Testing for Herpes Simplex Virus in Low-Volume Cerebrospinal Fluid Samples

HerpesSimplexVirus-960x540Detection of herpes simplex virus (HSV) types 1 or 2 in cerebrospinal fluid (CSF) is considered a medical emergency, especially in infants and young children. If untreated, HSV encephalitis (HSVE) can have a mortality rate of 70%.

Rapid and accurate detection of HSV is critical to ensure proper management of patients. However, it is relatively common for clinical microbiology laboratories to receive an inadequate volume of CSF to perform testing. In fact, at Mayo Clinic alone, this happens in an estimated 10% of CSF samples that are received. Therefore, there has been a significant amount of interest in how to properly test for HSV in low-volume CSF samples.

To determine the optimal method for detection of HSV-1/2, Mayo Clinic researchers compared the Mayo Medical Laboratories (MML) routine real-time PCR method to three protocols designed to analyze CSF samples: 1) heat extraction, 2) dilution, and 3) a recently FDA-cleared test, the Focus Simplexa® Direct HSV-1/2 (Focus Diagnostics, Cypress, CA). The study was published in the Journal of Clinical Microbiology.

The heat extraction protocol involves heating available CSF to 95 degrees Celsius to release the nucleic acid and subsequently testing 5 microliters of the heated sample. Alternatively, the dilution protocol dilutes available CSF to a total volume of 200 microliters, which is the amount of CSF that is needed for standard nucleic acid extraction on the automated platform used at MML. Finally, the new FDA-cleared test (Focus Simplexa Direct HSV-1/2) requires only 50 microliters of CSF, which can be pipetted directly into the supplied testing disc without the need for prior extraction. Importantly, the Focus Simplexa assay can detect and differentiate HSV-1 and HSV-2 in approximately one hour.

Matthew Binnicker, Ph.D.
Matthew Binnicker, Ph.D.

“Determining the optimal protocol to follow in these cases is important so that providers can obtain results and make informed decisions about how to manage their patients,” said Matt Binnicker, Ph.D., Director of the Clinical Virology Laboratory at Mayo Clinic and senior author of this study.

Identifying HSV-1/2generally dictates the course of treatment. Dr. Binnicker added, “The detection of HSV-1/2 in CSF often means that antibiotics can be discontinued and the patient can be treated with an antiviral. In contrast, when HSV-1/2 is not detected, antivirals may be discontinued and further testing can be performed to identify an alternative diagnosis.”

The study identified that the heat and dilution protocols had a sensitivity of only 73.2% and 71.6%, respectively, compared to MML’s routine real-time PCR method. In contrast, the FDA-cleared test showed a sensitivity of 97.8%. Additionally, the heat and dilution protocols yielded a high number of “invalid” results, while the FDA-cleared test did not produce any invalid results.

These results demonstrate that the Focus HSV-1/2 Direct assay provides superior sensitivity for testing low-volume CSF in comparison to the heat-extraction and dilution protocols.

“The Focus Simplexa Direct HSV-1/2 assay showed comparable sensitivity with MML’s routine real-time PCR protocol,” said Dr. Binnicker. “Our current protocol requires 200 microliters of CSF and has a turnaround time of approximately 4 hours. In contrast, the Focus Simplexa Direct HSV-1/2 assay requires only 50 microliters of CSF and successfully generates results in about 1 hour.”

Due in part to these findings, the Clinical Virology Laboratory at Mayo Clinic will be transitioning all HSV testing on CSF to the FDA-cleared test in early 2016.

Kelley Luedke

Kelley Luedke

Kelley Luedke is a Marketing Channel Manager at Mayo Clinic Laboratories. She is the principle editor and writer of Insights and leads social media and direct marketing strategy. Kelley has worked at Mayo Clinic since 2013. Outside of work, you can find Kelley running, traveling, playing with her kitty, and exploring new foods.