Syndromic Testing—The Evaluation of Four Novel Multiplex Real-Time PCR Panels
by
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 and
Naim Mahroum
1,* 1
Infectious Diseases and Clinical Microbiology, International School of Medicine, Istanbul Medipol University, Istanbul 34810, Turkey
2
Public Health General Directorate, Department of Microbiology Reference Laboratories and Biological Products, Ankara 06430, Turkey
3
Department of Clinical Microbiology, School of Medicine, Istanbul Medipol University, Istanbul 34810, Turkey
4
Aziz Sancar Institute of Experimental Medicine, Istanbul University, Istanbul 34093, Turkey
5
Infectious Diseases and Clinical Microbiology, Cerrahpasa Medical Faculty, Istanbul University, Istanbul 34810, Turkey
6
Department of Clinical Microbiology, Faculty of Medicine, Namik Kemal University, Tekirdag 59030, Turkey
*
Author to whom correspondence should be addressed.
Diagnostics 2025, 15(10), 1228; https://doi.org/10.3390/diagnostics15101228
Submission received: 13 March 2025
/
Revised: 1 May 2025
/
Accepted: 4 May 2025
/
Published: 13 May 2025
(This article belongs to the Section Pathology and Molecular Diagnostics)
Abstract
:Background: If used in the right clinical context, PCR carries great potential in rapidly diagnosing various infectious diseases. Objectives: To evaluate the clinical performance of four novel multiplex real-time PCR (qPCR) assays in 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 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, Istanbul Technical University Ari-3 Technopark 34475, Istanbul, Turkey), and LightCycler® 96 Instrument (400 Summer Street Boston MA 02210. USA) were used for all molecular testing. Results: All qPCR assays did not produce positive results for the samples spiked with the potential cross-reacting pathogens. Limit of detection (LOD) of the assays changed between 10 and 100 pathogens/mL-sample based on target and sample type. 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 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 health care providers to rapidly and efficiently diagnose and treat various infections.
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 the 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, 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 rapid application of directed treatment, halt the misuse of antimicrobials or 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, resources, and enhances the efficacy of microbiological labs 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 syndromic testing is expected to continue rising in terms of use and application, understanding the performance characteristics and limitations of multiplex assays is crucial. Therefore, we aimed hereby by our current study 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.
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), 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, and 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, 0.1 × LOD, 0.5 × LOD, LOD, 10 × LOD 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 in EDTA blood tubes for molecular analysis were collected 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 pathogens assays (Fast Track Diagnostics, Luxembourg), Allplex™ gastrointestinal and meningitis panel assays (Seegene, Seoul, Republic of Korea), and the qPCR-protocols of 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
All the Bio-Speedy® multiplex qPCR panels did not produce 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 eight false positives and eight 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. 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 were given in Table 7. The qPCR panels detected all the agents of co-infections. No co-infection 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.
It is necessary to point out 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 efficacy 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 quick assessment is provided by fully automated systems, the semi-automated platforms provide more testing per run with much lower costs per sample [12]. Thus, screening of a wider range of pathogens per assay is feasible.
All the off-panel targets of the Bio-Speedy® are clinically relevant pathogens [13]. Corynebacterium spp., C. lusitaniae and Micrococcus spp. are only in the GenMark’s sepsis panel. S. maltophilia is in the sepsis panels of GenMark and Seegene. Acinetobacter spp. is only in the Luminex’s sepsis panel. M. catarrhalis is only in the respiratory panel of Fast Track Diagnostics. Whereas 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 not any 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 5–7 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 contributing to the constellation of symptoms patients could suffer from makes syndromic testing a valuable method in clinical practice. The number and variety of samples evaluated in a single run using the Bio-Speedy® enables 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, among others, 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.
Funding
This research received no external funding.
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 de-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 is presented in the tables of the paper. Additional data is available from the authors. Due to privacy and ethical restrictions other data is not publicly available.
Conflicts of Interest
The authors declare no conflict of interests.
References
- Dien Bard, J.; McElvania, E. Panels and Syndromic Testing in Clinical Microbiology. Clin. Lab. Med. 2020, 40, 393–420. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- Kralik, P.; Ricchi, M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front. Microbiol. 2017, 8, 108. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- Biomerieux. BioFire® FilmArray® Panels 2025. Available online: https://www.biofiredx.com/products/the-filmarray-panels/ (accessed on 20 January 2025).
- DiaSorin. Infectious Diseases 2025. Available online: https://int.diasorin.com/en/immunodiagnostics/infectious-diseases (accessed on 20 January 2025).
- Seegene. Seegene Products 2025. Available online: https://www.seegene.com/product (accessed on 20 January 2025).
- Siemens Healthineers. Molecular Diagnostics 2025. Available online: https://www.siemens-healthineers.com/tr/about (accessed on 20 January 2025).
- Roche Diagnostics. cobas® Eplex System 2025. Available online: https://diagnostics.roche.com/us/en/products/systems/eplex-system-sys-356.html (accessed on 20 January 2025).
- Ausdiagnostics. Reliable Syndromic Testing 2025. Available online: https://www.ausdiagnostics.com/multiplexed-diagnostics/ (accessed on 20 January 2025).
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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.; ASM Press: Washington, DC, USA, 2019; Volume 2. [Google Scholar]
- Roche Diagnostics. Product Listing—LightCycler 2025. Available online: https://lifescience.roche.com/global/en/products/product-category/lightcycler.html (accessed on 20 January 2025).
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- Luyt, C.E.; Brechot, N.; Trouillet, J.L.; Chastre, J. Antibiotic stewardship in the intensive care unit. Crit. Care 2014, 18, 480. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
- 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. [Google Scholar] [CrossRef] [PubMed]
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.
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 | ||||
Table 2.
Multiple targets in 8-well qPCR strips of Bio-Speedy® qPCR panel for gastrointestinal samples, and results of the clinical performance study.
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 kontrol | - | - | |||||
| 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% | |||||
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.
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 | ||||
Table 4.
Multiple targets in 8-well qPCR strips of Bio-Speedy® qPCR panel for bloodstream samples, and results of the clinical performance study.
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 | ||||
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.
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 | ||||
Table 6.
Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the Bio-Speedy® qPCR panels.
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% |
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 |
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 |
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), blood and blood culture (BC) samples.
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), 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 panel |
| GI | 93.3–100%/99.2–100% | 14Bac/6Vir/5Par | ||||
| CNS | 100%/100% | 6Bac/12Vir | ||||
| Blood | 29%/95% | 24Bac/6Fun/3Res | ||||
| Fast track diagnostics | RP | >98% | 19Vir/12Bac | 2.5–3.5 h | Semi-automation | 8–10 samples, all types of panel |
| 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 panel |
| GI | >94.7%/98.9% | 6Bac/5Vir/3Par | ||||
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
MDPI and ACS Style
Yılmaz, M.; Kilic, S.; Bayrakdar, F.; Ötgün, S.N.; Tosun, A.I.; Zeybek, U.; Çelik, F.; Aygun, G.; Safak, B.; Mahroum, N. Syndromic Testing—The Evaluation of Four Novel Multiplex Real-Time PCR Panels. Diagnostics 2025, 15, 1228. https://doi.org/10.3390/diagnostics15101228
AMA Style
Yılmaz M, Kilic S, Bayrakdar F, Ötgün SN, Tosun AI, Zeybek U, Çelik F, Aygun G, Safak B, Mahroum N. Syndromic Testing—The Evaluation of Four Novel Multiplex Real-Time PCR Panels. Diagnostics. 2025; 15(10):1228. https://doi.org/10.3390/diagnostics15101228
Chicago/Turabian StyleYılmaz, Mesut, Selcuk Kilic, Fatma Bayrakdar, Selin Nar Ötgün, Ayse Istanbullu Tosun, Umit Zeybek, Faruk Çelik, Gokhan Aygun, Birol Safak, and Naim Mahroum. 2025. "Syndromic Testing—The Evaluation of Four Novel Multiplex Real-Time PCR Panels" Diagnostics 15, no. 10: 1228. https://doi.org/10.3390/diagnostics15101228
APA StyleYılmaz, M., Kilic, S., Bayrakdar, F., Ötgün, S. N., Tosun, A. I., Zeybek, U., Çelik, F., Aygun, G., Safak, B., & Mahroum, N. (2025). Syndromic Testing—The Evaluation of Four Novel Multiplex Real-Time PCR Panels. Diagnostics, 15(10), 1228. https://doi.org/10.3390/diagnostics15101228
Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.
