ERAGEN INFLUENZA REAL-TIME PCR ASSAY EVALUATION ON THE LIGHT CYCLER BY COMPARISON WITH EIA AND PMK SHELL VIAL

Background:  Rapid enzyme immunoassay (EIA) sensitivities in our laboratory were low for ’06 -‘07 (63/40%) for influenza A/B, as compared to culture.  In an effort to improve our respiratory virus diagnostic services, we are considering adding molecular testing for influenza to our virology laboratory menu.  The two Food and Drug Administration (FDA)- approved polymerase chain reaction (PCR) assays currently available are not appropriate for our situation – one is not a real-time assay, so is not useful for clinic patients, while the other cannot be used on our LightCycler platform.  We therefore chose to evaluate the EraGen MultiCode-RTx real-time PCR technology (EraGen Biosciences, Madison, WI).  This novel system with its unique bases measures a  decrease in fluorescence as the product is amplified. 

Methods:  Specimens were those submitted to the Diagnostic Virology Laboratory at the Louisiana State University Health Sciences Center at Shreveport for routine respiratory virus screen.  One aliquot was tested for influenza A and B antigen using Binax (Scarborough, Maine) EIA according to suggested manufacturer’s instructions.  Specimen was also inoculated onto two shell vials containing primary monkey kidney cells for culture and staining (Millipore – Chemicon – Light Diagnostics, Temecula, CA) at 24-72 hours.  A second specimen aliquot was frozen at -70 C until such time as the PCR could be performed.  Thus far, 51 of 100 test samples have been completed and the influenza results from the three methods, i.e. EIA, culture and PCR, compared. 

Results:  Twenty (39%) specimens were negative by all three assays.  Only 8 of the 29 (28%) PCR-positive specimens were positive by EIA, and none were positive for influenza B rapid testing.  Of those EIA positive samples, culture missed one that was overgrown with RSV.  Six PCR-positive specimens - 4 influenza A, 1 influenza B and 1 influenza A/B - were EIA negative, but culture-positive, with only influenza B indentified by culture in the dual infected specimen identified by PCR.  Seven specimens were culture-negative, PCR positive for influenza A; 4 specimens were culture-negative, PCR-positive for influenza B.  An additional 4 specimens were culture screen positive, not further identifiable, but PCR-identified as two influenza A and two influenza B. 

Conclusions:  Thus far, 11/51 (22%) specimens were found to be culture-negative, but positive for influenzavirus using EraGen PCR technology.  We plan to complete 100 specimens and to analyze discrepant samples by amplification of a second target.  Based upon those data, we hope to fully evaluate the EraGen system for next influenza season with the possibility of dropping culture from our menu.