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. 2025 May 13;15(10):1228. doi: 10.3390/diagnostics15101228

Syndromic Testing—The Evaluation of Four Novel Multiplex Real-Time Polymerase Chain Reaction Panels

Mesut Yılmaz 1, Selcuk Kilic 2, Fatma Bayrakdar 2, Selin Nar Ötgün 2, Ayse Istanbullu Tosun 3, Umit Zeybek 4, Faruk Çelik 4, Gokhan Aygun 5, Birol Safak 6, Naim Mahroum 1,*
Editor: Laurent Bélec
PMCID: PMC12109757  PMID: 40428221

Abstract

Background: If used in the right clinical context, PCRs carry great potential in rapidly diagnosing various infectious diseases. Objectives: We aimed to evaluate the clinical performance of four novel multiplex real-time PCR (qPCR) assays in the direct detection of pathogens in whole blood, cerebrospinal fluid, respiratory specimens, and stool samples. Methods: Spiked negative clinical specimens were used for the evaluation. Clinical samples for the comparative assessment of culture and molecular analyses were simultaneously examined. RINATM robotic nucleic acid isolation system and Bio-Speedy® multiplex qPCR panels (Bioeksen R&D Technologies, Turkey), and the LightCycler® 96 Instrument (Roche, USA) were used for the molecular testing. Results: No qPCR assays produced positive results for the samples spiked with the potential cross-reacting pathogens. The limit of detection (LOD) of the assays changed with the use of 10 and 100 pathogens/mL sample based on the target and sample type. The relative sensitivity and specificity of the assays were, respectively, 82% and 94% for blood, 97.1% and 99.3% for blood culture, 94% and 98% for stool, 96% and 97% for CSF, and 97% and 96% for respiratory specimens. Conclusions: The panels evaluated allow the direct molecular analysis of 10 samples from four clinical syndromes on the same run in 3 h with high clinical performance. The number and variety of samples in a single run enable healthcare providers to rapidly and efficiently diagnose and treat various infections.

Keywords: syndromic testing, real-time PCR, syndromic multiplex panels

1. Introduction

Since their advent more than a decade ago, the commercial panel-based molecular diagnostics for rapid pathogen detection in different biological samples have transformed clinical microbiology and practice [1]. Classified under syndromic panel testing, these molecular assays simultaneously detect and identify multiple pathogens associated with various syndromes related to bloodstream, respiratory, gastrointestinal (GI), or central nervous system (CNS) infections, which save valuable time and potentially improve healthcare outcomes. In practical life, for patients presenting with suspected infectious disease, with findings that overlap among numerous infectious agents (bacteria, viruses, and other pathogens), syndromic testing provides a simultaneous testing of a high number of pathogens from different biological samples [2]. The results detected aid in the rapid application of directed treatment, halt the misuse of antimicrobials or provide a much wider spectrum of coverage, which eventually enhances stewardship and decreases the risk of resistance among pathogens. What leaves no doubt is that this method of testing saves time, and resources, and enhances the efficacy of microbiological lab processes when compared to old and standard methods.

Nevertheless, molecular syndromic testing technologies also present several challenges, including cost, strategies of use, and the interpretation of results. For instance, current clinical practice guidelines do not always provide clear directions for result interpretation, and clinicians may not be familiar with all the detected organisms and/or resistance genes [3]. Additionally, fixed panel composition can be challenging in terms of application in clinical practice. The closed-system multiplex platforms also carry a contamination risk that may be difficult to identify. Additional challenges include integrating multiplex panels into laboratory workflows and monitoring result accuracy post-implementation.

As the use and application of syndromic testing are expected to continue to become more common, understanding the performance characteristics and limitations of multiplex assays is crucial. Therefore, we aimed to analyze the clinical performance of four novel multiplex real-time PCR (qPCR) assays for the identification of pathogens in clinical practice.

2. Materials and Methods

The study was conducted as a prospective observational diagnostic accuracy study. Samples were collected prospectively from patients with suspected infections, and each sample was tested both by culture-based gold standard methods and the novel qPCR panels. Trained clinical personnel at the participating centers collected the samples. For all molecular analyses, processing began within 2–3 h after collection. The detailed process is presented in the following manner:

2.1. Molecular Syndromic Testing

The RINATM-M14 robotic nucleic acid isolation system, Bio-Speedy® multiplex qPCR panels (Bioeksen, Sarıyer, Turkey), and LightCycler® 96 Instrument (Roche, Indianapolis, IN, USA) were used for all molecular syndromic tests in our study. Samples of blood, CSF, nasopharyngeal wash/aspirate, sputum, and bronchoalveolar lavage (BAL) were directly loaded into RINATM-M14 nucleic acid extraction cartridges. Oropharyngeal and nasopharyngeal swabs and approximately 30 mg of stool samples were transferred into 500 µL molecular grade water and homogenized before loading. The 75 min extraction protocol was employed for all extractions in the RINATM-M14 robot. Deionized water served as the negative control in each run.

The multiple targets in the pre-loaded and ready-to-use 8-well qPCR strips of the Bio-Speedy® qPCR panels are listed in Table 1, Table 2, Table 3, Table 4 and Table 5. A reaction containing a human DNA-targeted oligonucleotide set was used as an internal control to assess DNA extraction and PCR inhibition [4]. For each qPCR well, 5 µL of the nucleic acid extract was loaded into a qPCR well containing 15 µL of the target-specific multiplex qPCR mixture. The 90 min qPCR protocol was used for all assay types.

Table 1.

Multiple targets in 8-well qPCR strips of Bio-Speedy® qPCR panel for respiratory tract samples, and results of the clinical performance study.

Multiplex Reactions Respiratory Panel LOD
mL−1 		Precision True
+		 False
+		 True
-		 False
-
1A FAM Influenza A virus 66 100% 243 8 393 8
HEX Internal Control -
ROX Influenza A H1 virus 83 100% 177
CY5 Influenza B virus 88 98% 113 6 5
1B FAM Coronavirus 229E 62 96% 12
HEX Coronavirus OC43 68 100% 7
ROX Coronavirus NL63 94 98% 9
CY5 Coronavirus HKU1 86 100% 2
1C FAM Parainfluenza 1 virus 75 96% 8
HEX Parainfluenza 2 virus 91 100% 10
ROX Parainfluenza 3 virus 53 98% 1
CY5 Parainfluenza 4 virus 88 100% 6
1D FAM Metapneumovirus 64 96% 19
HEX MERS-CoV 92 100% 0
ROX Respiratory syncytial virus A/B 97 100% 8
CY5 Rhinovirus 97 98% 18
1E FAM Bocavirus 83 96% 17
HEX Enterovirus 71 100% 22 1
ROX Parechovirus 59 100% 4
CY5 Adenovirus 62 98% 28 1 1
1F FAM Legionella pneumophila 54 100% 0
HEX
ROX Mycoplasma pneumoniae 62 100% 9
CY5 Chlamydophila pneumoniae 58 98% 2
1G FAM Haemophilus influenzae 48 96% 11
HEX
ROX
CY5 Streptococcus pneumoniae 43 98% 31 1
1H FAM Bordetella pertussis 47 98% 2
HEX
ROX Bordetella parapertussis 63 96% 0
CY5 Bordetella holmesii 42 96% 0
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Table 2.

Multiple targets in 8-well qPCR strips of Bio-Speedy® qPCR panel for gastrointestinal samples, and results of the clinical performance study.

Multiplex Reactions Gastrointestinal Panel LOD
mL−1 		Precision True
+		 False
+		 True
-		 False
-
1A FAM Sapovirus (GI/GII/GIV/GV) 98 98% 1 95
HEX Internal Control - -
ROX
CY5
1B FAM Yersinia enterocolitica 74 98%
HEX Plesiomonas shigelloides 62 100% 2
ROX Entamoeba histolytica 58 100%
CY5 Cryptosporidium spp. 67 100% 1
1C FAM Giardia lamblia 92 100% 2
HEX
ROX
CY5 Cyclospora cayetanensis 88 96% 1
1D FAM Astrovirus 46 98% 2
HEX Norovirus (GI/GII) 31 98% 6
ROX Rotavirus (A) 24 98% 6 1
CY5 Adenovirus 62 100% 3
1E FAM Salmonella spp. 24 100% 14 1 1
HEX Campylobacter spp. 31 96% 10 1 1
ROX Vibrio parahaemolyticus 28 96%
CY5 Vibrio cholerae 63 96%
1F FAM Enteroinvasive E.coli 66 100% 2
HEX
ROX Enteroaggregative E. coli 54 98% 8
CY5 Shiga toxin producing E.coli 54 98% 2
1G FAM Enteropathogenic E.coli 74 98% 27 2
HEX
ROX
CY5 Enterotoxigenic E.coli 83 98% 2
1H FAM Clostridium difficile 62 98% 11 1
HEX C. difficile toxin B 68 100%
ROX C. difficile toxin A 52 98% 3
CY5 C. difficile binary toxin A/B 38 98%
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Table 3.

Multiple targets in 8-well qPCR strips of Bio-Speedy® qPCR panel for central nervous system samples, and results of the clinical performance study.

Multiplex Reactions Central Nervous System Panel LOD
mL−1 		Precision True
+		 False
+		 True
-		 False
-
1A FAM Mycobacterium tuberculosis 72 96% 182
HEX Internal Control - -
ROX
CY5
1B FAM Listeria monocytogenes 68 96% 2
HEX
ROX Neisseria meningitidis 91 98% 2
CY5 Streptococcus pneumoniae 74 96% 13 6
1C FAM Haemophilus influenzae 71 96% 1
HEX
ROX Streptococcus agalactiae 59 98% 0
CY5 Escherichia coli K1 77 96% 1
1D FAM Cytomegalovirus 63 96% 1
HEX Enterovirus 81 96% 3 1
ROX Parechovirus 82 100% 1
CY5 Varicella Zoster Virus 79 98% 1
1E FAM Human Herpesvirus 6 91 96% 1
HEX
ROX Human Herpesvirus 7 66 96% 0
CY5 Human Herpesvirus 8 56 98% 0
1F FAM Herpes simplex virus 1 32 100% 1
HEX
ROX
CY5 Herpes simplex virus 2 24 98% 0
1G FAM Cryptococcus gattii 33 98% 0
HEX
ROX
CY5 Cryptococcus neoformans 57 98% 0
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Table 4.

Multiple targets in 8-well qPCR strips of Bio-Speedy® qPCR panel for bloodstream samples, and results of the clinical performance study.

Multiplex Reactions Sepsis Panel LOD
mL−1 		Precision True
+		 False
+		 True
-		 False
-
1A FAM Staphylococcus spp. 92 100% 39 5 397 25
HEX Internal control - - -
ROX Brucella spp. 86 96% 1
CY5 Listeria monocytogenes 88 96% 1
1B FAM Staphylococcus aureus 94 96% 4
HEX Candida albicans 98 96% 1
ROX vanA/vanB—Vancomycin resistance 52/68 96% 5
CY5 Candida krusei 68 98% 2
1C FAM Pseudomonas aeruginosa 42 100% 4
HEX Aspergillus/Fusarium/Trichosporon spp. 86/88/94 96%
ROX Klebsiella pneumoniae 82 96% 12 1
CY5 Acinetobacter baumannii 96 96% 6
1D FAM Haemophilus influenzae 64 96% 1
HEX Klebsiella oxytoca 94 96% 3
ROX Candida parapsilosis 68 98% 0
CY5 OXA-48—Carbapenem resistance 74 100% 3
1E FAM KPC—Carbapenem resistance 78 98% 1
HEX NDM—Carbapenem resistance 82 96% 5
ROX VIM—Carbapenem resistance 78 98% 1
CY5 IMP—Carbapenem resistance 92 96% 1
1F FAM mcr-1—Colistin resistance 88 96%
HEX Candida glabrata 86 96% 1
ROX mecA/mecC—Methicillin resistance 92/97 98% 1
CY5 Candida tropicalis 76 96% 4
1G FAM Enterococcus spp. 78 98% 8
HEX Pseudomonas spp. 84 96% 4
ROX Enterobacteriaceae 88 96% 29
CY5 Streptococcus spp. 92 98% 4
1H FAM OXA-23/51/58—Carbapenem resistance 78/62/66 98% 4
HEX Escherichia coli 68 100% 9 1
ROX Neisseria meningitidis 86 96% 0
CY5 Streptococcus pneumoniae 76 96% 2
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Table 5.

Multiple targets in 8-well qPCR strips of Bio-Speedy® qPCR panel for positive blood culture samples, and results of the clinical performance study.

Multiplex Reactions Sepsis Panel LOD
mL−1 		Precision True
+		 False
+		 True
-		 False
-
1A FAM Staphylococcus spp. 6 100% 60 3 401 4
HEX Internal control - - -
ROX Brucella spp. 8 100% 1
CY5 Listeria monocytogenes 6 100% 1
1B FAM Staphylococcus aureus 7 100% 4
HEX Candida albicans 6 100% 1
ROX vanA/vanB—Vancomycin resistance 6 100% 5
CY5 Candida krusei 7 100% 2
1C FAM Pseudomonas aeruginosa 4 100% 4
HEX Aspergillus/Fusarium/Trichosporon spp. 8/8/2009 100%
ROX Klebsiella pneumoniae 8 100% 12
CY5 Acinetobacter baumannii 8 100% 6
1D FAM Haemophilus influenzae 4 100% 1
HEX Klebsiella oxytoca 8 100% 3
ROX Candida parapsilosis 9 100% 0
CY5 OXA-48—Carbapenem resistance 6 100% 3
1E FAM KPC—Carbapenem resistance 6 100% 1
HEX NDM—Carbapenem resistance 5 100% 5
ROX VIM—Carbapenem resistance 6 100% 1
CY5 IMP—Carbapenem resistance 7 100% 1
1F FAM mcr-1—Colistin resistance 8 100%
HEX Candida glabrata 5 100% 1
ROX mecA/mecC—Methicillin resistance 6/8 100% 1
CY5 Candida tropicalis 7 100% 4
1G FAM Enterococcus spp. 9 100% 8
HEX Pseudomonas spp. 6 100% 4
ROX Enterobacteriaceae 5 100% 29
CY5 Streptococcus spp. 5 100% 4
1H FAM OXA-23/51/58—Carbapenem resistance 8/9/2005 100% 4
HEX Escherichia coli 7 100% 9
ROX Neisseria meningitidis 6 100% 0
CY5 Streptococcus pneumoniae 6 100% 2
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2.2. Reference Sample Preparation

Reference strains and clinical isolates from culture-confirmed cases were obtained from various collection sites: The Department of Microbiology Reference Laboratories and Biological Products of Public Health General Directorate (HSGM), Istanbul Medipol University (IMU), the Aziz Sancar Institute of Experimental Medicine Istanbul University (DETAE), Istanbul University-Cerrahpasa Medical Faculty (CTF), and Namik Kemal University (NKU). Additional reference strains/materials were purchased from the American Type Culture Collection (ATCC) or NIBSC.

Reference strains: Corynebacterium diphtheriae, Neisseria pharynges, Moraxella catarrhalis, Streptococcus mutans, Streptococcus agalactiae, Streptococcus pyogenes, Staphylococcus haemolyticus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus simulans, Staphylococcus capitis, Staphylococcus lugdunensis, Stenotrophomonas maltophilia, Brucella melitensis, Brucella abortus, Brucella suis, Cryptosporidium parvum, Cryptosporidium hominis, Salmonella enterica serovar Enteritidis, S. enterica serovar Typhimurium, S. enterica serovar Infantis, Campylobacter jejuni, Campylobacter coli, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Fusarium solani, Fusarium oxysporum, Fusarium fujikuroi, Trichosporon asahii, Trichosporon asteroides, Trichosporon cutaneum, Trichosporon inkin, Trichosporon mucoides, Trichosporon ovoides, Enterococcus faecalis, Enterococcus faecium, Pseudomonas maltophilia, Serratia marcescens, Proteus mirabilis, and Proteus vulgaris.

The vector DNAs carrying the target DNA fragments were used as qPCR quantification standards [5]. The vectors were synthesized by GenScript (860 Centennial Ave, Piscataway, NJ 08854, USA). The vector quantity was checked using a 2100 Bioanalyzer (5301 Stevens Creek Blvd. Santa Clara, CA 95051, USA). Standard curves were generated using qPCRs that contained vector DNA copies between 106 and 100, with quantification cycle (Cq) values between 10 and 40. The standard dilutions and the extracted nucleic acid samples were run in duplicate for the quantification.

For all the target and non-target strains, 100–105 genome/mL dilutions in phosphate-buffered saline (PBS) were prepared. Clinical samples confirmed as negative via culture/PCR were spiked with the dilutions in PBS to obtain reference samples. The concentrations were confirmed via qPCR quantification.

2.3. Analytical Sensitivity and Specificity as Well as Repeatability

To determine the limit of detection (LOD) in PBS, 100–104 genome/mL dilutions were tested 12 times in the same run. Clinical samples containing 0, 0.1xLOD, 0.5xLOD, LOD, 10xLOD were prepared. In order to evaluate repeatability (precision), each dilution was tested two times in the same run; two different operators performed same-day testing; the tests were repeated on three consecutive days. A total of 12 replicates of all the dilutions were tested. Probit analysis was carried out to empirically determine LOD in the clinical samples. The analytical specificity was determined by testing the suspensions of the target and non-target strains with concentrations between LOD and 105 genome/mL in PBS.

2.4. Clinical Sampling and Processing

The study was conducted in accordance with the Declaration of Helsinki and approved by the research ethics committees of Istanbul University (13/08.06.2014) and Istanbul Medipol University (122/17.12.2013; 187/12.08.2014).

The clinical specimens were collected at IMU, IU and NKU Hospitals. No restrictions were placed on age, gender, medications or known pharmaceutical therapies. Between December 2015 and April 2018, 1929 patients in the intensive care unit (ICU) (63.6%) and non-ICU settings were enrolled in the study. Samples were obtained from patients with suspected bloodstream (532), central nervous system (216), gastrointestinal (190) and respiratory (991) infections.

CSF, stool, oropharyngeal and nasopharyngeal swabs, nasopharyngeal wash/aspirate, sputum, and bronchoalveolar lavage samples were used for both the culture and molecular analyses. Blood specimens for molecular analysis were collected in EDTA blood tubes simultaneously whenever blood cultures were taken. From the signal-positive blood culture tubes, 500 µL samples were also analyzed using the Bio-Speedy® qPCR panel for the bloodstream infections.

Routine clinical microbiology protocols were applied as the gold standard for the detection of bacterial, fungal, and parasitic pathogens. FTD respiratory pathogen assays (Fast Track Diagnostics, Luxembourg), Allplex™ gastrointestinal and meningitis panel assays (Seegene, Seoul, Republic of Korea), and the qPCR protocols of the Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) were used as the gold standard for the detection of viral pathogens.

3. Results

No Bio-Speedy® multiplex qPCR panels produced positive results for the samples spiked with the potential cross-reacting pathogens. LOD and repeatability of the assays were in the range of 10–100 pathogens/mL and 96–100%, respectively (Table 1, Table 2, Table 3, Table 4 and Table 5).

In the respiratory panel, a total of 243 true positives and 393 true negatives were recorded for Influenza A virus, with only 8 false positives and 8 false negatives, yielding a sensitivity of 97.3% and specificity of 96.3% (Table 1). Similarly, the GI panel achieved a sensitivity of 94.3% and specificity of 97.9%, effectively detecting common pathogens such as Salmonella spp., Norovirus, and Clostridium difficile (Table 2). The CNS panel demonstrated high sensitivity (96.4%) and specificity (96.8%) across targets including Streptococcus pneumoniae, Herpes simplex virus, and Cryptococcus neoformans (Table 3). The blood and blood culture panels showed improved performance, with specificity of >98% for all tested targets and sensitivity ranging from 82.0% in whole blood to 97.1% in blood culture-positive samples (Table 4 and Table 5).

The analysis of the true +/− and false +/− results is presented in Table 1, Table 2, Table 3, Table 4 and Table 5. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the qPCR panels are shown in Table 6. The statistics of the single and co-infections are given in Table 7. The qPCR panels detected all the agents of co-infections. No co-infection was detected for the CNS samples. The statistics of the detected off-panel organisms are illustrated in Table 8. There was no detected off-panel organism for the gastrointestinal panel.

Table 6.

Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Bio-Speedy® qPCR panels.

Panel Sensitivity Specificity PPV NPV
Respiratory 97.3% 96.3% 97.5% 96.1%
Gastrointestinal 94.3% 97.9% 98.0% 94.1%
Central Nervous System 96.4% 96.8% 81.8% 99.5%
Blood 82.0% 98.3% 94.2% 94.1%
Blood culture 97.1% 99.3% 97.8% 99.0%
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Table 7.

Statistics of the single and co-infections.

Panel Co-Infection Single-Infection
Agent True + True + False -
Respiratory S. pneumoniae + RSV 6 15 567 16
S. pneumoniae + Rhinovirus 3
RSV + Parainfluenza 1 1
RSV + Adenovirus 1
RSV + Rhinovirus 1
Parainfluenza 1 + Bocavirus 1
Bocavirus + Rhinovirus 1
RSV + Parainfluenza 1 + Rhinovirus 1
Gastrointestinal Campylobacter spp. + Adenovirus 2 13 87 6
Salmonella spp. + Astrovirus 2
Rotavirus + Enteroaggregative E. coli 2
P. shigelloides + Norovirus (GI/GII) 2
Norovirus (GI/GII) + Enteroaggregative E. coli 2
Salmonella spp. + Adenovirus 1
Campylobacter spp. + Rotavirus 1
Enteroaggregative E. coli + Astrovirus 1
Blood A. baumannii + P. aeruginosa 1 11 103 25
A. baumannii + P. aeruginosa + Staphylococcus spp. 1
Enterococcus spp. + Staphylococcus spp. 1
Enterococcus spp. + K. pneumoniae 3
E. coli + K. pneumoniae 1
Enterobacteriaceae + K. pneumoniae 4
Blood culture Same as the blood agents 11 11 124 4
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Table 8.

Statistics of the detected off-panel organisms.

Panel Target Positives Off-Panel Agent % in the Culture Positives
Blood Stenotrophomonas maltophilia 2 4.3%
Micrococcus spp. 1
Corynebacterium striatum 1
Candida lusitaniae 1
Acinetobacter lwoffii 1
Respiratory Moraxella catarrhalis 7 1.2%
Central nervous system Treponema pallidum 1 7.1%
Brucella spp. 1
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We must emphasize that the summary statistics presented in the Abstract were calculated using the aggregated performance values from these tables, notably from the comparative analysis against culture and reference PCR methods. Furthermore, the qPCR panels were able to detect all reported co-infections (Table 7) and identified several off-panel organisms (Table 8), although the CNS panel did not detect any co-infections.

4. Discussion

The performance of the Bio-Speedy® qPCR syndromic testing panels was sufficient and proved effective in terms of the identification of the causative pathogens tested.

In fact, the platforms used in the molecular panels evaluated in our study have related pros and cons seen in semi-automated platforms compared with fully automated ones [BioFire Diagnostics [6], Luminex (now named DiaSorin [7]), Seegene [8], Fast Track Diagnostics (now owned by Siemens Healthineers [9]), GenMark Diagnostics (now named cobas eplex system [10]), and Aus Diagnostics [11]] (Table 9). The semi-automated systems allow microbiology laboratories to manage numerous samples, as opposed to automated platforms, which are merely for emergency and point-of-care diagnostics. The total assay duration is between 1 and 2.5 h for the fully automated platforms, and between 2.5 and 4.5 h for the semi-automated platforms. While fully automated systems provide quick assessments, the semi-automated platforms provide more tests per run with a much lower cost per sample [12]. Thus, screening of a wider range of pathogens per assay is feasible.

Table 9.

Bacterial/fungal/viral/parasitic (Bac/Fun/Vir/Par) targets and the other testing properties of commercial syndromic testing panels for respiratory (RP), gastrointestinal (GI), central nervous system (CNS), and blood and blood culture (BC) samples.

Brand Panel Sensitivity/Specificity Targets Duration Run Capacity per Instrument
Biofire RP 97.1%/99.3% 3Bac/17Vir 1–1.5 h Full automation 1 sample, 1 type of panel
GI 98.5%/99.2% 13Bac/5Vir/4Par
CNS 94.2%/99.8% 6Bac/7Vir/3Fun
BC 98%/99.9% 19Bac/5Fun/3Res
Luminex RP 95.2%/99.6% 2Bac/17Vir 3.54 h Semi-automation 24 samples, 1 type of panel
GI 94.3%/98.5% 9Bac/3Vir/3Par
BC 89.6–90.5%/98.9–100% 22Bac/9Res 2–2.5 h Full automation 1 sample, 1 type of panel
Seegene RP 82.8–100%/95.5–100% 7Bac/19Vir 2.5–3.5 h Semi-automation 8–10 samples, all types of panels
GI 93.3–100%/99.2–100% 14Bac/6Vir/5Par
CNS 100%/100% 6Bac/12Vir
Blood 29%/95% 24Bac/6Fun/3Res
Fast RP >98% 19Vir/12Bac 2.5–3.5 h Semi-automation 8–10 samples, all types of panels
GI >99.5% 6Vir/6Bac/3Par
CNS >97.8% 6Vir/3Bac
GenMark RP >97.4% 15Vir/2Bac 2 h Full automation 3 samples, 1 type of panel
BC >94% 41Bac/14Res/15Fun
AUS RP >93.5%/99.7% 9Vir/5Bac 4.5 h Semi-automation 24 samples, all types of panels
GI >94.7%/98.9% 6Bac/5Vir/3Par
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All the off-panel targets of the Bio-Speedy® are clinically relevant pathogens [13]. Corynebacterium spp., C. lusitaniae and Micrococcus spp. are only in GenMark’s sepsis panel. S. maltophilia is in the sepsis panels of GenMark and Seegene. Acinetobacter spp. is only in Luminex’s sepsis panel. M. catarrhalis is only in the respiratory panel of Fast Track Diagnostics. In contrast, T. pallidum and Brucella spp. are not in any of the CNS panels. When the prevalence and pathogen-specific treatment options of the off-panel microorganisms [13] were evaluated together, it was concluded that the exclusion of Candida spp., Acinetobacter spp., and S. maltophilia in the Bio-Speedy® sepsis panel is a drawback.

In the assessed method in our study, the sensitivity and specificity for blood culture (BC), CNS, GI, and respiratory samples were better than previously reported (Table 9). In fact, Seegene’s Magicplex™ Sepsis Real-time Test (MSR) [8], Roche’s LightCycler® SeptiFast Test (LSF) [14] and the Bio-Speedy® qPCR sepsis panel are the only commercially available options for direct pathogen screening in whole blood via qPCR. Highly variable diagnostic performance has been reported for the most widely studied LSF [15]. There are no considerable differences between the specificities of the three platforms. The sensitivity of MSR [16] is much lower than that of the Bio-Speedy® qPCR sepsis panel and LSF. Lengthy hands-on time (ranging from 5 to 7 h) impedes the LSF efficacy. Whole blood identification may complement BC, with better results in less than 3 h. Rapid detection of a causative pathogen of bacteremia and severe sepsis leads to immediate initiation of a proper antibiotic regimen, subsequently reducing complication rates and reducing healthcare costs [17,18,19,20].

The main limitations of our study are summarized in the following points: 1. The analysis was conducted using archived clinical samples collected in a single country, which may limit the generalizability of results to other geographic regions or healthcare settings. 2. Although the panels covered a broad range of pathogens, some clinically relevant microorganisms such as Epstein Barr virus and parvovirus B19 were not included in the CNS panel, as well as Candida spp., Acinetobacter spp., and S. maltophilia. 3. While the panels demonstrated strong analytical performance, some off-panel organisms were not detected, reflecting the inherent limitations of fixed multiplex designs. 4. The diagnostic comparison was limited to standard culture and reference PCR methods; metagenomic sequencing was not used to resolve discordant results.

5. Conclusions

The ability to simultaneously detect possible pathogens that contribute to the constellation of symptoms that patients could suffer from makes syndromic testing a valuable method for use in clinical practice. The number and variety of samples evaluated in a single run using Bio-Speedy® enable the rapid diagnosis of various and relevant infections. Such a method of testing, which aids in the timely diagnosis of infectious diseases, influences critical decisions regarding antimicrobial therapy, improves stewardship, and assists greatly in managing infections. Having said that, microorganisms such as Candida spp., Acinetobacter spp., and S. maltophilia, should be added to the panel to improve detection.

Author Contributions

M.Y.: conceptualization, writing—review and editing; S.K.: investigation, writing—original draft; F.B.: investigation, writing—original draft; S.N.Ö.: investigation, writing—original draft; A.I.T.: investigation, writing—original draft; U.Z.: investigation, writing—original draft; F.Ç.: investigation, writing—original draft; G.A.: investigation, writing—original draft; B.S.: investigation, writing—original draft; N.M.: supervision, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the research ethics committees of Istanbul University (13/08.06.2014) on 8 June 2014 and Istanbul Medipol University (122/17.12.2013; 187/12.08.2014) on 12 August 2014.

Informed Consent Statement

Informed consent was not required for this study because all molecular analyses were performed on re-identified, archived clinical specimens that had been previously collected for routine diagnostic purposes. No personal identifiers or patient-related clinical data were used in the analysis. The use of stored, anonymized samples for method validation and assay performance evaluation is consistent with institutional ethical guidelines and international standards for biomedical research. The study protocol was approved by the ethics committees of Istanbul University and Istanbul Medipol University, which waived the requirement for informed consent due to the non-interventional nature of the research.

Data Availability Statement

Data regarding molecular testing are presented in the tables of the paper. Additional data are available from the authors. Due to privacy and ethical restrictions other data are not publicly available.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research received no external funding.

Footnotes

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References

  • 1.Dien Bard J., McElvania E. Panels and Syndromic Testing in Clinical Microbiology. Clin. Lab. Med. 2020;40:393–420. doi: 10.1016/j.cll.2020.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Dumkow L.E., Worden L.J., Rao S.N. Syndromic diagnostic testing: A new way to approach patient care in the treatment of infectious diseases. J. Antimicrob. Chemother. 2021;76((Suppl. S3)):iii4–iii11. doi: 10.1093/jac/dkab245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ramanan P., Bryson A.L., Binnicker M.J., Pritt B.S., Patel R. Syndromic Panel-Based Testing in Clinical Microbiology. Clin. Microbiol. Rev. 2018;31:e00024-17. doi: 10.1128/CMR.00024-17. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Kralik P., Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front. Microbiol. 2017;8:108. doi: 10.3389/fmicb.2017.00108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Oldham A.L., Duncan K.E. Similar gene estimates from circular and linear standards in quantitative PCR analyses using the prokaryotic 16S rRNA gene as a model. PLoS ONE. 2012;7:e51931. doi: 10.1371/journal.pone.0051931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Biomerieux BioFire® FilmArray® Panels 2025. [(accessed on 20 January 2025)]. Available online: https://www.biofiredx.com/products/the-filmarray-panels/
  • 7.DiaSorin Infectious Diseases 2025. [(accessed on 20 January 2025)]. Available online: https://int.diasorin.com/en/immunodiagnostics/infectious-diseases.
  • 8.Seegene Seegene Products 2025. [(accessed on 20 January 2025)]. Available online: https://www.seegene.com/product.
  • 9.Siemens Healthineers Molecular Diagnostics 2025. [(accessed on 20 January 2025)]. Available online: https://www.siemens-healthineers.com/tr/about.
  • 10.Roche Diagnostics cobas® Eplex System 2025. [(accessed on 20 January 2025)]. Available online: https://diagnostics.roche.com/us/en/products/systems/eplex-system-sys-356.html.
  • 11.Ausdiagnostics Reliable Syndromic Testing 2025. [(accessed on 20 January 2025)]. Available online: https://www.ausdiagnostics.com/multiplexed-diagnostics/
  • 12.Laakso S., Maki M. Assessment of a semi-automated protocol for multiplex analysis of sepsis-causing bacteria with spiked whole blood samples. Microbiologyopen. 2013;2:284–292. doi: 10.1002/mbo3.69. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Carroll K.C., Pfaller M.A., Landry M.L., McAdam A.J., Patel R., Richter S.S., Warnock D.W. Manual of Clinical Microbiology. 12th ed. Volume 2 ASM Press; Washington, DC, USA: 2019. [Google Scholar]
  • 14.Roche Diagnostics Product Listing—LightCycler 2025. [(accessed on 20 January 2025)]. Available online: https://lifescience.roche.com/global/en/products/product-category/lightcycler.html.
  • 15.Dark P., Blackwood B., Gates S., McAuley D., Perkins G.D., McMullan R., Wilson C., Graham D., Timms K., Warhurst G. Accuracy of LightCycler® SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: A systematic review and meta-analysis. Intensive Care Med. 2015;41:21–33. doi: 10.1007/s00134-014-3553-8. [DOI] [PubMed] [Google Scholar]
  • 16.Zboromyrska Y., Cillóniz C., Cobos-Trigueros N., Almela M., Hurtado J.C., Vergara A., Mata C., Soriano A., Mensa J., Marco F., et al. Evaluation of the Magicplex Sepsis Real-Time Test for the Rapid Diagnosis of Bloodstream Infections in Adults. Front. Cell Infect. Microbiol. 2019;9:56. doi: 10.3389/fcimb.2019.00056. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Garnacho-Montero J., Gutierrez-Pizarraya A., Escoresca-Ortega A., Fernandez-Delgado E., Lopez-Sanchez J.M. Adequate antibiotic therapy prior to ICU admission in patients with severe sepsis and septic shock reduces hospital mortality. Crit. Care. 2015;19:302. doi: 10.1186/s13054-015-1000-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Luyt C.E., Brechot N., Trouillet J.L., Chastre J. Antibiotic stewardship in the intensive care unit. Crit. Care. 2014;18:480. doi: 10.1186/s13054-014-0480-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Perez K.K., Olsen R.J., Musick W.L., Cernoch P.L., Davis J.R., Land G.A., Peterson L.E., Musser J.M. Integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs. Arch. Pathol. Lab. Med. 2013;137:1247–1254. doi: 10.5858/arpa.2012-0651-OA. [DOI] [PubMed] [Google Scholar]
  • 20.Reinhart K., Bauer M., Riedemann N.C., Hartog C.S. New approaches to sepsis: Molecular diagnostics and biomarkers. Clin. Microbiol. Rev. 2012;25:609–634. doi: 10.1128/CMR.00016-12. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

Data regarding molecular testing are presented in the tables of the paper. Additional data are available from the authors. Due to privacy and ethical restrictions other data are not publicly available.

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