Rapid and Direct Detection of Herpes Simplex Virus in Cerebrospinal Fluid by Use of a Commercial Real-Time PCR Assay

Abstract

Central nervous system infection due to herpes simplex virus (HSV) is a medical emergency and requires rapid diagnosis and initiation of therapy. In this study, we compared a routine real-time PCR assay for HSV types 1 (HSV-1) and 2 (HSV-2) to a recently FDA-approved direct PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) using cerebrospinal fluid samples (n = 100). The Simplexa HSV-1/2 assays demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively. In addition, the Simplexa assay does not require nucleic acid extraction, and the results are available in 60 min.

TEXT

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are a common cause of dermal, oral, and genital infections worldwide. Following primary infection, HSV-1 and HSV-2 establish latency in the dorsal root ganglia and persist there for the life of the host. In some cases, primary infection or reactivation of the virus can lead to central nervous system (CNS) disease (1). HSV-1 is responsible for approximately 10% of all cases of encephalitis and is the most common cause of fatal sporadic viral encephalitis worldwide. The mortality rate of HSV-1 encephalitis (HSE) may be >70% if untreated, and >95% of untreated survivors will suffer lifelong sequelae (2). Infection with HSV-2 may result in meningitis or meningoencephalitis, which may recur despite therapy (3). Neonatal infection with HSV-2 is especially devastating, and disseminated disease may occur in approximately 25% of cases (4).

Due to the high morbidity and mortality associated with HSV infection of the CNS, it is important to establish a diagnosis and initiate therapy as soon as possible. The detection of HSV-1 and HSV-2 in cerebrospinal fluid (CSF) using real-time PCR is now recognized as the gold-standard approach for diagnosing HSE and herpes meningitis (5, 6). A number of laboratory-developed real-time PCR assays for the detection and differentiation of HSV-1/2 have been described, with sensitivities and specificities typically of >95% (7–10). These methods have demonstrated superior performance compared to that of routine viral cell culture and have decreased the turnaround time in most situations to <8 h. However, laboratory-developed tests (LDTs) for the detection of HSV-1/2 in CSF lack standardization and require preanalytical nucleic acid extraction, which increases the turnaround time by up to several hours. Furthermore, the volume of CSF that is required for testing varies, and this can be an important factor in the diagnosis of neonatal HSV infection, when the amount of specimen recovered is typically small.

Recently, the Food and Drug Administration (FDA) approved the first real-time PCR assay (Simplexa HSV-1/2 Direct; Focus Diagnostics, Cypress, CA) for the detection and differentiation of HSV-1/2 from CSF. This assay does not require up-front nucleic acid extraction, and the results are available in approximately 60 min. In this study, we compared the performance of the Simplexa HSV-1/2 Direct assay to that of our routine real-time PCR (Roche HSV-1/2 analyte-specific reagents [ASR]; Roche Diagnostics, Indianapolis, IN) using a selected panel of clinical CSF samples (n = 100). The samples were submitted for routine testing by the Roche HSV-1/2 ASR on the LightCycler 2.0 (Roche), as previously described (11). This routine process includes preanalytic nucleic acid extraction on the MagNA Pure (Roche), which requires 200 μl of CSF. Following extraction, 5 μl of nucleic acid was tested by the Roche ASR and the results reported as positive or negative for HSV-1 and/or HSV-2, based on a melting curve analysis. In addition, a result of “HSV detected–type indeterminate” is possible with this assay when a melting curve is identified that falls between the expected ranges for HSV-1 and HSV-2. A prior study analyzed samples showing indeterminate results and using sequencing determined that HSV-1 or HSV-2 nucleic acid is present, but a 1- to 3-bp polymorphism in the probe region of the real-time PCR assay generates the abnormal melting curve result (11). Our internal validation of the Roche ASR demonstrated a limit of detection (LoD) for the CSF samples of ∼10 copies/μl. For this study, samples were selected following routine testing to ensure an adequate representation of positive (n = 52) and negative (n = 48) results. Among the 52 samples that were positive by our routine method, 37 (71.2%) were positive for HSV-2, 11 (21.2%) for HSV-1, and 4 (7.7%) were HSV detected–type indeterminate. The average crossing point (C_p) value of these positive CSF samples by the Roche ASR was 32.02 cycles (range, 27.25 to 34.39 cycles). Following routine testing, the samples were stored at 4°C and then tested in a blinded fashion by the Simplexa HSV-1/2 Direct assay within 48 h. Testing by the Simplexa assay was performed using the 3M Integrated Cycler (Focus), according to the manufacturer's FDA-cleared package insert. The Simplexa HSV-1/2 assay requires 50 μl of raw CSF, which is pipetted directly into the supplied Direct Amplification Disc (Focus). The samples showing discrepant results were stored at 4°C and tested within 24 h by a third real-time PCR assay (artus HSV-1/2 assay; Qiagen, Germantown, MD) (12), according to the manufacturer's European Conformity (CE)-marked product insert, with minor modifications. Briefly, nucleic acid extraction was performed using the MagNA Pure (Roche), and 5 μl of extract was combined with 15 μl of mastermix (Qiagen) and tested on the LightCycler 2.0 (Roche). According to the manufacturer, the LoD of the artus HSV-1/2 assay is 1 copy/μl.

Following the testing of 100 clinical CSF samples, the results of the Simplexa HSV-1/2 Direct assays were compared to those of our routine method (Roche HSV-1/2 ASR), which was established as the reference standard for this evaluation. The Simplexa HSV assays demonstrated a sensitivity of 100% (11/11) (95% confidence interval [CI], 70.0 to 100%) for HSV-1 and 100% (37/37) (95% CI, 88.8 to 100%) for HSV-2. Two samples were reported as HSV detected–type indeterminate by our routine method but were negative by the Simplexa HSV-1 and HSV-2 assays. The C_p values for these two samples were 32.3 and 32.7 cycles by the Roche ASR. Interestingly, these two samples were negative for HSV-1/2 by the artus real-time PCR assay and were also negative upon repeat testing by the Roche ASR. The specificities of the Simplexa HSV-1 and HSV-2 assays were 100% (85/85) (95% CI, 94.8 to 100%) and 98.3% (58/59) (95% CI, 90.2 to 99.9%), respectively (Table 1). One sample was found to be positive for HSV-2 by the Simplexa assay but was negative by our routine method. This sample demonstrated a C_p value of 40 cycles by the Simplexa assay and was negative for HSV-2 by artus, suggesting that the Simplexa result may have been falsely positive.

TABLE 1.

Comparison of the Focus Simplexa HSV-1/2 Direct assay to routine real-time PCR using cerebrospinal fluid samples^a

Results from Focus Simplexa HSV-1/2 Direct assay for	No. with Roche ASR HSV-1/2 result ofb:
	Positive	Negative	HSV Type Indeterminatec
HSV-1
Positive	11	0	1
Negative	0	85	3d
HSV-2
Positive	37	1d	1
Negative	0	58	3d

^an = 100. HSV-1/2, herpes simplex virus types 1 and 2.

^bASR, analyte-specific reagents.

^cHSV nucleic acid detected but unable to subtype as HSV-1 or HSV-2.

^dThese samples were negative by the artus HSV-1/2 real-time PCR assay. In addition, they were negative following repeat testing by the Roche HSV-1/2 analyte-specific reagents.

In this study, the recently FDA-approved Simplexa HSV-1/2 Direct assay demonstrated a combined sensitivity and specificity of 96.2% (50/52) and 97.9% (47/48), respectively, compared to the initial results of our routine method. Notably, the two samples that were negative by the Focus HSV-1/2 Direct assay but were HSV detected–type indeterminate by our routine method were repeated by the Roche ASR and found to be negative. These samples were also negative by the artus HSV-1/2 real-time PCR assay, suggesting that the initial results of our routine method may have been falsely positive. To our knowledge, this is the first report describing the performance of the Simplexa HSV-1/2 Direct CSF assay compared to that of the routine real-time PCR. Importantly, the Simplexa HSV-1/2 Direct assay does not require prior nucleic acid extraction, and it reduces the overall turnaround time by approximately 4-fold (∼4 h by our routine method compared to ∼1 h by the Simplexa assay). This may have a significant impact on the management of patients being evaluated for central nervous system disease, as a result in ∼1 h may allow for antimicrobial therapy to be rapidly adjusted. For example, a positive result for HSV-1/2 may prompt the discontinuation of antibiotic treatment, while a negative result may allow for acyclovir therapy to be discontinued and diagnostic testing to be focused on other potential etiologies. The Simplexa assay requires only 50 μl of CSF, which may be important in certain cases, including in the evaluation of neonatal CNS disease, where the recovery of CSF is often limited.