Study population and data collection
In this single center study, test results of swab samples taken from presumably herpesvirus-associated lesions on the skin and mucous membranes which had been analyzed by a routine PCR testing for HSV‑1, HSV‑2 and VZV, were retrospectively evaluated at the Department of Dermatology, Medical University of Vienna. An in-house herpes PCR technique was performed on a total of 3677 samples collected during a period of 4 years. A standardized data sheet was compiled of each case giving details of the suspected diagnosis, sample site, the test requested as well as demographic data of the patients including age, gender and immune status. Several types of viral genome were investigated in each sample concomitantly, in particular HSV 1 and 2, VZV, whereas CMV, EBV and HHV‑8 only when requested by the clinician. The results of the PCR analysis documented on these data sheets were anonymized for this analysis. Missing data were collected from archived patient charts. Finally, the clinical diagnoses were categorized based on similarities of the clinical manifestation mentioned on the order form. The data were then transferred to SPSS (Statistical Package for the Social Sciences, IBM® SPSS Statistics®, version 24, Chicago, IL, USA).
Sample and specimen collection:
Using a sterile cotton tip or disposable scalpel, swabs from vesicles, pustules, erosions on skin and mucous membranes were sampled after gently removing the roof of the vesicle or pustule in order to collect the fluid and cellular material at the base of the lesion. If the site was ulcerated, crusts of the lesions were removed. The obtained material was applied on a simple slide and passed to the laboratory. The DNA from biopsy samples was transferred to the laboratory in sterile tubes.
From the slides, the material was scraped off and transferred to sterile containers suspended in 1.0 ml sterile H2O, boiled for 15 min and stored at + 4 C. For all samples, DNA was extracted using a DNA extraction kit (Qiagen DNeasy blood and tissue kit [QIAGEN, Hildesheim, Germany]), a procedure carried out according to the manufacturer’s instruction.
PCR amplification for HSV1, HSV2, VZV, B-actin:
The amplification reaction was carried out in 50 µl reaction mixture, containing 5 µl of 10X PCR reaction buffer, 1 µl of 10 mM dNTPs, 3 µl MgCl2, 2.5 µl of 10 pmol forward primers, 2.5 µl of 10 pmol reverse primers, 0.1 µl of 5 U/µl Red hot Taq deoxyribonucleic acid (DNA) polymerase (Thermo Scientific, Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 5 µl DNA samples (100 ng). Amplification was carried out with a thermal profile at 94 °C for 4 min initial denaturation then 40 cycles (94 °C for 1 min, 58 °C for 30 s and 72 °C for 1.5 min) amplification with a final extension of 72°C for 8 min. The primer pair (B-actin‑s and B‑actin-as) was used to amplify (570 base pair) human housekeeping gene B‑actin, the primer pair (HSV1‑s and HSV1-as) was used to amplify (448 base pair) HSV1 DNA, the primer pair (HSV2‑s and HSV2-as) was used to amplify (239 base pair) HSV2 DNA, the primer pair (VZV‑s and VZV-as) was used to amplify (326 base pair) fragment of VZV DNA.
PCR amplification for HV8, CMV and EBV:
The amplification reaction was performed as described above. Amplification was carried out with a thermal profile at 94 °C for 4 min initial denaturation then 45 cycles (94°C for 1 min, 58°C for 30 s and 72°C for 1.5 min) amplification with a final extension of 72°C for 8 min. The primer pair (HV8‑s and HV8-as) was used to amplify (335 base pair) HV8 DNA, the primer pair (CMV‑s and CMV-as) was used to amplify (453 base pair) fragment of CMV DNA, the primer pair (EBV‑s and EBV-as) was used to amplify (200 base pare) fragment of EBV DNA.
The primer pairs that are illustrated in the following table (B-actin‑s and B‑actin-as, HSV1‑s and HSV-1as, HSV2‑s and HSV2-as, VZV‑s and VZV-as, HV8‑s and HV8-as, CMV‑s, CMV-as, EBV‑s and EBV-as) were used to amplify the requested herpes virus DNA (Table 1
Primers used in the amplification of different human herpesviruses
CAA CTA CCC CGA TCA TCA GTT A
ACA GTT GCC TCC CAT CCG AAA CCA A
CAT CGG CGG TAT TGC GTT TTG GGT A
CCT ATG AAC TGT CCT AGT TTC C
GCT CGT TGA GGA CAT CAA CCG TGT T
CAT CGT CGC TAT CGT CTT CAC CAC
ACG AGG GGC CAG GTA CAG GA
CAC CAT CTC TAT GTC TTG GC
CAG CAC CAT CCT CCT CTT CCT CTG G
CCA AGC GGC CTC TGA TAA CCA AGC C
ATG GAT CCC TCT GAC AAC CTT
CGT GGA TCC GTG TTG TCT ACG
TTC CCC TCC ATC GTG GGG CGC CCC AGG CAC CAG GGC
GGC GAC GTA GCA CAG CTT CTC
The statistical analysis was performed using SPSS 21.0 software for windows (SPSS Inc., Chicago, IL, USA). Metric data were described by mean values and standard deviations. Nonmetric data, namely clinical diagnosis, localization, immune status and sex were described by contingency tables and frequency analysis. Appropriate graphs were used to illustrate both types of variables. For the qualitative data analysis χ2-tests or Fisher’s exact tests were used. All p-values were two tailed and considered statistically significant at <0.005.