A-308 The Molecular Diagnosis of Varicella Zoster Virus (VZV): A Validation of a Real-Time PCR (qPCR) Assay for the Qualitative Detection
Authors: Mendonça C P , Zauli D A 
Published: September 2023

Abstract

Background
Varicella zoster virus (VZV) is the etiological agent of chicken pox and herpes zoster. After the primary infection, which usually occurs in childhood, VZV remains latent in sensory nerve roots for life. When reactivation occurs, especially in immunocompromised patients, the virus migrates to the skin and causes painful rashes that can evolute to a dermatome. Even after the end of the viral acute infection, some individuals may experience continued pain. Furthermore, VZV reactivation can lead to a wide spectrum of clinical manifestations such as self-limited radicular pain and central nervous system involvement. During acute illness, 90% of patients experience pain and 12% have symptoms similar to influenza infection (influenza-like). Immunocompromised patients may have an unfavorable course with disseminated infection, pneumonitis, hepatitis, and encephalitis. The cerebrospinal fluid polymerase chain reaction (PCR) test is indicated for suspected involvement of the central nervous system and complications of ocular or periocular lesions. Compared to other diagnostic methods, real-time PCR has great advantages as its high throughput, efficiency, and sensitivity. Therefore, the detection of VZV by real-time PCR presents great importance for the diagnosis and monitoring of the disease due to its precision and agility. Thus, we intended to validate a qualitative method for VZV detection by real-time PCR.

Methods
We evaluated the performance of the inventoried TaqMan assay (Vi06439647_s1) (Thermo Fisher Scientific). For this validation, we used 64 samples - whole blood (n = 31), cerebrospinal fluid (n = 32), vaginal lesion scraping (n = 1) - from the Genetics sector of the Pardini Group, previously tested by in-house PCR for the qualitative detection of VZV. Samples were extracted using the MagNA Pure 24 System platform (Roche Diagnostics) with the MagNA Pure 24 Total NA Isolation Kit, according to the manufacturer's instructions. All assays were performed using Taqman Universal Master Mix (Thermo Fisher Scientific). The inventoried B-actin Assay (assay ID: Hs01060665_g1) (Thermo Fisher Scientific) was used as endogenous control. Assay verification tests, evaluation of reaction efficiency, and determination of the detection limit (LOD) were performed using a synthetic control of known concentrations and a pool of negative samples (healthy volunteers). At the end of the tests, were made a comparison of the previous results with the results obtained in the ThermoFisher Real-Time PCR Assay.

Results
The Inventoriad Taqman Assay (VZV) had a limit of detection of 10 copies/µL and the Cq &gt; 35 was defined as a cut-off to consider a sample as positive. Whole blood samples had higher Cts than cerebrospinal fluid samples, indicating lower viremia. Whitin all evaluated samples, 11 samples had positive results detected by in-house PCR (9 from whole blood and 2 from cerebrospinal fluid). Of these, 10 had a result detected in Real-time PCR and the agreement with the positive results between compared methodologies was 91.0%. The divergent sample was whole blood and can be related to DNA degradation. The 47 negative samples evaluated were concordant in both methods.

Conclusion
The inventoried TaqMan Assay with Taqman Universal Master Mix protocol evaluated was sensitive, fast, and applicable as a tool for the qualitative detection of VZV.