2011 American Society for Microbiology Meeting Vicki Healer, Suzane Silbert, Joshua Kay, and Raymond Widen Department of Esoteric Testing/R&D, Tampa General Hospital, Tampa, FL 2011 American Society for Microbiology Meeting Comparative Evaluation of the Focus DX and Cepheid Real Time PCR ASRs for HSV1 and HSV2 Abstract Results CONCLUSIONS Background: Herpes simplex viruses 1 and 2 (HSV1/2) are able to infect various sites and cause disease ranging from relatively minor cold sores and genital lesions to serious infections such as ocular infections and meningitis. Laboratory methods for detecting these viruses include direct antigen testing (DFA), culture and molecular methods. Molecular methods such as real time PCR offer the advantage of relatively rapid results and very high sensitivity. For this reason, PCR is considered the gold standard for detecting CNS infection. The goal of this study was to compare the performance of two different ASRs for PCR based detection of HSV1/2: the HSV 1/2 Focus DX ASR (CA, USA) and Cepheid HSV Typing ASR (CA, USA) assays. Methods: A total of 101 samples were tested using both ASRs; 53 were tested prospectively on fresh samples and 48 were tested from archived patient samples. Sample types involved in this study included CSF, lesion swabs and blood. All of them were first spiked with the Focus DX and Cepheid internal controls and then, processed for DNA extraction using the NucliSENS EasyMag Extractor (Biomerieux, France). The Cepheid ASR was performed on the Smart Cycler Real Time instrument (Cepheid, USA) and the Focus Dx ASR was performed on the Simplexa Integrated Cycler (3M, USA). Both tests utilized 5 microliter of the sample, with the final reaction volume of 25 microliter in the Cepheid ASR and 10 microliter in the Focus DX ASR. Both ASRs combined the detection into a single reaction using 3 sets of reagents with different reporters for HSV1, HSV2 and the internal control. Results: There was 100% agreement of the Focus Dx and Cepheid ASRs on the 53 fresh samples tested. On the archived samples there was agreement on 24 (100%) of the HSV2 samples and in 23 (96%) out of the 24 HSV1 samples. The one discrepant sample was negative on the Focus Dx and positive on the Cepheid test. However, this sample had a Ct of 36.5 on the Cepheid test. Conclusion: The Focus Dx HSV 1/2 and the Cepheid HSV 1/2 ASRs performed equally well in detecting HSV in clinical samples. Table 3. Specificity challenge results. Nucleic acids extracted from each of the agents was tested with the Focus DX HSV1/HSV2 assay. Table 1. Comparative evaluation of the Focus DX and Cepheid HSV1 ASRs The Focus DX HSV1/HSV2 real time PCR ASR assay run on the 3M Integrated Cycler was able to accurately detect these viruses in clinical samples including CSF, lesion swabs in UTM and blood. Comparing the performance of the Focus DX HSV1/HSV2 real time PCR ASR assay with the Cepheid Smart Cycler HSV1/HSV2 ASR assay revealed excellent correlation for samples tested in parallel and for archived samples. The Focus DX HSV1/HSV2 real time PCR ASR displayed no cross reactivity when challenged with organisms likely to be in CSF samples. 4. The 3M Integrated Cycler with rapid cycling allows for an assay with total run time of approximately 1 hour. Focus DX PCR Ct value for each target Positive Negative 26 1a 74 Cepheid PCR Challenge HSV1 HSV2 Negative control Positive Control Mix 27.4 26.4 CMV EBV VZV Enterovirus JC Virus Adenovirus M. tuberculosis N. meningitidis S. pneumoniae S. agalactiae E. coli M. pneumoniae L. monocytogenes The single sample that was HSV1 positive on Cepheid and negative was a frozen archived sample with Ct of 36.5. Insufficient sample for retesting. HSV1 performance summary for the Focus DX ASR with Cepheid as reference assay: Sensitivity= 96% Specificity= 100% PPV= NPV= 99% Table 2. Comparative evaluation of the Focus DX and Cepheid HSV2 ASRs Focus DX PCR Positive Negative 28 73 Integrated Cycler 96 position reaction disk Cepheid PCR HSV1 performance summary for the Focus DX ASR with Cepheid as reference assay: Sensitivity= 100% Specificity= PPV= NPV=