Comparative Clinical Evaluation of a Novel FluA/FluB/SARS-CoV-2 Multiplex LAMP and Commercial FluA/FluB/SARS-CoV-2/RSV RT-qPCR Assays

Influenza and coronaviruses cause highly contagious respiratory diseases that cause millions of deaths worldwide. Public health measures implemented during the current coronavirus disease (COVID-19) pandemic have gradually reduced influenza circulation worldwide. As COVID-19 measures have relaxed, it is necessary to monitor and control seasonal influenza during this COVID-19 pandemic. In particular, the development of rapid and accurate diagnostic methods for influenza and COVID-19 is of paramount importance because both diseases have significant public health and economic impacts. To address this, we developed a multi-loop-mediated isothermal amplification (LAMP) kit capable of simultaneously detecting influenza A/B and SARS-CoV-2. The kit was optimized by testing various ratios of primer sets for influenza A/B (FluA/FluB) and SARS-CoV-2 and internal control (IC). The FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed 100% specificity for uninfected clinical samples and sensitivities of 90.6%, 86.89%, and 98.96% for LAMP kits against influenza A, influenza B, and SARS-CoV-2 clinical samples, respectively. Finally, the attribute agreement analysis for clinical tests indicated substantial agreement between the multiplex FluA/FluB/SARS-CoV-2/IC LAMP and commercial AllplexTM SARS-CoV-2/FluA/FluB/RSV assays.


Introduction
Influenza and coronaviruses are highly contagious respiratory diseases that cause millions of cases and deaths annually worldwide [1,2]. Influenza typically peaks during winter months and causes approximately 1 billion cases, 3-5 million severe cases, and up to 650,000 deaths [3]. However, public health measures and travel restrictions implemented during the COVID-19 pandemic have significantly curbed global influenza circulation [4]. The influenza B/Yamagata lineage has not been detected since April 2020, and other influenza viruses with considerably lower genetic diversity have circulated [5]. As the COVID-19 pandemic gradually weakens owing to various quarantine and vaccination measures, epidemic prevention, and control, global travel is gradually returning to preepidemic levels. Therefore, it is necessary to develop measures to monitor and control the spread of seasonal influenza during the COVID-19 pandemic.
In response to this challenge, various multi-diagnostic real-time reverse transcription polymerase chain reaction (RT-PCR) assays that can simultaneously detect multiple respiratory pathogens, including influenza and COVID-19, have been developed. This is particularly useful during the COVID-19 pandemic because it can simultaneously diagnose influenza and COVID-19, which induce similar symptoms. Bouassa et al. developed an RT-qPCR assay that could simultaneously diagnose influenza, COVID-19, and respiratory syncytial virus (RSV), and this kit exhibited 97.9, 89.5, 97.6, and 100% accuracy for influenza A, influenza B, SARS-CoV-2, and RSV, respectively [6]. Additionally, as another diagnostic method, various rapid antigen tests that are capable of detecting both influenza and COVID-19 antigens in a single test are being developed [7]. Although RT-qPCR is a commonly used gold-standard method of diagnosis, it is relatively slow and requires specialized equipment and skilled personnel, limiting its usefulness to rapid diagnosis in resource-constrained settings [8]. Rapid diagnostic tests, which use antibodies to detect viral antigens or antibodies in patient samples, have the advantage of being quick but have low sensitivity and tend to have a high false-negative rate [9]. Therefore, an alternative diagnostic technique capable of accurately and quickly diagnosing viral infections is required.
Loop-mediated isothermal amplification (LAMP) is a molecular diagnostic technique that enables the rapid and sensitive detection of specific RNA or DNA sequences in target pathogens [10]. This technique amplifies the target nucleic acid sequence at a constant temperature, using a set of four to six primers that recognize distinct regions of the target sequence. Among these, two are loop primers that allow the reaction to occur more efficiently by promoting the formation of stem-loop structures in the amplification product [11]. The LAMP assay has several advantages over other nucleic acid amplification methods, such as being simple, sensitive, and rapid [12]. In addition, LAMP assays can be performed using a variety of detection methods, including colorimetric, lateral flow, fluorescence, and fluorescent probe detection [13][14][15][16]. Among these methods, fluorescent probe detection is particularly useful for the real-time monitoring of the LAMP reaction. Among the various fluorescent probe techniques, the LAMP-assimilating probe consists of a fluorescently-tagged probe and complementary quencher-tagged probe. The assimilating probe was designed to hybridize with the target sequence during the LAMP reaction and was incorporated into the amplification products by Bst DNA polymerase, resulting in an increase in the fluorescent signal [17]. As amplification proceeds, the fluorescence intensity of the probe increases and can be monitored in real-time using a fluorescent detector to determine the presence or absence of the target pathogen. The use of LAMP-assimilating probes enables a highly sensitive and specific simultaneous detection of multiple pathogens in a single reaction, making them a powerful tool for molecular diagnostics in various settings [18].
In this study, we developed a FluA/FluB/SARS-CoV-2 multiplex LAMP assay that can detect influenza A, influenza B, SARS-CoV-2, and an internal control (actin beta) using LAMP primers and assimilating probes that were previously reported [19,20]. Among the five different ratios of LAMP primer sets evaluated, the ratio of 1:0.5:1:0.2 of influenza A, influenza B, SARS-CoV-2, and the internal control primer set showed the best performance in LAMP tests against the clinical samples of influenza A, influenza B, and SARS-CoV-2. Finally, the performance of the FluA/FluB/SARS-CoV-2 multiplex LAMP assay was compared with that of the commercial Allplex TM SARS-CoV-2/FluA/FluB/RSV assay (Seegene, Seoul, Korea) for clinical samples.

Clinical Samples and RNA Extraction
The Korea Disease Control and Prevention Agency (KDCA) measured the SARS-CoV-2 titers using the plaque-forming unit (PFU) test and provided 20 strains, including one wild-type sample and 19 mutant samples. Influenza A H1N1, H1N1pdm09, H3N2, and influenza B virus were cultured at the Department of Laboratory Medicine, the Korea University Guro Hospital, of which the viral titers were measured using the TCID50 method. For clinical sensitivity testing, we used clinical SARS-CoV-2 nasopharyngeal swab (NP) (n = 96), influenza A NP (n = 117), and influenza B NP (n = 61) samples collected from SARS-CoV-2-, influenza A-, and influenza B-infected patients (from February 2018 to July 2022) at Korea University Guro Hospital. All clinical samples were confirmed Diagnostics 2023, 13, 1432 3 of 13 using the Allplex TM SARS-CoV-2 assay (Seegene Inc., Seoul, South Korea) and Anyplex II RV16 Detection Kit (Seegene, Inc., Seoul, South Korea). To assess the specificity and crossreactivity, 135 NP swab specimens were tested from individuals with (102) and without (33) viral respiratory infections. Respiratory viral infections, as confirmed via PCR using the Anyplex II RV16 detection kit, included nine coronaviruses (KHU1, NL63, and 229E), three RSV A, three RSV B, three adenoviruses (AdV), three parainfluenza virus (PIV) types 1-4, three human bocaviruses (HboV), three human enteroviruses (HEV), three human rhinoviruses (HRV), and three metapneumoviruses (MPV). Nucleic acids were extracted from all samples using Zentrix (Biozenthech, Seoul, Korea), according to the manufacturer's instructions. Briefly, 200 µL of the sample was dispensed into a 96-well extraction plate and nucleic acid was extracted through the respiratory virus process program. The study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of Korea University Guro Hospital (approval number: 2021GR0547).

Primer Design
The influenza A, influenza B, SARS-CoV-2, and internal control RT-LAMP primer sets used in this study have been previously reported by our study group [19,20]. Primer sets for influenza A, influenza B, and SARS-CoV-2 were designed to target conserved regions of segment 7, the nucleoprotein, and the RdRP gene, respectively. The internal control primer set was designed within a conserved region of human actin beta mRNA (NM_001101.5:c.287-c.498) [19,20]. For the multiplex probe design, two types of additional synthetic oligonucleotide sequences were designed and added to the 5 end of the LB or LF primer of each LAMP primer set. The 5 end of the multiplex probe was tagged with a fluorescent marker. To quench the fluorophore multiplex probe, two types of complementary synthetic oligonucleotide sequences tagged with BHQ1 or BHQ2 at the 3 end were used. All LAMP primers and probes were synthesized by Macrogen Inc. (Seoul, Republic of Korea; Table 1).

RT-PCR
The performance of the FluA/FluB/SARS-CoV-2 multiplex LAMP assay was compared with that of the Allplex TM SARS-CoV-2/FluA/FluB/RSV assay (Seegene, Seoul, Republic of Korea) using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, California, USA). The Allplex TM SARS-CoV-2/FluA/FluB/RSV assay was performed according to the manufacturer's instructions. The PCR cycling conditions of the Allplex TM SARS-CoV-2/FluA/FluB/RSV assay were as follows: reverse transcription at 50 • C for 20 min, inactivation at 95 • C for 15 min, 2 cycles of 95 • C for 10 s, 60 • C for 40 s, and 72 • C for 20 s followed by 41 cycles of 95 • C for 10 s, 60 • C for 15 s, and 72 • C for 10 s with fluorescence detection at 60 • C and 72 • C.
Next, we calculated the agreement between the FluA/FluB/SARS-CoV-2 multiplex LAMP and Allplex™ SARS-CoV-2/FluA/FluB/RSV assays using SPSS 18 statistical package (SPSS 18 Inc., Chicago, IL, USA) ( Table 6). The kappa values for influenza A, influenza B, and SARS-CoV-2 NP clinical samples indicated substantial agreement between the two assays for these viruses. A kappa value of 0.845 for influenza A suggests almost perfect agreement, whereas a kappa value of 0.791 for influenza B suggests substantial agreement. The highest kappa value of 0.970 for SARS-CoV-2 NP clinical samples indicated an almost perfect agreement between the two assays for detecting SARS-CoV-2. Overall, these results suggest that the FluA/FluB/SARS-CoV-2 multiplex LAMP and Allplex™ SARS-CoV-2/FluA/FluB/RSV assays have good agreement for the detection of influenza A, influenza B, and SARS-CoV-2 NP clinical samples.

Cross-Reactivity Test
To confirm the absence of cross-reactivity of the FluA/FluB/SARS-CoV-2 multiplex LAMP assay with other common respiratory viruses, 33 NP swabs from patients with known infections with nine coronaviruses (229E, NL63, and OC43), three HEV, three AdV, three PIV, three MPV, three HboV, three HRV, and six RSV A/B were tested using the FluA/FluB/SARS-CoV-2 multiplex LAMP assay ( Table 7). The FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed negative results for Flu A, Flu B, and SARS-CoV-2 channels against 33 other common respiratory viruses. The internal controls of the kit were positive for all but three RSV B samples. These results suggest that the FluA/FluB/SARS-CoV-2 multiplex LAMP assay did not cross-react with other infectious viruses. Table 7. Cross-reactivity of the FluA/FluB/SARS-CoV-2 multiplex LAMP assay against other human infectious viruses.

SARS-CoV-2 Mutant Test
As the coronavirus continues to spread, new strains are emerging. To confirm that the developed FluA/FluB/SARS-CoV-2 multiplex LAMP assay can detect various SARS-CoV-2 mutant strains, one SARS-CoV-2 wild-type and 19 SARS-CoV-2 mutant samples distributed by the KDCA were used to evaluate the performance of the kit ( Table 8). The developed FluA/FluB/SARS-CoV-2 multiplex LAMP assay indicated positive reactions to all SARS-CoV-2 wild-type and mutant viruses tested.

Discussion
Presently, RT-qPCR is considered the most sensitive and specific molecular diagnostic test for infectious diseases, according to reports [22,23]. When new molecular diagnostic kits are developed, their performance is often evaluated using RT-qPCR as the reference standard [24,25]. Currently, LAMP is the most extensively researched and developed technique among various isothermal amplification methods. However, the detection limit of LAMP is similar or one to two orders of magnitude higher than that of RT-qPCR. Additionally, its sensitivity and specificity for clinical samples are reported to be similar to or slightly lower than those of RT-qPCR, ranging from 0 to 10% lower [23,26,27]. Although LAMP amplification can detect the target gene within 5-10 min when the target gene concentration is high, it does not demonstrate comparable sensitivity to RT-qPCR when the target gene concentration is low.
Despite these drawbacks, isothermal amplification methods remain attractive because of their isothermal amplification, rapidity, and ability to use relatively inexpensive equipment [27,28]. In fact, RT-qPCR kits take approximately 2-3 h to complete, but RT-LAMP analysis can detect several viruses simultaneously within 40 min because it does not require a reverse transcription step. Additionally, although the sensitivity and specificity of LAMP isothermal amplification may be lower than those of qPCR, it has higher sensitivity and specificity than rapid kits that mainly use antigen or antibody tests in the field [29,30]. Therefore, various methods incorporating isothermal amplification technology that can be used in facilities without the necessary equipment, or in the field, are being developed.
In this study, we optimized the primer set and reaction conditions of the FluA/FluB/ SARS-CoV-2 multiplex LAMP assay and evaluated its sensitivity and specificity compared to those of the commercially available Allplex TM SARS-CoV-2/FluA/FluB/RSV assay. The sensitivities of the FluA/FluB/SARS-CoV-2 multiplex LAMP assay for influenza A, influenza B, and SARS-CoV-2 NP clinical samples were 90.6%, 86.89%, and 98.96%, respectively. The specificity of the FluA/FluB/SARS-CoV-2 multiplex LAMP assay against non-infected samples was 100%, which was excellent in terms of ruling out false positives. When compared to the commercial Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (94.87%, 88.52%, and 97.92%), sensitivity levels of the FluA/FluB/SARS-CoV-2 multiplex LAMP assay were slightly lower but still quite accurate. In addition, the agreement between the FluA/FluB/SARS-CoV-2 multiplex LAMP assay and the Allplex TM SARS-CoV-2/FluA/FluB/RSV assay revealed high kappa values for influenza A, influenza B, and SARS-CoV-2 NP clinical samples, indicating substantial agreement between the two assays. Moreover, the FluA/FluB/SARS-CoV-2 multiplex LAMP assay showed no cross-reactivity with the other 11 respiratory viruses, which is a significant advantage over some commercial kits that have shown cross-reactivity with other viruses. Moreover, our LAMP kit demonstrated the ability to detect 20 SARS-CoV-2 mutants, including highly transmissible Delta and Omicron variants, which are crucial for identifying and controlling emerging COVID-19 outbreaks. These findings suggest that the multiplex LAMP assay is reliable for detecting influenza A, influenza B, and SARS-CoV-2 and can be used as an alternative to the Allplex TM SARS-CoV-2/FluA/FluB/RSV assay.
Generally, LAMP diagnostic kits are being developed as a middle ground between rapid antigen/antibody kits, which are available for field use but may have limited sensitivity, and the qPCR assays, which have high sensitivity but require complex equipment and trained personnel. In this study, a multiplex LAMP assay was optimized, which showed reliable and efficient diagnostic performance for detecting influenza A, influenza B, and SARS-CoV-2. The assay could diagnose both influenza and SARS-CoV-2 within an hour without requiring complex equipment. Additionally, it showed similar performance to commercial qPCR kits, making it a useful alternative to commercial qPCR kits that may not be suitable for use in the field.
In this study, we have demonstrated that developing a multiplex LAMP assay for detecting influenza and SARS-CoV-2 simultaneously could have some benefits. Our results suggest that the developed LAMP kit could provide a more comprehensive diagnosis of respiratory infections, especially during flu season when both viruses may be circulating. The ability to diagnose both influenza and SARS-CoV-2 using a single test could reduce the need for multiple tests and save time and resources. Additionally, the LAMP kit we developed could be used in resource-limited settings, where access to sophisticated equipment is limited, and could provide faster results compared to traditional PCR assays. This could be particularly useful in outbreak situations where quick and accurate diagnosis is crucial for effective control and management. Overall, our findings suggest that a multiplex LAMP assay has the potential to be a valuable tool for diagnosing respiratory infections, and could complement existing diagnostic methods in both clinical and public health settings.

Conclusions
In conclusion, the FluA/FluB/SARS-CoV-2 multiplex LAMP assay shows a similar level of sensitivity to the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay and commercial RT-qPCR in clinical testing, although the FluA/FluB/SARS-CoV-2 multiplex LAMP assay has a higher detection limit than that of the commercial RT-qPCR kit. Furthermore, the FluA/FluB/SARS-CoV-2 multiplex LAMP assay can detect multiple strains of SARS-CoV-2, including Delta and Omicron, without cross-reactivity to other respiratory viruses and demonstrates excellent specificity for uninfected samples. Additionally, the high kappa values obtained in the concordance assays using the Allplex TM SARS-CoV-2/FluA/FluB/RSV assay suggest that the FluA/FluB/SARS-CoV-2 multiplex LAMP assay can serve as an alternative to this commercial kit.  Institutional Review Board Statement: The study was conducted in accordance with the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Korea University Guro Hospital (IRB No. 2021GR0547, approval date: 2021.11.03).
Informed Consent Statement: Patient consent was waived by the Institutional Review Board of Korea University Guro Hospital because the identities of the subjects were completely anonymous, and there was minimal risk involved in the study.

Data Availability Statement:
The authors declare that all related data are available from the corresponding author upon reasonable request.

Conflicts of Interest:
The authors declare no conflict of interest.