Genotype Heterogeneity of Human Papillomavirus Infection and Associated Risk Factors in Northwest Ethiopia: Baseline Findings from Longitudinal Cohort Study

Background: Cervical cancer is a malignant tumor originating from the cells of the uterine cervix. High-risk Human Papillomavirus (HR-HPV) infection is the etiological agent of cervical cancer. The current study aimed to assess genotype heterogeneity human papillomaviruses and associated factors in Bahir Dar, northwest Ethiopia

Methods: A cross-sectional study nested from a prospective cohort was conducted among patients attending the gynecology unit. A total of 297 study participants were screened for HPV DNA and CD4 count was performed to investigate the immunity level. Data were collected using pre-prepared structured questionnaire. Statistical analyses were performed using SPSS version 26.0. 

Results: In this study, one hundred thirty-nine (46.8%) of study participants were positive for HPV infection. The HPV-16 genotype was the most frequent genotype, detected in 14.48% (43/297), followed by HPV-53, HPV-58, HPV-70, HPV-35, and HPV-52 infections, accounting for 10.44%, 7.07% (21/297), 5.72% (17/297), 4.71% (14/297), and 4.04% (12/297) respectively. Age between 30-50 years, parity of greater than six birth, age at first marriage, number of current sexual partner, and history of STI had significant association with HPV infection.

Conclusions: The proportion of HPV is high. Infection with HPV-16, HPV-58, HPV-35, and HPV-52 types require increased surveillance and follow-up. The major explanatory risk factors for HPV infection identified in this study include age, parity, age at first marriage, number of current sexual partner and sexually transmitted infections (STIs). To prevent and control HPV infection and cervical cancer in Amhara regional state and Ethiopia in general, there is a need to curb lack of awareness and poor screening practice.

Cervical cancer is a malignant tumor originating from the cells of the uterine cervix [1]. After lung, colorectal, and breast cancer, it is the fourth most frequent cancer in women worldwide. Each year, cervical cancer results in 340 000 fatalities and 600 000 new cases [2]. Crucially, 88% of all cervical cancer deaths and 83% of new cases are reported to occur in low- and middle-income countries (LMICs) respectively. In fact, 36 countries, including those in sub-Saharan Africa, Latin America, and India, have cervical cancer as the primary cause of cancer-related fatalities [3]. High-risk Human Papillomavirus (HR-HPV) infection is the primary etiological agent of cervical cancer [4]. Twelve HPV genotypes (-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, and -59) are identified by the International Agency for Cancer Research (IARC) as causal agents of cervical cancer, while eight more genotypes (-26, -53, -66, -67, -68, -70, -73, and -82) are classified as likely or possible causes of cervical cancer [5]. The most commonly found high-risk genotypes in a population-based study carried out in south central Ethiopia were HPV-16, -35, -52, -31 and -45 [6]. Persistent HR-HPV infection is an important risk factor for cervical cancer, which is closely linked to precancerous lesions and cervical cancer  [7].

Out of all cancer cases, cervical cancer is the most frequently diagnosed malignancy in Ethiopia (31.8%) and is on the rise (Assefa et al., 2019).. It has a significant impact on public health and affects the most vulnerable populations in the nation, including disadvantaged, rural, and HIV-positive women. The majority (over 80%) of cervical cancer cases in sub-Saharan Africa are detected in a late stage, which is linked to low survival rates. This is because there is a lack of knowledge about cervical cancer and preventive services [9]. On November 17, 2020, the World Health Organization (WHO) launched the "Global Strategy for accelerating the Elimination of Cervical Cancer as a Public Health Problem," and 194 nations pledged to eradicate cervical cancer for the first time (WHO, 2018). Cervical cancer can be associated with a number of factors other than persistent HPV infection. These include sexual behavior, high parity, oral hormonal contraceptive use, smoking tobacco (including cigarettes, cigars, pipes, hookah, and shisha), and co-infection with HIV. The two most significant of these are tobacco use and high parity, since they mediate the course of HR-HPV infection and lead to high incidences of cervical pre-cancer and carcinoma [11]. The current study was conducted to assess Genotype heterogeneity of human papillomavirus infection and associated factors among patients attending gynecology unit of Felege Hiwot Comprehensive Specialized Hospital. Bahir Dar, northwest Ethiopia.

Study setting and Population

A cross-sectional study nested from the baseline study of a prospective cohort was conducted among patients attending the gynecology unit of Felege Hiwot Comprehensive Specialized Hospital (FHCSH) in Bahir Dar, northwest Ethiopia. The study took place between January and December 2023. A total of 297 women were enrolled and all gave their informed consent to participate in the study. Data were collected using pre-prepared structured questionnaire. Sociodemographic, reproductive and sexual behavior data and whole blood sample were collected by clinical nurses whereas cervical swab, smear for PAP and punch biopsies were collected by senior gynecologist. All women who visited FHCSH gynecological unit, whose age above 30 years old and willing to participate were included in this study. Women aged less than 30 years old, if living with HIV and other chronic diseases were also included in the study. All women who took any kind of treatment for cervical cancer or any vaginal medication, vaginal contraceptives or douches 48 hours prior to the test, those who had sexual intercourse 24 hours before the test; women who were on menstruation, pregnant and women who had hysterectomy were excluded from the study.

Laboratory investigations

All 297 study participants were screened for HPV DNA and CD4 count was performed to investigate the immunity level. AnyplexTM II PCR System was used to detect human papillomavirus DNA in cervical swabs, based on a real-time multiplex PCR assay that allows simultaneous amplification, detection, and differentiation of target nucleic acids of 19 high-risk(HR) HPV types (-16, -18, -26, -31, -33, -35, -39, -45,-51, -52, -53, -56, -58, -59, -66, -68, -69, -73, -82) and 9 low-risk(LR) HPV types (-6, -11, -40, -42, -43, -44, -54, -61, -70).  CD4 count was analyzed using FACSPrestoTM near patient CD4 counter.BD FACSPresto technology is based on fluorescent photomicroscopy and light absorbance detection, on a multicolor platform. A single drop of capillary or venous blood was used to measure the percent and absolute CD4 T-lymphocytes using the FACSPresto, a portable CD4 counter with single disposable cartridges.

Statistical analysis

Statistical analyses were performed using SPSS version 26.0. Descriptive statistics such as frequency and cross tabulation were performed to summarize the data. Bivariate and multivariate logistic regression analyses were performed to calculate Crude Odds Ratio (COR) and Adjusted Odds Ratio (AOR) respectively at 95% confidence interval for factors associated with any HPV infection. Variables with a significance level of P less than 0.05 in the bivariate regression were included in the multivariable regression. Variables with a p-value of less than 0.05 were considered statistically significant.

Sociodemographic, sexual and reproductive characteristics of study participants

A total of 297 women attending gynecology unit of Felege Hiwot Comprehensive Specialized Hospital were participated in the study. About 61.3% (182/297) of the participants live in rural area, majority of study participants (84.5%) were illiterates, more than half of study participants (63%) were married and 52.2% (155/297) of them had 4 to 6 children.  One hundred thirty-three (44.8%) of study participants had their first sexual debut before the age of 18 years. Two hundred nineteen (73.7%) of them reported having two or more lifetime sexual partners and one hundred seventy-seven (59.6%) of study participants reported having two or more current sexual partners. One hundred seventy-six (59.3%) of the women reported never using a condom during sexual intercourse, while 20.9 % (62/297) reported using hormone contraceptives for more than five years. Most women 73.1% (217/297) had never heard about human papillomavirus, cervical cancer and its transmission. Furthermore 78.8% (234/297) of study participants had never been screened for HPV infection and precancerous lesion. History of sexually transmitted infections (STI) was reported by 35.4% (105/297). Two hundred six (69.4%) of study participants had CD4 count between 500 and 999 cells/mm3. Vaginal bleeding (35.69%), vaginal discharge (41.75%), abdominal pain (25.25%) and back pain (19.87%) were the chief complaints explained by the study participants. The socio-demographic, sexual and reproductive characteristics of study participants are summarized in Table 1.

Table 1: Sociodemographic, sexual and reproductive characteristics of women attending gynecology unit, Felege Hiwot Comprehensive Specialized Hospital, Bahir Dar, northwest Ethiopia.

*There were more than one sign and symptoms among participants; **Difficulty of urination, difficulty of defecation, protruded mass per vagina, burning sensation and itching, pain during sexual intercourse and urination

Proportion and type specific distribution of HPV

In this study, one hundred thirty-nine (46.8%) of study participants were positive for HPV infection. The HPV-16 genotype was the most frequent genotype, detected in 14.48% (43/297), followed by HPV-53, HPV-58, HPV-70, HPV-35, and HPV-52 infections, accounting for 10.44% (31/297), 7.07% ((21/297), 5.72% (17/297), 4.71% (14/297), and 4.04% (12/297) respectively. The four most prevalent high risk HPV types were HPV-16, HPV-58, HPV-35, and HPV-52 infections, with frequencies 14.48% (43/297), 7.07% (21/297), 4.71% (14/297), and 4.04% (12/297) respectively. Among the HPV-positive women, 52.52 % (73/139) were positive for a single HPV type, of which HPV-16, HPV-53, HPV-18, HPV-58, and HPV-70 accounting 23.29% (17/73), 13.69% (10/73), 8.22% (6/73), 6.85% (5/73), and 5.48% (4/73) respectively; 28.78% (40/139) had dual infections, 14.39% (20/139) had triple infections, and 4.32% (6/139) had four or more infections. The details are shown in Table 2.

Table 2: Proportion and type specific distribution of HPV

*16 of Type-53 occurs with Probable HPV, ** 8 of Type-70 occurs with probable HPV

Risk-factors associated with HPV infection

The association between HPV infection with sociodemographic, reproductive health, and sexual behavior was analyzed through bivariate and multivariate analysis using logistic regression. In bivariate analysis, the risk factors found to be significantly associated with HPV infection were age, monthly income, parity, age at first marriage, number of lifetime sexual partners, number of current sexual partner, Co-existing medical condition, hormonal contraceptive use for more than five years, history of STI, and CD4 count. In the multivariate analysis, age between 30-50 years, parity of greater than six, age at first marriage, number of current sexual partner, and history of STI had significant association with HPV infection. Women with age between 30 and 50 years were 4.3 times (AOR=4.3; 95% CI: 1.44-12.82) more likely at risk of HPV infection than those who were less than 30 years old. Women with parity greater than six were 2.66 times (AOR=2.66; 95%CI: 1.02-6.91) more likely to be infected with HPV. An association was found between HPV infection and age at first marriage more specifically, women who had married at age greater than 20 years were 64% (AOR=0.36; 95%CI: 0.16-0.81) less likely to acquire HPV infection than those women who married at the age less than 18 years. Likewise, the increasing number of sexual partners was associated with the increasing risk of HPV infection; women who had more than one current sexual partner had 7.72 times more risk to be infected with HPV (AOR = 7.72; 95%CI: 2.78-21.42) than those women who had only one current sexual partner. Moreover, women who had a history of sexually transmitted infection (STI) were 7.9 times (AOR=7.90; 95%CI: 3.92-15.93) more likely to have HPV infection than those women who did not have a history of STI (Table: 3).

Table 3: Bivariate and multivariate analysis of factors associated with HPV infection among women in Bahir Dar, northwest Ethiopia.

The primary goal of this study was to assess the proportion and type-specific distribution of HPV as well as potentially related sociodemographic, sexual behavior, and reproductive health risk factors of HPV infection among women who visited the gynecology unit of Felege Hiwot Specialized Referral Hospital. Cervical cancer can be caused by HPV infections however; it is not the only factor. According to Italian study [12], additional factors may also be involved in the persistence of HPV and progression to cervical cancer.  The finding of this study showed that the overall proportion of HPV among these study participants was 46.8% (139/297). This result is consistent with a Brazilian study that found 49% of people were infected with HPV [13]. However, our finding is lower compared to previous reports of studies done (81.82%) in Nigeria [14], (74.6%) in Italia [12], and (77.5%) in Ethiopia [15]. On the other hand, our finding on the HR-HPV proportion is higher than other studies conducted in South Africa which reported 32.2% (66/205) [16]. Similarly, our result is higher than study reports from different parts of Ethiopia, Addis Ababa (23.3%) (Bogale et al., 2022), Butajira (23.2%) (Teka et al., 2021), Oromia (22.7%) (Haile et al., 2019). The observed difference might be attributed to variations in the study's diagnostic test procedures, sample size, and cultural norms. Our study used AnyplexTM II PCR System for detection of human papillomavirus. In general, there is considerable variation in the distribution of HPV prevalence, suggesting worldwide regional differences. The compiled studies from cytologically healthy women revealed that sub-Saharan Africa (SSA) had a higher HPV prevalence (24.0%), with Eastern Africa having the highest prevalence (33.6%) (Kombe et al., 2021).

In our study, the four most prevalent high-risk types were HPV-16, HPV-58, HPV-35, and HPV-52 infections, with frequencies of 14.48% (43/297), 7.07% (21/297), 4.71% (14/297), and 4.04% (12/297) respectively. This finding is largely comparable with a systematic review done in Ethiopia [15] which indicated the top five were HPV-16 (37.3%; 95% CI 35.2.1–39.5%), HPV-52 (6.8%; 95% CI 5.8–8.0%), HPV-35 (4.8%; 95% CI 3.9–5.8%), HPV-18 (4.4%; 95% CI 3.5–5.3%) and HPV-56 (3.9%: 95% CI 3.1–4.9). This finding is also consistent with a systematic review and meta-analysis done in Nigeria which reported, the five most common HR-HPV genotypes, HPV-16, HPV-52, HPV-35, HPV-18, and HPV-58 [20]. Sociodemographic, sexual behavior, and reproductive health risk factors, have been investigated and shown to be associated with high-risk HPV infection. It is critical to understand these risk factors in order to target women who are most at risk of infection with screening programs, particularly in nations with limited screening resources.  In our study, five risk factors remained associated with HPV infection after multivariable regression analysis: age, parity, age at first marriage, number of current sexual partner and sexually transmitted infections (STIs). In the present study, 73.1% (217/297) of the study participants had never heard about HPV infection and cervical cancer and 78.8% (234/297) had never been screened for HPV and precancerous lesion. Our finding is consistent with a study done in Ethiopia which indicated 125 women (36%) were aware of cervical cancer and eight women (2.3%) had previously been screened [21]. Our results are also supported by a global assessment [22] which found women in Africa had the highest risk of cervical cancer and the lowest likelihood of screening for cervical cancer. These results underline the necessity of focused public health initiatives to raise awareness of HPV and cervical cancer and encourage routine screening. In this study, women with age between 30 and 50 years were 4.3 times (AOR=4.3; 95% CI: 1.44-12.82) more likely at risk of HPV infection than those who were less than 30 years old. The distribution of HPV genotypes varied among the three age groups: <30>50 years old. The highest proportion of HPV 74.8% (104/139) was seen in 30-50 years age group. This result is similar with a study done in Ghana [23] which had reported the prevalence of HPV infection increased with age up to the 41–60 age group, with a peak. [24] have reported the occurrence of a second peak in women who are less than 25 years old. 

Our study found that women with parity greater than six were 2.66 times (AOR=2.66; 95%CI: 1.02-6.91) more likely to be infected with HPV. This finding corresponds with other studies conducted in China that reported a high risk of HPV infection in women with high parity (Taku et al., 2020, Yu et al., 2022). Several other studies have confirmed this finding, that multigravida women were more likely to have HPV infection. These findings suggest a proportional association exists between multiparity and HPV infection. As previously mentioned, changes to the squamocolumnar junction occur during pregnancy due to increased hormone levels, especially progesterone. Such biological changes raises the possibility of coming into encounters with HPV, persistent of the infection and that eventually leads to cervical neoplasiathat progress to cervical cancer [16]. In this study, an association was found between HPV infection and age at first marriage more specifically, women who married after turning 20 were 64% (AOR=0.36; 95%CI: 0.16-0.81) less likely to get HPV infection than women who married before turning 18, consistent with studies that reported an early marriage is linked to HPV infection [22]. In the current study, the increasing number of sexual partner was associated with the increasing risk of HPV infection; women who had more than one current sexual partner had 7.72 times more risk to be infected with HPV (AOR = 7.72; 95%CI: 2.78-21.42) than those women who had only one current sexual partner, similar with other studies that reported more than two sexual partners was an independent factor for HR-HPV infection (Tangjitgamol et al., 2016, McHome et al., 2021). Additionally, a Japanese study found a link between HPV infection and the number of sexual partners [29].

In our study, women who had a history of sexually transmitted infection (STI) were 7.9 times (AOR=7.90; 95%CI: 3.92-15.93) more likely to have HPV infection than women without a history of STI., which is in agreement with a study conducted in Ethiopia that revealed higher odds of HPV positivity (COR = 1.56; 95% CI: 0.59–4.11, p = 0.36) among participants with a history of STIs [30].According to a study conducted in Brazil, the presence of any STI increases the prevalence of HPV infection, and having a history of STIs has been associated with either a decreased ability to clear HPV or an increased risk of contracting HPV infection (Kops et al., 2019). This finding may be crucial for the creation of behavioral and medical interventions, such as STI screening and treatment programs. Generally, HPV infection and cervical pre-cancer remains the major cause of cancer related deaths in women from developing countries like Ethiopia. Lack of awareness and screening along with other factors contribute for the death.

The proportion of HPV is high. Infection with HPV-16, HPV-58, HPV-35, and HPV-52 types require increased surveillance and follow-up. The major explanatory risk factors for HPV infection identified in this study include age, parity, age at first marriage, number of current sexual partner and sexually transmitted infections (STIs). The current study emphasizes poor awareness and screening history. To prevent and control HPV infection and cervical cancer in Amhara regional state and Ethiopia in general, there is a need to curb lack of awareness and poor screening practices. Some risk factors for the HPV infection which are preventable or means to early detection to reduce cervical cancer should be emphasized in the health system in Ethiopia. It is necessary to conduct further study to better understand the factors contributing to HPV infection and to develop effective prevention strategies.

Conflicts of Interest
The authors declare that they have no competing interests.

Ethics approval and consent to participate

Ethical approval was obtained from Institutional Review Board (IRB), Bahir Dar University. Written informed consent was ensured from all study participants to take part in the study voluntarily after they get informed about the objective and purpose of the study.

Funding

No funding was given to support this study

Author contributions

The corresponding author (Alemayehu Abate) was involved in the conception, design, drafted the present manuscript. Abaineh Munshea and Endalkachew Nibret were involved in the analysis and interpretation of data and critically revising the manuscript for important intellectual content.