Prevalence and Genotype Distribution of Human Papillomavirus among Women from Henan , China

Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in females worldwide (Jemal et al., 2011). More than 80% of the women diagnosed with cervical cancer live in the developing countries, such as China and Indian, the cervical cancer has been an important public health concern (Li et al., 2013; Rai et al., 2014). Several methods, such as visual inspection using acetic acid or Lugol’s iodine, the conventional Pap smear and human papillomavirus (HPV) DNA testing, had been implemented for the screen of cervical cancer. The sensitivity of visual inspection and Pap smear might depend on the experience and training of the health workers, however, HPV DNA testing would be an alternative method for objectively screening HPV infection with high sensitivity and moderate specific (Zhao et al., 2010). HPV infection has been proven to be the etiologic agent of cervical cancer (Ferlay et al., 2010). It is reported that more than 200 different HPV genotypes have been identified, and about 40 oncogenic subtypes are associated with the majority of cases of cervical cancer (Munoz et al., 2003). Although most of the HPV infections are transient and may resolve spontaneously, the persistent infections with subset of HPV genotypes are necessary for the development of cervical cancer and its precursors (Schlecht et al., 2001). Based on their association with


Introduction
Cervical cancer is the third most commonly diagnosed cancer and the fourth leading cause of cancer death in females worldwide (Jemal et al., 2011).More than 80% of the women diagnosed with cervical cancer live in the developing countries, such as China and Indian, the cervical cancer has been an important public health concern (Li et al., 2013;Rai et al., 2014).Several methods, such as visual inspection using acetic acid or Lugol's iodine, the conventional Pap smear and human papillomavirus (HPV) DNA testing, had been implemented for the screen of cervical cancer.The sensitivity of visual inspection and Pap smear might depend on the experience and training of the health workers, however, HPV DNA testing would be an alternative method for objectively screening HPV infection with high sensitivity and moderate specific (Zhao et al., 2010).
HPV infection has been proven to be the etiologic agent of cervical cancer (Ferlay et al., 2010).It is reported that more than 200 different HPV genotypes have been identified, and about 40 oncogenic subtypes are associated with the majority of cases of cervical cancer (Munoz et al., 2003).Although most of the HPV infections are transient and may resolve spontaneously, the persistent infections with subset of HPV genotypes are necessary for the development of cervical cancer and its precursors (Schlecht et al., 2001).Based on their association with
The principal objective of the present study is to investigate the prevalence and distribution of HPV genotypes among females from a hospital in Luoyang, Henan, by means of the commercially available HPV DNA Chip.

Sample Collection
From December 2008 to January 2014, consecutive samples of 578 cervical swabs from women attending the gynecological outpatient clinic were collected by healthcare workers in No. 150 Central Hospital of PLA.
Before collection, the woman was eligible to be in All the specimens and their corresponding clinical information were obtained under protocols approved by the Ethics Committee of the hospital, and the written informed consents were obtained from all the patients.
Every sample was obtained with a cytobrush from the ecto-and endocervix of the uterus of each woman, and then suspended in the physiologic saline and stored at -70°C until the HPV genotyping.

HPV Genotyping
After thrown, the supernatants were removed by centrifugation at 14000 rpm for 5 minutes and the pellets were collected for DNA extraction.Genomic DNA was extracted by alkalysis using DNA extraction kit (Chaozhou Hybribio Biotechnology Corp., China).The quality of the extracted DNA was confirmed by amplifying the β-globin gene as an internal control.The HPV DNA was amplified using the PCR kit (Chaozhou Hybribio Biotechnology Corp., China) with the general consensus primers PGMY09/PGMY11 to amplify the HPV L1 gene.According to the recommendation of the manufacturer, PCR was carried out in 25 μL reaction mixture per person in a thermal cycle (PE Applied Biosystems GeneAmp PCR System 9600) following parameters: initial denaturation at 95°C for 9 min, followed by 40 cycles at 95°C for 20s, 55°C for 30s, and 72°C for 30s, and a final extension at 72°C for 5 minutes.Distilled water and HPV 18 were presented separately as negative and positive control.
The genotyping was performed via hybridization of the PCR products to gene chip containing genotype-specific oligonucleotides that were immobilized on a nylon membrane.The amplicon was subsequently denatured at 95°C for 5 minutes, and then kept at 45°C for 10 minutes for hybridation on the platform of hybridization chamber (HMM2).After blocking with confining fluid, the chips were washed 3 times, and then the hybrids were detected by the addition of streptavidin-horse-radish peroxidase conjugate which binded to the biotinylated PCR products and incubated at 25°C for 3.5 minutes.After 4 times washing, the substrate NBT/BICP (nitroblue tetrozolium and 5-bromo-4-chloro-3-indoylphosphate) was added at 37°C for 5 minutes, followed by 4 times washing.The final results were detected by colorimetric change on the chip under direct visualization.

Statistical Analysis
SPSS version 19.0 (IBM, Armonk, NY, USA) was used to assess the significance of differences detected in the frequency of HPV infections among groups.The χ 2 test was used to compare the prevalence of HPV infection.A p-value <0.05 was considered statistically significant.

Prevalence of HPV Infection in Different age Groups
A total of 578 samples from females (ranging from  1).All cervical samples from females were found to be positive for β-globin, indicating that the extracted DNA was adequate to be analyzed further.Overall, 257 women (44.5%, 257/578) were found to be HPV positive for any HPV DNA, of whom 35.1% (203/578) were found to be HR-HPV infection, substantially higher than the LR-HPV infection (16.8%.97/578).Among the 257 women with HPV infection, there were two peaks of HPV infection among the women, the first was 71.4% in the ≤20 year-old group and the second was 65.0% in the >60 year-old groups (χ 2 =22.279, p<0.05).In different age groups, women infected with the HR-HPV or LR-HPV were significantly different (χ 2 =7.019, p<0.05).Among the women infected with the HR-HPV, the first peak was within the >60 yearold group (55.0%), and the second peak was within the 51-55 year-old group (50.0%) (χ 2 =19.497, p<0.05).The prevalence of the LR-HPV exhibited the first peak in the ≤20 age group, and decreased slowly until the second peak was in the >60 year-old group.

Discussion
In the present study, the prevalence of HPV infection among the females who underwent the gynecological outpatient clinic in Henan was evaluated by commercially available HPV DNA Chip.A high rate of HPV-DNA infection (44.4%) was observed in specimens taken from patients ranged from 17 to 79 years, and 35.1% was the HR-HPV types.The prevalence of HPV infection among females attending the cervical cancer screening varies in different regions of China, for example, in Shenzhen city, 13.8% of the volunteers was HPV positive; in Shanghai city, southeast of China, the infection rate was 30.2% (Xue et al., 2009;Wang et al., 2013).In the present study, the prevalence of HPV infection showed a significant difference among patients in the age groups (χ 2 =22.279, p<0.05).The first peak of HPV infection (71.4%) occurred in the ≤20 years group, might be due to be lack of adaptive immune responses and susceptible to the new LR-HPV or HR-HPV infection (Ye et al., 2010).

Xiao-Chuan Wang et al
The second peak (65.0%) was observed in the >60 years group, that may be partly explained by viral persistence or reactivation of latent HPV due to the physiologic and immunologic dysregulation caused by hormone fluctuations at menopausal transition (Althoff et al., 2009;Xue et al., 2009).The highest prevalence of the HR-HPV infection (55.0%) appeared in the >60 years group was more likely for viral persistence than for acquisition of new infection (Castle et al., 2005;Goodman et al., 2008).It was reported that the persistence rate of the HR-HPV infection was higher than LR-HPV infection, and it was a strong predictor for the development of CIN2/3 and invasive cervical cancer (Dalstein et al., 2003;Datta et al., 2012).The previous work found that the HR-HPV DNA was positive among 80% of patients with cervical cancer, so more inspections, including cytology and even colposcopy, should be proceed among women aged >60 years for the prevention of cervical cancer (Wang et al., 2013).
In summary, the rate of single-type HPV infection was detected in 68.5% (176/257), which was much higher than that of the multiple-type HPV infections (31.5%, 81/257).Females infected with multiple-type HPV could be divided into three categories: (i) high and low risk HPV; (ii) high and high risk HPV; (iii) low and low risk HPV.In the group of double infections, the rate of category (i) was 49.1% (26/53), (ii) was 47.2% (25/53), (iii) was 3.8% (2/53).In the triple infections group, the category (i) was 60% (9/15), the rest was (ii).Women infected with multiple-type HPV were at significantly increased risk of both CIN2+ and HSIL+, when compared with single infection (Dickson et al., 2014).