Evaluation of Human Papillomavirus Genotype Distribution in Cervical Samples.

BACKGROUND: The most common sexually transmitted infection in the world is human papillomavirus (HPV). HPV types 16 and 18 are responsible for 60-80% of cervical cancers and precancerous cervical lesions worldwide. AIM: In this study, it was aimed to evaluate the correlation of HPV genotype distribution with cervical cytology results in cervical smear samples and to contribute to HPV epidemiology.
MATERIALS AND METHODS: This study included 72 female patients. For detection of the HPV genotypes, a multiplex real-time polymerase chain reaction (PCR) method that could detect more than 25 different HPV types was used. The cervical cytology and histopathology results of the patients were also evaluated simultaneously.
RESULTS: The frequency of high-risk HPV was 35% (25/72). The most common types were HPV51 (10%), HPV16 (8%), and HPV66 (8%), respectively. The most common type HPV51 and multiple HPV types were seen in 21-34 age groups. HPV DNA was detected in 21 of 43 samples that had cervical smear diagnosis grouping. Twelve samples (26%) had normal cytology. Low grade squamous intraepithelial lesions were the most common cytological diagnosis in HPV DNA positive samples. The most common HPV types in the patients diagnosed low grade squamous intraepithelial lesions and high grade squamous intraepithelial lesions were HPV16 and HPV52.
CONCLUSIONS: In this study, the frequency of high-risk HPV genotypes was 35% as similar to reports of the other studies conducted in our country. The most common types were HPV51, HPV16, and HPV66, respectively. The follow-up of patients with HPV51 infection in our area could help to improve the natural course of the disease and effective prevention programs. Copyright:

INTRODUCTION

Human papillomavirus (HPV) is a small, non-enveloped, double-stranded DNA virus belonging to the Papillomaviridae family.[1] The most common sexually transmitted infection in the world is HPV.[2] HPV has been shown to be responsible for the etiology of cervical cancers.[3] Cervical cancer is the fourth most common neoplasm in women worldwide and is responsible for the death of 260,000 yearly with the majority of cases seen in developing countries.[4] The presence of high-risk HPV genotypes was demonstrated in 99.7% of cervical cancers worldwide.[5] Although more than 200 HPV types are described, it is known that approximately 40 HPV types can infect the epithelial layer in the anogenital region and the other mucosal surfaces of the body.[67] HPV is classified according to its nucleotide sequence as five strains (Alpha, Beta, Gamma, Mu, and Nu). High-risk HPV types are associated with cervical intraepithelial neoplasia (CIN) 2/3 and invasive cervical carcinomas and include types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, and 68. Low-risk HPV types are associated with genital warts and LSIL and includes HPV types 6, 11, 42, 43, and 44.[8] In the world, HPV types 16 and 18 are responsible for 60–80% of cervical cancers and precancerous cervical lesions.[4] After high-risk HPV infection, invasive cancer can develop by 1–3% and occurs after about 25–40 years.[9] In several studies on the distribution of HPV genotypes, the prevalence of HPV is estimated at 11.7%. The five most common HPV genotypes were reported as HPV16, 18, 31, 52, and 58. The most dominant genotypes were HPV16 and 18, followed by HPV52.[10]

The oncogenic potential of HPV16 and HPV18 relates to two early viral gene products, E6 and E7. E6 and E7 block tumor suppressor proteins p53 and retinoblastoma. As a result, a process that leads to the immortalization of damaged cells begins.[11] HPV infection is asymptomatic in most women and does not develop cancer. In 2 years, 91% of these infections will recover spontaneously.[2] HPV is more common in young sexually active women.[12] The persistence of HPV and its progression from infection to disease differ according to high-risk HPV genotypes.[13] Age is an important factor in disease progression. Up to the age of 50, 80% of women are infected with HPV and 35% of cervical cancer deaths occurs in women over 65 years of age.[14] In the early diagnosis of cervical cancer and precancerous lesions, cervical cytology and molecular tests are used as screening tests.[15] In this study, it was aimed to determine HPV genotype distribution in cervical smear samples, to evaluate their correlation with cervical cytology and biopsy results, and to contribute to HPV epidemiology.

MATERIALS AND METHODS

This retrospective study included 72 female patients who were analyzed HPV genotypes in the Division of Virology and Fundamental Immunology, Department of Medical Microbiology, Istanbul Faculty of Medicine, Istanbul University, Istanbul, Turkey between January 2016 and April 2018. The results of cervical cytology and biopsies sent to the pathology laboratory simultaneously were also included in this study. This study was approved by the Istanbul University, Istanbul Faculty of Medicine Ethics Committee (reference number: 2018/881/11).

Viral nucleic acid extraction in cervical swab samples was performed by the EZ1 Advanced XL (Qiagen, Germany) automatic extraction device using EZ1 virus mini kit V.2.0 (Qiagen, Germany). The extracted viral nucleic acid products were amplified by the Rotor-Gene Q device (Qiagen, Germany) using HPV Genotypes 14 Real-TM Quant kit (Nuclear Laser Medicine, Italy). The results were evaluated qualitatively by the Software program and the most common and oncogenic, 14 HPV genotypes including HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 were investigated.

Samples of cervical smears sent to the pathology laboratory for cytological examination were placed in the BD SurePathTM protective medium and evaluated in the light microscope after preparation according to the manufacturer's recommendations. Cervical biopsies of patients were investigated by used reference controls.

Statistical analysis was performed using SPSS 21 (SPSS Inc, Chicago, IL, USA). The normal distribution of variables was examined by visual methods (histogram and probability graphs) and Kolmogorov–Smirnov test. Student's t-test was used to compare between groups with normal distribution, and parameters with the non-normally distribution were compared using the Mann–Whitney U test. Chi-square or Fisher tests were used to compare qualitative variables between groups. P values less than 0.05 were considered statistically significant.

RESULTS

The mean age of 72 female patients in this study was 37.74 ± 10.17 (95%, CI: 40–35) years, ranging from 21 to 63 years. HPV was detected in 25 (35%) of 72 cervical samples. The mean age of HPV positive patients was 38.57 ± 9.65 and 36.16 ± 11.10 in HPV negative patients (P: 0.34). The frequency of HPV was 44% (12/27) in the 21–34 age group. It was found 32% (8/25) in the 35–44 age group and 25% (5/20) in the ≥45 age group. There was no statistically significant difference between the age groups in terms of HPV rates (P: 0.36) [Table 1].

Table 1

Distribution of single and multiple human papillomavirus types according to age groups

Age groups
HPV genotypes	21-34 n (%)*	35 -44 n (%)*	≥45 n (%)*	Total n (%)*	P
Negative	15 (56)	17 (68)	15 (75)	47 (65)
Positive	12 (44)	8 (32)	5 (25)	25 (35)	P:0.36
Single infection	5 (19)	5 (20)	5 (25)	15 (21)	P:0.16
Multiple infection	7 (30)	3 (12)	-	10 (14)
16	-	-	1 (5)	1 (1)
31	-	2 (8)	-	2 (3)
39	-	-	1 (5)	1 (1)
51	2 (7)	-	1 (5)	3 (4)
52	1 (4)	-	-	1 (1)
56	-	1 (4)	-	1 (1)
58	-	-	1 (5)	1 (1)
66	1 (4)	2 (8)	-	3 (4)
68	1 (4)	-	1 (5)	2 (3)
16,18	1 (4)	-	-	1 (1)
16,52	1 (4)	-	-	1 (1)
16,51	1 (4)	-	-	1 (1)
18,66	-	1 (4)	-	1 (1)
31,51	1 (4)	-	-	1 (1)
51,56	1 (4)	-	-	1 (1)
56,58	-	1 (4)	-	1 (1)
31,51,59	1 (4)	-	-	1 (1)
16,18,56,66	1 (4)	-	-	1 (1)
16,52,66,68	-	1 (4)	-	1 (1)

*Column percentage

In the 72 patients included in the study, the single infection rate with any HPV genotype was 21% (15/72) and the rate of multiple infections with multiple HPV genotypes was 14% (10/72) (P: 0.36). The median age of patients with single HPV infection was 39, whereas the median age of patients with multiple HPV infections was 28 (P < 0.01). Single and multiple HPV infection rates by age groups are shown in Figure 1. When these rates were evaluated, no statistically significant difference was found (P: 0.16).

Figure 1

Single and multiple human papillomavirus infection rates according to age groups

The most common HPV types detected in cervical specimens were HPV51, HPV16, and HPV66; 10% (7/72), 8% (6/72), and 8% (6/72), respectively. The most common HPV types according to age groups were HPV51 (22%) in 21–34 age group and HPV66 (16%) in 35–44 age group. In the ≥45 age group, HPV16, 39, 51, 58, and 68 were detected in equal rates (5%) [Figure 2].

Figure 2

Distribution of human papillomavirus genotypes according to age groups

In this study, 43 cervical smears and 14 cervical biopsies from 72 patients were investigated. No statistically significant difference was found in terms of normal Papanicolaou (Pap) smear and pathological Pap smear results in HPV genotype positive and negative patients (P: 0.18) [Table 2]. HPV DNA was detected in 21 of 43 samples that had cervical smear diagnosis grouping. Twelve samples (26%) had normal cytology. Low grade squamous intraepithelial lesions (LSIL) was the most common cytological diagnosis in HPV DNA positive samples [Table 2]. No significant difference was found in the frequency of single HPV infection between atypical squamous cells of undetermined significance (ASCUS), LSIL, and high grade squamous intraepithelial lesions (HSIL) cytological diagnosis groups. However, the most frequent HPV16 and HPV52 were detected in LSIL and HSIL. In the histopathological examination of eight clinical specimens, it was diagnosed CIN1 in two patients, CIN2 in five, and CIN3 in one [Table 3]. The most common histopathological stage was CIN2. In CIN2 stage, HPV16 and HPV52 were detected the most frequent genotypes [Table 4]. In the histopathological investigation of two patients with diagnosed HSIL, it was diagnosed CIN2 in one patient and CIN3 in the other. On the contrary, in the histopathological investigation of two of the four patients with diagnosed LSIL, it was diagnosed with CIN1 and CIN2. In a patient with diagnosed with ASCUS, it was diagnosed CIN2 [Table 3].

Table 2

Human papillomavirus genotype distribution according to cervical Papanicolaou smear cytology

Cervical cytology
	Normal n (%)	ASCUS n (%)	LSIL n (%)	HSIL n (%)	Total n (%)	P
HPV DNA negative	15 (32)	8 (17)	3 (6)	0	26 (55)	P: 0.18
HPV DNA positive	12 (26)	3 (6)	4 (9)	2 (4)	21 (45)
Multiple infection	4 (19)	1 (5)	4 (19)	-	9 (43)
HPV 31/51	-	-	1	-	1
HPV 16/51	1	-	-	-	1
HPV 51/56	1	-	-	-	1
HPV 16/18	1	-	-	-	1
HPV16/52	-	-	1	-	1
HPV 18/66	1	-	-	-	1
HPV 56/58	-	-	1	-	1
HPV 31,51,59	-	-	-	-	-
HPV16,18,56,66	-	1	-	-	1
HPV 16,52,66,68	-	-	1	-	1
Single infection	8 (38)	2 (10)	-	2 (10)	12 (57)
HPV 16	-	-	-	1	1
HPV 31	1	-	-	-	1
HPV 39	-	1	-	-	1
HPV 51	1	-	-	1	2
HPV 52	-	-	-	-	-
HPV 56	1	-	-	-	1
HPV 58	1	-	-	-	1
HPV 66	3	-	-	-	3
HPV 68	1	1	-	-	2

ASCUS: Atypical squamous cells of undetermined significance, LSIL: Low grade squamous intraepithelial lesions, HSIL: High grade squamous intraepithelial lesions

Table 3

The molecular, cervical cytology, and histopathology results of the patients detected HPV DNA

Patient no	Age groups	HPV type	Cervical cytology	Histopathology
1	21-34	52	-	CIN2
2		31,51	LSIL	-
3		16,51	Normal	Normal
4		16,18,56,66	ASCUS	-
5		51,56	Normal	-
6		16,18	Normal	Normal
7		68	Normal	-
8		16,52	LSIL	CIN2
9		51	-	Normal
10		31,51,59	-	-
11		51	Normal	Normal
12		66	Normal	-
13	35-44	16,52,66,68	LSIL	CIN2
14		18,66	Normal	CIN1
15		56,58	LSIL	CIN1
16		31	Normal	-
17		31	-	Normal
18		56	Normal	-
19		66	Normal	Normal
20		66	Normal	-
21	≥45	16	HSIL	CIN2
22		39	ASCUS	CIN2
23		58	Normal	-
24		51	HSIL	CIN3
25		68	ASCUS	-

CIN1: Cervical intraepithelial neoplasia grade 1; CIN2: Cervical intraepithelial neoplasia grade 2; CIN3: Cervical intraepithelial neoplasia grade 3

Table 4

Distribution of HPV genotypes in different histopathological stages

HPV genotype	CIN1 n (%)	CIN2 n (%)	CIN3 n (%)	Total n (%)
16	-	3 (21)	-	3 (21)
18	1 (7)	-	-	1 (7)
39	-	1 (7)	-	1 (7)
51	-	-	1 (7)	1 (7)
52	-	3 (21)	-	3 (21)
56	1 (7)	-	-	1 (7)
58	1 (7)	-	-	1 (7)
66	1 (7)	1 (7)	-	2 (14)
68		1 (7)	-	1 (7)
Total	4	9	1	14

CIN1: Cervical intraepithelial neoplasia grade 1; CIN2: Cervical intraepithelial neoplasia grade 2; CIN3: Cervical intraepithelial neoplasia grade 3.

DISCUSSION

In cervical screening programs around the world, the primary screening test is performed by HPV genotyping test with cytology or without cytology. HPV genotyping is important in low-grade cytology such as ASCUS and LSIL and for post-treatment follow-up of high-grade cervical neoplasia such as CIN2/CIN3.[16]

Cervical cancer is the second most common cause of cancer deaths after breast cancer in women in the European Union. According to Ministry of Health data, the eighth most common cancer in women is cervical cancer.[9] Risk factors for HPV are sex, age, and sexual activity, the number of sex partners, and the highest risk is sexually active women under 25 years of age. Other risk factors include smoking and immune status.[17]

The prevalence of HPV ranges from 2% to 44% worldwide. This rate in women with normal cervical cytology is 11.6–11.7%. It is reported to be as high as 21.4% in Eastern Europe, 16.1% in Latin America, and 24% in Sub-Saharan Africa.[101819] In a meta-analysis study conducted by the World Health Organization (WHO)/Catalan Oncology Institute Information Center, the prevalence of HPV in women with normal cytology was found to be highest in Africa (31.6%) and lowest in Southeast Asia (6.2%).[20] The prevalence of HPV infection in women has been reported to be between 3 and 38.9% in our country.[21222324] In this study, the frequency of high-risk HPV infection was 35%. The high peak rate (44%) seen in women aged 21–34 years was consistent with the other studies reported conducted in our country. The risk of HPV infection decreases with increasing age in women, whereas men have a risk for new HPV infections throughout their lives.[2526] Although the HPV DNA positivity rate was high in the 21–34 age group, no statistically significant difference was found between the age groups (P: 0.36). Higher rates in young women are associated with the natural course of HPV and the occurrence of transient infection in this age group.[27]

It is known that there are geographical differences in HPV type distribution.[19] HPV type 16 prevalence is the most common HPV type in worldwide, with a prevalence of 2.6%. HPV type 18 is the second most common type in the world after HPV type 16. Other HPV types vary according to age and geographical differences.[20] In this study, the most common HPV types were HPV51, HPV16, and HPV66; 10%, 8%, and 8%, respectively. In a study conducted in 323 cervical specimens in Italy, it was reported that the second most common HPV genotype after HPV16 was HPV51 with 22.7%.[28] In a meta-analysis study conducted worldwide, the most common oncogenic HPV types among women with normal cytological findings were 16, 18, 52, 31, 58, 39, 51, and 56.[29] Distribution differences in HPV types may be due to age and geographical characteristics as well as the number of samples in the study and cultural differences between countries.[30] The incidence of HPV is high in young women and often in the form of multiple infections. HPV DNA decrease rapidly in middle-age groups and a second increase in post-menopausal age groups is observed.[2] In this study, after the menopause as reported in the literature, a second peak could not be determined. The reason may be related to with sexual behavior, changes in the immune system, low socioeconomic status, and sociocultural factors.

In this study, the rate of single infection caused by one of HPV genotype was 21%, and the multiple infection rates caused by more than one HPV genotype was 14%. There was no statistically significant difference between the age groups in terms of single and multiple infection rates. However, multiple infections were not detected in ≥45 age group. Although the median age of the patients with single HPV infection was 39, the median age of the patients with multiple HPV infections was found to be 28. It is thought that infection with more than one type of HPV may result in regression over time.

Although it is known that HPV infection is responsible for cervical cancer etiology, multiple HPV infections are thought to be associated with a significant increase in high-grade neoplasia compared to single infection and increase the cumulative risk of cancer. Although HPV type 16 is the most common cause of cervical cancer, studies have shown that multiple HPV infections also increase the risk of cancer.[31] In this study, when we evaluated the cervical cytology and cervical biopsy results of 25 patients with HPV infection, in this regard, studies involving a larger number of cases are needed.

It is known that there is a strong relationship between cervical cancer and HPV infection. Therefore, oncogenic high-risk HPV genotypes are expected to be detected in abnormal cytological biopsy samples. In this study, HPV16 and HPV52 in LSIL and HSIL as well as in CIN2 were the most common HPV types. Although HPV51 was frequently detected in multiple infections in the 21–34 age group, it was determined as a single infection agent in a 56-year-old patient with CIN3. Early detection of HPV DNA before detecting abnormal cytological findings of patients could be useful for monitoring the disease. HPV DNA has been reported to have a higher sensitivity but lower specificity than cytology to detect high-grade cervical lesions.[32]

In conclusion, in this study, the frequency of high-risk HPV was 35% as similar to reports of the other studies conducted in our country. The most common HPV genotypes were HPV51, HPV16, and HPV66, respectively. The follow-up of patients with HPV51 infection in our area could help to improve the natural course of the disease and effective prevention programs.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.