Flow Cytometric Applications for Virus Detection in the Clinical Microbiology Laboratory: Enterovirus as a Prototype
RC She, SN Preobrazhensky, CA Petti, DW Bahler. University of Utah School of Medicine, Salt Lake City, UT; ARUP Laboratories, Salt Lake City, UT
Background: Culture and subsequent serotyping of human enteroviruses by fluorescence microscopy are both time-consuming and labor-intensive. The use of flow cytometry to detect virally infected cells has the potential of being more rapid, sensitive, and objective but has not been previously applied to enterovirus detection and serotyping.
Design: Primary monkey kidney (PMK) cells inoculated with several EV serotypes were trypsinized, fixed and permeabilized for staining with enterovirus-specific antibodies. Single color flow cytometry analysis was performed using an FC 500 instrument analyzing 5000 events. Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1-4 after inoculation and correlated with indirect fluorescence antibody (IFA) results.
Results: Echovirus 6, 11, and 30 infected cells were positive by flow cytometry for pan-enterovirus (Pan) and echovirus blend (EB) antibodies and negative for polio blend (PB), enterovirus blend (EVB), coxsackievirus B blend (CB), and isotype control (IC) antibodies. Coxsackievirus B1 infected cells were positive for Pan, CB, and coxsackievirus B1 antibodies and negative for PB, EVB, and IC antibodies. Results correlated with IFA in all cases but flow cytometry detected positive cells one day earlier than IFA. Figure 1. Rate of infection of PMK cells by coxsackievirus B1.
Conclusions: Flow cytometry can be effectively used for detecting enterovirus-infected cells in a laboratory setting. The potential advantages of flow cytometry over fluorescent microscopy include better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems. These could lead to faster laboratory identification of pathogenic viruses. Additional studies are still needed to extend this method to other viruses and optimize detection strategies.
Monday, March 9, 2009 1:00 PM
Poster Session II # 194, Monday Afternoon