Implementation and Evaluation of a Fully Automated Multiplex Real-Time PCR Assay on the BD Max Platform to Detect and Differentiate Herpesviridae from Cerebrospinal Fluids.

Thomas Köller,Daniel Kurze,Mirjam Lange,Martin Scherdin,Philipp Warnke,Andreas Podbielski

doi:10.1371/journal.pone.0153991

Abstract

A fully automated multiplex real-time PCR assay—including a sample process control and a plasmid based positive control—for the detection and differentiation of herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella-zoster virus (VZV) from cerebrospinal fluids (CSF) was developed on the BD Max platform. Performance was compared to an established accredited multiplex real time PCR protocol utilizing the easyMAG and the LightCycler 480/II, both very common devices in viral molecular diagnostics. For clinical validation, 123 CSF specimens and 40 reference samples from national interlaboratory comparisons were examined with both methods, resulting in 97.6% and 100% concordance for CSF and reference samples, respectively. Utilizing the BD Max platform revealed sensitivities of 173 (CI 95%, 88–258) copies/ml for HSV1, 171 (CI 95%, 148–194) copies/ml for HSV2 and 84 (CI 95%, 5–163) copies/ml for VZV. Cross reactivity could be excluded by checking 25 common viral, bacterial and fungal human pathogens. Workflow analyses displayed shorter test duration as well as remarkable fewer and easier preparation steps with the potential to reduce error rates occurring when manually assessing patient samples. This protocol allows for a fully automated PCR assay on the BD Max platform for the simultaneously detection of herpesviridae from CSF specimens. Singular or multiple infections due to HSV1, HSV2 and VZV can reliably be differentiated with good sensitivities. Control parameters are included within the assay, thereby rendering its suitability for current quality management requirements.

Highlights

Herpesviridae like Herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella zoster virus (VZV) are causative agents of skin or mucosal tissues lesions as well as of lifethreatening infections of the central nervous system (CNS) [1,2,3]
For clinical validation 123 cerebrospinal fluids (CSF) specimens were analyzed for the presence of HSV1, HSV2, and VZV genomes
The study focused on methodic benefits on routine diagnostic workflow, reproducibility, sensitivity, as well as the specificity of the individual species-directed PCR assays and compared the new method with an established accredited laboratory protocol employing

Summary

Introduction

Herpesviridae like Herpes simplex virus 1 (HSV1), herpes simplex virus 2 (HSV2) and varicella zoster virus (VZV) are causative agents of skin or mucosal tissues lesions as well as of lifethreatening infections of the central nervous system (CNS) [1,2,3]. PCR based diagnostics allow for a fast and sensitive detection of an increasing range of infectious agents and specimens [10]. Several commercially available and certified test-systems based on diverse protocols are available for separate HSV1, HSV2 and VZV detection from various clinical specimens [12]. In-house multiplex assays covering the simultaneous detection of up to 8 herpesviridae have been published [13,14,15,16]. For all these tests, numerous time consuming and error-prone manual working steps are necessary, i.e. preparation of working solutions, DNA extraction, transfer of DNA and mastermixes to the amplification devices

Methods

Results

Conclusion