key: cord-0711474-kvp2m5u9 authors: Ryu, Sook Won; Suh, In Bum; Ryu, Se‐Min; Shin, Kyu Sung; Kim, Hyon‐Suk; Kim, Juwon; Uh, Young; Yoon, Kap Jun; Lee, Jong‐Han title: Comparison of three rapid influenza diagnostic tests with digital readout systems and one conventional rapid influenza diagnostic test date: 2017-04-13 journal: Journal of Clinical Laboratory Analysis DOI: 10.1002/jcla.22234 sha: 7869d9bd96bbe92dff28bd42a77e92cd7d5a1e67 doc_id: 711474 cord_uid: kvp2m5u9 BACKGROUND: Rapid influenza diagnostic tests (RIDTs) show variable sensitivities in clinical settings. We aimed to compare three digital RIDTs and one conventional RIDT. METHODS: We assessed 218 nasopharyngeal swabs from patients between neonates and 90 years old in 2016. Three digital RIDTs were BUDDI, Sofia Influenza A+B Fluorescence Immunoassay, Veritor System Flu A+B assay. One conventional test was the SD Bioline Influenza Ag A/B/A(H1N1/2009). All test results were compared with those from the Anyplex Flu A/B Typing Real‐time Detection real‐time PCR. The four RIDTs were tested with diluted solutions from the National Institute for Biological Standards and Control (NIBSC) to compare lower detection limit. Cross‐reactivity of four RIDTs within other respiratory viruses was identified. RESULTS: For influenza A, BUDDI, Sofia, Veritor, and Bioline showed 87.7%, 94.5%, 87.7%, and 72.6% sensitivity, and 100%, 97.7%, 96.5%, and 100% specificity. For influenza B, BUDDI, Sofia, Veritor, and Bioline showed 81.7%, 91.7%, 81.7%, and 78.3% sensitivity, and 100%, 95.3%, 100%, and 100% specificity, respectively. Each RIDT could detect diluted NIBSC solution, according to the level of dilution and specific influenza subtypes. Cross‐reactivity of four RIDTs with other respiratory viruses was not noted. CONCLUSIONS: Sofia showed the highest sensitivity for influenza A and B detection. BUDDI and Veritor showed higher detection sensitivity than a conventional RIDT for influenza A detection, but similar results for influenza B detection. Further study is needed to compare the test performance of RIDTs according to specific, prevalent influenza subtypes. Seasonal influenza infection can be a burden on public health authorities. Rapid diagnosis is an important initial step in the appropriate management of influenza disease. Traditionally, viral cultures, serological tests, rapid antigen tests, and molecular methods have been used to diagnose influenza infection. 1 Rapid influenza diagnostic tests (RIDTs) have been used widely in the clinical setting because they can be handled readily and at relatively low cost, although they have low and variable detection sensitivities. Thus, a negative RIDT result does not confirm a status free of influenza virus infection. The clinical sensitivities of these tests have been reported to range from 20% to 90%, according to sample collection method, storage and transport, specimen type, swab and transport media used, and degree of adherence to manufacturers' recommendations for test procedures. 1 The performance of RIDTs is also dependent on the prevalence of the influenza viruses circulating in the population. 2, 3 Furthermore, clinicians should be cautious about using RIDTs in certain patient populations and with respect to how the results should be interpreted. [3] [4] [5] Thus, further validation of RIDTs should be provided before routine clinical use. In this study, we compared three RIDTs with digital readout systems, and one conventional RIDT, with respect to the detection sensitivity and relative limit of detection according to influenza subtype. We included 218 left-over nasopharyngeal swab specimens from non- BUDDI (NanoEnTek) is a newly developed digital readout system to diagnose influenza virus. The method is based on immunochromatography and a digital readout system. Briefly, an antibody colloidal gold probe was applied on the conjugate pad, and influenza antibody was immobilized to a nitrocellulose membrane as the capture reagent to prepare the RIDT strip test. Equal volumes of specimen in UTM (75 μL) and reagent buffer (75 μL) were mixed thoroughly and five drops (100 μL) of mixed sample was loaded into the test device. In 15 minutes, the BUDDI device reads the intensity of the reaction band. If the band intensity is higher than a cut-off level, BUDDI reports a positive result of influenza infection. This test required less than 30 minutes to accomplish the qualitative detection of influenza antigen. The results of the BUDDI system can be transferred to a central laboratory or the Centers for Disease Control (CDC) via an appropriate wireless network. The Sofia Influenza A+B Fluorescence Immunoassay (Quidel) is a rapid diagnostic kit that uses immunofluorescence technology to enhance its sensitivity. It detects the nucleoprotein of the influenza virus and can discriminate strain A from B in nasal swabs, aspirates, and nasopharyngeal swab specimens. This test uses a lateral flow design with location-dependent lines and zones. The Sofia Analyzer scans the test strip and displays the results after using methodspecific algorithms. This assay can be completed within 15 minutes, and results are reported as positive or negative for influenza virus A or B, without providing the numerical value assigned for each specimen. Briefly, equal volumes of specimen in UTM (250 μL) and reagent buffer (prepared by reconstituting lyophilized buffer with detergent and reducing agent, 250 μL) were mixed in a reaction tube. A final volume of 120 μL of sample was placed in a Sofia reaction cassette using a premeasured pipette, incubated for 15 minutes, and analyzed using the Sofia fluorescent reader. The results also can be interfaced directly using a local area network (LAN) to a laboratory information system. BD Veritor System Flu A+B assay (BD Diagnostics) is a rapid qualitative chromatographic immunoassay for the detection of influenza A and B viral nucleoprotein antigens from nasal and nasopharyngeal swabs of patients with a time-to-result of 10 minutes. The system comprises a unique reagent tube containing mucolytic agents and a detergent solution, a test device, and a digital reader. When nasal or nasopharyngeal swab specimens are processed with the reagent tube and added to the test device, influenza A or B viral antigens bind to anti-influenza antibodies conjugated to detector particles in the test strip. The antigen-conjugate complex migrates across the test strip to the reaction area, and is captured by antibodies against influenza A, influenza B, and a control antibody on three separate membranes. 6 For this study, 75 μL of specimen in UTM was mixed with an equal volume of lysis buffer (75 μL). Three drops (80 μL) of the mixed sample were placed into a Veritor test device cassette and allowed 10 minutes for incubation. Then, the cassette was placed into the colorimetric reader for analysis. is an immunochromatographic assay for the qualitative detection of influenza virus types A and type B using embedded mouse monoclonal anti-influenza A and anti-influenza B antibodies on the test strip. 7 Briefly, 75 μL of nasopharyngeal specimen in UTM was mixed with the same volume of reagent solution. The test strip was inserted into a tube containing the total volume of 150 μL of the reaction mixture. Test results were examined visually and interpreted after 10-15 minutes. Viral RNAs were extracted from 530 μL of the nasopharyngeal specimen using the SeePrep12 Viral NA kit (Seegene) and the SeePrep12 Viral NA instrument (Seegene). This is an automated nucleic acid (NA) extraction system for downstream detection, for extracting and purifying NAs using a magnetic bead method. An internal control (10 μL) was added to each specimen before the extraction step to confirm the entire process, from NA extraction to PCR. initial incubation at 50°C for 20 minutes and 95°C for 15 minutes, followed by 45 cycles of 95°C for 30 seconds and 55°C for 60 seconds. A comparison study was performed to compare the influenza subtype detection capacity among four different RIDT assays using 20 different influenza antigen reagents from the National Institute for Biological Standards and Control (NIBSC). First, the stock solution of each subtype reagent was diluted in the recommended buffer to 512 ng/mL. Then, the 512 ng/mL NIBCS influenza A subtypes were diluted serially to 128, 64, 16, 8, 4, 2, and 1 ng/mL. The 512 ng/mL NIBCS influenza B subtypes were diluted serially to 128, 64, 16, and 8 ng/mL. The diluted subtype reagents were measured to determine and compare the detection limit of each RIDT. The nonspecific positive results of four kinds of RIDTs with other respiratory viruses excluding influenza viruses were identified. All of 29 respiratory swab specimens were confirmed by real-time PCR (Seegene RV16 real-time PCR, Seegene Seoul, Korea) using manufacturer's recommended real-time PCR protocol. The RV16 realtime PCR has been updated from previous reports. The four RIDTs were compared according to sample volume, assay time, discrimination of influenza A and B, recommended specimen types, and test principles. We briefly describe the specifications of each RIDT in Table 1 . Also, the 218 enrolled samples were classified as 73 cases of influenza A, 60 of influenza B, and 85 negatives ( Table 2 ). Serially diluted NIBSC solutions showed some differences in low-level detection power between the RIDTs. Overall, the digitalized influenza detection tests had a higher detection power than those of the con- We identified that four RIDTs did not show cross-reactivity results in 29 specimens which had other respiratory viruses excluding influenza viruses. These differences could be caused by different coverage levels of monoclonal antibodies to specific influenza subtypes. RIDTs should be updated to target prevalent influenza subtypes and to identify many influenza subtypes (to increase detection sensitivity). This study had some limitations. First, we did not include large number of samples and various influenza subtypes. Second, the mean age of the enrolled individuals was relatively young, at 10-32.2 years, for a random selection within a specific period. Cruz et al. 2 reported that the performance of RIDTs have been known to be better in children compared with adults (approximately 13% higher), maybe due to higher viral loads and longer viral shedding in children compared with adults. We also identified the higher sensitivity of four RIDTs in children group than those of adults in our study. Third, test results were compared only with those of real-time PCR, which could have false positives and negatives. Also, only nasopharyngeal swab specimens were included, without considering specimen variations. However, for the first time, this study presented the detection sensitivity of a newly developed digital readout system, BUDDI. The BUDDI had the same detection sensitivity as a previously launched RIDT (Veritor), and higher sensitivity than a conventional RIDT. However, BUDDI showed less sensitivity than Sofia. Also, BUDDI could detect more than other RIDTs in terms of specific influenza biotypes, such as influenza A A/Texas/36/91, A/Beijing/262/95, and A/Sydney/5/97. In conclusion, RIDTs with digital readout systems showed higher detection sensitivity than a conventional rapid test. The fluorescence technique of Sofia gave it the highest detection sensitivity, but there were differences in low-level detection power according to influenza subtype. Further well-designed prospective studies are needed for additional assessment of the value of updated RIDTs according to specific influenza subtypes. Update on influenza diagnostics: lessons from the novel H1N1 influenza A pandemic Performance of a rapid influenza test in children during the H1N1 2009 influenza a outbreak Seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the Infectious Diseases Society of America World Health Organization (WHO) Use of influenza rapid diagnostic tests Guidance for clinicians on the use of rapid influenza diagnostic tests Clinical performance evaluation of the BD Veritor System Flu A+B assay Comparison of two new generation influenza rapid diagnostic tests with instrument-based digital readout systems for influenza virus detection Comparison of rapid antigen test and real-time reverse transcription PCR for the detection of influenza B virus Detection of 12 respiratory viruses with two-set multiplex reverse transcriptase-PCR assay using a dual priming oligonucleotide system Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene Evaluation of Seeplex RV detection kit for detecting rhinovirus, human metapneumovirus, and coronavirus Evaluation of multiple commercial molecular and conventional diagnostic assays for the detection of respiratory viruses in children Influenza-associated hospitalizations in the United States Mortality associated with influenza and respiratory syncytial virus in the United States Influenza vaccination among the elderly in the United States Broadly protective adenovirusbased multivalent vaccines against highly pathogenic avian influenza viruses for pandemic preparedness Production of adenovirus vectors and their use as a delivery system for influenza vaccines Current approaches for diagnosis of influenza virus infections in humans Accuracy of rapid influenza diagnostic tests: a meta-analysis Antiviral agents for the treatment and chemoprophylaxis of influenza -recommendations of the Advisory Committee on Immunization Practices (ACIP) World Health Organization (WHO) Geneva: Recommendations on the use of rapid testing for influenza diagnosis We thank NanoEnTek and Quidel corporation for providing test kits and NIBSC for the influenza subtype samples.