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

<div><p>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.</p></div

Reproducibility.

<p>Reproducibility.</p

Primers and probes.

<p>Primers and probes.</p

Summary of CSF analyses with BD Max.

<p>Summary of CSF analyses with BD Max.</p

Scheme of workflow.

<p>Abbreviations: p/p, primers/probes; PC, positive control; MM, master mix; NC, negative control.</p

Plasmids used as positive control.

<p>Plasmids used as positive control.</p

Interlaboratory comparison/reference samples.

<p>Interlaboratory comparison/reference samples.</p

Detection of third-generation cephalosporin-resistant Enterobacteriaceae in returning soldiers.

<p>Detection of third-generation cephalosporin-resistant Enterobacteriaceae in returning soldiers.</p

Detection of enteric colonization with third-generation cephalosporin-resistant Enterobacteriaceae in returned soldiers and previously reported detection rates in selected African and Asian countries (in alphabetic order).

<p>Detection of enteric colonization with third-generation cephalosporin-resistant Enterobacteriaceae in returned soldiers and previously reported detection rates in selected African and Asian countries (in alphabetic order).</p

Detection of third-generation cephalosporin-resistant Enterobacteriaceae per year and yearly percentage of respective detections.

<p>Detection of third-generation cephalosporin-resistant Enterobacteriaceae per year and yearly percentage of respective detections.</p