Prevalence and Type Distribution of Human Papillomavirus in Invasive Cervical Cancer in Aba, South-East Nigeria: A Sentinel Study to Encourage and Guide HPV Vaccination

Abstract

Background: Despite the established link between human papillomavirus (HPV) and the pathogenesis of invasive cervical cancer and its precursor lesions, HPV vaccine acceptance faces cultural and religious biases in this region. There is no available local epidemiological study to support the current advocacy of HPV vaccination for the primary prevention of cervical cancer in this region. In this cross-sectional epidemiological study, we determined the prevalence and types of HPV in invasive cervical cancer so as to encourage and guide HPV vaccination for the primary prevention of cervical cancer in this south-east region of Nigeria.

Methology: Two-hundred archived formalin-fixed, paraffin-embedded tissue blocks with confirmed diagnoses of invasive cervical cancer during the study period (2015-2024) were retrieved and examined. DNA was extracted by proteinase k lysis procedure while HPV DNA amplification, detection and typing was done using E7 type-specific multiplex genotyping (E7-MPG), which combines multiplex polymerase chain reaction (PCR) and hybridization to type-specific oligonucleotide probes on fluorescent beads.

Result: Fourteen blocks were excluded from the analysis for inadequacy. A total of 79% (147/186) invasive cervical cancers were positive for single or multiple high-risk HPV types. HPV 16 was the predominant high risk HPV-type, being present in 87.3% of the cases in single and multiple infections. The five most common high risk HPV types seen in single infections are HPV 16 (31.3%), HPV 18 (19.7%), HPV 35 (5.4%), HPV 31 (1.4%) and HPV 52 (1.4 %).

Conclusion: There is high prevalence of HPV-DNA in invasive cervical cancers in this region, with HPV 16, HPV 18, HPV 35, HPV 31, and HPV 52 being the predominant types. HPV vaccination and testing will greatly reduce the burden of cervical cancer in this geographical region.

Introduction

Cervical cancer is the commonest cancer of the female genital tract in Nigeria and the second most common cancer in women worldwide [1, 2]. It is a major cause of cancer-related morbidity and mortality in women in developing countries [3, 4]. It is estimated that 15 out of every 1000 women in developing countries will develop cervical cancer before the age of 65 years [5].

Persistent infection with certain types of human papilloma virus (HPV) is the major etiologic factor in the development of cervical cancer and its precursor lesions [6]. There are over 200 genotypes of HPV classified into high and low risk types [7]. About 40 types can infect the uterine cervix of which types 16, 18, 31, 33, 35, 39, 45, 51,

52, 56, 58, 59 and 66 are classified as group 1 carcinogens [7]. Types 16 and 18 are the commonest worldwide and have been associated with more than 70 % of invasive cervical cancer (ICC) [7, 8]. Some HPV types tend to be transmitted together causing concurrent infections with different HPV types in some ICC [7].

The high prevalence of HPV DNA in cervical cancers and its precursor lesions has led to the advocacy for a widespread population-based HPV vaccination to prevent cervical cancers. The great reduction in the incidence of cervical cancer in developed nations has been attributed to organised screening programmes and prophylactic HPV vaccination. But there are concerns that there is regional variation in the distribution of HPV types and the proportion of cervical cancer preventable by HPV vaccine might be lower in sub-saharan africa [9]. Also, vaccine implementation in Nigeria has met with barriers including religious and cultural biases. This study aims to determine the prevalence and type of HPV DNA associated with cervical cancer in this region. To the best of our knowledge, this is a sentinel study in this state and will provide a local epidemiological data to encourage and guide the implementation of HPV vaccination for the primary prevention of cervical cancer in this region.

Materials and Methods

Study design and ethical considerations

This cross-sectional, point prevalence, hospital-based epidemiological study was carried out in Abia state university teaching hospital (ABSUTH), Aba, Abia state, south-east geopolitical zone of Nigeria. The pathology department of the hospital serves patients of ABSUTH and other private hospitals in Aba. The study was approved by the institutional health ethical research committee (IHERC) of ABSUTH in line with the declaration of helsinki and good clinical practice guidelines.

Study Specimen

The study specimens are biopsies of the uterine cervix in paraffin blocks. Two hundred, archival, formalin-fixed, paraffin-embedded (FFPE) tissue blocks of cervical cancer cases diagnosed between August 2015 and May 2024 were randomly retrieved for the study. Patient information was retrieved from the original request forms. The original biopsy specimens were obtained by a gynecologist from women who presented with cervical lesions suspicious of invasive cervical cancer at ABSUTH and other private hospitals in Aba as part of routine investigative procedures. The wet specimens were immediately fixed in 10% buffered formalin and processed for paraffin wax embedding using standard protocol. The histologic diagnosis of invasive cervical cancer was determined by light microscopic examination of hematoxylin and eosin (H/E) stained sections. The tissue blocks were stored at room temperature.

Parafin sections for tumour evaluation

To confirm the initial diagnosis and classify the tumour in line with WHO classification, fresh sections were taken from the tissue blocks for light microscopic examination of H/E stained sections. Two hundred suitable blocks with confirmed diagnosis of invasive cervical cancer were used for the HPV DNA detection and typing.

Parafin Sections for DNA Extraction

Three 10-mm, thick sections were taken from each block and put in a 1.5 ml safe-lock, sterile, eppendorf tube (Germany) with the aid of millipore tweezers. To prevent cross-contamination between specimens, the microtome and microtome blade were extensively cleaned after sectioning each block with a gauze soaked in DNA cleaner (DNA away, molecular bioproducts, ref: 7010) followed by 70 percent ethanol. The tubes containing the tissue sections were shipped to IARC lab (Lyon, France) for DNA extraction and typing.

DNA Extraction

DNA was extracted by overnight incubation of the paraffin tissue sections in 250 μl of digestion buffer (10 mm Tris/Hcl at ph 7.4, 0.5 mg/ml proteinase k, and 0.4% tween 20) at 37℃. To inactivate proteinase k and to separate paraffin from the aqueous phase, samples were incubated at 95 °C for 10 min, centrifuged, and chilled on ice. The aqueous phase is then transferred to a new tube. The solution is purified by the use of a high pure PCR template preparation kit (roche, basel, switzerland), according to the manufacturer’s instructions. The DNA is eluted in 0.2 ml of elution buffer (10 mm tris, ph 8.5) and stored at −20°C until further use.

Amplification and DNA typing

HPV-DNA positivity was determined using a type- specific multiplex genotyping (E7-MPG) assay, which combines multiplex PCR and bead-based luminex technology (luminex corporation, Austin, TX) [10]. This assay detects twelve HR-HPVs (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), seven probable HR (pHR)-HPVs (HPV26, 53, 66, 68, 70, 73, 82), and two low-risk (LR) HPVs (HPV6 and 11), as well as cellular beta-globin gene which is used to for DNA quality. The PCRs were performed with the QIAGEN multiplex pcr kit according to the instructions of the manufacturer. After PCR amplification (TS-PCR), 10 µl of each reaction mixture was analysed by multiplex HPV genotyping (MPG) using luminex technology (luminex corporation, Austin, TX).

Data Analysis

The data collated were analysed using statistical package for social sciences version 20 (spss 20) and the results presented as Tables and charts. Statistical comparison was done using chi-square with the level of significance set at p less or equal to 0.05. The findings of this study were compared with those of previous studies.

Results

Descriptive Data

Two hundred invasive cervical cancer cases seen between august 2015 and may 2024 were used for the study. Out of the 200 cases, 14 cases were excluded from the analysis since they were β-globin negative. The patients were aged from 23 to 86 years. majority of the patients were above 50 years; the mean age was 54.54 (SD 12.87).

Types of cervical cancer

Majority of the cervical cancers seen in this study were squamous cell carcinoma and its variants (80.10%), followed by adenocarcinoma and its variants (14 %). adenosquamous carcinoma, carcinosarcoma and sarcomas make up less than 6% of the cervical cancer types seen.

Prevalence and

Out of the 186 cases used for statistical analysis, 147 cases (79%) were positive for HPV-specific DNA. Nine [9] different high-risk HPV genotypes were detected (Table 1).

They included HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 45, HPV 51, HPV 52, and HPV 82. HPV 16 is the most common genotype detected (53.2% of cases), followed by HPV 18 (37.6%). Less common high-risk genotypes seen include HPV 31 and HPV 33. This highlights the dominance of high-risk HPV strains, particularly HPV 16 and HPV 18.

Single and multiple high-risk HPV co-infections were observed (Table 2).

There were 90 (48.4%) cases of single HPV infection and 96 (51.6%) cases of multiple HPV infections. A co-infection with low-risk HPV 11 was seen in five cases (4.08%). In both single and multiple HPV infections, the three most frequent high-risk HPV genotypes detected were HPV 16 (53.2%), HPV 18 (37.6%) and HPV 35 (13.4%). HPV 16 and HPV 18 accounted for 90.8%. In multiple HPV infections, double infections with HPV 16 and 18 was the most frequent, observed in 28 (19.05%) cases. Triple and quadruple HPV infections were also seen.

When we considered the type of cervical cancer, the highest HPV positivity was seen in squamous cell carcinoma (Figure 1).

Figure 1. Shows the Relationship between HPV Positivity and the Type of Cervical Cancer Seen in This Study. The highest positivity is observed in Squamous cell carcinoma (83.9%), with significant variability among other types, reflected in a p-value of 0.007. SQC=Squamous cell carcinoma; ADC=Adenocarcinoma; ASC=Adenosquamous carcinoma; CSA=Carcinosarcoma; SAR=Sarcoma.

Considering the HPV positivity across the age groups used in this study shows that HPV 16 is consistently dominant across all age groups (Figure 2).

Figure 2. Examines HPV Strain Distribution by Age Group, with HPV16 Being Consistently Dominant Across all Age Categories. There is no significant differences in strain distribution across ages suggesting that the prevalence of specific HPV types remains steady, regardless of age. This points to widespread exposure across the lifespan.

Discussion

It has been established that infection with high-risk HPV is the most important aetiologic agent in the pathogenesis of cervical cancer [6, 7]. Determining the HPV type distribution in cervical cancers in different geographical regions is an essential step in the development of population-specific prophylactic HPV vaccine strategies. Several studies have led to the advocacy for prophylactic HPV vaccines against the development of HPV-associated cervical pre-malignant lesions [11]. Despite the fact that cervical cancer is the commonest gynaelogical cancer in Nigeria and contribute significantly to cancer related deaths in women [2], only few studies have been conducted in this country to determine the prevalence and type distribution of HPV in cervical cancer [12-16]. To the best of our knowledge, no such study has been done in this state. In addition, from our experience, HPV vaccine reception in this part of the world has religious and cultural sentiments as barriers. To encourage and guide HPV vaccination for the primary prevention of cervical cancer in this region, we have determined the HPV prevalence and type distribution in cervical cancer in this South-East region of Nigeria.

Worldwide, there are geographical variations in the prevalence and type distribution of HPV-DNA in cervical cancers [11]. The overall prevalence has been reported to be in the range of 90-100% [17, 18]. In this study, HPV positivity in squamous cell carcinoma is 83.9%. A total prevalence of 79% was recorded when all the cancer types were considered. This result is comparable to results obtained in other studies conducted in Nigeria [15, 16], Ethiopia [19], and Serbia [20]. Some studies have reported prevalence rates of greater than 90%. These includes studies carried out in Nigeria [12, 21], Ghana [22], Malawi [23], South Africa [12], India [24], and USA [25]. Also, relatively low HPV-DNA prevalence rates have been observed in cervical cancer in studies done in iran [26] and poland [27]. These differences in the prevalence rates of HPV-DNA in cervical cancers have been attributed to geographical variations [28], quantity and quality of the tissue specimen [22], method of DNA extraction and sensitivity of the method [29], the specificity of the detection method [30] and type of cervical cancer studied. HPV infects mucosal surfaces [7]. This can explain why studies using only squamous cell carcinomas (the commonest type of cervical cancer) obtained higher prevalences as seen in some of the studies mentioned above. in this study, HPV positivity was seen in 125/149 (83.9%) of squamous cell carcinomas.

The spectrum of HPV-DNA detected in cervical cancers also show regional variations. All over the world, HPV-16 and -18 are the predominant HR-HPV types detected [12]. In this study, eight different high-risk (HPVs-16, 18, 31, 33, 35, 45, 51, and 52) and one

probable high-risk (HPV-82) hpv types were identified. A co-infection with low-risk HPV 11 was observed in five cases. Both single and multiple HPV infections were observed. The three most prevalent high-risk HPV genotypes in this study in decreasing order of frequency were HPV 16, HPV 18, HPV 35. HPV 16 and HPV 18 accounted for 40.3% and 70% of single and multiple infections, respectively. This finding is consistent with several studies across the world [12, 15, 24, 25]. The high prevalence of multiple infections has been attributed to factors like multiple sexual partners and immunosuppression [21], and may be explained by the fact that prior infection with one HPV-type does not decrease the risk of infection by another HPV-type and supports the use of multivalent vaccines [31]. There is conflicting evidence on the significance of multiple infections. Some studies have shown that infection with multiple HPV-types exhibit synergism and additivity of effect and are associated with persistence of infection, increased risk of disease progression, disease severity and poorer outcomes [32, 33]. This is contrary to the findings by salazar et al [34] which shows a reduced rate of high-grade cervical lesions in multiple infections due to intergenotypic competition and/or effective immune response.

Despite the recognition of geographical variation in the prevalence of HPV types [35], HPV-16 and HPV-18 are the most prevalent and the most potent carcinogenic viruses with a higher probability of persistence and disease progression [6, 34]. The four major stages in cervical cancer pathogenesis includes infection of a metaplastic epithelium at the transformation zone, high-risk HPV viral persistence, progression of the persistently infected epithelium to precancer, and invasion of the basement membrane of the epithelium [7].

Because prior infection with one HPV-type does not decrease the risk of infection by another HPV-type [31], multivalent HPV vaccines hold great promise to reduce the burden of ICC globally. It can be deduced from this study that the currently available bivalent (against HPV 16 and HPV 18), quadrivalent (against HPV 6, HPV 11, HPV 16 and HPV 18), and nonavalent (against HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52 and HPV 58) HPV vaccines can prevent majority of the HPV infections in this environment. This is because in this study, the combined proportions of positivity for HPV 16, HPV 18, HPV 31, HPV 33, HPV 45, HPV 52 and HPV 11 is greater than 80%. Reducing the incidence of hpv infections will also protect unvaccinated individuals through herd immunity [14]. Also, because Pap test has relatively high specificity but low sensitivity in detecting HPV infections [36], introducing HPV DNA testing as part of the protocol for cervical cancer screening will improve the sensitivity of Pap test for cervical cancer screening in women above 30 years in this region.

In conclusion, there is high prevalence of HPV-DNA in invasive cervical cancers in this region, with HPV 16, HPV 18, HPV 35, HPV 31, and HPV 52 being the predominant types. This observed high prevalence of HPV in invasive cervical cancers and the predominance of HPV 16 and HPV 18 is a worldwide phenomenon consistent with several studies. The high proportion of multiple HPV types detected in the samples supports multiple sexual partners as a risk factor for cervical cancer in this environment. Being a sentinel study in this state, the findings of this study will serve as baseline data. HPV vaccination and testing will greatly reduce the burden of cervical cancer in this geographical region. The currently available HPV vaccines will be beneficial in this region.

Acknowledgements

The authors sincerely acknowledge and thank surepath pathology consultants’ personnel who contributed to the conduct of this study. Special thanks to Dr. Ayodele Ajayi and Mr. Idongesit Ikpe for providing statistical and technical assistance, respectively. We are immensely grateful to dr tarik gheit of the IARC for making this work a reality.

Conflict of Interest

None

Ethical declaration

The study was approved by the institutional health ethical research committee (IHERC) of absuth in line with the declaration of helsinki and good clinical practice guidelines.

Authors contribution

AE and OC conceived the study. all authors were involved in the conduct of the study. OC and UC were involved in drafting the manuscript. all authors read and approved the final version of the manuscript.

References

References

1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA: a cancer journal for clinicians. 2021; 71(3)DOI
2. Yakasai I. A., Ugwa E. A., Otubu J.. Gynecological malignancies in Aminu Kano Teaching Hospital Kano: a 3 year review. Nigerian Journal of Clinical Practice. 2013; 16(1)DOI
3. Singh D, Vignat J, Lorenzoni V, Eslahi M, Ginsburg O, Lauby-Secretan B, Arbyn M, et al. Global estimates of incidence and mortality of cervical cancer in 2020: a baseline analysis of the WHO Global Cervical Cancer Elimination Initiative. The Lancet. Global Health. 2023; 11(2)DOI
4. The Iarc Perspective On Cervical Cancer Screening | New England Journal Of Medicine [Internet]. [Cited 2025 Jan 4]. Available From: Https://Www.Nejm.Org/Doi/Full/10.1056/Nejmsr2030640.
5. Nigeria: Human Papillomavirus And Related Cancers, Fact Sheet 2023. Fact Sheet. 2023.
6. Kabuga AI, Nejati A, Arero AG, Jalilvand S, Mokhtari-Azad T, Sighaldeh SS, Wali UH, Shahmahmoodi S, El Zowalaty ME. Prevalence and Type Distribution of Human Papillomavirus Recovered from the Uterine Cervix of Nigerian Women: A Systematic Review and Meta-Analysis. Asian Pacific journal of cancer prevention: APJCP. 2020; 21(10)DOI
7. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet (London, England). 2007; 370(9590)DOI
8. Wheeler CM, Castellsagué X, Garland SM, Szarewski A, Paavonen J, Naud P, Salmerón J, et al. Cross-protective efficacy of HPV-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by non-vaccine oncogenic HPV types: 4-year end-of-study analysis of the randomised, double-blind PATRICIA trial. The Lancet. Oncology. 2012; 13(1)DOI
9. Chauhan SC, Jaggi M, Bell MC, Verma M, Kumar D. Epidemiology of Human Papilloma Virus (HPV) in Cervical Mucosa. Methods in Molecular Biology (Clifton, N.J.). 2009; 471DOI
10. Schmitt M, Dondog B, Waterboer T, Pawlita M, Tommasino M, Gheit T. Abundance of multiple high-risk human papillomavirus (HPV) infections found in cervical cells analyzed by use of an ultrasensitive HPV genotyping assay. Journal of Clinical Microbiology. 2010; 48(1)DOI
11. Clifford G. M., Smith J. S., Plummer M., Muñoz N., Franceschi S.. Human papillomavirus types in invasive cervical cancer worldwide: a meta-analysis. British Journal of Cancer. 2003; 88(1)DOI
12. Denny L, Adewole I, Anorlu R, Dreyer G, Moodley M, Smith T, Snyman L, et al. Human papillomavirus prevalence and type distribution in invasive cervical cancer in sub-Saharan Africa. International Journal of Cancer. 2014; 134(6)DOI
13. Mohammed U, Banjo A. a. F., Abdullahi K., Umar A. G., Hassan M., Ango I. G., Magaji B. A., et al. Prevalence and Genotype of HPV in Cervical Smear in Sokoto, North-Western Nigeria: A Sentinel Study to Guide Vaccination. Annals of Tropical Pathology. 2023; 14(1)
14. Manga MM, Fowotade A, Abdullahi YM, El-Nafaty AU, Adamu DB, Pindiga HU, Bakare RA, Osoba AO. Epidemiological patterns of cervical human papillomavirus infection among women presenting for cervical cancer screening in North-Eastern Nigeria. Infectious Agents and Cancer. 2015; 10DOI
15. Orah NO, Banjo AA. Prevalence and Distribution of High Risk Human Papillomavirus Subtypes in Invasive Cervical Cancer in South‑West Nigeria. Annals of Tropical Pathology. 2018; 9(2)
16. Kabir A, Bukar M, Nggada HA, Rann HB, Gidado A, Musa AB. Prevalence of human papillomavirus genotypes in cervical cancer in Maiduguri, Nigeria. The Pan African Medical Journal. 2019; 33DOI
17. Hadzisejdić I, Simat M, Bosak A, Krasević M, Grahovac B. Prevalence of human papillomavirus genotypes in cervical cancer and precursor lesions. Collegium Antropologicum. 2006; 30(4)
18. Natural History And Epidemiology Of Hpv Infection And Cervical Cancer - Pubmed [Internet]. [Cited 2025 Jan 14]. Available From: Https://Pubmed.Ncbi.Nlm.Nih.Gov/18760711/.
19. Bekele A., Baay M., Mekonnen Z., Suleman S., Chatterjee S.. Human papillomavirus type distribution among women with cervical pathology - a study over 4 years at Jimma Hospital, southwest Ethiopia. Tropical medicine & international health: TM & IH. 2010; 15(8)DOI
20. Stamenković M., Knežević A, Kuzmanović I., Karalić D, Jovanović T. Distribution of Human papilloma virus genotypes in cervical cancer tissues. Archives of Biological Sciences. 2014; 66(2)
21. Okolo C, Franceschi S, Adewole I, Thomas JO, Follen M, Snijders PJ, Meijer CJ, Clifford GM. Human papillomavirus infection in women with and without cervical cancer in Ibadan, Nigeria. Infectious Agents and Cancer. 2010; 5(1)DOI
22. Awua A. K., Sackey S. T., Osei Y. D., Asmah R. H., Wiredu E. K.. Prevalence of human papillomavirus genotypes among women with cervical cancer in Ghana. Infectious Agents and Cancer. 2016; 11DOI
23. Howitt BE, Herfs M, Tomoka T, Kamiza S, Gheit T, Tommasino M, Delvenne P, Crum CP, Milner D. Comprehensive Human Papillomavirus Genotyping in Cervical Squamous Cell Carcinomas and Its Relevance to Cervical Cancer Prevention in Malawian Women. Journal of Global Oncology. 2017; 3(3)DOI
24. Gheit T, Vaccarella S, Schmitt M, Pawlita M, Franceschi S, Sankaranarayanan R, Sylla BS, Tommasino M, Gangane N. Prevalence of human papillomavirus types in cervical and oral cancers in central India. Vaccine. 2009; 27(5)DOI
25. Hopenhayn C, Christian A, Christian WJ, Watson M, Unger ER, Lynch CF, Peters ES, et al. Prevalence of human papillomavirus types in invasive cervical cancers from 7 US cancer registries before vaccine introduction. Journal of Lower Genital Tract Disease. 2014; 18(2)DOI
26. Pcr Detection And High Risk Typing Of Human Papillomavirus Dna In Cervical Cancer In Iranian Women | Semantic Scholar [Internet]. [Cited 2025 Jan 14]. Available From: Https://Www.Semanticscholar.Org/Paper/Pcr-Detection-And-High-Risk-Typing-Of-Human-Dna-In-Eslami-Golshani/7bba1a1ceacd560f691db79758ed344a7f912f6.
27. Dybikowska A, Licznerski P, Podhajska A. HPV detection in cervical cancer patients in northern Poland. Oncology Reports. 2002; 9(4)
28. Castellsagué X. Natural history and epidemiology of HPV infection and cervical cancer. Gynecologic Oncology. 2008; 110(3 Suppl 2)DOI
29. Biedermann K, Dandachi N, Trattner M, Vogl G, Doppelmayr H, Moré E, Staudach A, et al. Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue. Journal of Clinical Microbiology. 2004; 42(8)DOI
30. Detection Of Hpv Dna In Paraffin-Embedded Cervical Samples: A Comparison Of Four Genotyping Methods | Bmc Infectious Diseases | Full Text [Internet]. [Cited 2025 Jan 14]. Available From: Https://Bmcinfectdis.Biomedcentral.Com/Articles/10.1186/S12879-015-1281-5.
31. Koutsky LA, Ault KA, Wheeler CM, Brown DR, Barr E, Alvarez FB, Chiacchierini LM, Jansen KU. A controlled trial of a human papillomavirus type 16 vaccine. The New England Journal of Medicine. 2002; 347(21)DOI
32. Lopalco PL. Spotlight on the 9-valent HPV vaccine. Drug Design, Development and Therapy. 2017; 11DOI
33. Munagala R, Donà MG, Rai SN, Jenson AB, Bala N, Ghim SJ, Gupta RC. Significance of multiple HPV infection in cervical cancer patients and its impact on treatment response. International Journal of Oncology. 2009; 34(1)
34. Salazar KL, Zhou HS, Xu J, Peterson LE, Schwartz MR, Mody DR, Ge Y. Multiple Human Papilloma Virus Infections and Their Impact on the Development of High-Risk Cervical Lesions. Acta Cytologica. 2015; 59(5)DOI
35. Nogueira Dias Genta ML, Martins TR, Mendoza Lopez RV, Sadalla JC, Carvalho JPM, Baracat EC, Levi JE, Carvalho JP. Multiple HPV genotype infection impact on invasive cervical cancer presentation and survival. PloS One. 2017; 12(8)DOI
36. Comparative Analysis Of Cervical Human Papillomavirus Dna Testing And Cytological Findings Among Women Presenting For “Pap” Smear In A Tertiary Health Centre In Northern Nigeria [Internet]. [Cited 2025 Jan 15]. Available From: Https://Www.Researchgate.Net/Publication/287799749_Comparative_Analysis_Of_Cervical_Human_Papillomavirus_Dna_Testing_And_Cytological_Findings_Among_Women_Presenting_For_Pap_Smear_In_A_Tertiary_Health_Centre_In_Northern_Nigeria.