• GI-Bacteria(I) Assay: Detects 7 bacterial pathogens including Aeromonas spp., Campylobacter spp., Clostridium difficile toxin B, Salmonella spp., Shigella spp./Enteroinvasive E. coli (EIEC), Vibrio spp., and Yersinia enterocolitica [14].
• GI-Bacteria(II) Assay: Identifies 6 additional diarrheagenic E. coli pathotypes and hypervirulent C. difficile, including Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC), E. coli O157, Enterotoxigenic E. coli (ETEC), Hypervirulent C. difficile, and Shiga toxin-producing E. coli (STEC) [13].
• GI-Parasite Assay: Detects 6 parasitic pathogens including Blastocystis hominis, Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, and Giardia lamblia [16].
• GI-Virus Assay: Identifies 6 viral pathogens including Adenovirus, Astrovirus, Norovirus GI, Norovirus GII, Rotavirus, and Sapovirus [13].

A key innovation of the Allplex system is its MuDT (Multiple Detection Temperature) technology, which enables reporting of multiple Ct values for different analytes in a single fluorescence channel, facilitating the detection of co-infections within the same panel [13] [14]. The automated Seegene Viewer software provides standardized interpretation and laboratory information system interoperability, reducing technical variability in result reporting.

Comparative Multiplex PCR Systems

Several other multiplex PCR systems are widely used for gastrointestinal pathogen detection, with varying target ranges and methodological approaches:

The Luminex NxTAG Gastrointestinal Pathogen Panel (Luminex Corporation, Austin, Texas) utilizes a bead-based array technology to detect 15 common gastrointestinal pathogens in a single reaction tube [7]. The panel covers 9 bacterial, 3 viral, and 3 parasitic targets, with the advantage of higher throughput capacity compared to some other systems.

The BioFire FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT) employs a nested PCR approach within a closed pouch system to detect 22 gastrointestinal pathogens [22] [23]. The fully integrated system performs nucleic acid extraction, amplification, and detection in approximately one hour, with minimal hands-on technical time required.

The BD MAX Enteric Panel (Becton Dickinson, Franklin Lakes, NJ) provides a more focused approach, detecting 5 bacterial and 3 parasitic pathogens commonly associated with gastroenteritis [24]. This system may be suitable for laboratories with more specific testing requirements rather than comprehensive syndromic testing.

Laboratory Processing Protocols

The analytical workflow for multiplex PCR testing follows a standardized process across different platforms, though specific details vary by manufacturer. The following diagram illustrates the general workflow for processing stool samples using these syndromic panels:

Figure 1. Workflow for Multiplex PCR Testing of Stool Specimens. This generalized protocol illustrates the key steps from sample collection to result interpretation used in syndromic testing for gastrointestinal pathogens.

Sample collection and transport typically involves preservation of freshly excreted stool in Cary-Blair transport medium, which maintains pathogen viability and nucleic acid integrity during transport to the laboratory [7] [23]. For the Allplex system, 140-180 mg of stool is suspended in the transport medium, vigorously mixed, and centrifuged to remove particulate matter before extraction [16].

Nucleic acid extraction is performed using automated systems such as the HAMILTON STARlet (Hamilton Company, USA) or the Seegene NIMBUS, with extraction protocols optimized for the diverse range of pathogens targeted [16] [7]. The Allplex system is specifically validated for use with Seegene's automated platforms, ensuring optimal performance through standardized protocols.

Amplification and detection parameters vary by platform. The Allplex system utilizes a one-step real-time RT-PCR approach with four separate reaction tubes to cover the complete pathogen panel [7]. In contrast, systems like the BioFire FilmArray incorporate all reagents into a single disposable pouch that performs nested PCR with endpoint melting curve analysis [22]. The Luminex NxTAG system employs a unique tag-based array detection system that allows multiplexing of all targets in a single reaction well [7].

Analytical Performance Characteristics

Multiplex PCR assays demonstrate generally high sensitivity and specificity compared to traditional diagnostic methods. The Allplex GI-Bacteria assay showed sensitivity and specificity values exceeding 95% for most targets when compared to culture-based methods, with the exception of Aeromonas spp., which demonstrated 81% sensitivity and 99% specificity [1]. Similar performance has been observed for the parasite panel, with sensitivities of 81% for Giardia duodenalis and Dientamoeba fragilis, and 100% for Cryptosporidium spp., Blastocystis hominis, and Cyclospora cayetanensis [16].

Table 1. Performance Characteristics of Multiplex PCR Assays for Selected Gastrointestinal Pathogens

Pathogen	Sensitivity (%)	Specificity (%)	Assay System	Study
Campylobacter spp.	>95%	>95%	Allplex GI-Bacteria	[1]
Salmonella spp.	>95%	>95%	Allplex GI-Bacteria	[1]
Aeromonas spp.	81%	99%	Allplex GI-Bacteria	[1]
Giardia duodenalis	81-100%	>95%	Allplex GI-Parasite	[16]
Dientamoeba fragilis	81-97.2%	>95%	Allplex GI-Parasite	[16]
Cryptosporidium spp.	100%	>95%	Allplex GI-Parasite	[16]
Blastocystis hominis	99.4%	>95%	Allplex GI-Parasite	[16]
Norovirus	93-99%	96-99%	Multiple Assays	[7] [24]

Frequency and Patterns of Mixed Infections

Multiplex PCR testing has revealed that concurrent detection of multiple gastrointestinal pathogens is substantially more common than previously recognized. The frequency of co-infections varies considerably across different patient populations and geographic settings, with studies reporting rates ranging from 16% to as high as 59% in positive stool specimens [22] [23] [24].

A comprehensive evaluation of the Allplex GI panel demonstrated its capacity to detect multiple pathogens simultaneously, with the assay identifying 261 positive samples (66.2%) out of 394 tested, compared to only 109 (27.7%) detected by conventional culture methods [1]. Importantly, this enhanced detection capability revealed numerous mixed infections that would have been missed by traditional testing approaches. In a direct comparison of multiplex systems, the Allplex assay identified 16 cases of multiple pathogen detection, while the Luminex xTAG system detected 51 cases, and the BD MAX system identified only one case from the same sample set [24].

The frequency of co-infections appears to be influenced by several epidemiological factors. A large retrospective cohort study conducted at Columbia University Irving Medical Center found that among 1,341 patients with positive GI PCR tests, 356 (26.5%) had multiple pathogens detected [23]. This study identified Hispanic ethnicity and chronic kidney disease as factors significantly associated with increased likelihood of multiple pathogen detection, while surprisingly, immunosuppression was not independently associated with co-infections after adjusting for other variables.

Table 2. Prevalence of Mixed Infections Across Different Study Populations

Study Population	Sample Size	Co-infection Rate	Most Common Pathogen Combinations	Testing Method
South African children [22]	275	59% (161/275)	EAEC + EPEC + Viruses	BioFire FilmArray
Spanish hospital patients [1]	394	Significantly higher than culture	Campylobacter + Salmonella	Allplex GI Panel
US adult patients [23]	1,341	26.5% (356/1,341)	EPEC + Norovirus + EAEC	BioFire FilmArray
Comparative study [24]	858	Varied by assay (16-51 cases)	Dependent on assay sensitivity	Multiple assays

Specific Pathogen Combinations in Mixed Infections

Analysis of co-infection patterns reveals that certain pathogen combinations occur more frequently than others, potentially reflecting shared transmission routes, similar seasonal patterns, or biological interactions between pathogens.

Viral-viral co-infections are commonly detected in pediatric populations. An 11-year investigation of children hospitalized with acute gastroenteritis in Italy found that 8.3% of virus-positive patients had mixed viral infections, with rotavirus and norovirus being the most frequently identified combination, accounting for 70.6% of viral co-infections [25]. Another study reported that rotavirus and norovirus co-infections were particularly prevalent, followed by rotavirus and astrovirus combinations [25].

Bacterial-bacterial co-infections frequently involve diarrheagenic E. coli pathotypes in combination with other bacterial pathogens. In South African children under five years, EAEC and EPEC were commonly detected together in multiple pathogen combinations [22]. The Allplex GI-Bacteria assay has demonstrated particular utility in detecting co-infections with Campylobacter and Salmonella species, which were frequently missed by conventional culture methods [1].

Viral-bacterial mixed infections represent a particularly important category with potential clinical implications. Research on gut microbiota in children with acute gastroenteritis has demonstrated that viral-bacterial co-infections are associated with more severe clinical presentations compared to single-pathogen infections [21]. Specifically, combinations of rotavirus or norovirus with pathogenic E. coli strains (EAEC or EPEC) resulted in significantly higher severity scores, with notable alterations in gut microbiota composition characterized by reduced Bacteroides proportions and increased Bifidobacteriaceae richness [21].

Geographic and Demographic Variations

The epidemiology of mixed gastrointestinal infections demonstrates substantial geographic variation, influenced by factors such as sanitation infrastructure, climate, population immunity, and local circulating pathogen strains.

In developing regions such as sub-Saharan Africa, co-infection rates appear particularly high. A study conducted in rural and peri-urban communities of South Africa's Vhembe district found that 59% of stool specimens from children with diarrhea contained multiple pathogens, with 25% of positive samples containing four or more different pathogens [22]. This high rate of multiple infections likely reflects environmental conditions conducive to fecal-oral transmission and higher exposure densities in these settings.

In developed countries, mixed infection rates, while lower than in developing regions, still represent a substantial proportion of cases. A US-based study reported that 26.5% of positive GI PCR tests detected multiple pathogens [23]. The most commonly detected pathogens in this population included Enteropathogenic E. coli (EPEC, 27%), norovirus (17%), and Enteroaggregative E. coli (EAEC, 14%), with similar prevalence patterns in both singly- and multiply-infected patients.

Age-related patterns are also evident in mixed infection epidemiology. Children under five years consistently demonstrate higher rates of co-infections compared to adults, possibly reflecting their immunologically naive status and increased exposure risks through exploratory behaviors and group settings such as daycare centers [22] [25]. The Italian study that spanned 11 years of surveillance found that viral co-infections were predominantly detected in children under five years hospitalized with acute gastroenteritis [25].

Comparative Performance of Multiplex Assays in Detecting Co-infections

Agreement Between Testing Platforms

The detection of mixed infections presents particular challenges for diagnostic assays, as sensitivity for individual targets must be maintained within a multiplex reaction environment. Studies comparing different GI PCR panels have generally found good overall agreement, though notable differences exist for specific pathogens.

A 2023 comparison of the Seegene Allplex and Luminex NxTAG panels demonstrated high overall concordance, with Negative Percentage Agreement (NPA) consistently above 95% and kappa values exceeding 0.8 for most pathogens [7]. Similarly, a 2019 comparative evaluation reported overall positive percentage agreements of 94% for Seegene Allplex, 92% for Luminex xTAG, and 78% for BD MAX when using a consensus definition of positivity from at least two tests [24].

However, discordant results were observed for certain pathogens, particularly Salmonella species and Cryptosporidium [7]. The Luminex xTAG system demonstrated frequent false positives for Salmonella with low median fluorescent intensity values, while both platforms showed lower agreement for Cryptosporidium detection compared to other targets [7] [24]. These findings highlight the importance of understanding platform-specific performance characteristics when interpreting co-infection results.

Detection of Co-infections Across Platforms

Different multiplex systems vary in their capacity to detect multiple pathogens in a single specimen, influenced by factors such as assay design, amplification efficiency, and target selection. In the comparative evaluation by Yoo et al., the number of cases with multiple pathogens detected differed substantially across platforms: 16 cases with Seegene, 51 cases with Luminex, and only 1 case with BD MAX [24]. Importantly, only 3 of these co-infection cases were identified as consensus positives across multiple platforms, suggesting that careful interpretation of positive results for multiple pathogens is required.

The following diagram illustrates the comparative detection rates of co-infections across different testing platforms:

Figure 2. Comparative Detection of Co-infections Across Multiplex PCR Platforms. The varying detection rates highlight platform-specific differences in sensitivity for identifying multiple pathogens in individual specimens. PPA: Positive Percentage Agreement.

These discrepancies in co-infection detection may be attributed to several factors. The number of targets included in each panel differs, with the Allplex system covering 25 pathogens, the BioFire FilmArray detecting 22, the Luminex NxTAG targeting 15, and the BD MAX Enteric panel focusing on 8 primary pathogens [13] [24]. Additionally, technical variations in nucleic acid extraction efficiency, amplification conditions, and detection methodologies contribute to differences in co-infection identification.

Impact of Assay Choice on Co-infection Rates

The selection of a specific multiplex PCR platform directly influences the observed epidemiology of mixed infections in a patient population. Assays with broader pathogen coverage, such as the Allplex and BioFire systems, naturally detect more co-infections simply by virtue of targeting more potential pathogens.

The analytical sensitivity for individual targets varies between platforms, potentially affecting the detection of pathogens present at lower concentrations in mixed infections. For example, the Allplex GI-Parasite assay demonstrated 81% sensitivity for Giardia duodenalis and Dientamoeba fragilis, with false-negative results primarily occurring in samples with low parasitic loads [16]. This suggests that co-infections involving low abundance pathogens might be missed by some assays.

The threshold determination algorithms also differ between platforms, potentially affecting the detection of multiple pathogens. Some systems utilize predetermined cycle threshold (Ct) cutoffs, while others employ melting curve analysis or target-specific fluorescence thresholds. These technical differences can impact whether a pathogen present at lower levels is reported as detected, particularly in the context of co-infections where competitive amplification might occur.

Clinical Implications of Mixed Infections

Severity and Outcomes

The clinical significance of detecting multiple gastrointestinal pathogens remains an area of active investigation, with emerging evidence suggesting that mixed infections may indeed influence disease severity and patient outcomes.

Several studies have demonstrated that viral-bacterial co-infections are associated with more severe clinical presentations compared to single-pathogen infections. Research on gut microbiome in children with acute gastroenteritis found significantly higher severity scores in those with mixed viral-bacterial infections compared to those with viral infections alone [21]. Specifically, co-infections involving rotavirus or norovirus in combination with pathogenic E. coli strains (EAEC or EPEC) resulted in more pronounced clinical manifestations.

A large retrospective cohort study examining outcomes in patients with multiple pathogens detected on GI PCR testing found that these individuals had increased healthcare utilization in the 90 days following testing, with 40% requiring emergency department visits compared to 32% of patients with single pathogen detection [23]. However, no significant differences were observed in mortality or hospitalization rates between the two groups, suggesting that while co-infections may increase morbidity, they do not necessarily lead to more grave outcomes in generally resilient patient populations.

The direction of causality in the association between multiple pathogen detection and worse clinical outcomes remains unclear. It is possible that more severe gastrointestinal illness creates conditions favorable for colonization or infection with multiple pathogens, rather than the co-infection itself driving severity. Alternatively, certain host factors (such as immunodeficiency or compromised gut integrity) might simultaneously predispose individuals to both more severe illness and acquisition of multiple pathogens.

Interpretation Challenges in Clinical Practice

The detection of multiple gastrointestinal pathogens presents significant interpretation challenges for clinicians, who must determine whether all detected pathogens are clinically relevant or if some represent incidental colonization.

A key consideration is the epidemiological context of detected pathogens. While some microorganisms are established primary pathogens (e.g., Salmonella, Shigella, Campylobacter, norovirus, rotavirus), others such as EAEC and EPEC have a more ambiguous pathogenic significance and may represent colonization in some cases [23]. This distinction becomes particularly challenging when multiple pathogens of varying clinical significance are detected simultaneously.

The quantitative aspect of pathogen detection may provide valuable clues for clinical interpretation. Some studies have suggested that using cycle threshold (Ct) values as a proxy for pathogen load could help distinguish true infections from colonization [25]. In viral co-infections, rotavirus has been shown to be generally detected at higher levels (lower Ct values) in co-infected patients compared to other viruses, potentially indicating its primary role in the clinical presentation [25].

The patient's immune status also influences the interpretation of multiple detections. While immunosuppression might be expected to increase susceptibility to multiple infections, one large study surprisingly found that immunosuppression was not independently associated with multiple pathogen detection after adjusting for other variables [23]. However, clinical experience suggests that immunocompromised patients may be more likely to have prolonged shedding of multiple enteric pathogens, potentially complicating the distinction between active infection and colonization.

Research Applications and Essential Methodologies

The Scientist's Toolkit: Key Research Reagents and Platforms

Conducting comprehensive research on mixed gastrointestinal infections requires specialized reagents, platforms, and methodological approaches. The following table outlines essential components of the research toolkit for investigating co-infections:

Table 3. Essential Research Reagents and Platforms for Studying Mixed Gastrointestinal Infections

Category	Specific Tools	Research Applications	Key Features
Multiplex PCR Panels	Allplex GI Panels, BioFire FilmArray GI Panel, Luminex NxTAG GPP	Comprehensive pathogen detection, Co-infection screening	Simultaneous detection of multiple targets, High sensitivity compared to culture
Automated Nucleic Acid Extraction Systems	HAMILTON STARlet, Seegene NIMBUS	Standardized DNA/RNA extraction	Reproducible recovery of nucleic acids from diverse pathogens, Integration with PCR platforms
Specialized Transport Media	Cary-Blair Medium, FecalSwab with Cary-Blair	Sample preservation for molecular testing	Maintains pathogen nucleic acid integrity during transport and storage
Reference Materials	External quality control panels, Quantified pathogen standards	Assay validation, Quality assurance	Verification of assay performance, Standardization across laboratories
Data Analysis Software	Seegene Viewer, BioFire System Software, Custom bioinformatics pipelines	Result interpretation, Epidemiological analysis	Automated pathogen identification, Co-infection pattern recognition

Advanced Methodologies for Co-infection Research

Beyond standard multiplex PCR testing, several advanced methodological approaches provide deeper insights into the epidemiology and clinical significance of mixed gastrointestinal infections:

Molecular genotyping and sequencing techniques enable researchers to distinguish between strains within a single pathogen species, providing crucial information about whether co-infections involve genetically distinct variants of the same species. Studies on viral gastroenteritis have employed genotyping to demonstrate the diversity of viruses detected in co-infections, with some patients harboring multiple genotypes of the same virus [25].

Quantitative PCR approaches offer advantages over qualitative multiplex panels by providing information about pathogen load, which may help distinguish clinically significant infections from incidental detections. Research on viral co-infections has utilized Ct values as a proxy for viral load, finding that rotavirus typically presents with higher loads in co-infected patients compared to other viruses [25].

Microbiome analysis through 16S rRNA sequencing or shotgun metagenomics provides a broader context for understanding how pathogen co-infections interact with the commensal gut microbiota. Studies have demonstrated that mixed infections significantly alter gut microbiota composition, with characteristic reductions in Bacteroides and increased Bifidobacteriaceae richness in viral-bacterial co-infections [21].

Longitudinal sampling designs are particularly valuable for understanding the dynamics of mixed infections over time, including the sequence of pathogen acquisition, duration of shedding, and how the presence of one pathogen might influence susceptibility to others.

Multiplex PCR testing has fundamentally transformed our understanding of mixed gastrointestinal infections, revealing that co-detection of multiple pathogens is substantially more common than previously recognized. The Allplex Gastrointestinal Panel and similar syndromic assays have demonstrated excellent performance characteristics for comprehensive pathogen detection, with sensitivity and specificity exceeding 95% for most targets. These advanced molecular tools have uncovered complex patterns of co-infection that vary by geographic region, patient age, and underlying health status.

The clinical significance of detecting multiple pathogens continues to be defined, with evidence suggesting that mixed infections, particularly viral-bacterial combinations, may be associated with more severe disease presentations and increased healthcare utilization. However, interpretation challenges remain, as not all detected pathogens necessarily contribute equally to clinical illness. The use of quantitative measures such as Ct values and integration with clinical data may help distinguish true co-infections from colonization with multiple organisms.

Future research directions should focus on elucidating the biological interactions between co-infecting pathogens, their collective impact on the gut microbiome, and the development of standardized approaches for interpreting mixed infection results in clinical practice. As multiplex PCR testing becomes more widespread, ongoing surveillance of co-infection patterns will provide valuable insights for clinical management, public health interventions, and vaccine development strategies aimed at reducing the global burden of infectious gastroenteritis.

Regional Epidemiological Variations in Enteric Pathogen Prevalence

Enteric infections represent a significant global health challenge, contributing substantially to morbidity and mortality worldwide. The effective management and prevention of these infections are critical for controlling gastrointestinal diseases, with accurate and rapid diagnosis being essential for patient management [7]. This guide objectively evaluates the performance of the Seegene AllPlex Gastrointestinal Panel against other diagnostic alternatives, framing the analysis within large patient cohort research. The global burden of enteric infections remains serious, particularly in children under 5 years and in low and low-middle socio-demographic index (SDI) regions [26]. Understanding regional variations in pathogen prevalence is fundamental for developing targeted interventions, optimizing laboratory diagnostics, and guiding public health policy.

Molecular diagnostic methods have revolutionized the detection of gastrointestinal pathogens, overcoming key limitations of conventional methods such as specimen degradation and prolonged turnaround times [7]. Multiplex PCR tests allow for simultaneous detection of multiple gastrointestinal microorganisms, offering advantages including faster turnaround times, comprehensive pathogen identification, and improved diagnostic accuracy [7]. This evaluation assesses the AllPlex panel's performance across diverse geographical settings and patient populations, providing researchers and clinical microbiologists with evidence-based comparisons to inform diagnostic selection and implementation.

Global Burden and Regional Epidemiology of Enteric Infections

Global Prevalence and Geographic Distribution

The global burden of enteric infections remains substantial, with recent data revealing significant geographical disparities. In 2021, the global age-standardized prevalence rate for enteric infections was 879.58 per 100,000 population, with an age-standardized incidence rate of 57,721.08 per 100,000 [27]. Between 1990 and 2021, while mortality rates showed significant improvement, incidence rates remained persistently high, highlighting the ongoing challenge of enteric infections worldwide [27] [26].

Regions with low SDI consistently demonstrate the highest burden of enteric infections, while high-SDI regions show the lowest rates [26]. This disparity reflects differences in healthcare infrastructure, sanitation, food safety standards, and access to clean water [19]. From 1990 to 2019, decreasing trends in age-standardized incidence rates were observed in low and low-middle SDI areas, particularly in Central Latin America, South Asia, and Central Europe [26]. Conversely, increasing trends were identified in 14 regions, with the most pronounced increases in North Africa and the Middle East, Andean Latin America, and Central Sub-Saharan Africa [26].

Table 1: Global Burden of Enteric Infections (2021)

Metric	Value	Rate Change (1990-2021)
Global Prevalence	67,826,600	-0.18
Age-Standardized Prevalence Rate (per 100,000)	879.58	-0.18
Global Incident Cases	4,448,407,870	-0.12
Age-Standardized Incidence Rate (per 100,000)	57,721.08	-0.12
Global Deaths	1,336,220	-0.73
Age-Standardized Mortality Rate (per 100,000)	17.83	-0.73
Global DALYs	71,929,008	-0.72
Age-Standardized DALY Rate (per 100,000)	1,020.15	-0.72

Data sourced from Global Burden of Disease 2021 analysis [27].

Regional Variations in Pathogen Distribution

Epidemiological studies conducted in different global regions reveal distinct patterns of enteric pathogen distribution, influenced by climatic, socioeconomic, and demographic factors.

In Lebanon, a multicenter study utilizing the AllPlex Gastrointestinal Assay found enteropathogens in 71% of enrolled cases with acute gastroenteritis, with 54% of positive cases showing mixed infections [19]. Bacterial pathogens dominated across all age groups (48%), followed by parasites (12%) and viruses (11%). Diarrheagenic Escherichia coli pathotypes were most prevalent, with Enteroaggregative E. coli (EAEC) at 26.5%, Enterotoxigenic E. coli (ETEC) at 23.2%, and Enteropathogenic E. coli (EPEC) at 20.3% [19]. The highest hospitalization rates occurred with Rotavirus (63%), ETEC (50%), and Blastocystis hominis (45%).

A six-year active surveillance study in a teaching hospital in southern Italy reported different prevalence patterns, with bacteria isolated in 62.2% of positive samples, followed by fungi (29.0%), viruses (8.2%), and parasites (0.6%) [28]. EPEC was the most prevalent bacterial target (11.1%), followed by C. difficile toxin A/B-producing strains (8.3%) and C. jejuni (2.5%) [28]. Norovirus and Candida spp. were most prevalent in pediatric patients.

Table 2: Regional Variations in Enteric Pathogen Prevalence

Pathogen	Lebanon (2020-2021) [19]	Southern Italy (2018-2023) [28]	Spain (2023) [7]
EAEC	26.5%	3.13%	Not specified
ETEC	23.2%	~1%	Detected
EPEC	20.3%	11.1%	Not specified
C. difficile	6.3%	8.3%	Detected
Campylobacter spp.	2.6%	2.5%	Detected
Salmonella spp.	Less common	~1%	Detected
Blastocystis hominis	15.5%	Not specified	Not specified
Rotavirus	7.7%	Not specified	Detected
Norovirus	Not specified	Most prevalent virus in pediatrics	Detected
Overall Detection Rate	71%	23-25% annually	High concordance

Research has also identified challenges in detecting certain pathogens across multiple regions. A Spanish study comparing multiplex PCR panels noted that lower agreement was observed for Cryptosporidium spp. (86.6%) and discrepancies were primarily observed for certain pathogens, such as Salmonella spp. and Cryptosporidium spp., highlighting the diagnostic challenges associated with these targets [7].

Comparative Performance of Diagnostic Panels

AllPlex GI Panel Performance Characteristics

The Seegene AllPlex Gastrointestinal Panel is a comprehensive multiplex PCR system that detects a wide range of gastrointestinal pathogens through multiple discrete assays: GI-Bacteria (I) Assay, GI-Bacteria (II) Assay, GI-Parasite Assay, and GI-Virus Assay [7]. The panel utilizes Seegene's proprietary Multiple Detection Temperature (MuDT) technology and Dual Priming Oligonucleotide (DPO) systems to enable highly specific multiplex detection [19].

Multiple studies have validated the performance characteristics of the AllPlex panels in clinical settings. A multicentric Italian study evaluating the AllPlex GI-Parasite Assay demonstrated exceptional performance for detecting common intestinal protozoa [29]. Compared to traditional parasitological methods, the assay showed sensitivity and specificity of 100% and 100% for Entamoeba histolytica, 100% and 99.2% for Giardia duodenalis, 97.2% and 100% for Dientamoeba fragilis, and 100% and 99.7% for Cryptosporidium spp., respectively [29].

The complete AllPlex Gastrointestinal Assay was implemented in a Lebanese study, where it significantly improved detection rates compared to conventional methods used previously in the region [19]. The comprehensive panel identified pathogens that would typically be missed by routine testing algorithms, particularly mixed infections which accounted for 54% of positive cases [19].

Comparison with Alternative Multiplex Panels

A direct comparative study conducted at a Spanish hospital in 2023 evaluated the diagnostic performance of the Seegene AllPlex Gastrointestinal Panels against the Luminex NxTAG Gastrointestinal Pathogen Panel [7]. The prospective and retrospective analysis of 196 stool samples found that both assays demonstrated high overall concordance, with negative percentage agreement (NPA) values consistently above 95% and overall Kappa values exceeding 0.8 for most pathogens [7].

The average positive percentage agreement (PPA) was greater than 89% for nearly all targets, indicating strong agreement between the two methods for pathogen detection [7]. However, the study design highlighted differences in workflow requirements between the platforms. The AllPlex assay requires multiple tubes (typically four) to complete full panel detection, while the Luminex NxTAG GPP requires only a single tube per sample for comprehensive pathogen detection [7].

Table 3: Multiplex PCR Panel Comparison [7]

Parameter	Seegene AllPlex GI Panels	Luminex NxTAG GPP
Format	Multiple panels (4 tubes)	Single panel (1 tube)
Pathogen Coverage	25+ targets across bacteria, viruses, parasites	Comprehensive coverage of bacteria, viruses, parasites
Positive Percentage Agreement (PPA)	>89% for nearly all targets	>89% for nearly all targets
Negative Percentage Agreement (NPA)	>95%	>95%
Overall Concordance	High (Kappa >0.8 for most pathogens)	High (Kappa >0.8 for most pathogens)
Challenging Targets	Cryptosporidium spp. (86.6% agreement)	Cryptosporidium spp. (86.6% agreement)
Extraction System	HAMILTON STARlet	HAMILTON STARlet
Sample Pretreatment	Not specified	Required per package insert

Another study adapting a gastrointestinal panel for high-throughput qPCR systems provided comparative data for enteric virus detection. The validation of a laboratory-developed test on Roche cobas systems showed that when compared to the Allplex GI-Virus Assay, specificity and sensitivity ranged between 98.2-100.0% and 85.7-100.0%, respectively, across various viral targets [30].

Experimental Protocols and Methodologies

Sample Processing and Nucleic Acid Extraction

The studies reviewed employed standardized protocols for sample processing and nucleic acid extraction to ensure reproducible results across testing platforms. In the comparative study of AllPlex and Luminex panels, all stool samples were preserved in Cary-Blair medium upon arrival and underwent genetic material extraction using the HAMILTON STARlet extraction system [7]. This standardized extraction method across both platforms minimized variability in sample preparation, enabling robust comparison of the two PCR platforms.

For the AllPlex GI-Parasite Assay evaluation, researchers employed a specific protocol: 50-100 mg of stool specimens was collected and suspended in 1 mL of stool lysis buffer (ASL buffer; Qiagen) [29]. After pulse vortexing for 1 minute and incubation at room temperature for 10 minutes, the tubes were centrifuged at full speed (14,000 rpm) for 2 minutes. The supernatant was used for nucleic acid extraction using the Microlab Nimbus IVD system, which automatically performed the nucleic acid processing and PCR setup [29].

In the Lebanon study implementing the full AllPlex Gastrointestinal Assay, the QIAamp DNA Mini Kit (Qiagen) was used for extraction according to the manufacturer's protocol [19]. A FLOQ swab collected stool from the specimen and was suspended in 1 ml of stool lysis buffer (ASL buffer) and incubated for 10 minutes at room temperature. Two hundred microliters of each sample was then extracted, with internal controls added to all samples prior to extraction. Nucleic acids were concentrated in 50 µl of elution buffer, and 5 µl of nucleic acid was used for each reaction [19].

Figure 1: Experimental Workflow for Enteric Pathogen Detection. This diagram illustrates the standardized protocols for sample processing, nucleic acid extraction, and PCR amplification used in comparative studies of gastrointestinal pathogen detection panels.

PCR Amplification and Detection Protocols

The AllPlex assays employ a one-step real-time PCR multiplex approach with specific cycling conditions optimized for each panel. For the full gastrointestinal assay, PCR was performed under the following cycling conditions: 20 minutes at 50°C for 1 cycle; 15 minutes at 95°C for 1 cycle; 10 seconds at 95°C, 1 minute at 60°C and 30 seconds at 72°C for 45 cycles; 10 seconds at 95°C, 44 more times [19]. Detection and data analysis utilized Seegene Viewer Software, with samples reported as positive at a cycle threshold value of <40 [19].

For the GI-Parasite Assay, DNA extracts were amplified with one-step real-time PCR multiplex using the CFX96 Real-time PCR system with CFX Manager 1.6 software [29]. Fluorescence was detected at two temperatures (60°C and 72°C), and a positive test result was defined as a sharp exponential fluorescence curve that intersected the crossing threshold (Ct) at a value of less than 45 for individual targets. Positive and negative controls were included in each run, and results were interpreted using Seegene Viewer software [29].

The adaptation of a gastrointestinal panel for high-throughput systems on Roche cobas platforms demonstrated an alternative approach, with the assay providing a fast, fully automated and easily scalable high-throughput solution for gastrointestinal routine virus testing [30]. This implementation highlights the flexibility of molecular detection systems for different laboratory workflows and throughput requirements.

The Scientist's Toolkit: Research Reagent Solutions

Table 4: Essential Research Reagents and Platforms for Enteric Pathogen Detection

Reagent/Platform	Function	Application in Studies
AllPlex GI Panels (Seegene)	Multiplex PCR detection of gastrointestinal pathogens	Comprehensive detection of bacteria, viruses, parasites in clinical samples [7] [19]
Luminex NxTAG GPP (Diasorin)	Multiplex PCR panel for gastrointestinal pathogens	Comparative performance evaluation with AllPlex [7]
HAMILTON STARlet	Automated nucleic acid extraction system	Standardized extraction for multiple PCR platforms [7]
Microlab Nimbus IVD	Automated nucleic acid processing and PCR setup	High-throughput processing for parasite detection [29]
QIAamp DNA Mini Kit (Qiagen)	Manual nucleic acid extraction from stool samples	DNA extraction for AllPlex panel implementation [19]
CFX96 Real-Time PCR (Bio-Rad)	Real-time PCR detection system	Amplification and detection for AllPlex assays [29] [19]
Cary-Blair Medium	Stool sample transport and preservation	Maintains specimen integrity during transport and storage [7]
ASL Buffer (Qiagen)	Stool lysis buffer	Preparation of stool samples for nucleic acid extraction [29] [19]

The regional epidemiological variations in enteric pathogen prevalence highlight the importance of context-specific diagnostic approaches and the value of comprehensive multiplex PCR panels like the Seegene AllPlex Gastrointestinal system. The performance evaluation across multiple global studies demonstrates that the AllPlex panels provide reliable detection of gastrointestinal pathogens with high sensitivity and specificity, contributing to improved patient management and epidemiological surveillance.

The comparative data shows that while multiple multiplex PCR platforms demonstrate high concordance, the selection of an appropriate diagnostic technique should consider specific laboratory needs, including workflow requirements, target pathogens of regional importance, and throughput considerations. The implementation of syndromic testing panels like AllPlex has significantly improved detection rates for multiple pathogens, particularly in identifying mixed infections that would be missed by conventional diagnostic algorithms.

Future directions in enteric pathogen detection should focus on improving detection accuracy for challenging targets like Cryptosporidium and Salmonella species, expanding target panels to include emerging pathogens, and enhancing accessibility in resource-limited settings where the burden of enteric infections remains highest. The comprehensive evaluation of the AllPlex panel within large patient cohort research provides valuable insights for researchers, clinical microbiologists, and public health professionals working to reduce the global burden of gastrointestinal diseases.

Comparison of Detection Rates Between Inpatient and Outpatient Populations

The accurate and rapid identification of gastrointestinal pathogens is crucial for effective patient management, infection control, and antimicrobial stewardship. This guide provides an objective comparison of detection rates for multiplex gastrointestinal PCR panels across inpatient and outpatient populations, with particular focus on the AllPlex Gastrointestinal Panel Assays (AGPA). We synthesize experimental data from multiple clinical studies to evaluate analytical performance, diagnostic yield, and technological characteristics of leading syndromic panels in diverse clinical settings.

Infectious gastroenteritis represents a significant global health burden, causing substantial morbidity and mortality, particularly in vulnerable populations such as young children, the elderly, and immunocompromised individuals [31] [32]. Traditional diagnostic methods for gastrointestinal pathogens—including stool culture, enzyme immunoassays, and microscopic examination—are characterized by limited sensitivity, extended turnaround times (typically 48-72 hours), and labor-intensive processes [31] [17]. The emergence of multiplex PCR syndromic panels has revolutionized gastrointestinal pathogen detection by enabling simultaneous identification of multiple bacterial, viral, and parasitic pathogens with significantly improved sensitivity and reduced turnaround times [17] [32] [33].

The transition from conventional methods to molecular diagnostics has revealed substantially higher pathogen detection rates, with multiplex panels identifying over twice as many pathogens compared to traditional approaches [17]. This enhanced detection capability has important implications for clinical management, particularly in distinguishing between infectious and non-infectious causes of gastroenteritis, guiding appropriate antimicrobial therapy, and implementing effective infection control measures [32]. This guide systematically evaluates the performance of multiplex GI panels across healthcare settings, with specific attention to detection rate disparities between inpatient and outpatient populations.

Technology Platforms and Methodologies

Table 1: Comparative Analysis of Multiplex Gastrointestinal PCR Panels

Platform	Manufacturer	Targets	Run Time	Throughput	Key Features
AllPlex GI Panel Assays (AGPA)	Seegene	13 bacteria, 6 viruses, 6 parasites	~4 hours	Medium	Multiple multiplex reactions; detects diarrheagenic E. coli strains
FilmArray GI Panel	BioFire Diagnostics	22 pathogens	~1 hour	Low	Fully automated; sample-to-result integrated system
NxTAG GPP	Luminex	16 pathogens	~5 hours	High	Bead-based technology; moderate throughput capability
QIAstat-Dx Gastrointestinal Panel	QIAGEN	23 pathogens	76 minutes	Medium	Provides Ct values; automated extraction and detection
LiquidArray Gastrointestinal VER 1.0	Bruker	26 pathogens	~5 hours	High (48 samples)	Combines RT-PCR and melting curve analysis; minimal hands-on time

The methodological approaches vary significantly across platforms. The AllPlex GI Panel Assays employ multiple multiplex real-time PCR reactions (two bacterial, one viral, one parasitic) performed on standard real-time PCR instruments [17]. In contrast, the FilmArray GI Panel utilizes a fully integrated, sample-to-result system that automates nucleic acid extraction, nested multiplex PCR amplification, and detection in a single pouch [34]. The NxTAG Gastrointestinal Pathogen Panel employs reverse transcriptase PCR combined with the Luminex tag sorting system on the MAGPIX instrument [31], while the LiquidArray technology integrates automated nucleic acid extraction with PCR amplification and melting curve analysis [33].

Experimental Workflow for Multiplex GI Testing

The following diagram illustrates the general workflow for gastrointestinal pathogen detection using multiplex PCR panels:

AllPlex Gastrointestinal Panel Performance Analysis

Detection Rates in Mixed Patient Populations

The AllPlex Gastrointestinal Panel Assays (AGPA) demonstrate significantly higher detection rates compared to conventional diagnostic methods. In a comprehensive evaluation involving 135 prospective stool samples, the AGPA detected pathogens in 60/135 (44.4%) specimens, whereas conventional methods (culture and electron microscopy) identified pathogens in only 24/135 (17.8%) samples [17]. This represents a 2.5-fold increase in detection rate compared to traditional approaches.

The AGPA exhibited particularly strong performance for bacterial pathogens, correctly detecting 46/48 (96%) pathogens in archived culture-positive samples [17]. By organism, the panel demonstrated excellent sensitivity for Salmonella spp. (26/27, 96.3%), Campylobacter spp. (9/9, 100%), Shigella spp. (6/6, 100%), E. coli O157 (4/4, 100%), and Aeromonas spp. (1/1, 100%) [17]. Additionally, the AGPA detected several types of diarrheagenic E. coli that are not routinely identified through conventional culture methods, including enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and non-O157 Shiga toxin-producing E. coli (STEC) [17].

Comparison with Other Multiplex GI Panels

Table 2: Detection Rate Comparison Across Multiplex GI Panels in Clinical Studies

Platform	Study	Sample Size	Overall Detection Rate	Bacterial Detection	Viral Detection	Co-infection Rate
AllPlex GI Panel	Buss et al. [17]	135	44.4% (60/135)	96% in culture-positive samples	High for norovirus GII	23.3% (14/60)
FilmArray GI Panel	Buss et al. [34]	168	54.8% (92/168)	71.7% single pathogens	28.2% multiple pathogens	28.2% (26/92)
NxTAG GPP	Jones et al. [31]	159	28.3% (45/159)	Campylobacter most common (28.9%)	Norovirus 15.4% of infections	11.1% (5/45)
QIAstat-Dx	Hassan et al. [32]	110	62.7% (69/110)	Comparable to FilmArray	Comparable to FilmArray	20.9% (23/110)
LiquidArray	Davies et al. [33]	1512	High sensitivity (>90% most targets)	Specificity >99% all targets	Specificity >99% all targets	Not specified

When compared directly with other multiplex panels, the FilmArray GI Panel demonstrated the highest detection rate in a coded panel of 300 stool samples, identifying viruses in 199/207 (96.1%) true positive samples, compared to 100/127 (78.7%) for the GPP and 172/207 (83.1%) for the TaqMan Array Card (TAC) system [35]. The FilmArray also showed the highest clinical accuracy (98%) followed by TAC (97.2%) and GPP (96.9%) [35].

Inpatient vs. Outpatient Detection Rates: Comparative Analysis

While most studies do not explicitly stratify detection rates by inpatient versus outpatient status, several provide insights into performance across different care settings:

In a district general hospital evaluation of the NxTAG GPP assay, positive results were detected in 45/159 specimens (28.3%), with the panel identifying a higher positivity rate compared to traditional diagnostic methods which detected 31/159 (19.5%) positive infections [31]. This study included specimens from both hospital inpatients and outpatient clinics, though specific breakdown between these populations was not provided.

The AllPlex GI Panel evaluation included 70 adult and 65 pediatric samples, with specimens originating from both hospital and community settings [17]. The significantly higher detection rate of the AGPA (44.4%) compared to conventional methods (17.8%) was consistent across the study population, suggesting improved diagnostic yield in both inpatient and outpatient settings [17].

A study comparing QIAstat-Dx and BioFire FilmArray GI Panels in a pediatric population included 48 (43.6%) samples from inpatient units and 62 (56.4%) from outpatient clinics and the emergency department [32]. While detection rates between the two platforms were comparable (89.1% concordance), the study did not report separate detection rates for inpatient versus outpatient specimens [32].

Detailed Experimental Protocols

AllPlex GI Panel Testing Methodology

Specimen Preparation:

• Stool samples are collected in sterile containers or enteric transport medium (modified Cary-Blair medium)
• For nucleic acid extraction, 300 μL of stool is added to 1 mL ASL Stool Lysis Buffer (Qiagen) to create a sample suspension
• The mixture is centrifuged, and 400 μL of the supernatant is extracted using the MagNA Pure Compact System (Roche Molecular Systems) with a final elution volume of 100 μL [17]

PCR Amplification and Detection:

• The AGPA multiplex PCR is performed following manufacturer's recommendations on a CFX96 Real Time Detection System (Bio-Rad, Hercules, CA, USA)
• The assay comprises four separate multiplex reactions:
  • First bacterial panel: Shigella spp./enteroinvasive E. coli, Campylobacter spp., Yersinia enterocolitica, Vibrio spp., Clostridium difficile toxin B, Aeromonas spp., Salmonella spp.
  • Second bacterial panel: Shiga toxin-producing E. coli (STEC)/enterohemorrhagic E. coli (EHEC), E. coli O157, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), hypervirulent C. difficile
  • Viral panel: Norovirus genogroups I and II (GI/GII), rotavirus A, adenovirus F (serotype 40/41), astrovirus, sapovirus
  • Parasite panel: Detects 6 parasitic pathogens [17]

Discrepant Analysis Protocol:

• For organisms detected by AGPA but not by conventional tests, monoplex 5' exonuclease probe PCR assays are performed
• Results are considered true positives if positive by conventional methods or if both the AGPA and monoplex PCR assays are positive [17]

Comparative Study Design

The analytical performance of the AllPlex GI Panel was evaluated through a comprehensive study comparing it to conventional diagnostic methods [17]. The study included:

• Consecutive stool samples submitted for both bacterial and viral testing
• Prospective collection of samples between January and July 2017
• Inclusion of archived bacterial culture-positive samples to supplement prospective samples with limited positive results
• Statistical analysis using McNemar test to compare results for paired samples

Conventional comparator methods included:

• Bacterial culture using selective and differential media with 10 μL of stool inoculated per medium
• Electron microscopy for virus detection using a JEM 1010 Electron Microscope
• C. difficile testing using enzyme immunoassay for glutamate dehydrogenase (GDH) followed by Toxin B PCR if GDH-positive [17]

Research Reagent Solutions and Essential Materials

Table 3: Key Research Reagents for Multiplex GI Panel Testing

Reagent/Equipment	Manufacturer	Function	Application in Protocol
ASL Stool Lysis Buffer	Qiagen	Stool sample homogenization and pathogen lysis	Initial sample processing step for nucleic acid release
MagNA Pure Compact System	Roche Molecular Systems	Automated nucleic acid extraction	Isolation of pathogen DNA/RNA from stool samples
CFX96 Real Time Detection System	Bio-Rad	Real-time PCR amplification and detection	Platform for AllPlex GI Panel multiplex reactions
Cary-Blair Transport Medium	Various	Preserves stool specimens during transport	Maintains pathogen viability and nucleic acid integrity
AllPlex GI Panel Assays	Seegene	Multiplex PCR reagents	Contains primers/probes for pathogen detection
FilmArray GI Panel	BioFire Diagnostics	Integrated testing pouch	All-in-one detection platform for 22 pathogens
QIAstat-Dx Gastrointestinal Panel	QIAGEN	Cartridge-based testing	Integrated extraction and detection for 23 targets

Discussion and Clinical Implications

Detection Rate Disparities: Analytical Considerations

The substantially higher detection rates of multiplex GI panels compared to conventional methods can be attributed to several factors. First, molecular methods detect nucleic acids rather than relying on pathogen viability or expression of specific antigens, enabling identification of fastidious organisms that may not grow in culture [17]. Second, multiplex panels simultaneously test for a broad range of pathogens that would require multiple individual tests with conventional approaches [31] [17]. Third, PCR methods demonstrate superior sensitivity for pathogens that are present in low concentrations or intermittently shed in stool [34].

The clinical significance of these detection rate disparities is substantial. Higher detection rates facilitate more accurate diagnosis, appropriate treatment selection, and effective infection control measures [32]. This is particularly important for vulnerable patient populations, where delayed or incorrect diagnosis can lead to severe complications. Additionally, the identification of coinfections—detected in 11.1-28.2% of positive samples across studies [31] [17] [34]—may inform understanding of disease severity and treatment response.

Limitations and Future Directions

While multiplex GI panels offer significant advantages, several limitations warrant consideration. These assays detect nucleic acid from both viable and non-viable organisms, potentially leading to false positives in patients with recent infections [33]. Additionally, most panels do not provide information on antimicrobial susceptibility, which may still require reflex culture [31]. The clinical significance of detecting certain pathogens at low concentrations remains uncertain, particularly in asymptomatic individuals or in cases of co-detection with other pathogens.

Future development should focus on:

• Quantitative or semi-quantitative reporting to help distinguish active infection from carriage
• Integration of antimicrobial resistance markers
• Refined pathogen targets to improve clinical specificity
• Cost-effectiveness analyses across different healthcare settings
• Standardized protocols for result interpretation and reporting

Multiplex gastrointestinal PCR panels represent a significant advancement in the diagnosis of infectious gastroenteritis, demonstrating consistently higher detection rates compared to conventional methods across diverse patient populations. The AllPlex Gastrointestinal Panel Assays show excellent performance characteristics, with overall detection rates of 44.4% compared to 17.8% for conventional methods in a mixed patient population [17]. While direct comparative data specifically stratified by inpatient versus outpatient status remains limited, the consistent pattern of enhanced detection capability across healthcare settings supports the utility of these platforms for improving diagnostic accuracy and patient management.

The selection of an appropriate multiplex GI panel should consider factors beyond mere detection rates, including turnaround time, throughput requirements, cost, and the specific pathogen targets most relevant to the patient population served. As these technologies continue to evolve, further research explicitly examining performance differences between inpatient and outpatient populations will enhance our understanding of their optimal application across the healthcare continuum.

Impact on Diagnostic Yield Compared to Conventional Testing Methods

The accurate and timely identification of pathogens causing acute gastroenteritis is fundamental for effective patient management, antimicrobial stewardship, and public health surveillance [7] [19]. For decades, conventional methods such as stool culture for bacteria, microscopy for parasites, and antigen tests or electron microscopy for viruses have formed the cornerstone of diagnostic workflows. However, these techniques are often characterized by longer turnaround times, lower sensitivity, and a limited scope of detectable pathogens [7] [36]. The advent of multiplex PCR syndromic panels, like the Allplex Gastrointestinal Panel (AGPA, Seegene, Seoul, Korea), represents a paradigm shift in diagnostic microbiology. This guide objectively evaluates the impact of the Allplex GI Panel on diagnostic yield compared to conventional testing methods, synthesizing evidence from large patient cohort studies to provide researchers and clinicians with a clear comparison of performance metrics and methodological considerations.

Comparative Diagnostic Performance: A Data-Driven Analysis

Evidence from multiple clinical studies consistently demonstrates that the Allplex GI Panel significantly enhances the detection of gastrointestinal pathogens compared to conventional methods.

Table 1: Overall Pathogen Detection Yield: Allplex vs. Conventional Methods

Study Cohort Description	Sample Size	Positive by Conventional Methods	Positive by Allplex GI Panel	P-value / Statistical Significance
Prospective Study (Canada) [17]	135	24 (17.8%)	60 (44.4%)	P < 0.001
Patients with Acute Gastroenteritis (Korea) [3]	432	27 (for Salmonella spp. by OBC)	40 (for Salmonella spp.)	P = 0.002 (for any target)
Multicenter Study (Lebanon) [19]	271	Not Reported	192 (71%)	Not Applicable

A 2018 study by T. Brewer et al. found that the Allplex panel detected over twice as many pathogens as conventional culture and electron microscopy (44.4% vs. 17.8%) [17]. Discrepant analysis confirmed that the majority of the additional positives detected by the molecular panel were true infections, underscoring its superior sensitivity [17]. Similarly, a 2024 Korean study reported a significantly higher positivity rate for the Allplex panel (54.9%) compared to a conventional multiplex PCR panel (48.8%) [3].

The increased detection yield is particularly pronounced for specific pathogen groups that are challenging to culture or identify morphologically.

Table 2: Detection of Specific Pathogen Groups

Pathogen Group / Species	Conventional Method	Performance of Conventional Methods	Performance of Allplex GI Panel
Diarrheagenic E. coli	Culture (largely unavailable)	Often not detected [17]	Detects EAEC, EPEC, ETEC, EIEC, STEC; identified in 9.3% to over 23% of samples [3] [19]
Salmonella spp.	Stool Culture	Lower sensitivity; detected 8-27 isolates [3] [17]	Higher sensitivity; detected 40 isolates [3]
Intestinal Protozoa	Microscopy	Low sensitivity (e.g., 14.1% for D. fragilis, 44.2% for B. hominis) [16]	High sensitivity (e.g., 97.2%-100% for G. duodenalis, D. fragilis, B. hominis) [18] [16] [5]

The Allplex panel provides a distinct advantage in detecting diarrheagenic E. coli pathotypes, which are not routinely identifiable with standard culture [17]. In a Lebanese cohort, enteroaggregative (EAEC), enterotoxigenic (ETEC), and enteropathogenic (EPEC) E. coli were the three most frequently identified pathogens, collectively found in over two-thirds of positive samples [19]. For parasites, a large prospective study on 3,500 stool samples concluded that "the multiplex PCR proved more efficient to detect protozoan parasites" than microscopic examination [18].

Detailed Experimental Protocols from Key Studies

To assess the validity of the comparative data, it is essential to understand the experimental methodologies from which they are derived.

Protocol: Comparative Evaluation of Two Multiplex Panels in AGE

This 2024 study provides a direct comparison between the Allplex panel and an earlier multiplex PCR panel (Seeplex) alongside ordinary bacterial culture (OBC) [3].

• Sample Collection: A total of 432 stool specimens were randomly collected from both inpatients and outpatients with acute gastroenteritis between January 2021 and July 2022.
• Conventional Culture (OBC): Ordinary bacterial stool culture was performed using Hektoen Enteric / Salmonella-Shigella agar, targeting only Salmonella and Shigella species.
• Molecular Testing: Nucleic acids were extracted from all samples using the Microlab STARlet system (Hamilton Robotics). The extracts were then tested using both the Allplex Gastrointestinal V/B1/B2 Assays and the Seeplex Diarrhea V/B1/B2 ACE Detection Assays (a conventional multiplex PCR requiring electrophoresis).
• Data Analysis: The overall percent agreement (OPA) between the two molecular panels was calculated for common targets. Detection rates for all targets were compared, with a specific focus on Salmonella spp., Shigella spp., Clostridium perfringens, and pathogenic E. coli.

Protocol: Prospective Evaluation of Allplex vs. Conventional Methods

This 2018 study prospectively compared the Allplex panel to the standard of care methods in a Canadian setting [17].

• Sample Collection: 135 consecutive stool samples submitted for both bacterial and viral testing were included prospectively. An additional set of archived bacterial culture-positive samples was also tested to assess sensitivity.
• Conventional Methods:
  • Bacterial Culture: Stools were cultured in enteric transport medium using selective and differential media, with organisms identified by standard methods.
  • Viral Electron Microscopy (EM): Viruses were detected using a JEM 1010 Electron Microscope following standard procedures.
  • C. difficile Testing: An enzyme immunoassay for glutamate dehydrogenase (GDH) followed by Toxin B PCR if GDH-positive.
• Molecular Testing: Nucleic acid was extracted using the MagNA Pure Compact System (Roche). The Allplex GI Assay was performed on a CFX96 Real-Time Detection System (Bio-Rad), targeting 13 bacteria and 6 viruses.
• Discrepancy Analysis: Samples positive by Allplex but negative by conventional methods were tested with monoplex 5'exonuclease probe PCR assays. Results were considered true positives if positive by conventional methods or by both the Allplex and monoplex PCR assays.

Protocol: Multicenter Evaluation of the Allplex GI-Parasite Assay

This 2025 Italian study evaluated the parasitic component of the panel against traditional parasitological techniques across 12 laboratories [5].

• Sample Collection and Reference Testing: 368 samples collected for routine diagnosis were analyzed using conventional techniques per WHO and CDC guidelines. These included macroscopic examination, microscopic examination after concentration, Giemsa or Trichrome staining, antigen detection tests for Giardia duodenalis, Entamoeba histolytica/dispar, and Cryptosporidium spp., and amoebae culture.
• Molecular Testing: Samples were stored frozen, then retrospectively extracted using the Microlab Nimbus IVD system (Hamilton). The Allplex GI-Parasite Assay was run on a CFX96 Real-Time PCR System (Bio-Rad).
• Data Analysis: Sensitivity and specificity were calculated for each target (G. duodenalis, D. fragilis, E. histolytica, B. hominis, C. cayetanensis, and Cryptosporidium spp.) using the conventional method results as the baseline comparator.

Workflow and Performance Visualization

The transition to syndromic PCR testing represents a fundamental shift in the laboratory workflow for diagnosing gastroenteritis, which directly contributes to its enhanced diagnostic yield.

The streamlined molecular workflow not only accelerates time-to-result but also systematically addresses the primary limitations of conventional methods. The following performance summary visualizes the key advantages:

Table 4: Key Performance Advantages of the Allplex GI Panel

Aspect	Conventional Methods	Allplex GI Panel	Impact on Diagnostic Yield
Turnaround Time	2-3 days (culture) to several days (full workup) [7]	~4 hours post-extraction [17]	Enables same-day clinical decision-making
Pathogen Spectrum	Limited to cultivable bacteria, visible parasites [17]	25+ targets, including diarrheagenic E. coli and viruses [19]	Directly increases detection of pathogens with no routine conventional tests
Analytical Sensitivity	Variable and often low (e.g., microscopy for D. fragilis) [16]	High and consistent (e.g., 97.2% for D. fragilis) [5]	Reduces false negatives, especially in low pathogen load cases
Automation & Standardization	High technologist dependence and subjective interpretation	High degree of automation and software-based interpretation [17] [5]	Improves reproducibility and reduces inter-operator variability

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 5: Key Research Reagent Solutions for Allplex GI Panel Implementation

Item	Function in Experimental Protocol	Example from Search Results
Automated Nucleic Acid Extractor	Standardizes the extraction of DNA/RNA from complex stool matrices, reducing manual labor and contamination risk.	Hamilton MICROLAB STARlet [3], Hamilton MICROLAB Nimbus IVD [5]
Real-Time PCR Instrument	Performs the multiplex PCR amplification and fluorescence detection for multiple targets simultaneously.	CFX96 Real-Time Detection System (Bio-Rad) [17] [16] [5]
Stool Transport/Lysis Buffer	Preserves nucleic acids and prepares the stool sample for automated extraction.	ASL Stool Lysis Buffer (Qiagen) [17] [5] [19], Cary-Blair Medium (e.g., FecalSwab) [36] [5]
Software for Result Interpretation	Automates the interpretation of complex multiplex PCR results, assigning positivity based on Ct values.	Seegene Viewer Software [17] [5] [19]

The collective evidence from large patient cohort studies firmly establishes that the Allplex Gastrointestinal Panel significantly improves the diagnostic yield for acute gastroenteritis compared to conventional testing methods. The panel consistently demonstrates a two-fold or greater increase in pathogen detection rates by overcoming the critical limitations of traditional techniques: their low sensitivity for fastidious bacteria and parasites, and their inability to routinely detect important pathogen groups like diarrheagenic E. coli. For researchers and clinicians, the implementation of this syndromic molecular approach translates to more accurate epidemiological data, enhanced patient management through etiologic diagnosis, and robust tools for public health surveillance.

Implementation Strategies and Analytical Performance in High-Throughput Diagnostic Settings

The adoption of multiplex molecular panels for the diagnosis of gastrointestinal infections represents a significant advancement in clinical microbiology. These systems, which integrate automated nucleic acid extraction and PCR setup, have demonstrated potential to streamline laboratory workflows while maintaining diagnostic accuracy. This guide objectively compares the workflow efficiency of the Seegene AllPlex Gastrointestinal Panel system against alternative methodologies, drawing from large-scale clinical evaluations to provide evidence-based insights for researchers and laboratory professionals making platform selection decisions.

Automated Workflow Architecture & Technological Foundations

Automated molecular diagnostic systems integrate several key technological components that collectively contribute to workflow efficiency. The fundamental architecture typically consists of three interconnected modules: sample preparation, nucleic acid extraction, and PCR setup. Systems compatible with the Seegene AllPlex panels, such as the Hamilton STARlet and Seegene NIMBUS platforms, automate these processes through robotic liquid handling systems that transfer samples and reagents with minimal manual intervention [13] [16].

These automated platforms utilize specialized cartridge-based extraction chemistry, such as the STARMag 96 Universal Cartridge kit, which processes samples in batch formats ranging from 25 to 96 reactions depending on the system configuration [16]. The integration of upstream sample pre-treatment steps varies between systems, with some platforms requiring manual suspension of stool samples in transport media like Cary-Blair or eNAT before automated processing [7] [6]. This architectural variation represents a critical differentiator in total hands-on time requirements between competing systems.

The software integration layer represents another key component, with systems like the Seegene platform incorporating automated data interpretation through Seegene Viewer software that interfaces directly with Laboratory Information Systems (LIS) [13]. This eliminates manual result interpretation steps required by some conventional PCR systems that rely on electrophoresis and visual reading [3].

Comparative Performance Metrics Across Automated Platforms

Throughput and Processing Time Analysis

Table 1: Automated Platform Processing Capabilities Comparison

Platform	Extraction & PCR Setup Time	Maximum Batch Size	Total Hands-On Time (Estimated)	Compatibility with AllPlex Panels
Seegene STARlet	~4 hours (total processing)	96 samples	30-45 minutes	Full compatibility [16]
Hamilton STARlet	~4 hours (total processing)	96 samples	30-45 minutes	Full compatibility [7] [6]
Manual processing	24-72 hours (culture) + additional PCR setup	Limited by manual capacity	2-3 hours (active hands-on)	Not applicable
BD MAX Enteric	Not specified	Not specified	Not specified	Limited comparison [24]
Luminex xTAG/NxTAG	Not fully automated	Not specified	Significant manual steps	Limited comparison [24] [7]

The data from comparative studies demonstrates that automated extraction and PCR setup systems significantly compress total processing time compared to conventional methods. The AllPlex system with automated extraction requires approximately 4 hours from sample to result for molecular detection, compared to 24-72 hours for conventional culture methods [17]. This represents a 83-94% reduction in turnaround time, enabling same-day result reporting that significantly impacts clinical decision-making for gastroenteritis management.

Diagnostic Performance in Large Cohort Studies

Table 2: Detection Performance in Comparative Studies

Evaluation Parameter	AllPlex with Automation	Conventional Methods	Alternative Molecular Panels
Overall pathogen detection rate	44.4-66.2% [17] [1]	17.8-27.7% [17] [1]	48.8% (Seeplex) [3]
Positive percentage agreement	94% [24]	Reference	92% (Luminex), 78% (BD MAX) [24]
Sensitivity for protozoa detection	81-100% [16]	14.1-77.5% [6] [16]	Not specified
Co-detection capability	23.3% samples with >1 pathogen [17]	Limited	11.8% (Seeplex) [3]

The implementation of automated AllPlex systems demonstrates not only workflow efficiency but also enhanced diagnostic sensitivity across large-scale evaluations. One prospective study on 3,500 stool samples conducted over three years confirmed the superior detection capability of the multiplex PCR approach for intestinal protozoa compared to classical microscopy [18]. Similarly, a study on 394 diarrheic stool samples found the AllPlex system detected 66.2% positive samples compared to 27.7% with conventional culture methods [1].

Experimental Protocols for Workflow Efficiency Assessment

Standardized Automated Processing Protocol

The following methodology was adapted from multiple large-scale evaluations of the AllPlex system with automated extraction [7] [16]:

• Sample Preparation: Suspend 140-180 mg of stool specimen in Cary-Blair transport medium (FecalSwab) and vortex thoroughly. After 10-minute incubation at room temperature, centrifuge at 2000×g for 10 minutes.

• Automated Extraction: Transfer 50 μL of supernatant to the extraction plate on the automated system (Hamilton STARlet or equivalent). Utilize the STARMag 96 Universal Cartridge kit with a final elution volume of 100 μL. The internal control DNA is added to the medium before extraction as quality control.

• PCR Setup: The automated system dispenses 5 μL of DNA extract into pre-plated PCR mixtures containing AllPlex master mix and primers. The system utilizes barcoded tube strips for sample tracking.

• Amplification and Analysis: Perform multiplex real-time PCR on a CFX96 system (Bio-Rad) with the following cycling parameters: 50°C for 20 minutes, 95°C for 15 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Automated result interpretation is performed using Seegene Viewer software with data export to LIS.

Workflow Efficiency Measurement Parameters

Studies comparing automated versus manual workflows typically assess the following metrics [3] [17] [1]:

• Total Hands-on Time: Measured in minutes from sample receipt to PCR initiation
• Total Turnaround Time: From sample receipt to result reporting
• Batch Processing Capacity: Number of samples processed per technologist per 8-hour shift
• Error Rates: Frequency of invalid results requiring retesting
• Result Concordance: Percentage agreement between automated and manual result interpretation

Research Reagent Solutions for Automated Workflow Implementation

Table 3: Essential Research Reagents and Platforms

Component	Specific Product Examples	Function in Workflow	Compatibility Notes
Automated Extraction System	Hamilton STARlet, Seegene NIMBUS	Nucleic acid purification	Hamilton STARlet used in multiple studies [7] [16]
Extraction Chemistry	STARMag 96 Universal Cartridge	DNA/RNA isolation	Compatible with STARlet system [16]
Transport Medium	Cary-Blair, eNAT	Sample preservation & transport	Enables sample stability [7] [16]
Real-time PCR System	CFX96 (Bio-Rad)	Amplification & detection	Standard platform for AllPlex [6] [16]
Master Mix	AllPlex GI Panel Assays	Multiplex PCR reaction	Target-specific primer/probe sets [13]
Analysis Software	Seegene Viewer	Automated result interpretation	Reduces manual analysis time [13]

Operational Impact Analysis

Hands-On Time Reduction Metrics

Implementation of fully automated extraction and PCR setup systems demonstrates significant reduction in active technologist time. Studies indicate that automated processing reduces hands-on time from approximately 2-3 hours with conventional methods to 30-45 minutes with systems like the Hamilton STARlet [17] [1]. This 75-85% reduction in active hands-on time enables laboratory staff to perform other value-added activities while processing occurs automatically.

The automation of PCR setup particularly contributes to this efficiency gain by eliminating manual reagent aliquoting and reaction assembly, which are not only time-consuming but also potential sources of pre-analytical error. The integrated approach also reduces material costs through minimized reagent dead volumes and decreased repeat testing due to improved process standardization [3].

Limitations and Implementation Considerations

Despite the demonstrated efficiencies, automated systems present certain limitations that warrant consideration. The AllPlex GI-Helminth assay has shown suboptimal performance compared to microscopy (59.1% sensitivity vs. 100% with conventional methods) [6], indicating that complementary traditional techniques remain necessary for comprehensive parasitology evaluation in specific patient populations.

Additionally, the high throughput capabilities of automated systems (up to 96 samples per run) may be underutilized in lower-volume settings, potentially affecting cost-efficiency. Platform selection must therefore align with testing volumes and specific pathogen detection requirements of the patient population served.

Automated extraction and PCR setup systems, particularly when implemented with comprehensive multiplex panels like the Seegene AllPlex Gastrointestinal assays, demonstrate substantial advantages in workflow efficiency and hands-on time reduction compared to conventional methods and less integrated molecular platforms. Evidence from large-scale clinical evaluations confirms that these systems can reduce active technologist time by 75-85% while simultaneously improving pathogen detection rates and maintaining high diagnostic accuracy. Laboratory directors and researchers should consider testing volume, required pathogen targets, and existing platform infrastructure when selecting automated systems to maximize operational efficiencies while maintaining diagnostic comprehensiveness.

This guide provides an objective comparison of the analytical and clinical performance of the AllPlex Gastrointestinal Panel (Seegene) against conventional diagnostic methods and other molecular panels. The data is framed within the context of large patient cohort research to aid researchers, scientists, and drug development professionals in evaluating this syndromic testing platform.

The following tables summarize the detection capabilities and clinical performance of the AllPlex GI Panel for bacterial, viral, and parasitic targets, as evidenced by multiple independent studies.

Table 1: Pathogen Coverage of the AllPlex GI Panel Assays [13]

Panel Name	Targets Detected	Number of Targets
GI-Bacteria(I) Assay	Aeromonas spp., Campylobacter spp., Clostridium difficile toxin B, Salmonella spp., Shigella spp./Enteroinvasive E. coli (EIEC), Vibrio spp., Yersinia enterocolitica	7
GI-Bacteria(II) Assay	Enteroaggregative E. coli (EAEC), Enteropathogenic E. coli (EPEC), E. coli O157, Enterotoxigenic E. coli (ETEC), Hypervirulent C. difficile, Shiga toxin-producing E. coli (STEC)	6
GI-Virus Assay	Adenovirus, Astrovirus, Norovirus GI, Norovirus GII, Rotavirus A, Sapovirus	6
GI-Parasite Assay	Blastocystis hominis, Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia	6

Table 2: Clinical Sensitivity and Specificity from Large Cohort Studies

Target Pathogen	Sensitivity (%)	Specificity (%)	Study Details
Giardia duodenalis	100	99.2 - 100	Prospective study (n=586) and Italian multicentric study (n=368) [37] [5]
Cryptosporidium spp.	100	99.7 - 100	Retrospective (n=26) & prospective studies; detected 6 different species [37] [5]
Entamoeba histolytica	100	100	Italian multicentric study (n=368) [5]
Dientamoeba fragilis	97.2 - 100	100	Italian multicentric study (n=368) and prospective evaluation [37] [5]
Blastocystis hominis	99.4	N/R	Prospective study (n=588); significantly higher than microscopy (44.2%) [37]
Cyclospora cayetanensis	100	N/R	Retrospective evaluation (n=4) [37]
Bacterial Panel (Overall)	>95*	>95*	Evaluation on 394 samples; *Except Aeromonas spp. (Sens: 81%) [1]

Comparative Diagnostic Performance

Versus Conventional Methods

Molecular methods like the AllPlex GI Panel demonstrate significantly higher sensitivity compared to traditional techniques, which is critical for accurate diagnosis and patient management.

• Bacterial Detection: A 2018 study comparing the AllPlex GI Panel to conventional culture methods found that the molecular assay detected over two-fold more bacterial and viral pathogens (44.4% vs. 17.8% positive samples). The multiplex PCR was also significantly faster, providing results in approximately 4 hours compared to the 24–72 hours required for culture. [17]
• Parasitic Detection: A large prospective study on 3,500 stool samples over three years concluded that multiplex PCR was more efficient at detecting protozoan parasites than microscopic examination. PCR detected protozoa in 909 samples, compared to only 286 samples detected by microscopy. The study noted that microscopy remains necessary for detecting parasites not included in the PCR panel, such as Cystoisospora belli and helminths. [18]
• Detection of Diarrheagenic E. coli: The AllPlex GI Panel reliably detects types of E. coli that are difficult to identify with conventional culture, including Enteropathogenic E. coli (EPEC), Enteroaggregative E. coli (EAEC), and non-O157 Shiga toxin-producing E. coli (STEC). [17]

Versus Other Molecular Panels

A 2025 study provided a direct comparison between the Seegene AllPlex and the Luminex NxTAG Gastrointestinal Pathogen Panel. [7] [38]

• Overall Concordance: Both assays demonstrated high overall concordance, with Negative Percentage Agreement (NPA) values consistently above 95% and overall Kappa values exceeding 0.8 for most pathogens.
• Positive Percentage Agreement (PPA): The average PPA was greater than 89% for nearly all targets. Lower agreement was observed for specific pathogens like Cryptosporidium spp. (86.6%), highlighting particular diagnostic challenges.
• Workflow Difference: A key distinction is that the AllPlex assay requires four separate tubes to complete the full panel testing, whereas the Luminex NxTAG panel requires only a single tube per sample. [7]

Experimental Protocols and Workflows

The methodology from key cited studies provides a reproducible framework for evaluating the AllPlex GI Panel's performance in a research setting.

Sample Processing and DNA Extraction

The following workflow, based on standardized protocols from multiple studies [7] [37] [5], ensures consistent and comparable results.

Key Steps in the Experimental Workflow:

• Sample Collection and Preservation: Stool samples are collected and preserved in Cary-Blair transport medium. Studies have shown that DNA in these suspensions remains stable for up to 7 days at both room temperature and +4°C, facilitating batch processing. [37] [16]
• Nucleic Acid Extraction: The process is automated using systems like the HAMILTON STARlet or NIMBUS with specific extraction kits (e.g., STARMag 96). An internal control DNA is added to the lysis buffer to monitor extraction efficiency and PCR inhibition. [37] [5]
• Multiplex PCR Amplification: The extracted DNA is amplified using the four AllPlex panels on a real-time PCR instrument like the Bio-Rad CFX96. The assay utilizes Seegene's MuDT technology to report multiple Ct values for different targets in a single channel. [13]
• Data Analysis: Results are automatically interpreted using Seegene Viewer software, which provides Ct values and identifies positive/negative results based on predefined thresholds. [37] [5]

Discrepancy Analysis Protocol

In studies comparing the AllPlex GI Panel to conventional methods, a rigorous discrepancy analysis was employed to confirm true positives and negatives. [17] [1]

• Confirmatory Techniques: Results that disagreed between the multiplex PCR and conventional methods were resolved using a third, confirmatory technique. This often included:
  • Monoplex PCR assays designed to target the specific gene of the discrepant organism.
  • Microbial culture with plate-based identification for bacterial targets.
  • Alternative commercial PCR kits (e.g., VIASURE Real-Time PCR Detection Kit, G-DiaParaTM assay for E. histolytica).

The Scientist's Toolkit: Key Research Reagents and Materials

Table 3: Essential Materials for AllPlex GI Panel Evaluation [37] [13] [5]

Item	Function in the Protocol
AllPlex GI Panel Assays	Four multiplex real-time PCR kits for detecting bacteria, viruses, and parasites.
Cary-Blair Transport Medium	Preserves stool sample integrity during transport and storage.
Lysis Buffer (e.g., Qiagen ASL Buffer)	Lyses pathogen cells and (oo)cysts to release nucleic acids.
Automated Nucleic Acid Extraction System	Standardizes DNA/RNA purification (e.g., HAMILTON STARlet, NIMBUS).
Internal Control DNA	Monitors nucleic acid extraction efficiency and detects PCR inhibition.
Real-Time PCR Instrument	Performs amplification and fluorescence detection (e.g., Bio-Rad CFX96).
Seegene Viewer Software	Automates data analysis, interpretation, and result reporting.

Evaluation of the AllPlex GI Panel across large patient cohorts demonstrates its high analytical and clinical sensitivity for a comprehensive range of gastrointestinal pathogens. The panel significantly outperforms conventional microscopy and culture, particularly for protozoa like Dientamoeba fragilis and Blastocystis hominis, and for diarrheagenic E. coli strains. When compared directly with another leading multiplex PCR panel, it shows high overall concordance, with performance variations for specific targets like Cryptosporidium serving as a focus for further assay refinement. The standardized protocols and reagents outlined provide a robust framework for its implementation in clinical and research laboratories.

Multiplex real-time PCR has revolutionized molecular diagnostics by enabling simultaneous detection of multiple pathogens in a single reaction. A significant technological advancement in this field is Multi-Ct technology in single channels, which allows for the reporting of multiple cycle threshold (Ct) values for different analytes within a single fluorescence channel. This technical innovation, exemplified by Seegene's proprietary MuDT (Multiple Detection Temperature) system, represents a substantial leap beyond conventional multiplex PCR where each target requires a dedicated channel. The technology addresses a critical bottleneck in molecular assay design by dramatically expanding multiplexing capacity without requiring additional hardware or fluorescence channels. This review examines the technical foundations, advantages, and data interpretation challenges of Multi-Ct technology, with a specific focus on its implementation in gastrointestinal pathogen detection through evaluation of the AllPlex Gastrointestinal Panel in large patient cohort research. We objectively compare this technology's performance against alternative diagnostic approaches and provide supporting experimental data to inform researchers, scientists, and drug development professionals working in the molecular diagnostics field.

Technical foundations of Multi-Ct technology

Core principles and mechanism

Multi-Ct technology represents a sophisticated approach to multiplex real-time PCR that enables detection and differentiation of multiple targets within a single fluorescent channel. Conventional real-time PCR assays typically require a dedicated channel for each target, fundamentally limiting multiplexing capacity based on the available fluorescence channels on the detection instrument. Multi-Ct technology overcomes this limitation through probe chemistry innovations and temperature-resolution algorithms that allow distinct detection of multiple targets sharing the same fluorescent dye.

The underlying mechanism, commercially implemented in Seegene's MuDT technology, utilizes specially designed probes with different melting temperatures (Tm) for each target within the same channel. During the real-time PCR amplification process, fluorescence measurements are taken at multiple specific temperatures rather than at a single standardized reading point. This temperature-resolution approach generates unique fluorescence signatures for each target based on their probe hybridization characteristics at different temperatures. The resulting data provides individual Ct values for each target in the shared channel, enabling both detection and semi-quantification of multiple analytes simultaneously [39].

This technical approach effectively decouples the relationship between fluorescence channels and detection capacity, allowing development of highly multiplexed assays without requiring instrumentation with numerous optical channels. The technology maintains the practical advantages of real-time PCR while significantly expanding its multiplexing capabilities for comprehensive pathogen detection.

Visualization of Multi-Ct technology workflow

The following diagram illustrates the procedural workflow and key technological differentiators of Multi-Ct analysis in gastrointestinal pathogen detection:

This workflow demonstrates how Multi-Ct technology integrates with automated laboratory systems while providing distinctive advantages over conventional diagnostic approaches. The temperature-resolved detection phase represents the core innovation that enables multiple target discrimination within shared fluorescence channels.

Research reagents and materials for Multi-Ct experiments

Implementation of Multi-Ct technology requires specific research reagents and laboratory equipment optimized for this sophisticated detection methodology. The following table details essential materials and their functions based on current research applications:

Table 1: Essential research reagents and materials for Multi-Ct experimental workflows

Item	Function	Implementation Example
Allplex GI-Parasite Assay	Multiplex real-time PCR detection of 6 protozoan parasites	Targets: Blastocystis hominis, Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia [39] [5]
Automated DNA Extraction System	Nucleic acid purification with minimal inhibitor carryover	Microlab Nimbus IVD system; STARlet automated extractor [6] [5]
Real-time PCR Instrument	Amplification and multi-temperature fluorescence detection	CFX96 Real-time PCR System (Bio-Rad) [6] [5]
Data Interpretation Software	Automated analysis of multi-Ct results and co-infection detection	Seegene Viewer software (version 3.28.000+) [5]
Stool Lysis Buffer	Initial sample processing and pathogen disruption	ASL buffer (Qiagen) for stool specimen homogenization [5]
UDG System	Prevention of PCR amplicon contamination	Uracil-DNA glycosylase incorporation to degrade carryover amplicons [39]
Internal Control	Monitoring of extraction and amplification efficiency	Included in Allplex assays to identify inhibition issues [39]

The integration of these specialized reagents and equipment creates an optimized system for Multi-Ct applications. The automated DNA extraction and PCR setup are particularly critical for maintaining reproducibility in high-throughput research environments, while the specialized detection chemistry enables the core multi-analyte discrimination capability.

Performance evaluation in large patient cohorts

Experimental methodology for comparative studies

Robust evaluation of Multi-Ct technology requires carefully designed experimental protocols that compare its performance against conventional diagnostic methods. Recent multicenter studies have established standardized methodologies for these comparative assessments. The following diagram illustrates a representative study design for evaluating Multi-Ct-based parasite detection:

In a representative Italian multicenter study, 368 stool samples were collected from patients suspected of enteric parasitic infections across 12 laboratories [5]. Samples were examined using conventional techniques including macro- and microscopic examination after concentration, Giemsa or Trichrome staining, antigen detection for Giardia duodenalis, Entamoeba histolytica/dispar, and Cryptosporidium spp., and amoebae culture according to WHO and CDC guidelines. These same samples were stored frozen, then retrospectively extracted and tested using the Allplex GI-Parasite Assay with automated nucleic acid extraction and PCR setup on the Microlab Nimbus IVD system [5]. For discrepant results between methods, samples were retested using both approaches to establish final classification. This rigorous methodology ensures unbiased performance comparison between conventional techniques and the Multi-Ct technology approach.

Comparative performance data

The following tables summarize quantitative performance data for Multi-Ct technology compared to alternative diagnostic approaches from recent multicenter studies:

Table 2: Performance comparison of Allplex GI-Parasite Assay versus conventional methods

Parasite	Sensitivity (%)	Specificity (%)	Comparative Advantage
Entamoeba histolytica	100	100	Species-specific identification unlike microscopy [5]
Giardia duodenalis	100	99.2	Superior to microscopic detection in multi-center studies [5]
Dientamoeba fragilis	97.2	100	Significant improvement over microscopy (47.4% sensitivity) [6] [5]
Cryptosporidium spp.	100	99.7	Equivalent to antigen testing with higher throughput capacity [5]
Blastocystis hominis	95	N/A	Superior to conventional workflow (77.5% sensitivity) [6]

Table 3: Methodological comparison across gastrointestinal pathogen detection platforms

Parameter	Multi-Ct Technology	Conventional Microscopy	Other Molecular Panels
Multiplexing Capacity	6 protozoa in single tube	Limited by technologist expertise	Varies by platform (3-15 targets) [24]
Analytical Sensitivity	High (detection at low parasite burden)	Variable (requires skilled technician)	Generally high [24] [6]
Species Differentiation	Excellent (e.g., E. histolytica vs E. dispar)	Limited	Varies by platform [5]
Throughput	High (automation compatible)	Low (labor-intensive)	Moderate to high [24]
Co-infection Detection	Excellent (individual Ct values)	Challenging (requires multiple techniques)	Platform-dependent [39]
Turnaround Time	~3-4 hours (batch processing)	30-60 minutes per sample (skilled staff dependent)	2-6 hours [24]

The performance data demonstrates consistent advantages of Multi-Ct technology across multiple parasite targets, with particularly notable improvements for pathogens like Dientamoeba fragilis that are difficult to identify by conventional microscopy. The technology maintains excellent specificity while significantly enhancing detection sensitivity compared to traditional methods.

Data interpretation in Multi-Ct systems

Specialized software and analysis approaches

Data interpretation from Multi-Ct experiments requires specialized software capable of resolving multiple targets within individual fluorescence channels. The Seegene Viewer software provides automated interpretation of the complex fluorescence data generated by Multi-Ct assays [39] [5]. This software analyzes fluorescence curves at different detection temperatures to generate individual Ct values for each target in the shared channels, with positive results typically defined as sharp exponential fluorescence curves crossing the threshold at Ct values <45 for individual targets [5].

The software facilitates detection of co-infections by presenting multiple Ct values within the same channel, as demonstrated in a case showing co-infection of B. hominis (Ct 20.10) and G. lamblia (Ct 31.10) in the FAM channel [39]. This capability for semi-quantitative assessment of multiple targets in co-infection scenarios represents a significant advantage over conventional methods that often struggle with mixed infections.

Handling complex results and limitations

Despite sophisticated software, interpretation of Multi-Ct data requires careful consideration of several factors. Weak positive results (Ct values ≥38) may need confirmation through retesting, as implemented in the Italian multicenter study where such samples were retested and considered negative if the second result was negative [5]. Additionally, while Multi-Ct assays demonstrate excellent performance for protozoan detection, their performance for helminth identification has been more variable, with one study showing the Allplex GI-Helminth assay correctly identified only 59.1% of pathogenic helminths compared to conventional microscopy which identified 100% [6]. This highlights that Multi-Ct technology, while powerful, must be applied in context with the specific targets of interest and may require supplemental methods for comprehensive parasite detection.

Multi-Ct technology in single channels represents a significant advancement in multiplex molecular diagnostics, offering expanded detection capabilities within existing instrument platforms. The technology demonstrates excellent performance characteristics for protozoan parasite detection, with sensitivity and specificity frequently exceeding 95% in well-controlled multicenter studies [5]. When implemented with automated extraction and analysis systems, Multi-Ct technology provides substantial advantages in throughput, sensitivity, and co-infection detection compared to conventional microscopic methods. However, researchers should recognize that optimal application of this technology requires understanding its limitations, particularly for helminth detection, and should implement appropriate confirmatory testing protocols for borderline results. As molecular diagnostics continues to evolve, Multi-Ct technology provides a powerful approach for comprehensive pathogen detection in research and clinical laboratory settings.

The integrity of biological specimens is a cornerstone of reliable diagnostic and research data, particularly in large patient cohort studies. The choice between using fresh or frozen samples can significantly influence assay performance, impacting the sensitivity, specificity, and overall validity of experimental findings. Within the context of evaluating the AllPlex Gastrointestinal (GI) Panel—a multiplex real-time PCR assay for detecting intestinal protozoa—understanding the implications of sample processing protocols is paramount. This guide objectively compares the performance of fresh versus frozen specimens across various analytical domains, providing researchers and scientists with evidence-based data to inform their study designs in drug development and clinical research. The analysis is framed by a growing body of literature that directly investigates how pre-analytical handling affects downstream results, enabling a critical assessment of optimal specimen management for large-scale research initiatives.

Performance Data at a Glance

The following tables summarize key experimental data comparing the performance of fresh and frozen specimens across different diagnostic assays and sample types, highlighting the impact on analytical outcomes.

Table 1: Diagnostic Performance of Fresh vs. Frozen Stool Samples in FIT for Colorectal Cancer Screening [40]

Parameter	Frozen Samples	Fresh Samples	Notes
Positivity Rate (at 17 μg/g cutoff)	12.8% (95% CI, 11.3%–14.5%)	8.7% (95% CI, 7.5%–10.1%)	P < .001 for difference
Sensitivity for Advanced Neoplasms	27.8% (95% CI, 21.4%–35.1%)	25.6% (95% CI, 19.8%–32.1%)	At adjusted cutoff for 5% positivity rate
Specificity for Advanced Neoplasms	97.7% (95% CI, 96.8%–98.4%)	97.6% (95% CI, 96.7%–98.3%)	At adjusted cutoff for 5% positivity rate
Conclusion	Comparable performance to fresh samples after adjusting the hemoglobin cutoff value.

Table 2: Stability of Clinical Chemistry Analytes in Serum After Multiple Freeze-Thaw Cycles [41]

Analyte Stability	Representative Analytes	Key Finding
Stable	Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Glucose, Creatinine, Cholesterol, Triglycerides	No significant change after 10 freeze-thaw cycles.
Unstable	Lactate dehydrogenase (LD), Blood urea nitrogen (BUN), Uric acid, Total protein, Albumin, Total bilirubin, Calcium	Changed significantly (P < 0.005) after freeze-thaw cycles.
Conclusion	Many common chemistry analytes show adequate stability, but variability of unstable analytes must be considered.

Table 3: Diagnostic Performance of the AllPlex GI-Parasite Assay on Frozen Samples in a Multicentric Italian Study [5]

Parasite Target	Sensitivity	Specificity
Entamoeba histolytica	100%	100%
Giardia duodenalis	100%	99.2%
Dientamoeba fragilis	97.2%	100%
Cryptosporidium spp.	100%	99.7%
Conclusion	The assay exhibited excellent performance for detecting common enteric protozoa from frozen samples.

Detailed Experimental Protocols

To critically appraise the data on fresh versus frozen specimens, it is essential to understand the methodologies from which these findings are derived. The protocols below detail the key experiments cited in this guide.

Protocol 1: Comparative FIT Performance in a Screening Cohort

This prospective study directly compared the effect of fecal sampling methods on a quantitative fecal immunochemical test (FIT) for colorectal cancer screening [40].

• Study Population & Design: The analysis involved 3,466 participants (mean age 62) undergoing screening colonoscopy at 20 practices in Germany. Up to February 2012, participants collected and froze raw stool samples (n=1,644). After this date, participants collected fresh stool samples directly into buffer-filled tubes (n=1,822). All participants underwent colonoscopy, the results of which served as the reference standard.
• Sample Processing: For the frozen group, participants stored collected stool samples in their home freezers. These were later transported on ice packs to the laboratory, where they were stored at -80°C until analysis. For the fresh group, samples were collected directly into a tube containing a hemoglobin-stabilizing buffer, obviating the need for freezing.
• Laboratory Analysis: Hemoglobin (Hgb) concentrations were measured in all samples using a commercial, quantitative FIT. A positive result was initially defined as ≥17 μg Hgb/g feces, per the manufacturer's recommendation.
• Data Analysis: Test performance (sensitivity and specificity) for detecting colorectal neoplasms (cancer, advanced adenoma, non-advanced adenoma) was calculated against the colonoscopy findings. The Hgb cutoff was subsequently adjusted to generate equal positivity rates (10% and 5%) between the two sample types to enable a direct comparison of test characteristics.

Protocol 2: Multicenter Evaluation of the AllPlex GI-Parasite Assay

This study evaluated the performance of a multiplex PCR on frozen samples compared to conventional techniques, providing a relevant protocol for the AllPlex panel in a large cohort [5].

• Study Design & Sample Collection: A total of 368 stool samples were collected from 12 Italian laboratories during routine diagnostic workups for suspected enteric parasitic infections. This was a retrospective, multicentric study.
• Reference Method (Conventional Techniques): All samples were initially examined using a combination of traditional methods, including macroscopic inspection, microscopic examination of direct and concentrated smears, special stains (Giemsa, Trichrome), antigen detection assays for Giardia, Cryptosporidium, and Entamoeba histolytica/dispar, and in some cases, amoebic culture.
• Sample Storage & Molecular Testing: After conventional analysis, samples were frozen at -20°C or -80°C by the participating labs and later shipped to a central laboratory. There, nucleic acids were automatically extracted using the Microlab Nimbus IVD system. The extracts were tested using the AllPlex GI-Parasite Assay on a CFX96 Real-time PCR system.
• Data Analysis & Discrepancy Resolution: The sensitivity and specificity of the PCR assay were calculated for each parasite target using the conventional method results as the baseline. In cases of discrepant results, samples were re-tested with both the real-time PCR and the traditional methods to resolve the final status.

Workflow and Pathway Diagrams

The following diagram illustrates the logical decision-making pathway and experimental workflow for selecting and processing fresh versus frozen specimens in a gastrointestinal pathogen study, based on the cited methodologies.

The Scientist's Toolkit: Key Research Reagent Solutions

Successful execution of experiments comparing fresh and frozen specimens relies on specific materials and reagents. The following table details essential items used in the featured studies.

Table 4: Essential Research Reagents and Materials for Specimen Processing

Item	Function/Application	Example from Literature
Cary-Blair Transport Medium	A non-nutritive transport medium designed to preserve enteric pathogens in stool samples during transit.	Used for fresh stool samples in GI panel PCR tests to maintain pathogen nucleic acid integrity at ambient or refrigerated temperatures for up to 4 days [42].
Hemoglobin-Stabilizing Buffer	A chemical solution added to fecal immunochemical test (FIT) tubes that prevents degradation of human hemoglobin, allowing for accurate quantification.	Enabled reliable performance of fresh samples in the German FIT cohort study without the need for freezing [40].
Nucleic Acid Stabilization Solution (e.g., RNAlater, eNAT)	A reagent that rapidly permeates tissues or cells to stabilize and protect RNA and DNA at room temperature, minimizing degradation prior to extraction.	eNAT medium was used to suspend stool samples prior to automated DNA extraction for the Seegene Allplex assay [6].
Automated Nucleic Acid Extraction System	Instrumentation that performs automated, high-throughput purification of DNA and/or RNA from various sample matrices, improving consistency and yield.	The Microlab Nimbus IVD system was used for automated extraction and PCR setup in the Italian multicentric evaluation of the AllPlex assay [5].
Multiplex Real-Time PCR Master Mix	A optimized reagent mixture containing enzymes, dNTPs, and buffers, specially formulated for the simultaneous amplification of multiple targets in a single reaction.	The AllPlex GI-Parasite Assay and other commercial panels use such master mixes for the detection of multiple parasite targets [6] [5].

The comparative analysis of fresh and frozen specimen performance reveals a nuanced landscape for researchers designing large patient cohort studies. For the evaluation of the AllPlex GI Panel and similar molecular assays, frozen samples demonstrate excellent and reliable performance for detecting protozoan targets, making them a robust choice for retrospective and batched analyses [5]. The central tenet emerging from the data is that the choice between fresh and frozen is not about inherent superiority, but about context and appropriate protocol adjustment. Key findings indicate that frozen specimens can perform comparably to fresh ones when analytical cutoffs are calibrated for the sample type [40], and that many analytes remain stable through multiple freeze-thaw cycles, though some require careful consideration [41]. Ultimately, the decision should be guided by the study's primary objective, logistical constraints, and target analytes, with the understanding that both pathways are viable when supported by a rigorously controlled and well-documented specimen processing protocol.

Internal Control Systems and Quality Assurance in High-Volume Testing

The shift toward syndromic molecular testing for infectious diseases represents a significant advancement in clinical diagnostics, particularly for gastrointestinal infections characterized by diverse and non-specific clinical presentations. These multiplex panels simultaneously detect numerous bacterial, viral, and parasitic pathogens from a single specimen, revolutionizing laboratory workflows and patient management [7]. However, this technological progression introduces substantial quality assurance challenges, especially in high-volume testing environments where analytical accuracy, process monitoring, and result reliability are paramount.

Internal control systems serve as the foundation for verifying successful nucleic acid extraction, amplification, and detection throughout the testing process. These controls are particularly critical for stool samples, which contain inhibitory substances that can compromise molecular assays. The integration of robust,全程 (whole-process) internal controls allows laboratories to distinguish true negative results from false negatives caused by amplification inhibition or extraction failures [43] [13]. This evaluation examines the internal control systems and quality assurance measures of the AllPlex Gastrointestinal Panel Assays (Seegene, Seoul, Korea) within the context of large patient cohort studies, comparing its performance with alternative multiplex platforms to assess reliability in high-throughput diagnostic settings.

Comparative Platform Technologies and Internal Control Methodologies

AllPlex Gastrointestinal Panel Assays Architecture

The AllPlex Gastrointestinal Panel Assays utilize a multiplex one-step real-time PCR platform capable of detecting 25 gastrointestinal pathogens (13 bacteria, 6 viruses, and 6 parasites) across four coordinated reactions [13]. Key technological features supporting its quality assurance system include:

• MuDT Technology: Enables reporting of multiple Ct values for different analytes within a single fluorescence channel, allowing comprehensive internal control monitoring without channel limitations [13].
• UDG System: Incorporates uracil-DNA glycosylase to prevent carry-over contamination, a critical quality feature in high-volume environments where amplicon contamination can cause false positives [13].
• Whole Process Control: Validates the entire testing process from extraction through amplification, providing end-to-end quality verification [13].
• Automated Interpretation: Seegene Viewer software automatically analyzes results and interfaces with Laboratory Information Systems, reducing subjective interpretation errors [13].

Alternative Platform Control Systems

Comparative platforms utilize different approaches to quality control:

• Luminex NxTAG GPP: Utilizes a single-tube multiplex format with internal controls for extraction and amplification, though with fewer targeted pathogens (15 total) than the AllPlex system [7].
• RIDAGENE Parasitic Stool Panel: Employs a four-plex real-time PCR format with internal controls but lacks the UDG contamination prevention system present in the AllPlex assay [43].
• G-DiaParaTrio: Uses a tri-plex real-time PCR approach with extraction and amplification controls but covers a more limited pathogen spectrum focused solely on parasites [43].

Table 1: Internal Control System Comparison Across Multiplex GI Panels

Platform	Control Type	Contamination Prevention	Automation Compatibility	Pathogen Coverage
AllPlex GI Panels	Whole process control	UDG system	Full automation with Seegene platforms	25 targets
Luminex NxTAG GPP	Extraction/amplification controls	Not specified	Compatible with standard extraction systems	15 targets
RIDAGENE Stool Panel	Extraction/amplification controls	None specified	Compatible with standard extraction systems	Limited parasite targets
G-DiaParaTrio	Extraction/amplification controls	No UDG system	Compatible with multiple thermocyclers	3 primary parasites

Experimental Protocols for Performance Validation in Large Cohorts

Standardized Testing Workflows

Large-scale validation studies have employed standardized protocols to ensure comparable results across testing platforms. The typical workflow for evaluating the AllPlex system encompasses:

Specimen Preparation: Fresh stool samples are collected and suspended in Cary-Blair transport medium (FecalSwab) [16] [2]. For optimal DNA preservation, samples are processed within specified timeframes when stored at room temperature or 4°C, with studies demonstrating stable CT values for up to 7 days under proper conditions [16].

Nucleic Acid Extraction: Automated extraction systems, particularly the Hamilton MICROLAB STARlet with STARMag 96 Universal Cartridge kit or QIASymphony, process 50-400μL of supernatant with elution volumes of 85-100μL [16] [43]. Internal control DNA is added to the lysis buffer prior to extraction to monitor the entire process [16].

Amplification and Detection: PCR reactions are performed on CFX96 Real-Time Detection Systems (Bio-Rad) with cycling parameters of 50°C for 20 minutes, 95°C for 15 minutes, followed by 45 cycles of 95°C for 10 seconds, 60°C for 1 minute, and 72°C for 30 seconds [19]. The internal control amplification is monitored alongside pathogen targets with automatic interpretation via Seegene Viewer software [13].

Diagram 1: AllPlex GI Panel Testing Workflow with Quality Control Checkpoints

Large Cohort Study Designs

Recent prospective studies evaluating the AllPlex system have implemented rigorous methodologies suitable for high-volume settings:

• Robert-Gangneux et al. (2025): Conducted a 3-year prospective study analyzing 3,495 stool samples from 2,127 patients, comparing AllPlex multiplex PCR against classical microscopy with concentration methods [18]. This large-scale design provided substantial power for statistical analysis of assay performance.

• Multi-center Evaluation (2024): Implemented a cross-sectional study of 271 samples from outpatient and inpatient settings, utilizing the full AllPlex gastrointestinal assay to investigate epidemiology while validating the assay's reliability in diverse clinical scenarios [19].

• Comparative Platform Study (2025): Analyzed 196 stool samples using both AllPlex and Luminex NxTAG panels, employing retrospective and prospective approaches to assess concordance through Positive Percentage Agreement (PPA) and Negative Percentage Agreement (NPA) metrics [7].

Performance Metrics in High-Volume Settings

Analytical Sensitivity and Specificity

Large-scale studies demonstrate that the AllPlex system maintains high sensitivity and specificity across diverse pathogen targets, with particular strengths in protozoan detection:

Table 2: AllPlex Performance Metrics for Protozoan Detection in Large Cohort Studies

Pathogen	Sensitivity vs. Microscopy	Specificity vs. Microscopy	Study Cohort Size
Giardia duodenalis	100% (prospective) [16]	100% (prospective) [16]	588 samples [16]
Cryptosporidium spp.	100% (retrospective) [16]	100% (multiple studies) [16] [43]	103 samples [16]
Entamoeba histolytica	100% (prospective) [16]	100% (confirmed cases) [16]	588 samples [16]
Dientamoeba fragilis	97.2% (prospective) [16]	High (study-dependent) [16]	588 samples [16]
Blastocystis hominis	99.4% (prospective) [16]	High (study-dependent) [16]	588 samples [16]

The AllPlex system demonstrated significantly higher sensitivity compared to microscopy, particularly for Dientamoeba fragilis (97.2% vs. 14.1%) and Blastocystis hominis (99.4% vs. 44.2%) in prospective evaluations [16]. This enhanced detection capability directly impacts patient management in high-throughput environments where microscopic screening efficiency varies with technologist expertise.

Comparative Platform Performance

Head-to-head comparisons with alternative molecular platforms reveal important differences in operational reliability:

• AllPlex vs. Luminex xTAG: A comprehensive evaluation of 858 stool samples demonstrated overall positive percentage agreements of 94% for Seegene AllPlex compared to 92% for Luminex xTAG and 78% for BD MAX Enteric panels [24]. The AllPlex system showed fewer false positives for Salmonella compared to Luminex, which exhibited low negative percentage agreement due to frequent false positives with low median fluorescent intensity [24].

• Multi-platform Parasite Detection: A comparative study of three commercial multiplex PCR assays demonstrated overall sensitivity/specificity of 96.5%/98.3% for AllPlex GI parasite assay compared to 93.2%/100% for G-DiaParaTrio and 89.6%/98.3% for RIDAGENE parasitic stool panel [43]. The composite reference method (microscopy) showed substantially lower overall sensitivity of 59.6%, highlighting the superior detection capability of molecular methods [43].

• Bacterial Target Detection: In a prospective study of 432 specimens, the AllPlex system detected bacterial pathogens in 25.2% of specimens compared to 11.9% by conventional methods, with enhanced detection of diarrheagenic E. coli strains [2]. The overall agreement between AllPlex and Seeplex panels exceeded 95% for common targets [3].

Essential Research Reagent Solutions

Implementation of reliable internal control systems requires specific reagent solutions that maintain integrity throughout high-volume processing:

Table 3: Essential Research Reagents for Quality-Assured GI Pathogen Detection

Reagent Solution	Function in Quality Assurance	Implementation Example
Cary-Blair Transport Medium	Preserves nucleic acid integrity during storage and transport	FecalSwab system for room temperature or refrigerated storage [16]
Universal Extraction Kits	Standardized nucleic acid purification with minimal inhibitors	STARMag 96 Universal Cartridge kit on Hamilton systems [16]
Process Control DNA	Monitors extraction efficiency and amplification competence	Included in AllPlex kits, added to lysis buffer pre-extraction [16] [13]
UDG Reaction Components	Prevents carry-over contamination in high-throughput settings	Incorporated in AllPlex master mixes [13]
Multiplex Master Mix	Enables simultaneous amplification of multiple targets with controls	AllPlex proprietary formulations with DPO and TOCE technologies [19] [13]

Discussion: Implications for High-Volume Laboratory Operations

The integration of robust internal control systems in multiplex GI panels like the AllPlex system has substantial implications for high-volume laboratory operations. The whole-process validation approach, which monitors extraction, amplification, and detection within a unified system, provides laboratories with greater confidence in negative results—particularly valuable in exclusion diagnostics [13]. The implementation of UDG contamination prevention proves essential in high-throughput environments where amplicon accumulation poses significant risks to result validity [13].

Large cohort studies confirm that the AllPlex system maintains operational reliability across diverse sample types and storage conditions. The consistency of CT values for samples stored in Cary-Blair medium at both room temperature and 4°C for up to 7 days enables batch testing workflows that improve efficiency in high-volume settings [16]. This stability, combined with automated interpretation systems, reduces technologist-dependent variability—a crucial advantage over microscopy-based methods that show significant sensitivity fluctuations based on operator expertise [18] [16].

While the AllPlex system demonstrates superior sensitivity for most protozoan targets, laboratories must recognize that comprehensive parasitic diagnosis may still require supplementary methods in specific clinical scenarios. As evidenced in the 3,500-sample prospective study, microscopy remains necessary for detecting parasites not included in the multiplex panel, particularly Cystoisospora belli in immunocompromised patients and helminths in returning travelers or migrant populations [18]. This underscores that even advanced molecular systems with robust internal controls function most effectively within a diagnostic algorithm tailored to patient populations and clinical presentations.

Internal control systems represent a critical component of high-volume molecular testing for gastrointestinal pathogens. The AllPlex Gastrointestinal Panel Assays incorporate comprehensive quality assurance features, including whole-process controls, UDG-mediated contamination prevention, and automated result interpretation, that maintain analytical performance across large patient cohorts. Comparative studies demonstrate excellent agreement with alternative molecular platforms while revealing advantages in pathogen coverage and operational reliability.

The implementation of these sophisticated quality systems enables clinical laboratories to meet the challenges of syndromic testing, where result accuracy directly impacts patient management and public health interventions. As molecular diagnostics continues to evolve, further refinement of internal control methodologies will enhance their utility in increasingly complex multiplex assays, ultimately supporting more effective diagnosis and treatment of gastrointestinal infections worldwide.

For clinical laboratories processing hundreds of stool samples, the Seegene Allplex Gastrointestinal Panel Assays offer a distinct high-throughput advantage through automated, batched molecular testing. This guide objectively compares its throughput capabilities against alternative syndromic panels, providing researchers with experimental data on scalability for large patient cohort studies.

Automated High-Throughput Workflow

The core throughput capability of the Seegene system stems from its integration with automated liquid handling platforms and a batched PCR approach.

• Full Process Automation: The system validates the entire diagnostic process from nucleic acid extraction to PCR setup using platforms like the Hamilton MICROLAB STARlet [4] [44]. This integration minimizes hands-on time and reduces procedural variability, which is critical for large-scale studies.
• Batch Processing Design: Unlike single-sample cartridge systems, the Allplex assays are designed for batch analysis [30]. A single 96-well plate can process multiple patient samples simultaneously for a comprehensive pathogen panel, significantly increasing daily testing capacity [7].
• Defined Throughput Metrics: One validation study clocked the total pre-analytical and analytical time at under 7 hours per batch, demonstrating a substantial reduction in turnaround time compared to manual methods [44].

Comparative Throughput and Performance Data

When evaluated against other commercial panels, the Allplex system demonstrates particular strengths in batch scalability, though with variations in per-sample setup complexity.

Table 1: Comparative Throughput Analysis of Multiplex GI Panels

Platform (Manufacturer)	Batch Size Capacity	Samples per Full Run	Targets per Sample	Hands-on Time (per sample)	Total Processing Time
Allplex GI Panels (Seegene) [13] [7]	96-well plate	94 patient samples + 2 controls	Up to 25 pathogens across 4 tubes	~1 minute [33]	~5 hours per batch [33] [44]
LiquidArray Gastrointestinal VER 1.0 [33]	48 samples per run	48 samples	26 pathogens in single tube	<1 minute	~5 hours
BioFire FilmArray GI Panel [45] [46]	Single cartridge system	1 sample per cartridge	~22 pathogens in single tube	Minimal	~1 hour per sample
Luminex NxTAG GPP [7]	96-well plate compatible	Variable	21 pathogens in single tube	Not specified	Not specified

Table 2: Key Performance Metrics in Validation Studies

Performance Measure	Allplex GI Panels	LiquidArray VER 1.0 [33]	Comparative Agreement
Invalid Rate	Not specified	0.5% (initial), 0% (after repeat)	N/A
Sensitivity	>90% for most protozoa [4] [47]	>90% for most targets	86.8-100% with BioFire FilmArray [45] [46]
Specificity	>98% for most targets [4] [44]	>99% for all targets	>95% NPA with Luminex NxTAG [7]
Co-detection Capability	Excellent for protozoa [4]	Excellent co-amplification	Good concordance for multiple pathogens [7]

Detailed Experimental Protocols for Throughput Validation

To ensure reproducible results in large cohort studies, the following validated methodologies from recent studies should be implemented.

Automated Nucleic Acid Extraction Protocol

The DNA extraction process has been specifically optimized for high-throughput processing in studies involving hundreds of samples [4] [44]:

• Sample Preparation: Inoculate one swab of stool into 2 mL of Cary-Blair transport medium (e.g., FecalSwab tubes) and vortex for 10 seconds [44].
• Automated Extraction: Load samples into the Hamilton STARlet system using the STARMag 96 × 4 Universal Cartridge kit [44]. The system automatically processes 50 µL of stool suspension and elutes nucleic acids in 100 µL of elution buffer.
• Quality Control: Incorporate an internal control DNA into the transport medium prior to extraction to monitor extraction efficiency and PCR inhibition [4].

This automated extraction process enables processing of up to 94 patient samples plus controls in a single 96-well plate run, making it particularly suitable for large-scale studies [7].

Multiplex PCR Amplification and Analysis

The amplification protocol has been validated for consistent results across large sample batches [4] [30] [44]:

• PCR Setup: The Hamilton STARlet automatically aliquots 20 µL of PCR master mix (containing 5 µL 5X GI-P MOM primer, 10 µL RNase-free water, and 5 µL EM2) into reaction tubes, then adds 5 µL of extracted DNA [44].
• Thermal Cycling: Run real-time PCR on a Bio-Rad CFX96 system with the following profile [44]:
  • Denaturation: 95°C for 10 seconds
  • Annealing/Extension: 60°C for 1 minute
  • Repeat for 45 cycles
• Result Interpretation: Analyze amplification curves using Seegene Viewer software, which automatically interprets results and provides Ct values. Samples are considered positive at Ct ≤43 according to manufacturer specifications [44].

The Researcher's Toolkit: Essential Reagent Solutions

Table 3: Key Research Reagents for High-Throughput Implementation

Reagent/Kit	Function	Throughput Consideration
STARMag 96 × 4 Universal Cartridge Kit [44]	Automated nucleic acid extraction	Enables simultaneous processing of 96 samples in approximately 2 hours
Allplex GI-Bacteria(I), (II), Virus, Parasite Assays [13] [7]	Multiplex pathogen detection	Four-tube approach requires more setup but enables comprehensive pathogen coverage
Cary-Blair Transport Medium [4] [44]	Sample preservation & transport	Maintains DNA stability for up to 7 days at 4°C, facilitating batch testing
FecalSwab Tubes [4] [44]	Standardized sample collection	Ensures consistent sample input volume for automated processing
Seegene Viewer Software [13] [45]	Automated result interpretation	Reduces analysis time and standardizes calling for large datasets

For large-scale gastrointestinal pathogen surveillance studies, the Seegene Allplex GI Panels provide a viable high-throughput solution with demonstrated capacity for processing hundreds of samples daily through automated batching. While the requirement for multiple reactions per complete pathogen profile increases setup complexity compared to single-tube alternatives, the platform's 96-well format and integration with automated liquid handling systems offer distinct scalability advantages for research cohorts requiring comprehensive pathogen detection. Researchers should weigh the multiplexing capacity and batch efficiency against the operational complexity of multiple reactions when selecting platforms for large cohort studies.

Integration with Laboratory Information Systems and Result Reporting

The diagnosis of gastrointestinal infections presents a complex challenge for clinical laboratories, requiring the accurate detection of diverse bacterial, viral, and parasitic pathogens from a single specimen. Traditional methods relying on microscopy, culture, and antigen detection are time-consuming, labor-intensive, and often lack sensitivity [7]. The integration of multiplex PCR panels like the AllPlex Gastrointestinal Panel represents a paradigm shift toward syndromic testing approaches that can simultaneously detect numerous pathogens from a single stool sample [2]. This transition necessitates sophisticated laboratory information systems (LIS) capable of handling complex result reporting structures while maintaining clarity for clinical decision-making. The implementation of these molecular platforms has demonstrated significant improvements in detection rates - one study showed pathogens were detected in 71% of cases using multiplex PCR compared to lower rates with conventional methods [19]. This article examines the integration and reporting considerations for the AllPlex Gastrointestinal Panel within the context of large patient cohort research, comparing its performance with alternative diagnostic approaches.

Multiplex PCR Technology and Workflow Integration

AllPlex Panel Configurations and Target Pathogens

The AllPlex Gastrointestinal Panel system employs multiple configurations to comprehensively cover gastrointestinal pathogens:

• GI-Bacteria (I) Assay: Detects Aeromonas spp., Campylobacter spp., Clostridioides difficile toxin B, Salmonella spp., Enteroinvasive Escherichia coli / Shigella spp., Vibrio spp., and Yersinia enterocolitica [7]
• GI-Bacteria (II) Assay: Identifies diarrheagenic E. coli pathotypes including Enteroaggregative (EAEC), Enteropathogenic (EPEC), E. coli O157, Enterotoxigenic (ETEC), Enterohemorrhagic (EHEC), plus hypervirulent C. difficile [7]
• GI-Parasite Assay: Detects Blastocystis hominis, Giardia lamblia, Dientamoeba fragilis, Entamoeba histolytica, Cyclospora cayetanensis, and Cryptosporidium spp. [7] [16]
• GI-Virus Assay: Identifies Astrovirus, Sapovirus, Rotavirus A, Norovirus GI-GII, and Adenovirus F [7]

This comprehensive coverage enables laboratories to replace multiple stand-alone tests with a unified workflow, though it requires careful LIS configuration to manage the complex result structures.

Automated Workflow and LIS Integration

The integration of the AllPlex system into laboratory workflows follows a standardized process that can be automated to maximize efficiency:

Automated Laboratory Workflow for AllPlex GI Panel Testing

The workflow begins with stool sample collection in Cary-Blair transport medium, followed by automated nucleic acid extraction using systems such as the Hamilton STARlet or Hamilton Nimbus [7] [5]. The extracted nucleic acids are then subjected to multiplex PCR amplification using Seegene's proprietary technologies including Dual Priming Oligonucleotide (DPO) and Multiple Detection Temperature (MuDT) [19]. Results are automatically interpreted using Seegene Viewer software, which interfaces with laboratory information systems for final result reporting [43] [5].

Key Research Reagent Solutions

Table 1: Essential Research Materials for AllPlex GI Panel Implementation

Component	Function	Implementation Considerations
AllPlex Gastrointestinal Panel Assays (4 panels)	Simultaneous detection of 13 bacteria, 5 viruses, 6 parasites	Requires separate testing tubes; comprehensive pathogen coverage [7]
Nucleic Acid Extraction System (Hamilton STARlet/Nimbus)	Automated nucleic acid purification from stool samples	Integrated protocol for stool samples; reduces hands-on time [7] [5]
Cary-Blair Transport Medium	Sample preservation and transport	Maintains nucleic acid integrity for up to 7 days at 4°C or room temperature [16]
Seegene Viewer Software	Automated result interpretation and analysis	Interfaces with LIS; provides standardized reporting templates [43]
Internal Control DNA	Monitoring extraction and amplification efficiency	Added to each sample prior to extraction; identifies inhibition [16]
Real-time PCR Instrument (CFX96)	Amplification and detection	Compatible with MuDT technology for multiple target discrimination [16] [19]

Comparative Performance Data from Large Cohort Studies

Detection Sensitivity and Specificity Across Pathogen Categories

Large-scale studies have validated the performance of the AllPlex panels across diverse patient populations and geographic regions:

Table 2: Comparative Performance of AllPlex GI Panel in Large Cohort Studies

Study Characteristics	Bacterial Detection	Viral Detection	Parasitic Detection	Key Findings
3,495 stools over 3 years [18]	N/A	N/A	Sensitivity: G. duodenalis (100%), Cryptosporidium (100%), E. histolytica (100%), D. fragilis (97.2%)	Significantly higher detection compared to microscopy; PCR detected protozoa in 909 samples vs. 286 by microscopy
368 samples, multicentric Italian study [5]	N/A	N/A	Sensitivity/Specificity: E. histolytica (100%/100%), G. duodenalis (100%/99.2%), D. fragilis (97.2%/100%), Cryptosporidium (100%/99.7%)	Excellent performance for common enteric protozoa; suitable for routine diagnosis
858 samples comparative evaluation [24]	Overall PPA: 94%	PPA: 99%, NPA: 96%	Included in overall performance	Higher overall positive percentage agreement vs. Luminex (92%) and BD MAX (78%)
196 samples, Seegene vs. Luminex [7]	Average PPA >89% for most targets	Similar high performance	Lower agreement for Cryptosporidium (86.6%)	Overall concordance >95%; Kappa values >0.8 for most pathogens
271 samples, Lebanon implementation [19]	Detected in 48% of cases	Detected in 11% of cases	Detected in 12% of cases	Enteric pathogens detected in 71% of cases; 46% single and 54% mixed infections

Impact on Detection Rates Compared to Conventional Methods

The transition to multiplex PCR significantly enhances pathogen detection capabilities:

Table 3: Detection Rate Comparison Between AllPlex and Conventional Methods

Study	Sample Size	Conventional Method Detection Rate	AllPlex Detection Rate	Fold Increase
Ottawa, Canada [2]	135	24/135 (17.8%)	60/135 (44.4%)	2.5x
French Prospective [16]	586	95/586 (16.2%)	207/586 (35.3%)	2.2x
Spanish Comparison [7]	196	Variable by pathogen	Significantly higher for multiple targets	1.5-3x depending on pathogen

Result Reporting Frameworks and LIS Configuration

Structured Reporting Protocols

The complex nature of multiplex PCR results requires careful consideration of reporting frameworks to ensure clinical utility:

Single versus Multiple Pathogen Detection: Studies consistently show a high rate of multiple pathogen detection with the AllPlex system, ranging from 11.8% to 54% of positive samples [24] [19]. This necessitates clear reporting guidelines to assist clinicians in interpreting potential co-infections versus colonization.

Semi-Quantitative Reporting: The AllPlex system provides Cycle Threshold (Ct) values that can correlate with pathogen load [16]. While not truly quantitative, these values can be structured in reporting to provide clinical context, particularly for pathogens where load may correlate with disease severity.

Reflex Testing Protocols: Implementation of the AllPlex system benefits from established reflex testing algorithms. For example, positive results for C. difficile may require toxin testing, while Salmonella or Shigella positives may require reflex to culture for susceptibility testing [2].

LIS Configuration Considerations

Successful integration requires specific LIS configurations:

• Orderable Test Setup: Creating a unified "GI Panel" orderable that replaces multiple individual test orders
• Cascading Result Fields: Structured result fields that accommodate single or multiple pathogen detection with appropriate comment fields
• Interpretive Comment Libraries: Standardized comments for common detection patterns and clinical correlations
• Epidemiological Reporting Tools: Aggregate data capabilities for infection control and public health reporting

Comparative Performance with Alternative Platforms

Methodological Comparison Studies

Several studies have directly compared the AllPlex system with alternative molecular platforms:

AllPlex versus Luminex Platforms: A 2025 comparison of AllPlex with Luminex NxTAG GPP demonstrated high overall concordance, with Negative Percentage Agreement (NPA) consistently above 95% and Kappa values exceeding 0.8 for most pathogens [7]. Discrepancies were noted for specific pathogens including Salmonella spp. and Cryptosporidium spp., highlighting pathogen-specific performance variations.

AllPlex versus Seeplex Platforms: A prospective comparison between AllPlex and the earlier Seeplex platform from the same manufacturer demonstrated superior detection with AllPlex (54.9% vs. 48.8% positive, p=0.002) [3]. The Allplex system identified 40 samples positive for Salmonella spp. compared to 27 with Seeplex and only 8 with conventional culture.

Multi-platform Evaluation: A 2019 comparative evaluation of Seegene Allplex, Luminex xTAG, and BD MAX systems found Allplex showed the highest overall positive percentage agreement at 94%, compared to 92% for Luminex and 78% for BD MAX [24].

Workflow Efficiency Comparisons

The implementation of multiplex PCR panels significantly reduces turnaround time compared to conventional methods. One study noted that detection of bacterial pathogens by AllPlex required approximately 4 hours versus 24-72 hours for conventional culture and identification [2]. This accelerated time-to-result has important implications for patient management and infection control.

Discussion: Implementation Challenges and Considerations

Analytical Strengths and Limitations

The AllPlex system demonstrates particular strengths in sensitive detection of pathogens that are challenging for conventional methods. For parasitic detection, it shows significantly higher sensitivity compared to microscopy, especially for Dientamoeba fragilis (97.2% vs. 14.1%) and Blastocystis hominis (99.4% vs. 44.2%) [16]. For bacterial targets, it reliably detects diarrheagenic E. coli pathotypes that are not routinely identified through culture methods [2].

Limitations include the inability to distinguish Shigella spp. from enteroinvasive E. coli, lack of detection for some less common pathogens like Plesiomonas shigelloides, and the challenge of interpreting multiple detections [2]. Additionally, the detection of nucleic acid rather than viable organisms may limit utility in some clinical scenarios.

Integration with Conventional Methods

Despite the advantages of multiplex PCR, conventional methods retain importance in specific scenarios. Microscopy remains necessary to detect helminths and parasites not included in the panel, such as Cystoisospora belli [18]. Culture is still required for antibiotic susceptibility testing and outbreak investigation of bacterial pathogens [2]. An optimal diagnostic algorithm incorporates both molecular and conventional methods based on clinical presentation and patient factors.

The integration of the AllPlex Gastrointestinal Panel into laboratory information systems represents a significant advancement in gastrointestinal pathogen detection. Data from large patient cohort research demonstrates superior sensitivity and comprehensive detection capabilities compared to both conventional methods and some alternative molecular platforms. The implementation requires careful consideration of LIS configuration, result reporting frameworks, and reflexive testing protocols. When properly integrated, the system provides clinically actionable results with improved turnaround times, ultimately enhancing patient management and supporting public health surveillance efforts. Future developments should focus on expanding pathogen targets, further automating workflows, and refining interpretive reporting for complex detection patterns.

Performance Challenges and Optimization Strategies for Enhanced Detection Accuracy

Multiplex PCR panels have revolutionized the detection of gastrointestinal pathogens in clinical and research settings, offering a high-throughput alternative to traditional microscopic examination. However, their performance is not uniform across all targets, raising important questions about diagnostic reliability. Entamoeba histolytica, the causative agent of amebiasis, represents a particular challenge due to its morphological similarity to non-pathogenic species and its significant health burden, causing approximately 100,000 deaths annually worldwide [48]. This evaluation examines the performance characteristics of the Allplex Gastrointestinal Panel, with a specific focus on its capacity to accurately detect E. histolytica amidst sensitivity concerns and variable performance reports across different study conditions and pathogen targets.

Performance Comparison of Multiplex PCR Assays

Allplex GI-Parasite Assay Performance Metrics

Table 1: Performance of the Allplex GI-Parasite Assay for Key Protozoan Pathogens

Pathogen	Sensitivity (%)	Specificity (%)	Study Characteristics
Entamoeba histolytica	100	100	Multicentric Italian study (2025), n=368 samples [5]
Giardia duodenalis	100	99.2	Multicentric Italian study (2025), n=368 samples [5]
Dientamoeba fragilis	97.2	100	Multicentric Italian study (2025), n=368 samples [5]
Cryptosporidium spp.	100	99.7	Multicentric Italian study (2025), n=368 samples [5]
Blastocystis hominis	99.4	N/R	Prospective study (2020), n=586 samples [49]
Cyclospora cayetanensis	100	N/R	Retrospective study (2020), n=4 positive samples [49]

N/R = Not Reported

Comparative Performance Against Alternative Molecular Methods

Table 2: Comparative Diagnostic Performance of Commercial Multiplex PCR Assays

Method	Manufacturer	E. histolytica Detection	Overall Sensitivity	Overall Specificity
Allplex GI-Parasite Assay	Seegene	Specific detection	96.5%	98.3%
G-DiaParaTrio	Diagenode Diagnostics	Specific detection	93.2%	100%
RIDAGENE Parasitic Stool Panel	R-Biopharm	Specific detection	89.6%	98.3%
FTD Stool Parasites	Fast Track	Specific detection	N/R	N/R
Microscopy (Composite Reference)	-	Cannot differentiate from E. dispar	59.6%	99.8%

N/R = Not Reported in the available search results [43] [50]

Experimental Protocols and Methodologies

Sample Processing and DNA Extraction

The consistently high sensitivity for E. histolytica detection reported in the 2025 multicentric study was achieved through a standardized protocol across 12 participating Italian laboratories [5]:

• Sample Collection: Stool specimens were collected during routine parasitological diagnostic procedures from patients suspected of enteric parasitic infection.

• Sample Preparation: Between 50-100 mg of stool specimens were suspended in 1 mL of stool lysis buffer (ASL buffer; Qiagen). After pulse vortexing for 1 minute and incubation at room temperature for 10 minutes, tubes were centrifuged at 14,000 rpm for 2 minutes. The supernatant was used for nucleic acid extraction.

• Automated Nucleic Acid Extraction: The Microlab Nimbus IVD system (Hamilton) automatically performed nucleic acid processing and PCR setup, ensuring standardization across sites.

• Amplification Conditions: DNA extracts were amplified with one-step real-time PCR multiplex (CFX96 Real-time PCR, Bio-Rad) using the Allplex GI-Parasite Assay. Fluorescence was detected at two temperatures (60°C and 72°C), with a positive test result defined as a sharp exponential fluorescence curve intersecting the crossing threshold (Ct) at a value of less than 45 for individual targets.

Discrepancy Resolution Methodology

In cases of discordant results between real-time PCR analysis and traditional investigations, samples were retested with both real-time PCR and traditional methods to resolve discrepancies—a critical methodology that strengthens the validity of the reported performance metrics [5].

Critical Analysis of Variable Performance Factors

Sample Quality and Storage Conditions

The retrospective evaluation by Ghozzi et al. (2025) demonstrated optimal performance with 100% sensitivity and specificity for E. histolytica detection, which can be attributed to several methodological strengths [5]:

• Standardized DNA Extraction: The use of automated nucleic acid extraction with the Microlab Nimbus IVD system minimized technical variability across sites.
• Multi-site Validation: Participation of 12 laboratories across Italy provided robust, generalizable performance data.
• Discrepancy Resolution: Systematic retesting of discordant results strengthened the accuracy of final performance metrics.

In contrast, earlier studies noted variable performance for other targets in the panel, particularly for G. duodenalis and D. fragilis, where sensitivity was influenced by parasitic load, with false-negative results associated with low parasite concentrations [49].

Technological Advancements in E. histolytica Detection

The superior performance of molecular methods like the Allplex GI-Parasite Assay for E. histolytica detection must be understood in the context of limitations inherent to traditional diagnostic approaches:

• Microscopy Limitations: Conventional microscopy cannot differentiate between pathogenic E. histolytica and non-pathogenic E. dispar and E. moshkovskii due to morphological similarities [43] [5]. This fundamental limitation has historically complicated accurate diagnosis and treatment decisions.

• Species Differentiation: Molecular methods provide specific identification of E. histolytica through genetic markers, eliminating confusion with non-pathogenic species [51] [5]. Recent advancements in qPCR-HRM (high-resolution melting) analysis have further refined this capability, with studies demonstrating distinct melting peaks for E. histolytica (80±2°C), E. dispar (69±2°C), and E. moshkovskii (82±2°C) [51].

Complementary Detection Methodologies

While the Allplex panel demonstrates strong performance for E. histolytica, researchers should be aware of emerging complementary technologies:

• Serological Approaches: A 2025 study described a gradient-based digital immunoassay using a recombinant Igl-C fragment that detects specific anti-Igl-C antibodies in serum, providing an alternative diagnostic approach within approximately 15 minutes [52].

• Pathogen-Specific Enhancements: For targets with variable performance, supplementary assays may be necessary. For example, the RIDAGENE Parasitic Stool Panel has shown superior sensitivity for Cryptosporidium detection (87.5%), while the FTD Stool Parasites method excelled for G. duodenalis (100% sensitivity) [50].

Research Reagent Solutions

Table 3: Essential Research Materials for Intestinal Protozoa Detection

Reagent/Equipment	Manufacturer	Function in Protocol
Allplex GI-Parasite Assay	Seegene	Multiplex detection of 6 major protozoa
Microlab Nimbus IVD System	Hamilton	Automated nucleic acid extraction and PCR setup
ASL Stool Lysis Buffer	Qiagen	Stool specimen processing and initial lysation
CFX96 Real-time PCR System	Bio-Rad	Amplification and fluorescence detection
Seegene Viewer Software	Seegene	Automated data interpretation and analysis
FecalSwab with Cary-Blair Medium	Copan Diagnostics	Sample preservation and transport

The Allplex GI-Parasite Assay demonstrates excellent performance characteristics for the detection of E. histolytica, with recent multicentric studies confirming 100% sensitivity and specificity when optimal laboratory protocols are followed. The primary advantage of this molecular approach lies in its ability to specifically differentiate pathogenic E. histolytica from non-pathogenic Entamoeba species, a critical diagnostic capability not possible with conventional microscopy. Researchers should implement standardized DNA extraction protocols and be mindful of target-specific performance variations when selecting diagnostic approaches for comprehensive gastrointestinal pathogen detection. The integration of multiplex PCR panels into diagnostic algorithms represents a significant advancement for precise epidemiological studies and clinical management of amebiasis.

Helminth Detection Limitations and Complementary Testing Approaches

Soil-transmitted helminths (STH), including roundworms (Ascaris lumbricoides), whipworms (Trichuris trichiura), hookworms (Necator americanus and Ancylostoma spp.), and threadworms (Strongyloides stercoralis), infect more than 1.5 billion people globally, creating a substantial health burden in tropical and subtropical regions [53]. Accurate detection of these parasites remains challenging despite decades of diagnostic advancement. The World Health Organization (WHO) has established 2030 targets for STH control that explicitly acknowledge the need for improved diagnostic tools to monitor intervention programs effectively [54]. While molecular methods like the AllPlex Gastrointestinal Panel (Seegene, Seoul, South Korea) have revolutionized detection of gastrointestinal bacterial and viral pathogens, their application to helminth detection presents unique challenges that require complementary testing approaches [17] [18].

This review examines the limitations of current helminth detection methods, with specific attention to the AllPlex Gastrointestinal Panel's capabilities and constraints within large-scale research settings. We analyze comparative performance data across diagnostic platforms, detail experimental methodologies from key studies, and present integrated approaches that address the diagnostic gaps in helminth detection for research and clinical practice.

Detection Method Limitations: Microscopy to Molecular Assays

Conventional Microscopy-Based Techniques

Microscopic examination of stool samples remains the most widely used diagnostic approach for helminth infections in resource-limited settings, despite significant limitations. These methods include direct wet mounts, formalin-ether concentration (FEC), Kato-Katz thick smears, McMaster technique, Baermann method, and agar plate culture [55] [53]. Each technique varies considerably in sensitivity, required expertise, and suitability for different helminth species.

Table 1: Performance Characteristics of Microscopy-Based Helminth Detection Methods

Method	Target Helminths	Sensitivity Range	Advantages	Limitations
Kato-Katz	A. lumbricoides, T. trichiura, hookworms	52-83.3% for A. lumbricoides, 12.5-75% for T. trichiura, 37.9-85.7% for hookworms [53]	Low cost, quantitative (egg counts), WHO-recommended	Low sensitivity in low-intensity infections, not suitable for S. stercoralis
Formalin-Ether Concentration	Broad spectrum of helminths	32.5-81.4% for A. lumbricoides, 57.8-75% for T. trichiura, 64.2-72.4% for hookworms [53]	Concentrates parasites, improves detection	Requires multiple steps, chemical handling
Baermann Technique	Strongyloides stercoralis	70% [55]	Specific for larvae detection	Requires specialized equipment, 24-hour incubation
McMaster	A. lumbricoides, hookworms	62% for A. lumbricoides, lower for hookworms [55]	Quantitative, determines infection intensity	Lower sensitivity than sedimentation methods
Agar Plate Culture	Strongyloides stercoralis	Less sensitive than Baermann [55]	Allows larval development	Requires 2-3 days incubation, contamination risk

Microscopy-based methods suffer from several fundamental limitations. Sensitivity is highly dependent on infection intensity, technician expertise, and parasite egg-shedding patterns [53]. A study comparing coprological analysis to adult worm burden in badgers demonstrated that fecal examination consistently underreported infections, with only 41% sensitivity for hookworm and 10% for lungworm compared to actual worm burdens [56]. Similar challenges exist in human diagnostics, particularly as mass drug administration programs reduce infection prevalence and intensity in endemic populations.

Molecular Detection Methods

Nucleic acid amplification tests (NAATs) have emerged as promising alternatives to microscopy, offering potentially higher sensitivity and specificity, particularly in low-intensity infections [57] [54]. Quantitative real-time PCR (qPCR) platforms can detect helminth DNA even when microscopic examination is negative, revealing a significant burden of previously undiagnosed infections [57].

The AllPlex Gastrointestinal Panel Assays (AGPA) represent a comprehensive syndromic testing approach that detects 13 bacteria, 6 viruses, and 6 parasites in four multiplex PCR reactions [17]. However, its utility for helminth detection is limited, as the panel primarily targets protozoan parasites (Giardia intestinalis, Cryptosporidium spp., Entamoeba histolytica, Dientamoeba fragilis, Blastocystis spp., and Cyclospora cayetanensis) with minimal coverage of helminths [18] [43]. This creates a significant diagnostic gap for STH detection in both clinical and research settings.

Table 2: Helminth Detection Capabilities of Commercial Multiplex PCR Panels

Molecular Panel	Helminth Targets	Sensitivity	Specificity	Limitations
AllPlex GI Panel	Limited to protozoan parasites; does not cover STH [18] [43]	96.5% overall for protozoa [43]	98.3% overall for protozoa [43]	No coverage of STH, requires supplementary methods
G-DiaParaTrio	E. histolytica only among parasites [43]	93.2% overall [43]	100% overall [43]	Very limited parasite targets
RIDAGENE Parasitic Stool Panel	E. histolytica, D. fragilis (protozoa) [43]	89.6% overall [43]	98.3% overall [43]	No STH coverage
Research qPCR Assays	Comprehensive STH detection [57] [54]	Varies by target and platform	Varies by target and platform	Not standardized, limited commercial availability

A 5-year international External Quality Assessment Scheme (HEMQAS) for NAATs detecting STH and schistosomiasis revealed substantial inter-laboratory variability in quantitative results (Cq-values), primarily due to differences in sample pre-treatment, DNA isolation methods, and amplification procedures [54]. While false-positive results were rare (<2%), false-negative results occurred in 15% of stool samples and 5% of DNA samples, with the highest rate (29%) for Trichuris trichiura [54].

Comparative Performance Data: AllPlex GI Panel in Large Cohort Studies

Large-scale prospective studies provide the most reliable data for evaluating diagnostic performance in real-world settings. A 3-year prospective study comparing multiplex qPCR (AllPlex GI Panel) to classical microscopy on 3,500 stool samples demonstrated significantly higher detection rates for protozoan parasites with molecular methods [18]. The AllPlex panel detected Blastocystis spp. in 19.25% of samples compared to 6.55% by microscopy, and Dientamoeba fragilis in 8.86% versus 0.63% by microscopy [18].

However, this study also highlighted critical limitations of the AllPlex panel for comprehensive parasitological diagnosis. Microscopy identified additional parasites not targeted by the multiplex PCR panel, including 5 cases of Cystoisospora belli (significant in HIV-infected patients), 331 samples with non-pathogenic protozoa, and 68 samples with helminths [18]. This detection gap underscores the necessity of maintaining microscopic techniques in specific clinical contexts, particularly for immunocompromised patients, migrants, and travelers from helminth-endemic regions.

The analytical performance of the AllPlex GI Panel was directly compared to two other commercial multiplex PCR assays (G-DiaParaTrio and RIDAGENE) in a retrospective study of 184 stool samples [43]. The AllPlex panel demonstrated superior overall sensitivity (96.5%) compared to G-DiaParaTrio (93.2%) and RIDAGENE (89.6%) for protozoan detection, with specificity of 98.3% [43]. Importantly, the composite reference method (microscopy plus E. histolytica-specific adhesion testing) showed markedly lower sensitivity (59.6%), confirming the superior detection capability of molecular approaches for protozoan parasites [43].

For STH detection specifically, research qPCR assays have demonstrated strong correlation between DNA quantity and egg counts in spiked samples. A comparative study of two qPCR platforms (targeting ribosomal sequences versus highly repetitive genomic elements) showed strong correlations for T. trichiura (Tau-b 0.86-0.87) and A. lumbricoides (Tau-b 0.60-0.63), though weaker for A. duodenale (Tau-b 0.41) and S. stercoralis (Tau-b 0.48-0.65) [57]. Field sample testing revealed only moderate agreement between the different qPCR assays (kappa 0.28-0.45 for most STH), highlighting the need for standardization in molecular helminth diagnostics [57].

Experimental Protocols and Methodologies

Sample Collection and Processing

Large-scale studies require standardized protocols for sample collection, transport, and processing to ensure reliable results. The 3-year prospective study on 3,500 stool samples implemented strict standardized procedures [18]. Fresh stool samples were collected without preservatives and processed within 24 hours. Each sample underwent parallel testing using multiplex qPCR (AllPlex GI Panel) and microscopic examination with two concentration methods (sedimentation and flotation). When Cryptosporidium detection was specifically requested, acid-fast staining was additionally performed [18].

For molecular testing, nucleic acid extraction followed manufacturer recommendations with consistent input materials. In the AllPlex GI Panel evaluation, 300 μL of stool was added to 1 mL ASL Stool Lysis Buffer to create a sample suspension [17]. After centrifugation, 400 μL of supernatant was extracted using the MagNA Pure Compact System (Roche Molecular Systems) with a final elution volume of 100 μL [17]. This standardized extraction protocol minimized variability and ensured reproducible results across the study period.

DNA Extraction and Amplification Procedures

The critical importance of DNA extraction efficiency for helminth detection was highlighted in the HEMQAS study, which distributed both stool and DNA samples to participating laboratories [54]. False-negative results were significantly more common for stool samples (15%) than for DNA samples (5%), emphasizing the challenge of efficient DNA release from resilient helminth eggs and larvae [54]. The study identified that proper stool homogenization and rigorous disruption of helminth egg shells are essential steps for reliable DNA amplification.

For the AllPlex GI Panel, multiplex PCR was performed following manufacturer's recommendations on a CFX96 Real Time Detection System (Bio-Rad, Hercules, CA, USA) [17]. The panel utilizes MuDT (Multiple Detection Temperature) technology to detect multiple targets in a single reaction. The parasitic panel specifically targets Giardia intestinalis, Cryptosporidium spp., Entamoeba histolytica, Dientamoeba fragilis, Blastocystis spp., and Cyclospora cayetanensis [43]. Each PCR run includes internal controls to monitor inhibition, with diluted re-testing performed when inhibition is detected [43].

Discrepant Analysis and Resolution

Robust studies incorporate discrepant analysis to resolve conflicting results between diagnostic methods. In the AllPlex GI Panel evaluation, when results disagreed with conventional methods, monoplex 5' exonuclease probe PCR assays were performed as a tiebreaker [17]. Results were considered true positives if positive by conventional methods or if both the AGPA and monoplex PCR assays were positive [17]. Statistical analysis using McNemar's test for paired samples confirmed that the increased detection rate with AGPA was statistically significant (p<0.001) [17].

Figure 1: Diagnostic Workflow for Discrepant Analysis in Helminth Detection Studies

Complementary Testing Approaches

Given the limitations of individual diagnostic methods, integrated approaches provide the most comprehensive detection strategy for helminth infections. The complementary strengths of molecular and conventional methods can be leveraged to maximize diagnostic sensitivity and specificity while maintaining the ability to detect unexpected pathogens.

Integrated Diagnostic Algorithm

An effective diagnostic algorithm begins with clinical assessment to determine pre-test probability based on factors such as immune status, travel history, and symptom profile. For high-risk populations (immunocompromised patients, migrants, travelers from endemic areas), simultaneous testing using both molecular and microscopic methods is recommended [18]. This approach leverages the high sensitivity of PCR for protozoan detection while maintaining the broad detection capability of microscopy for helminths and uncommon parasites.

For STH-specific detection in endemic areas, a cost-effective approach involves initial screening with a sensitive sedimentation/concentration method followed by species-specific qPCR for confirmation and species identification [55] [57]. The Baermann technique remains essential for S. stercoralis detection, as this parasite is frequently missed by both conventional microscopy and molecular panels that lack specific targeting [55].

Figure 2: Complementary Testing Algorithm for Comprehensive Helminth Detection

Method Combinations for Optimal Detection

Research indicates that specific method combinations maximize detection sensitivity for different helminth species:

• For comprehensive STH detection: Sedimentation/concentration combined with Baermann techniques provides the most sensitive microscopic approach [55]. This combination detected A. lumbricoides with 96% sensitivity, hookworms with 87% sensitivity, and S. stercoralis with 70% sensitivity [55].

• For protozoan parasites: Multiplex PCR (AllPlex GI Panel) alone provides superior sensitivity to microscopy, though supplementary microscopy detects non-targeted parasites [18] [43].

• For species-specific identification and quantification: Research qPCR assays targeting highly repetitive genomic elements offer the highest sensitivity, particularly in low-intensity infections [57].

Essential Research Reagent Solutions

Table 3: Key Research Reagents and Materials for Helminth Detection Studies

Reagent/Equipment	Application	Function	Examples/Specifications
ASL Stool Lysis Buffer	Nucleic acid extraction	Stool homogenization and initial lysis	Qiagen ASL Buffer [17]
MagNA Pure Compact System	Automated nucleic acid extraction	High-throughput DNA/RNA purification	Roche MagNA Pure Compact [17]
CFX96 Real Time Detection System	qPCR amplification	Real-time PCR detection and quantification	Bio-Rad CFX96 [17]
Seegene AllPlex GI Panel	Multiplex PCR detection	Simultaneous detection of 25 GI pathogens	Seegene AllPlex GI Assays [17] [18]
Formalin-Ether Solution	Stool concentration	Parasite concentration and preservation	10% formalin with diethyl ether [53]
Baermann Apparatus	S. stercoralis detection	Isolation of live larvae from stool	Funnel, sieve, rubber tube, clamp [55]
Agar Plates	Larval culture	Strongyloides and hookworm cultivation	Non-nutrient agar for parasite culture [55]
Internal Control Assays	PCR quality control	Detection of amplification inhibition	Exogenous internal controls [43]

Helminth detection remains challenging due to the limitations of individual diagnostic methods. The AllPlex Gastrointestinal Panel offers excellent sensitivity for protozoan parasites but does not cover soil-transmitted helminths, creating a significant detection gap. Microscopic methods, while broad in scope, suffer from variable sensitivity and operator dependence. Research qPCR assays demonstrate superior sensitivity for STH detection but lack standardization and commercial availability.

Complementary testing approaches that leverage the strengths of multiple methods provide the most comprehensive solution for helminth detection in research settings. For large cohort studies, an algorithm combining multiplex PCR for protozoan detection with optimized concentration methods and Baermann techniques for STH detection offers an effective strategy. As molecular technologies continue to evolve, integration of STH targets into commercial panels and standardization of extraction protocols will further improve diagnostic capabilities for these neglected tropical diseases.

In the field of molecular diagnostics, effective sample pre-treatment and dilution strategies are critical for overcoming inhibition in gastrointestinal pathogen testing. Inhibition can significantly compromise assay sensitivity and lead to false-negative results, particularly when dealing with complex sample matrices like stool. Within the context of large patient cohort research utilizing the AllPlex Gastrointestinal Panel, resolving inhibition is paramount for data accuracy and reliability. This guide examines the experimental approaches and comparative performance data that inform optimal inhibition resolution strategies, providing researchers with evidence-based protocols for maximizing detection efficacy.

Sample Pre-treatment Methodologies for Inhibition Resolution

Nucleic Acid Extraction Protocols

The foundation of effective inhibition resolution begins with robust nucleic acid extraction. Research across multiple clinical studies has consistently utilized automated extraction systems to ensure standardized sample processing and inhibitor removal.

Hamilton Microlab Nimbus Protocol: In a large prospective study analyzing 3,500 stool samples, researchers employed the Hamilton Microlab Nimbus IVD system for automated nucleic acid extraction [18]. This system automatically performed nucleic acid processing and PCR setup, demonstrating excellent inhibition control across a vast sample set. The protocol involved suspending 50-100 mg of stool specimens in 1 mL of stool lysis buffer (ASL buffer; Qiagen), followed by pulse vortexing for 1 minute and incubation at room temperature for 10 minutes [5]. After centrifugation at full speed (14,000 rpm) for 2 minutes, the supernatant was used for nucleic acid extraction.

Hamilton Microlab STARlet System: Another comparative evaluation of multiplex panels utilized the Hamilton Microlab STARlet for nucleic acid extraction from 432 clinical stool samples [3]. This automated approach supported the high-throughput needs of large cohort research while effectively managing inhibitors present in stool matrices.

Sample Dilution Strategies

When initial extraction fails to resolve inhibition, strategic sample dilution provides an effective secondary approach. While the specific dilution factors aren't detailed in the available studies, the consistent reporting of high sensitivity and specificity across large sample sets suggests that standardized dilution protocols were implemented when necessary.

Retesting Protocols: The multicentric Italian study implementing the AllPlex GI-Parasite Assay established a clear protocol for discrepancy resolution [5]. When results between real-time PCR analysis and traditional investigations conflicted, samples were retested with both methods. This systematic approach to resolving discordant results inherently addresses inhibition concerns through repeated testing, which may incorporate dilution strategies.

Comparative Performance Data in Large Cohort Studies

The effectiveness of pre-treatment and inhibition resolution strategies is ultimately reflected in assay performance metrics across substantial patient populations. The following table summarizes key findings from large-scale evaluations of the AllPlex Gastrointestinal Panel system:

Table 1: Performance Metrics of AllPlex Panels in Large Cohort Studies

Study Description	Sample Size	Target Pathogens	Sensitivity	Specificity	Key Findings
Prospective study over 3 years [18]	3,495 stools from 2,127 patients	G. intestinalis, Cryptosporidium spp., E. histolytica, D. fragilis, Blastocystis spp.	G. duodenalis: 100%Cryptosporidium: 100%D. fragilis: 97.2%E. histolytica: 100%	G. duodenalis: 99.2%Cryptosporidium: 99.7%D. fragilis: 100%E. histolytica: 100%	Multiplex PCR proved more efficient for protozoan detection than microscopy; effective inhibition resolution enabled detection on first stool sample in vast majority of cases
Multicentric Italian study [5]	368 samples from 12 laboratories	G. duodenalis, D. fragilis, E. histolytica, B. hominis, C. cayetanensis, Cryptosporidium spp.	G. duodenalis: 100%E. histolytica: 100%D. fragilis: 97.2%Cryptosporidium: 100%	G. duodenalis: 99.2%E. histolytica: 100%D. fragilis: 100%Cryptosporidium: 99.7%	Excellent performance in detecting common enteric protozoa; validated extraction and inhibition control protocols across multiple sites
Comparative evaluation with Seeplex panels [3]	432 specimens	Bacterial and viral targets for acute gastroenteritis	Overall detection: 54.9% positive for any target	Significantly higher detection than conventional methods (P=0.002)	Superior detection ability for Salmonella and pathogenic E. coli; effective inhibition management in diverse sample types

Research Reagent Solutions for Inhibition Management

Table 2: Essential Research Reagents for Effective Inhibition Resolution

Reagent/Equipment	Manufacturer	Function in Inhibition Resolution
AllPlex Gastrointestinal Panel Assays	Seegene Inc.	Multiplex real-time PCR detection of gastrointestinal pathogens with optimized master mixes containing inhibition-resistant components
AllPlex GI-Parasite Assay	Seegene Inc.	Specific detection of intestinal protozoa with validated resistance to common stool-derived inhibitors
Hamilton Microlab Nimbus IVD	Hamilton Company	Automated nucleic acid extraction system that standardizes inhibitor removal across large sample sets
Hamilton Microlab STARlet	Hamilton Company	High-throughput automated nucleic acid extraction system for processing numerous clinical samples
ASL Buffer	Qiagen	Stool lysis buffer that initiates inhibitor breakdown and prepares samples for nucleic acid extraction
Seegene Viewer Software	Seegene Inc.	Results interpretation software that aids in identifying potential inhibition through curve analysis and Ct value assessment

Workflow Visualization for Inhibition Resolution

Complementary Diagnostic Approaches

Despite the effectiveness of molecular methods with proper inhibition controls, traditional techniques remain valuable in specific scenarios. Microscopy maintains relevance for detecting parasites not targeted by multiplex panels, such as Cystoisospora belli in immunocompromised patients or helminths in migrant and traveler populations [18]. This complementary approach provides a safety net when inhibition might compromise molecular detection of less common pathogens.

Effective inhibition resolution through optimized sample pre-treatment and strategic dilution protocols is fundamental to successful gastrointestinal pathogen detection in large cohort research. The consolidated data from multiple substantial studies demonstrates that the AllPlex Gastrointestinal Panel system, when combined with automated extraction platforms and standardized processing protocols, delivers exceptional sensitivity and specificity across diverse sample types. The implementation of these evidence-based inhibition resolution strategies ensures data reliability and supports the expanding role of multiplex molecular assays in clinical and research settings. As molecular diagnostics continues to evolve, maintaining rigorous attention to sample pre-treatment fundamentals remains essential for accurate pathogen detection and meaningful research outcomes.

Molecular syndromic panels like the Allplex Gastrointestinal Panel (Seegene) represent a significant advancement in diagnosing infectious gastroenteritis. However, their implementation requires robust algorithms to manage discrepancies when their results conflict with those from conventional methods such as culture and microscopy. This guide outlines a data-driven approach for resolving these conflicts, based on performance evaluations from clinical studies.

Multiplex PCR panels detect pathogen nucleic acids with high sensitivity, often identifying pathogens missed by conventional techniques. Consequently, laboratories frequently encounter scenarios where a molecular test is positive but the conventional method is negative. One study found that while conventional methods detected pathogens in 17.8% of specimens, the Allplex assay detected pathogens in 44.4% of the same samples, with the majority of discrepancies confirmed as true positives by additional molecular testing [2]. Establishing a systematic process is therefore essential for accurate patient management and reporting.

Performance Data: Allplex vs. Conventional Methods

The following tables summarize key performance metrics from comparative studies, providing a foundation for assessing likely true positives and false positives.

Table 1: Overall Detection Rate Comparison Between Allplex and Conventional Methods

Study Reference	Specimen Type	Positive by Conventional Methods	Positive by Allplex GI Panel	Key Findings
Yoo et al. (2019) [24]	Clinical stool specimens	N/A	N/A	Overall Positive Percentage Agreement: 94%
Ahn et al. (2018) [2]	Prospective stool samples	24/135 (17.8%)	60/135 (44.4%)	AGPA detected >2-fold more pathogens; 33/37 discrepant positives confirmed as true positives
Paulos et al. (2019) [9]	DNA from stool specimens	N/A	N/A	Excellent sensitivity/specificity for protozoa (e.g., 100%/100% for E. histolytica; 100%/99.2% for G. duodenalis)

Table 2: Pathogen-Specific Performance of Allplex GI Panel

Pathogen Category	Example Pathogens	Common Discrepancy Scenarios	Recommended Action
Bacteria	Salmonella spp., Campylobacter spp., Diarrheagenic E. coli (EPEC, EAEC, ETEC) [2]	Allplex positive, culture negative. Frequent for diarrheagenic E. coli not routinely cultured [2].	Accept Allplex result. If antibiotic susceptibility is needed, inform lab to culture from PCR-positive specimen.
Viruses	Norovirus, Rotavirus, Adenovirus [2]	Allplex positive, electron microscopy (EM) negative. EM is significantly less sensitive [2].	Accept Allplex result. Be aware of potential false positives with other panels (e.g., BioFire) [58].
Parasites	Giardia duodenalis, Dientamoeba fragilis, Cryptosporidium spp. [43] [5] [9]	Allplex positive, microscopy negative. Microscopy has low sensitivity and cannot differentiate E. histolytica from non-pathogenic amoebae [43] [5].	Accept Allplex result. Molecular methods are superior for sensitive and specific parasite identification [5].

Experimental Protocols for Discrepancy Resolution

The following workflow synthesizes methodologies from cited studies to validate and investigate conflicting results systematically.

Detailed Protocol Steps

• Initial Verification: Repeat the Allplex assay using the original nucleic acid extract or, preferably, a new extract from the original stool specimen to rule out technical errors or cross-contamination [2] [5].
• Confirmatory Testing with an Alternate Molecular Method: If the initial result is verified, test the sample using a different molecular technique. This could be:
  • Laboratory-Developed Tests (LDTs) or Singleplex PCRs: Used in multiple studies to confirm targets like norovirus or specific bacterial genes [2] [58].
  • Different Commercial Multiplex Panels: For example, using the Xpert Norovirus or BD MAX Enteric Virus assays to confirm a norovirus result [58].
  • Species-Specific PCRs from Reference Laboratories: Used for definitive confirmation, especially for parasites [7] [43].
• Discrepancy Analysis and Final Interpretation: Consider the results of confirmatory testing alongside clinical and epidemiological data to make a final diagnosis. A confirmed positive result should typically supersede a negative conventional result.

The Scientist's Toolkit: Key Reagents & Materials

Table 3: Essential Research Reagents and Kits for Method Comparison

Item Name	Function/Description	Example Use Case
Stool Lysis Buffer (e.g., ASL Buffer, Qiagen)	Homogenizes and lyses stool specimens for nucleic acid release.	Initial sample preparation for DNA extraction [2] [5].
Automated Nucleic Acid Extraction System (e.g., Hamilton STARlet, MagNA Pure Compact)	Standardizes and purifies nucleic acids from complex stool matrices.	Critical step to remove PCR inhibitors and ensure consistent results across compared methods [7] [2] [3].
Monoplex/Singleplex PCR Assays	Target-specific PCR tests for individual pathogens.	Used for discrepant analysis to confirm positive results from the multiplex panel [2].
Internal Control	Non-human DNA sequence added to each sample.	Monitors both extraction efficiency and PCR amplification to identify inhibition [43] [39].
Seegene Viewer Software	Automated data interpretation software.	Analyzes multiplex real-time PCR results, determines Ct values, and checks for PCR inhibition [43] [39].

Visualizing Comparative Performance

The chart below synthesizes data from multiple studies to illustrate the enhanced detection capability of the Allplex panel compared to conventional techniques across different pathogen types.

Managing discrepancies between the Allplex GI Panel and conventional methods is a critical component of modern laboratory practice. Evidence consistently shows that the Allplex assay offers superior sensitivity for detecting a broad range of gastrointestinal pathogens. A structured algorithm prioritizing molecular confirmation, as outlined in this guide, ensures accurate results, enhances patient care, and maximizes the diagnostic value of syndromic panels.

The accurate diagnosis of gastrointestinal pathogens is crucial for effective patient management, particularly for parasitic infections that cause significant global morbidity and mortality [16]. The shift from traditional microscopic examination to molecular methods like multiplex PCR has transformed diagnostic paradigms, making optimal specimen handling a critical component of reliable test results [5]. Within this context, the AllPlex Gastrointestinal Panel (Seegene Inc., Seoul, South Korea) has emerged as a comprehensive solution for detecting diverse enteric pathogens, though its performance is intimately tied to appropriate pre-analytical specimen management [16] [5]. This evaluation examines the storage conditions and transport media requirements for the AllPlex system, analyzing data from large patient cohorts to establish evidence-based protocols that maximize diagnostic sensitivity and specificity while accommodating practical laboratory workflow needs.

Specimen Handling Protocols for the AllPlex GI Panel

Transport Media and Initial Processing

The AllPlex GI Panel system utilizes a standardized approach to specimen preparation across its various target panels (protozoa, helminths, bacteria, and viruses). The recommended protocol begins with suspending approximately 140-180 mg of stool in Cary-Blair transport medium using the provided FecalSwab system [16] [59]. After vigorous mixing, the suspension should be incubated for 10 minutes at room temperature, followed by centrifugation at 2000 g for 10 minutes [16] [59]. The resulting supernatant is then processed for automated DNA extraction using systems such as the MICROLAB STARlet with STARMag 96 Universal Cartridge reagents [16] [59] [5]. For nucleic acid extraction, 50-200 μL of supernatant is typically used, with final elution volumes of 100 μL [16] [17]. This standardized approach to initial specimen processing ensures optimal DNA yield while minimizing PCR inhibitors present in stool samples.

DNA Preservation in Cary-Blair Suspension

The stability of DNA in Cary-Blair transport medium is essential for flexible laboratory workflows, particularly when batch testing is required. A comprehensive evaluation tested signal intensities (expressed as CT values) for samples positive for various parasites after storage under different conditions [16]. The results demonstrated that FecalSwab stool suspensions in Cary-Blair medium maintained stable DNA signals when stored at both room temperature and +4°C for up to 7 days [16]. Statistical analysis comparing cycle threshold (CT) values before storage (CT(D0)) and after storage (CT(DX)) found no significant differences, indicating excellent DNA preservation across these conditions [16]. This storage flexibility is particularly valuable for laboratories that process specimens in batches or need to transport samples between facilities.

Table 1: DNA Stability in Cary-Blair Transport Medium Under Different Storage Conditions

Storage Duration	Storage Temperature	Organisms Tested	Result	Statistical Significance
Up to 7 days	Room temperature	B. hominis, D. fragilis, G. duodenalis, Cryptosporidium sp., E. histolytica	Stable CT values	No significant difference (p-value not specified)
Up to 7 days	+4°C	B. hominis, D. fragilis, G. duodenalis, Cryptosporidium sp., E. histolytica	Stable CT values	No significant difference (p-value not specified)

Long-Term Storage Considerations

For longer-term storage of specimens prior to DNA extraction, the evidence supports freezing stool samples at -80°C without preservatives as an effective preservation method [16] [59]. Multiple studies utilizing retrospective sample collections stored at -80°C for several years demonstrated successful PCR amplification, indicating that this method effectively preserves nucleic acid integrity [16] [59]. Some studies have also reported successful analysis of samples stored at -20°C, though -80°C is preferred for extended storage periods [5]. For DNA extracts themselves, storage at -20°C is generally sufficient to maintain stability for subsequent molecular analyses [9].

Comparative Performance Data from Large Cohort Studies

Sensitivity and Specificity in Protozoa Detection

Large-scale prospective studies have demonstrated the superior sensitivity of the AllPlex GI-Parasite Assay compared to conventional microscopy. A comprehensive 3-year prospective study analyzing 3,495 stool samples found significantly higher detection rates for most protozoa using the multiplex PCR approach [18].

Table 2: Detection Rates of Intestinal Protozoa by Multiplex PCR vs. Microscopy in a 3-Year Prospective Study (n=3,495 samples)

Organism	Detection by Multiplex PCR	Detection by Microscopy	Relative Improvement
Giardia intestinalis	45 (1.28%)	25 (0.7%)	80% increase
Cryptosporidium spp.	30 (0.85%)	8 (0.23%)	275% increase
Entamoeba histolytica	9 (0.25%)	24 (0.68%)*	*Microscopy cannot differentiate E. histolytica from non-pathogenic E. dispar
Dientamoeba fragilis	310 (8.86%)	22 (0.63%)	1309% increase
Blastocystis spp.	673 (19.25%)	229 (6.55%)	194% increase

Note: Microscopy detection for Entamoeba histolytica actually represents E. histolytica/dispar/moshkovskii complex [16] [18].

A recent Italian multicentric study involving 368 samples corroborated these findings, reporting excellent performance metrics for the AllPlex GI-Parasite Assay with sensitivities of 100% for E. histolytica, 100% for G. duodenalis, 97.2% for D. fragilis, and 100% for Cryptosporidium spp. [5]. The specificities were equally robust, ranging from 99.2% to 100% for these pathogens [5].

Impact of Bead-Beating Pretreatment on Helminth Detection

The detection of helminths presents unique challenges due to the robust structure of eggs and larvae, which can impede DNA extraction. An evaluation of the AllPlex GI-Helminth(I) Assay demonstrated that mechanical lysis through bead-beating pretreatment significantly improves detection sensitivity for certain helminths [59].

Table 3: Impact of Bead-Beating Pretreatment on Helminth Detection Sensitivity

Organism	Sensitivity Without Bead-Beating	Sensitivity With Bead-Beating	Improvement
Trichuris trichiura	9% (1/11)	91% (10/11)	82% increase
Ascaris spp.	88% (7/8)	100% (8/8)	12% increase
Hymenolepis spp.	90% (19/21)	100% (21/21)	10% increase
Hookworms	46% (6/13)	Not specified	Moderate improvement
Strongyloides spp.	80% (28/35)	Not specified	Moderate improvement
Enterobius vermicularis	64% (7/11)	Not specified	Moderate improvement

The need for bead-beating varies by parasite, with the most dramatic improvement observed for Trichuris trichiura, where sensitivity increased from 9% to 91% [59]. This pretreatment involves using MagNA Lyser Green Beads tubes and the MagNA Lyser system for 35 seconds at full speed before proceeding with standard DNA extraction [59].

Workflow Integration and Automation

Automated Workflow Systems

The AllPlex system is compatible with fully automated platforms such as the STARlet All-In-One System (AIOS), which integrates the complete molecular diagnostic workflow from nucleic acid extraction to PCR setup and amplification [60]. This system significantly reduces hands-on time and enables walk-away testing, with studies demonstrating excellent concordance (96%) with laboratory-developed tests for viral gastroenteritis pathogens [60]. The automated workflow minimizes technical variability and increases throughput, making it particularly valuable for high-volume clinical laboratories.

Figure 1: Automated Workflow for AllPlex GI Panel Testing

Comparative Performance with Alternative Methods

When compared to other commercial multiplex PCR assays, the AllPlex system demonstrates favorable performance characteristics. A comparative evaluation of four commercial multiplex real-time PCR assays found that the AllPlex Gastrointestinal Panel showed overall positive percentage agreements of 94% compared to consensus results, outperforming both the Luminex xTAG GPP (92%) and BD MAX Enteric Panel (78%) [24]. Similarly, another study comparing the AllPlex panel with conventional methods found a greater than two-fold higher detection rate (44.4% vs. 17.8%) for bacterial and viral pathogens [17].

The Scientist's Toolkit: Essential Research Reagents

Table 4: Essential Research Reagents for AllPlex GI Panel Implementation

Reagent/Equipment	Function	Application Notes
Cary-Blair Transport Medium (FecalSwab)	Preserves specimen integrity during transport and storage	Maintains DNA stability for up to 7 days at both room temperature and +4°C [16]
STARMag 96 Universal Cartridge	Automated nucleic acid extraction	Used with MICROLAB STARlet system [16] [59]
MagNA Lyser Green Beads	Mechanical lysis for difficult-to-lyse organisms	Critical for helminth detection, especially Trichuris trichiura [59]
AllPlex GI Panel Assays	Multiplex PCR detection of pathogens	Available as separate panels for parasites, helminths, bacteria, and viruses [16] [17] [59]
CFX96 Real-Time PCR System	Amplification and detection	Compatible with Seegene Viewer software for automated result interpretation [16] [5]

The optimal specimen handling for the AllPlex Gastrointestinal Panel requires strict adherence to protocols involving Cary-Blair transport medium, with demonstrated stability for up to 7 days at both room temperature and 4°C. For long-term storage, freezing at -80°C preserves nucleic acid integrity effectively. The implementation of bead-beating pretreatment is essential for adequate detection of certain helminths, particularly Trichuris trichiura. Evidence from large patient cohorts confirms that these optimized specimen handling conditions, combined with the automated workflow capabilities of the AllPlex system, contribute to significantly higher detection rates compared to conventional methods, with sensitivity improvements exceeding 1000% for challenging-to-detect pathogens like Dientamoeba fragilis. These advances position multiplex PCR as a transformative technology in gastrointestinal pathogen diagnosis, though microscopy retains complementary value for detecting parasites not included in molecular panels and for helminth surveillance in specific patient populations.

Automation Challenges and Solutions in High-Volume Laboratory Settings

Clinical laboratories worldwide are undergoing a profound transformation driven by the critical need for enhanced efficiency, accuracy, and throughput in diagnostic testing. The global lab automation market, valued at US $6.36 billion in 2025, is projected to grow at a robust CAGR of 7.2% from 2025 to 2030, culminating in a valuation of US $9.01 billion by 2030 [61]. This surge reflects the rising adoption of automated lab systems, AI-driven research, and smart laboratories across the healthcare spectrum. In high-volume laboratory settings, particularly those processing hundreds of stool samples for gastrointestinal pathogen detection, automation has evolved from a luxury to an absolute necessity. Laboratories face intensifying pressure from growing test volumes, staffing constraints, and the demand for rapid results, pushing leaders to seek technological solutions that can streamline workflows while maintaining diagnostic accuracy [62] [63].

The integration of automation technologies presents both unprecedented opportunities and significant challenges. According to recent surveys, 95% of laboratory professionals believe that adopting automated technologies will improve their ability to deliver patient care, with 89% agreeing that automation is vital to keep up with demand [62]. However, successful implementation requires careful strategic planning, consideration of technical compatibility, and attention to human factors. This article examines these challenges and solutions within the specific context of high-volume gastrointestinal pathogen testing, with a particular focus on the performance of multiplex PCR panels like the AllPlex Gastrointestinal system in automated laboratory environments.

Laboratory Automation Landscape: Current Technologies and Trends

Automation in clinical laboratories extends far beyond simple mechanization, encompassing an integrated ecosystem of advanced technologies including robotics, artificial intelligence, cloud computing, and the Internet of Medical Things (IoMT). These systems automate repetitive and time-consuming processes like sample preparation, liquid handling, and data analysis, delivering substantial benefits in reduced manual effort, minimized human error, standardized processes, and accelerated time-to-results [61].

Several key trends are shaping the laboratory automation landscape in 2025. Process automation is expanding beyond analytical phases to encompass pre-analytical steps like manual aliquoting, with IoMT-enabled devices communicating seamlessly to automate entire workflows [62]. Enhanced data analytics and visualization tools are becoming increasingly available, helping laboratories manage complex datasets, identify trends, and improve clinical decision-making [62]. There is also growing emphasis on sustainability, with laboratories seeking energy-efficient equipment, waste reduction, and greener processes that align with environmental goals while offering long-term savings [62].

For infectious disease diagnostics, including gastrointestinal pathogen detection, automation often centers around syndromic testing panels that integrate nucleic acid extraction, PCR amplification, and analysis in streamlined workflows. These systems differ in their level of automation, throughput capability, hands-on processing time, and turnaround time to result [33]. Platforms like Seegene's AllPlex Gastrointestinal panels can be paired with automated extraction systems and PCR setup instruments like the NIMBUS & STARlet to create semi-automated workflows [13], while other systems offer fully integrated solutions in single cartridges.

Table 1: Key Laboratory Automation Technologies and Their Applications

Technology Category	Specific Applications	Benefits in High-Volume Settings
Robotics	Sample sorting, aliquoting, pipetting, tube opening/closing	Reduced manual labor, increased throughput, minimized repetitive strain injuries
AI and Machine Learning	Data analytics, predictive modeling, image analysis, quality control	Enhanced decision-making, error reduction, operational efficiency
Cloud and IoT Platforms	Remote monitoring, digital collaboration, data integration	Real-time workflow optimization, seamless data sharing, enhanced compliance
Multiplex PCR Systems	Syndromic testing for gastrointestinal, respiratory, CNS infections	Comprehensive pathogen detection, reduced turnaround time, streamlined workflow

Critical Challenges in Implementing Laboratory Automation

Financial and Integration Barriers

The initial capital investment required for advanced automation systems represents the most significant barrier for many laboratories. Automated platforms for molecular diagnostics often carry substantial price tags, with costs extending beyond equipment purchase to include specialized consumables, maintenance contracts, and potential infrastructure modifications [61] [63]. This financial hurdle is particularly challenging for smaller laboratories or those with limited capital budgets. Additionally, integration with existing legacy systems presents technical challenges, as compatibility issues may arise between new automated platforms and established laboratory information systems (LIS) or middleware solutions [61]. Without seamless integration, laboratories risk creating operational silos where automated modules function in isolation rather than as part of a cohesive workflow, potentially limiting the overall efficiency gains.

Operational and Workforce Challenges

From an operational perspective, laboratories must navigate the complex process of reengineering established workflows to incorporate automation effectively. This often requires reevaluating and modifying long-standing procedures, which can disrupt operations during the transition period [63]. The human element presents equally critical challenges, as staff may experience anxiety about job security or struggle to adapt to new technologies and workflows. A survey of laboratory professionals indicates that concerns about automation replacing human jobs remain prevalent, potentially affecting staff morale and cooperation during implementation [63]. Furthermore, operating and maintaining automated systems typically requires specialized technical skills that existing staff may lack, necessitating comprehensive training programs and potentially creating dependency on external technical support [61].

Comparative Performance of Gastrointestinal PCR Panels in Automated Settings

Multiplex PCR panels for gastrointestinal pathogen detection represent a compelling application of automation in high-volume laboratory settings. These panels simultaneously detect numerous bacterial, viral, and parasitic pathogens from stool specimens, dramatically reducing turnaround times compared to traditional culture methods while significantly expanding diagnostic coverage [33]. Several studies have directly compared the performance of different automated and semi-automated GI panels, providing valuable insights for laboratories considering implementation.

A comprehensive 2019 comparative study evaluated three molecular assays: Seegene Allplex Gastrointestinal (24 targets), Luminex xTAG Gastrointestinal Pathogen Panel (15 targets), and BD MAX Enteric panel (5 bacteria and 3 parasites). The researchers analyzed 858 stool samples (554 for bacterial/parasite detection and 304 for viral pathogens) and found that the Seegene Allplex system demonstrated 94% overall positive percentage agreement (258 of 275), outperforming Luminex (92%) and BD MAX (78%) in this metric [24]. The study also revealed that these multiplex molecular assays identified significantly more pathogens compared to routine microbiology testing, with Seegene, Luminex, and BD MAX detecting 39, 40, and 12 additional pathogens respectively [24]. This enhanced detection capability comes with an important caveat: careful interpretation of positive results for multiple pathogens is required, as only 3 of 61 multi-pathogen detections were consensus positives across platforms [24].

More recent studies continue to validate the performance of automated GI panels. A 2024 evaluation of the LiquidArray Gastrointestinal VER 1.0 assay, which detects 25 nucleic acid targets covering 26 pathogens, demonstrated excellent performance characteristics with >90% sensitivity for most targets and >99% specificity for all targets [33]. The study noted a very low invalid rate (0.5% at initial testing, down to 0% after repeat) and highlighted the assay's suitability for implementation in clinical routine for rapid and accurate diagnosis of infectious gastroenteritis [33].

A 2025 study evaluating the Biosynex AMPLIQUICK Fecal Bacteriology kit further reinforces the value of automated syndromic panels, showing positive, negative, and overall agreement rates ranging from 98.24% to 100% for most bacterial targets [64]. This research emphasized that syndromic PCR kits can detect bacteria that are difficult to identify or isolate using conventional methods, thereby expanding the diagnostic coverage of diarrheal syndromes [64].

Table 2: Performance Comparison of Automated Gastrointestinal PCR Panels

Platform	Targets	Overall Positive Agreement	Key Advantages	Limitations
Seegene Allplex	25 targets (13 bacteria, 5 viruses, 6 parasites, 1 internal control) [13]	94% [24]	Comprehensive pathogen coverage, compatibility with automated systems	Requires careful interpretation of multi-pathogen detections [24]
Luminex xTAG GPP	15 targets (9 bacteria, 3 viruses, 3 parasites) [24]	92% [24]	Established platform, good performance for common pathogens	Lower target coverage than some newer panels
BD MAX Enteric	8 targets (5 bacteria, 3 parasites) [24]	78% [24]	Simpler panel, may be sufficient for basic screening	Limited target range may miss less common pathogens
LiquidArray VER 1.0	25 targets covering 26 pathogens [33]	>90% sensitivity for most targets [33]	Low invalid rate, high throughput capability	Newer platform with less established track record

Strategic Implementation Framework for Automation Solutions

Pre-Implementation Planning

Successful automation implementation begins with thorough strategic planning that aligns technological solutions with specific laboratory needs. Rather than adopting automation for its own sake, laboratory leaders should first conduct a comprehensive assessment to identify workflow bottlenecks, staffing pain points, and quality issues that automation might address [63]. This diagnostic phase should include detailed workflow analysis to quantify the time and resources currently devoted to specific tasks, helping to prioritize automation opportunities with the greatest potential return on investment. Engagement with frontline staff during this planning stage is crucial, as these individuals possess intimate knowledge of daily operational challenges and can provide valuable insights into which automation solutions would deliver the most significant benefits [63]. Financially, laboratories should develop realistic total-cost-of-ownership models that account not only for equipment acquisition but also for implementation services, maintenance, consumables, and staff training.

Implementation and Optimization

The implementation phase requires careful change management to ensure smooth transition and staff adoption. Open and transparent communication with frontline staff throughout the process helps alleviate anxiety and build engagement, with clearly articulated information about how automation will affect different roles and responsibilities [63]. Laboratories should develop structured training programs that progress from theoretical understanding to hands-on operation, with particular emphasis on troubleshooting and quality control procedures specific to the new automated systems [63]. From a technical perspective, implementation should follow a phased approach, beginning with parallel testing where automated and manual methods run concurrently to validate performance and build confidence in the new system. Process optimization should focus on the complete end-to-end workflow rather than isolated steps, ensuring that automation delivers comprehensive benefits rather than simply shifting bottlenecks to different parts of the testing process [65]. Successful implementations typically designate automation champions among staff who receive advanced training and can provide peer support, while also establishing clear metrics to quantitatively evaluate the impact of automation on turnaround times, error rates, and operational costs.

Experimental Protocols and Methodologies for Panel Evaluation

Rigorous evaluation of automated gastrointestinal panels requires standardized methodologies to ensure comparable and reproducible results across different laboratory settings. The following protocols represent consolidated approaches from recent studies assessing the performance of syndromic GI panels:

Sample Preparation and Nucleic Acid Extraction: Stool samples, either prospectively collected fresh samples or archived samples stored frozen at -80°C, are aliquoted and added to specific stool transport buffers [33] [64]. Nucleic acid extraction is typically performed using automated systems such as the Microlab STARlet (Hamilton Robotics) or platform-specific extraction modules [64] [3]. The extraction process generally incorporates an internal control to monitor extraction efficiency and detect potential inhibition [13].

Amplification and Detection: For the AllPlex Gastrointestinal Panel, amplification occurs in a one-step real-time RT-PCR format that detects and identifies 25 gastrointestinal pathogens (13 bacteria, 6 viruses, and 6 parasites) across multiple panels [13]. The assay utilizes Seegene's proprietary MuDT technology, which reports multiple Ct values for each pathogen in a single channel using standard real-time PCR instruments [13]. The UDG system is incorporated to prevent carry-over contamination [13]. For fully automated systems like the BD MAX Enteric Panel, extraction and amplification occur within a single integrated cartridge [64].

Data Analysis and Interpretation: Results are automatically interpreted using manufacturer-provided software (e.g., Seegene Viewer for the AllPlex system), which provides automated data interpretation and LIS interlocking capabilities [13]. For comparative studies, consensus positive/negative status is typically defined as concordant results from at least two different testing methods, with discordant resolutions performed using an additional reference method [24] [33].

Statistical Analysis: Performance metrics including positive percentage agreement (PPA), negative percentage agreement (NPA), overall agreement, and kappa values for inter-assay agreement are calculated according to standard statistical methods [24] [33] [64]. For targets with low positive rates, composite reference standards (CRS) may be employed in the absence of a perfect gold standard method [64].

Workflow Visualization of Automated GI Pathogen Testing

The transition from conventional to automated testing methodologies represents a significant restructuring of laboratory workflows for gastrointestinal pathogen detection. The following diagram illustrates the key stages in this automated process:

Automated vs. Conventional GI Pathogen Detection Workflow

This workflow visualization highlights the streamlined nature of automated multiplex PCR testing compared to conventional methods. The automated pathway significantly reduces manual intervention points, decreases hands-on time, and integrates previously disconnected testing processes into a cohesive workflow [13] [65]. For high-volume laboratories, this consolidation translates to substantially improved efficiency, with some automated systems capable of processing up to 48 samples in approximately five hours with less than one minute of hands-on time per sample [33].

Essential Research Reagents and Materials for Automated GI Testing

Successful implementation and operation of automated gastrointestinal pathogen detection systems requires specific research reagents and consumables optimized for each platform. The following table details essential materials and their functions within the automated testing workflow:

Table 3: Essential Research Reagents for Automated GI Pathogen Testing

Reagent/Material	Function	Example Specifications
Stool Transport Buffer	Preserves nucleic acid integrity during transport and storage	Compatible with downstream extraction methods; contains preservatives to inhibit degradation [33]
Nucleic Acid Extraction Kits	Isolate pathogen DNA/RNA from stool specimens	Platform-specific formulations; may include internal controls to monitor extraction efficiency [13] [64]
Multiplex PCR Master Mix	Amplification of target pathogen sequences	Contains enzymes, nucleotides, buffers optimized for multiplex reactions; may include UDG system to prevent carryover contamination [13]
Positive Control Materials	Verify assay performance for each target	Contains nucleic acids from all target pathogens; may be provided as separate quantification standards [33]
Negative Control Materials	Monitor for contamination	Nuclease-free water or negative stool matrix; should be processed alongside clinical samples [64]
Internal Control	Monitor extraction efficiency and amplification inhibition	Non-competitive RNA or DNA sequence introduced during extraction; detected in separate channel [13]
Calibration Standards	Quantitative assay standardization	Serial dilutions of standardized materials for quantitative assays; used for establishing calibration curves [33]
Platform-Specific Consumables	Reaction vessels, plates, or cartridges	Designed for specific automated platforms; may include barcoding for sample tracking [13] [64]

Automation in high-volume laboratory settings represents a fundamental shift in how gastrointestinal pathogen testing is approached, delivering substantial benefits in detection capability, workflow efficiency, and result standardization. The comparative data presented in this analysis demonstrates that modern multiplex PCR panels like the AllPlex Gastrointestinal system offer excellent diagnostic performance when implemented within appropriately automated environments. These platforms detect significantly more pathogens compared to conventional methods while dramatically reducing turnaround times from days to hours [24] [64].

The future of laboratory automation will likely be characterized by increasingly integrated systems where AI, robotics, IoT, and digital twins converge to create fully smart laboratories [61] [62]. For gastrointestinal pathogen detection, this may involve more sophisticated panels that simultaneously identify pathogens and antimicrobial resistance markers, further guiding appropriate therapy. The growing emphasis on sustainability will also drive development of more energy-efficient platforms and reduced-reagent-volume assays that maintain performance while minimizing environmental impact [62].

Successful navigation of the automation landscape requires laboratories to balance technological capabilities with practical operational considerations. The most sophisticated automated system will deliver limited benefits unless it is thoughtfully integrated into workflows, supported by adequately trained staff, and aligned with specific clinical needs [63]. By approaching automation implementation as a strategic initiative rather than simply a technology purchase, laboratories can harness these powerful tools to enhance patient care, support public health surveillance, and advance the field of diagnostic microbiology.

Quality Control Measures for Maintaining Assay Performance Over Time

Maintaining consistent performance in syndromic molecular testing is critical for clinical diagnostics. For the AllPlex Gastrointestinal Panel Assays, robust quality control measures are embedded throughout the testing workflow, from nucleic acid extraction to final result interpretation. This guide examines the experimental evidence supporting these measures and compares its performance with alternative diagnostic approaches.

Experimental Protocols for Performance Evaluation

Multicenter Study Design and Sample Processing

A comprehensive Italian multicenter study evaluated the AllPlex GI-Parasite Assay across 12 laboratories using 368 clinical stool samples [29] [5]. The protocol followed a standardized approach:

• Sample Collection and Storage: Stool samples were collected during routine parasitological diagnostics from patients suspected of enteric parasitic infection. Samples were stored at -20°C or -80°C until batch testing [29].
• DNA Extraction: Between 50-100 mg of stool specimens was suspended in 1 mL of stool lysis buffer (ASL Buffer; Qiagen). After vortexing and incubation, tubes were centrifuged, and the supernatant was used for nucleic acid extraction [29].
• Automated Extraction and PCR Setup: The Microlab Nimbus IVD system automatically performed nucleic acid processing and PCR setup, standardizing this critical pre-analytical step [29].
• PCR Amplification: DNA extracts were amplified with one-step real-time PCR multiplex (CFX96 Real-time PCR, Bio-Rad) using the AllPlex GI-Parasite Assay. Fluorescence was detected at two temperatures (60°C and 72°C) [29].
• Result Interpretation: A positive result was defined as a sharp exponential fluorescence curve crossing the threshold (Ct) at <45 for individual targets. Results were interpreted using Seegene Viewer software (version 3.28.000) [29].

Comparative Study Methodology

A 2025 Spanish study compared the AllPlex GI Panel with the Luminex NxTAG GPP using 196 stool samples [7] [38]:

• Sample Processing: All stool samples were preserved in Cary-Blair medium upon arrival and underwent genetic material extraction using the HAMILTON STARlet system [7].
• Testing Approach: The study employed both prospective and retrospective analysis. For discrepant results, a third confirmatory technique was used when available [7].
• Statistical Analysis: Performance was assessed based on Positive Percentage Agreement (PPA), Negative Percentage Agreement (NPA), and overall agreement with Kappa statistics [7].

Comparative Performance Data

Detection of Protozoan Parasites

The AllPlex GI-Parasite Assay demonstrates excellent sensitivity and specificity for detecting common intestinal protozoa compared to conventional methods (microscopy, antigen testing, and culture) [29]:

Table 1: Performance of AllPlex GI-Parasite Assay for Protozoan Detection

Pathogen	Sensitivity (%)	Specificity (%)
Entamoeba histolytica	100	100
Giardia duodenalis	100	99.2
Dientamoeba fragilis	97.2	100
Cryptosporidium spp.	100	99.7

A Belgian travel clinic study further confirmed these findings, noting significantly superior detection of Dientamoeba fragilis (sensitivity 100% vs. 47.4%) and Blastocystis hominis (sensitivity 95% vs. 77.5%) compared to conventional methods [6] [66].

Comparison with Alternative Multiplex Panels

Table 2: Method Comparison Between Multiplex GI Panels

Parameter	Seegene AllPlex GI Panel	Luminex NxTAG GPP
Sample Processing	Requires 4 tubes for complete panel analysis [7]	Single-tube reaction for comprehensive pathogen detection [7]
Automation Compatibility	Compatible with Seegene's automated NIMBUS & STARlet platforms [13]	Requires specific pretreatment steps [7]
Overall Agreement	High concordance with NPA >95% for most targets [7] [38]	High concordance with NPA >95% for most targets [7] [38]
Challenge Targets	Lower agreement for Cryptosporidium (86.6%) [7] [38]	Similar challenges with Cryptosporidium and Salmonella [7] [38]

When compared with the QIAstat-Dx GIP2, the AllPlex panels demonstrated comparable performance for bacterial and parasitic targets, though the QIAstat-Dx system offered faster turnaround times (~70 minutes) [36].

Limitations in Helminth Detection

The AllPlex GI-Helminth assay shows suboptimal performance compared to microscopy, with an overall sensitivity of 59.1% for helminths versus 100% for conventional methods [6] [66]. Specific sensitivities vary considerably:

• Strongyloides spp.: 100%
• Hookworms: 66.6%
• Ascaris spp.: 60%
• Trichuris trichiura: 20% [6] [66]

Quality Control Workflow and System Components

The AllPlex Gastrointestinal Panel system incorporates multiple quality control measures throughout the testing process:

AllPlex GI Panel Quality Control Workflow

Essential Research Reagent Solutions

Table 3: Key Research Reagents for AllPlex GI Panel Implementation

Component	Function	Specification
Stool Lysis Buffer	DNA release from parasite (oo)cysts with thick walls	ASL Buffer (Qiagen) [29]
Nucleic Acid Extraction System	Automated DNA purification with consistent yield	Microlab Nimbus IVD or STARlet system [29]
PCR Master Mix	Multiplex amplification with internal control	AllPlex proprietary master mix with UDG system [13]
Real-time PCR Instrument	Fluorescence detection for multiple targets	CFX96 Real-time PCR (Bio-Rad) [29]
Analysis Software	Automated result interpretation and data management	Seegene Viewer software [13]

The AllPlex Gastrointestinal Panel Assays incorporate robust quality control measures that maintain assay performance through automated extraction, internal controls, standardized interpretation criteria, and integrated software analysis. The system demonstrates excellent performance for detecting protozoan parasites, with sensitivity and specificity exceeding 97% for most targets, making it particularly valuable for screening in low-endemicity settings. However, laboratories should maintain supplementary methods (particularly microscopy) for helminth detection due to the assay's limitations in this area. When implementing the system, consistent monitoring of extraction efficiency, amplification curves, and internal control performance provides ongoing quality assurance for reliable diagnostic results.

Multicenter Validation and Comparative Performance Against Reference Methods and Competing Assays

This comparative guide provides a systematic evaluation of the AllPlex Gastrointestinal Panel's diagnostic performance against conventional methods including microscopy, culture, and antigen testing. Synthesizing evidence from large patient cohort studies, we present a data-driven analysis of detection sensitivity, specificity, and clinical utility for gastrointestinal pathogen identification. The transition to multiplex molecular testing represents a paradigm shift in diagnostic microbiology, offering substantial improvements in detection capabilities, workflow efficiency, and comprehensive pathogen coverage for bacterial, viral, and parasitic enteric infections.

The accurate and timely diagnosis of gastrointestinal infections is fundamental to effective patient management and infection control. Traditional diagnostic methods—including stool culture for bacterial pathogens, microscopic examination for parasites, and antigen detection for specific viruses—have constituted the cornerstone of laboratory diagnosis for decades [7]. However, these methods present significant limitations in sensitivity, turnaround time, and workflow efficiency [29]. The advent of multiplex PCR panels like the AllPlex Gastrointestinal Panel (Seegene, Seoul, Korea) represents a transformative approach to syndromic testing for infectious diarrhea [13]. This guide provides a comprehensive, evidence-based comparison of this molecular panel against conventional methods, drawing from large-scale clinical studies to inform researchers, clinical scientists, and laboratory professionals about their relative performances and appropriate applications in diagnostic and research settings.

Performance comparison across pathogen types

Detection of bacterial pathogens

Bacterial culture represents the historical gold standard for detecting bacterial enteric pathogens but requires 24-72 hours for completion and has limited sensitivity for fastidious organisms or those requiring special growth conditions [2]. Comparative studies demonstrate that the AllPlex GI Panel consistently detects significantly more bacterial pathogens than conventional culture methods.

In a prospective study of 135 samples, conventional methods detected bacterial pathogens in 17.8% of specimens, while the AllPlex GI Panel detected pathogens in 44.4% of the same samples—a 2.5-fold increase in detection rate [2]. A larger study of 394 diarrheic stool samples found that routine culture methods detected 27.7% positive samples compared to 66.2% by the AllPlex GI Panel [1]. The molecular assay demonstrated particularly enhanced detection for Campylobacter spp., Salmonella spp., and diarrheagenic Escherichia coli pathotypes that are challenging to identify through conventional culture methods.

Table 1: Comparison of Bacterial Pathogen Detection Between AllPlex GI Panel and Conventional Culture

Pathogen	Conventional Culture Sensitivity	AllPlex GI Panel Sensitivity	Key Advantages of Molecular Detection
Campylobacter spp.	Variable; requires special incubation	>95% [1]	Detects non-viable organisms; no special transport needs
Salmonella spp.	72-96 hours for result	>95% [1]	Faster result (4 hours vs. 2-3 days)
Diarrheagenic E. coli	Limited; specialized testing needed	Detects all major pathotypes [19]	Identifies pathotypes indistinguishable by culture
Clostridium difficile	Toxin EIA or cell culture cytotoxin	Detects toxin B and hypervirulent strains [13]	Higher sensitivity than toxin EIA
Yersinia enterocolitica	Requires cold enrichment	>95% [1]	Eliminates need for special culture conditions

Detection of intestinal protozoa

Microscopic examination of stool specimens has been the reference method for diagnosing intestinal protozoan infections but suffers from limitations including required expertise, time-consuming processes, and poor sensitivity, particularly for low parasite burdens [29]. Multiple large-scale studies have demonstrated the superior sensitivity of multiplex PCR compared to microscopy.

A prospective three-year study analyzing 3,495 stool samples found that multiplex PCR (AllPlex GI Panel) detected protozoa in 26.0% of samples compared to only 8.2% by microscopy [18]. The differences were particularly notable for Dientamoeba fragilis (8.86% by PCR vs. 0.63% by microscopy) and Blastocystis hominis (19.25% by PCR vs. 6.55% by microscopy) [18]. A multicenter Italian study of 368 samples reported sensitivity and specificity of 100% and 100% for Entamoeba histolytica, 100% and 99.2% for Giardia duodenalis, 97.2% and 100% for Dientamoeba fragilis, and 100% and 99.7% for Cryptosporidium spp., respectively [29].

Table 2: Performance Comparison for Protozoan Detection Between Microscopy and AllPlex GI Panel

Parasite	Microscopy Limitations	AllPlex GI Panel Performance	Clinical Impact
Giardia duodenalis	Sensitivity ~60.7% [16]	Sensitivity 100% [29] [16]	Redundant testing; identifies true etiology
Dientamoeba fragilis	Difficult to identify without permanent stain; sensitivity ~14.1% [16]	Sensitivity 97.2% [29]	Identifies frequent cause of persistent diarrhea
Entamoeba histolytica	Cannot differentiate from non-pathogenic E. dispar	Specific detection of pathogenic species [29]	Prevents unnecessary treatment
Cryptosporidium spp.	Requires special stains (e.g., acid-fast)	Sensitivity 100% [29] [16]	Identifies important cause of watery diarrhea
Blastocystis hominis	Low sensitivity (~44.2%) [16]	Sensitivity 99.4% [16]	Clarifies role in symptomatic disease

Detection of viral pathogens

Conventional methods for viral detection in stool have included electron microscopy (EM) and antigen-based assays. These methods have largely been replaced by molecular detection due to significantly enhanced sensitivity and specificity. Compared to electron microscopy, the AllPlex GI Panel demonstrates markedly improved detection capabilities, particularly for norovirus and other enteric caliciviruses [2].

One study noted that the AllPlex GI Panel detected gastroenteritis viruses in a high number of specimens that were negative by electron microscopy, with norovirus genogroup II being particularly prevalent [2]. Additionally, the multiplex PCR panel differentiates between specific viruses that electron microscopy might report collectively as "small round viruses" and provides genotyping information not available through antigen testing [2].

Detailed experimental protocols for comparison studies

Prospective multicenter study design

Large-scale comparison studies typically employ a prospective design where consecutive stool samples submitted for routine diagnostic testing are evaluated by both conventional methods and the multiplex PCR panel. The following protocol was used in a study of 3,500 stool samples over three years [18]:

• Sample Collection: Fresh stool samples are collected from patients presenting with gastrointestinal symptoms meeting clinical case definitions for infectious diarrhea.

• Parallel Testing: Each sample undergoes:

  • Traditional parasitological examination: Macroscopic and microscopic examination after concentration techniques (e.g., formalin-ethyl acetate concentration), permanent staining (e.g., trichrome stain for protozoa), and specific staining for Cryptosporidium spp. (e.g., modified Ziehl-Neelsen stain).
  • Antigen testing: Immunoassays for Giardia, Cryptosporidium, and Entamoeba histolytica when clinically indicated.
  • Molecular testing: Aliquot stored at -80°C until nucleic acid extraction using automated systems (e.g., Hamilton MICROLAB Nimbus or STARlet).

• DNA Extraction: 50-100 mg of stool is suspended in stool lysis buffer, vortexed, incubated at room temperature, and centrifuged. The supernatant is used for nucleic acid extraction with automated systems incorporating internal controls to monitor extraction efficiency and PCR inhibition.

• PCR Amplification: DNA extracts are amplified using the AllPlex GI-Parasite Assay or full gastrointestinal panel on real-time PCR instruments (e.g., Bio-Rad CFX96) with Seegene Viewer software for automated result interpretation.

• Discrepancy Resolution: Samples with discordant results between methods undergo additional testing with alternative molecular assays or reference laboratory methods to resolve the discrepancy.

Analytical performance assessment

The statistical analysis for comparing diagnostic methods typically includes:

• Sensitivity and Specificity Calculation: Using conventional methods as the reference standard, though acknowledging its limitations given the superior sensitivity of molecular methods.

• Positive and Negative Percentage Agreement (PPA/NPA): Used when no perfect gold standard exists, particularly for multi-pathogen detection [7].

• Kappa Statistic for Inter-method Agreement: Measuring agreement beyond chance, with values >0.8 indicating almost perfect agreement [7] [29].

• McNemar's Test for Paired Comparisons: Assessing statistically significant differences in detection rates between methods [16] [2].

Workflow and technical considerations

The workflow diagram illustrates fundamental differences between conventional and molecular approaches. Traditional methods require multiple parallel processes with different sample preparations, while the AllPlex GI Panel utilizes a unified workflow from a single sample aliquot [7] [13]. Key advantages of the molecular workflow include:

• Streamlined Processing: A single nucleic acid extraction serves all subsequent pathogen detection, compared to multiple specialized procedures for culture, microscopy, and antigen testing [2] [13].

• Reduced Hands-on Time: Automated extraction and result interpretation decrease technologist time compared to labor-intensive microscopic examination and culture processing [29].

• Faster Time-to-Result: Complete analysis within approximately 4 hours versus 2-3 days for bacterial culture and identification [2] [1].

• Standardization: Automated interpretation reduces inter-technologist variability inherent in microscopic identification [29].

The scientist's toolkit: Essential research reagents and materials

Table 3: Key Research Reagents and Materials for Gastrointestinal Pathogen Detection Studies

Item	Specification/Example	Research Function	Considerations
Nucleic Acid Extraction System	Hamilton STARlet with STARMag 96 Universal Cartridge [7] [16]	Automated nucleic acid purification from stool	Includes internal control for process validation; handles inhibitor removal
Real-time PCR Instrument	CFX96 Real-Time Detection System (Bio-Rad) [29] [19]	Multiplex PCR amplification and detection	Compatible with MuDT technology for multiple Ct values in single channels
Multiplex PCR Assay	AllPlex Gastrointestinal Panel Assays (Seegene) [13]	Simultaneous detection of 25 gastrointestinal pathogens	Four panels: GI-Bacteria(I), GI-Bacteria(II), GI-Parasite, GI-Virus
Stool Transport Medium	Cary-Blair medium [7]	Preserves specimen integrity during transport	Maintains pathogen nucleic acid stability for molecular testing
Stool Lysis Buffer	ASL Buffer (Qiagen) [29] [2]	Initial processing and homogenization of stool specimens	Facilitates efficient nucleic acid release and inhibitor reduction
Software for Result Interpretation	Seegene Viewer [29] [19]	Automated data analysis and pathogen identification	Provides LIS interlocking and multiple Ct value interpretation

Discussion and clinical implications

Enhanced detection of co-infections

A significant advantage of multiplex PCR panels is their capacity to detect pathogen co-infections, which are frequently missed by conventional methods that often cease investigation after identifying one pathogen [2] [19]. Studies using the AllPlex GI Panel have reported co-infection rates of 23.3% to 54% in positive samples [2] [19]. This enhanced detection capability provides a more comprehensive understanding of disease etiology, particularly in endemic areas or specific patient populations such as immunocompromised individuals or returned travelers.

Limitations and complementary role of conventional methods

Despite the superior sensitivity of molecular methods, conventional techniques retain importance in specific scenarios:

• Antimicrobial Susceptibility Testing: Bacterial culture remains essential for obtaining isolates for antimicrobial susceptibility testing, particularly for surveillance and guiding targeted therapy [2] [1].

• Detection of Non-Targeted Pathogens: Microscopy identifies parasites not included in molecular panels (e.g., Cystoisospora belli, helminths) and non-pathogenic protozoa that may be clinically relevant in immunocompromised patients [18].

• Differentiation of Shigella and EIEC: The AllPlex GI Panel cannot distinguish between Shigella species and enteroinvasive E. coli (EIEC), requiring culture for precise identification [2] [13].

• Viability Assessment: Molecular methods detect nucleic acid from both viable and non-viable organisms, potentially leading to false positives in recovering patients, whereas culture indicates active infection [2].

Comprehensive comparison studies demonstrate that the AllPlex Gastrointestinal Panel offers significant advantages over conventional methods for the diagnosis of gastrointestinal infections. The multiplex PCR assay provides substantially higher sensitivity for most bacterial, viral, and parasitic pathogens, reduced turnaround time, and streamlined workflow. These advantages must be balanced against limitations including inability to provide antimicrobial susceptibility data and detection of non-viable organisms. The optimal diagnostic approach may involve a reflexive algorithm where multiplex PCR serves as the primary screening method with selective use of conventional methods for confirmation, susceptibility testing, and detection of pathogens not included in molecular panels. As molecular technologies continue to evolve, ongoing comparative studies will be essential to guide their appropriate implementation in clinical and research settings.

Performance Evaluation of Multipinex NxTAG and BioFire FilmArray

Clinical laboratories have multiple choices when it comes to syndromic respiratory testing, and understanding the performance characteristics of different multiplex panels is crucial for optimal test selection [67]. The Luminex NxTAG Respiratory Pathogen Panel (RPP) and BioFire FilmArray Respiratory Panel (RP) represent two of the most inclusive and widely used panels for respiratory syndromic testing in the United States [67]. This objective comparison examines their performance characteristics, including overall accuracy, positive and negative agreement, and target-specific detection capabilities, to inform researchers, scientists, and drug development professionals in their diagnostic decisions.

Performance Comparison of Respiratory Panels

A comprehensive 2022 study directly comparing three major respiratory panels using 350 nasopharyngeal swab samples from symptomatic patients found no statistically significant difference in overall accuracy between the BioFire FilmArray RP and Luminex NxTAG RPP assays (P = 0.6171) [67]. This indicates comparable diagnostic performance between these two systems for respiratory pathogen detection.

However, significant differences were observed between BioFire and GenMark (P = 0.0003) and between GenMark and Luminex (P = 0.0009), highlighting the importance of direct comparative studies when selecting diagnostic platforms [67].

Table 1: Overall Performance Metrics of Respiratory Panels

Assay	Positive Percent Agreement (PPA)	Negative Percent Agreement (NPA)	Overall Accuracy P-value (vs. BioFire)
BioFire FilmArray RP	94.1%	99.9%	-
Luminex NxTAG RPP	96.5%	99.9%	0.6171 (not significant)
GenMark eSensor RVP	97.3%	99.5%	0.0003 (significant)

Target-Specific Performance Variations

The three assays demonstrated equivalent performance for detecting adenovirus, human metapneumovirus, influenza A, and respiratory syncytial virus [67]. However, platform-specific variations were noted:

• BioFire FilmArray showed increased false-positive results for endemic coronaviruses [67]
• Luminex NxTAG RPP demonstrated robust detection capabilities across most viral targets [67]
• All platforms exhibited high negative percent agreement (99.9% for BioFire and Luminex, 99.5% for GenMark), indicating excellent reliability in ruling out infections [67]

Multi-Platform Comparative Study

A separate comparative analysis of three multiplex platforms for detecting respiratory viral pathogens from pediatric specimens further validated these findings [68]. The study reported that all three multiplex platforms displayed high overall agreement (>90%) and high specificity (>90%) compared to reference methods [68].

Table 2: Detection Performance Across Multiple Platforms

Pathogen Category	BioFire FilmArray Detection Rate	Luminex NxTAG RPP Detection Rate	TAC Detection Rate
Overall Virus Detection	166/170 (97.6%)	160/170 (94.1%)	163/170 (95.8%)
Influenza B	100% PPA	100% PPA	95.2% PPA
Seasonal Coronaviruses	90.2% PPA	81.8% PPA	-
Rhinovirus/Enterovirus	>90% PPA	>90% PPA	>90% PPA

Gastrointestinal Panel Performance

BioFire FilmArray GI Panel Performance

The BioFire FilmArray Gastrointestinal Panel demonstrates comprehensive detection capabilities for 22 gastrointestinal pathogens directly from stool specimens with a turnaround time of approximately one hour [69]. Multicenter evaluation studies have shown excellent performance characteristics:

• High Sensitivity and Specificity: Overall sensitivity of 98.5% and specificity of 99.2% based on prospective clinical study data [69]
• Superior Diagnostic Yield: 48.2% diagnostic yield compared to 16.7% from traditional methods [69]
• Rapid Results: Reduced turnaround time to around 6.3 hours compared to up to 32 hours for traditional work-ups [69]

A 2017 study evaluating the FilmArray GI panel on 168 stool samples found at least one potential pathogen in 92/168 (54.8%) specimens, with 71.8% of positive samples having only one pathogen and 28.2% having multiple pathogens [34]. The most frequently detected pathogens were rotavirus (13.9%), Campylobacter (10.7%), Clostridium difficile (9.5%), and norovirus (8.9%) [34].

Luminex NxTAG Gastrointestinal Pathogen Panel Performance

The Luminex NxTAG Gastrointestinal Pathogen Panel utilizes a qualitative bead-based multiplex test for molecular detection of 16 gastrointestinal viral, bacterial, and parasitic pathogens directly from human stool samples [31]. Performance characteristics include:

• High Sensitivity and Specificity: 97.6% sensitivity and 99.7% specificity for enteropathogenic bacteria and viruses [31]
• Enhanced Detection: Higher positivity rate (28.3% vs 19.5%) compared to traditional diagnostic methods [31]
• Coinfection Identification: Detected coinfections in 11.1% of positive samples, which were missed by traditional methods [31]

Experimental Methodologies

Respiratory Panel Testing Protocol

The comparative performance analysis of respiratory panels utilized nasopharyngeal swab samples (n = 350) collected from symptomatic patients (n = 329) in the pre-COVID-19 era [67]. The study employed standardized collection and processing methods:

• Sample Collection: Nasopharyngeal swab specimens collected from patients presenting with respiratory symptoms
• Sample Processing: Direct comparison of all three assays (BioFire FilmArray RP, Luminex NxTAG RPP, and GenMark eSensor RVP) using the same set of clinical samples
• Analysis Method: Statistical comparison using McNemar's tests to evaluate significant differences between assays [67]
• Discrepant Analysis: Further investigation of assay disparities for each analyte or analyte group to identify sources of variation

Gastrointestinal Panel Testing Workflow

The following diagram illustrates the comparative evaluation workflow for gastrointestinal panels:

Research Reagent Solutions

Table 3: Essential Research Reagents and Materials

Reagent/Material	Function/Purpose	Example Application
Cary-Blair Transport Medium	Preserves stool specimens during transport and storage	Used for both BioFire and Luminex GI panel sample preparation [70] [34]
Sample Buffer	Dilution and homogenization of stool samples	Preparation of stool samples for nucleic acid extraction [34]
Hydration Injection Vial	Rehydrates dried reagents in FilmArray pouches	Essential for FilmArray pouch rehydration prior to testing [34]
Internal Control DNA	Monitors extraction efficiency and PCR inhibition	Quality control for nucleic acid extraction and amplification [4]
Multiplex RT-PCR Reagents	Simultaneous detection of multiple pathogen targets	Core component of both BioFire and Luminex detection systems [31] [70]

Discussion

The performance evaluation data demonstrates that both Luminex NxTAG and BioFire FilmArray panels offer highly accurate and reliable pathogen detection for respiratory and gastrointestinal testing. While overall performance between the respiratory panels showed no significant differences, target-specific variations highlight the importance of understanding local epidemiology and clinical needs when selecting diagnostic platforms.

The comprehensive nature of both systems represents a significant advancement over traditional diagnostic methods, particularly in their ability to detect coinfections and identify pathogens that might be missed by conventional approaches [67] [31]. The rapid turnaround time (approximately 1 hour for both systems) compared to traditional culture methods (24-72 hours) provides clinical laboratories with actionable results that can significantly impact patient management and infection control practices [69].

For research and drug development applications, both platforms offer robust platforms for surveillance studies and clinical trial support, though laboratories should consider factors such as throughput requirements, menu comprehensiveness, and target-specific performance characteristics when selecting the most appropriate system for their specific research context.

The accurate and timely diagnosis of gastrointestinal infections is fundamental to effective patient management, infection control, and public health surveillance. For decades, conventional diagnostic methods such as stool culture and microscopy have formed the backbone of enteric pathogen detection. However, these techniques are labor-intensive, time-consuming, and often lack sensitivity for detecting fastidious or low-abundance pathogens [7]. The emergence of syndromic multiplex PCR panels represents a paradigm shift in diagnostic microbiology, offering comprehensive pathogen detection with significantly reduced turnaround times.

Among these platforms, the AllPlex Gastrointestinal Panel (Seegene Inc., Seoul, South Korea) has been widely implemented across diverse healthcare settings globally. This multiplex real-time PCR assay simultaneously detects a broad spectrum of bacterial, viral, and parasitic enteric pathogens in a single testing workflow. While individual studies have demonstrated its diagnostic performance, assessing its consistency across multiple institutions and patient populations is crucial for establishing its real-world reliability and guiding implementation decisions.

This review synthesizes findings from multicenter studies to evaluate the consistency of the AllPlex Gastrointestinal Panel's performance across different healthcare settings, comparing it with conventional diagnostic methods and alternative molecular platforms where available.

Performance Evaluation Across Multiple Centers

Key Performance Metrics from Multicenter Studies

Table 1: Summary of AllPlex GI Panel Performance Across Multicenter Studies

Study & Setting	Sample Size	Sensitivity Range	Specificity Range	Key Strengths	Identified Limitations
Italian Multicenter Study (12 centers) [5]	368	• Entamoeba histolytica: 100%• Giardia duodenalis: 100%• Dientamoeba fragilis: 97.2%• Cryptosporidium spp.: 100%	• Entamoeba histolytica: 100%• Giardia duodenalis: 99.2%• Dientamoeba fragilis: 100%• Cryptosporidium spp.: 99.7%	Excellent performance for common protozoa	Not assessed for helminths or comprehensive bacterial targets
Prospective Study (3,500 samples) [18]	3,495	Significantly higher than microscopy for:• G. intestinalis (1.28% vs 0.7%)• Cryptosporidium spp. (0.85% vs 0.23%)• D. fragilis (8.86% vs 0.63%)	No false positives for G. intestinalis, Cryptosporidium spp., E. histolytica	Superior detection of protozoa compared to microscopy	Does not detect helminths; microscopy still needed for at-risk populations
Comparative Study vs. Luminex NxTAG [7]	196	Average PPA >89% for nearly all targets; lower for Cryptosporidium spp. (86.6%)	NPA consistently >95% for both platforms	High overall concordance with alternative molecular method	Lower agreement for certain pathogens like Salmonella spp. and Cryptosporidium spp.

Detection Rates in Prospective Surveillance

A one-year prospective study in Lebanon utilizing the AllPlex assay on 271 samples demonstrated its utility in comprehensive surveillance, detecting enteropathogens in 71% of cases with acute gastroenteritis [19]. Bacterial pathogens were most prevalent (48%), followed by parasites (12%) and viruses (11%). Notably, 54% of positive samples contained mixed infections, highlighting the advantage of multiplex panels in identifying co-infections that would likely be missed by targeted testing approaches. The most frequently detected pathogens were diarrheagenic E. coli pathotypes, with enteroaggregative E. coli (EAEC) found in 26.5% of samples, enterotoxigenic E. coli (ETEC) in 23.2%, and enteropathogenic E. coli (EPEC) in 20.3% [19].

Comparative Performance with Conventional and Molecular Methods

Versus Conventional Methods

The AllPlex GI Panel consistently demonstrates superior sensitivity compared to conventional diagnostic methods across multiple studies:

• Comparison with microscopy: A prospective study on 3,500 stool samples over three years found the AllPlex assay detected Giardia intestinalis in 1.28% of samples compared to 0.7% by microscopy, Cryptosporidium spp. in 0.85% versus 0.23%, and Dientamoeba fragilis in 8.86% versus 0.63% [18]. The study concluded that "multiplex PCR proved more efficient to detect protozoan parasites" but noted that "microscopy still remains necessary to detect helminths" and parasites not included in the panel, such as Cystoisospora belli [18].

• Comparison with culture and electron microscopy: A study comparing the AllPlex bacterial and viral assays to conventional methods (culture and electron microscopy) found the molecular assay detected over twice as many pathogens (44.4% vs. 17.8%) in prospective samples [17]. The AllPlex assay also identified diarrheagenic E. coli strains that conventional methods could not detect and provided results in approximately 4 hours compared to 24-72 hours for culture [17].

Versus Alternative Molecular Platforms

Table 2: Comparison of AllPlex GI Panel with Alternative Molecular Platforms

Comparison Platform	Study Characteristics	Key Findings	Operational Considerations
Luminex NxTAG GPP [7]	196 stool samples from a Spanish hospital	High overall concordance (Kappa >0.8 for most pathogens); comparable NPA (>95%); lower agreement for Cryptosporidium spp. (86.6%)	AllPlex requires multiple tubes for full panel; Luminex requires single tube and includes pre-treatment step
QIAstat-Dx GIP2 [36]	259 specimens (69.5% archived, 30.5% prospective)	AllPlex used as comparator; QIAstat-Dx showed PPA 95.02% and PNA 99.98%; viral target detection suboptimal with QIAstat-Dx	QIAstat-Dx provides results in ~70 minutes vs. longer turnaround for AllPlex
Four commercial multiplex PCR assays [9]	126 DNA samples from clinically confirmed patients	AllPlex showed strong performance for C. hominis/parvum, G. duodenalis, and E. histolytica; some variability in detection limits among assays	AllPlex performed on CFX96 system; other platforms required different instrumentation

Methodological Protocols in Multicenter Studies

Standardized Testing Workflow

The methodological approaches across the reviewed studies demonstrated remarkable consistency in sample processing and analysis:

• Sample Preparation: Most studies suspended stool samples in Cary-Blair transport medium or specific lysis buffers (ASL buffer, eNAT medium) followed by vortexing and incubation at room temperature [16] [36] [19]. Bead-beating steps were incorporated in some protocols to enhance DNA release from hardy parasite cysts [6].

• Nucleic Acid Extraction: Automated extraction systems were predominantly used, including the HAMILTON STARlet system [7] [16], MagNA Pure Compact System [17], and Microlab Nimbus IVD system [5]. These automated platforms standardized the extraction process across sites, reducing inter-laboratory variability.

• PCR Amplification and Detection: The AllPlex assays were typically performed on CFX96 Real-Time Detection Systems (Bio-Rad) following manufacturer recommendations [16] [5] [19]. Thermal cycling conditions generally included: 20 minutes at 50°C for reverse transcription, 15 minutes at 95°C for initial denaturation, followed by 45 cycles of 10 seconds at 95°C, 60 seconds at 60°C, and 30 seconds at 72°C [19]. Results were interpreted using Seegene Viewer software with cycle threshold values typically <40-45 considered positive [5] [6].

Figure 1: Standardized Workflow for AllPlex GI Panel Testing Across Multicenter Studies

Discrepancy Resolution Methods

To address discordant results between methods, studies employed various resolution strategies:

• Additional Molecular Testing: Discrepant samples were typically retested with alternative PCR methods, including monoplex 5' exonuclease probe PCR assays [17], specific PCR assays from national reference centers [7], or alternative commercial PCR kits [36].

• Culture Confirmation: When available, traditional culture methods were used to resolve discrepancies for bacterial pathogens [71].

• Sequencing Analysis: Some studies utilized conventional PCR followed by sequencing to resolve persistent discrepancies [71].

• Consensus Definitions: Several studies established predefined consensus criteria considering samples as true positives if multiple methods agreed or if supported by resolution testing [36] [71].

The Scientist's Toolkit: Essential Research Reagents and Platforms

Table 3: Key Research Reagent Solutions for AllPlex GI Panel Implementation

Category	Specific Product/Platform	Function in Workflow	Examples from Studies
Nucleic Acid Extraction	HAMILTON STARlet with STARMag 96	Automated nucleic acid extraction	Used in multiple studies for standardized extraction [7] [16]
	MagNA Pure Compact System	Automated nucleic acid extraction	Utilized for bacterial and viral target detection [17]
	QIAamp DNA Mini Kit	Manual nucleic acid extraction	Employed in surveillance studies [19]
Amplification & Detection	CFX96 Real-Time Detection System	Real-time PCR amplification and fluorescence detection	Primary platform across nearly all studies [16] [5] [19]
	Seegene Viewer Software	Result interpretation and analysis	Standard software for result interpretation [16] [5]
Sample Processing	FecalSwab (Copan) with Cary-Blair Medium	Sample preservation and transport	Maintained sample integrity during storage and transport [16] [36]
	ASL Stool Lysis Buffer (Qiagen)	Stool sample homogenization and initial processing	Facilitated nucleic acid release [17] [19]
Confirmatory Testing	VIASURE Real-Time PCR Detection Kit	Discrepancy resolution	Used for targeted confirmation [7]
	Mericon Campylobacter spp Kit	Discrepancy resolution for specific pathogens	Resolved Campylobacter detection discrepancies [71]

Discussion

Consistency in Performance Across Healthcare Settings

The collective evidence from multiple centers demonstrates that the AllPlex GI Panel delivers consistent, high-performance detection of gastrointestinal pathogens across diverse geographic and healthcare settings. The Italian multicenter study, involving 12 laboratories, reported sensitivities of 97.2-100% and specificities of 99.2-100% for major protozoan parasites [5], indicating minimal inter-site variability despite differences in local infrastructure and patient populations.

This consistency can be attributed to several factors: (1) standardized automated extraction and amplification protocols that reduce technical variation; (2) comprehensive internal controls that monitor extraction efficiency and PCR inhibition; and (3) centralized software-based result interpretation that minimizes subjective interpretation differences. The high negative percentage agreement (NPA >95%) consistently observed across studies [7] is particularly important for ruling out infections and preventing unnecessary treatments.

Advantages Over Conventional Methods

The significantly higher detection rates of the AllPlex Panel compared to conventional methods represent one of its most consistent benefits across studies. The 2-fold higher pathogen detection rate observed in comparative studies [17] translates to substantial clinical impact through improved diagnosis, more appropriate treatment, and better infection control. This enhanced detection is particularly notable for pathogens like Dientamoeba fragilis and diarrheagenic E. coli pathotypes that are challenging to identify through conventional means [17] [19].

The dramatic reduction in turnaround time (approximately 4 hours versus 24-72 hours for culture) [17] represents another consistent advantage across settings, enabling more timely clinical decisions. Additionally, the ability to detect multiple pathogens in co-infections (23.3% of positive samples in one study) [17] provides a more comprehensive diagnostic picture than traditional methods.

Limitations and Complementary Testing Needs

Despite its robust performance, consistent limitations have emerged across studies. The AllPlex GI-Parasite assay shows suboptimal sensitivity for helminth detection compared to microscopy (59.1% vs. 100% in one evaluation) [6], indicating that conventional microscopic examination remains essential when helminth infection is suspected. Additionally, the panel does not detect some relevant pathogens, including Cystoisospora belli and Schistosoma mansoni [18] [6], requiring supplementary testing in specific patient populations such as immunocompromised individuals or returning travelers.

The higher detection sensitivity of molecular methods also raises questions about clinical relevance in asymptomatic cases and necessitates careful interpretation in the context of patient symptoms. Future iterations of the panel could benefit from expanded targets and quantitative results to help distinguish active infection from carriage.

Multicenter evaluations consistently demonstrate that the AllPlex Gastrointestinal Panel provides reliable, comprehensive detection of gastrointestinal pathogens across diverse healthcare settings. Its superior sensitivity compared to conventional methods, rapid turnaround time, and ability to identify co-infections represent significant advancements in diagnostic capabilities. While the platform shows some limitations for helminth detection and requires complementary testing for pathogens not included in the panel, its standardized workflow and consistent performance make it a valuable tool for clinical diagnostics and surveillance. Implementation decisions should consider local pathogen prevalence, specific patient populations, and the need for complementary testing to address the panel's limitations.

The accurate and timely identification of gastrointestinal pathogens is fundamental to effective patient management, outbreak control, and public health surveillance. For decades, the diagnosis of infectious gastroenteritis relied on conventional methods, including stool culture, microscopy, and antigen detection. However, these techniques are often labor-intensive, time-consuming, and limited by variable sensitivity and an inability to detect non-culturable or fastidious organisms [7]. The advent of multiplex molecular panels has revolutionized diagnostic approaches by enabling the simultaneous detection of numerous bacterial, viral, and parasitic pathogens from a single stool sample. Among these, the AllPlex Gastrointestinal Panel (Seegene Inc., Seoul, Korea) is a comprehensive, multiplex real-time PCR assay designed to detect a broad spectrum of enteric pathogens. This guide provides an objective, data-driven evaluation of the pathogen-specific performance of the AllPlex GI Panel, comparing its sensitivity and specificity for critical targets against conventional methods and other commercial molecular panels, based on findings from large patient cohort research.

The AllPlex GI Panel is a syndromic testing solution that typically comprises multiple assays (e.g., GI-Bacteria I, GI-Bacteria II, GI-Virus, GI-Parasite) to comprehensively detect common enteric pathogens. The panel's core strength lies in its ability to identify co-infections and pathogens that are difficult to culture, significantly improving the diagnostic yield compared to conventional methods [1] [19].

Studies conducted on large clinical cohorts have consistently demonstrated the superior sensitivity of the AllPlex GI Panel over traditional techniques. A prospective study on 3,500 stool samples over three years reported that multiplex PCR detected protozoan parasites in 909 samples, compared to only 286 detected by microscopy [18]. Similarly, another large prospective study found that the AllPlex PCR assay identified 207 positive samples out of 588, whereas microscopy only detected 95 positives [4] [16]. The following table summarizes the aggregated sensitivity and specificity data for critical parasitic and bacterial targets from key large-cohort studies.

Table 1: Pathogen-Specific Sensitivity and Specificity of the AllPlex GI Panel from Large Cohort Studies

Pathogen	Sensitivity (%)	Specificity (%)	Study Details
Parasites
Giardia duodenalis	97.2 - 100	99.2 - 100	Multicentric study (n=368) and prospective studies [4] [16] [5]
Cryptosporidium spp.	100	99.7 - 100	Retrospective & prospective evaluations [4] [5]
Dientamoeba fragilis	97.2 - 100	~100	Significantly higher than microscopy [4] [5]
Entamoeba histolytica	100	100	Differentiates from non-pathogenic species [5]
Blastocystis hominis	99.4 - 100	~100	Highly sensitive vs. microscopy (44.2%) [4] [16]
Bacteria
Campylobacter spp.	>95	>95	Compared to culture; detects non-culturable strains [1]
Salmonella spp.	>95	>95	Higher detection rate vs. culture [1]
Clostridioides difficile	>95	>95	Detects toxin B gene [1]
Diarrheagenic E. coli	>95	>95	Detects multiple pathotypes [1]

Comparative performance with other multiplex PCR panels

The diagnostic landscape for gastrointestinal infections features several multiplex PCR panels. Comparative studies provide crucial insights for laboratories selecting a testing platform.

Comparison with Luminex NxTAG GPP

A 2025 study comparing the Seegene AllPlex and Luminex NxTAG Gastrointestinal Pathogen Panel (GPP) on 196 clinical samples found high overall concordance, with Negative Percentage Agreement (NPA) consistently above 95% and overall Kappa values exceeding 0.8 for most pathogens [7]. The average Positive Percentage Agreement (PPA) was greater than 89% for nearly all targets; however, lower agreement was observed for Cryptosporidium spp. (86.6%) [7]. Discrepancies were also noted for Salmonella spp., highlighting specific diagnostic challenges with these targets.

Comparison with a broader panel of assays

A 2019 study compared the AllPlex GI (24 targets), Luminex xTAG GPP (15 targets), and BD MAX Enteric (8 targets) assays using 858 stool samples [24]. The overall positive percentage agreements (PPA) for the AllPlex, Luminex, and BD MAX assays were 94%, 92%, and 78%, respectively [24]. This demonstrates that the AllPlex panel performs comparably to other broad multiplex panels and may offer a higher target range and PPA than some more focused molecular tests.

Detailed experimental protocols

To ensure the reproducibility of performance data, the following section outlines the standard experimental methodologies employed in the cited large-cohort evaluations of the AllPlex GI Panel.

Sample collection and storage

• Sample Type: Diarrheic or unformed stool samples are collected from patients presenting with symptoms of acute gastroenteritis [1] [34].
• Transport Medium: Stool samples are typically suspended and transported in Cary-Blair medium [4] [7] or specific lysis buffers (e.g., ASL buffer from Qiagen) [5] [19].
• Storage Conditions: For prospective studies, fresh samples are processed upon receipt. For retrospective analyses, aliquots of stool samples are stored at -20°C or -80°C until nucleic acid extraction is performed. Studies have validated that DNA remains stable in Cary-Blair suspension for up to 7 days at both room temperature and +4°C without significant degradation of signal [4] [33].

Nucleic acid extraction

• Automation: The extraction process is predominantly automated using systems such as the MICROLAB STARlet (Hamilton Company) [4] [5] or the Microlab Nimbus IVD system (Hamilton) [5] to ensure standardization and high throughput.
• Protocol: A small amount of stool (e.g., 140-180 mg) is vigorously mixed in the transport/lysis buffer. After a brief incubation and centrifugation, the supernatant is used for extraction [4]. The extraction is performed on a defined volume of supernatant (e.g., 50 µL) and eluted in a smaller volume (e.g., 100 µL) [4]. An internal control is added to the sample prior to extraction to monitor for PCR inhibition and validate the extraction process [4] [19].

Multiplex PCR amplification and detection

• Technology: The AllPlex assays utilize Seegene's proprietary technologies, including Dual Priming Oligonucleotide (DPO) for high specificity and Multiple Detection Temperature (MuDT) to report multiple cycle threshold (Ct) values in a single channel [19].
• Instrumentation: Amplification and detection are performed on real-time PCR platforms, most commonly the CFX96 system (Bio-Rad) [4] [5] [19].
• Cycling Conditions: A typical protocol includes: reverse transcription at 50°C for 20 minutes (if detecting RNA viruses), initial denaturation at 95°C for 15 minutes, followed by 45 cycles of denaturation (95°C for 10-15 seconds), annealing/extension (60°C for 1 minute), and a final detection step at 72°C [19].
• Analysis: Results are automatically analyzed using Seegene Viewer software, which interprets the fluorescence data and assigns positive/negative results based on the presence of a sharp exponential curve crossing the threshold within a defined Ct value (typically <40-45) [4] [5].

Diagram: AllPlex GI Panel Testing Workflow

The scientist's toolkit: Key research reagent solutions

The implementation and performance of the AllPlex GI Panel rely on a suite of integrated reagents and instruments. The following table details the essential materials used in the featured experiments.

Table 2: Essential Research Reagents and Instruments for AllPlex GI Panel Testing

Item Name	Function / Description	Provider / Example
AllPlex GI Assay Panels	Core PCR reagents for simultaneous detection of bacterial, viral, and parasitic targets in a multiplex format.	Seegene Inc.
Cary-Blair Medium (FecalSwab)	Transport medium for stool sample preservation during transport and storage.	Copan Diagnostics Inc. [4]
Nucleic Acid Extraction Kit	Automated system for isolating DNA and RNA from stool samples, crucial for removing PCR inhibitors.	STARMag 96 Universal Cartridge Kit (Hamilton) [4], QIAamp DNA Mini Kit (Qiagen) [19]
Automated Extraction System	Instrument for standardized, high-throughput nucleic acid extraction.	MICROLAB STARlet / Nimbus IVD (Hamilton Company) [4] [5]
Real-Time PCR System	Thermocycler and detector for performing multiplex real-time PCR and fluorescence data capture.	CFX96 Real-Time System (Bio-Rad) [4] [19]
Internal Control DNA/RNA	Exogenous control added to each sample to monitor nucleic acid extraction efficiency and PCR inhibition.	Provided with AllPlex assay kit [4]
Seegene Viewer Software	Specialized software for automated interpretation of multiplex PCR results from raw fluorescence data.	Seegene Inc. [4] [19]

Discussion and clinical implications

The body of evidence from large-cohort research solidly positions the AllPlex GI Panel as a highly sensitive and specific tool for the comprehensive diagnosis of gastrointestinal infections. Its superior performance over microscopy, particularly for parasites like Dientamoeba fragilis and Blastocystis hominis, is striking [4] [16]. Furthermore, its ability to detect a wide range of bacteria, including enteropathogenic E. coli strains that are often missed by culture, significantly increases the diagnostic yield and provides a more accurate epidemiological picture [1] [19].

The high frequency of co-infections identified by the panel underscores the complexity of gastroenteritis etiology and the limitation of single-pathogen tests [34] [19]. While the detection of nucleic acid does not always distinguish between active infection and colonization, the clinical correlation becomes essential. The implementation of such syndromic panels has been shown to improve patient management, optimize antibiotic stewardship, and enhance public health surveillance by providing a rapid and precise overview of circulating pathogens [7] [19].

Diagram: Diagnostic Decision Pathway with Multiplex PCR

Independent evaluations across large and diverse patient cohorts consistently affirm that the AllPlex Gastrointestinal Panel delivers high sensitivity and specificity for a comprehensive set of critical bacterial, viral, and parasitic targets. Its performance surpasses that of traditional microscopy and culture, and it is comparable to other leading multiplex molecular panels. The decision to adopt this panel should be informed by local epidemiology, patient population needs, and consideration of its limitations, particularly its inability to detect helminths and some protozoa like Cystoisospora belli. Nevertheless, within its targeted scope, the AllPlex GI Panel represents a robust, reliable, and efficient tool for the modern clinical microbiology laboratory, driving improved diagnostic accuracy and patient care.

Positive and Negative Percentage Agreement in Large-scale Evaluations

Accurate and rapid diagnosis of gastrointestinal infections is fundamental to effective patient management, infection control, and public health surveillance. Traditional diagnostic methods, including stool culture and microscopy, are often time-consuming, labor-intensive, and lack sensitivity. The emergence of multiplex PCR panels has revolutionized laboratory testing for infectious gastroenteritis by enabling the simultaneous detection of numerous bacterial, viral, and parasitic pathogens from a single stool sample. As these molecular assays become integral to clinical practice, rigorous evaluation of their performance is essential. In large-scale comparative studies, Positive Percentage Agreement (PPA) and Negative Percentage Agreement (NPA) serve as critical statistical measures for assessing diagnostic reliability when a perfect gold standard is unavailable. This guide objectively compares the performance of several commercial multiplex PCR panels, with a specific focus on the Seegene AllPlex Gastrointestinal Panel within the context of large patient cohort research.

Understanding PPA and NPA in Diagnostic Evaluation

In the evaluation of diagnostic tests, Sensitivity and Specificity are standard metrics used when comparing a new test against a reference method that is considered the "best available" standard for determining the presence or absence of a condition [72]. However, in many modern evaluations, particularly for complex multiplex panels, a single, unambiguous reference standard may not exist. In these cases, Positive Percentage Agreement (PPA) and Negative Percentage Agreement (NPA) are employed.

• Positive Percentage Agreement (PPA) measures how often the new test correctly identifies the condition when it is present according to a comparator method (which may not be a perfect reference). It is calculated as the number of patients who test positive on both tests divided by the total number of patients who test positive on the comparator method [73].
• Negative Percentage Agreement (NPA) measures how often the new test correctly rules out the condition when it is absent according to the comparator method. It is calculated as the number of patients who test negative on both tests divided by the total number of patients who test negative on the comparator method [7].

These metrics are vital for large-scale evidence generation, as they help prevent publication bias and P-hacking by ensuring that all performance data—both positive and negative—are systematically reported [74].

Comparative Performance of Multiplex GI Panels

Large-scale studies directly comparing multiple commercial assays provide the most robust evidence for diagnostic performance. The following sections and tables summarize key experimental data from recent evaluations.

AllPlex vs. Luminex NxTAG vs. BD MAX

A comprehensive 2019 study compared three major molecular assays using 858 clinical stool samples. A result was considered a consensus positive or negative if at least two of the three tests agreed [75] [24].

Table 1: Overall Agreement with Consensus Results from a 2019 Study (858 samples)

Assay	Targets Detected	Overall Positive Percentage Agreement (PPA)	Remarks
Seegene AllPlex	24 targets (13 bacteria, 5 viruses, 6 parasites)	94% (258/275)	Additionally identified 39 pathogens missed by routine microbiology.
Luminex xTAG GPP	15 targets (9 bacteria, 3 viruses, 3 parasites)	92% (254/275)	Showed low NPA for Salmonella due to frequent false positives; identified 40 additional pathogens.
BD MAX Enteric	8 targets (5 bacteria, 3 parasites)	78% (46/59)	Identified 12 additional pathogens.

For viral targets, the Seegene AllPlex and Luminex xTAG demonstrated high performance, with PPA/NPA of 99%/96% and 93%/99%, respectively [24]. The study also noted that while these panels frequently detected multiple pathogens, only a small fraction of these co-detections were confirmed by a second assay, highlighting the need for careful clinical interpretation [75] [24].

AllPlex vs. Luminex NxTAG: A 2025 Perspective

A more recent 2025 study with 196 samples reinforced the high concordance between updated versions of these panels, reporting overall NPA values consistently above 95% and Kappa values exceeding 0.8 for most pathogens [7]. The average PPA was greater than 89% for nearly all targets, with slightly lower agreement observed for Cryptosporidium (86.6%). Discrepancies were most notable for Salmonella and Cryptosporidium, pointing to ongoing diagnostic challenges with these specific pathogens [7].

AllPlex as a Reference Method

The Seegene AllPlex panel itself is often used as a comparator in the validation of newer assays. For instance, a 2025 evaluation of the Biosynex AMPLIQUICK Fecal Bacteriology kit used the Seegene panels to resolve discordant results, reporting positive and negative agreement rates between 98.24% and 100% for key bacterial targets like Campylobacter, Salmonella, and Shigella/EIEC [76]. Similarly, a 2024 study of the LiquidArray Gastrointestinal VER 1.0 assay used multiple Seegene AllPlex panels as a comparator, with the new assay demonstrating high sensitivity (>90% for most targets) and specificity (>99% for all targets) [33].

Experimental Protocols for Large-scale Evaluation

The methodology from the 2025 comparative study provides a robust template for large-scale evaluation of GI panels [7].

Sample Collection and Study Design

• Sample Source: A total of 196 human fecal samples were collected from patients at a Spanish hospital.
• Preservation: Upon arrival, all stool samples were preserved in Cary-Blair transport medium.
• Study Phases: The study was divided into two phases:
  • Retrospective: 60 samples previously characterized by the Seegene AllPlex assay were re-analyzed using the Luminex NxTAG GPP.
  • Prospective: 136 samples were processed in parallel using both the Seegene AllPlex and Luminex NxTAG GPP assays simultaneously.

Nucleic Acid Extraction and PCR Workflow

• Automated Extraction: Genetic material from all samples was extracted using the HAMILTON STARlet automated system with the STARMag 96 × 4 Universal Cartridge kit [7] [44].
• Assay-specific Protocols:
  • Seegene AllPlex: Requires four separate PCR tubes to run the full suite of GI-Bacteria(I), GI-Bacteria(II), GI-Parasite, and GI-Virus panels [7] [13].
  • Luminex NxTAG GPP: Requires a specific sample pre-treatment step before nucleic acid extraction. The subsequent PCR and detection are completed in a single tube per sample [7].

The workflow for a typical large-scale comparison is summarized in the diagram below.

Discrepancy Resolution and Statistical Analysis

• Resolving Discordant Results: In cases where the two assays produced conflicting results, a third confirmatory technique was employed when possible. This could include microbial culture, targeted PCR from a national reference center, or an alternative real-time PCR kit [7] [33].
• Statistical Metrics: Performance was primarily assessed using PPA and NPA. The Kappa statistic was also used to measure agreement beyond chance, with values over 0.8 indicating strong agreement [7] [24].

The Scientist's Toolkit: Essential Research Reagents & Platforms

The following table details key materials and platforms used in the featured large-scale evaluations.

Table 2: Essential Research Reagents and Platforms for GI Panel Evaluation

Item Name	Function / Description	Example Use in Evaluation
Cary-Blair Transport Medium	Preserves microbial DNA/RNA in stool specimens during transport and storage.	Used for all 196 samples in the 2025 study to maintain sample integrity [7].
HAMILTON STARlet	An automated liquid handling system for high-throughput, standardized nucleic acid extraction.	Used for DNA extraction in both the 2025 and 2025 parasitic validation studies [7] [44].
STARMag Universal Cartridge	A magnetic bead-based chemistry kit for nucleic acid purification, compatible with the HAMILTON system.	The extraction kit of choice in the referenced validation studies [44].
Seegene AllPlex Panels	A suite of four multiplex real-time PCR assays detecting 25 GI pathogens from bacteria, viruses, and parasites.	Served as both the test platform and a comparator method in multiple studies [7] [33] [13].
Bio-Rad CFX96	A real-time PCR detection system for amplifying and detecting nucleic acid targets.	Used for thermocycling and fluorescence detection in the AllPlex GI-Parasite assay validation [44].
Composite Reference Standard (CRS)	A method that combines results from multiple tests to define a "true" positive or negative in the absence of a perfect gold standard.	Used in the Biosynex evaluation, where discordant results were resolved with Seegene panels or targeted PCR [76].

Large-scale comparative evaluations consistently demonstrate that multiplex PCR panels like the Seegene AllPlex Gastrointestinal assay offer rapid, comprehensive, and reliable detection of gastrointestinal pathogens. When assessed using PPA and NPA, these panels show high overall agreement with each other and can identify significantly more pathogens than traditional methods. The Seegene AllPlex platform, with its broad target coverage, has proven to be a robust tool in both clinical diagnostics and as a comparator in validation studies for newer assays. Key considerations for researchers include the observation that certain pathogens like Salmonella and Cryptosporidium may present persistent detection challenges across platforms. Furthermore, the high sensitivity of these panels can lead to frequent co-detections, which require cautious clinical interpretation. Future efforts should focus on standardizing evaluation protocols and expanding pathogen panels to include emerging targets, thereby further enhancing the management of infectious gastroenteritis.

Cross-Reactivity Assessment with Non-Target Organisms

Syndromic molecular panels for gastrointestinal pathogens represent a significant advancement in diagnostic microbiology, offering the potential for rapid and comprehensive pathogen detection. A critical metric for evaluating these multiplex assays is analytical specificity, which refers to the assay's ability to exclusively identify the intended targets without cross-reacting with non-target organisms. This assessment is particularly crucial for the AllPlex Gastrointestinal Panel (Seegene, Seoul, South Korea), which detects a broad spectrum of bacterial, viral, and parasitic enteric pathogens. False-positive results due to cross-reactivity can lead to misdiagnosis, inappropriate treatment, and unnecessary public health measures. Within the context of a broader thesis evaluating the AllPlex GI Panel through large patient cohort research, this review systematically examines evidence regarding its cross-reactivity with non-target organisms, providing researchers and clinical microbiologists with a comprehensive evidence-based assessment of the assay's specificity and reliability.

Experimental Protocols for Cross-Reactivity Assessment

Standardized Cross-Reactivity Testing Methodologies

The assessment of cross-reactivity for the AllPlex GI Panel follows rigorous experimental protocols designed to challenge the assay with a wide array of non-target organisms. These methodologies are consistent with established guidelines for molecular assay validation.

• Panel Composition: Cross-reactivity panels typically include a diverse collection of pathogens and commensal organisms that may be present in the gastrointestinal tract or could potentially cause similar clinical presentations. One comprehensive study evaluated the AllPlex GI-Virus Assay against 169 different pathogens, comprising 43 viruses and 126 bacterial strains [77]. This extensive panel ensures broad coverage of potential cross-reactants.

• Testing Procedure: Each potential cross-reactant is tested in replicates (typically three times) using the same nucleic acid extraction and amplification procedures applied to clinical samples [77]. The process involves extracting nucleic acids from purified cultures of non-target organisms and testing them undiluted at high concentrations to maximize the potential for detecting any cross-reactivity.

• Data Interpretation: The absence of amplification signals (cycle threshold values exceeding the cut-off) for non-target organisms indicates no cross-reactivity. Valid positive controls for the actual targets and internal extraction controls are run concurrently to ensure assay validity [78].

Target-Specific Validation Approaches

Different components of the AllPlex GI Panel require specialized validation approaches tailored to the pathogen types:

• Viral Targets: For the viral panel, cross-reactivity testing includes other enteric viruses not targeted by the assay, as well as common respiratory viruses and herpesviruses that could theoretically be present in clinical specimens [77].

• Bacterial Targets: Bacterial cross-reactivity assessments focus on closely related species within the same genera, as well as common gastrointestinal flora that could generate false-positive signals [1].

• Parasitic Targets: Parasite panel validation includes testing against other stool parasites not included in the panel, particularly those with morphological similarities that could lead to diagnostic confusion [16] [5].

Table 1: Standard Experimental Protocol for Cross-Reactivity Assessment

Protocol Component	Specifications	Quality Control Measures
Organism Panel	169 pathogens (43 viruses, 126 bacteria)	Includes phylogenetically related species and common commensals
Testing Replicates	Minimum of three replicates per organism	Ensures reproducibility of results
Sample Concentration	High concentration preparations	Challenges assay with potential cross-reactants at extreme concentrations
Control Elements	Positive target controls, internal extraction controls	Verifies assay performance and nucleic acid quality
Data Threshold	No amplification signal above background	Clear cutoff for cross-reactivity determination

Cross-Reactivity Assessment Findings

Viral Panel Specificity

The AllPlex GI-Virus Assay, which detects norovirus GI, norovirus GII, rotavirus A, adenovirus F, astrovirus, and sapovirus, has demonstrated excellent analytical specificity in multiple studies.

• Comprehensive Specificity: In a rigorous evaluation, the assay showed no cross-reactivity with any of the 169 non-target pathogens tested, which included 24 target-related viruses and 145 unrelated organisms [77]. This demonstrates the high specificity of the primer and probe designs employed in the assay.

• Comparative Performance: When compared to the Seeplex Diarrhea-V ACE Detection assay, the AllPlex assay showed high agreement rates (93.3-99.6% for various targets) while additionally detecting sapoviruses, which were not targeted by the comparator assay [77]. The implementation of the Multiple Detection Temperature (MuDT) technique allows for simultaneous amplification and detection of multiple targets in a single channel without cross-reactivity [78].

• Genotype Coverage: The assay effectively detected multiple genotypes of each target virus without cross-reacting with non-target genotypes. Evaluation studies confirmed detection of various norovirus GI and GII genotypes, rotavirus genotypes, adenovirus genotypes, astrovirus genotypes, and sapovirus genotypes [77].

Bacterial Panel Specificity

The bacterial components of the AllPlex GI Panel (GI-Bacteria I and II Assays) detect 13 major bacterial pathogens, including Salmonella spp., Shigella spp./enteroinvasive E. coli, Campylobacter spp., Yersinia enterocolitica, Vibrio spp., Clostridium difficile toxin B, Aeromonas spp., E. coli O157, and various diarrheagenic E. coli pathotypes.

• High Specificity Rates: For all bacterial targets, the percentages of specificity were greater than 95%, with most approaching 99%, when compared to culture methods and resolved discrepancies by alternative molecular methods [1]. The assay demonstrated particular strength in detecting pathogens that are frequently missed by conventional culture methods, such as enteropathogenic E. coli (EPEC), enteroaggregative E. coli (EAEC), and non-O157 Shiga toxin-producing E. coli (STEC) [17].

• Discordant Resolution: In cases of initial discordance between AllPlex and conventional culture results, follow-up testing using monoplex PCR assays confirmed that the majority (89.2%) of AllPlex-positive/culture-negative results represented true infections rather than cross-reactivity [17]. This highlights the assay's superior sensitivity rather than non-specificity.

Parasitic Panel Specificity

The AllPlex GI-Parasite Assay targets Giardia duodenalis, Cryptosporidium spp., Entamoeba histolytica, Dientamoeba fragilis, Blastocystis hominis, and Cyclospora cayetanensis.

• Species Differentiation: The parasitic panel demonstrates excellent specificity in differentiating pathogenic from non-pathogenic species, particularly for Entamoeba histolytica versus non-pathogenic E. dispar, which is impossible by microscopic examination alone [16] [5]. This specific differentiation has important clinical implications for treatment decisions.

• Multicenter Validation: A large Italian multicenter study demonstrated specificity values of 100% for Entamoeba histolytica, 99.2% for Giardia duodenalis, 100% for Dientamoeba fragilis, and 99.7% for Cryptosporidium spp. when compared to conventional parasitological methods [5].

• Cryptosporidium Species Detection: The assay successfully detected multiple Cryptosporidium species (C. parvum, C. hominis, C. felis, C. canis, C. cuniculus, C. meleagridis) without cross-reactivity between species [16].

Table 2: Cross-Reactivity Assessment Results by Pathogen Category

Pathogen Category	Number of Non-Target Organisms Tested	Cross-Reactivity Results	Key Supporting Studies
Viral Pathogens	169 pathogens (43 viruses, 126 bacteria)	No cross-reactivity detected	[77] [78]
Bacterial Pathogens	Not explicitly stated (comprehensive panel)	Specificity >95% for all targets	[17] [1]
Parasitic Pathogens	Evaluated against microscopic examination	Specificity 99.2-100% for major targets	[16] [5]

Comparative Performance with Alternative Methods

Comparison with Conventional Diagnostic Methods

When compared to traditional diagnostic approaches, the AllPlex GI Panel demonstrates significantly improved detection rates while maintaining high specificity.

• Enhanced Detection Yield: A comprehensive study comparing the AllPlex GI Panel to conventional methods (stool culture for bacteria and electron microscopy for viruses) found that the multiplex PCR assay had a greater than two-fold higher detection rate (44.4% vs. 17.8%) without evidence of false positivity due to cross-reactivity [17]. This increased detection primarily resulted from identifying pathogens that conventional methods typically miss, such as various E. coli pathotypes and viruses undetectable by electron microscopy.

• Detection of Co-infections: The AllPlex GI Panel identified mixed infections in 23.3% of positive samples [17], a finding that would be challenging to ascertain with conventional methods due to their limited multiplexing capability and the dominance of one pathogen in culture.

Comparison with Other Molecular Panels

The AllPlex GI Panel shows favorable performance characteristics when compared to other commercial molecular gastrointestinal panels.

• Versus Seeplex Panels: In a prospective comparison, the AllPlex system demonstrated a significantly higher overall detection rate (54.9% vs. 48.8%, p=0.002) compared to Seeplex Diarrhea V/B1/B2 ACE Detection assays [3]. The overall percent agreement between the two molecular methods exceeded 95% for common targets, indicating consistent specificity despite differences in panel composition and detection technology.

• Automated Interpretation Advantage: Unlike conventional multiplex PCR systems that require capillary electrophoresis and manual interpretation, the AllPlex system provides automated result interpretation using its proprietary viewer software, reducing subjective interpretation errors [3].

• LiquidArray Comparison: In a comprehensive evaluation comparing multiple platforms, the AllPlex panels were used as comparator methods for validating the new LiquidArray Gastrointestinal VER 1.0 assay, confirming their established reliability in the field [33].

Research Reagent Solutions

Table 3: Essential Research Reagents and Platforms for AllPlex GI Panel Implementation

Reagent/Platform	Function	Specification/Application
AllPlex GI-Virus Assay	Detection of 6 major viral pathogens	Identifies ASV, SV, ROV, NVG1, NVG2, ADV-F
AllPlex GI-Bacteria (I) Assay	Detection of 7 bacterial targets	Identifies Shigella/EIEC, Campylobacter, Yersinia, Vibrio, C. difficile toxin B, Aeromonas, Salmonella
AllPlex GI-Bacteria (II) Assay	Detection of 6 bacterial targets	Identifies STEC/EHEC, E. coli O157, EPEC, ETEC, EAEC, hypervirulent C. difficile
AllPlex GI-Parasite Assay	Detection of 6 parasitic targets	Identifies G. duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, B. hominis, C. cayetanensis
Nucleic Acid Extraction Systems	Automated nucleic acid purification	Compatible with Seegene STARlet-AIOS, NIMBUS, Maelstrom 9600, NucliSENS EasyMag
Real-time PCR Instruments	Amplification and detection	CFX96 Dx, CFX96 systems
ASL Stool Lysis Buffer	Stool sample processing	Initial processing of stool specimens for nucleic acid extraction
Cary-Blair Medium	Transport medium	Preservation of stool specimens during transport

Technological Basis for High Specificity

The high specificity and minimal cross-reactivity of the AllPlex GI Panel can be attributed to its underlying technological innovations:

• MuDT (Multiple Detection Temperature) Technique: This proprietary technology enables the simultaneous detection of multiple targets in a single fluorescence channel by analyzing differences in fluorescence signals at specific detection temperatures [78]. The MuDT technique allows for specific target identification based on cycle threshold (Ct) values without requiring melting curve analysis, reducing the potential for cross-reactivity between similarly sequenced targets.

• TOCE (Tagging Oligonucleotide Cleavage and Extension) Technology: This method forms the basis for the MuDT system, enabling highly specific target identification and differentiation through cleavage and extension of tagging oligonucleotides [78].

• Automated Workflow Integration: The compatibility of AllPlex assays with fully automated systems such as the Seegene STARlet-AIOS minimizes manual handling errors and cross-contamination potential, contributing to more specific and reliable results [15].

The following diagram illustrates the experimental workflow for cross-reactivity assessment:

Figure 1: Experimental Workflow for Cross-Reactivity Assessment

The comprehensive analysis of cross-reactivity assessment for the AllPlex Gastrointestinal Panel demonstrates exceptional analytical specificity across viral, bacterial, and parasitic targets. Extensive evaluation against 169 non-target organisms revealed no cross-reactivity, while clinical studies consistently reported specificity exceeding 95% for all targets, with many approaching 100%. The panel's innovative MuDT technology, combined with rigorous primer and probe design, effectively minimizes non-specific amplification while maintaining high sensitivity for intended targets. When compared to both conventional methods and alternative molecular panels, the AllPlex GI Panel shows superior detection capabilities without compromising specificity. These findings, derived from multiple large-scale studies, support the reliability of the AllPlex GI Panel for clinical and research applications where distinguishing between closely related organisms is crucial for accurate diagnosis and appropriate patient management. The demonstrated lack of cross-reactivity with non-target organisms positions this syndromic panel as a valuable tool for comprehensive gastrointestinal pathogen detection in diverse clinical and research settings.

Infectious gastroenteritis remains a major global cause of morbidity and mortality, particularly in children, the elderly, and immunocompromised patients [33]. Accurate and rapid pathogen identification is crucial for effective patient management, antimicrobial stewardship, and public health interventions [7] [36]. Traditional diagnostic methods based on stool culture and microscopy are time-consuming, labor-intensive, and lack sensitivity for many pathogens [7] [18] [33]. The emergence of multiplex PCR syndromic panels has revolutionized diagnostic approaches by enabling simultaneous detection of numerous bacteria, viruses, and parasites from a single stool sample [7] [33]. Among these, the Seegene AllPlex Gastrointestinal Panel has been widely implemented, but understanding its clinical impact on patient outcomes and treatment decisions requires critical evaluation against competing technologies and assessment of its performance in large patient cohorts.

Comparative Performance Analysis of Multiplex GI Panels

Detection Accuracy Across Pathogen Categories

Table 1: Overall Detection Performance of Multiplex GI Panels

Multiplex Panel	Target Coverage	Overall PPA	Overall NPA	Key Strengths	Key Limitations
Seegene AllPlex GI	24 targets (13 bacteria, 5 viruses, 6 parasites)	94% [24]	>95% [7]	Excellent protozoa detection [6] [18]	Lower performance for helminths [6]
Luminex NxTAG GPP	15 targets (9 bacteria, 3 viruses, 3 parasites)	92% [24]	>95% [7]	Single-tube reaction [7]	Lower PPA for viruses [24]
BD MAX Enteric	8 targets (5 bacteria, 3 parasites)	78% [24]	Not reported	Automated system	Limited target coverage
LiquidArray GI VER 1.0	26 targets	>90% (most targets) [33]	>99% [33]	Low invalid rate (0%) [33]	Limited published data
QIAstat-Dx GIP2	24 targets	95.02% [36]	99.98% [36]	Rapid results (~70 min) [36]	Suboptimal virus detection (PPA 88.3%) [36]

Table 2: Pathogen-Specific Performance of Seegene AllPlex Panel

Pathogen Category	Specific Pathogens	Sensitivity	Specificity	Clinical Impact
Protozoa	Giardia duodenalis	100% [29]	99.2% [29]	Enables targeted antiprotozoal therapy
	Cryptosporidium spp.	100% [29]	99.7% [29]	Identifies opportunistic infections in immunocompromised
	Entamoeba histolytica	100% [29]	100% [29]	Distinguishes pathogenic from non-pathogenic species
	Dientamoeba fragilis	97.2-100% [6] [29]	99.3-100% [6] [29]	Detects often-missed cause of persistent diarrhea
	Blastocystis hominis	93-95% [6] [44]	98.3% [44]	Clinical significance interpretation needed
Bacteria	Campylobacter spp.	High agreement [7]	High agreement [7]	Guides antibiotic selection
	Salmonella spp.	High agreement [7]	High agreement [7]	Impacts public health measures
	Clostridioides difficile	High agreement [7]	High agreement [7]	Directs appropriate antimicrobial therapy
Viruses	Norovirus	99% [24]	96% [24]	Reduces unnecessary antibiotic use
	Rotavirus	99% [24]	96% [24]	Supports infection control measures
Helminths	Multiple species	59.1% [6]	Not reported	Suboptimal vs. microscopy (100%) [6]

Impact on Diagnostic Yield and Turnaround Time

The implementation of the Seegene AllPlex panel has demonstrated significant improvements in diagnostic yield compared to conventional methods. In a prospective study of 3,500 stool samples over three years, multiplex PCR detected enteric protozoa in 26% of samples compared to only 8.2% by microscopy [18]. Specifically, for Dientamoeba fragilis, PCR detected 310 positive samples (8.86%) versus only 22 (0.63%) by microscopy, while Blastocystis spp. was detected in 673 samples (19.25%) by PCR compared to 229 (6.55%) by microscopy [18].

Turnaround time is significantly reduced with the AllPlex system. One validation study reported that the molecular platform reduced pre-analytical and analytical testing time by 7 hours per batch compared to conventional methods [44]. This acceleration directly impacts patient management by enabling same-day result availability compared to the 2-4 days typically required for culture-based methods [64].

Experimental Protocols and Methodologies

Standardized Testing Workflow

Figure 1: Automated Workflow for AllPlex GI Panel Testing

Key Methodological Considerations

The analytical performance of the Seegene AllPlex panels has been validated across multiple large-scale studies. In a comparative evaluation of 858 stool samples, the AllPlex system demonstrated 94% positive percentage agreement (PPA) compared to 92% for Luminex xTAG GPP and 78% for BD MAX Enteric Panel [24]. Sample processing follows a standardized protocol: approximately 1g of stool is suspended in 2mL of eNAT medium, vortexed, and incubated for 10 minutes at room temperature [6]. After transfer to bead-beating tubes and additional vortexing, DNA extraction is performed using automated systems such as the Hamilton STARlet with STARMag 96 × 4 Universal Cartridge kits [6] [44].

PCR amplification utilizes the Bio-Rad CFX96 thermal cycler with four fluorophores (FAM, HEX, Cal Red 610, and Quasar 670) and a 45-cycle protocol [44]. Samples are considered positive at cycle threshold (Ct) values ≤43 according to manufacturer specifications [44]. This standardized approach enables detection of pathogens that are difficult to culture or identify microscopically, particularly enteric protozoa.

Discrepancy Resolution Protocols

In cases of discordant results between multiplex PCR and conventional methods, studies employed third-line confirmatory testing. Techniques included microbial culture with plate-based identification, specific PCR assays from reference laboratories, and alternative commercial PCR kits such as the VIASURE Real-Time PCR Detection Kit [7]. This rigorous approach to discrepancy analysis strengthens the validity of performance assessments.

The Scientist's Toolkit: Essential Research Reagents and Materials

Table 3: Key Research Reagent Solutions for GI Panel Evaluation

Reagent/Equipment	Manufacturer	Specific Function	Application in Validation
Hamilton STARlet	Hamilton Company	Automated nucleic acid extraction	Standardized DNA extraction across studies [7] [44]
STARMag 96 × 4 Universal Cartridge	Seegene Inc.	Bead-based nucleic acid purification	Integrated extraction for AllPlex panels [44]
Bio-Rad CFX96	Bio-Rad	Real-time PCR amplification	Detection system for AllPlex assays [6] [44]
AllPlex GI-Parasite Assay	Seegene Inc.	Multiplex detection of 6 protozoa	Targeted parasite identification [6] [18] [44]
AllPlex GI-Bacteria (I) & (II)	Seegene Inc.	Comprehensive bacterial pathogen detection	Bacterial target coverage [7] [64]
AllPlex GI-Virus Assay	Seegene Inc.	Detection of 5 viral pathogens	Viral gastroenteritis diagnosis [7]
AllPlex GI-Helminth Assay	Seegene Inc.	Detection of 8 helminths	Helminth identification (limited sensitivity) [6]
FecalSwab Tubes with Cary-Blair	COPAN Diagnostics	Sample preservation and transport	Maintains specimen integrity [44]
eNAT Medium	COPAN Diagnostics	Nucleic acid preservation	Stabilizes genetic material pre-extraction [6]

Clinical Correlation with Patient Outcomes

Impact on Treatment Decisions and Antimicrobial Stewardship

The comprehensive pathogen detection capability of the AllPlex system directly influences treatment decisions. Traditional methods typically target only the most common bacterial pathogens (Campylobacter, Salmonella, Shigella), potentially missing clinically relevant pathogens such as diarrheagenic E. coli, parasites, and viruses [64]. The AllPlex panel's broad coverage enables identification of these additional pathogens, facilitating targeted therapy.

For bacterial infections, detection of specific pathogens guides appropriate antibiotic selection. For parasitic infections, the high sensitivity for Giardia duodenalis (100%) and Cryptosporidium spp. (100%) enables timely administration of appropriate antiprotozoal agents [29]. Conversely, identification of viral pathogens reduces inappropriate antibiotic prescribing, supporting antimicrobial stewardship efforts [36].

Management of Persistent and Chronic Gastroenteritis

The AllPlex panel demonstrates particular clinical value in detecting pathogens frequently missed by conventional methods. Dientamoeba fragilis, an often-overlooked cause of persistent gastrointestinal symptoms, was detected with 100% sensitivity compared to only 47.4% by conventional microscopy [6]. Similarly, Blastocystis hominis was detected with 95% sensitivity compared to 77.5% by conventional methods [6]. This enhanced detection directly impacts patients with unexplained chronic gastrointestinal symptoms who may have previously undergone extensive negative workups.

Public Health and Infection Control Implications

Rapid identification of outbreak-related pathogens represents another significant clinical impact. The AllPlex system's ability to detect norovirus and other enteric viruses with high sensitivity (99% PPA) enables prompt implementation of infection control measures in healthcare settings [24]. Similarly, detection of bacterial pathogens such as Salmonella spp. and Shiga toxin-producing E. coli triggers public health reporting and investigation, potentially limiting community transmission [64].

Limitations and Complementary Diagnostic Approaches

Despite its advantages, the AllPlex system has limitations that impact its clinical utility. Most notably, the AllPlex GI-Helminth assay demonstrates suboptimal performance with 59.1% sensitivity compared to 100% for conventional microscopy [6]. This limitation is particularly relevant for travelers and migrants from endemic areas who may harbor helminthic infections [18].

Additionally, the panel does not detect some less common pathogens, including Cystoisospora belli, which is particularly relevant for HIV-infected patients [18]. The detection of nucleic acid rather than viable organisms may also complicate interpretation in settings where colonization must be distinguished from active infection [33].

These limitations highlight the need for complementary diagnostic approaches. Microscopy remains necessary when helminth infection is suspected, and culture may still be required for antibiotic susceptibility testing in specific clinical scenarios [18] [64].

The Seegene AllPlex Gastrointestinal Panel represents a significant advancement in the diagnosis of infectious gastroenteritis, with demonstrated impact on patient outcomes and treatment decisions. Its high sensitivity for most bacterial, viral, and protozoan pathogens enables rapid, targeted therapy while supporting antimicrobial stewardship. The panel's limitations in helminth detection and inability to provide antibiotic susceptibility data necessitate complementary approaches in specific clinical scenarios. Future developments should focus on expanding target panels, improving detection accuracy for challenging pathogens, and further integrating these technologies with clinical decision support systems to optimize patient management.

Conclusion

The Allplex Gastrointestinal Panel represents a significant advancement in syndromic testing for infectious gastroenteritis, demonstrating high overall diagnostic accuracy and substantially improved detection rates compared to conventional methods. Implementation in large patient cohorts reveals critical insights into pathogen epidemiology, mixed infections, and demographic variations. While the assay shows exceptional performance for most bacterial, viral, and protozoan targets, particularly for Dientamoeba fragilis, Giardia duodenalis, and Cryptosporidium species, limitations in helminth detection and variable Entamoeba histolytica sensitivity necessitate complementary testing approaches in specific clinical scenarios. The transition to automated, high-throughput molecular testing reduces turnaround time by approximately 7 hours per batch, significantly impacting patient management and public health surveillance. Future research should focus on expanding target panels for challenging pathogens, conducting cost-effectiveness analyses across healthcare systems, and developing standardized algorithms for integrating multiplex PCR results into antimicrobial stewardship programs and public health reporting systems.

References

• 1. Evaluation of the multiplex PCR Allplex-GI assay in ... [https://www.sciencedirect.com/science/article/abs/pii/S0167701217302890]
• 2. A comparison of the Allplex™ bacterial and viral assays to ... [https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-018-3645-6]
• 3. Comparative evaluation of two molecular multiplex ... [https://www.sciencedirect.com/science/article/abs/pii/S0732889324000403]
• 4. Evaluation of the AllplexTM Gastrointestinal Panel ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7232139/]
• 5. Evaluation of Allplex™ GI-Parasite Assay—A Multiplex ... [https://www.mdpi.com/2414-6366/10/8/234]
• 6. Evaluation of the Allplex GI Parasite and Helminth PCR ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11430856/]
• 7. Seegene Allplex™ vs. Luminex NxTAG® in clinical stool ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12116839/]
• 8. Multiplex Polymerase Chain Reaction Panels for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12541719/]
• 9. Comparative performance evaluation of four commercial ... [https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215068]
• 10. Comparative epidemiology of diarrhoeal organisms in high ... [https://www.sciencedirect.com/science/article/abs/pii/S1198743X25004021]
• 11. Detection of gastrointestinal pathogens in stool samples ... [https://www.sciencedirect.com/science/article/abs/pii/S0732889324003699]
• 12. A Comparison of Pathogen Detection and Risk Factors ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11215998/]
• 13. Allplex™ Gastrointestinal Panel Assays [https://www.seegene.com/assays/allplex_gastrointestinal_panel_assays]
• 14. Allplex™ GI-Bacteria(I) Assay [https://www.seegene.com/assays/allplex_gi_bacteria_i_assay]
• 15. Allplex™ GI-Virus Assay [https://www.seegene.com/assays/allplex_gi_virus_assay]
• 16. Evaluation of the Allplex TM Gastrointestinal Panel ... [https://www.mdpi.com/2076-2607/8/4/569]
• 17. A comparison of the Allplex™ bacterial and viral assays to ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6064067/]
• 18. a prospective study on 3500 stool samples over 3 years [https://pubmed.ncbi.nlm.nih.gov/40162804/]
• 19. Enhancing enteric pathogen detection: implementation and ... [https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-024-09047-z]
• 20. Gastrointestinal Pathogen Trends | bioMérieux - bioMerieux [https://www.biomerieux.com/us/en/blog/diagnostic-digest/gastrointestinal-pathogen-trends.html]
• 21. Mixed Viral-Bacterial Infections and Their Effects on Gut ... [https://www.nature.com/articles/s41598-018-37162-w]
• 22. High Burden of Co-Infection with Multiple Enteric Pathogens in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9959912/]
• 23. Risk factors and clinical outcomes associated with multiple ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11365154/]
• 24. Comparative Evaluation of Seegene Allplex Gastrointestinal, Luminex xTAG Gastrointestinal Pathogen Panel, and BD MAX Enteric Assays for Detection of Gastrointestinal Pathogens in Clinical Stool Specimens - PubMed [https://pubmed.ncbi.nlm.nih.gov/30763118/]
• 25. Assessing the burden of viral co-infections in acute ... [https://www.sciencedirect.com/science/article/abs/pii/S1386653220302559]
• 26. Global, regional, and national incidence and mortality for ... [https://bmcpublichealth.biomedcentral.com/articles/10.1186/s12889-024-21270-6]
• 27. Global burden, subtype, risk factors and etiological analysis of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11965617/]
• 28. Prevalence of Enteric Pathogens and Antibiotic Resistance [https://www.mdpi.com/2079-6382/13/8/726]
• 29. Evaluation of Allplex™ GI-Parasite Assay—A Multiplex Real ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12390207/]
• 30. Adaptation and validation of a gastrointestinal panel to ... [https://link.springer.com/article/10.1007/s00430-025-00837-z]
• 31. Comparison of the Luminex® NxTAG® Gastrointestinal ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12585152/]
• 32. Comparison of QIAstat-Dx and BioFire FilmArray ... [https://www.mdpi.com/2076-2607/12/11/2282]
• 33. Clinical Performance of the LiquidArray® Gastrointestinal ... [https://www.mdpi.com/2075-4418/14/21/2377]
• 34. FilmArray™ GI panel performance for the diagnosis of acute ... [https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-017-1018-2]
• 35. Comparison of three multiplex gastrointestinal platforms for ... [https://www.sciencedirect.com/science/article/abs/pii/S1386653217302445]
• 36. Evaluation of the QIAstat-Dx® Gastrointestinal Panel 2 for ... [https://www.sciencedirect.com/science/article/abs/pii/S0732889325004511]
• 37. Evaluation of the AllplexTM Gastrointestinal Panel-Parasite ... [https://pubmed.ncbi.nlm.nih.gov/32326453/]
• 38. Seegene Allplex™ vs. Luminex NxTAG® in clinical stool ... [https://pubmed.ncbi.nlm.nih.gov/40095260/]
• 39. Allplex™ GI-Parasite Assay [https://www.seegene.com/assays/allplex_gi_parasite_assay]
• 40. Fresh vs Frozen Samples and Ambient Temperature Have ... [https://www.sciencedirect.com/science/article/abs/pii/S154235651630982X]
• 41. The effect of storage time and freeze-thaw cycles on ... [https://pubmed.ncbi.nlm.nih.gov/23457767/]
• 42. GIP - Overview: Gastrointestinal Pathogen Panel, PCR, ... [https://www.mayocliniclabs.com/test-catalog/overview/63169]
• 43. a comparative study of Allplex® (Seegene ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8826582/]
• 44. Validation of an Automated High-Throughput Multiplex ... [https://www.mdpi.com/2673-947X/5/1/8]
• 45. A comparison of two multiplex-PCR assays for the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7888160/]
• 46. A comparison of two multiplex-PCR assays for the diagnosis ... [https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-021-05885-3]
• 47. Evaluation of the Allplex GI Parasite and Helminth PCR ... [https://pubmed.ncbi.nlm.nih.gov/39335677/]
• 48. Revisiting Drug Development Against the Neglected ... [https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2020.628257/full]
• 49. Evaluation of the AllplexTM Gastrointestinal Panel—Parasite Assay for Protozoa Detection in Stool Samples: A Retrospective and Prospective Study - PMC [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7232139/]
• 50. Comparative performance evaluation of four commercial multiplex real-time PCR assays for the detection of the diarrhoea-causing protozoa Cryptosporidium hominis/parvum, Giardia duodenalis and Entamoeba histolytica - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC6453453/]
• 51. Real-time PCR-HRM assay for precise identification of ... [https://pubmed.ncbi.nlm.nih.gov/40471554]
• 52. Serologic diagnosis of Entamoeba histolytica infection ... [https://www.sciencedirect.com/science/article/pii/S000326702401403X]
• 53. Advantages and Limitations of Microscopy and Molecular ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9979072/]
• 54. Detection of soil-transmitted helminths and Schistosoma spp ... [https://journals.plos.org/plosntds/article?id=10.1371/journal.pntd.0012404]
• 55. Comparison of parasitological methods for the ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11321430/]
• 56. A comparison of helminth infections as assessed through ... [https://www.sciencedirect.com/science/article/pii/S2213224418300956]
• 57. Comparison of multi-parallel quantitative real-time PCRs ... [https://parasitesandvectors.biomedcentral.com/articles/10.1186/s13071-024-06464-6]
• 58. Investigation of suspected false positive norovirus results ... [https://www.sciencedirect.com/science/article/abs/pii/S1386653224000945]
• 59. Evaluation of the Allplex™ GI-Helminth(I) Assay, the first ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8019563/]
• 60. Diagnosing viral gastro-enteritis using the fully automated ... [https://www.sciencedirect.com/science/article/pii/S0166093424001095]
• 61. Lab Automation: Transforming Modern Laboratories in 2025 [https://www.marketsandmarkets.com/blog/HC/lab-automation-systems-complete-guide]
• 62. Top lab trends for 2025 | LabLeaders [https://diagnostics.roche.com/global/en/article-listing/lab-leaders/article/top-lab-trends-2025.html]
• 63. Clinical Lab Automation: 3 Key Considerations for ... [https://www.clinicallab.com/clinical-lab-automation-3-key-considerations-for-successful-implementation-28298]
• 64. Performance evaluation of the Biosynex AMPLIQUICK ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12220677/]
• 65. How Lab Automation Unlocks New Levels of Productivity [https://www.wakoautomation.com/scaling-your-lab-how-automation-unlocks-new-levels-of-productivity]
• 66. Evaluation of the Allplex GI Parasite and Helminth PCR ... [https://www.mdpi.com/2075-4418/14/18/1998]
• 67. Comparative Performance of the Luminex NxTAG ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9431521/]
• 68. Comparative analysis of three multiplex platforms for the ... [https://www.sciencedirect.com/science/article/abs/pii/S1386653222002062]
• 69. BIOFIRE® FILMARRAY® Gastrointestinal (GI) Panel [https://www.biomerieux.com/us/en/our-offer/clinical-products/biofire-gastrointestinal-panel.html]
• 70. Multicenter Evaluation of the BioFire FilmArray ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4390666/]
• 71. Comparison of several Real-Time PCR Kits versus a ... [https://www.nature.com/articles/s41598-020-61202-z]
• 72. Sensitivity and Specificity or Positive and Negative Percent ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5654895/]
• 73. What Does Positive Percent Agreement Mean [https://arletstars.com/what-does-positive-percent-agreement-mean/]
• 74. Principles of Large-scale Evidence Generation and Evaluation ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7481029/]
• 75. Comparative Evaluation of Seegene Allplex ... Gastrointestinal [https://www.sigmaaldrich.cn/CN/en/tech-docs/paper/1228489]
• 76. Performance evaluation of the Biosynex AMPLIQUICK Fecal ... [https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-025-04118-w]
• 77. Evaluation of a New Multiplex Real-Time PCR Assay for ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC5820066/]
• 78. Evaluation of an Assay Based On Multiple Detection ... [https://brieflands.com/journals/jjm/articles/57024]