PREVALENCE OF HUMAN PAPILLOMA VIRUS IN CERVICAL CELLS AMONG PATIENTS WITH ABNORMAL PAP SMEARS

Mohammed Ibraheem Mezaal 1 , Dr. Ismail Husein Aziz 2 , Dr. Maad Mahdi Shalal 3 and Dr. Nada Alwan 4 . 1. Ph.D. student Genetic Engineering and Biotechnology Institute for Postgraduate Studies / University of Baghdad. 2. Asst.Prof.Genetic Engineering and Biotechnology Institute for Postgraduate Studies / University of Baghdad. 3. Prof.Director of National Center for Cancer Research/ University of Baghdad. 4. Prof.College of medicine / University of Baghdad. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

The collected cells are examined under a microscope to look for abnormalities. The test aims to detect potentially pre-cancerous changes (called cervical intraepithelial neoplasia (CIN), cervical dysplasia or squamous intraepithelial lesion system (SIL) according to the Bethesda Terminology. These lesions are often caused by sexually transmitted human papilloma viruses [6].
Genomic DNA isolation:-Total DNA (genomic, mitochondrial, and viral) isolated from the Pap smear sample for molecular studies was applied using genomic DNA purification kits of Qiagen ( QIAamp DNA Mini Kit / Germany) . This kit has been used by other researchers in similar study [7]. Agarose Gel Electrophoresis:-After genomic DNA extraction, agarose gel electrophoresis was adopted to confirm the presence and integrity of the extracted DNA [8].

Multiplex Polymerase Chain Reaction (PCR) for HPV detections:-Primer selections:-
Multiplex polymerase Chain Reaction (PCR) for HPV type 16 and 18 -E6 gene detections were done by using a specific primer designed in our study.
The detections of L1 region for HPV as general (HPV-high risk) by using a specific primers [9].
The positive control for human beta-globin gene used in this study by using a specific primers (TAKARA company, Japan). Primers sequences are listed below, table (1) . Primers have been designed in this study based on the Bioinformatics tools by using the international databases (NCBI,EMBL-EBI and DDBJ) and a number of tools that are available on website (online tools and software).
PCR Reaction setups:-Multiplex PCR was performed in a 30 μl total volume, as described in Table (2).

End point RT-PCR for HPV detections:-
The last part of the designed PCR was end point RT-PCR for HPV detections , This part was conducted for the purpose of high precision and to make sure the true positive and true negative results.
The end point RT-PCR for HPV detections part includes the same targets of the multiplex PCR (E6 gene for each HPV type 16 and type 18 , L1 gene for HPV-HR and human beta glubin) but it was replaced PCR Mastermix (Bioland) with SYBR green Mastermix (Bioland) for real time PCR with same Cycling conditions for each gene (Table 4).  Sub group II (Healthy Control Group) included 10 healthy individuals with normal Pap smear findings.

Age distribution of the samples:-
The age of all women was categorized as those who were less than 30 years old versus those equal to and over 30. (Table 5) revealed that 28% of the study women in general were less than 30 years old while 72 % were equal or over 30. This points out that may leads to conclude that group 2 constituted the greatest number groups in the present study .They were two subgroups in this study , the first group the women with abnormal pap smear revealed that 28.88% of the women were less than 30 ( < 30) years and followed by 71.12% of women whose age about more than 30 years old ( >30) years. The second group the healthy women (with normal pap smear ) revealed that 20% of the women were about ( <30) years and followed by 80% of women whose age about ( >30) years. The results agreed with those reported by Wright (2014) who displayed that the aging is a risk factor for persistent infection. The rate of persistent high-risk infection for women older than age 40 is 50%, compared with a persistence rate of 20% in women younger than age 25.   Fifty years ago, for squamous histology, the cervical cellular abnormalities viewed as the precursor of cervical cancer were termed mild, moderate or severe dysplasia; severe dysplasia was distinguished from the more severe diagnosis of carcinoma in situ. In the late 1960s, Richart proposed the concept of intraepithelial neoplasia [9]. CIN3 encompassed severe dysplasia and carcinoma in situ, CIN2 replaced moderate dysplasia, and CIN1 later came to include both the cytologic evidence of HPV infection (koilocytotic atypia) and mild dysplasia. The severity of the diagnosis was based on the degree of replacement of the normal stratified epithelium with mitotically active basallike epithelium (≤1/3 = CIN1, ≤2/3 = CIN2, >2/3 = CIN3). CIN was viewed as a stepwise progression, with a high probability of transition from the more minor to more serious cancer precursors [10].

Designed PCR -Multiplex Polymerase Chain Reaction and End point RT-PCR for HPV detections:-
The present study used Multiplex Polymerase Chain Reaction and end point RT-PCR technique for HPV detections. The multiplex PCR results revealed that identical bands related to the (E6) gene for HPV type 16 and type 18 , L1 region for HPV high risk and human beta glubin were present. (E6-HPV16) PCR amplified regions showed a molecular weight of 204 bp , (E6-HPV16) showed a molecular weight of 151 bp , (L1-HR-HPV) showed 450 bp and human beta glubin about 345 bp (Figure 8) .

Emanuella et al.,(2014)
showed that the L1 HPV conventional PCR products were 450 bp and their findings were in line with the results of this study [11].
PCR is being increasingly used in clinical laboratories to diagnose HPV. In another study that used conventional PCR for HPV detections a wide range .Studies in the literature indicate variables rates of HPV detection. . This differences in detecting DNA of HPV suggest a potential difference in the ability to amplify fragments of different sizes and specific types of HPV, depending on the methods used for DNA detection, and may also be attributable to 1398 the difference in types of studied material (smears, frozen material, paraffin material), anatomical localization, population issue, and design of oligonucleotides [12].  (Table 8).  Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore. The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase. Then the reaction is run in a real-time PCR instrument, and after each cycle, the intensity of fluorescence is measured with a detector; the dye only fluoresces ( such as dsDNA dyes -SYBR Green) will bind to all dsDNA PCR products .This method has the advantage of only needing a pair of primers to carry out the amplification, which keeps costs down and more sensitivity and high accuracy for real time PCR than conventional PCR [12].
Conclusions:- 1. In this study a new method has been been developed of high precision and more sensitivity for early detection of human papilloma virus (HPV) the main cause of cervical cancer compared with the global diagnosis kit (Sacace, Italy). 2. The study found variation in the results of PCR (RT-PCR /Sacace, Italy and designed PCR) for HPV detection depending on the histological and Cytological examinations .