Quantitative Detection of Human Herpes Virus 1 & 2 in Clinical Samples Using the Real Time PCR STAT-NAT HSV1 & 2 Assays.

Introduction: Herpes simplex virus 1 and 2 (HSV- 1 and HSV-2) are 2 members of the human Herpesviridae family. Both HSV-1 and HSV-2 are very common, lifelong infections that affect up to 90% of adults in the world, depending on socioeconomic status. HSV-1 can cause a wide variety of diseases, spanning from common cold sores and genital herpes to the more severe encephalitis, pneumonitis, hepatitis, and many ocular pathologies, especially in immunocompromised patients. HSV-2 is responsible for one of the most common sexually transmitted infections, can be contracted during pregnancy, and can be transmitted to newborns. Both infections are often asymptomatic or subclinical, and due to the peculiar mechanism of viral latency, can cause recurrent episodes. Methods: Both assays developed are in a readyto- use test format containing all the required elements for the amplification of both HSV-1 or HSV-2 DNA fragments and human beta-globin genes as internal controls. Two sets of primers and probes are combined in a lyophilized and ready-to-use mix, co-amplified, and detected by a Real-Time PCR instrument. Several clinical samples (n = 60) obtained from L. Sacco Hospital (ASST FBF Sacco - Milan, Italy), previously tested with InGenius HSV-1/HSV-2 ELITe MGB Kits (ELITech), were investigated. PCR reactions were performed on nucleic acids extracted from plasma, swabs, liquor, and vitreous humor. Analytical assessment studies were conducted including: LoD and LoQ, precision, reproducibility, linear range, and specificity using 2 plasmids (IDT) containing regions for both HSV-1/HSV-2 targets. Results: These detection kits proved to be specific for HSV-1 and HSV-2, and did not crossreact with the other human herpesvirus tested. The test performances showed a lower LoQ of 10 genome copies/reaction and an LoD of 10 genome copies/reaction; a linear range between 107 and 101 genome copies/reaction, and a precision CV <5% for both assays. The new freeze-dried, ready-to-use assays demonstrated robust and accurate target amplification, according to the data obtained at L. Sacco Hospital. The quantification of the samples was precise, reproducible, and comparable to that obtained with the predicted device. Conclusions: The described Real-Time PCR assays proved their effectiveness for the detection and quantitation of HSV-1 and HSV-2 DNA in clinical samples from different matrices. The high sensitivity and specificity, linearity, and quantitation performances of these assays, associated with the ready-to-use and room temperature storage, would have a direct impact on the early and correct management of affected patients.