|Year : 2018 | Volume
| Issue : 3 | Page : 78-82
|Genotyping the human papillomavirus infection in Iranian women referred to Shahid Motahari Hospital, in Urmia, with real-time polymerase chain reaction techniques
Jalhe Bagheri Hamzyan Olia1, Mohamad Hasan Khadem Ansari2, Parichehreh Yaghmaei3, Haleh Ayatollahi4, Hamid Reza Khalkhali5
1 Department of Biology, Science and Research Branch, Islamic Azad University, Iran
2 Nephrology and Kidney Transplant Research Center, Urmia University of Medical Sciences, Urmia, Iran
3 Department of Biochemistry, Faculty Member of Islamic Azad University, Science and Research Branch of Tehran, Iran
4 Solid Tumor Research Center, Urmia University of Medical Sciences, Urmia, Iran
5 Department of Biostatistics and Epidemiology, Patient Safety Research Center, School of Medicine, Urmia University of Medical Sciences, Urmia, Iran
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|Date of Web Publication||10-Dec-2019|
| Abstract|| |
Objective(s): Human papillomavirus (HPV) is in viridae papillomavirus family. These viruses have been classified based on the DNA sequences. At least 120 types of these viruses have been identified. Different types of HPV genotypes are associated with lower genital tract infection. This study aimed to identify the genotypes of HPV that cause vaginal infections in women with infection. Materials and Methods: This cross-sectional study was performed on eighty patients with vaginal infection. Extracting viral DNA was performed automatically using MagCore Nucleic Acid Extractor (made in Taiwan) and MagCor® Viral Nucleic Acid Extraction kit (Cartridge code 202) made by MogCore Co. Taiwan, and genotyping the samples was carried out using the real-time polymerase chain reaction (PCR) technique in the Light Cycler 96 System (made in Germany) and real-quality relative quantification-HPV infection high-risk (HR)/low-risk (LR) Multiplex Kit made by AB Analitica® Co. Italy. Results: Among the 80 samples, 30 people (37.5%) were positive for infection with HPV; evaluating the positive genotypes, it was found that of these, 16 people (53.4%) were infected with LR genotypes of HPV, seven patients (23.3%) were infected with only HR HPV genotype, and seven people (23.3%) were infected with both LR and HR genotypes of HPV (coinfection). Conclusion: Considering that HPV infection is asymptomatic, and it has a high prevalence in Iran, early diagnosis and prevention of progressing the infection can prevent the malignancies of the uterus. Molecular techniques, particularly real-time PCR, are reliable methods for detecting the HPV infection seven with quite low viral loads.
Keywords: Human papillomavirus, real-time polymerase chain reaction, vaginal infection
|How to cite this article:|
Olia JB, Ansari MH, Yaghmaei P, Ayatollahi H, Khalkhali HR. Genotyping the human papillomavirus infection in Iranian women referred to Shahid Motahari Hospital, in Urmia, with real-time polymerase chain reaction techniques. Ann Trop Med Public Health 2018;11:78-82
|How to cite this URL:|
Olia JB, Ansari MH, Yaghmaei P, Ayatollahi H, Khalkhali HR. Genotyping the human papillomavirus infection in Iranian women referred to Shahid Motahari Hospital, in Urmia, with real-time polymerase chain reaction techniques. Ann Trop Med Public Health [serial online] 2018 [cited 2020 Apr 10];11:78-82. Available from: http://www.atmph.org/text.asp?2018/11/3/78/272548
| Introduction|| |
Human papillomaviruses (HPVs) with more than 100 genotypes of HPV with worldwide distribution are in the family of HPV viridae with the structure of a protein and double-stranded circular and uncoated genome. HPV infection is the most common sexually transmitted infections and is a significant source of mortality caused by cancer., Many HPV infections occur dormant, they can be identified on healthy skin, and these infections are largely transient and frequently asymptomatic and in most cases are without clinical results. Infection with certain types of HPVs is the main cause for a variety of diseases and cancers. World Health Organization described 12 types of HPV with 16, 18, 31, 33, 35, 39, 45, 51, 56, 52, 58, and 59 genotypes as high-risk (HR) types or carcinogenic and some other subtypes with 68, 73, 26, 30, 53, 66, 67, 69, 82, 85 genotype as those that may be carcinogenic. Nearly 71% of these HPV16 and HPV18 were identified as worldwide cervical cancer cases. Other types of HPV genotypes including genotypes of 6, 11, 13, 40, 42, 43, 44, 54, 61, 70, 72, 81, and 89, mostly are related to genital warts and were classified as a low-risk (LR) group. The prevalence of HPV infection was exhibited differently, in different regions and countries and even in different demographic subgroups of a country. However, the global prevalence of HPV was reported, 7/11%. Distribution of certain types of HPV infection is different in various geographic areas., Some types of HPV in the Asia-Pacific region are more common than in other regions.
Several risk factors were identified for the development and persistence of HPV infection. Age is an important factor although significant differences exist in age-HPV curve in different areas.,, In some countries, the genital HPV infections were detected in 20%–40% of sexually active young women that the prevalence of the infection is reduced by increasing the age. Other factors include the existence of sexual partners, age of starting sexual activity, socioeconomic status, using condoms, using contraceptives, smoking,,, using of public baths, and training. The number of pregnancies and childbirth were identified as a risk factor for HPV infection.
Currently, the best way to prevent these infections and related diseases is to perform routine cytological screening and treatment of precancerous lesions, if necessary., However, due to the cost, performance challenges, and complexities in preparation for screening and treatment, this method has restrictions in Iran. The HPV vaccine was prepared against the most common types (types 16 and 18), and a vaccine was prepared for types of genital warts (HPV 6 and 11). Studies have indicated that the vaccine used against genital warts has the protective effect on the infection of papillomavirus 16 and 18.,, In other words, HPV vaccine can prevent cervical cancer potentially, particularly in centers with poor socioeconomic status. According to the surveys conducted by the researcher, this study is the first epidemiological study, which was designed to determine the prevalence of HPV infection of the genital tract in Iranian women living in West Azerbaijan, so that if the spread of this viral infection which is the main cause of cervical cancer in the population of women is high, provisions are considered to implement the vaccination and prevention programs against cervical cancer.
This cross-sectional study was conducted from December 2015 to December 2016. In this study, 80 married women between the ages of 45 and 25 years were investigated who had a vaginal infection and referred to the Kosar women subspecialty clinic of Motahari Hospital, in Urmia, for vaginal examination. Sampling was performed by a gynecologist using sterile swabs from the vaginal secretions and cervical mouth of each patient under sterile conditions. The samples were placed in 2 ml of transport medium (Pasto Cyto Prep) (prepared by the Pasteur Institute of Iran) and were transferred to molecular laboratories. The swaps related to vaginal secretions were stored in the transport medium at −20°C after stirring with a vortex.
Extraction of DNA
To perform the molecular test, separating the DNA of HPVs from vaginal secretions was conducted automatically using MagCore® Nucleic Acid Extractor (made in Taiwan) and MagCor® Viral Nucleic Acid Extraction kit (Cartridge code 202) made by MagCore Co. Taiwan. To extract DNA from vaginal specimens, K proteinase, RNA carrier and the mentioned kit were used and the amount of extracted DNA was adjusted to 60 μl. The concentration of extracted DNAs was measured using NanoDrop ND – 1000, Thermo Fisher Scientific USA Co. at 260 nm in terms of ng/ml. The extracted DNAs were stored at −20°C until implementing the real time-polymerase chain reaction (PCR) reactions.
DNA genotyping using real-time polymerase chain reaction technique
Genotyping the samples through real-time PCR technique was performed using real-quality relative quantification-HPV HR/LR Multiplex Kit made by Light Cycler® Co. (Italy) in for typing 22 HPV type including HR HPV genotypes (16, 18, 31, 35, 39, 45, 51, 52, 56, 59, 66, and 68) and LR HPV genotypes (6, 11, 26, 53, 67, 70, 73, and 82) in Light Cycler® 96 Roche system (made by Germany) using specific primers of the kit. The kit was designed to use an internal control that allows the operator to detect inhibition of the PCR reaction, monitor the extraction process, as well as identify false-negative samples. The internal control (β-globin gene, amplification control) is amplified in multiplex with the target pathogen. The positive controls supplied in this kit contain DNA fragments that correspond to the amplified gene regions of HPV 16, 18, and 33 and the β-globin gene(PC HPV RX-1) and of HPV 6, 11, and 26 and the β-globin gene(PC HPV RX-2). For genotyping with this technique, all four channels of the device were used; FAM channel was used to identify HR genotypes (31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) and LR genotypes (26, 53, 67, 70, 73, 82), Hex channel identifies the HR genotype: 16 and LR: 6 genotype, Texas Red channel identifies internal control, and Cy5 channel identifies the HR genotype: 18, and LR genotype: 11.
| Results|| |
In a study performed on women with vaginal infection who referred to a specialized clinic of Motahhari Hospital, in Urmia, among 80 samples, 30 people were infected with HPV, of these 16 people had LR genotypes of HPV (6, 11, 26, 53, 67), seven people had only HR HPV genotype (16, 31, 58, 18), and seven patients had both LR and HR genotypes of HPV (coinfection) (6, 33, 53, 39, 68, 70, 52, 35, 26, 51, 16). Hence, in terms of frequency, the prevalence of HPV is 37.5% [Figure 1]. Of which, 53.4% was with LR HPV, 23.3% with HR HPV, and 23.3% with coinfection of HPV [Figure 2]. The positive correlation was observed between age and HPV Among the patients with HPV, 18 people (54.5%) were within the age of 25–30 years, seven patients (43.8%) were within the age of 35–31 years, and five patients (16.1%) were within the age of 36–45, respectively. With increasing age, the proportion of positive HPV significantly decreased (P = 0.006) [Table 1].
|Figure 1: Percentage of positive or negative frequencies of human papillomavirus in the samples|
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|Figure 2: The percentage of low-risk human papillomavirus, high-risk human papillomavirus, and coinfection of human papillomavirus in samples|
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| Discussion|| |
More than 450,000 cases of cervical cancer occur in the world annually. Although the mortality rate has been decreased in the last 30 years compared to the past, each year, almost 200,000 deaths caused by cervical cancer occur that this rate is higher than 12,000 new cases of cervical cancer in the developed countries annually. Of this rate, 4000 deaths are associated with this disease. HPV is certainly considered as the main cause of uterine malignancies and progressive cancer of the uterus.
In the study of Mahmudi et al. which was conducted to determine the HPV genotypes in cervical cancer samples, the certain types of HPV and their correlation with cervical malignancies in Yazd province were detected. In this study, in 70% of the samples, HPV genome was detected that the 16 and 18 types with the frequency of 70% and 16.7%, respectively, were diagnosed as the most common genotypes. Other identified types were HPV 33, 45 and 73. In another study in this field which was conducted in Isfahan, the prevalence of HPV in married women with the age of 18–60 years with normal Pap smear was investigated, who referred to gynecology clinics affiliated with the University of Medical Sciences of Isfahan. In this study of 46 positive HPVs, 15.21% was genotype 16 (7 samples), 13.04% was genotype 18 (6 samples), and 21.74% was genotype 11 or 6. In the study of Monsefi et al (2013) which was conducted in Kerman on 20,000 genotyping Pap smear samples, in which in 93.75% of the people, positive HPV type 16 was identified, and type 18 was identified in just 6.25%. In the study of Ghaffari et al. (2006), the prevalence of HPV genotypes in Iranian women was evaluated to determine the important role of HPV infection in progressing uterus cancer. This study aimed to determine the genotype of the common HPV in women with normal and abnormal cervical cytology. Genotyping was performed by PCR, and the results showed that the infection with HPV type 16 in patients was significantly higher compared to the normal group (42% vs. 11.6%). Similarly, HPV genotype type 18 was more common in the abnormal group. In this study, a significant correlation was found between genotypes of HPV and cervical pathological changes. Other study in this regard conducted by Khodakarami et al. (2012) regarding the prevalence of HPV infection in women living in the areas covered by health regions of Shahid Beheshti University of Medical Sciences, Tehran, aimed to determine the prevalence of HPV infection and its role in the incidence of cervical cancer in Iran. The genotyping of HPV in this study was performed by PCR, and the results showed that although the prevalence of HPV infection in patients under 25 years was higher than other groups slightly, it was not significant statistically, and the prevalence of infection in the samples was 7.8%, of which 5.1% was HR, and the most common HPV was type 16 as well. In other study performed by Sohrabi et al. based on evaluating of HPV as the primary cause of cervical cancer, the different genotypes of HPV, especially HR genotypes, were the main causes of cancer. In this study, the documentation of about 100 papers was reviewed in scientific bases and molecular diagnostic methods such as PCR and hybridization were introduced as valid methods for detecting and identifying the genotypes. The results of this study showed that increasing the genital infections, sexually transmitted diseases, and cancers caused by microorganisms, especially HPV, demands cooperation with the international community to perform more comprehensive studies, particularly in developing countries.
In a population-based study performed by Nahar et al. regarding the genital infection with HPV in women in Bangladesh, aimed to determine the prevalence of HPV genotypes in an urban and a rural area in Bangladesh, HPV genotyping was performed by PCR techniques. In this study, the prevalence of HPV was 7.7% and there was no significant difference between rural and urban women. The most common HR genotypes were HPV 31, 40, 18, 66, 16, and 53. The maid urban women were at HR for HPV infection compared to homemakers or garment workers. Rural women whose spouses were living abroad were almost two times more likely to develop HPV in comparison with the rest. The results of this study showed that the prevalence of infection in Bangladeshi women was similar to that in other Asian regions. However, they were different in terms of HPV genotypes. The study of Kuzaran et al. (2015) was performed based on HPV infection in the cervix and its continuation in 300 women in public health care clinics in a region of Brazil, and the genotyping was performed based on PCR. Cytology analysis was performed by Pap smear test, and HPV infection was diagnosed in 47 women. The results of this study showed that HPV infection is more common in young women aged ៲ years and in low-income individuals and the persistence of viral infection in this group was lead to precancerous lesions in the cervix. HR HPV genotypes were found in precancerous lesions.
Xi et al. studied the incidence of certain types of HPV infections in squamous intraepithelial lesions of the cervix tissue in the women over 35 years in West Africa. In this study, the women over 35 years and older were tested for cytologic abnormalities of the cervix using Pap smear and PCR, respectively. HPV infection was found in 18% of women with negative cytologic abnormalities. The prevalence of HR and LR HPVs increases with age. HPV16 and HPV58 were predominant types in this study. The results showed a significant association between HPV types and severity of cervical tissue lesions. In other study performed by Clifford et al. based on the distribution of genotypes of HPV in low-grade squamous intraepithelial lesion (LSIL) and comparison of different geographical position and their correlation with cervical cancer. The prevalence of HPV in cervical lesions with low intensity (LSIL) in women in North America was 80%, and it was % in other areas. This reflects the regional differences in diagnosing LSIL. Among the 5910 HPV-positive samples, the most prevalent genotype was for HPV type 16, and then, the most common genotypes were HPV31 (11.5%), HPV51 (10.6%), and HPV53 (10.2%). The positive HPV type 16 in African women was 2 times less than infected women in Europe, and in North America, papillomavirus type 18 infections was higher than in Europe and South and Central America.
| Conclusion|| |
Despite the fact that Iran, as a developing country, is exposed to prevalence and incidence of malignancies caused by HPV infection and resulted deaths, comprehensive statistics regarding the distribution of HPV types in the country and regional distribution in the city of Urmia has not been published. Hence, the need for determining a regional genotyping is felt to prevent the infection more accurately and cost-effectively. According to studies, this study is the first study which was conducted in West Azerbaijan. So that if the spread of the infection is high which is the main cause of cervical cancer, appropriate provision should be considered regarding the preventing programs, including training, screening, immunizing, and treatment.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Prof. Mohamad Hasan Khadem Ansari
Nephrology and Kidney Transplant Research Center, Urmia University of Medical Sciences, Urmia
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2]