Detection of Herpes Simplex Virus and Varicella-Zoster Virus by Traditional and Multiplex Molecular Methods

Dejan Nikolic, MD, PhD; Debra Kohn; Belinda Yen-Lieberman, PhD; Gary W Procop, MD, MS


Am J Clin Pathol. 2018;151(1):122-126. 

In This Article


Culture and DFA have been the mainstays of diagnostics for the detection of HSV and VZV from vesicular, non-CSF specimens for many years. HSV grows rapidly in permissive cell lines, and detection is straightforward. The development of the HSV ELVIS Test System, which uses a genetically engineered cell line, improved upon the speed and accuracy of detection of this virus.[16] Numerous nucleic acid amplification assays have been developed for the detection of HSV or VZV, some of which have been FDA cleared for the detection of HSV from non-CSF specimens; one assay has received FDA clearance for CSF specimens.[12–14,17,18]

Gitman and colleagues[14] compared a laboratory-developed PCR and a commercially available PCR assay with DFA and culture for the detection of HSV from swab specimens; they reported the following sensitivities for the cytospin-DFA, culture, laboratory-developed PCR, and commercially available PCR, respectively: 86.2%, 87.9%, 98.3%, and 94.8%. Similarly, when Wilson et al[15] evaluated culture, DFA, and PCR for the detection of VZV from non-CSF specimens, they found PCR to be superior to traditional methods and the sensitivity of DFA to be 87.8%, and they recommended the elimination of culture, given its poor performance.

There is currently only one commercially available nucleic acid amplification assay that detects and differentiates HSV and VZV, the Solana HSV1 + 2/VZV Assay. We compared this assay with traditional methods, as well as to a laboratory-developed HSV/VZV assay, the ARIES HSV 1&2/VZV Combination Assay. In this study, both of these assays significantly outperformed the traditional methods for the detection of HSV and VZV, detecting more true-positive specimens. The sensitivity of the HSV ELVIS Test System was only 71.1%, whereas the nucleic acid amplification assays had sensitivities from 94.7% to 94.9%. These multiplex assays both provided typing results for HSV (ie, HSV-1 and HSV-2) without additional testing. Similarly, the VZV DFA had a sensitivity of only 71.4% in this study, whereas the sensitivities of the amplification assays ranged from 94.3% to 100%. These and previously reported findings support the transition from traditional virologic methods, such as culture and DFA, to nucleic acid amplification–based methods.

The reactivation of VZV in the genital area is an uncommon presentation but has been reported. Birch and colleagues,[19] in a 2003 published study using a laboratory-developed, multiplex assay for HSV-1, HSV-2, and VZV, were able to detect VZV in 2.9% of genital specimens. More recently, Granato et al,[2] using a commercially available isothermal amplification assay, detected VZV in 14 (11.1%) of 126 specimens from genital sites. These findings suggest that VZV is present more frequently than expected in genital lesions and that both viruses should remain in the differential diagnosis of vesicular lesions, unless the clinical presentation is characteristic (eg, obvious dermatomal distribution).

In this study, we demonstrated that 8.4% (8/95) of the specimens submitted for HSV testing were found to contain VZV. Half of these (4.2%; 4/95) were from genital sites, and importantly, three of these four were not clinically suspected to contain VZV (ie, a VZV DFA was not concomitantly ordered with the HSV culture order). Similarly, but perhaps more surprising, was that 19.4% (18/93) of specimens submitted for VZV DFA had HSV present as determined by both molecular methods. The HSV was an unexpected finding in 5.4% (5/93) of the specimens submitted for VZV testing (ie, a concomitant HSV culture was not ordered with the VZV DFA). The findings of unexpected HSV in specimens submitted for VZV and vice versa suggest that clinical differentiation of HSV and VZV disease is not always apparent. Therefore, the use of a combination HSV/VZV molecular detection assay is supported.