|Year : 2019 | Volume
| Issue : 4 | Page : 125-130
Prevalence of high-risk human papillomavirus types 16 and 18 among libyan women in tripoli libya
Hani Alzaquzi1, Lubna Almaghur2, Ahmed Eshagrouni3, Omar Elahmer4, Abdulla Bashein5
1 Department of Life Sciences, Division of Microbiology, School of Basic Sciences; National Centre for Disease Control, University of Tripoli, Tripoli, Libya
2 Department of Gynecology and Obstetrics, Faculty of Medicine, University of Tripoli, Tripoli, Libya
3 ATTASAMI for Diagnostic Services, University of Tripoli, Tripoli, Libya
4 National Centre for Disease Control, University of Tripoli, Tripoli, Libya
5 National Centre for DiMsease Control; Department of Biochemistry, Faculty of Medicine, University of Tripoli, Tripoli, Libya
|Date of Submission||24-Jul-2019|
|Date of Acceptance||07-Dec-2019|
|Date of Web Publication||26-Dec-2019|
Dr. Hani Alzaquzi
Health Protection and Promotion Directorate, National Centre for Disease Control, Gorji Near Sports City, P. O. BOX: 71171, Tripoli
Source of Support: None, Conflict of Interest: None
Background and Aims: Despite the well-known association between human papillomavirus (HPV) and cervical cancer, yet there are no available data concerning the prevalence of HPV and its type distribution among Libyan women. The aim of this study was to investigate the prevalence of the most common high-risk HPV types 16 and 18 among Libyan women in Tripoli and to compare it with the cytological findings of the cervix. Methods: A total of 132 cervical samples were collected from women who sought medical attention at the gynecology outpatient clinic of the Tripoli University Hospital and other gynecology private clinics in Tripoli region. Cervical cytological status was classified according to the Bethesda System 2014. Quantitative polymerase chain reaction was used to facilitate the specific detection of HPV types 16 and/or 18. Results: The cytopathological examination showed that 92.4% of women had normal cervical cytology (n = 122/132) and 7.5% (n = 10/132) of them had cervical lesions. The overall prevalence of the most common oncogenic HPV types was 4.5%, as only six samples (n = 6/132) were confirmed of harboring HPV-DNA. Concerning the cytological status of the cervix, HPV-DNA was not found (0%) in women with a normal cervix, and it was present in 60% of women with cervical lesions. The high-risk HPV type 16 was the exclusive type among our all positive samples, with no detection of HPV type 18 among all our recruited subjects. Conclusion: Even though our findings showed a low overall prevalence of high-risk HPV types among Libyan women, the burden of HPV 16 among women with cervical lesions highlights the need to raise attention toward expanding research about HPV and adopt measures to prevent cervical cancer by vaccination and national screening program. The introduction of HPV-DNA testing in cervical cancer management will greatly benefit early-stage HPV detection and help prevent cervical lesions from progression to cancer.
Keywords: Cervical cancer, cytology, human papillomavirus, Libya, quantitative polymerase chain reaction
|How to cite this article:|
Alzaquzi H, Almaghur L, Eshagrouni A, Elahmer O, Bashein A. Prevalence of high-risk human papillomavirus types 16 and 18 among libyan women in tripoli libya. Libyan J Med Sci 2019;3:125-30
|How to cite this URL:|
Alzaquzi H, Almaghur L, Eshagrouni A, Elahmer O, Bashein A. Prevalence of high-risk human papillomavirus types 16 and 18 among libyan women in tripoli libya. Libyan J Med Sci [serial online] 2019 [cited 2020 Jul 6];3:125-30. Available from: http://www.ljmsonline.com/text.asp?2019/3/4/125/274100
| Introduction|| |
Human papillomavirus (HPV) is considered one of the most sexually transmitted diseases worldwide,, and many epidemiological and clinical studies have recognized this virus as a causative agent of squamous cell carcinoma (SCC) of the cervix uteri and many other epithelial cancers of the vagina, vulva, penis, anus, and oropharynges., More than 40 types of mucosal HPVs have been identified that infect genital tracts, of those 13 types are classified according to their oncogenic potential into high-risk HPV types which implicated in the development of squamous intraepithelial lesions that may progress to cervical cancer in case of persistence.,, The overall prevalence of high-risk HPV in women with cervical cancer has been reported to be 99.7%. The HPV types 16 and 18 are accounted as the most frequent high-risk types and estimated responsible for about 70% of cervical cancer worldwide, with the valuation of HPV type 16 being the most oncogenic type of all., The majority of infected women with high-risk HPV usually do not develop cancer as most HPV infections are transient and resolved spontaneously. However, women with normal cervical epithelium infected with high-risk HPV types have about a 100-fold increased risk of progression to cervical cancer compared to uninfected women. The International Agency for Research on Cancer and its project GLOBOCAN 2012 have estimated 528,000 new cervical cancer cases globally and ranked the cervical cancer as the 4th most common cancer in women and 7th overall. Furthermore, the majority of global incidence (85%) falls in less developed regions, with estimated deaths of 266,000 from cervical cancer in 2012, accounted for 7.5% of all female deaths from cancer.
In Libya, 241 are diagnosed with cervical cancer annually according to the report of the HPV information center. The same report ranked cervical cancer as the 3rd leading cause of female cancer in the country. About 95 cervical cancer deaths occur a year in Libya, ranked it as the 4th leading cause of female cancer deaths. Worldwide, the effective management of cervical cancer depends on early detection of the disease by national screening programs, or on a prophylactic vaccination, which is found to be an interesting primary prevention procedure against cervical cancer.,, Although the data on the prevalence of high-risk HPV types and the strains circulating among the Libyan population are scarce, the quadrivalent vaccine has been introduced in Libya since 2013 of Gardasil® 4. The aim of the current study was to investigate the prevalence of the HPV types 16 and 18 among Libyan women in Tripoli and to compare it with the cytological findings of the cervix.
| Methods|| |
This cross-sectional descriptive study was conducted during January to July 2017. A total of 132 consecutive cervical samples were collected from women seeking routine gynecological care or advice on clinical symptoms such as vaginal discharge or bleeding, menorrhagia, irregular menstruation, or lower back pain who were attending the outpatient department (OPD) of gynecology clinic at the Tripoli University Hospital, Algouri and Almoktar private gynecology clinics in Tripoli region. The samples were collected consecutively from the three clinics once a week in a specific day.
Data and sample collection
The data were collected from the female subjects by a specialized gynecologist. Inclusion criteria included married nonpregnant women. The exclusion criteria included women who had undergone a hysterectomy, cervical conization, mentally impaired or immunocompromised women. All participants were interviewed face to face to collect information about their residence, age, parity, and symptoms using a standardized questionnaire. The liquid-based cervical samples were collected using cervical brushes and preserved directly in stabilizing solution of EasyFix Q Path by VWR Chemicals, France. All samples were sent to Attasami Diagnostic Services (private laboratory) for cytology diagnosis, part of the sample was used for the Pap test, and the remaining was transferred to the National Centre for Disease Control (NCDC) referral virology laboratory for molecular HPV-DNA analysis.
The liquid-based Pap smear More Details assay was conducted blindly by a senior pathologist at Attasami laboratory. The cervical cytology findings were reported according to the Bethesda System 2001 updated in 2014 and classified as follows: Negative for Intra-epithelial Lesion or Malignancy (NILM) Atypical squamous cells of undetermined significance (ASC-US); Atypical squamous cells – cannot exclude HSIL (ASC-H) Low-grade squamous intra-epithelial lesion (LSIL); High grade squamous intra-epithelial lesion (HSIL), Squamous cell carcinoma (SCC).
DNA extraction and quantification
The DNA purification from cervical samples was carried out using QIAamp DNA Minikit (Qiagen, CA, USA). This kit is a silica membrane-dependent technique. The manufacturer's instruction was strictly followed. Then, the extracted genomic DNA was divided into two aliquots: one was stored at −20°C for near-time processing and the other at −80°C for backup storage. All samples were tested for DNA concentration and purity measurement using NanoDrop introduced by Thermo Scientific Ltd., Co. Most of the samples showed a DNA concentration at A260 ranged between 3.3 and 48 ng/μl, and purity at A260/A280 between 1.22 and 1.96. The samples with high DNA concentration were diluted to be suited for quantitative polymerase chain reaction (qPCR).
Human papillomavirus types 16 and 18 detection and genotyping
To evaluate the DNA integrity, extraction, and proper sample collection, all samples were amplified for cytochrome P4502C9 (CYP2) using the conventional PCR technique and all samples showed amplification after it had been visualization by means of electrophoresis on 2% agarose gel containing ethidium bromide (0.5 mg/ml).
The genesig® advanced kit introduced by Primerdesign™ Ltd., Co., Southampton, UK, was used for both detection and genotyping. This kit relies on the TaqMan® method and has a primer and probe mix that hybridizes specifically with HPV 16 and/or 18 E6 oncogene. Furthermore, the kit has an internal DNA extraction control which was multiplexed with the template at the same PCR run that was added during the DNA extraction procedure. The successful detection of the DNA control on the VIC channel indicates that the PCR inhibitors are at low concentration levels. As instructed by the kit provider, the PCR reaction was performed in a total volume of 20 μl of the reaction mixture containing 10 μl MasterMix, 1 μl primer/probe of HPV 16 or 18, internal extraction DNA control Primer/Probe of 1 μl, 3 μl RNAs/DNAs free water, and 5 μl of the sample with DNA concentration of 25–50 ng/μl. The positive and negative controls were included in each PCR reaction for an indication that the reagents worked properly on that particular experimental scenario. The PCR amplification was performed in a Rotor-Gene Q, the thermocycler introduced by Qiagen, Hilden, Germany. The PCR thermal cycle was adjusted as the kit manufacturer instructed and was started with the initial step of enzyme activator at 95°C for 2 min, followed by 50 cycles of denaturation step at 95°C for 10 s and the extension step was for 60 s at 60°C. The fluorogenic data were collected through the FAM (template) and VIC (internal extraction DNA control) channels during the extension phase.
This study was approved by the Libyan National Committee for Biosafety and Bioethics. For all subjects, a written Arabic consent and an additional informative sheet on HPV and cervical cancer were provided to be signed by the candidate if she agreed to participate in the study.
Data were analyzed using IBM SPSS software package version 24.0 (Chicago, IL, USA). Qualitative data were presented using frequency and percentage. Quantitative data were described by mean. Fisher's exact test was used to examine the studied risk factors associated with HPV infection among our subjects. The results were considered to be significant when P <0.05.
| Results|| |
One hundred thirty-two female subjects seeking a routine gynecological care or medical attention on clinical symptoms at the OPD clinic of gynecology department at Tripoli University Hospital and the private gynecology clinics of Algouri and Almoktar in Tripoli region were recruited to collect the cervical samples.
The mean age in the group of the study was of 41.73 years, and the age range among our recruited subjects was well represented, ranged across the sample between 20 and 73 years old [Table 1]. Of the 132 participants, 3.79% (n = 5/132) underwent routine Pap smear test, and 96.21% (n = 127/132) were advised on clinical symptoms. In addition, based on cytology pap smear test, 92.42% (n = 122/132) of women tested had a normal cytology results without lesions or malignancy, and a minority of 7.58% (n = 10/132) had abnormal cytology results, with ASC-US and LSIL indicating mild dysplasia, HSIL reflecting severe dysplasia, and SCC representing invasive cervical cancer [Table 2].
Human papillomavirus prevalence and human papillomavirus positivity compared to cytology diagnosis
The overall prevalence of HPV was 4.55%, that only six samples harbored HPV-DNA (n = 6/132). The HPV prevalence was identified as 0.0% (n = 0/132) among women with normal cytology. Moreover, among women with abnormal cytology, the prevalence of HPV was found to be as high as 60%, that the HPV-DNA was detected in 6 out of 10 samples who had cervical abnormalities (n = 6/10). The HPV 16 was the only type detected in all positive cases, and HPV type 18 was not detected among all the 132 samples that had been tested. The distribution of HPV according to cervical abnormalities showed that HPV-DNA was found in 3 cases graded as ASC-US (n = 3/6), in 1 case of HISIL (n = 1/6), and 2 cases diagnosed as SCC (n = 2/6), that represented by 50%, 16.67%, and 33.33%, respectively [Table 3]. Because the HPV 16 was the only type that was detected in positive samples, the above distribution is true for HPV 16 only.
|Table 3: Distribution of HPV genotypes 16 and 18 according to cytological diagnosis|
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The risk factors considered in the study
To evaluate the relationship between the risk factors and HPV infection, the multivariant logistic regression was applied. The prevalence of HPV was highest in the age group of 45–54 years (n = 3/6), followed by the age group of 33–44 years (n = 2/6), that represented by 50% and 33.3%, respectively, and this distribution was statistically insignificant (P = 0.664).
The parity of more than 3 showed to be the only peaked group among others that were positive for HPV-DNA, and when statistically assessed, it showed no statistical significance between negative and positive cases with P = 0.220. The clinical findings were statistically insignificant too with P = 0.615 when compared to HPV positivity [Table 4].
|Table 4: Some risk factors associated with HPV infection in the Libyan women|
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| Discussion|| |
To our knowledge, this is the first study to investigate the high-risk HPV types 16 and 18 among Libyan women compared to cervical cytological changes. It has used the powerful molecular technique of qPCR in HPV-DNA detection and genotyping and has hired the liquid-based Pap smear to investigate cervical abnormalities, which is one of the most accurate and sensitive in the field of pathology. Furthermore, this study by far is the first with such sample size that has been collected in Tripoli city, which is considered one of the highest in population and its residents represent most of the Libyan tribes and regions.
Our study revealed that the HPV overall prevalence was of 4.5%. It was found to be lower than the prevalence reported in the North Africa region, which was estimated at 12% in the low-risk population. In more detailed comparison to neighboring countries, our prevalence was found to be less than Tunisia, wherein Ardhaoui et al. reported in 2013 that the HPV prevalence was of 13%, in Morocco 15.7%, and of 40% in Egypt.,, On the other hand, the HPV prevalence in our study was found to be agreed with what was documented in Algeria of 6.3% in a study done by Hammouda et al. in 2011 and it was the same in Qatar with overall HPV prevalence of 6.3%. However, our results showed high disparity in HPV prevalence compared to Sub-Saharan countries, where it ranked as the highest globally estimated of 24%, as when we compared the prevalence of 4.5% found in our study to what was reported in Gabon of 60% in a study done by Zoa Assoumou et al. in 2016.,
Remarkably, the HPV prevalence among our subjects with normal cervical findings was found to be 0% (n = 0/122) that was not agreed with what was found in Morocco of HPV prevalence with 2.9% among women who had normal cervical findings, in Algeria of 9%, in Tunisia of 5.2%, and in Egypt of 5.1%. The result of no detection of HPV among our recruited subjects without cervical lesions could be attributed to the fact that this study was cross-sectional and the HPV infection is transient and resolves spontaneously over a time period of 6–12 months, and a small portion of infected individuals might develop a persistent infection that could progress to cervical cancer. In this study, the HPV prevalence among women with cervical abnormalities was found to be 60%, that's less than what was reported in the Extended Middle East and North Africa region where the general prevalence was 98%. In Egypt, the prevalence of HPV among women with cervical abnormalities was found to be of 82.3% in a study done by Youssef et al., which is higher than the prevalence of HPV among women with cervical lesions that our study revealed. On the other hand, our study finding was found to be higher than what was documented in Morocco of 46.7%, in Algeria of 41.8%, and Tunisia of 45.6%. The relatively high prevalence of HPV among women with cervical abnormalities found in our study is consistent with the incidence of cervical cancer in Libya, where it was reported as the third leading cause of female cancer, ninth leading deaths of female cancers annually, with estimation deaths of 95 females in 2012 as reported by HPV center in 2017.
In our study, the HPV genotyping showed dominance for HPV 16 in all samples that were positive to HPV-DNA, this finding is well consolidated with what was previously reported from many parts of the world, and it agrees with the most common HPV types outlined in North Africa region, where the HPV 16 is predominant type. Likewise, similarity is true when compared to what was revealed by a study done in Egypt by Yossef et al. in 2011, where the HPV 16 was found to be the predominant type, also the predominance of HPV 16 was reported in Tunisia, Morocco, and Gabon.,, However, in some African studies, HPV 16 is not the predominant high-risk HPV type, as outlined in Algeria where the HPV 31 was detected as the most common type in a study done by Hammouda et al. in 2014. Remarkably, the HPV 18 was not detected in all our samples tested among women with or without cervical abnormalities, even though the HPV 18 is considered to be the second prevalent high-risk HPV type globally. Our finding resembles what was reported by Ardhaoui et al., in Tunisia 2016, where they revealed that the HPV 18 was not the second common oncogenic HPV type infecting Tunisian women. In Egypt, a study done by Shaltout et al. 2014, reported that the HPV 31 and 52 were the second predominant high-risk types among Egyptian women, which also agrees with our study results.
The infection peaked in the age group of 45–54 years, which contrasts of what was reported globally, as it peaked among young women following their first sexual activity. Our findings could be due to the conservative religious upbringing of Libyan girls from being infected early by HPV. The age as a risk factor was found to have no relationship with the HPV-positive cases and it was statistically insignificant. Notably, all the positive cases fall in the category of multiparous women that may predict parity as a risk factor among our selected group; however, no correlation to HPV positivity was found when the statistical analysis was conducted. Furthermore, all positive cases were collected from women presented to the gynecology clinic seeking advice on clinical symptoms that suggests a lack of awareness toward the routine Pap smear testing as an efficient tool for early detection of the cervical epithelial changes, which could progress to cervical cancer. The results of insignificance correlation between the risk factors tested and HPV-positive results could be attributed to the small number of HPV-positive cases and the efficient statistical assessment for the HPV infection and its risk factors among the Libyan population needs a larger number of samples.
Due to the small sample size. In addition to the fact that the samples were gathered only from adult females who were attending the outpatient gynecology clinics for advice on clinical complaints or as routine visits. The Libyan women's general population might not be represented well in our study. The actuality that the samples were collected only from married women due to religious and societal considerations that might lead to selection bias. The previous could be considered as study limitations. On the other hand, the strength of our study relies on the fact that the clinical examination and sample collection was done by a senior gynecologist. The liquid-based cytology technique was applied to the pathology diagnosis, performed by a consultant pathologist. Furthermore, the HPV-DNA detection and genotyping were done by employing qPCR at the referral laboratory of the National Centre for Disease Control-Libya.
| Conclusion|| |
Although our study showed a low overall prevalence of 4.5% of HPV infection compared to other countries, yet there was enough evidence of the high rate burden of HPV type 16 among Libyan women with cervical lesions to warrant public health intervention as the HPV vaccine should continue to be scheduled in the Libyan national vaccination program. A national cervical cancer screening program should be considered to reduce the increasing incidence of cervical cancer. The expanding of research on the HPV prevalence and type distribution among the Libyan population would be of great aid for the medical authorities to assess the impact of the current HPV vaccination and to efficiently design a national comprehensive cervical cancer screening program.
We are grateful for all the participant women who provided their time, biological samples, and personal information. Special thanks to all the National Centre for Disease Control laboratory's staff who were of great assistance in preparing the samples for molecular testing. I would very much like to acknowledge the teaching staff member of the Libyan Academy Dr. Mahmoud Buazzi whom I was greatly benefited from the valuable bits of advice that he provided during the conducting of this study.
Financial support and sponsorship
The National Centre for Disease Control has offered the kits and the equipments needed for the molecular testing.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]