Detection of herpes simplex viruses type I and II in dermal, genital, oral, cerebrospinal fluid, and lower respiratory specimens by a Roche HSV I/II analytic specific reagent kit on real-time polymerase chain reaction
Abstract number: p646
Liu S., Tichota-Lee J., DeBoer D.S., Marum S.A., Van Santen R.J., Koch M.R.
To contrast traditional cell culture and real-time polymerase chain reaction (PCR) methods for detecting herpes simplex virus type I and II (HSV I/II) in several types of clinical specimens.
A total of 112 specimens [dermal (n = 22), genital (n = 34), oral (n = 11), cerebrospinal fluid (n = 7), and lower respiratory (bronchial wash, n = 32 and bronchoalveolar lavage n = 6)] were tested blindly by using both traditional viral cultural and a HSV I/II analytic specific reagent (ASR) kit (Roche, Indianapolis, IN) at the same time. DNA was extracted by using a MagaZorb mini-prep kit (Cortex Biochem, San Leandro, CA) and the PCR was performed on Roche LightCycler with 45 cycles used. 20-ml of total volume (15-ml master mixture and 5-ml sample) was used in the PCR. Data analysis was carried out either through describing quantization issues (crossing point) or by charting the melting curves. PCR results were compared to culture findings to determine sensitivity and specificity.
The positive control on PCR was established by using Roche HSV I/II template DNA. The positive diagnosis on PCR was made by using the cut off of 100 copies of the template DNA (which will be adjusted in the near future because of the ASR qualification test for the HSV). Among all of the 112 patients' samples, 68/112 showed negative results by both methods; 25/112 showed positive by both; 6/112 showed clearly positive by PCR, but not viral culture; 11/112 showed peaks on the PCR designed HSV I/II positive ranges, but were unable to be diagnosed as positive compared to the cut off of the100 copies; 1/112 viral culture showed positive, but not PCR. 1/112 showed positive on viral culture and low peak on PCR.
Real-time PCR is a rapid test for HSV I/II diagnosis in the clinical virology laboratory with high sensitivity (25/27) compared with the traditional viral culture method. For the group which showed positive on PCR but not viral culture, DNA sequencing would be performed. This experiment is in progress and the results will be shared in the future.
|Session name:||XXIst ISTH Congress|
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