الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Cervical Scrapings from the posterior vaginal pool and Squamoepithelial junction were collected using technique for Papanicolaou smear (Pap) with cytospatula. The scrapings were detected serologically by HPV ELIZA; RTPCR and PCR. Human Papilloma Virus (HPV) antigen was detected serologically in eight samples of serum of infected patients out of fifty one cases tested. ELISA test was used to detect antibodies of HPV DNA in blood serum of women cases studied. Those samples were collected from women attending Benghazi Gynecological Clinics in hospitals and outpatient settings. Positive reaction was defined if the optical density (OD) of the test sample exceed that of the Cut off value; ≥ 0.24. Histopathological findings were detected in nine Cervical Smears of infected patients out of the fifty one cases studied. The TP Imaging System is an automated imaging and review system indicated for primary screening of Pap tests. Cervical scrapings from the posterior vaginal pool and squamo-epithelial junction were collected for Papanicolaou smear using cytospatula. Papanicolaou smears were evaluated by a pathologist according to the Bethesda diagnostic criteria. Normal superficial cells are large and flat with pyknotic nuclei and eosinophilic cytoplasm. Normal intermediate cells are large cells with small vesicular nuclei and cyanophilic or eosinophilic cytoplasm. Normal parabasal cells are usually singly dispersed, have a large uniform nucleus, and distinct cytoplasmic borders (liquid-based Thin Prep; Papanicolaou stain). Low-grade squamous intraepithelial lesion on Pap smear showed koilocytic atypia with clearing of central cytoplasm, condensation of peripheral cytoplasm, nuclear enlargement, and hyperchromasia. Squamous cell carcinoma and neoplastic cells showed cytoplasmic keratinization, cellular pleomorphism and background tumor diathesis in a Pap smear. The two major histologic types of cervical cancer, adenocarcinoma and squamous cell carcinoma, and the preinvasive disease that corresponds with these histologies share many of the same risk factors. Most of these are associated with an increased risk of acquiring or having appropriate compromised immune response to infection with Human Papilloma Virus (HPV), the etiologic agent of most cervical cancers. Of these risk factors; the age, parity and age at marriage with other demographic factors seem to be independent factors taking into consideration that the sample size is small. The age group of the high risk HPV positive samples in this study was between 28 to 49 years with an average at 41 years. The prevalence of HPV infection in the study was 21%. The prevalence of high risk HPV types in this sample was 100%. The age at first conception was on average 20 years. Smoking was not noted in any of the cases nor was use of oral contraceptive pills. The initial examination of the cytology at the laboratories using Papnicolaou staining technique showed severe dysplasia in all smears that were positive for HPV pathogenic types with one showing changes that needed referral to the colposcopy clinic and biopsies. Real Time (RT) PCR showed eleven infected patients cases out of fifty one cases which gave positive reaction with RT PCR with (21.6%) in Cervical Smear and blood serum of women cases collected attending Benghazi gynecological clinics in hospitals and outpatient settings. HPV DNA was detected by real time polymerase chain reaction in 11 women patient out of 51 belong to 31, 16,18,35, 39, 45, 51, 58, 33, 52, and 66 HPV types. High risk HPV types 16 and 18 were found in all 11 specimens and the other high risk types were also positive in all specimens. The prevalence of HPV infection in the study was 21%. The prevalence of high risk HPV types in this sample was 100%. Taking into consideration that the sample size is small, precision of the sample size was performed but will not be accurate even though it was calculated at 1.95%. Ideally it should be at least 5% using the equation for sample size. The age group of the high risk HPV positive samples was between 28 to 49 years with an average at 41 years. DNA extracted from infected patient with HPV types 16, 31, 15, and 51 have the expected size ~150 bp fragment of L1 gene, was successfully amplified by nested-PCR assay. To confirm the specificity of the assay, the nested-PCR assay was also performed with DNA extracted from negative HPV cervical samples. The PCR products of L1 BMC gene DNA were eluted from agarose gel using the clean gel DNA extraction kit as mentioned in material and methods. Amplicons forming the PCR were allowed for sequencing reaction through cycle sequencing method. The DNA amplicons returned as electropherogram files. Electropherogram showed distinct peaks for each base cell as well as high values for each cells. The Primers were easily identified in either the forward or reverse direction in each sequence fragment and easily used to piece together the individual sequence. Sequences obtained for each primer for each isolate had sufficient overlap between them and used to form one continuous sequence (Contag). The partial nucleotide sequence of 150 bp L1 BMC gene DNA for 4 HPV isolated from infected patient serum were done to determine the typing with other recommended L1 BMC gene HPV strains registered in Gene Bank. A phylogenetic tree of 4 HPV isolates revealed 85% a moderate degree of similarity amongst them. The homology matrix of 4 HPV isolated was divided to four groups. The first group included Human papillomavirus type 16 (P1) isolated from serum (patient -1) where 100% similarity with 3 HPV stains was recorded in the Gene Bank (NC001528; KX247760 and KU951195) and 81% similarity with the second group. The second group included Human papillomavirus type 31 (P2) isolated from serum (patient -2) where 100% similarity with (JN617892) ; 44% with (KU163578) and 57% with (KU163583) HPV stains was recorded in gene bank. The third group included Human papillomavirus type 15 (P3) isolated from serum (patient -3) where 77% similarity with (KU707835 and KT164597); 50% with (GU344762) HPV stains recorded in the Gene Bank. The fourth group included Human papillomavirus type 51 (P4) isolated from serum (patient -4) where 100% similarity with (KU050115 and KU050116) ; 77% with (KF548859) HPV stains was recorded in Gene Bank.