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EVALUATION OF DIAGNOSTIC HYBRIDS NEW D3 METAPNEUMOVIRUS MONOCLONAL ANTIBODY STAIN IN DIRECT SPECIMEN TESTING AND R-MIX TOO SHELL VIAL CULTURES Richard Henry, Kathy A. Fauntleroy, Davise H. Larone, Weill Cornell Medical Center/New York-Presbyterian Hospital, New York, NY Abstract

Methods

Results (Cont)

Background: Detection of Metapneumovirus (MPV) in clinical specimens can be accomplished by either direct fluorescent antigen staining of patient cells (DFA), tissue culture, or RT-PCR. A new 15-minute stain has been developed by Diagnostic Hybrids, Inc. (Athens, OH [DHI]) for use with both DFA and cell culture. Objective: The goal of this study was to evaluate the new DHI D3 MPV stain for use in DFA and R-Mix Too shell vial cultures (DHI). Methods: A total of 86 nasopharyngeal swabs and aspirates from children less than 3 years of age received in viral transport medium were included in the study. Specimens were centrifuged at 2000 rpm for 10 minutes. The cell sediment from the specimens were washed once in PBS, and cell spot preparations were made for the direct antigen staining. The slides were fixed in acetone, stained, and read according to the DHI D3 MPV instructions. The R-Mix Too culture vials were inoculated with 200 µL of supernatant from the specimens above according to the R-Mix Too package insert. The cultures were incubated for 24 hours at 35ºC and then fixed, stained, and read according to the DHI D3 MPV instructions. Results: Of the 86 specimens, 3 were positive for MPV by both DFA and culture. A fourth specimen was positive for both Influenza A and MPV by both DFA and culture. The staining intensity for the DFA preparations was between 2+ and 3+ fluorescence with little to no background staining. The R-Mix Too cultures showed a 3+ fluorescent intensity of infected cells with no background staining. Conclusions: The DHI D3 MPV stain shows great promise for both direct antigen and pre-CPE culture identification. The stain demonstrates little to no background staining when used for either DFA or culture confirmation. The ability of the stain to detect the virus by DFA or 24-hour culture allows a significant improvement over the current 10 to 14 days required for development of CPE in culture. The stain should prove to be a valuable tool in the clinical virology laboratory and allow rapid, straightforward detection of this new respiratory pathogen.

A total of 86 nasopharyngeal swabs and aspirates from children less than 3 years of age received in viral transport medium were included in the study. The specimens tested were received between 01/01/06 and 02/07/06. Each specimen was processed as follows:

Of the 86 specimens tested 4 (5%) were positive for MPV (1 specimen was a dual infection with influenza A). The R-Mix Too cultures showed a 3+ fluorescent intensity of infected cells with no background staining.

Background Human metapneumovirus (MPV) is a newly described viral respiratory pathogen. It has been describe as a causative agent of both upper and lower respiratory tract infections in the pediatric and geriatric populations. Published methods of detection for MPV include LLC-Mk2 tissue culture or reverse-transcriptase PCR. Recently the use of monoclonal antibodies for rapid detection of MPV from either direct specimens or centrifuged-enhanced cell culture has been described. The ability to detect infected cells from the patient’s specimen can give same day results without the need to PCR. The use of centrifugedenhanced cell culture improves the time to detection by cell culture from 14days to as fast as 1-day. The goal of this study was to evaluate the new DHI D3 MPV stain for use in DFA and R-Mix Too shell vial cultures (DHI) for the detection of MPV.

R-Mix Too Cultures

DIRECT SPECIMEN 1. Specimens were centrifuged at 2000 rpm for 10 minutes. 2. The supernatant was removed and saved for culture. 3. The cell sediment from the specimens were washed once with 1-mL of PBS. 4. Cell spots were prepared and allowed to air dry. 5. The slides were fixed in acetone for 5-minutes. 6. Each cell spot was stained with 25-µL of the D3 MPV stain. 7. The slides were incubated for 15-minutes at 35ºC. 8. Each slide was read for the presence of fluorescing cells. R-MIX TOO CULTURE 1. R-Mix Too shell vials were processed for culture according to the PI. 2. 200-µL of the specimen supernatant was added to 1-R-Mix Too shell vial. 3. The inoculated shell vial was centrifuged for 60-minutes at 700xg. 4. The vials were incubated for 18-hours at 35ºC. 5. The vials were fixed in acetone for 5-minutes. 6. The vials were stained with 200-µL of the D3 MPV stain and incubated for 15minutes. 7. The cover slips were removed from the vials and read for the presence of fluorescent cells.

Results DIRECT SPECIMEN Of the 86 specimens tested 4 (5%) were positive for MPV (1 specimen was a dual infection with influenza A). The staining intensity for the DFA preparations was between 2+ and 3+ fluorescence with little to no background staining.

Conclusion: The DHI D3 MPV stain shows great promise for both direct antigen and pre-CPE culture identification. The stain demonstrates little to no background staining when used for either DFA or culture confirmation. The ability of the stain to detect the virus by DFA or 24-hour culture allows a significant improvement over the current 10 to 14 days required for development of CPE in culture. The stain should prove to be a valuable tool in the clinical virology laboratory and allow rapid, straightforward detection of this new respiratory pathogen.


http://www.dhiusa.com/websites/website1/uploads/file/New_York_MPV_poster_CVS2006  

http://www.dhiusa.com/websites/website1/uploads/file/New_York_MPV_poster_CVS2006.pdf

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