Clinical Validation of the Lyra Direct HSV 1+2/VZV Assay for Simultaneous Detection and Differentiation of Three Herpesviruses in Cutaneous and Mucocutaneous Lesions

Abstract

We evaluated the Lyra Direct HSV 1؉2/VZV multiplex real-time PCR assay for the detection and differentiation of herpes simplex virus 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) on 695 consecutive cutaneous and mucocutaneous lesion specimens. The intra-assay and interassay coefficient of variation values for the Lyra assay were 0.29 to 1.30% and 2.33 to 2.61%, respectively. The sensitivities, specificities, and positive and negative predictive values were 93.4 to 95.0%, 96.1 to 96.8%, 78.0 to 80.3%, and 99.0 to 99.1%, respectively, in comparison to those of viral culture. The values were further improved when a resolution analysis was performed with a laboratory-developed PCR assay. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2, respectively) and varicella-zoster virus (VZV) cause serious cutaneous and mucocutaneous lesions at every stage in immunocompromised patients (1). Due to the similarities in the clinical presentations of infections caused by these three herpesviruses, a clinical diagnosis needs to be confirmed by laboratory testing to prevent misdiagnosis (2). The need for accurate and specific laboratory diagnostics is especially important in immunocompromised patients who may present with atypical lesions confounding the clinical diagnosis and delaying the institution of appropriate antiviral therapy (1, 3, 4). Consequently, etiologic diagnosis and differentiation of HSV-1, HSV-2, and VZV infections is critical for both patient care and infection control (3, 4).Traditional laboratory methods for the diagnosis of cutaneous and mucocutaneous HSV-1, HSV-2, and VZV infections include the Tzanck smear, direct fluorescent assay (DFA), and cell culture (including shell vial culture) (3). Several reports have been published on the increased sensitivity of real-time PCR assays for detecting HSV/VZV over the traditional methods described above (5-7). Most published molecular assays utilize separate reactions for either HSV or VZV, and limited multiplex formats for the simultaneous detection and differentiation of HSV-1, HSV-2, and VZV have been reported (7-9). Currently, four molecular assays have been cleared by the Food and Drug Administration (FDA) for the detection and/or typing of HSV-1 and HSV-2 in genital and/or oral lesions: the MultiCode HSV-1&2 assay (Luminex Corporation, Austin, TX), the ProbeTec HSV QX amplified DNA assay (BD Diagnostics, Sparks, MD), the IsoAmp HSV assay (BioHelix Corporation, Beverly, MA), and the AmpliVue HSV 1ϩ2 assay (Quidel Corporation, San Diego, CA) (10).(This study was presented in part at the 114th Annual Meeting of the American Society for Microbiology, Boston, MA, 17 to 20 May 2014.)In May 2014, the FDA cleared the Lyra Direct HSV 1ϩ2/VZV assay (Quidel Corporation, San Diego, CA). This is the first FDAcleared commercial multiplex real-time PCR assay that simultaneously detects and differentiates HSV-1/2 and VZV DNA in both cutaneous and mucocutaneous lesion specimens. The objectives of this preclinical study were to validate the performance of the Lyra assay on cuta...

Development and Validation of a Laboratory-Developed Multiplex Real-Time PCR Assay on the BD Max System for Detection of Herpes Simplex Virus and Varicella-Zoster Virus DNA in Various Clinical Specimens

“…For HSV, both the routine and BD Max assays targeted the viral DNA polymerase gene and were able to detect HSV-1 and HSV-2 without type differentiation, as previously described (31). For VZV, the BD Max assay targeted the viral DNA polymerase gene (32) whereas the routine assay targeted glycoprotein 19 (11). For the BD Max multiplex assay, primers targeting the sample process control (SPC) were added to the PCR master mix; this internal control consists of a fragment of Drosophila melanogaster genome (GenBank sequence accession no.…”

“…This trend is due to the multiple advantages of nucleic acid testing (NAT), including the rapid availability of results, its adaptation to a wide diversity of clinical specimens, the capacity for automation, and, mostly, its high sensitivity and specificity. Several FDA-approved or CE-marked in vitro diagnostic (IVD) kits are available for detecting these viruses in various specimens (9)(10)(11). However, all of them propose separate detection for HSV and VZV DNA, and only a few multiplex assays including both agents have been published as laboratory-developed tests (LDTs) (10,12).…”

Evaluation of 2 multiplex real-time PCR assays for the detection of HSV-1/2 and Varicella zoster virus directly from clinical samples

“…Multiplex assays offer the potential to detect multiple pathogens in a single test, which may reduce the amount of sample required, the hands-on time, and turnaround time for results. In this study, we compared 2 commercial assays (Lyra™ HSV 1 + 2/VZV assay [Quidel, San Diego, CA, USA] (Fan et al, 2014) and Simplexa™ HSV-1/2 and VZV combined analyte specific reagents [Focus Diagnostics, Cypress, CA, USA]) (Burrel et al, 2012) to routine real-time PCR for HSV-1, HSV-2, and VZV.…”

“…Briefly, 100 μL of sample was heated for 5 minutes at 60°C followed by the addition of 25 μL of process buffer. Subsequently, 5 μL of each sample and 15 μL of mastermix were added to each SmartCycler cuvette and then tested on a SmartCycler (Cepheid, Sunnyvale, CA, USA) (Fan et al, 2014).…”

Microbiology laboratory and the management of mother-child varicella-zoster virus infection

“…Molecular biology tests are also useful in the case of varicella appearing 7-42 d after vaccination, in the case of herpes zoster appearing 42 d after vaccination and, in the case of the suspected transmission of the vaccine virus, can distinguish virus vaccine, wild-type virus and potential recombinants of vaccine and wild-type viruses [30,451,452,454,455,[458][459][460][461][462][463] , although some tests have proved to be less appropriate over time for these purposes [30] . There are also some multiplex assays that can differentiate HSV-1, HSV-2 and VZV in cutaneous and mucocutaneous samples, making them very useful in the case of skin lesions of unclear etiology or immunosuppressed patients [429,464,465] . Multiplex tests are rapid, need only a small volume of sample, and can simultaneously detect different viral agents at the same time in a single, closed-tube reaction [466][467][468] .…”