GinPolMedProject © 3 (61) 2021 : 001-07 • ORIGINAL RESEARCH The High-risk Human Papilloma Virus (Hr-HPV) in abnormal pap smears Ihab I. Samaha1 (CDEF), Ibrahim A. Abdelazim 2,3* (ABDE), Tamer E. El-Ghazaly2 (CDEF) 1 Department of Obstetrics and Gynecology, Faculty of Medicine, Helwan University, Cairo, Egypt. Department of Obstetrics and Gynecology, Faculty of Medicine, Ain Shams University, Cairo, Egypt. 3 Department of Obstetrics and Gynecology, Kuwait Oil Company (KOC), Ahmadi, Kuwait. SUMMARY 2 AUTHORS’ CONTRIBUTION: (A) Study Design · (B) Data Collection . (C) Statistical Analysis · (D) Data Interpretation · (E) Manuscript Preparation · (F) Literature Search · (G) Funds Collection Background: The Human papilloma virus (HPV) is the commonest sexually transmitted disease (STD) in low- and middle-income countries and it is the most powerful carcinogens implicated in cervical cancer. Objectives: To detect types of Hr-HPV in abnormal Pap smears and cervical cytology abnormalities associated with Hr-HPV infection. M ethods: Women eligible for Pap smears and HPV-co-testing according to hospital protocol were included in this study. Pap smears were done using the liquid-based cytology (LBC) and evaluated according to the Bethesda system. HPV-DNA co-testing was done using the Anyplex-II. Collected data were statistically analyzed to detect types of Hr-HPV in abnormal Pap smears, and cervical cytology abnormalities associated with Hr-HPV infection. Results: The prevalence of Hr-HPV in abnormal Pap smears was 48.2% (106/220). The Hr-HPV detected in abnormal Pap smears were HPV-16 (36.8% (39/106)), HPV-31 (12.3% (13/106)), HPV-58 (11.3% (12/106)), HPV-66 (10.4% (11/106)), HPV-51 (9.4% (10/106)), HPV-18 (7.5% (8/106)), while multiple Hr-HPVs-35, -33, -52, and 58 were detected in 12.3% (13/106) of abnormal Pap smears. The Hr- HPV detected in 39.6% (42/106) of ASCUS, in 80.2% (85/106) of LSIL, in 81.1% (86/106) of ASC-H, and in 83.01 (88/106) of HSIL. The Hr-HPV detection rate was significantly higher in LSIL, ASC-H, and HSIL compared to ASCUS (P=0.002, 0.001 and 0.001, respectively). Conclusion: The prevalence of Hr-HPV in abnormal Pap smears was 48.2%. The low prevalence of HPV-18 in this study confirms the variations in genotype distribution of Hr-HPV. The Hr-HPV detection rate was significantly higher in LSIL, ASC-H, and HSIL compared to ASCUS. This finding supports the recommendation of HPV co-testing with routine Pap smear in women aged 30-65 years. Keyw ords: High-Risk; HPV; Cervical; Pap; Cytology Address for correspondence: Ibrahim A. Abdelazim Department of Obstetrics and Gynecology, Kuwait Oil Company (KOC), Ahmadi, Kuwait. Tel: +965-66551300 E-mail: dr.ibrahimanwar@gmail.com Word count: 1674 Tables: 2 Figures: 0 References: 24 Received: 20.08.2021 Accepted: 06.09.2021 Published: 28.09.2021 INTRODUCTION Cancer cervix is an ideal malignancy for screening and prevention because it can be diagnosed and treated at the pre-cancerous lesions before development of invasive disease [1-3]. Most cervical cancers develop from infected cells with high-risk human papilloma virus (Hr-HPV), originated from the squamo-columnar junction of the cervix [4]. The HPV is the commonest sexually transmitted disease (STD) in low- and middle-income countries and it is the most powerful carcinogens implicated in cervical cancer [5]. Since the causal link between cervical cancer and Hr-HPV infection was established, much effort has been devoted for diagnosis and prevention of HPV infection [6,7]. The liquid-based cytology (LBC) approved by the FDA in 1996 as an alternative to conventional cervical smear (CCS) [8,9]. The polymerase chain reaction (PCR) based methods for detection of HPV deoxyribonucleic acid (DNA) is highly sensitive and specific, and it has the additional advantages of detecting small viral concentrations [10]. Initially, the Hr-HPV co-testing was recommended for ASC-US [11]. Based on the recommendations from National Institute of Health (NIH), American Society for Colposcopy and Cervical Pathology (ASCCP), and American Cancer Society (ACS) [12], the FDA in 2014 approved the Hr-HPV co-testing as primary screening of cervical cancer in women ≥25 years at intervals ≥3 years [13]. The joint recommendation released in 2012 advocates HPV co-testing in conjunction with routine Pap smear in women aged 30-65 years [14]. The co-testing increases the detection of cervical-intraepithelial lesion-2 (CIN2) or greater lesions at baseline screening, and significantly decreases the detection of CIN2/CIN3 lesions at subsequent screening compared to cytology alone [15]. Variation in the genotype distribution of Hr-HPV was reported in previous studies [16-18]. Therefore, this study design to detect types of Hr-HPV in abnormal Pap smears and cervical cytology abnormalities associated with Hr-HPV infection. OBJECTIVES To detect types of Hr-HPV in abnormal Pap smears and cervical smear abnormalities associated with Hr-HPV infection. −1 © GinPolMedProject 3 (61) 2021: 001-007 MATERIALS AND METHODS Sample Size Women eligible for Pap smears and HPV-co-testing according to hospitals protocol were included in this prospective study which was conducted over two years from January 2019 till January 2021, after informed consents in accordance with Helsinki declaration to detect the types of Hr-HPV in abnormal Pap smears and cervical smear abnormalities associated with Hr-HPV infection. The required sample size calculated using G Power software version 3.1.9.7 for sample size calculation, setting α -error probability at 0.05, power (1-β error probability) at 0.95%, and effective sample size (w) at 0.5. An effective sample ≥220 Pap smears was needed to produce a statistically accepted acceptable figure. Eligibility to Pap smear and HPV-co-testing include sexually active women, between 25-65 years old. Women received HPV vaccine; previous abnormal Pap smear, previous hysterectomy, previous genital malignancy, women under treatment for genital tract malignancy (i.e., either chemo and/or radiotherapy) or women refused to participate and/or to give consent were excluded from this study. Pap smears were done using the LBC and evaluated according to the Bethesda system. HPV-DNA testing was done using the Anyplex-II (Anyplex, Seoul, South Korea) which is a semi-quantitative real-time PCR test utilizing dual priming oligonucleotide and tagging oligonucleotide cleavage. Anyplex-II provides sensitive and specific genotyping information of 28 HPV types (19 Hr-HPV types and 9 low-risks HPV (Lr-HPV)) in single reaction. The abnormal cervical cytology was classified as atypical squamous cells of undetermined significance (ASCUS), low-grade and high-grade squamous intraepithelial lesion (LSIL and HSIL), atypical squamous cells cannot exclude HSIL (ASC-H), squamous cell carcinoma (SCC), atypical glandular cells not otherwise specified (AGC-NOS), atypical glandular cells suspicious of adenocarcinoma or in situ carcinoma (AGC neoplastic) [1]. Women with abnormal Pap smears and/or Hr-HPV positivity were offered colposcopy examination and cervical biopsy. The biopsy specimens were examined and graded as CIN1, CIN2, CIN3, in situ carcinoma or invasive cancer. Collected data were statistically analyzed to detect types of Hr-HPV in abnormal Pap smears (primary outcome) and cervical cytology abnormalities associated with Hr-HPV infection (secondary outcome). Tab. 1. Types of Hr-HPV and Lr-HPV detected in abnormal Pap smears. Statistical Analysis Categorical variables will be presented as number and percentage. Chi-square test (x2) was used for analysis of qualitative variables to detect types of Hr-HPV in abnormal Pap smears (primary outcome) and cervical cytology abnormalities associated with Hr-HPV infection (secondary outcome). P<0.05 was considered significant. RESULTS Prevalence of HPV in abnormal Pap smears: Four hundred and thirty (430) cervical cytology specimens were collected from January 2019 to January 2021 for Pap smears and HPV co-testing, 51.2% (220/430) of the collected cervical cytology specimens had abnormal Pap smear results, and 55.5% (122/220) of the abnormal Pap smears were positive for HPV (Hr-HPV and Lr-HPV). The prevalence of Hr-HPV in abnormal Pap smears was 48.2% (106/220). Types of Hr-HPV in abnormal Pap smears: The Hr-HPV detected in abnormal Pap smears were HPV-16 (36.8% (39/106)), HPV-31 (12.3% (13/106)), HPV-58 (11.3% (12/106)), HPV-66 (10.4% (11/106)), HPV-51 (9.4% (10/106)), HPV-18 (7.5% (8/106)), while multiple Hr-HPVs-35, -33, -52, and 58 were detected in 12.3% (13/106) of abnormal Pap smears (Tab. 1.). Lr-HPV detected in abnormal Pap smears include HPV-6 (37.5% (6/16)), HPV-40 (25% (4/16)), HPV-44 (18.75% (3/16)), and HPV-61 (18.75% (3/16)) (Tab. 1.). The cervical cytology abnormalities associated with Hr-HPV infection: The Hr- HPV detected in 39.6% (42/106) of ASCUS, in 80.2% (85/106) of LSIL, in 81.1% (86/106) of ASC-H, and in 83.01 (88/106) of HSIL. The Hr-HPV detection rate was significantly higher in LSIL, Type of HPV Number and (%) Hr-HPV HPV-16 36.8% (39/106) HPV-31 12.3% (13/106) HPV-58 11.3% (12/106) HPV-66 10.4% (11/106) HPV-51 9.4% (10/106) HPV-18 7.5% (8/106) Multiple Hr-HPV-35, -33, -52, and 58 12.3% (13/106) Lr-HPV HPV-6 2− 37.5% (6/16) HPV-40 25% (4/16) HPV-44 18.75% (3/16) HPV-61 18.75% (3/16) © GinPolMedProject 3 (61) 2021: 001-007 ASC-H, and HSIL compared to ASCUS (P=0.002, 0.001 and 0.001, respectively (Tab. 2.). Although, the ARTISTIC trial reported HPV-16, -18, -31, -51, and -52 as the most common Hr-HPV in UK [20]. Correlation between abnormal Pap smears and CIN: Women with abnormal Pap smears and/or HrHPV positivity were offered colposcopy examination and cervical biopsy. Out of 122 colposcopy examinations, the CIN1 was diagnosed in 36.9% (45/122) and CIN2 was diagnosed in 21.3% (26/122) of cases. A Chinese study reported HPV-18, followed by HPV52, -16, -58, -33, and -53 as the most common Hr-HPV in China [22]. In addition, this study, found the most common Hr-HPV in Kuwait were HPV-16 (36.8%), HPV-31 (12.3%), HPV-58 (11.3%), HPV-66 (10.4%), HPV-51 (9.4%), and HPV-18 (7.5%). DISCUSSION The LBC approved by the FDA in 1996 as an alternative to CSS. A systematic review showed that the LBC had an equivalent performance to CCS [9]. The detection of HPVDNA using PCR based techniques are highly sensitive and specific, and it has the additional advantages of detecting small viral concentrations [10]. Variation in the genotype distribution of Hr-HPV was reported in previous studies [16-18]. Therefore, this study designed to detect types of Hr-HPV in abnormal Pap smears and cervical smear abnormalities associated with Hr-HPV infection. The prevalence of Hr-HPV in abnormal Pap smears was 48.2% (106/220), which was higher than that (18.4%) observed in Qatar, and lower than that (76.4%) observed in Kazakhstan [16,19]. This can explained by the conservative socio-cultural norms in Qatar, and by method of HPV-DNA testing used in this study (Anyplex-II), which is highly sensitive and specific semi-quantitative real-time PCR method for detection of HPV-DNA at small viral concentrations [10]. Like other studies [20-22], this study found that the Hr-HPV detection rate was significantly higher in LSIL, ASC-H, and HSIL compared to ASCUS (P=0.002, 0.001 and 0.001, respectively), which support the need for HPV co-testing during cervical cancer screening. The low prevalence of HPV-18 was not only observed in this study, but also observed in studies performed in Qatar and South Korea [16-18]. Variations in the genotype distribution of Hr-HPV were also reported in South Korea studies [17,18]. In this study, multiple Hr-HPVs infection (HPV-35, -33, -52, and 58) were detected in 12.3% of abnormal Pap smears which is lower than the 44.7% observed in Brazil [23] and 38.2% in Houston [24]. This could be explained by the concurrent Hr-HPV infection which induces effective local or humoral immune response than that triggered by a single infection, with an overall stronger immunity against other Hr-HPV infection. The study from Brazil shows that women with multiple high-risk HPV infection tend to have high-grade or persistent CIN lesions [23]. So larger future studies are needed to confirm the association between multiple HPV infection and cervical pre-cancerous and/or cancerous lesions. This study found that the prevalence of Hr-HPV in abnormal Pap smears was 48.2% (106/220). The most common Hr-HPV detected in this study was HPV-16 (36.8%), HPV-31 (12.3%), HPV-58 (11.3%), HPV-66 (10.4%), HPV-51 (9.4%), and HPV-18 (7.5%). The HrHPV detected in 39.6% of ASCUS, in 80.2% of LSIL, in 81.1% (86/106) of ASC-H, and in 83.01 (88/106) of HSIL. The Hr-HPV detection rate was significantly higher in LSIL, ASC-H, and HSIL compared to ASCUS. The ATHENA trial found that about 10% of HPV16 and HPV-18 positive women had high-grade CINs (≥CIN2) [21]. This study was the first prospective study conducted in Kuwait and confirms the variations in genotype distribution of Hr-HPV because of the low prevalence of HPV-18 observed in this study. Initially, the Hr-HPV co-testing test recommended for ASC-US [11]. Based on the recommendations from the NIH, ASCCP, and ACS [12], the FDA in 2014 approved the Hr-HPV co-testing as primary screening of cervical cancer in women ≥25 years at intervals ≥3 years [13]. This study was also the first prospective study conducted in Kuwait to detect types of Hr-HPV in abnormal Pap smears using the Anyplex-II which is highly sensitive and specific PCR method for detection of HPV-DNA at small viral concentrations. Tab. 2. The cervical cytology abnormalities associated with HrHPV infection. Hr-HPV in ASCUS Hr-HPV in ASCUS Hr-HPV in ASCUS 39.6 (42/106) 39.6 (42/106) 39.6 (42/106) Hr-HPV LSIL 80.2% (85/106) - - Hr-HPV in ASC-H - 81.1% (86/106) - Hr-HPV in HSIL - - 83.01% (88/106) P value (X2 test) 0.002 0.001 0.001 ASC-H: Atypical squamous cells cannot exclude HSIL. ASCUS: Atypical squamous cells of undetermined significance. Chi-square (X2) test used for statistical analysis Data presented as number and percentage (%) Hr-HPV: High-risk Human papilloma virus HSIL: High-grade squamous intraepithelial lesion. LSIL: Low-grade squamous intraepithelial lesion. 3− REFERENCES Farghali MM, et al. – Effect of Labor Epidural Analgesia on Delivery of Second Twin: A Prospective Observational Study… Women refused to participate and/or give consent was the only limitation faced during this study. Larger future studies are needed to confirm the association between multiple HPV infection and cervical pre-cancerous and/or cancerous lesions. CONFLICT OF INTERESTS CONCLUSION FINANCIAL SUPPORT Authors declare no conflict of interests related to this study. Nil. The prevalence of Hr-HPV in abnormal Pap smears was 48.2%. The low prevalence of HPV-18 in this study confirms the variations in genotype distribution of Hr-HPV. ETHICS APPROVAL The Hr-HPV detection rate was significantly higher in LSIL, ASC-H, and HSIL compared to ASCUS. This finding supports the recommendation of HPV co-testing with routine Pap smear in women aged 30-65 years. 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