Comparison of the Focus Diagnostics Simplexa™ Flu A/B & RSV Direct Assay with the Prodesse® ProFlu™ + Assay for Detection of Influenza A Virus (FluA), Influenza B Virus (FluB), and Respiratory Syncytial Virus (RSV) in Clinical Specimens Bobbie Collett Sutton MD PhD, Kevin Maggert MT(ASCP), Elizabeth Rowell MLT, and Renee Etter BS. South Bend Medical Foundation, Inc., South Bend, IN Introduction/Background. Influenza is an acute respiratory illness caused by infection with Influenza virus, which typically has two primary types: A and B (FluA and FluB). Influenza epidemics occur yearly, although FluA usually predominates. It affects all ages, but more complications occur in the very young, the aged, and immunocompromised individuals. It is estimated that influenza infection results in as many as 200,000 hospitalizations and up to 49,000 deaths in the United States every year (1). Another common human respiratory virus, Human Respiratory Syncytial Virus (RSV), is the leading cause of lower respiratory tract infection in infants and children. Like Influenza virus, there are two main types, A and B. Yearly epidemics also occur with RSV and typically show a mixture of types A and B. RSV infection causes significant morbidity in the United States, where RSV associated pneumonia or bronchiolitis results in up to 125,000 pediatric hospitalizations per year (1). Adults with impaired immunity or those 65 and older are also at risk for severe respiratory disease caused by RSV (1). Viral infections of all types have proven to be a difficult target for clinical laboratories as viruses typically require a tissue-based cultivation system. However, with the advent of PCR testing, confirmation of viruses in clinical specimens, including Influenza and RSV, has become routine. Typically, the first step in this process is extraction of nucleic acid from the specimen prior to use as starting material for PCR. This requires added technical steps and increases overall turnaround time for the test. However, other assay designs are available. We compared two Real-Time PCR (RT-PCR) tests that detect FluA and B and RSV A and B in clinical samples. The first, the Prodesse® ProFlu™ + assay (Prodesse® ProFlu™), requires extraction of nucleic acid from samples, whereas the second, the Focus Simplexa™ Flu A/B & RSV Direct assay (Focus Simplexa™), does not. These assays performed comparably and showed similar sensitivities when applied to archived clinical respiratory specimens. Table 4. Viral Dilution Studies for Assessment of Assay Sensitivity Results. Time to completion of each phase of the testing process, beginning with an unaltered sample and ending with data entry into the Laboratory Information System (Turnaround Time), was calculated for each assay (See Table 2). In addition to no requirement for nucleic acid extraction of the sample prior to starting the test, the Focus Simplexa™ kit supplies premixed reagents and requires fewer pipetting steps for the operator, both of which save substantial technical time. For an eight sample run (the limiting variable being 8 specimens to fill a Focus Simplexa™ disc), the Focus Simplexa™ was a significantly faster test, with a total time to completion in our laboratory of 1.75 hours, versus 4.08 hours for the Prodesse® ProFlu™ test. Table 2. Determination of Assay Turnaround Time Material and Methods. Sixty-eight clinical nasopharyngeal specimens collected in Remel MicroTest M6 transport media (M6; Thermo Fisher Scientific Remel Products, Lenexa, KS ) were tested by various methods and stored frozen at -80oC for up to 6 months. These were retrieved, thawed at room temperature, thoroughly vortexed, and divided into two aliquots (See Table 1, below). One aliquot was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit and the MagNA Pure 1.0 Instrument (Roche Diagnostics Corporation, Indianapolis, IN) . The resultant nucleic acid was interrogated using the Prodesse® ProFlu™ assay on a SmartCycler instrument (Cepheid, Sunnyvale, CA). The second aliquot was used unaltered in the Focus Simplexa™ assay per manufacturer’s instructions using a 3M™ Integrated Cycler instrument (Focus Diagnostics, Cypress, CA). Three nonclinical known positive FluB samples were also compared in the two assays. In order to address assay sensitivity, stock cultures of FluA (Influenza A virus [H1N1] [ATCC® VR-1520™]), FluB (Influenza B virus [ATCC® VR-1535™]), RSV A (Human respiratory syncytial virus [ATCC® VR-1540™]) and B (Human respiratory syncytial virus [ATCC® VR-1400™]) were diluted with M6 and run in duplicate on each platform. Additionally, a positive clinical FluB sample was also diluted with M6 and tested. Both assays perform reverse transcription of virus-specific RNA targets into complementary DNA copies (cDNA), with subsequent PCR amplification of cDNA and detection of amplicons using a flurophore based system. The Prodesse® ProFlu™ assay targets virus-specific sequences in the matrix, polymerase, and non-structural NS1 and NS2 genes for FluA, RSV A&B, and FluB viruses, respectively. The Focus Simplexa™ assay targets sequences in the matrix gene (Flu A&B) and the RSV M gene. With each cycle of the PCR, amplicons accumulate and increase the amount of fluorescent signal, with different targets being discriminated by the absorbance peak of a specific fluorophore. This generates an amplification curve for each analyte, with the threshold cycle (Ct) representing the intersection between the amplification curve and a threshold of detection for a positive result set by the manufacturer. The Ct is also a relative measure of the concentration of target viral sequence in the reaction. There was 100% concordance between previously recorded PCR test results and repeat testing of aliquots from 68 cases performed on the two platforms (See Table 3). The recorded Ct values generated by the Prodesse® ProFlu™ test changed very little when the assay was repeated following specimen storage and a single freeze-thaw cycle (data not shown). In the initial tests using the Prodesse® ProFlu™ assay, three specimens yielded an unresolved result, which according to the package insert could be due to PCR inhibition or reagent failure. Two of these aliquots gave a negative result on repeat testing while the third remained unresolved using the Prodesse® ProFlu™ assay. All three aliquots yielded a negative result on testing with the Focus Simplexa™ assay. A 4th previously negative specimen was also unresolved on repeat testing with the Prodesse® ProFlu™ assay, while the initial repeat test with the Focus Simplexa™ assay gave a negative result. Because of the infrequency of positive FluB tests in our laboratory, three known FluB positive non-clinical samples were also included in this project and all three tested positive for FluB on both platforms. Both assays performed equally well in viral dilution series, with end points for assay sensitivity reproducibly falling within one dilution for the two platforms (Table 4). Table 3. Samples Assessed Table 1. Workflow Process 1. Focus Simplexa™ Flu A/B & RSV Direct Assay. Prodesse® ProFlu™ Assay. ND: Not Detected. Runs 1 & 2 used stock Influenza B virus. Run 3 used a high Ct positive clinical Influenza B sample as starting material. Identify and Retrieve Stored Frozen Specimens Thaw at Room Temperature. Mix thoroughly. Make Aliquots: Extract Nucleic Acid Focus Simplexa™ Assay Prodesse® ProFlu™ Assay Aliquot #1 Aliquot #2 Conclusions The Prodesse® ProFlu™ and the Focus Simplexa™ Flu A/B & RSV Direct assays performed similarly and detected all positive FluA, FluB, RSV A and RSV B clinical nasopharyngeal specimens used in this study. The Prodesse® ProFlu™ and the Focus Simplexa™ assays showed similar sensitivities in viral dilution studies. The Focus Simplexa™ assay has a significantly faster turnaround time in our laboratory than the Prodesse® ProFlu™ assay. 1. A small number of cases were tested originally either by serology (enzyme linked immunoassay for influenza virus), or using the FilmArray respiratory virus panel (Biofire Diagnostics, Inc., Salt Lake City, UT). Three cases were known positive Flu B specimens that did not originate from clinical samples. Per manufacturer’s instructions. See Results section for more information. References http://www.cdc.gov Focus Simplexa™ Flu A/B & RSV Direct Assay product insert. Prodesse® ProFlu™ + Assay product insert.