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EVALUATION OF DIAGNOSTIC HYBRIDS NEW D3 HERPES SIMPLEX VIRUS TYPING KIT FOR CULTURE CONFIRMATION Kathleen H. Sullivan, Ph.D., D.(ABMM), Mary Ann Kohlmiller, MT(ASCP), Lois J. Legnasky, MT(ASCP), Deborah A. Mc Andrew, M.S., MT(ASCP), Associated Clinical Laboratories, a Quest Diagnostics affiliate, Erie, PA.

Abstract

Background: Identification and typing of Herpes simplex virus (HSV) isolates in cell culture from clinical specimens is accomplished by staining infected cells with virus type-specific fluorescent labeled monoclonal antibodies (MAbs). The level of expression of the viral antigens needed for this confirmation will vary from culture to culture depending on the virus and its titer in the specimen. To allow accurate confirmation and identification of the HSV isolate the MAbs used in the stain must produce detectable fluorescence even at the lowest level of antigen expression. The MAbs must also have a high degree of specificity to allow for the correct identification of either HSV type 1 or HSV type 2. This study evaluates a new confirmation stain that has been developed by Diagnostic Hybrids, Inc. (Athens, OH [DHI]). Objective: The goal of this study was to compare the new DHI D3 HSV Typing stain with the Trinity HSV 1/HSV 2 stain (Trinity Biotech, Carlsbad, CA and Wicklow, Ireland). The stains were used for culture confirmation of infected MRC-5 cells (DHI). Design/Methods: A total of 56 clinical isolates were included in the study. The isolates were inoculated into a MRC-5 tube culture. The cultures were incubated until CPE was observed. The culture tubes were processed according to each manufacturer’s package insert. The processed cell spots were then fixed, stained and read according to each manufacturer’s package insert. Results: The percent correlation between the two reagents was 98.3%. One HSV-2 isolate did not type in the Trinity reagent. The new D3 HSV typing reagent showed increased fluorescence in many of the isolates, however, the intensity of both reagents was sufficient to type all but one of the isolates. Conclusions: The D3 HSV Typing reagent produced increased fluorescent intensity compared to the Trinity reagent when used for isolate typing in tissue culture. It identifies both HSV-1 and HSV-2 equally well with no cross-reactivity, and the 15-30 minute staining time enhances workflow.

Methods A total of 56 isolates were included in the study. 1. The isolates were inoculated into a MRC-5 tube culture. 2. The cultures were incubated at 35°C for up to 5-days or until CPE was observed. 3. Four cell spots were made from the culture tubes showing CPE. 4. The processed cell spots were then fixed in acetone for 10-minutes. 5. The cell spots were stained according to each manufacturer’s package insert: DHI 30-minutes, Trinity 30-minutes. 6. The cells were interpreted for both the number of positive cells and the intensity of the fluorescence.

Trinity HSV-1

DHI HSV-1

Results The percent correlation between the two reagents was 98.3%. One HSV-2 isolate did not type in the Trinity reagent. The new D3 HSV typing reagent showed increased fluorescence in many of the isolates, however, the intensity of both reagents was sufficient to type all but one of the isolates. Trinity HSV-2

Background Background: Identification and typing of Herpes simplex virus (HSV) isolates in cell culture from clinical specimens is accomplished by staining infected cells with virus type-specific fluorescent labeled monoclonal antibodies (MAbs). The level of expression of the viral antigens needed for this confirmation will vary from culture to culture depending on the virus and its titer in the specimen. To allow accurate confirmation and identification of the HSV isolate the MAbs used in the stain must produce detectable fluorescence even at the lowest level of antigen expression. The MAbs must also have a high degree of specificity to allow for the correct identification of either HSV type 1 or HSV type 2. This study evaluates a new confirmation stain that has been developed by Diagnostic Hybrids, Inc. (Athens, OH [DHI]).

DHI HSV-2 Conclusion: The D3 HSV Typing reagent produced increased fluorescent intensity compared to the Trinity reagent when used for isolate typing in tissue culture. It identifies both HSV-1 and HSV-2 equally well with no cross-reactivity, and the 15-30 minute staining time enhances workflow.

Evaluation of Diagnostic Hbyrids New D3 Herpes Simplex Virus Typing Kit for Culture Confirmation  

http://www.dhiusa.com/upload_files/files/ACL_Typing_Final_poster_CVS2006.pdf

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