Supervisors info:
Λουκάς Ιωάννης, Αναπληρωτής Καθηγητής, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Παντερή Ειρήνη, Αναπληρώτρια Καθηγήτρια, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ντότσικας Ιωάννης, Επίκουρος Καθηγητής,Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
The burden of new infections rates continues to escalate and HPV virus is rapidly becoming the leading infection type worldwide. More than 130 types of HPV so far have been identified and divided into low-risk (LR) and high risk (HR) genotypes, based on their ability to lead to oncogenic transformation. Early detection and identification of HPV can contribute to the choice of treatment and improve the monitoring of patients after treatment.
In the present study, a total of 410 patients (380 women and 30 men) participated and detection and identification of HPV was performed by PCR-RFLP and Real-Time PCR. Sampling was performed by qualified staff as well as by the process of self-sampling. DNA was isolated by three in house procedures (DNA isolation with chloroform: isoamylalcohol 24: 1, DNA isolation from tissues and paraffin blocks, DNA isolation from Swabs) and the DNA isolation protocol of «Genomic Kit DNA from tissue» of MACHEREY-NAGEL company.
In PCR-RFLP analysis the genetic material was amplified using PCR followed by electrophoresis and specimens were examined. The positive on HPV samples were amplified by subsequent PCR, followed by DNA digestion using the restriction enzymes PstI, HaeIII, Rsal, DdeI (RFLP analysis). Identification of the HPV isoforms was performed by observing the restriction fragments through a second electrophoresis. To evaluate the efficiency of the analysis with the restriction RFPL method, β-globin was used as endogenous control.
AmpliSens® HPV HCR genotype-titre-FRT PCR kit was used for the identification by Real-Time PCR. The method relies on the simultaneous amplification in real time (multiplex PCR) of the DNA fragments of HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and a DNA fragment of β-globin gene in a tube. The DNA fragment of β-globin gene was used as endogenous control.
Subsequently, we compared the two techniques concluding that PCR-RFLP analysis, despite being labor-intensive and with difficulty in interpreting results from multiple infections, is a simple, cost-effective, reliable and sensitive method suitable for the routinely detection of HPV. In contrast, Real-Time PCR has greater sensitivity, selectivity and gives reproducible results as well. HPV prevalence was found to be relatively high in Greece with the HR-HPV types being the most frequent types of infection, especially in the age group 16-26, where vaccination can be used and help reduce the risk of infection. Finally, the most common findings were HPV infection, LGSIL / CIN I (Low Grade Cervical Lesions), ASCUS (Atypical Squamous Cells of Undetermined Significance), HGSIL / CIN II / CIN III (High Grade Cervical Lesions) and genital warts.
