Prevalence of human papillomavirus and Chlamydia trachomatis infection in paired urine and cervical smear samples of Palestinian young women. Walid Salim Basha, PhD Faculty of Human Medicine An-Najah National University
A nonmotile, gram-negative obligate intracellular bacterial pathogen, classified serologically into 15 serovars. Pathogenesis Chlamydia trachomatis 1- Trachoma serovars A-C 2- Genital infections serovars D-K 3- Lymphogranuloma venereum serovars L1, L2, L3
Diagnosis Fluorescent antibody assay Frei test (delayed hypersensitivity) for LGV Growth in tissue culture DNA probe test PCR LGV- Lymphogranuloma venereum
C. trachomatis new detection method PCR-SB Cryptic plasmid PCR hybridization assay DNA purification Cervical and Urine Samples PCR (DNA amplification) Hybridization (Oligo-probes)
PCR (DNA amplification) Two primers named CT-CPF: 5'-TGATTGTACAAGGGATCCGTAAGT-3' (start at nt. 7089) and CT-CPR: 5'-TCGATGAAAGACAGGAAATACG-3' (end at nt. 7465) (X07547, GenBank) Amplify 376 bps CPF CPR Cryptic plasmid homology to some other genes of Human, Drosophila melanogaster and S.cerevisiae.
Hybridization (Oligo-probes) C. trachomatis-specific anti-sense probe named CP35 (nt.7335- nt. 7360) The oligoprobe, CP-35 has also sequence homology in many sequences of human.
Comparison of the results with IDEIA, PCR-MWP and PCR-SB assays in 16 samples from urethritis patients Case No. IDEIA Amplicore MWP (OD) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 - - - - - - - - + + + + + + + + 0.06 0.65 0.15 0.14 2.47 0.09 0.10 1.74 3.42 3.13 0.81 3.23 2.93 0.79 3.37 3.36 PCR - SB 400 (bps) 300 (bps) Agarose gel 400 (bps) 300 (bps)
Human papillomavirus Non-enveloped small double-stranded DNA viruses, classified into more than 100 genotypes. Pathogenesis 1- Warts HPV 1, 2, 3 & 7 2- Condyloma acuminata HPV 6 & 11 3- Intraepithelial neoplasia HPV 11, 16, 31& 52 4- Cervical cancer HPV 16 & 18
HPV TYPES Low-risk HPV types High risk HPV types Develop genital wart Common, Causes no symptoms HPV-6 or HPV-11 Develop cervical intraepithelial neoplasia (CIN) Common regress spontaneously Minority of lesions progress to highgrade dysplasias Then to carcinomas in situ before becoming invasive cancers. HPV-16 or HPV-18
HPV-induced cancers Cervix Anus Vagina/Vulva Penis Mouth Throat 100,000 200,000 300,000 400,000 500,000 Annual number of cases worldwide
Open Reading Frames HPV genome - schematic representation 1 2 3 E6 E7 E1 E2 E4 E5 L2 Poly - A L1 URR (LCR) Poly - A 0 1 2 3 4 5 6 7 8 Kilobase
Noncoding Upstream Regulatory Region Contributes to the control of DNA replication and transcription of ORFs Late region 1 L1 protein is the major capsid protein Late region 2 L2 protein is the minor capsid protein Early region 5 E5 protein is located in the cellular membrane, prevents acidification of endosomes, and can stimulate the transformating activity of epidermal growth factor receptor and contribute to oncogenicity L1 L2 Early region 6 E6 protein of oncogenic HPVs binds to the p53 tumor suppressor gene product and abrogates it activity by accelerating its degradation URR Poly - A Poly - A E6 E5 / E4 / E2 E7 E1 Early region 4 E4 proteins form filamentous cytoplasmic networks and share the same cellular distribution. They appear to play a role in viral replication Early region 7 E7 protein of oncogenic HPVs bind to the tumor suppressor gene product Retinoblastoma protein (RB) and related proteins, thus inhibiting their function Early region 1 E1 protein involved in viral plasmid replication Early region 2 E2 protein is an important modulator of viral transcription and play a role in viral replication HPV Genome Structure
HPV infection and replication in cervical epithelial cells Columnar epithelial cell Infection Shedding L1 & L2 expression Squamous epithelial cell Transformation zone Basement membrane Basal epithelial cell E1, E2, & E4-E7 expression
Malignant transformation of the cervix E7 E2F RB E7 RB E6 E6 P53 E2F P53 HPV transformed epithelial cell Cervical intraepithelial neoplasia (CIN) Invasive cervical cancer
PCR (DNA amplification) LCR - E7 PCR LCR-1,2,3,4 E7R-1,2,3,4 LCR E6 E7
A. Hybridization result for HPV screening M A B C D E F G H I J 500 bps B. Hybridization result for HPV cutting pattern Sample No. HPV type M B H I (HPV16) (Unknown) (HPV16-33) Cutting enzymes AvaII RsaI DdeI AvaII RsaI DdeI AvaII RsaI DdeI 500 bps
Aim of work To determine the prevalence of HPV infection in Palestine To determine the age group considered as a risk factor Is there a correlation between C. trachomatis and HPV infection Is there a correlation between HPV infection and cervical abnormalities in West Bank Genotyping for detected HPV in West Bank Recommendations in order to improve women s health care services ( quality of care).
Subjects 418 women visited governmental or private obstetrics and gynecology clinics for various reasons in the West Bank 309 Cervical samples 109 Cervical & Urine Samples 277 Asymptomatic 32 Symptomatic 28 Asymptomatic 81 Symptomatic No cervico-vaginitis cervico-vaginitis No cervico-vaginitis cervico-vaginitis
Percentage Prevalence of of HPV infection among symptomatic and asymptomatic women 8.6% 3.6% 9 8 7 6 5 4 3 2 1 0 Symptomatic Asymptomatic
Prevalence Prevalence of HPV Types Low risk High risk 25 23.0% 23.0% 20 17.2% 15 11.3% 11.1% 10 5.2% 5.2% 5 0 HPV 6 HPV 11 HPV 16 HPV 33 HPV 52 HPV 66 UN HPV Types
Percentage Prevalence of C. trachomatis among symptomatic and asymptomatic women 12.3% 3.6% 14 12 10 8 6 4 2 0 Symptomatic Asymptomatic
Comparison between cervical and urine samples for HPV detection Cervix alone Cervix and Urine Urine alone 0.8% 3.7 % 2.8% 7.3%
Comparison between cervical and urine samples for Chlamydia trachomatis detection Cervix alone Cervix and Urine Urine alone 0.8% 6.4 % 2.8% 10%
Recommendations STDs are a public health problem in Palestine Dealing with STDs should receive more attention by MOH and other health providers. Health education programs should be developed, carried out, and implemented by national health education committee. Medical and community awareness should be increased by all means.
Recommendations Introduction of HPV/DNA testing greatly facilitate the identification of women at risk for cervical cancer. This strategy minimizes unnecessary follow-up visits and invasive procedures without compromising the detection of disease. The urine samples can t be an alternative test for cervical samples for HPV detection but could be for CT screening