Poster # T 71 EVALUATION OF RAPID IDENTIFICATION ASSAYS FOR VIRAL MARKERS AND DETERMINING OPTIMAL SAMPLE SUBMISSION D. Hathaway, T. Hayes, D. Jaskot, J. Krahn, S. Milburn, A. Tirolese, R. Wheeler, and R. Lannigan Virology Department, London Laboratory Services Group, London, Ontario Abstract Objective: Rapid detection of viral pathogens is a staple of most clinical Microbiology laboratories. Methods to achieve this goal vary according to the depth of expertise and equipment available in each of these laboratories. Products to suit most (if not all) laboratory operational levels are available to provide timely and accurate clinical information for the front line care provider. Purpose: Laboratory protocols are continuously under pressure to provide rapid turnaround-time results coupled with a diverse scope of investigation. An evaluation of current and prominent rapid antigen detection methodologies was evaluated. This laboratories current protocol of direct fluorescent antibody investigation was compared to lateral-flow immunoassays. A secondary investigation regarding optimal volume of transport media utilized to submit a sample was also investigated. Method: Published sample handling and processing testing protocols were followed for both the rapid immunoassays techniques (Quidel: QuickVue / Meridian TRU FLU / TRU RSV) and direct immunofluorescence as currently performed by the investigating laboratory. Spiked respiratory samples were produced in-house and used to evaluate the two differing technologies. A second aspect to the investigation was the volume of transport media (Starplex Multitrans S-160) utilized to submit a patient sample. Results: The comparison of differing technologies for a similar purpose illuminates inherent advantages and disadvantages for each process. Most rapid immunoassays kits on the market provide comparable results when it comes to sensitivity and specificity. Direct fluorescent techniques allow for expanded testing algorithms, but fall victim to subjective evaluation and may also produce a false negative result for the patient sample that only contains a minimal number of infected cells or a large percentage of cell-free viruses. Analysis indicated that rapid immunoassays had a higher level of sensitivity than DFA techniques, but both comparisons to individual manufacturers products and volume of transport media utilized were not conclusive. Conclusions: Rapid and accurate analysis of respiratory infections in the hospital and community setting are of major concern. Depending on the scope and needs of the laboratory service, differing technologies may be utilized. In the case of cell-free viral samples the immunoassay technique has a distinct advantage over DFA, but the scope of testing offered by DFA is a certain advantage if required. Sample volumes related to transport media can, at the extremes, play a role but this needs to be evaluated by each testing site based on the extent of laboratory examination performed. Objective Diagnostic laboratories need to provide the critical patient information required to treat or control the presence of infectious agents. Many new technologies have been introduced in the past few years to challenge some of the identified standard techniques and some of the improving methodologies that have been established for years. Many laboratories have chosen to refer certain work out or stand pat on techniques utilized for many years without question. In light of some recent findings our laboratory decided to evaluate some competing assay technologies, different manufacturer’s products and differing sample submission conditions. Purpose The laboratory initiated an evaluation of current and common direct antigen detection methods. The current testing protocol involves respiratory samples tested by direct fluorescent techniques for eight common viral pathogens (Flu A/B, PF 1/2/3, AV, RSV and MPV). Along with culture backup and introduction of molecular based assays the laboratory identified failures in the last two consecutive External Quality Assurance challenges, which target DFA processes. In both cases the molecular method was able to properly identify all the challenge material. On the last examination we were also aided by the availability of some evaluation kits of lateral-flow immunoassays. It was also discovered that some manufacturers of the rapid kit methods are evaluating a sample submission volume of Viral Transport (Starplex Multitrans S-160, used by this lab) significantly reduced from the standard produced. Results Meridian Tru A+B, RSV Method for swabs in transport media. 1) Remove conjugate tube from foil pouch – label tube, remove and discard cap. 2) Using a kit supplied transfer pipette, add 100 uL (second mark from the tip) of sample diluent. Swirl or vortex 10 seconds. 3) Add 100 uL of well mixed patient sample. Vortex or mix with pipette 10 seconds. 4) Add test strip and secure in place. Incubate at 20 – 25 degrees Celsius for 15 minutes. Read results within 1 minute. QuickVue /RSV test Method / transport media 1) Add extraction reagent to fill line on supplied test tube (250 uL) 2) Fill supplied pipette with patient sample. Note the pipette is designed to collect and dispense the correct amount of liquid sample. 3) Add sample to tube. Swirl or shake. Wait one minute before placing test strip into tube. 4) Incubate 15 minutes at 20 – 25 degrees Celsius. QuickVue Influenzae A + B Method / transport media 1) Dispense all of the extraction reagent solution from the reagent tube. Gently swirl the extraction tube to dissolve its contents. 2) Fill the supplied pipette to uppermost notch. Add entire contents to tube. 3) Place test strip into tube. Incubate 10 minutes at 20 – 25 degrees Celsius. Chart 2 Influenza A/B Rapid Immunoassay Evaluation RSV Evaluation Specimen Panel – Lot 500557.026 Expected Results 1 Neg 2 Pos 3 Pos 4 Pos 5 Neg 6 Neg 7 Pos 8 Neg 9 Neg 10 Pos 11 Neg 12 Neg 13 Neg 14 Pos 15 Pos 16 Pos 17 Neg 18 Pos 19 Neg 20 Pos Methods [email protected] Typical Visual Observations: The following charts summarize the findings from evaluating an EQA panel and spiked samples created in the lab utilizing two different volumes of VTM. Chart 1 A Joint Venture between London Health Sciences Centre and St. Joseph’s Health Care Centre, London, Ontario, Canada Quidel QuickVue RSV – Lot 705336 exp. 2010.07.31 Meridian Tru RSV - Lot 751330.022 exp. 2010.04.20 Neg Pos Pos Pos Neg Neg Pos Neg Neg Pos Neg Neg Neg Pos Pos Pos Neg Pos Neg Pos Neg Pos Pos Pos Neg Neg Pos Neg Neg Pos Neg Neg Neg Pos Pos Pos Neg Pos Neg Pos Flu A/B Evaluation Specimen Panel – Lot 500556.024 Expected Results 1 Neg 2 A Pos 3 A Pos 4 B Pos 5 Neg 6 Neg 7 B Pos 8 Neg 9 Neg 10 A Pos 11 Neg 12 Neg 13 Neg 14 B Pos 15 A Pos 16 A Pos 17 Neg 18 B Pos 19 Neg 20 B Pos Quidel QuickVue Influenzae A/B – Lot 705425 exp 2010.09.10 Neg A Pos A Pos B Pos Neg Neg B Pos Neg Neg A Pos Neg Neg Neg B Pos A Pos A Weak Pos Neg B Pos Neg B Pos Meridian Tru Flu Influenzae A/B – Lot 751230.037 exp 2010.02.26 Neg A Weak Pos A Weak Pos B Pos Neg Neg B Pos Neg Neg A Pos Neg Neg Neg B Pos A Weak Pos A Weak Pos Neg B Pos Neg B Pos Conclusions Strongly positive samples as indicated by immunoassay and DFA analysis Respiratory Syncytial Virus Evaluation of Transport Media Volume Serial Dilution Quidel QuickVue Starplex VTM 2.7 mL Quidel QuickVue Starplex VTM 1.0 mL Meridian Tru Flu Starplex VTM 2.7 mL Meridian Tru RSV Starplex VTM 1.0 mL Neat A Pos B Pos RSV Pos A Pos B Pos RSV Pos A Pos B Pos RSV Pos A Pos B Pos RSV Pos 10^2 Weak Pos Weak Pos Pos Pos Pos Pos Pos Weak Pos Neg Pos Pos Pos 10^4 Weak Pos Weak Pos Pos Pos Pos Pos Neg Weak Pos Neg Weak Pos Weak Pos Weak Pos 10^6 *Very weak POS *Very weak POS Neg Weak Pos Weak Pos Pos Neg Neg Neg Neg Neg Neg 10^8 Neg Neg Neg Trace POS Neg Trace POS Neg Neg Neg Neg Neg Neg 10^10 Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg Neg *Very weak positive… very difficult to read. Strong positives and weak reactions which can be easily interpreted as negative by DFA Many factors need to be considered when a testing facility adopts an algorithm for the testing of patient samples. Commercial companies have recently expanded the considerations regarding the direct detection of primarily viral pathogens, but will also impact secondary analysis of expanded microorganisms by predominantly molecular based assays. Ease of performance and interpretation coupled with rapid turnaround-time favor the lateral flow immunoassays. Our study compared only two of the kits on the market and there were distinguishable sensitivity differences when in-house prepared materials were utilized. When External Quality Control (supplied by Somagen Diagnostics Inc.) was utilized as test material, results were comparable. Direct Fluorescent Antibody analysis allows the lab to easily expand a testing menu to include other pathogens of choice, but is dependent on the availability of Fluorescent Microscopy and expertise in interpretation. Also noted was the fact that some samples submitted lack an adequate number of intact and appropriate cells for analysis which lead to false negative or indeterminate conclusions, when compared to immunoassays or molecular techniques. This was primarily evident for Influenza B in this study, but historically also included Influenza A and RSV. The amount of Viral Transport used in sample collection has some noticeable effect on the positive/negative interpretation to a degree, but varied slightly between viruses analyzed and manufacturer of kit used which did not qualify for statistical analysis. The determination of VTM volume to utilize should be based on the extent of testing that will be performed post direct analysis as our findings showed only some advantages to more concentrated samples. Those labs still performing viral culture techniques or molecular testing for both viral and bacterial targets may be limited to the larger volume, but should not witness significant reduction in performance.
© Copyright 2024