UTILITY OF MULTIPLEX REVERSE TRANSCRIPTION/REAL TIME PCR FOR DETECTION OF RSV, INFLUENZA A, AND INFLUENZA B IN NASOPHARYNGEAL ASPIRATES

Introduction: Commercial reagents for the detection of respiratory viruses including RSV, influenza A, and influenza B by nucleic acid amplification are becoming increasingly available and a number of studies have indicated that diagnostic yield of these assays is increased compared to conventional antigen detection and culture methods. The goal of this study was to determine the diagnostic utility of one such assay (ProFlu+ Research Use Only kit, Prodesse, Waukesha, WI) by testing a panel of nasopharyngeal aspirates (NPAs) containing known negative and positive samples, as identified by the laboratory’s current respiratory testing algorithm.

Method: Residual frozen NPA supernatants were selected for study and were categorized according to result obtained in the course of clinical testing. A tiered testing algorithm, consisting of immunochromatography (IC) for RSV, FluA, and FluB (BinaxNow, Inverness Biomedical, Scarborough, ME) followed by DFA (D³ Ultra Screening and ID reagents, Diagnostic Hybrids, Athens, OH) and culture only if IC results are negative, is employed during respiratory season. Viral RNAs were extracted using a BioRobot M48 and the Virus Mini protocol (Qiagen, Gaithersburg, MD). Multiplex PCR was performed according to the manufacturer’s instructions using a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA).

Results: A panel of 489 NPAs were tested including 298 that were negative by all conventional methods (IC, DFA, shell vial, and tube culture) and 191 that were positive for RSV (N=137), FluA (N=42), and FluB (N=12) as determined by either IC, DFA, or culture. In the negative NPA panel, valid PCR results were obtained for 294/298 specimens. 4 samples (1.3%) were invalid due to internal control amplification failure. 13 additional positives (4.4% of negative NPAs) were identified by ProFlu+, including 10 RSV (5 in children <18 yrs, 5 in adults), 3 FluA (2 in children, 1 in an adult). In the positive panel, 114/135 (86%) of specimens were confirmed as positive for RSV. 19/135 NPAs (14%) were negative by ProFlu+ (8/112 conventional positive results in children, 11/23 conventional positive results in adults). PCR failure (internal control inhibition) was observed in 2/135 NPAs. For FluA positive NPAs, 30/42 (71%) were confirmed positive by ProFlu+ ; 12/42 (29%) were negative for FluA by ProFlu+ (10 adult and 7 pediatric NPAs, with 2/7 pediatric NPAs positive for RSV rather than FluA). All FluB samples were confirmed as positive for FluB by ProFlu+. All conventional positive results that could not be confirmed by ProFlu+ were obtained by IC.

Conclusions:  A small increment of diagnostic yield was observed with ProFlu+. Surprisingly, the greater gain appeared to be the identification of false positive results obtained by IC. Further investigation revealed that the original results were obtained primarily during one particular shift. A quality assurance action plan consisting of re-training, technologist proficiency assessment, and sporadic re-testing of IC positive samples with ProFlu+ to monitor false positivity rates has been enacted to ensure the integrity of results obtained with the respiratory testing algorithm.