Published online 12 September 2022
DOI: 10.2147/IDR.S373748
Puja Neopane, Jerome Nypaver, Rojeet Shrestha, and Safedin Beqaj
Patients Choice Laboratories, Indianapolis, IN, 46278, USA
Copyright © 2022 Neopane et al.
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Abstract
Purpose To detect and differentiate co-infection with influenza and respiratory syncytial virus during the COVID pandemic, a rapid method that can detect multiple pathogens in a single test is a significant diagnostic advance to analyze the outcomes and clinical implications of co-infection. Therefore, we validated and evaluated the performance characteristics of TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay for the detection of SARS-CoV-2, Flu A/B, and RSV using nasopharyngeal and saliva samples.
Materials and Methods The method validation was performed by using culture fluids of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009), RSV A2 cpts-248, SARS-CoV-2 (USA-WA1/2020) and quantitative RNA controls of Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ), RSV A2 (VR-1540DQ) and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) from ATCC and Zeptometrix, NY, USA. A total of 110 nasopharyngeal specimens and 70 saliva samples were used for the SARS-CoV-2 detection, and a total of 70 nasopharyngeal specimens were used for Influenza and RSV detection. Total RNA was extracted from all the samples and multiplex PCR was performed using TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay. The assay was used for SARS-CoV-2 variant (B.1.1.7_601443, B.1.617.1_1662307, P.1_792683, B.1.351_678597, B.1.1.529/BA.1).
Results Validation controls showed accurate and precise results. The correlation study found the accuracy of 96.38 to 100% (95% CI) in nasopharyngeal and 94.87 to 100% (95% CI) in saliva for SARS-CoV-2 and 91.1 to 100% (95% CI) for both Influenza A/B and RSV. The diagnostic efficiency of this assay was not affected by SARS-CoV-2 variant, including Omicron.
Conclusion The TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay is a rapid method to detect and differentiate SAR-CoV-2, Flu A and B, and RSV in nasopharyngeal and saliva samples. It has a significant role in the diagnosis and management of respiratory illnesses and the clinical implications of co-infection.
Keywords: TaqMan, SARS-CoV-2, Flu A/B, respiratory syncytial virus, RSV, multiplex
Introduction
Respiratory tract infections remain a significant public threat with high morbidity and mortality worldwide.1 Viruses are responsible for about 90% of upper respiratory tract infections and, about 30% of lower respiratory tract infections.2 The respiratory syndrome can be caused by several viruses, including rhinoviruses, respiratory syncytial viruses, adenoviruses, influenza viruses, and parainfluenza viruses.1 Most of the viruses have characteristic seasonal patterns, including Influenza virus and respiratory syncytial virus (RSV), which rise mainly in the winter. In addition, the coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) added the potential threats of respiratory viruses. During this pandemic, co-infection with influenza and RSV could pose a challenge to healthcare providers due to their overlapping clinical presentations. Influenza and COVID-19 share very similar symptoms. However, the incubation period of COVID-19 is longer (2–14 days), and people stay infectious longer than with the flu. These viruses can cause fever, chills, headaches, cough, muscle soreness, fatigue, vomiting, diarrhea, shortness of breath, runny nose, and sore throat. The only symptom that is unique to COVID-19 is loss of taste or smell.3 In the case of RSV, symptoms appear four to six days after infection and are usually self-limiting. However, in infants and the elderly, symptoms tend to be more severe and can include fever and wheezing.4
The respiratory illness caused by the influenza virus, SARS-CoV-2 virus, and RSV are all highly contagious. In addition, there is a chance of co-infection with all multiple viruses at the same time complicating the situation.5
The SARS-CoV-2 virus is a newly discovered novel coronavirus, causing a global pandemic of COVID-19. In severe conditions, it can cause severe lower respiratory disease including pneumonia and respiratory failure and, in some cases, death. The SARS-CoV-2 virus is the seventh known coronavirus belonging to the 2B group of the Betacoronavirus family, which is the same family as SARS-CoV and MERS-CoV, and has a 70% similarity in genetic sequence to SARS. Each SARS-CoV-2 virion is 50–200 nm in diameter and composed of several proteins including spike (S), envelope (E), membrane (M), and nucleocapsid (N): the N protein holds the RNA genome, and S, E, and M proteins together create the viral envelope. The spike protein is a key player in binding angiotensin-converting enzyme 2 (ACE2) receptors and mediating membrane fusion and virus entry into host cells.6
Influenza, or the flu, is a contagious viral infection caused primarily by the influenza virus A, B, or C and transmitted through respiratory droplets or airborne. It affects mainly the upper respiratory tracts (ie, the nose, throat, bronchi, and infrequently, lungs) but other organs such as the heart, brain, and muscles can be involved. Influenza infection can increase morbidity and mortality in pediatric, elderly, and immunocompromised populations by serious complications of pneumonia. Influenza viruses are segmented negative-strand RNA viruses belonging to the Orthomyxoviridae family with diverse antigenic characteristics.7 Influenza A (Flu A) is the most common type of influenza virus in humans and is generally responsible for seasonal flu epidemics and potential pandemics. Influenza A virus can be further classified into subtypes based on two surface proteins: hemagglutinin (H) and neuraminidase (N). Seasonal flu is normally caused by influenza A subtypes H1, H2, H3, N1, and N2. The influenza B virus has a similar viral structure to type A, but no subtypes due to the fixed antigenic characteristics of HA and NA.8
RSV is a non-segmented negative-strand RNA virus and a member of the Pneumoviridae family (formerly Paramyxoviridae), consisting of two strains (subgroups A and B).9 It is also a contagious disease that affects primarily infants with a mortality rate of 0.3% in hospitalized children and immunocompromised elderly, making them at the highest risk for death.10
Due to the similarity in symptoms, laboratory investigation is the best way to diagnose these viral diseases. Multiplex RT-PCR assays are fast with increased sensitivity for the detection of a wider range of respiratory viruses than immunofluorescence (DFA) and viral culture. The ability to detect multiple pathogens in a single test is an important diagnostic advance as it provides an approach to better understanding the outcomes and clinical significance of co-infection. There are some commercially available kits and CDC’s developed assay (FDA-emergency use authorization) that detect either SARS-CoV-2 only, SARS-CoV-2 and Influenza A/B, SARS-CoV-2, Flu & RSV, or Multiplex respiratory panel.11 They can detect a limited number of samples and take a long time if needed to test a larger volume of samples. Therefore, the rapid, accurate, and economical diagnostic assay is highly desirable in diagnostic laboratories that can detect and differentiate SARS-CoV-2, influenza, and RSV even in a larger volume of samples. The TaqMan SARS-CoV-2, Flu A/B, RSV RT-PCR multiplex assay is a newly developed molecular diagnostic assay that enables the detection and differentiation of SARS-CoV-2, influenza A and B, and RSV in respiratory specimens. This is a multiplex real-time PCR assay containing primer and probe sets specific to SARS-CoV-2 (N gene and S gene), Flu A/B, RSV, and MS2 for the detection of RNA from the SARS-CoV-2 virus, influenza A and B viruses, and RSV subtypes A and B, respectively.
In this study, we evaluated the performance characteristics of TaqMan SARS-CoV-2, Flu A/B, and RSV RT-PCR multiplex assay for the detection of SARS-CoV-2, RSV, and Influenza viruses using nasopharyngeal and saliva samples.
Materials and Methods
Validation Controls
A total of four different culture fluids with known controls of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009), and RSV A2 cpts-248 from ATCC and SARS-CoV-2 (USA-WA1/2020) from Zeptometrix, NY, USA, were used for determining the accuracy, linearity, efficiency, and reproducibility of the TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay. The quantitative genomic RNA, namely Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ) and RSV A2 (VR-1540DQ) from ATCC and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) from Twist bioscience were used for the accuracy, precision, and limit of detection of the assay.
Extraction of RNA from Controls
Total RNA was extracted from the controls using Omega Bio-Tek Mag-Bind Viral RNA Xpress Extraction Kit on the automated liquid handling Hamilton STAR according to the manufacturer’s instruction.12
Multiplex Reverse Transcriptase PCR
Extracted RNA (17.5 μL) was mixed with a 7.5 μL reaction mixer containing TaqPath 1-Step Multiplex Master Mix (No ROX™) and TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay per reaction according to the manufacturer’s instruction.13 This multiplexed assay includes primer and probe set specific to SARS-CoV-2 (N gene and S gene), Flu A/B, RSV, and MS2 for the detection and differentiation of SARS-CoV-2, Flu A/B, and RSV viruses. The PCR reaction was incubated at 50 ℃ for 10 min, followed by 95 ℃ for 2 min and 46 cycles of 95 ℃ for 3s and 60 ℃ for 30s and run in 96 well plates by standard curve mode in QuantStudio 12K flex. The threshold level was determined for each target based on the exponential phase of the amplification curve above the background signal of the highest dilution series.
Accuracy Using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay
Validation controls using culture fluid of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009), RSV A2 cpts-248 and SARS-CoV-2 (USA-WA1/2020), and quantitative RNA of Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ), RSV A2 (VR-1540DQ), and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) were used for multiplex PCR using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay on QuantStudio 12K Flex to detect SARS-CoV-2, Flu A and B and RSV to check the accuracy of the controls.
Precision or Reproducibility Using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay
The intra-assay and inter-assay reproducibility or variability of the TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay was assessed by using quantitative genomic RNA from Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ), RSV A2 (VR-1540DQ), and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) in triplicates on three runs.
Standard Curve Using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay
To determine the dynamic range of the one-step multiplex RT-PCR assay, four different controls of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009), and RSV A2 cpts-248 from ATCC and SARS-CoV-2 (USA-WA1/2020) from Zeptometrix were serially diluted in 10-fold with nuclease-free water (108 to 1 copies/reaction) for each target to obtain standard curves and amplification plots of the multiplex RT-PCR assays. The total RNA was extracted from diluted controls, and RT-PCR was run using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay. The standard curve was generated, and cutoff values were established based on the lowest detected copy numbers or the highest Ct. Cycle threshold (Ct) values were plotted against quantities of RNA to establish a standard curve, and linear regression analysis was performed for the four targets, allowing the determination of the correlation coefficient (R2). The amplification efficiencies (E) of the reactions were calculated from the curves using the equation E = −1+10(−1/slope).
Analytical Specificity Using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay
The species-specific sample that includes human coronavirus 229E, MERS, rhinovirus, and negative specimens were used to verify the specificity of TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay.
Validation Using Patients Samples
The TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay were assessed by using two different types of specimens – nasopharyngeal and saliva samples. The nasopharyngeal swab was collected in a disposable virus sampling tube containing 3 mL of virus transport medium. Saliva samples were collected into Omni tubes containing ceramic beads for homogenization in the Omni Bead Ruptor Elite (OMNI International, Perkin Elmer company). All the clinical specimens were assessed in accordance with the Declaration of Helsinki in Patients Choice Laboratories, IN, USA. The study was approved by the Sterling Institutional Review Board (IRB ID-9190) and includes de-identified samples.
A total of 110 de-identified nasopharyngeal specimens, of which 50 were positive and 60 were negative for SARS-CoV-2 by reference method (Taqpath Combo kit, Applied Biosystems), were used for SARS-CoV-2 detection using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay. A total of 70 de-identified saliva samples were included in the study. Of them, five samples were previously positive for SARS-CoV2 (as determined by the reference method). Due to the low number of saliva samples (n = 5) positive for SARS-CoV2, we spiked saliva with positive nasopharyngeal specimens (n = 30) to evaluate the performance of the TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay. In brief, the nasopharyngeal specimens previously tested positive for SARS-COV-2 (reference method) were centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and the collected pellet was reconstituted with 30 negative saliva samples. Further, an additional 35 negative saliva samples were used as negative specimens to verify the specificity of assays.
A total of 60 de-identified nasopharyngeal specimens, of which 20 were positive for Flu A/B and 20 were positive for RSV, and 20 were negative for both by reference method (multiplex RT-PCR open array technology, Applied Biosystems) was used for detecting Influenza A/B and RSV.
A total of 15 nasopharyngeal samples, of which 6 were previously positive for SARS-CoV-2, Influenza, and RSV, 3 were positive for SARS-CoV-2 and RSV and 6 were positive for SARS-CoV-2 and Influenza by the reference method were included for co-infection detection by TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay.
Furthermore, a total of 20 nasopharyngeal specimens and 25 saliva samples that were previously tested negative for SARS-CoV-2, Flu A/B, and RSV were selected and spiked with culture fluids of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009), RSV A2 cpts-248, and SARS-CoV-2 (USA-WA1/2020) controls to check the accuracy of the assay in the co-infection.
Extraction of RNA from the Patient’s Samples
Total RNA was extracted from 200 μL samples in the viral transport medium and 200 μL homogenized saliva samples using Omega Bio-Tek Mag-Bind Viral RNA Xpress Extraction Kit on the automated liquid handling Hamilton STAR according to the manufacturer’s instruction.12
Reference Methods
Taqpath Combo Kit Assay
After total RNA was extracted from nasopharyngeal and saliva specimens, SARS-CoV-2 was detected by the FDA-EUA-approved method validated at Patients Choice Laboratories using the Taqpath combo kit (Applied Biosystems, Thermo Fisher Scientific). This multiplex PCR method includes primer and probe sets specific to the N gene, S gene, and Orf ab gene for detecting a single SARS-CoV-2. The master mix was prepared from TaqPath 1-Step Multiplex Master Mix (No ROX™), COVID-19 Real-Time PCR Assay Multiplex, and Nuclease-free water. Ten microliters of Master mix was dispensed into the 384 well plates followed by the addition of 2.5 μL of the eluted specimen to the appropriate well. Each run includes a SARS-CoV-2 positive control and negative control. The PCR reaction includes an incubation at 50 ℃ for 10 min, followed by 95 ℃ for 2 min and 40 cycles of 95 ℃ for 3 s and 60 ℃ for 30s and run by standard mode in QuantStudio 12K flex.
Multiplex RT-PCR Open Array Technology
After total RNA was extracted from respiratory samples, RNA was reverse transcribed to cDNA in the thermocycler. The Influenza A/B and RSV were determined by multiplex RT-PCR using open array technology method and validated at Patients Choice Laboratories using TaqMan open array master mix and customized respiratory panel open array chips (Applied Biosystems, Thermo Fisher Scientific). The open array chip is a microscope slide–sized plate with 48 sub arrays, each with 64 through-holes. It has multiple targets of respiratory viruses and bacteria including influenza and RSV. The readymade open array master mix is mixed with the sample in the 1:1 ratio in the 384 well plates, and samples are transferred into open array chips with the help of Accufill. Each through hole receives 33 nl sample. The open array is loaded into QuantStudio 12K flex instrument for RT-PCR.
Analytical Sensitivity (Limit of Detection)
The lowest RNA concentration of Influenza A and B, RSV, and SARS-CoV-2 that allows the multiplex RT-PCR to determine the correct target is considered as LOD for TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay. The quantitative genomic RNA from Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ), RSV A2 (VR-1540DQ), and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) were serially diluted in 10-fold with pooled negative nasopharyngeal specimens from 1 x 107copies/μL to 1copy/μL for LOD. The standard curve was generated, and cutoff values were established based on the lowest detected copy numbers or the highest Ct. LOD is considered after >95% positive results from 19/20 replicates.
For LOD in saliva, a ten-fold dilution series of Influenza A virus (H3N2) (A/Wisconsin/67/2005), RSV A2 cpts-248, and SARS-CoV-2 (USA-WA1/2020) was spiked into pooled negative saliva samples.
Data Analysis
Data were analyzed using QuantStudioTM Design and Analysis Software v2.5 for TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay method and SDS software v1.4.1 for reference method. Basic statistical analysis, including mean, standard deviation, and coefficient of variation of the mean Ct value, were calculated using Excel (Microsoft Corp., Redmond, WA, USA). The standard curve was generated and efficiency, R2, and slope were calculated using QuantStudioTM Design and Analysis Software v2.5. Correlation analysis was performed using the MedCalc statistical software, version 16.4.3 (Ostend, Belgium). The cycle threshold (Ct) values of a positive sample between reference method assays and TaqMan multiplex RT-PCR assay by simple regression were assessed using the Pearson correlation coefficient for comparative analysis.
Results
Validation of Controls
Accuracy
Validation controls of Influenza A virus (H3N2) (A/Wisconsin/67/2005), Influenza B virus (B/Virginia/ATCC4/2009) and Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ) showed positive for Flu A and B, RSV A2 cpts-248 and RSV A2 (VR-1540DQ) showed positive for RSV and SARS-CoV-2 (USA-WA1/2020) and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) showed positive for SARS-CoV-2 as expected for known controls when tested in triplicates (Supplementary Table 1).
Precision
The inter-assay and intra-assay precision study using RNA controls of Influenza A virus (H1N1) strain A/PR/8/34 (VR-95DQ), RSV A2 (VR-1540DQ), and SARS-CoV-2 (MN908947.3 Wuhan-Hu-1) was performed in triplicates. Precision was determined by calculating the mean, SD, and CV using cycle threshold (Ct) or Cq. Results from precision showed acceptable reproducibility and repeatability with a CV of less than 5% when Ct was used (Supplementary Table 2).
Standard Curve Using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay
The standard curve using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay showed a linear efficiency between 90 and 110% and R2 values with 0.99 for SARS-CoV-2, Flu A/B, and RSV (Figure 1).
Correlation of Two Methods
For SARS-CoV-2
The correlation of the reference method (Taqpath combo kit) and TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay method for Sars-CoV-2 was determined in nasopharyngeal and saliva samples. Considering the reference method as standard, the sensitivity, specificity, and accuracy using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay method in NP specimens are 92.89% to 100%, 92.89% to 100%, and 96.38–100% (95% CI), respectively (Table 1). Similarly, the sensitivity, specificity, and accuracy using TaqMan SARS-CoV-2, Flu A/B, and RSV Multiplex Assay methods in saliva samples are 90% to 100%, 90% to 100%, and 94.87% to 100% (95% CI), respectively, when compared with the reference method (Table 2).
The correlation between the cycle threshold (Ct) value of positive SARS-CoV-2 samples by reference method and TaqMan multiplex assays (n = 33) is shown in Figure 2. There was a strong positive correlation between CT values of SARS-CoV-2 positive samples by two assay methods with Pearson correlation (r) of 0.98.
