Comparison of SmartCycler Real-Time Reverse Transcription-PCR Assay in a Public Health Laboratory with Direct Immunofluorescence and Cell Culture Assays in a Medical Center for Detection of Influenza A Virus

Nadia F. Habib-Bein,^1,2 William H. Beckwith III,³ Donald Mayo,^1,3 and Marie L. Landry^1,2*

Yale University School of Medicine,¹ Yale-New Haven Hospital, New Haven, Connecticut,² Connecticut Department of Public Health Laboratory, Hartford, Connecticut³

Received 17 March 2003/ Returned for modification 24 April 2003/ Accepted 23 May 2003

A single-tube real-time (fluorogenic) reverse transcription (RT)-PCR with the SmartCycler instrument (SmartCycler RT-PCR) for influenza A virus detection was evaluated with 238 respiratory specimens. Direct immunofluorescence antibody staining (DFA) and primary rhesus monkey kidney cell culture were performed on-site at Yale-New Haven Hospital. Specimens were transported to the Connecticut Department of Public Health Laboratory for real-time RT-PCR. Cell culture detected influenza A virus in all 150 influenza A virus-positive specimens, DFA detected the virus in 148 influenza A virus-positive specimens, and SmartCycler RT-PCR detected the virus 143 influenza A virus-positive specimens. The sensitivity and specificity of RT-PCR were 95.3 and 100%, respectively. The high sensitivity and specificity and the rapid turnaround time made the SmartCycler RT-PCR valuable for the rapid diagnosis of influenza A, especially in a public health laboratory. The closed real-time RT-PCR system avoided cross-contamination possible with RT-PCR and the excessive manipulations required for conventional RT-PCR analysis and saved time and labor as well. In a medical center, rapid diagnosis by DFA was labor intensive but was 98.7% sensitive and 100% specific compared to the results of culture and provided results within 2 h throughout operating hours, helping with bed allocation on admission and patient management.

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* Corresponding author. Mailing address: Department of Laboratory Medicine, P.O. Box 208035, Yale University School of Medicine, New Haven, CT 06520-8035. Phone: (203) 688-3475. Fax: (203) 688-8177. E-mail: marie.landry@yale.edu.