High rate of non-vaccine-targeted high-risk HPV genotypes in Ethiopia: Its implication in future vaccine selection

doi:10.21203/rs.3.rs-3111550/v1

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High rate of non-vaccine-targeted high-risk HPV genotypes in Ethiopia: Its implication in future vaccine selection

https://doi.org/10.21203/rs.3.rs-3111550/v1

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published 10 Jan, 2024

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Since the distribution of high-risk HPV genotypes varies across countries, genotype-based vaccination is widely recommended to control the burden of cervical cancer. As of 2018, HPV vaccination program is underway in Ethiopia for girls aged 9–14 years against HPV-6, HPV-11, HPV-16 and HPV-18. However, the rate and distribution of non-vaccine-targeted genotypes are not well characterized. Therefore, by determining the prevalence and characterizing their genotypes, we assessed the level of multiple infections with other vaccine-targeted genotypes in Ethiopia. A health facility-based cross-sectional study including 110 women with a positive HPV DNA results was conducted from April to August 2021. We used a structured questionnaire to collect demographic and clinical data and collected cervical swabs using L-shaped FLOQSwabs®. We, then, stored them in eNAT nucleic acid preservation and transport® medium. Women's cytological profile was determined based on Pap smear teat results, and we made automated nucleic acid extraction using STARMag 96 ProPrep Universal Extraction Kit. We have used a real-time amplification assay to amplify and identify the HPV Late 1 [L1] gene used for genotyping. After the collected data has entered into Epi data version 3.1 software, the analysis was done with STATA version 14. Among 901 women who underwent HPV DNA testing, only 110 women [age range 30 to 60 years, mean age = 36.5 years and SD ± 6.9] had positive HPV DNA results and were included in the study. Among these, 108 women had valid co-testing [Pap test and HPV DNA test] results for further analysis, and the results of the remaining 2 women were rejected. Overall, the prevalence of non-vaccine-targeted HPV was 51.8% (95% CI: 0.424–0.611), of which 28 women (25.4%, 95% CI: 0.181–0.345) had a single non-vaccine HPV genotype infection. The remaining 29 women (26.4%, 95% CI: 0.190–0.355) experienced multiple infections. The non-vaccine-targeted genotypes of HPV-35 (10%, 95% CI: 0.056–0.173), HPV-68 (8.2%, 95% CI: 0.043–0.151), HPV-56 (7.3%, 95% CI: 0.036–0.140), and HPV-66 (7.3%, 95% CI: 0.036–0.140) were found in higher numbers. In addition, out of these 108 women, 93 (86.1%, 95% CI: 0.781–0.915) had low-grade squamous intraepithelial lesions, 13 (12%, 95%CI: 0.071–0.198) no intraepithelial lesion or malignancy, and two (1.9%, 95%CI: 0.004–0 .072) high-grade squamous intraepithelial lesions. Furthermore, there was no statistical difference (p = 0.755) between vaccine-targeted and non-vaccine-targeted genotypes as the primary cause of cervical injury. In Ethiopia, non-vaccine-targeted HPV genotypes are highly prevalent, including HPV-35, HPV-68, HPV-56, and HPV-68. More than a quarter of women had multiple infections, which increase their risk of developing cervical cancer. Therefore, changing from the current vaccine that protects against four HPV types to the vaccine that covers seven HPV genotypes will have better outcome in preventing cervical cancer.

Health sciences/Medical research/Epidemiology

Biological sciences/Molecular biology

Human papillomavirus is one of the most common carcinogens that cause cervical cancer[1]. Despite most infections clearing up within 2 years without showing any symptoms, HPV genital infection can lead to different types of clinical diseases [2]. Persistent infection due to the hr HPV occurs in almost all cases of cervical cancer [3]. However, the level of persistence, progression to high-grade squamous intraepithelial lesion (HSIL), and cervical cancer depends on the hr HPV genotype [4]. Globally, cervical cancer is the fourth most common cause of cancer in women and its prevalence of the disease is very high among low- and middle-income countries (LMICs)[5–7]. Countries are using two preventive measures to prevent the spread of the virus. The first level of prevention is the HPV vaccine and the second level of prevention is the cervical cancer screening program. Although these are effective prevention methods, their application only helps to prevent cervical cancer [8, 9].

Currently, there are six types of HPV vaccines approved by the World Health Organization: three bivalent, two quadrivalent and one nonavalent vaccine[10]. Globally, HPV-16 and HPV-18 are responsible for approximately 70% of cervical cancer cases. So, all the vaccines that have been used so far are very effective in preventing infections caused by HPV-16 and HPV-18. In addition to these two genotypes, quadrivalent vaccines are highly effective in preventing two additional warts-causing HPV genotypes, HPV-6 and HPV-11. In addition to HPV-16 and HPV-18, the nonavalent vaccine is highly effective against the two low-risk (HPV-6 and HPV-11) and five high-risk (HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58) HPV genotypes [10, 11]. In Ethiopia, a two-dose quadrivalent vaccination program has been implemented for girls 9 to 14 years of age since 2018 [12]. The results of previous vaccine trials and post-implementation evaluations showed that the bivalent [13] and quadrivalent vaccines provided some additional levels of protection against additional HPV genotypes not directly targeted by the vaccines. But, the level of cross-protection varies with vaccine type, HPV genotype, and study [14]. In connection with this, observations of a reduction in HPV prevalence in women after the implementation of vaccination programs in some countries serve as a first indication of the effectiveness of the program. However, they also reported that there was no significant change in non-vaccine-targeted HPV prevalence in these countries [15, 16].

The molecular assays of HPV are capable of partial, extended, or complete genotyping of the virus [17–19]. Partial genotyping is the ability of an assay to report HPV-16 and HPV-18 individually and the remaining 12 high-risk genotypes in one group while extended genotyping refers to assays that individually detect at least 6 hr HPV genotypes and the remaining in one or more groups. In addition, complete genotyping reflects assays that report all hr HPV genotypes individually[17].

In addition to the low-level and inconsistent cross-protection, CC is often associated with multiple HPV genotype infections. Therefore, the non-vaccine-targeted genotypes may develop carcinogenesis more rapidly and more extensively when infected together with vaccine-targeted genotypes (For example HPV-51 and HPV-58%) [20–22]. The non-vaccine-targeted HPV genotypes, HPV-35/ HPV-39/ HPV-51/ HPV-56/ HPV-59, account for 7% of cervical cancers worldwide. However, in some areas, it was found that their prevalence in women aged 15 to 26 years is from 17 to 48.6%. Indeed, they are not prevalent at the stage of HSIL and invasive cancer [23–26].

Although there is some information on the prevalence of HPV genotypes not targeted by vaccines in other parts of the world, there is a serious information gap in Ethiopia. This study was conducted to fill the information gap and address the lack of data.

Study setting and design

This study was conducted from April to August 2021 at Hiwot Fana Specialized University Hospital (Harar), Dil-Chora Referral Hospital (Dire-Dawa), and Shiek Hassan Yabare Referral Hospital (Jigjiga) in Eastern Ethiopia.

Population and Eligibility criteria

This study included women who met the following inclusion criteria and who sought medical care for gynecological problems. Women who have lived permanently in the study area for at least 6 months, aged between 30 and 65 years and signed their consent to participate in the study were included in the study. However, women who have had sexual intercourse within the 24 hours prior to clinical examination, or who have had heavy menstrual bleeding and it is difficult to perform an appropriate screening test, or who have had physical or mental problems for various reasons and who have had difficulty conducting interviews, or who have had a history of gynecological surgery were not included in this study.

Sample size

Among 901 study participants who went through DNA tests in the study, all 110 women who had positive DNA test results were included in the study and underwent the genotyping.

Operational definitions

Cervarix vaccine-targeted high-risk HPV genotypes are the hr HPVs genotypes which are targeted and able to be prevented by the Cervarix vaccine. These are HPV-16 and HPV-18

Quadrivalent vaccine-targeted high-risk HPV genotypes are the hr HPVs genotypes which are targeted and able to be prevented by the Quadrivalent vaccine. These are HPV-6, HPV-11, HPV-16 and, HPV-18.

Nonavalent vaccine-targeted high-risk HPV genotypes are the hr HPVs genotypes which are targeted and able to be prevented by the Nonavalent vaccine. These are HPV-6, HPV HPV-11, HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58)

Non-vaccine targeted high-risk types are the hr HPVs not targeted and prevented directly by Cervarix or Gardasil or Gardasil 9 vaccines. These are HPV-35, HPV-39, HPV-51, HPV-56, HPV-59, and HPV-68

Unsatisfactory Pap test result means a clinical specimen that does not have enough cells or a specimen in which the cells appear clumped together or are obscured by blood or mucus and are difficult to examine.

Data collection

Socio-demographic and other relevant bio-behavioral and clinical data were collected through a face-to-face interview using pre-designed and pre-tested structured questionnaire.

Cytological examinations

We first visually screened women who sought medical care for HPV infection and met the inclusion criteria using the acetic acid [27]according to the WHO visual inspection with acetic acid [VIA] screening guidelines. The exfoliated ectocervical and endocervical cells were collected using L-shaped Endo/Esocervical FLOQSwab® [Copan Italia SpA, Brescia, Italy] and used to make a smear on the slide. The smear was fixed on the slide using ethanol and stained according to standard protocols[28]. Then, the cytological features of cells were read and results were recorded on standardized forms according to the 2015 Bethesda System[29]. Additional descriptions related to cytological test result reporting and decision-making can be seen in our research work published earlier[30]

Liquid-based cervical swabs collection and storage

Following the manufacturer's instructions, we collected cervical swabs from the cervical canal using L-Shaped Endo/Esocervical FLOQSwabs® [Copan Italia SpA, Brescia Italy] and placed them in a 2 mL tube of eNAT® medium. The medium can store the collected nucleic acids for a month at room or higher temperature for HPV DNA detection and genotyping. The collected cervical samples were transported to Child Health and Mortality Surveillance [CHAMPS] Ethiopia project, Haramaya University, and Armauer Hansen Research Institute [AHRI], Addis Ababa laboratories and stored at -80^OC until further processed.

HPV DNA Extraction, Detection and Genotyping

We performed HPV DNA extraction, detection and genotyping procedures according to the manufacturer's instructions [Seegene, South Korea]. The detailed description and process are described in our previously published research work[30] .

Statistical Methods

We checked the collected data for completeness before entering the data into the database. Then, we cleaned and coded the collected data and entered it into Epi data version 3.1 software [The EpiData Association, Odense, Denmark], and export it to STATA version 14 [StataCorp LP., College Station, Texas 77845 USA] for analysis. Frequencies, proportions, and summary statistics were used to describe the study population with relevant variables. For the categorical and discrete variables under the first and third objectives, proportions were estimated using the Pearson Chi-squared test to make comparisons. However, the data we used under the second objective did not meet the statistical assumptions that led us to use Pearson's chi-square test, so we used Fisher's exact test to compare proportions as needed. The binary logistic regression model was used to identify factors associated with HPV infection and odds ratio with 95% CI were used to assess the degree of association. We used variables with p < 0.25 to select candidates for multivariate logistic regression, and variables with p < 0.05 were considered statistically significant associations.

Ethical considerations

The study protocol got ethical approval from the Institutional Health Research Ethics Review Committee [IHRERC] of the College of Health and Medical Sciences, Haramaya University, Ethiopia [Reference Number: IHRERC/212/2020] and the Armauer Hansen Research Institute Ethics Committee [Reference Number: PO/20/20]. Before data collection, we obtained signed informed consent from the study participants. During the course of the study, we linked women with positive HPV DNA in their samples to an Oncologist and Gynecologist for optimal and ongoing treatment. We also coded the names and other identifiers of study participants to protect the confidentiality of the results. The study was conducted in accordance with the Helsinki Declaration[31] and national research ethics review guidelines of Ethiopia[32].

Sociodemographic characteristics

Of the total 110 HPV positive participants, 80 (72.7%) women had a single HPV genotype. Majority of the study participants were urban dwellers (90%), married (73.6%), aged 18 years and above (63.5%), and had more than one sexual partner (80.9%) (Table 1).

Table 1

Socio-demographic characteristics of the study participants
Variable	Category	Single infection, N (%)	Multiple infections, N (%)	Total, N (%)
Residence	Urban	71 (88.7)	28 (93.3)	99 (90)
Residence	Rural	9 (11.2)	2 (6.7)	11 (10)
Age (in years)	< 31	25 (31.2)	8 (26.7)	33 (30)
	31–35	22 (27.5)	8 (26.7)	30 (27.3)
	36–40	21 (26.2)	4 (13.3)	25 (22.7)
	41–45	5 (6.2)	6 (20)	11 (10)
	46–50	4 (5)	3 (10)	7 (6.4)
	56–60	3 (3.7)	1 (3.3)	4 (3.6)
Marital status	Married	60 (75)	21 (70)	81 (73.6)
	Never married	6 (7.5)	3 (10)	9 (8.2)
	Widowed	3 (3.7)	3 (10)	6 (4.4)
	Divorced	10 (12.5)	3 (10)	13 (11.8)
	Separated	1 (1.2)	-	1 (0.9)
Age at first Marriage [ in years]	< 15	7 (8.7)	4 (13.3)	11 (10)
	15–17	19 (23.7)	10 (33.3)	29 (26.5)
	≥ 18	54 (67.5)	16 (3.3)	70 (63.5)
Current occupational status	Full time employee	22 (27.5)	9 (30)	31 (28.2)
	Part-time employee	9 (11.2)	4 (13.3)	13 (11.8)
	Unemployed	42 (52.5)	13 (43.3)	55 (50)
	Student	2 (2.5)	2 (6.7)	4 (3.6)
	Retired	1 (1.2)	1 (3.3)	2 (1.8)
	Other	4 (5)	1 (3.3)	5 (4.5)
Educational status	Unable to read and write	35 (43.7)	8 (26.7)	43 (39.1)
	Between 1 to 8 grades	23 (28.7)	13 (43.3)	36 (32.7)
	Between 9 to 12 grades	10 (12.5)	4 (13.3)	14 (12.7)
	Diploma and above	12 (15)	5 (16.7)	17 (15.4)
Number of sexual partners	Single	17 (21.2)	4 (13.3)	21 (19.1)
Number of sexual partners	Multiple	63 (78.7)	26 (86.7)	89 (80.9)

Note

A single infection means that only one type of HPV is detected at the time of testing, and multiple infection means that more than one type of HPV is present at the time of testing.

The overall distribution of hr HPV genotype proportions

Among the 110 hr HPV genotypes detected in the study, HPV-16 (32.7%, 95%CI: 0.245–0.422), HPV-31 (19.1%, 95%CI: 0.127–0.277), HPV-52 (11.8%, 95%CI: 0.069–0.194), HPV-58 (10.9%, 95% CI: 0.062–0.184), and HPV-35 (10%, 95% CI: 0.056–0.173) were placed in the first top-five ranks. HPV-35 is one of the non-vaccine-targeted genotypes.

In addition, the non-vaccine-targeted genotypes, HPV-68 (8.2%, 95%CI: 0.043–0.151), HPV-56 (7.3%, 95%CI: 0.036–0.140), and HPV-66 (7.3%, 95%CI: 0.036–0.140) were within the highest three ranks after HPV-35 (Fig. 1).

The distribution and composition of multiple infections among the study participants

A total of 157 hr HPV genotypes [including multiple infections] were detected in 110 women. Of the women with hr HPV detected, 30 (27.3%, 95% CI: 0.197–0.365) women had multiple infections while the remaining were analysed for single infection.

Among women with multiple infections, the occurrence of vaccine-targeted hr HPV-16 (16.7%) and hr HPV-31 (6.7%) with the non-vaccine targeted genotypes and the non-vaccine targeted hr HPV-35 (6.7%) with other vaccine-targeted genotype, placed them in the top three positions (Table 2).

Table 2

The occurrence of HPV-16, HPV-31, and HPV-33 with other genotypes among the study participants
Genotype	Other genotypes detected with HPV-16			Number (n = 30)
HPV-16*	HPV-31*	HPV-52*	HPV-66**	1
HPV-16*	HPV-31*	HPV-68**		1
HPV-16*	HPV-45*	HPV-59**		1
HPV-16*	HPV-51**	HPV-59**		1
HPV-16*	HPV-52*	HPV-56**		1
HPV-16*	HPV-45*			2
HPV-16*	HPV-31*			1
HPV-16*	HPV-58*			1
HPV-31*	HPV-58*			3
HPV-31*	HPV-33*			2
HPV-31*	HPV-66**			2
HPV-31*	HPV-39**			1
HPV-31*	HPV-45*			1
HPV-35**	HPV-39**	HPV-52*	HPV-58*	1
HPV-35**	HPV-56**	HPV-66**		1
HPV-35**	HPV-52*			1
HPV-35**	HPV-56**			1
HPV-35**	HPV-68**			2
* Vaccine-targeted hr HPV genotype **Non-vaccine-targeted hr HPV genotype

Distribution of non-vaccine-targeted genotypes based on types of infection

Besides the HPV genotypes that can be prevented through vaccines approved by the World Health Organization (WHO) [Cervarix, Gardasil, and Gardasil 9], we detected other non-vaccine-targeted HPV genotypes [HPV-35, HPV-39, HPV-51, HPV-56, HPV-66, and HPV-68] from women who participated in the study.

Thus, among the 57 women (51.8%, 95% CI: 0.424–0.611) with non-vaccine-targeted genotypes [including multiple infection], 28/110 (25.4%, 95% CI: 0.181–0.345) women had a single non-vaccine-targeted hr HPV genotype infection, while the remaining 29/110 (26.4%, 95% CI: 0.190–0.355) women had multiple infection (Table 2). In addition, HPV-35 (10%, 95% CI: 0.056–0.173), HPV-68 (8.2%, 95% CI: 0.043–0.151), HPV-56 (7.3%, 95% CI: 0.036–0.140), and HPV-66 (7.3%, 95% CI: 0.036–0.140) were found in higher numbers (Table 3).

Table 3

Non-vaccine-targeted genotypes distribution based on types of infections
hr HPV* (n = 110)			95% CI	Single infection			Multiple infection
Genotype	N	%	95% CI	N	%	95% CI	N	%	95% CI
HPV-35	11	10	0.055–0.173	3	2.7	0.008–0.082	8	7.3	0.038–0.140
HPV-39	7	6.4	0.030–0.129	4	3.6	0.013–0.094	3	2.7	0.009–0.082
HPV-51	7	6.4	0.030–0.129	5	4.5	0.019–0.106	2	1.8	0.004–0.071
HPV-56	8	7.3	0.036–0.140	3	2.7	0.009–0.082	5	4.5	0.019–0.106
HPV-59	7	6.4	0.030–0.129	4	3.6	0.013–0.094	3	2.7	0.009–0.082
HPV-66	8	7.3	0.036–0.140	4	3.6	0.013–0.094	4	3.6	0.013–0.094
HPV-68	9	8.2	0.043–0.151	5	4.5	0.019–0.106	4	3.6	0.013–0.094
*high-risk Human papillomavirus

Distribution of vaccine-targeted genotypes based on types of infections

In addition to the non-vaccine-targeted hr HPV genotypes, the study has also determined the three vaccines [Cervarix, Gardasil, and Gardasil 9] targeted hr HPV genotypes.

Accordingly, HPV-16 and − 18 were detected in a total of 39 (35.4%, 95% CI: 0.270–0.450) women. Among these, HPV-16 was detected in 36 (32.7%, 95%CI: 0.245–0.422) while HPV-18 was detected in only 3 (2.7%, 95% CI: 0.009–0.082) women. Additionally, 25 (22.7%) of 36 women with HPV-16 had a single infection (95%CI: 0.157–0.316) and in the remaining 11 (10%) women it was found to be multiple infections (95% CI: 0.055–0.173).

Similarly, the hr HPV genotypes targeted by nonavalent vaccine, namely HPV-16, HPV-18, HPV-31, HPV-33, HPV-45, HPV-52, and HPV-58 were found in 80 (72.7%, 95% CI: 0.635–0.803) women [including multiple infection], of whom nine (8.2%, 95% CI: 0.043–0.151) women had multiple infections (Table 4).

Table 4

Vaccine-targeted genotype distribution based on types of infections
Vaccine	hr HPV (n = 110)			95% CI	Single infection			Multiple infection
	Genotype	N	%		N	%	95% CI	N	%	95% CI
Cervarix, Gardasil⁺, and Gardasil 9⁺	HPV-16	36	32.7	0.245–0.422	25	22.7	0.157–0.316	11	10	0.055–0.173
Cervarix, Gardasil⁺, and Gardasil 9⁺	HPV-18	3	2.7	0.009–0.082	2	1.8	0.001–0.071	1	0.9	0.001–0.063
Gardasil 9⁺	HPV-31	21	19.1	0.127–0.277	13	11.8	0.069–0.194	8	7.3	0.036–0.140
	HPV-33	3	2.7	0.009–0.082	2	1.8	0.004–0.071	1	0.9	0.001–0.063
	HPV-45	7	6.4	0.030–0.129	4	3.6	0.013–0.094	3	2.7	0.009–0.082
	HPV-52	13	11.8	0.070–0.194	6	5.4	0.024–0.117	7	6.4	0.030–0.129
	HPV-58	12	10.9	0.062–0.184	7	6.4	0.030–0.129	5	4.5	0.019–0.106

+ Targets additional two low-risk HPV genotypes [HPV-6 and HPV-11]

Distribution of non-vaccine-targeted genotypes based on cytology results

Among 110 women who participated in the study to determine the prevalence of vaccine and non-vaccine-targeted genotypes based on cytology results, two women were excluded from further analyses due to unsatisfactory Pap test results. Therefore, we used the test results of 108 women for further analysis.

Among the 108 women, 93 (86.1%, 95% CI: 0.781–0.915) women had Low-grade squamous intraepithelial lesions [LSIL], 13 (12%, 95%CI: 0.071–0.198) women had no intraepithelial lesion or malignancy [NILM], and two (1.9%, 95%CI: 0.004–0 .072) women had cytological results of high-grade squamous intraepithelial lesions [HSIL]. Bearing in mind multiple infections during analysis, among 108 women, the hr HPV genotypes targeted by vaccines in use were detected in 61 (56.5%, 95%CI: 0.468–0.6563) women, and surprisingly, genotypes not targeted by vaccines in use so far were detected in the remaining 47 (43.5%, 95% CI: 0.344–0.531) women (Fig. 2).

Non-vaccine targeted genotypes association with cervical lesions

In addition to descriptive statistics, we examined the association between the incidence of cervical lesions and the vaccine- and non-vaccine-targeted genotypes. Accordingly, we found no statistically significant difference between vaccine-targeted and non-vaccine-targeted genotypes that could be classified as a primary cause of cervical lesions. This means that non-vaccine-targeted genotypes are equally likely to be classified as primary causes of cervical lesions as vaccine-targeted genotypes. Thus, it is important to consider the prevalence of non-targeted genotypes in vaccines.

To the best of our knowledge, no study has been conducted to determine the prevalence of non-vaccine-targeted HPV genotypes in Ethiopia. Thus, this study is at the forefront in determining the prevalence of non-vaccine-targeted HPV genotypes in Ethiopia.

In this study, close to half of the study participants had non-vaccine-targeted genotypes, and similarly, we found multiple infections which are among the main risk factors for cervical cancer in a higher number. Furthermore, we could observe that both vaccine-targeted and non-vaccine-targeted genotypes could be a risk factor in the incidence of cervical lesions.

The prevalence of non-vaccine-targeted HPV genotypes was almost close to the prevalence of vaccine-targeted HPV genotypes among women participating in the study (43.5% versus 56.5%). It was comparable to Mónica Saccucci et al.'s clinical trial results (48.6%) in 2018 [26]. In contrast, it was higher than the results from clinical trials in France (21.6%) and the Philippines (5.1%) published by Daron G. Ferris et al., 2020[33]. This data set points out that the prevalence of hr HPV genotypes not targeted by vaccines is high in Ethiopia. Therefore, determining the prevalence of non-vaccine-targeted genotypes can serve as baseline data for future studies using a longitudinal study design. In addition, the results of this study will encourage other researchers to evaluate the role of non-vaccine-targeted genotypes in combination with other genotypes in the development of cervical cancer, as well as the effects of high numbers of non-vaccine genotypes such as HPV-35 alone and in combination with other cervical cancer-causing genotypes on cervical cancer development.

The HPV vaccine [Gardasil®] currently being administered in Ethiopia which targets HPV-16 and HPV-18 [high-risk group] and HPV-6 and HPV-11 [low-risk group] has a chance to curtail the transmission of other non-vaccine-targeted HPV genotypes [34–36], the cross-protection immunity provided by the vaccine will decrease over time [37]. For this reason, as stated in some studies, there is a possibility of becoming a major public health problem if preventive measures including HPV genotypes that are not the target of the vaccine are not implemented. To further support this idea, results from a 2014 clinical trial showed that non-vaccine-targeted genotypes, namely HPV35/39/51/56/59, accounted for 23–30% of CIN1, 7–14% for CIN2, 3–4% Incidence of CIN3 lesions, were the primary causes [24]. Thus, such information will greatly contribute to the selection of the type of vaccine we intend to use for cervical cancer prevention and to better cover the genotype.

Even though this is a point that needs further research, according to the results of cross-sectional and longitudinal studies conducted at different locations and times, multiple infections with high-risk HPV genotypes increase the risk of cervical cancer [38–42]. Thus, multiple HPV infections are common among women infected with HPV, ranging from 25.1–34.5% [43, 44]. In the present study, its prevalence (27.3%) was comparable to the previous studies in other places. In addition, including non-vaccine-targeted genotypes, this study observed a high prevalence of multiple infections among HPV-16, HPV-31, and HPV-35 with other genotype/s. The results of this study are similar to the results of other longitudinal studies. According to the results of the studies [45–47], many types of infections associated with HPV-16, HPV-18, HPV-35, HPV-52, and HPV-45 can persist and evade the immune system. This is a prerequisite to the development of cancer.

Therefore, knowing the level of multiple infections serves as good information about the measures that should be taken during early cervical cancer prevention, screening and cancer treatment.

Limitation of the study

The study was cross-sectional and did not investigate a cause-effect relationship. Nevertheless, the application of an extended genotyping method of diagnosis strengthens the findings.

In the study, the prevalence of non-vaccine-targeted HPV genotypes was high, with the predominant non-vaccine-targeted genotypes of HPV-35, HPV-68, HPV-56, HPV-66, HPV-39, HPV-51, and HPV-59. More than a quarter of the study participants had multiple infections. The non-vaccine-targeted hr HPV genotypes are equally likely to be a cause of cervical lesions.

Revising the partial genotyping method currently implemented in Ethiopia with an extended or complete genotyping method would be important in determining the contemporary genotype heterogeneity level, rate of multiple infections, and the non-vaccine-targeted genotype prevalence. This will be helpful to provide evidence-based selective treatment to the patient and to evaluate the role of the non-vaccine-targeted genotype co-existence in the process of carcinogenesis.

Most importantly, in addition to the currently used qHPV vaccine, revision of the partial genotyping method will have a significant role in providing additional evidence to review and modify the current vaccination guidelines as necessary.

Data availability

All data supporting the results reported in the article are include in the manuscript

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This study was funded by Amauer Hnasen Research Institute (AHRI) and Haramaya University.

Author contributions

AS, AdM, AnM, BS, RH, and AbA participated in proposal development, data collection, laboratory works, data analysis, and manuscript writing. TG conducted clinical examination, sample collection, and supervision of midwives during sample collection. AdA and AB participated in the cytological examination. DA and DH participated in nucleic acid extraction, laboratory protocol review, HPV detection, and HPV genotyping. All authors contributed to the article and approved the submitted version.

Acknowledgments

First of all, we would like to thank the Armauer Hansen Research Institute [AHRI] for providing financial, laboratory and monitoring support for this study. We would also like to thank Haramaya University for providing support to conduct this research. We would like to express our gratitude to Santina Castriciano and COPAN Italia SpA, Brescia Italy, for donating us the L-Shaped FLOQSwabs® and 2 ml eNAT nucleic acid preservation and transportation medium that are playing a major role for the successful accomplishing of this study. We would also like to extend our thanks to the management of Jugol Hospital, Harar General Hospital, Dire-Dawa Dil Chora Hospital, and Sheikh Hassan Yebere Hospital for their cooperation in collecting data from the study participants. Finally, we would like to express our gratitude to the study participants who gave their consent to participate in this study, and to Mussie Brhane and Tamrayehu Seyoum for their professional support.

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No competing interests reported.

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High rate of non-vaccine-targeted high-risk HPV genotypes in Ethiopia: Its implication in future vaccine selection

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Journal Publication

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Abstract

Figures

Introduction

Materials and methods

Study setting and design

Population and Eligibility criteria

Sample size

Operational definitions

Data collection

Cytological examinations

Liquid-based cervical swabs collection and storage

HPV DNA Extraction, Detection and Genotyping

Statistical Methods

Ethical considerations

Results

Sociodemographic characteristics

The overall distribution of hr HPV genotype proportions

The distribution and composition of multiple infections among the study participants

Distribution of non-vaccine-targeted genotypes based on types of infection

Distribution of vaccine-targeted genotypes based on types of infections

Distribution of non-vaccine-targeted genotypes based on cytology results

Non-vaccine targeted genotypes association with cervical lesions

Discussion

Limitation of the study

Conclusion

Declarations

Conflict of interest

Funding

Author contributions

Acknowledgments

References

Additional Declarations

Status:

Journal Publication

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