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Multiplex Molecular Point-of-Care Test for Syndromic Infectious Diseases

Multiplex Molecular Point-of-Care Test for Syndromic Infectious Diseases Point-of-care (POC) molecular diagnostics for clinical microbiology and virology has primarily focused on the detection of a single pathogen. More recently, it has transitioned into a comprehensive syndromic approach that employs multiplex capabilities, including the simultaneous detection of two or more pathogens. Multiplex POC tests provide higher accuracy to for actionable decisionmaking in critical care, which leads to pathogen-specific treatment and standardized usages of anti- biotics that help prevent unnecessary processes. In addition, these tests can be simple enough to operate at the primary care level and in remote settings where there is no laboratory infrastructure. This review focuses on state-of-the-art multiplexed molecular point-of-care tests (POCT) for infectious diseases and efforts to overcome their limitations, especially related to inadequate throughput for the identification of syndromic diseases. We also discuss promising and imperative clinical POC approaches, as well as the possible hurdles of their practical applications as front-line diagnostic tests. Keywords Multiplex molecular point-of-care testing · Syndromic infectious disease · Molecular diagnosis · POCT 1 Introduction A personalized medicine and treatment regimen, which establishes clinical plans on a patient-by-patient basis in the treatment of infectious diseases, is a trending topic in the field of clinical microbiology and virology [1 –5]. In * Doo-Ryeon Chung general, patients with infectious illness present common dr.chung@samsung.com symptoms. Symptoms including fever, cough, vomiting, * Minhee Kang abdominal pain, myalgia, and headache are often not suf- minhee.kang@samsung.com ficiently specific to differentiate the exact etiology for an Biomedical Engineering Research Center, Smart Healthcare infectious disease. In the paradigm of personalized medi- Research Institute, Samsung Medical Center, Seoul 06351, cine, the sample in and answer out approach of point-of-care South Korea (POC) diagnostics has the potential to empower physicians Department of Medical Device Management and Research, with the ability to make early evidence-based treatment deci- SAIHST (Samsung Advanced Institute for Health Sciences sions so that the right medication can be administered to the & Technology), Sungkyunkwan University, Seoul 06355, South Korea patient earlier, which can improve the prognosis. In fact, the treatment of infectious diseases are rarely considered to Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School be model applications of personalized medicine; however, of Medicine, Seoul 06351, South Korea this perception is gradually changing due to a substantial Center for Infection Prevention and Control, Samsung increase in antimicrobial resistance (AMR) to antibiotics Medical Center, Seoul 06351, South Korea due to unnecessary usage [6–9]. Since experience-based Asia Pacific Foundation for Infectious Diseases (APFID), empiric treatment is a possible driver of over-broad and Seoul 06367, South Korea unnecessary antibiotic usage, rapid POC diagnostic testing Division of Infectious Diseases, Department of Internal has become a promising solution to this problem. Molecular Medicine, Samsung Medical Center, Sungkyunkwan tests, such as polymerase chain reaction (PCR) and other University School of Medicine, Seoul 06351, South Korea Vol:.(1234567890) 1 3 BioChip Journal (2021) 15:14–22 15 nucleic acid-based amplification technologies (NAATs), [22]. Infectious disease MDx requires especially careful have gradually replaced or augmented traditional laboratory handling of specimens and test procedures to prevent the techniques for pathogen identification in the form of POC spread of viral molecules and aerosol from the sample and testing, because these tests can detect fastidious or unculti- cross-contamination in the community [23, 24]. Infectious vable microorganisms that indicate possible poly-microbial disease MDx POCT is also lacking in the number of patho- infection [10–12]. The successful migration of molecular gens that can be identified at once and has insufficient phe- diagnostics from universal laboratories to the clinical set- notypic information on pathogens. Fortunately, many studies ting. could dramatically improve the accuracy and sensi- have recently highlighted the significance of the multiplex tivity of molecular POC testing (MDx POCT) [13–17]. In capability of molecular POCT, because it provides rapid addition, it could lead to pathogen detection derived from a and high-accuracy answers that support quicker therapeu- small amount of specimen, with delivery of results in just a tic decisions for the individual and at the public level [24]. few hours [13, 15, 17, 18]. Those rapid-throughput results Ultimately, multiplexed molecular POCT leads to pathogen- would give providers confidence in prescribing the proper specific treatment and the use of narrow-spectrum antibiotics treatment (Fig.  1). Ultimately, exclusive molecular POC instead of excessive use of broad-spectrum antibiotics. technologies may be implemented in the central care method This review focuses on up-to-date progress in multiplexed in limited-resource settings. molecular POCT for infectious diseases and efforts to over - Clinical microbiological and virological POC molecu- come limitations. We have summarized information on lar diagnostics have initially addressed the optimization of recently approved devices and state-of-the-art developments technologies from centralized laboratories to the medical through a comprehensive literature review. We also discuss checkup field, regardless of the number of detectable patho- the potential clinical impact of the recent POC approach and gens [19]. The field has now expanded to encompass a com- the possible hurdles that impede their practical application prehensive syndromic approach that simultaneously detects as a leading diagnostic test. several similar pathogens. A few molecular POC tests exploit the early diagnosis of frequent infections, such as 1.1 Multiplex Molecular POC Testing: The Next influenza [20] and healthcare-associated infections (HAIs) Phase of Molecular Diagnostics caused by S. aureus, C. difficile, enterococci or multi-resist- ant enterobacteria [21]. However, innovative multiplexed The implementation of NAATs has been a huge leap for- molecular POC testing has yet to be applied to infectious ward in test sensitivity and time-to-results. There are sev- diseases because of the complex sample preparation required eral molecular-based assays that meet the criteria for POC and the low throughput diagnosis. For instance, molecular application and have been approved or are clinically avail- diagnostics (MDx) involves numerous steps and reagents able for the detection of single-key pathogens, such as influ- for sample preparation, including extractions and the puri- enza, C. difficile, or methicillin-resistant Staphylococcus fication of nucleic acids from specimens, amplification of a aureus (MRSA). The first molecular test designed for POC specific sequence, and confirmation of the target molecules was approved by the U.S. Food and Drug Administration Fig. 1 Clinical diagnosis of infection according to a diagnostic platform. The implementation of a multiplex point-of-care (POC) test reduces diagnostic cycles for faster and better treatment decisions 1 3 16 BioChip Journal (2021) 15:14–22 (FDA) in 2015. ID NOW™ Influenza A & B 2, formerly targets. The microfluidic pouch system connects to a module known as Alere I influenza A & B 2, (Alere Scarborough for DNA extraction and purification and employs a post-PCR Inc., Scarborough, ME, USA), provides highly sensitive melting curve analysis that differentiates targets based on results in 13 min or less. It is based on isothermal nucleic the distinct melting temperature (T ) peaks for each target. acid amplification technology, which uses nicking enzyme This allows for the multiplexed detection of multiple targets. amplification reaction (NEAR) technology. As many emerg- Another example is the FDA-cleared Verigene (Nanosphere, ing isothermal amplified POC applications have reached Northbrook, IL, USA) system, which that uses gold nanopar- the proof-of-concept stage, ID NOW™ has successfully ticle probes and a microarray for the detection of bacterial entered the infectious disease POC market for influenza A pathogens and several resistance markers. However, neither & B, Strep A and respiratory syncytial virus (RSV) test- platform is classified as a POC test. ing [25–27]. ID NOW™, cobas Liat influenza A/B/RSV assays (Roche Molecular Systems, Pleasanton, CA, USA) 1.2 Recent Multiplex Molecular POC Testing and GeneXpert Xpress Flu/RSV (Cepheid, Sunnyvale, for the Broad Detection of pathogens: The Way CA, USA) are known as Clinical Laboratory Improvement Forward Amendments (CLIA)-waived (CW) assays that enable non- training personnel to obtain results within 30–50 min and are Microfluidic devices for molecular diagnostics have been suitable for POCT [28]. However, ID NOW™ using NEAR less commercialized over the last few years compared to the technology is limited to the simultaneous detection and dis- immense academic interest focusing on the miniaturization crimination of multiple targets, which means that it includes of molecular biological techniques, including target gene individual assay panels for either influenza or RSV rather amplification onto microfluidic devices. However, there is than a multiplexed test, whereas cobas® Liat influenza A/B/ an increasing need to integrate microfluidic devices with RSV assays and GeneXper t Xpress Flu/RSV are based on other applications, such as sample pre-treatment, fluid con- real-time reverse transcription-polymerase chain reaction trol, and multiplex capability (Fig.  2). For example, Liu (rtRT-PCR), which can detect both influenza A/B and RSV et al. reported a microfluidic cassette that is able to capture, during a single run [29–31]. The binx io (binx Health Diag- concentrate, amplify, and optically detect the Zika virus nostic, Boston, MA, USA), which combines PCR amplifica- (ZIKV) in a single sample-to-answer chip [36]. They have tion and electrochemical detection technology, has recently established a silica-based nucleic acid isolation membrane received 510(k) clearance from the U.S. FDA for the dual in a microchannel and thermally insulated a portable heat- targets of chlamydia (CT) and gonorrhea (NG) [32, 33]. ing cup for PCR reactions in an emergency. Even though These above-mentioned platforms have adopted single-use the POC molecular test is composed of a modest set with a test units for sample preparation; nucleic acid extraction, chip and heater, the obtained result was reasonable, with a purification and amplification, and the detection of biomark - high sensitivity of 5 PFU ZIKV per sample within 40 min. ers. While the laboratories have consistently demonstrated In accordance with the emergence of nucleic acid amplifi - that the upgraded test kits have added target molecules, there cation technologies, multiplex microfluidics have also been are still significant challenges for the robust deployment of developed with the goal of eliminating the heating cycle molecular POC systems; in particular, there is relatively low apparatus. Lee et  al. demonstrated a self-powered inte- throughput in terms of simultaneous detection of copious grated microfluidic POC low-cost enabling (SIMPLE) chip pathogens. This limitation mainly arises from interference [37]. It contains an isothermal recombinase polymerase between multiple sets of primers and optical sources for tar- amplification initiator patterning on the well of the chan- get analysis. In detail, a large number of primers are required nel, autonomous plasma separation into 224 microwells for for simultaneous multi-pathogen detection, which results in digital nucleic acid amplification, and a vacuum battery on preferential amplification between co-amplified genes and the chip. The integrated options have been developed as a non-specific amplifications, such as primer dimer and mis- lab-on-a-chip for molecular testing that automatically pre- priming artifacts [34]. Moreover, the optics of current ana- pares plasma from whole blood into hundreds of microwells, lytical instruments and sequencing probes (e.g., fluorescence directly amplifies the independent target in every well due to label) bring a limited number of targets to deflect from an micropatterning of the initiator, and can run without a power overlapping wavelength in a single reaction and laser source source or external pumps. The SIMPLE chip also detects [35]. The novel design of probe formats and combination Staphylococcus aureus DNA from human blood samples in of microfluidic technologies has been proposed to be the 30 min with separation, amplification, and digital quantita- solution for improved simultaneous detection of a larger tive nucleic acid readouts. The result simplifies molecular number of targets. For instance, FilmArray (BioFire, Salt testing, making it portable, and available for rapid medi- Lake City, UT, USA) exemplifies this idea of a technical cal decisionmaking. On the other hand, NAATs that utilize breakthrough enabling simultaneous detection of multiple paper substrate matrices provide a low-cost, easy-to-store, 1 3 BioChip Journal (2021) 15:14–22 17 Fig. 2 Integrated molecular diagnostic assay-based microfluidic Sci Adv 2017, 3 (3), e1501645 under open access license CC BY-NC, devices. a SIMPLE chip for blood sample prep, simultaneous digital reprinted with permission from AAAS/b Reprinted from Lee, N. Y. amplification, and quantitative nucleic acid testing with minimal han- et  al., Sens. Actuators B Chem 2020, 314, 128,057. Copyright by dling and no-power source. b A foldable all-in-one microdevice for Elsevier/ (C) From Lee, H. et  al., Sci Adv 2016, 2 (5), e1600300 the entire process of multiplex pathogen molecular test by a folding under open access license CC BY-NC, Reprinted with permission and stacking motion. c Polarized anisotropy diagnostics (PAD) sys- AAAS) tem for the detection of bacterial total RNA. (a From Lee, L.P. et al., and portable platform. Klapperich’s et al. developed a single (HPV) DNA directly from cervical specimens in less than paper-fluidic-chip-based lateral flow assay, which allows for 1 h with an integrated on-chip assay that produces an imme- the completion of NAAT steps from sample to result [38]. diate visual readout. The device drives the isolation of DNA with reagents on the Numerous studies have highlighted the importance of chip, followed by isothermal loop-mediated amplification a syndromic approach in a multiplex molecular diagnostic and a pump-free immunochromatographic assay. All pro- test [39–41]. A syndromic approach in molecular diagno- cesses are operated on paper, which passively move fluids sis achieves the simultaneous detection of genetic material through wicking, capillary force, and sample handling. The from pathogens of different species and even different taxo- authors have detected 16 subtypes of human papillomavirus nomic levels in a single test. For a comprehensive group 1 3 18 BioChip Journal (2021) 15:14–22 of pathogens that could cause a specific syndrome, such the GenePOC technology platform (Meridian Bioscience, as food poisoning or an upper respiratory infection, it may Inc., Quebec, Canada) integrates a microfluidic centripetal help effectively manage outbreaks with seasonal pathogen device to provide molecular diagnostic technologies in POC screening and detect community- and healthcare-associated [48, 49]. Within 1 h and with less than 1 min of hands-on outbreaks [42, 43]. Lee et al. presented a foldable all-in-one time, the GenePOC platform enables fully automated nucleic microdevice for simultaneous detection of three foodborne acid-based testing for infectious microorganisms. Overall, pathogens, Salmonella spp., Staphylococcus aureus, and it can process a wide range of clinical samples with up to Escherichia coli O157:H7, and a multi-drug-resistant bac- 12 targets. Culture-independent direct pathogen detection teria Acinetobacter baumannii [44]. The platform is feasible approaches are included in most advanced technologies, for accomplishing PCR preparation, amplification, and mul- such as Abbott’s IRIDICA and T2 Biosystems’ magnetic tiplex optical detection of pathogens in less than 2 h. The resonance technology, which are capable of detecting many thin polymethyl methacrylate (PMMA)-molded microdevice microorganisms. Abbott’s IRIDICA combines a set of consists of PCR reagent pre-stored filter paper disks and broad-range PCRs and electrospray ionization-mass spec- Flinders Technology Associates cards for cell lysis, protein trometry (PCR/ESI–MS). Herein, the PCRs amplify gene denaturation, and DNA extraction. Consequently, adding sil- encoding 16S ribosomal RNA and detect the housekeeping ver nitrate results in the multiplex pathogen detection system gene region. Otherwise, T2 relies on changes in a sample’s for direct colorimetric detection without an expert analytical T2 magnetic resonance (T2MR ) signal, which is caused tool. To quantify the bacterial cell, Mu et al. reported an by hybridization of the PCR-amplified pathogen DNA and integrated multiplex digital recombinase polymerase ampli- the capture of probe-decorated nanoparticles [50]. The T2 fication (imdRPA) microfluidic chip, which enables to detect system requires 1 mL uncultured whole blood and provides 1 × 10 foodborne bacterial cells of each species within results in approximately 3 h, with a claimed limit of detec- 45 min [45]. They adopted a magnetic bead-based DNA tion as low as 1 colony-forming unit (CFU)/ml. However, extraction component with a digital RPA region including this assay has currently been confirmed to detect only the 12,800 chambers for simultaneous detection of the targets five most common Candida species (which account for 95% and control, and then quantitatively analyzed the multiplex of candidemia) but is able to provide antifungal susceptibil- foodborne pathogen from the fluorescence signal of the ity data [51]. Due to growing demand for multiplex molecu- DNA probe. These microdevices are highly suitable for the lar POC testing, major companies in the in vitro diagnostics detection of numerous pathogens by increasing the number (IVD) market are focused on pipeline expansion and fortify- of channels in a chip for a syndromic diagnosis approach. ing their partnerships. For instance, the molecular diagnostic Among various emerging breakthrough technologies, Park company Qiagen (Hilden, Germany) has agreed to acquire et al. validated a new detection system based on polarization Stat-Dx (Barcelona, Spain), which has developed a fully anisotropy diagnostics (PAD), which measures changes in integrated one-step molecular test for common syndromes. fluorescence anisotropy when detection probes recognize the QIAstat-Dx (formerly Stat-Dx DiagCORE ) is a multiplex nucleic acid of the target bacteria [46]. This assay supports molecular diagnostic system that enables fast, cost-effective, a universal capture key that targets a conserved region of and flexible syndromic testing. The system has received 16S rRNA in different bacterial species. PAD was applied CE-IVD certification for a respiratory panel that detects 21 on-site in clinical HAI diagnostics and achieved an accuracy pathogens and will be able to process up to 48 molecular comparable to that of bacterial culture, and with a shorter targets at once, which will permit the diagnosis of serious turnaround time (~ 2 h). One of the commercial molecular respiratory and gastrointestinal infections. POCs, an STD Direct Flow Chip Kit (Master Diagnóstica, Granada, Spain), was evaluated for the detection of up to 1.3 Persistent Challenges nine different pathogen species of sexually transmitted dis- eases (STDs) from clinical specimens in a single reaction 47. A few years ago, molecular POC diagnosis was limited The kit achieves molecular diagnosis based on a multiplex to testing only the most common pathogens. This lack in PCR and automatic hybridization onto a microarray with diagnostics capability led to further downstream tests or specific oligo probes and broad clinical specimens while unnecessary prescription of antibiotics. The recently com- avoiding DNA purification steps. From a total of 633 speci- mercialized multiplex molecular assays enable simultaneous mens, the direct PCR-analysis results were 98.4% and 99.9% detection and identification of multiple pathogens associ - for sensitivity and specificity, respectively. ated with clinical syndromes with regard to bloodstream, Numerous studies involving multiplex molecular POC are respiratory, gastrointestinal (GI), or central nervous sys- now moving toward extensive diagnosis capability, but few tem infections [40]. As the demand for syndromic testing kits have been approved for the detection of multiple patho- in molecular diagnostics increases, there is an ongoing gens based on clinical syndromes (Table 1). For example, debate about whether these tests will be applied as front-line 1 3 BioChip Journal (2021) 15:14–22 19 Table 1 Analytical spectrum of molecular near-patient and point-of-care testing for infectious diseases System Multiplexity Virology Microbiology ID NOW™ Single FLU A/B, RSV, GAS Abbott SARS-CoV-2 ® ® cobas Liat Single FLU A/B GAS, CDI Roche Multiple FLU A/B + RSV GeneXpert Omni Single EBO, EV, FLU A/B, HPV, HBV, HCV, HIV, SARS-CoV-2* GAS, TV, CT, GBS, CDI, Cepheid CARBA-R, MRSA, NORO, VRE Multiple FLU A/B + RSV MTB + RIF, CT + NG, MRSA + SA binx io Single HPV, HIV, CT, NG, TV, TP, MG binx health, inc HCV, HBV, HSV-2 Multiple CT + NG, CT + NG + TV, CT + NG + TV + MG FilmArray Multiple Respiratory Panel: 18 viruses, 4 bacteria BioMerieux Blood Culture Identification 2 (BCID2) Panel: 26 bacteria, 7 yeast, 10 antibiotic resistance genes Gastrointestinal (GI) Panel: 5 viruses, 7 bacteria, 6 Diarrheagenic E. coli/Shigella, 4 parasites Meningitis-Encephalitis (ME) Panel: 7 viruses, 6 bacteria, 1 yeast Pneumonia plus Panel: 18 bacteria, 7 antibiotic resistance genes, 9 viruses Verigene Nanosphere Single CDI Multiple Enteric Pathogens Test: 2 viruses, 6 bacteria, 2 toxins Respiratory Pathogens Flex test: 13 viruses, 2 bacteria Blood Culture Nucleic Acid Testing Panels: 1. Gram-positive: 8 species, 1 group, 4 genus, 3 resistance markers 2. Gram-negative: 5 species, 4 genus, 6 resistance markers GenePOC Single CDI, GBS Meridian Bioscience, inc Q-POC Single HPV, MDR-TB QuantuMDx Multiple CT + NG QIAstat-Dx Multiple Respiratory Panel: 18 viral, 3 bacterial Qiagen pathogens Respiratory SARS-CoV-2 panel*: 19 viral pathogens including SARS-CoV-2, 3 bacterial pathogens Gastrointestinal Panel: 6 viral, 14 bacterial, 4 parasitic pathogens T2MR Single Candida T2 Biosystems Multiple ESKAPE FLU A/B Influenza A/B, RSV respiratory syncytial virus, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, GAS group A strepto- cocci, CDI Clostridium difficile, EBO Ebola virus, EV enterovirus, HPV human papillomavirus, HBV Hepatitis B virus, HCV Hepatitis C virus, HIV Human immunodeficiency virus, TV Trichomonas vaginalis, CT Chlamydia trachomatis, GBS group B streptococci, CARBA-R carbap- enem-resistant Enterobacteriaceae, MRSA methicillin-resistant Staphylococcus aureous, NORO Norovirus, VRE Vancomycin-resistant entero- cocci, MTB Mycobacterium tuberculosis, RIF Rifampicin resistance, NG Neisseria gonorrhoeae, SA Staphylococcus, HSV-2 Herpes simplex virus type two, TP Treponema pallidum, MG Mycoplasma genitalium, MDR-TB Multi-drug resistance tuberculosis, ESKAPE Enterococcus fae- cium/Staphylococcus aureus/Klebsiella pneumoniae/Pseudomonas aeruginosa/Escherichia coli For use under an Emergency-Use Authorization (EUA, 2020.09.24) Not POC diagnostics techniques for all patients or will be limited to to confirm the clinical syndromes, it may be ineffective cost- specific patients and regions [39, 52]. Even if multiplex test wise to test all specimens. Thus, syndromic testing must be menus, i.e., combinations of pathogens, are properly linked customizable and personalized for patient-specific needs to 1 3 20 BioChip Journal (2021) 15:14–22 exclude over-testing and excess patient medical bills. Conse- practice, such as moderate test performance, complexity quently, an optimized technical model should be applied in that make it difficult to use in many clinical settings, limited the development of POC tests using a syndromic approach. access to testing, and high costs. Considering that count- In addition, incorporating multiplex molecular POC tests in less researchers have encouraged finding a way to overcome clinical practice may encourage a change in the workflow of these challenges, we believe that multiplex molecular POC existing central laboratory tests. Currently, it is important to will become a powerful principal diagnostic test. consider whether multiplex MDx POCT would replace or be Acknowledgements This work was supported by the National conducted in addition to existing tests. For instance, many Research Foundation of Korea (NRF) grant funded by the Korean gov- studies have applied ultrafast thermocycling approaches that ernment (the Ministry of Science and ICT) [No. 2019R1A2C2087631, perform efficient PCR processes through rapid tempera- No. 2016M3A9B6919189, No. 2016M3A9B6919187], by the Korea ture control and a high transfer rate of heating and cool- Medical Device Development Fund grant funded by the Korea govern- ment (the Ministry of Science and ICT; the Ministry of Trade, Industry, ing, resulting in a small reaction volume and photophysical and Energy; the Ministry of Health & Welfare, Republic of Korea; the effect [11, 53, 54]. The developed factors consist of minia- Ministry of Food and Drug Safety) [No. 202011A04] and by Samsung turized thermocyclers for ultrafast PCR, including capillary Research Funding & Incubation Center of Samsung Electronics [No. tubes with air heating, a microfluidic device, and a photonic SRFC-IT1902-05]. microwave-based resistor-mediated or convective thermocy- Author Contributions MK and DRC designed and determined the cler [55–59]. In fact, the key prerequisite is that performance study concept and fully accessed all the data in the study and hold should be equal in terms of sensitivity and specificity when responsibility for the integrity of the data, accuracy of the data analysis, it is compared to tests that are conducted in a central labora- and obtained funding. MK, HK, HJH, and EP acquired the data. MK and HK analyzed and interpreted the data, drafted the manuscript, and tory. Another consideration is that it should be available to obtained administrative, technical, or material support. use at the primary care level, even in resource-limited set- tings. Although the sensitivity and specificity are similar to Compliance with Ethical Standards the gold standard method, performance should be achieved equally, even in a resource-constrained setting. When a com- Conflict of interest No potential conflicts of interest relevant to this prehensive test such as the multiplex MDx POC test is actu- article were reported. alized to administer medical checkup performance, it has the potential to become a dominant diagnostic test. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, 2 Conclusion provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated Evolving POC molecular diagnostics are closer to reali- otherwise in a credit line to the material. If material is not included in zation in the medical field. The successful migration of a the article’s Creative Commons licence and your intended use is not molecular POC test has changed the trends of centralized permitted by statutory regulation or exceeds the permitted use, you will laboratory testing from general molecular diagnostics with need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. highly skilled experts and complex equipment to the more comprehensive molecular diagnostics in a clinical set- ting using simpler instruments. The emergence of a new CLIA-waived molecular POC platform has received major References attention in the global in vitro diagnostics (IVD) market, because traditional POC tests, e.g., lateral flow, are com- 1. de la Fuente-Nunez, C., Torres, M.D., Mojica, F.J., Lu, T.K.: monly known to lack sensitivity. 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Multiplex Molecular Point-of-Care Test for Syndromic Infectious Diseases

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Springer Journals
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Copyright © The Korean BioChip Society 2021
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1976-0280
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2092-7843
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10.1007/s13206-021-00004-5
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Abstract

Point-of-care (POC) molecular diagnostics for clinical microbiology and virology has primarily focused on the detection of a single pathogen. More recently, it has transitioned into a comprehensive syndromic approach that employs multiplex capabilities, including the simultaneous detection of two or more pathogens. Multiplex POC tests provide higher accuracy to for actionable decisionmaking in critical care, which leads to pathogen-specific treatment and standardized usages of anti- biotics that help prevent unnecessary processes. In addition, these tests can be simple enough to operate at the primary care level and in remote settings where there is no laboratory infrastructure. This review focuses on state-of-the-art multiplexed molecular point-of-care tests (POCT) for infectious diseases and efforts to overcome their limitations, especially related to inadequate throughput for the identification of syndromic diseases. We also discuss promising and imperative clinical POC approaches, as well as the possible hurdles of their practical applications as front-line diagnostic tests. Keywords Multiplex molecular point-of-care testing · Syndromic infectious disease · Molecular diagnosis · POCT 1 Introduction A personalized medicine and treatment regimen, which establishes clinical plans on a patient-by-patient basis in the treatment of infectious diseases, is a trending topic in the field of clinical microbiology and virology [1 –5]. In * Doo-Ryeon Chung general, patients with infectious illness present common dr.chung@samsung.com symptoms. Symptoms including fever, cough, vomiting, * Minhee Kang abdominal pain, myalgia, and headache are often not suf- minhee.kang@samsung.com ficiently specific to differentiate the exact etiology for an Biomedical Engineering Research Center, Smart Healthcare infectious disease. In the paradigm of personalized medi- Research Institute, Samsung Medical Center, Seoul 06351, cine, the sample in and answer out approach of point-of-care South Korea (POC) diagnostics has the potential to empower physicians Department of Medical Device Management and Research, with the ability to make early evidence-based treatment deci- SAIHST (Samsung Advanced Institute for Health Sciences sions so that the right medication can be administered to the & Technology), Sungkyunkwan University, Seoul 06355, South Korea patient earlier, which can improve the prognosis. In fact, the treatment of infectious diseases are rarely considered to Department of Laboratory Medicine and Genetics, Samsung Medical Center, Sungkyunkwan University School be model applications of personalized medicine; however, of Medicine, Seoul 06351, South Korea this perception is gradually changing due to a substantial Center for Infection Prevention and Control, Samsung increase in antimicrobial resistance (AMR) to antibiotics Medical Center, Seoul 06351, South Korea due to unnecessary usage [6–9]. Since experience-based Asia Pacific Foundation for Infectious Diseases (APFID), empiric treatment is a possible driver of over-broad and Seoul 06367, South Korea unnecessary antibiotic usage, rapid POC diagnostic testing Division of Infectious Diseases, Department of Internal has become a promising solution to this problem. Molecular Medicine, Samsung Medical Center, Sungkyunkwan tests, such as polymerase chain reaction (PCR) and other University School of Medicine, Seoul 06351, South Korea Vol:.(1234567890) 1 3 BioChip Journal (2021) 15:14–22 15 nucleic acid-based amplification technologies (NAATs), [22]. Infectious disease MDx requires especially careful have gradually replaced or augmented traditional laboratory handling of specimens and test procedures to prevent the techniques for pathogen identification in the form of POC spread of viral molecules and aerosol from the sample and testing, because these tests can detect fastidious or unculti- cross-contamination in the community [23, 24]. Infectious vable microorganisms that indicate possible poly-microbial disease MDx POCT is also lacking in the number of patho- infection [10–12]. The successful migration of molecular gens that can be identified at once and has insufficient phe- diagnostics from universal laboratories to the clinical set- notypic information on pathogens. Fortunately, many studies ting. could dramatically improve the accuracy and sensi- have recently highlighted the significance of the multiplex tivity of molecular POC testing (MDx POCT) [13–17]. In capability of molecular POCT, because it provides rapid addition, it could lead to pathogen detection derived from a and high-accuracy answers that support quicker therapeu- small amount of specimen, with delivery of results in just a tic decisions for the individual and at the public level [24]. few hours [13, 15, 17, 18]. Those rapid-throughput results Ultimately, multiplexed molecular POCT leads to pathogen- would give providers confidence in prescribing the proper specific treatment and the use of narrow-spectrum antibiotics treatment (Fig.  1). Ultimately, exclusive molecular POC instead of excessive use of broad-spectrum antibiotics. technologies may be implemented in the central care method This review focuses on up-to-date progress in multiplexed in limited-resource settings. molecular POCT for infectious diseases and efforts to over - Clinical microbiological and virological POC molecu- come limitations. We have summarized information on lar diagnostics have initially addressed the optimization of recently approved devices and state-of-the-art developments technologies from centralized laboratories to the medical through a comprehensive literature review. We also discuss checkup field, regardless of the number of detectable patho- the potential clinical impact of the recent POC approach and gens [19]. The field has now expanded to encompass a com- the possible hurdles that impede their practical application prehensive syndromic approach that simultaneously detects as a leading diagnostic test. several similar pathogens. A few molecular POC tests exploit the early diagnosis of frequent infections, such as 1.1 Multiplex Molecular POC Testing: The Next influenza [20] and healthcare-associated infections (HAIs) Phase of Molecular Diagnostics caused by S. aureus, C. difficile, enterococci or multi-resist- ant enterobacteria [21]. However, innovative multiplexed The implementation of NAATs has been a huge leap for- molecular POC testing has yet to be applied to infectious ward in test sensitivity and time-to-results. There are sev- diseases because of the complex sample preparation required eral molecular-based assays that meet the criteria for POC and the low throughput diagnosis. For instance, molecular application and have been approved or are clinically avail- diagnostics (MDx) involves numerous steps and reagents able for the detection of single-key pathogens, such as influ- for sample preparation, including extractions and the puri- enza, C. difficile, or methicillin-resistant Staphylococcus fication of nucleic acids from specimens, amplification of a aureus (MRSA). The first molecular test designed for POC specific sequence, and confirmation of the target molecules was approved by the U.S. Food and Drug Administration Fig. 1 Clinical diagnosis of infection according to a diagnostic platform. The implementation of a multiplex point-of-care (POC) test reduces diagnostic cycles for faster and better treatment decisions 1 3 16 BioChip Journal (2021) 15:14–22 (FDA) in 2015. ID NOW™ Influenza A & B 2, formerly targets. The microfluidic pouch system connects to a module known as Alere I influenza A & B 2, (Alere Scarborough for DNA extraction and purification and employs a post-PCR Inc., Scarborough, ME, USA), provides highly sensitive melting curve analysis that differentiates targets based on results in 13 min or less. It is based on isothermal nucleic the distinct melting temperature (T ) peaks for each target. acid amplification technology, which uses nicking enzyme This allows for the multiplexed detection of multiple targets. amplification reaction (NEAR) technology. As many emerg- Another example is the FDA-cleared Verigene (Nanosphere, ing isothermal amplified POC applications have reached Northbrook, IL, USA) system, which that uses gold nanopar- the proof-of-concept stage, ID NOW™ has successfully ticle probes and a microarray for the detection of bacterial entered the infectious disease POC market for influenza A pathogens and several resistance markers. However, neither & B, Strep A and respiratory syncytial virus (RSV) test- platform is classified as a POC test. ing [25–27]. ID NOW™, cobas Liat influenza A/B/RSV assays (Roche Molecular Systems, Pleasanton, CA, USA) 1.2 Recent Multiplex Molecular POC Testing and GeneXpert Xpress Flu/RSV (Cepheid, Sunnyvale, for the Broad Detection of pathogens: The Way CA, USA) are known as Clinical Laboratory Improvement Forward Amendments (CLIA)-waived (CW) assays that enable non- training personnel to obtain results within 30–50 min and are Microfluidic devices for molecular diagnostics have been suitable for POCT [28]. However, ID NOW™ using NEAR less commercialized over the last few years compared to the technology is limited to the simultaneous detection and dis- immense academic interest focusing on the miniaturization crimination of multiple targets, which means that it includes of molecular biological techniques, including target gene individual assay panels for either influenza or RSV rather amplification onto microfluidic devices. However, there is than a multiplexed test, whereas cobas® Liat influenza A/B/ an increasing need to integrate microfluidic devices with RSV assays and GeneXper t Xpress Flu/RSV are based on other applications, such as sample pre-treatment, fluid con- real-time reverse transcription-polymerase chain reaction trol, and multiplex capability (Fig.  2). For example, Liu (rtRT-PCR), which can detect both influenza A/B and RSV et al. reported a microfluidic cassette that is able to capture, during a single run [29–31]. The binx io (binx Health Diag- concentrate, amplify, and optically detect the Zika virus nostic, Boston, MA, USA), which combines PCR amplifica- (ZIKV) in a single sample-to-answer chip [36]. They have tion and electrochemical detection technology, has recently established a silica-based nucleic acid isolation membrane received 510(k) clearance from the U.S. FDA for the dual in a microchannel and thermally insulated a portable heat- targets of chlamydia (CT) and gonorrhea (NG) [32, 33]. ing cup for PCR reactions in an emergency. Even though These above-mentioned platforms have adopted single-use the POC molecular test is composed of a modest set with a test units for sample preparation; nucleic acid extraction, chip and heater, the obtained result was reasonable, with a purification and amplification, and the detection of biomark - high sensitivity of 5 PFU ZIKV per sample within 40 min. ers. While the laboratories have consistently demonstrated In accordance with the emergence of nucleic acid amplifi - that the upgraded test kits have added target molecules, there cation technologies, multiplex microfluidics have also been are still significant challenges for the robust deployment of developed with the goal of eliminating the heating cycle molecular POC systems; in particular, there is relatively low apparatus. Lee et  al. demonstrated a self-powered inte- throughput in terms of simultaneous detection of copious grated microfluidic POC low-cost enabling (SIMPLE) chip pathogens. This limitation mainly arises from interference [37]. It contains an isothermal recombinase polymerase between multiple sets of primers and optical sources for tar- amplification initiator patterning on the well of the chan- get analysis. In detail, a large number of primers are required nel, autonomous plasma separation into 224 microwells for for simultaneous multi-pathogen detection, which results in digital nucleic acid amplification, and a vacuum battery on preferential amplification between co-amplified genes and the chip. The integrated options have been developed as a non-specific amplifications, such as primer dimer and mis- lab-on-a-chip for molecular testing that automatically pre- priming artifacts [34]. Moreover, the optics of current ana- pares plasma from whole blood into hundreds of microwells, lytical instruments and sequencing probes (e.g., fluorescence directly amplifies the independent target in every well due to label) bring a limited number of targets to deflect from an micropatterning of the initiator, and can run without a power overlapping wavelength in a single reaction and laser source source or external pumps. The SIMPLE chip also detects [35]. The novel design of probe formats and combination Staphylococcus aureus DNA from human blood samples in of microfluidic technologies has been proposed to be the 30 min with separation, amplification, and digital quantita- solution for improved simultaneous detection of a larger tive nucleic acid readouts. The result simplifies molecular number of targets. For instance, FilmArray (BioFire, Salt testing, making it portable, and available for rapid medi- Lake City, UT, USA) exemplifies this idea of a technical cal decisionmaking. On the other hand, NAATs that utilize breakthrough enabling simultaneous detection of multiple paper substrate matrices provide a low-cost, easy-to-store, 1 3 BioChip Journal (2021) 15:14–22 17 Fig. 2 Integrated molecular diagnostic assay-based microfluidic Sci Adv 2017, 3 (3), e1501645 under open access license CC BY-NC, devices. a SIMPLE chip for blood sample prep, simultaneous digital reprinted with permission from AAAS/b Reprinted from Lee, N. Y. amplification, and quantitative nucleic acid testing with minimal han- et  al., Sens. Actuators B Chem 2020, 314, 128,057. Copyright by dling and no-power source. b A foldable all-in-one microdevice for Elsevier/ (C) From Lee, H. et  al., Sci Adv 2016, 2 (5), e1600300 the entire process of multiplex pathogen molecular test by a folding under open access license CC BY-NC, Reprinted with permission and stacking motion. c Polarized anisotropy diagnostics (PAD) sys- AAAS) tem for the detection of bacterial total RNA. (a From Lee, L.P. et al., and portable platform. Klapperich’s et al. developed a single (HPV) DNA directly from cervical specimens in less than paper-fluidic-chip-based lateral flow assay, which allows for 1 h with an integrated on-chip assay that produces an imme- the completion of NAAT steps from sample to result [38]. diate visual readout. The device drives the isolation of DNA with reagents on the Numerous studies have highlighted the importance of chip, followed by isothermal loop-mediated amplification a syndromic approach in a multiplex molecular diagnostic and a pump-free immunochromatographic assay. All pro- test [39–41]. A syndromic approach in molecular diagno- cesses are operated on paper, which passively move fluids sis achieves the simultaneous detection of genetic material through wicking, capillary force, and sample handling. The from pathogens of different species and even different taxo- authors have detected 16 subtypes of human papillomavirus nomic levels in a single test. For a comprehensive group 1 3 18 BioChip Journal (2021) 15:14–22 of pathogens that could cause a specific syndrome, such the GenePOC technology platform (Meridian Bioscience, as food poisoning or an upper respiratory infection, it may Inc., Quebec, Canada) integrates a microfluidic centripetal help effectively manage outbreaks with seasonal pathogen device to provide molecular diagnostic technologies in POC screening and detect community- and healthcare-associated [48, 49]. Within 1 h and with less than 1 min of hands-on outbreaks [42, 43]. Lee et al. presented a foldable all-in-one time, the GenePOC platform enables fully automated nucleic microdevice for simultaneous detection of three foodborne acid-based testing for infectious microorganisms. Overall, pathogens, Salmonella spp., Staphylococcus aureus, and it can process a wide range of clinical samples with up to Escherichia coli O157:H7, and a multi-drug-resistant bac- 12 targets. Culture-independent direct pathogen detection teria Acinetobacter baumannii [44]. The platform is feasible approaches are included in most advanced technologies, for accomplishing PCR preparation, amplification, and mul- such as Abbott’s IRIDICA and T2 Biosystems’ magnetic tiplex optical detection of pathogens in less than 2 h. The resonance technology, which are capable of detecting many thin polymethyl methacrylate (PMMA)-molded microdevice microorganisms. Abbott’s IRIDICA combines a set of consists of PCR reagent pre-stored filter paper disks and broad-range PCRs and electrospray ionization-mass spec- Flinders Technology Associates cards for cell lysis, protein trometry (PCR/ESI–MS). Herein, the PCRs amplify gene denaturation, and DNA extraction. Consequently, adding sil- encoding 16S ribosomal RNA and detect the housekeeping ver nitrate results in the multiplex pathogen detection system gene region. Otherwise, T2 relies on changes in a sample’s for direct colorimetric detection without an expert analytical T2 magnetic resonance (T2MR ) signal, which is caused tool. To quantify the bacterial cell, Mu et al. reported an by hybridization of the PCR-amplified pathogen DNA and integrated multiplex digital recombinase polymerase ampli- the capture of probe-decorated nanoparticles [50]. The T2 fication (imdRPA) microfluidic chip, which enables to detect system requires 1 mL uncultured whole blood and provides 1 × 10 foodborne bacterial cells of each species within results in approximately 3 h, with a claimed limit of detec- 45 min [45]. They adopted a magnetic bead-based DNA tion as low as 1 colony-forming unit (CFU)/ml. However, extraction component with a digital RPA region including this assay has currently been confirmed to detect only the 12,800 chambers for simultaneous detection of the targets five most common Candida species (which account for 95% and control, and then quantitatively analyzed the multiplex of candidemia) but is able to provide antifungal susceptibil- foodborne pathogen from the fluorescence signal of the ity data [51]. Due to growing demand for multiplex molecu- DNA probe. These microdevices are highly suitable for the lar POC testing, major companies in the in vitro diagnostics detection of numerous pathogens by increasing the number (IVD) market are focused on pipeline expansion and fortify- of channels in a chip for a syndromic diagnosis approach. ing their partnerships. For instance, the molecular diagnostic Among various emerging breakthrough technologies, Park company Qiagen (Hilden, Germany) has agreed to acquire et al. validated a new detection system based on polarization Stat-Dx (Barcelona, Spain), which has developed a fully anisotropy diagnostics (PAD), which measures changes in integrated one-step molecular test for common syndromes. fluorescence anisotropy when detection probes recognize the QIAstat-Dx (formerly Stat-Dx DiagCORE ) is a multiplex nucleic acid of the target bacteria [46]. This assay supports molecular diagnostic system that enables fast, cost-effective, a universal capture key that targets a conserved region of and flexible syndromic testing. The system has received 16S rRNA in different bacterial species. PAD was applied CE-IVD certification for a respiratory panel that detects 21 on-site in clinical HAI diagnostics and achieved an accuracy pathogens and will be able to process up to 48 molecular comparable to that of bacterial culture, and with a shorter targets at once, which will permit the diagnosis of serious turnaround time (~ 2 h). One of the commercial molecular respiratory and gastrointestinal infections. POCs, an STD Direct Flow Chip Kit (Master Diagnóstica, Granada, Spain), was evaluated for the detection of up to 1.3 Persistent Challenges nine different pathogen species of sexually transmitted dis- eases (STDs) from clinical specimens in a single reaction 47. A few years ago, molecular POC diagnosis was limited The kit achieves molecular diagnosis based on a multiplex to testing only the most common pathogens. This lack in PCR and automatic hybridization onto a microarray with diagnostics capability led to further downstream tests or specific oligo probes and broad clinical specimens while unnecessary prescription of antibiotics. The recently com- avoiding DNA purification steps. From a total of 633 speci- mercialized multiplex molecular assays enable simultaneous mens, the direct PCR-analysis results were 98.4% and 99.9% detection and identification of multiple pathogens associ - for sensitivity and specificity, respectively. ated with clinical syndromes with regard to bloodstream, Numerous studies involving multiplex molecular POC are respiratory, gastrointestinal (GI), or central nervous sys- now moving toward extensive diagnosis capability, but few tem infections [40]. As the demand for syndromic testing kits have been approved for the detection of multiple patho- in molecular diagnostics increases, there is an ongoing gens based on clinical syndromes (Table 1). For example, debate about whether these tests will be applied as front-line 1 3 BioChip Journal (2021) 15:14–22 19 Table 1 Analytical spectrum of molecular near-patient and point-of-care testing for infectious diseases System Multiplexity Virology Microbiology ID NOW™ Single FLU A/B, RSV, GAS Abbott SARS-CoV-2 ® ® cobas Liat Single FLU A/B GAS, CDI Roche Multiple FLU A/B + RSV GeneXpert Omni Single EBO, EV, FLU A/B, HPV, HBV, HCV, HIV, SARS-CoV-2* GAS, TV, CT, GBS, CDI, Cepheid CARBA-R, MRSA, NORO, VRE Multiple FLU A/B + RSV MTB + RIF, CT + NG, MRSA + SA binx io Single HPV, HIV, CT, NG, TV, TP, MG binx health, inc HCV, HBV, HSV-2 Multiple CT + NG, CT + NG + TV, CT + NG + TV + MG FilmArray Multiple Respiratory Panel: 18 viruses, 4 bacteria BioMerieux Blood Culture Identification 2 (BCID2) Panel: 26 bacteria, 7 yeast, 10 antibiotic resistance genes Gastrointestinal (GI) Panel: 5 viruses, 7 bacteria, 6 Diarrheagenic E. coli/Shigella, 4 parasites Meningitis-Encephalitis (ME) Panel: 7 viruses, 6 bacteria, 1 yeast Pneumonia plus Panel: 18 bacteria, 7 antibiotic resistance genes, 9 viruses Verigene Nanosphere Single CDI Multiple Enteric Pathogens Test: 2 viruses, 6 bacteria, 2 toxins Respiratory Pathogens Flex test: 13 viruses, 2 bacteria Blood Culture Nucleic Acid Testing Panels: 1. Gram-positive: 8 species, 1 group, 4 genus, 3 resistance markers 2. Gram-negative: 5 species, 4 genus, 6 resistance markers GenePOC Single CDI, GBS Meridian Bioscience, inc Q-POC Single HPV, MDR-TB QuantuMDx Multiple CT + NG QIAstat-Dx Multiple Respiratory Panel: 18 viral, 3 bacterial Qiagen pathogens Respiratory SARS-CoV-2 panel*: 19 viral pathogens including SARS-CoV-2, 3 bacterial pathogens Gastrointestinal Panel: 6 viral, 14 bacterial, 4 parasitic pathogens T2MR Single Candida T2 Biosystems Multiple ESKAPE FLU A/B Influenza A/B, RSV respiratory syncytial virus, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, GAS group A strepto- cocci, CDI Clostridium difficile, EBO Ebola virus, EV enterovirus, HPV human papillomavirus, HBV Hepatitis B virus, HCV Hepatitis C virus, HIV Human immunodeficiency virus, TV Trichomonas vaginalis, CT Chlamydia trachomatis, GBS group B streptococci, CARBA-R carbap- enem-resistant Enterobacteriaceae, MRSA methicillin-resistant Staphylococcus aureous, NORO Norovirus, VRE Vancomycin-resistant entero- cocci, MTB Mycobacterium tuberculosis, RIF Rifampicin resistance, NG Neisseria gonorrhoeae, SA Staphylococcus, HSV-2 Herpes simplex virus type two, TP Treponema pallidum, MG Mycoplasma genitalium, MDR-TB Multi-drug resistance tuberculosis, ESKAPE Enterococcus fae- cium/Staphylococcus aureus/Klebsiella pneumoniae/Pseudomonas aeruginosa/Escherichia coli For use under an Emergency-Use Authorization (EUA, 2020.09.24) Not POC diagnostics techniques for all patients or will be limited to to confirm the clinical syndromes, it may be ineffective cost- specific patients and regions [39, 52]. Even if multiplex test wise to test all specimens. Thus, syndromic testing must be menus, i.e., combinations of pathogens, are properly linked customizable and personalized for patient-specific needs to 1 3 20 BioChip Journal (2021) 15:14–22 exclude over-testing and excess patient medical bills. Conse- practice, such as moderate test performance, complexity quently, an optimized technical model should be applied in that make it difficult to use in many clinical settings, limited the development of POC tests using a syndromic approach. access to testing, and high costs. Considering that count- In addition, incorporating multiplex molecular POC tests in less researchers have encouraged finding a way to overcome clinical practice may encourage a change in the workflow of these challenges, we believe that multiplex molecular POC existing central laboratory tests. Currently, it is important to will become a powerful principal diagnostic test. consider whether multiplex MDx POCT would replace or be Acknowledgements This work was supported by the National conducted in addition to existing tests. For instance, many Research Foundation of Korea (NRF) grant funded by the Korean gov- studies have applied ultrafast thermocycling approaches that ernment (the Ministry of Science and ICT) [No. 2019R1A2C2087631, perform efficient PCR processes through rapid tempera- No. 2016M3A9B6919189, No. 2016M3A9B6919187], by the Korea ture control and a high transfer rate of heating and cool- Medical Device Development Fund grant funded by the Korea govern- ment (the Ministry of Science and ICT; the Ministry of Trade, Industry, ing, resulting in a small reaction volume and photophysical and Energy; the Ministry of Health & Welfare, Republic of Korea; the effect [11, 53, 54]. The developed factors consist of minia- Ministry of Food and Drug Safety) [No. 202011A04] and by Samsung turized thermocyclers for ultrafast PCR, including capillary Research Funding & Incubation Center of Samsung Electronics [No. tubes with air heating, a microfluidic device, and a photonic SRFC-IT1902-05]. microwave-based resistor-mediated or convective thermocy- Author Contributions MK and DRC designed and determined the cler [55–59]. In fact, the key prerequisite is that performance study concept and fully accessed all the data in the study and hold should be equal in terms of sensitivity and specificity when responsibility for the integrity of the data, accuracy of the data analysis, it is compared to tests that are conducted in a central labora- and obtained funding. MK, HK, HJH, and EP acquired the data. MK and HK analyzed and interpreted the data, drafted the manuscript, and tory. Another consideration is that it should be available to obtained administrative, technical, or material support. use at the primary care level, even in resource-limited set- tings. Although the sensitivity and specificity are similar to Compliance with Ethical Standards the gold standard method, performance should be achieved equally, even in a resource-constrained setting. When a com- Conflict of interest No potential conflicts of interest relevant to this prehensive test such as the multiplex MDx POC test is actu- article were reported. alized to administer medical checkup performance, it has the potential to become a dominant diagnostic test. Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, 2 Conclusion provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated Evolving POC molecular diagnostics are closer to reali- otherwise in a credit line to the material. If material is not included in zation in the medical field. The successful migration of a the article’s Creative Commons licence and your intended use is not molecular POC test has changed the trends of centralized permitted by statutory regulation or exceeds the permitted use, you will laboratory testing from general molecular diagnostics with need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativ ecommons .or g/licenses/b y/4.0/. highly skilled experts and complex equipment to the more comprehensive molecular diagnostics in a clinical set- ting using simpler instruments. The emergence of a new CLIA-waived molecular POC platform has received major References attention in the global in vitro diagnostics (IVD) market, because traditional POC tests, e.g., lateral flow, are com- 1. de la Fuente-Nunez, C., Torres, M.D., Mojica, F.J., Lu, T.K.: monly known to lack sensitivity. 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Published: Feb 15, 2021

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