Human Papillomavirus Prevalence and Genotyping in Paraffin-Embedded Cervical Tumor Samples from Women in Kordofan State, Western Sudan

doi:10.21203/rs.3.rs-4437388/v1

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Human Papillomavirus Prevalence and Genotyping in Paraffin-Embedded Cervical Tumor Samples from Women in Kordofan State, Western Sudan

https://doi.org/10.21203/rs.3.rs-4437388/v1

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Background

This study aimed to assess HPV prevalence and genotypic distribution in Sudanese cervical tumors, filling a global data gap and aiding screening and prevention efforts.

Methods

121 FFPE blocks of cervical tumors with a mean age of 53 ± 14 years were collected. Despite 41 samples having degraded DNA, all underwent amplification with HPV-specific primers and human HLA-DQA1 gene as an internal control. DNA microarray chips was used to detected 41 HPV genotypes.

Results

A 121 cervical tumor specimens were analyzed using DNA microarray chip techniques to identify 41 HPV genotypes. The majority of age group (46.3%) falling into the ≥ 60 years age group. we found that the most predominant tumor is SCC (69.3%), with a significant age-tumor progression correlation (P = 0.001).

Human Papillomavirus (HPV)

HPV genotyping

DNA

VissionArray HPV DNA Chip 1.0

Cervical tumors

Prevalence

Cervical cancer is a major global health issue, ranking fourth in both the number of diagnoses and cancer-related deaths among women. In 2018, there were approximately 570,000 cases and 311,000 deaths worldwide [1]. Without significant intervention, the global burden of cervical cancer is expected to increase to nearly 700,000 cases and 400,000 deaths by 2030 [2] In Sudan, there are approximately 12.7 million women aged 15 and older who are at risk of developing cervical cancer. Each year, an estimated 833 Sudanese women are diagnosed with cervical cancer, resulting in 534 deaths from the disease [3]. According to the 2014 National Cancer Registry report, there were 6771 new cancer cases registered in Khartoum, Sudan. Of these cases, 53.8% were in women, with breast cancer being the most common, followed by leukemia, cervical cancer, and ovarian cancer. The incidence rate of cervical cancer was 4.0 per 100,000 population and 8.5 per 100,000 women, with the highest incidence among women aged 55–64 and those aged 65 and older. [4]. The Human Papillomavirus (HPV) family comprises over 200 genotypes, with more than 40 HPV types capable of genital tract transmission [6]. These types fall into five genera: Alfa, Beta, Gamma, Mu, and Nu, with the largest groups being Alfa, Beta, and Gamma. Alfa can infect both mucous membranes and skin, while the others are cutaneous. Within the Alfa-papillomaviruses genus, HPV types are classified as low-risk cutaneous (e.g., HPV 27, 28, 29, 106, 114), low-risk mucosal (e.g., HPV 6, 11, 13, 44), or high-risk based on their association with cancer development. High-risk HPV types include confirmed human carcinogens (e.g., HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68) and probable human carcinogens (e.g., HPV 26, 30, 34, 53, 66) [7].

It is well known that the initial causing agent of cervical cancer is human papillomaviruses. It is reported that more than 99% of cervical cancers contain HPV DNA [8]. In Sudan, few published studies explored the association between HPV and cervical cancer in Sudanese women. In Northern Africa, including Sudan, approximately 10.7% of women in the general population carry cervical HPV, with HPVs 16 or 18 responsible for about 78.4% of invasive cervical cancers. Notably, tobacco use is significantly linked to cervical cancer in Sudanese women. [9].

Cervical cancer originates from cells at the cervix's squamocolumnar junction persistently infected with approximately 13 HPV genotypes. Most HPV infections lead to low-grade squamous epithelial lesions, with over 90% regressing spontaneously, but about 10% progress to high-grade lesions and rarely to invasive cancer. Tumor progression involves increased expression of E6 and E7 genes from high-risk HPVs, which deactivate p53 and pRb oncosuppressors, viral DNA integration into the host genome, disrupting E2 viral genes and host chromosomal loci, and molecular changes in cell cycle regulators. [10].

HPV is a highly prevalent sexually transmitted infection, with genotypes 16, 18, 52, 58, 31, 51, and 56 commonly found in women with normal cervical cytology. Globally, about 70% of cervical cancers are caused by genotypes 16 and/or 18, which are the focus of first-generation HPV vaccines like Gardasil® and Cervarix®. The 9-valent vaccine, Gardasil® 9, targets genotypes 16, 18, 31, 33, 45, 52, or 58, which collectively account for 90% of cervical cancers and are also associated with most HPV-related vulvar and vaginal cancers. [12]. in Africa, age-specific HPV is observed in both < 25 years and over 65 years age groups. Unfortunately, there is no specific data available for age-specific HPV distribution in Sudan. The HPV Information Center on HPV and Cancer (ICO) summarized a comparison between the most frequent HPV oncogenic types and the degree of cervical lesion as follow:

In the world, the most oncogenic types of HPV found in women with normal cytology are 16 (2.9%), 52 (1.5%), 31& 53 (1.3%) and 18 (1.2%). While in low-grade lesion 16 (19.3%), 52 (8.9%), 51 (8.8%), 31 (7.7%) and 53 (6.7%). The most distributed HPV-types among women with high-grade lesion are 16 (45.1%), 52 (11.0%), 31 (10.4%), 58 (8.1%), 33 (7.3%) and 18 (6.8%). In cervical cancer (any histology) 16 (55.2%), 18 14.2%), 45 (5.0%), 33 (4.2%), 58 (3.9%) and 31 (3.5%). While squamous cell carcinoma 16 (56.8%), 18 (11.6%), 45 (4.8%), 33 (4.3%) and 58 (3.9%). In adenocarcinoma the distribution is 16 (36.1%), 18 (34.9), 45 (5.7%), 33 (1.8%) and 31 (1.6%).

In less developed countries the HPV-genotypes distribution among women with squamous cell carcinoma was found as follow; HPV16 (56.1%), HPV18 (12.4%), HPV45 (5.8%), HPV58 (4.7%) and HPV52 (4.1%), while is adenocarcinoma found as; HPV18 (36.2%), HPV16 (36.1%), HPV45 (5.8%), HPV33 (2.4%) and HPV52 (1.9%) [13].

In Sudan, Abate and his colleges found that 94% of the subjects (80/85) harbored high risk and 11.7% (10/85) harbored low risk HPV genotypes. HPV 16 represent (82.5%, 66/80) followed by HPV 18, 45, and 52 [14]. In second study of 40 cases, Elasbali and his colleges found that both hr-HPV 16 and 18 were identified in 40% of the samples, of them hr-HPV 16 represent 50% while hr-HPV 18 represent 37.5% and co-infection found in 12.5% [15]. In a third study done by Salih and his colleges in a cytological sample, 60.7% of samples were ß globin positive (HPV positive), three of them classified as HPV X because they HPV\DNA positive with gel electrophoresis and negative with ELISA. HPV 58 was the most common type associated with mild and moderate dyskaryosis followed by HPV 16 and HPV 42 which associated with inflammation and normal cytological smear respectively [16]. In fourth study used cytological smears and ISH technique done by Gafar and his colleges, hr-HPV DNA was detected in 27.4% of the subjects. Of them 17% were detected with cytological evidences of HPV infection (presence of Koilocytes) on Pap smears assessment. In this study, only one smear was detected with Koilocytes but revealed a negative finding in ISH. These results giving the Pap test a specificity of 98.7% and a sensitivity of 20.7%. Nevertheless, of the 130 subjects, only 38/130 (29.2%) have claimed doing regular Pap. test, of whom 13/38(34.2%) were found positive for HPV [17]. In other study aimed only for identification of hr-HPV 18 from 50 cervical cancer biopsies using real time PCR and SYBER green for E6 gene, 18 (36%) specimens revealed positive [18].

Study samples:

This retrospective descriptive study, conducted in Kordofan state, western Sudan, aimed to detect and identify HPV genotypes in cervical tumors. We included cervical formalin-fixed paraffin-embedded (FFPE) archival blocks diagnosed between 2013 and 2017 at Kordofan University Diagnostic Center, whether as benign or malignant tumors of any histological grade.

A total 121 FFPE blocks were collected with benign tumor (n = 25), SCC (n = 84), adenocarcinoma (n = 6), undifferentiated carcinoma (n = 6). Then the blocks were sectioned under clean conditions and 2–3 10 µm ribbons were collected in sterile, labeled 1.5 ml Eppendorf tube.

Extraction and Concentration:

DNA Extraction was done according to VisionArray FFPE Extraction Kit; REF: VI-0001-50. DNA concentration was measured by Eppendorf BioSpektrometer kinetic to check the quantity of DNA. Each sample should have a DNA concentration of at least 15 ng/µl with a high degree of purity (260/280: ~1.8).

PCR Amplification:

A consensus primer (GP5+\GP6+) set used to amplify and biotinylated the L1 gene, a highly conserved region of HPV, in addition to primers against the human HLA-DQA1 gene used as an internal positive control. Depending on the HPV genotype, the amplification results in PCR products of 139–148 bp fragment length. PreCise Taq DNA polymerase utilizes the dNTP/dUTP in reagent number for synthesis of new complementary DNA strands. In order to avoid contamination with PCR amplification products, uracil nucleotides were included into the VisionArray HPV Primer Kit. By performing an Uracil-DNA-Glycosylase step prior to the PCR, all sequences that contain uracil bases and therefore possible contaminations with PCR products from previous VisionArray PCRs can be removed. The Uracil-DNA-Glycosylase is inactivated by temperatures above 95°C so that the PCR reaction can be performed as usual. According to VisionArray thermal profile, the final denaturation repeated once for 1 minute at 95°C in order to ensure of DNA is a single stranded. The cooled at 8°C to stabilize DNA.

Quality Control and Gel Electrophoresis:

A duplicated human HLA-DQA1 gene internal control was used to ensure successful amplification, while biotinylated sequences confirmed successful hybridization, and agarose gel electrophoresis with a 140 bp fragment length verified the presence of HPV in positive samples. Although, gel electrophoresis isn't necessary in the VisionArray system due to favorable PCR conditions, it was performed here to confirm the degradation and bad quality of 41 sample's DNA and to document that for research issue. Nevertheless, all samples proceeded to PCR and chip hybridization.

Detection System and Interpretation of results

The VisionArray Detection Kit, used with a Primer Kit and DNA Chip, detects specific DNA sequences. After amplification and biotinylation, DNA fragments are hybridized with immobilized catchers on a chip figure (1). Stringent washing removes unbound fragments, and biotinylated sequences are marked and stained by tetramethylbenzidine (TMB). The automated evaluation of the chip is performed with the VisionArray Analysis Package.

Statistical analysis

Study sample data and results were computerized and analyzed with IBM SPSS 23. Age differences were assessed using One Way ANOVA, and correlations were explored using Chi-squared and Spearman rank correlation coefficients. Significance was set at P < 0.05 for all tests.

Incitation and Bibliography

Mendely Desktop version 1.17.12 was used in references incitation and bibliography.

In this descriptive retrospective study, a total of 121 previously diagnosed specimens with diverse sorts of the cervical tumor were tested by DNA microarray chip techniques for detection of 41 HPV genotypes. Out of all subjects, the age extended from 17 to 90 years with a mean of (53 ± 14 years). Most of the subjects amassed in ≥ 60 years age group representing 56 (46.3%) taken after by an age group 31–45 years representing 38 (31.4%) and age group 46–59 years 21 (17.4%). Dissemination of study subjects by age group outlined by figure (2).

Benign cervical tumors comprised 25 (20.7%), squamous cell carcinoma 84 (69.3%), adenocarcinoma and undifferentiated carcinoma 6 (5%) for each as outlined in figure (3).

Most benign tumors amassed in age group 31–45 years and represented 19\38 (50%) of group cases, while the group aged ≥ 60 years harbored 54\96 (56.3%) of all malignant cases, most of them were squamous carcinoma 44\54 (81.5%) in addition to 83.3% of adenocarcinomas amassed in age group ≥ 60 years. The relationship between age, type, and progression of the cervical tumor was found significant as outlined in the table (1) (P = 0.001).

However, all the 121 samples were proceeded for genotyping by HPV DNA Chip, as it were 80 (66%) samples gave substantial results while the remaining 41 (34%) tests gave invalid results due to their degraded and bad quality DNA. Out of 80 valid samples, HPV DNA was detected in 47 (58.8%) and was negative in 33 (41.2%). Three high-risk HPV genotypes were identified in this study which are; HPV 16 the major genotype 44\47 (93.6%), followed by HPV 18, HPV 35 which represented by 1\47 (2.1%) for each one. In addition to one probably high-risk HPV 26 (2.1%). Neither low-risk nor multi-infection was identified in this study. Distribution of HPV infection among the subjects showed in figure (4) and photos (1) (2) (3) (4) (5).

HPV DNA was most frequently detected in ≥ 60 years group 23\80 (28.8%) followed by 46–59 years group 12\80 (15%) and age group 31–45 10\80 (12.5%). Fifty % (22\44) of HPV 16 were identified in age group ≥ 60 years followed by age group 46–59 which represent 25% (11\44) and age group 31–45 which represent 20.5% (9\44). Nevertheless, there was no correlation between subjects' ages and detection of HPV DNA\genotypes (P = 0.64) as shown in table (2).

Detection of high-risk HPV DNA can differentiate between benign and malignant cervical tumors (P = 0.002). Benign tumors harbor 1\47 (2.1%) at the same time as malignant tumor contained the majority (46\47, 98%) of HPV DNA positivity. 66.1% (41\62) of the squamous carcinoma diagnosed samples were positive for HPV DNA, while the HPV DNA positivity in adenocarcinomas, undifferentiated carcinoma, and benign tumors was represented as 50% (2\4), 60% (3\5) and 11.1% (1\9) respectively.

The majority of positivity for HPV DNA was detected in squamous cell carcinoma 41\47 (87.2%). The four HPV genotypes identified in this study were associated with squamous cell carcinoma and represented as follow; HPV 16 38\41 (93%), HPV18, 35 and 26 were represented by 1\41 (2.4%) for each one. HPV 16 was detected in 3\47 (6.4%) of undifferentiated carcinoma and 2\47 (4.3%) detected in adenocarcinoma. Only HPV16 was identified in all cervical lesions as Illustrated in the table (4).

It is well known that HPV is the initial causing agent of cervical cancer. its miles stated that more than 99% of cervical cancers and extra than 90% of their precursor lesions (SIL) comprise HPV DNA [8]. HPV alone was insufficient for the progression of cervical pre-neoplastic lesion to invasive cervical cancer [19]. Despite the fact that chronic hrHPV infection is a vital prerequisite for CIN, the vast majority of viruses have been cleared via the hosts' immune system, without clinical intervention and only 1–2% of the ultimate 10% grow to be continual resulting in cervical most cancers [20]. Cervical cancer accounted for 8.2% of all most cancer types and ranked the second pinnacle in Sudanese women, and the gender-specific age-standardized incidence of cervical cancer in Sudan is 8.5 per 100,000 ladies [4]. Considering that, the epidemiological traits of the HPV genotypes various by using geographic place [21], therefore, the present study aimed to screen cervical tumor biopsies for the presence of HPV genotypes and correlate this presence to the histological diagnosis and age in order to roadbed the creation of prophylactic vaccines.

Preceded by a study used Line probe assay (Inno-LiPA) in 2013 [14], this is the second study in Sudan using DNA microarray technology and first one that using (VissionArray HPV DNA Chip 1.0) for genotyping of 41 genotypes from 121 formalin-fixed paraffin-embedded (FFPE) cervical blocks. Only 80 samples (66%) gave substantial results represented as; 62 (77.5%) for squamous cell carcinoma, 4 (5%) for adenocarcinoma, 5 (6.3%) for undifferentiated carcinoma and 9 (11.3%) for benign lesions. As there was a chance for hybridization, the residual 41 degraded and fragmented DNA samples were proceeded for amplification and chip hybridization and gave invalid results. Bad fixation, the temperature of paraffin and long storage may have led to this result. FFPE samples do not offer the most suitable material for PCR-based assays because formalin fixation induces fragmentation of the nucleic acids, accordingly lowering the quantity of DNA appropriate as a template. Using of small primer to extend HPV L1 area will reduce invalid end outcome.[22] and [21].

The overall prevalence of HPV infection in this study turned to be 47\80 (58.8%), which was consistent to previous observation from Sudan (60.7%) [23] and located higher than previous studies additionally from Sudan 40% (14), 27% [24] and 34.2% [25] and lower than different observation from Sudan and Ethiopia (94%) and (93%) respectively [14]. Concerning the neighboring countries, HPV prevalence among study samples turned to be located higher than studies from Yemen 53% [26], Egypt 40.8% [24], the Saudi Arabia Kingdom 43% [27] and Cape town 25.4% [28]. From a large African meta-analysis (2015), the HPV prevalence in this study is consistent with that of Southern Africa region (57.3%) and higher than that of eastern Africa region (42.2%), Western Africa region (27.8%), and northern Africa (12.8%) [29].

In the present study, three hr-HPV genotypes (16, 18, 35) and one probably hr-HPV (26) were identified. HPV 16 represent the most frequent genotype 44\47 (93.6%), while HPV 18, HPV 35 and HPV 26 were represented by 1\47 (2.1%) for each one. The three hr-HPV identified in this study were in concord with the most four frequent HPV genotypes in Sub-Saharan Africa {HPV 16, 18, 45 and 35} [30] and worldwide largest cross-sectional study [31]. The high prevalence of HPV 16 in this study was agreed with previous study from Ethiopian (91%, 136/149) and Sudanese (82.5%, 66/80) women done by Abate and his college in 2013 [14] and higher than worldwide (53%) [26], India (71.7%) [19], Egypt (42%) [24], Yemen (37%) [26] and KSA (30%) [25]. In most published literature, HPV 18 represents the second most prevalent genotypes. Its prevalence in this study (2.1%) was found high than that reported in Egypt (0.7%) [24] and lower than previous study from Sudan (37.5%) [15], KSA (8.0%) [25], Yemen (16%) [26], another Egyptian study (29%) [24]. Worldwide the prevalence of HPV 18 in high grade and cervical cancer was found (6.8%) and (14.2%) respectively [27]. The hr-HPV genotypes identified in this study were in concord with [29] and disagree with [14] and [23].

In this study, it's the first time that probably hr-HPV 26 was identified from Sudanese cervical cancer biopsies and represent 1\47 (1.3%) of total HPV positivity and associated with 1\62 (1.6%) of SCC cases which is higher than worldwide prevalence (0.3%) [27]. This result may be due to small sample size. One probably carcinogenic (HPV 68) was identified form Egyptian cervical samples [24]. In the present study HPV16 and 18 were responsible for 62% of cervical cancers which is near to South Africa (64.2%) [28] and lower than worldwide (about 70%) and African (67.7%) [29].

Neither low-risk HPV nor multi-infection was identified in this study. This may be due to a small number of benign samples included in this study and DNA degradation. In addition to impairment of multi-infection identification from FFPE biopsy (9), high-risk HPV types were persisted longer than low-risk types by resisting host immunity (3).

It is believed that more than 95% of the cervical cancer cases are associated with a history of persistent infection by HR-HPV [29]. In this study, 65% (46\71) of cervical cancer samples were harbored HPV DNA and only one benign case contained HPV DNA 11.1% (1\9). This prevalence was found higher than Egypt (22.5%) [24], and located lower than Morocco (92.5%) [32], Ghana (89.8%) [33] and international meta-analysis report (85%) [31]. HPV16 alone identified in (60.6%) of all cervical cancer which is in higher than the prevalence of the world (55.2%), less developed countries (55.7%) and more developed countries (55.8%). And in concord with Guinea (60%), Algeria (61.4%) and South Africa (59%) and lower than Sudan (79.5%), Zimbabwe (95%) and Kenya (80%) [27]. Other co-factors of the progression of HPV infection to cervical cancer like low immunity, pregnancy, or immunosuppressive chemotherapy, nutrition, parity, and chronic STD infections could be required as a carcinogenic to advance HPV-infected cells toward neoplastic progression [24]. The superiority of HPV (particularly HPV16) can appreciably differentiate between benign and malignant cervical tumor (P = 0.002) and severity or progression of cervical malignancy (P = 0.003). The direct proportional determined displaying HPV as an essential chance trouble for development of the disease [8], [34].

In this study, 66.1% (41\62) of the squamous carcinoma diagnosed samples were positive for HPV DNA, while the HPV DNA positivity in adenocarcinomas, undifferentiated\invasive cancer, and benign tumors was represented as 50% (2\4), 60% (3\5) and 11.1% (1\9) separately. These findings were located lower than a report from the largest international cross-sectional study in 2010 identified HPV DNA in (87%) of squamous cell carcinoma and (62%) of adenocarcinoma [31]. These finding also were located lower than Sudan; squamous cell carcinoma 94% (73/78) and adenocarcinoma 100% (6\6), Ethiopia: squamous cell carcinoma 94% (120/128) and adenocarcinoma 88% (7\8) [14], and Morocco; 92.2% of squamous cell carcinomas and 100% of adenocarcinomas).

The prevalence of HPV16 displaying a significant correlation (P = 0.011) with the histological diagnosis, we found that HPV16 was identified in all types of cervical lesions. 61.3% (43\62) of squamous cells carcinoma harbored HPV16 which is agreement with international study (61%) [34] and more developed countries (59%) [27] and higher than the world prevalence (56.8%), less developed countries (56.1%) [27], India (42.4%) [19] and Yemen (57.5%) [26]. While in adenocarcinoma HPV16 identified in 50% (2\4) which is higher than world and less developed countries prevalence (36.1%), more developed countries (37.2%) [27], Yemen (26.3%) [26], and India (20%) [19]. This high prevalence may be due to low representation. Regarding the undifferentiated carcinoma, HPV16 represent 60% (3\5) which agrees with the prevalence in the world and less developed countries (59%) and (60.5%) respectively [27].

HR-HPV35 identified in 1.6% (1\62) of squamous carcinoma cases which agrees to the prevalence in the world (1.7%), less developed countries (2.0%) and more developed countries (1.4%) [27]. HPV 35 was considered as one of the most four frequent hr-HPV genotypes in sub-Sahara Africa [30] and one of the most ten frequent hr-HPV genotypes worldwide [27]. In Sudan, there was only one report from unpublished these done in 2010 by Moneira Fedail identified HPV 35 from cytological smears as the third most common genotype (26\400, 6.5%) [35].

For HPV negative results this may have been due to the absence of HPV DNA or false negative PCR results either due to the integration of HPV DNA in cervical carcinoma cells which may lead to loss of L1 ORF hence PCR primer target sequence disrupted or due to low viral load extracted from FFPE.

This study confirms that HPV, particularly HPV16, is a significant factor in cervical cancer in Sudan, with an overall prevalence of 58.8%. Notably, this is the first identification of HPV26 in cervical cancer cases in Sudan. These findings highlight the urgent need for HPV vaccination programs to reduce cervical cancer rates in the region.

Bp: Base pairs

CIN: Cervical Intraepithelial Neoplasia

dNTP: Deoxyribonucleotide Triphosphate

dUTP:DeoxyUridine Triphosphate

DNA: Deoxyribonucleic Acid

ELISA: Enzyme-linked Immunosorbent Assay

FFPF: Formalin-fixed Paraffin Embedded

HPV: Human Papillomavirus

hr-HPV: High Risk Human Papillomavirus

HLA: Human Leucocyte Antigen

ICO: Information Center on HPV and Cancer

ISH: In Situ Hyperidization

KSA: King Saudi Arabia

Pap: Papanicolaou

PCR: Polymerase Chain Reaction

SCC: Squamous Cell Carcinoma

SIL: Squamous Intraepithelial Lesion

SPSS: Statistical Package for the Social Sciences

STD: Sexual Transmitted Disease

TMB: Tetramethylbenzidine

Consent to Participate and Publication

This research involved the retrospective collection of formalin-fixed paraffin-embedded (FFPE) blocks from the Kordofan University Diagnostic Center. The study was approved by the center's director, who also serves as the main supervisor of the study. No direct contact with patients occurred, and no patient-related information will be published. All data presented in the publication are anonymized, ensuring that no patient identifiers are disclosed. The focus of the publication is on the detection of HPV genotypes and their correlation with cervical tumors and patient age.

Ethical Approval

Ethical approval for this research was obtained following the ethical standards outlined in the Helsinki Declaration and international guidelines. Approval was granted by the University of Alzaiem Alzhari, Faculty of Medical Laboratory Sciences. Additionally, a letter of consent was sent to the administration of the Kordofan University Diagnostic Center to secure their approval for the retrospective collection of blocks and data. This process ensures that all ethical considerations, including the protection of patient confidentiality and the responsible use of clinical data, were thoroughly addressed and adhered to.

Availability of data and materials:

If data and material are need, they will be available at the following link:

https://drive.google.com/drive/folders/1xNkVNttXT68fYzjFrcjYZ-XcLtkroF17?usp=share_link

Competing Interests:

The author(s) declare that they have no financial or non-financial competing interests.

Funding

This research received no external funding. The work was supported solely by the author's own resources.

Authors Contributions:

Dr. Hussien Hamid: the principal investigator, this paper was extracted from my Ph.D. project

Prof: Ahmed A. A. Eldour: The main supervisor of my project, he is a consultant pathologist, has histopathology clinic, previous president of Cordovan University, and previous head of Kordofan University diagnostic center, from where archived biopsies were taken. he did a great effort to support me during my Ph.D. journey.

Prof: Elwaleed M. Elamin: the Co-supervisor of my project, he is a professor of Histopathology, the previous president of Alzaiem Alazhari University, where I registered my Ph.D., he played a vital role in editing preparation of the manuscript

Acknowledgements:

I would like to extend my heartfelt gratitude to Zytovision Array GmbH for their invaluable support in the technical aspects of my research. Their advanced technology in HPV genotyping played a crucial role in the success of this project. The expertise and resources provided by Zytovision Array GmbH significantly enhanced the quality and precision of our work, enabling us to achieve our research objectives efficiently. Thank you for your commitment to excellence and for contributing to the advancement of HPV research.

F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre, and A. Jemal, “Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries,” CA Cancer J Clin, vol. 68, no. 6, pp. 394–424, Nov. 2018, doi: 10.3322/CAAC.21492.
M. Zhao et al., “Global, regional, and national burden of cervical cancer for 195 countries and territories, 2007–2017: findings from the Global Burden of Disease Study 2017,” BMC Womens Health, vol. 21, no. 1, pp. 1–13, Dec. 2021, doi: 10.1186/S12905-021-01571-3/FIGURES/4.
L. Bruni et al., “Human Papillomavirus and Related Diseases in Sudan. Summary Report,” HPV Centre, no. Afghanistan, p. iii, 2017.
I. E. Saeed, H.-Y. Weng, K. H. Mohamed, and S. I. Mohammed, “Cancer incidence in Khartoum, Sudan: first results from the Cancer Registry, 2009-2010.,” Cancer Med, vol. 3, no. 4, pp. 1075–84, Aug. 2014, doi: 10.1002/cam4.254.
A. K. Adams, T. M. Wise-Draper, and S. I. Wells, “Human Papillomavirus Induced Transformation in Cervical and Head and Neck Cancers,” Cancers, vol. 6, pp. 1793–1820, 2014, doi: 10.3390/cancers6031793.
E. M. Burd, “Human Papillomavirus Laboratory Testing: the Changing Paradigm.,” Clinical microbiology reviews, vol. 29, no. 2, pp. 291–319, Apr. 2016, doi: 10.1128/CMR.00013-15.
N. Egawa, K. Egawa, H. Griffin, and J. Doorbar, “Human Papillomaviruses; Epithelial Tropisms, and the Development of Neoplasia,” Viruses, vol. 7, pp. 3863–3890, 2015, doi: 10.3390/v7072802.
A. Khan, M. Moshahid, and A. Rizvi, “Therapeutic action of curcumin loaded chitosan nanoparticles for cervical cancer,” in Current Research & Reviews on Cervical Cancer, 2017.
A. Elamin, M. E. Ibrahim, D. Abuidris, K. E. H. Mohamed, and S. I. Mohammed, “Part I: cancer in Sudan—burden, distribution, and trends breast, gynecological, and prostate cancers.,” Cancer medicine, vol. 4, no. 3, pp. 447–56, Mar. 2015, doi: 10.1002/cam4.378.
M. L. Tornesello, L. Buonaguro, P. Giorgi-Rossi, and F. M. Buonaguro, “Viral and cellular biomarkers in the diagnosis of cervical intraepithelial neoplasia and cancer.,” BioMed research international, vol. 2013, p. 519619, Dec. 2013, doi: 10.1155/2013/519619.
X.-X. Zeng et al., “Prevalence and genotype distribution of human papillomavirus among Hakka women in China.,” Annals of translational medicine, vol. 4, no. 15, p. 276, Aug. 2016, doi: 10.21037/atm.2016.05.49.
M. Wagner, L. Bennetts, H. Patel, S. Welner, S. de Sanjose, and T. W. Weiss, “Global availability of data on HPV genotype-distribution in cervical, vulvar and vaginal disease and genotype-specific prevalence and incidence of HPV infection in females.,” Infectious agents and cancer, vol. 10, p. 13, 2015, doi: 10.1186/s13027-015-0008-y.
World HPV Information Center, “Human Papillomavirus and Related Diseases Report,” HPV Information Centre report, no. Indonesia, p. 60, 2017.
E. Abate et al., “Genotyping of human papillomavirus in paraffin embedded cervical tissue samples from women in ethiopia and the Sudan,” Journal of Medical Virology, vol. 85, no. 2. pp. 282–287, 2013. doi: 10.1002/jmv.23437.
A. M. Elasbali, A. H. S. El Din, R. A. H. Abdallah, and H. G. Ahmed, “Cervical and Oral Screening for HR-HPV types 16 and 18 among Sudanese Women Cervical Lesions.,” Infect Agent Cancer, vol. 7, no. 1, p. 17, 2012, doi: 10.1186/1750-9378-7-17.
M. M. Salih, M. El Safi, K. Hart, K. Tobi, and I. Adam, “Genotypes of human papilloma virus in Sudanese women with cervical pathology.,” Infectious agents and cancer, vol. 5, p. 26, Dec. 2010, doi: 10.1186/1750-9378-5-26.
S. E. GAFAR, H. G. AHMED, S. A. A. HAROUN, and E. M. MOHAMMED, “SCREENING FOR HR-HPV AMONGST SUDANESE WOMEN VISITING GYNECOLOGIC CLINIC BY ISH AND PAP.TEST,” Management in Health, vol. 17, no. 2, 2013, doi: 10.5233/MIH.V17I2.268.
Ibrahim, H. E. Hammad, and W. I. E. A. Elrahman, “Molecular Detection of Human Papillomavirus Type- 18 DNA in Cervical Cancer Tissue Biopsies from Sudanese Patients Attended Some Hospitals in Khartoum State during April to September 2012,” SUST REPOSITORY, Oct. 2012.
S. Srivastava, U. P. Shahi, A. Dibya, S. Gupta, and J. K. Roy, “Distribution of HPV Genotypes and Involvement of Risk Factors in Cervical Lesions and Invasive Cervical Cancer: A Study in an Indian Population.,” Int J Mol Cell Med, vol. 3, no. 2, pp. 61–73, 2014.
D. Song, H. Li, H. Li, and J. Dai, “Effect of human papillomavirus infection on the immune system and its role in the course of cervical cancer.,” Oncol Lett, vol. 10, no. 2, pp. 600–606, Aug. 2015, doi: 10.3892/ol.2015.3295.
M. G. Donà et al., “Performance of the linear array HPV genotyping test on paired cytological and formalin-fixed, paraffin-embedded cervical samples,” Journal of Molecular Diagnostics, vol. 15, no. 3. pp. 373–379, 2013. doi: 10.1016/j.jmoldx.2013.01.002.
E. Herraez-Hernandez, O. Preda, S. Alonso, R. S. Pardo, and A. Olmo, “Detection and Genotyping of Human Papillomavirus DNA in Formalin-Fixed Paraffin-Embedded Specimens with the HPV Direct Flow CHIP System.,” Open Virol J, vol. 7, pp. 91–5, 2013, doi: 10.2174/1874357920130927004.
M. M. Salih, M. El Safi, K. Hart, K. Tobi, and I. Adam, “Genotypes of human papilloma virus in Sudanese women with cervical pathology.,” Infect Agent Cancer, vol. 5, p. 26, Dec. 2010, doi: 10.1186/1750-9378-5-26.
M. A. Youssef et al., “Prevalence of human papilloma virus (HPV) and its genotypes in cervical specimens of Egyptian women by linear array HPV genotyping test,” Infect Agent Cancer, vol. 11, no. 1, p. 6, 2016, doi: 10.1186/s13027-016-0053-1.
R. Turki, K. Sait, N. Anfinan, S. S. Sohrab, and A. M. Abuzenadah, “Prevalence of human papillomavirus in women from Saudi Arabia.,” Asian Pac J Cancer Prev, vol. 14, no. 5, pp. 3177–81, 2013.
H. G. Ahmed et al., “Prevalence of Human Papilloma virus subtypes 16 and 18 among Yemeni Patients with Cervical Cancer,” Asian Pac J Cancer Prev, vol. 18, no. 6, p. 6, 2017, doi: 10.22034/APJCP.2017.18.6.1543.
ICO HPV Information Centre, “Human Papillomavirus and Related Diseases Report - World,” HPV Information Centre, no. Albania, p. 60, 2017.
I. H. I. Center, “South Africa: Human Papillomavirus and Related Cancers, Fact Sheet,” Hpvcentre.Net, vol. HPV Inform, no. 1, pp. 263–268, 2017.
R. K. Ogembo et al., “Prevalence of Human Papillomavirus Genotypes among African Women with Normal Cervical Cytology and Neoplasia: A Systematic Review and Meta-Analysis,” PLoS One, vol. 10, no. 4, p. e0122488, 2015, doi: 10.1371/journal.pone.0122488.
D. V. H. A. L. L. C. C. C. S. V. B. C. D. L. P. GP, “The burden of human papillomavirus infections and related diseases in sub-Saharan Africa. TT -,” Vaccine, vol. 31, no. S5. pp. F32–F46, 2013. doi: http://dx.doi.org/10.1016/j.vaccine.2012.07.092.
S. de Sanjose et al., “Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study,” Lancet Oncol, vol. 11, no. 11, pp. 1048–1056, Nov. 2010, doi: 10.1016/S1470-2045(10)70230-8.
M. Berraho et al., “HPV and cofactors for invasive cervical cancer in Morocco: a multicentre case- control study,” BMC Cancer, vol. 17, no. 435, 2017, doi: 10.1186/s12885-017-3425-z.
A. K. Awua, S. T. Sackey, Y. D. Osei, R. H. Asmah, and E. K. Wiredu, “Prevalence of human papillomavirus genotypes among women with cervical cancer in Ghana,” Infect Agent Cancer, vol. 11, no. 1, p. 4, Dec. 2016, doi: 10.1186/s13027-016-0050-4.
R. Murillo, R. Herrero, M. S. Sierra, and D. Forman, “Etiology of cervical cancer ( C53 ) in Central and South America,” Cancer in Central and South America. Lyon: International Agency for Research on Cancer, 2016.
M. A. A. M. Fedail, “Cervical Cancer and its Association with Human Papilloma Virus (HPV) Genotypes in Khartoum State.,” SUST Repository, 2010.

Age group			Diagnosis
Age group	Squam. Ca	Adeno-carcinoma	Undifferentiated- carcinoma	Benign Tumor	Total	Percentage %
15 – 30	4	1	0	1	6	5
31 – 45	19	0	0	19	38	31.4
46 - 59	17	0	1	3	21	17.3
>60	44	5	5	2	56	46.3
Total	84	6	6	25	121	100

Table (1): Shows the Correlation between Age-group and Lesion Type (P= 0.001)

	HPV Genotypes
Age group	Negative	HPV16	HPV18	HPV26	HPV35	Total	Percentage %
15 - 30	1	2	0	0	0	3	3.80%
31 - 45	11	9	0	1	0	21	26.30%
46 - 59	5	11	1	0	0	17	21.30%
≥60	16	22	0	0	1	39	48.80%
Total	33	44	1	1	1	80	100%

Table (2): Shows the Correlation between Age group and HPV Genotypes (P= 0.64)

HPV DNA	Histological Diagnosis
HPV DNA	SCC	Adeno-carcinoma	Undifferentiated- carcinoma	Benign tumor	Total Percentage %
Positive %	41	2	3	1	47
Positive %	61.10%	50%	60%	11.10%	58.80%
Negative %	21	2	2	8	33
Negative %	33.90%	50%	40%	88.90%	41.30%
Total %	62	4	5	9	80
Total %	100%	100%	100%	100%	100%

Table (3): Shows the Correlation between HPV Positivity and the Histological Diagnosis of Cervical Biopsies (P= 0.002)

HPV Genotypes

Histological Diagnosis

Total

Percentage %

SSC

Adeno-carcinoma

Undifferentiated-

carcinoma

Benign tumor

Negative count

33.9%

50%

40%

88.9%

41.2%

HPV 16 count

61.3%

50%

60%

11.1%

55%

HPV 18 count

1.6%

0.0%

1.3%

HPV 35 count

1.6%

0.0%

1.3%

HPV 26 count

1.6%

0.0%

1.3%

Total count

100%

Table (4): Shows the Correlation between HPV Genotypes and the Histological Diagnosis of Cervical Biopsies (P= 0.003)

Photos are available in the Supplementary Files section.

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Human Papillomavirus Prevalence and Genotyping in Paraffin-Embedded Cervical Tumor Samples from Women in Kordofan State, Western Sudan

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