“U” shape of age-specific prevalence of high-risk human papillomavirus infection in women attending hospitals in Shanghai, China

Results Human papillomavirus was detected in 5173 of 17,148 women (30.2%) aged 17–79 years. The highest prevalence appeared in the ≤20-year age group (45.2%). After age 20, the prevalence declined rapidly and then ascended slowly from 28.5% in the 21–30-year age group to 38.0% in the ≥61-year group. The mean viral load was 294.12 ± 511.66 relative light units/positive control in total human papillomavirus positive cases. The viral load of the 21–30-year age group was the lowest (271.99 ± 499.24 relative light units/positive control) and the highest was found in the ≥61-year interval (560.30 ± 672.87 relative light units/positive control). No significant correlation was found between viral load and the severity of cervical lesions. Conclusions Our study showed a “U” shape of age-specific prevalence of high-risk human papillomavirus infection occurring in women attending hospitals in Shanghai, China, similar to worldwide figures. Keywords High-risk human papillomavirus Cervical cancer Cervical intraepithelial neoplasia Prevalence Age interval 1 Introduction Cervical cancer is one of the most common female genital cancers worldwide. There is strong epidemiological evidence indicating that human papillomavirus (HPV) plays a central role in the etiology of cervical cancer [1] . Women who test positive for HPV DNA have a greater risk of developing cervical cancer than those without HPV DNA. Anogenital HPVs have been divided into high and low risk groups according to the frequency of their presence in cervical carcinomas. High-risk HPV (HR-HPV) is thought to be oncogenic, and it is the key causative event for the development of cervical precancerous lesions and cervical cancer. Although HPV infection is common among young women, only a small minority go on to develop cervical cancer. Viral persistence of oncogenic HPV, possibly for 10–20 years, appears to be crucial for the development of cervical cancer [2] . HPV vaccine has been used clinically and satisfactory results have been achieved. Optimal selection of the age when the vaccine is delivered can supply efficient protection before exposure to HPV. Characteristics of the prevalence of HR-HPV infection vary with the age of the patient [3] . It is therefore important to study the distribution and prevalence of HR-HPV infection in different age groups. Hybrid capture II (HC-2) is one of the methods used to detect HR-HPV DNA that supplies semi-qualitative viral DNA load analysis by the resultant relative light units (RLUs) and the ratio of RLUs to positive control (RLU/PC). The high sensitivity and negative predictive value reported by many studies [4–6] indicated that it was a very good adjuvant assay or a substitute for cytological screening. The test detects any of 13 HR-HPV types and is currently one of the most frequently applied methods to detect HR-HPV in China. This study investigated the distribution of HR-HPV infection prevalence and the HR-HPV viral load by HC-2 assay in different age groups of women who attended the gynecological clinic for anogenital diseases in two hospitals in Shanghai, China. Our study should help improve our understanding of HPV infection prevalence trends among women in Shanghai, determine the current uncertainty regarding the age-specific rate of decline of HPV incidence and provide baseline data for future applications of HPV vaccine in China. 2 Materials and methods 2.1 Study population A retrospective analysis was performed on the HC-2 of 17,148 patients attending the Sixth People's Hospital, Shanghai Jiao Tong University and the Obstetrics & Gynecology Hospital, Fudan University, Shanghai, between January 2005 and February 2007. Patients who attended the gynecological clinic in these two hospitals for different anogenital diseases are included in this retrospective analysis. All the patients were sexually active. Patients who had previous gynecological neoplasm after the therapies and pregnant women were excluded. Patients who had received several results of HC-2 during a follow-up of HPV infection on a cervical lesion were enrolled into the study and the first result of HC-2 (the positive one) was noted. Follow-up data were analyzed. Patients were divided into 6 age groups: ≤20 years, 21–30 years, 31–40 years, 41–50 years, 51–60 years and ≥61 years. The prevalence of HR-HPV infection was estimated in each age group and in different histological diagnoses. The HR-HPV viral load was evaluated in HPV-positive cases of each group. The study was approved by the local ethics committee. 2.2 Detection of HR-HPV DNA with HC-2 Sample was obtained from the ectocervix and endocervix with a cervical cytobrush by a gynecologist. The specimens were tested using the HC-2 assay according to the manufacturer's protocol (Digene Diagnostics Inc., USA). Denatured DNA from the sample was hybridized with a cocktail of 13 RNA probes for oncogenic HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. Hybrids were captured with alkaline phosphatase-conjugated antibodies specific to HPV–DNA–RNA hybrids. A dioxetane-based chemiluminescent substrate was added and the resultant relative light units (RLUs) were measured in a luminometer. Carrier DNA in specimen transport medium (100 mg/ml) was used as a negative control. Triplicate specimens of HPV 16 DNAs at 1.0 pg/ml served as the positive controls for high-risk probes. All RLU measurements for specimens were divided by the mean RLU of the three corresponding positive controls (PCs) to give a ratio of specimen RLU/PC. We used 1.00, 10.00, and 100.00 as cut-off levels. A ratio of 1 or greater was defined as positive for HPV DNA, and a ratio of less than 1 was regarded as negative. Ratios of 1.00–10.00, 10.01–100.00, and more than 100.00 are regarded as low, medium, and high viral loads, respectively. 2.3 Statistical analysis The difference in HR-HPV infection prevalence among age groups was analyzed by the Chi-square test. The viral load of each age group was compared with statistical analysis software SPSS 11.0. The means were assessed using one-way analysis of variance. P -value < 0.05 was considered statistically significant. 3 Results 3.1 Prevalence of HR-HPV infection in different age groups The mean age of all 17,148 women was 34.4 ± 3.4 years, ranging from 18 to 79 years. All women were Chinese and 83.5% were married. All 17,148 women underwent HC-2 and 30.2% (5173/17,148) had HR-HPV infections. The highest prevalence of HR-HPV infection occurred within the ≤20-year-old group (45.2%) and the lowest occurred in the 21–30-year-old group (28.5%). The prevalence of HR-HPV maintained a plateau in the 21–30- and 31–40-year-old age groups, and then it ascended slowly from the 51–60-year-old group to 61+ years group to a level of 38.0% HR-HPV prevalence ( P = 0.000). The distribution of HR-HPV by age is shown in left half of Table 1 . The trend in the prevalence of HR-HPV by age intervals is shown in Fig. 1 . 3.2 HR-HPV viral load in different age groups The HR-HPV viral load was estimated on the features of RLU/PC and the HR-HPV viral load level among different age groups. The mean of RLU/PC was 294.12 ± 511.66 in all HR-HPV-positive cases. The group with the highest RLU/PC was the ≥61-year-old group (560.30 ± 672.87). Forty percent of the ≥61-year-old cases (12/30) had a high viral load. The second highest RLU/PC was in ≤20-year group (382.87 ± 599.06). The lowest RLU/PC was noted in the 21–30-year-old group (271.99 ± 499.24). There was a significant difference between age groups on the features of RLU/PC and HR-HPV viral load level (right half of Table 1 , Fig. 2 ). 3.3 Follow-up of patients with HR-HPV infection in different age groups Multiple HR-HPV detection was done on 359 patients. Among them, the HR-HPV viral load was shown to decrease in 78.8% of patients, increase in 19.5% of patients, and remain unchanged in 1.7% of patients. In the 21–30-, 31–40- and 41–50-year-old age groups the proportions of decreasing load (73.9–81.8%) were almost similar, as were the proportions of increasing (17.5–24.5%) HR-HPV viral load. For 51–60-year-old age group the decreasing percentage was less as compared to the other age categories (66.7%) ( Table 2 ). 3.4 Prevalence of HR-HPV infection by type of histological diagnosis Of the 17,148 women, there were 4617 women with high-risk factors who underwent colposcopic biopsy for histological results. Among them 71.9% (3321/4617) of biopsies showed no pathological abnormality and 18.3% (846/4617) showed HPV infection. The incidences of CIN1, CIN2, CIN3 and cervical cancer in the biopsy group were 4.1% (189/4617), 3.6% (167/4617, 1.1% (49/4617) and 1.0% (45/4617), respectively. The prevalence of HR-HPV infection in these 4617 women was 35.5% (1640/4617). In different cervical lesions the HR-HPV positive rates were 64.3% for HPV infection (544/846), 71.4% for CIN (289/405), and 95.6% for cervical cancer (43/45). The correlation between viral load and cervical lesion severity was not statistically significant ( P = 0.075). The mean viral load in women with CIN1 was 366.55 ± 539.38, which was slightly higher than the mean load in women with CIN2 (259.47 ± 443.04), CIN3 (281.69 ± 339.65), or cervical cancer (217.18 ± 229.92), but the difference among the mean viral loads was not statistically significant ( P = 0.082). 4 Comment In many studies, HPV testing for cervical intraepithelial neoplasia (CIN) 2 and CIN 3+ (CIN 2 or worse) had high sensitivity, being 96.1% overall (94.2–97.4% and 93.6–97.6%, respectively). The higher sensitivity of HPV testing also leads to a higher negative predictive value, suggesting that the screening interval can be safely lengthened if HPV testing is used. Cytological screening has moderate sensitivity (44–78%), but the overall specificity for cytology was 96.3% (96.1–96.5%) and it has a higher positive predictive value than HPV testing, which reduces the costs associated with referral for colposcopy. Many groups recommend the use of HPV testing in conjunction with cytology as a way of extending safe screening intervals [7] . However, currently the Chinese Cancer Society guideline about cervical cancer screening recommends the three-stage technique including conventional Pap smear or liquid-based cytology for large scale screening annually, colposcopy and biopsy according to the situation of patients. HPV DNA is only done in several big cities in China. Because the explanation and advice about cervical screening given by doctors was considered more confident and less ambiguous, gynecologists tend to promote HC-2 simultaneous with cytology as a primary screening method in many countries [7] . The prevalence of HR-HPV infection is highly dependent on the study sample and the detection method used [8] . A study of large samples showed that HPV DNA was present in 83.7% of cervical cancers in China. A recent meta-analysis investigated HPV prevalence among women with normal cytology and the results showed the adjusted prevalence was 10.41% globally, and 7.95% in Asia. Trottier et al. [9] reported that the prevalence of HR-HPV ranges from 2% to 44% among asymptomatic women in the general population. Our study retrospectively analyzed the HC-2 results of 17,148 women attending hospital. The total prevalence of HR-HPV in this group was 30.2%. In CIN and cervical cancer patients, the prevalence of HR-HPV was 71.4% and 95.6%, which are similar to the worldwide range. The total HPV prevalence results were higher in our study, and they did not represent HPV infection among general women in Shanghai, but rather our results partly reflect the status of HR-HPV infection of women in Shanghai who attend hospital for different medical reasons. In the ≤20-year age group, 157 patients were enrolled in the study (17 years, 1 case; 18 years, 9 cases; 19 years, 27 cases; 20 years, 120 cases) and the total infection rate was 45.2%. Similar to many studies, the peak of HPV prevalence appeared among younger women. All the women in our study were sexually active. Sexual transmission is considered a major route of HPV infection [10,11] . Multiple studies have found that HPV is the most prevalent sexually transmitted infection in adolescents and the infection rate ranged from 54% to 90% [12–14] . ASC-US cytology in young women and adolescents is associated with a high rate of HPV positivity but a low prevalence of high-grade cervical disease [15–17] . This suggests that HPV triage is a useful method for young women with ASC-US so that fewer women would be referred for colposcopic evaluation [18,19] . Earlier sexual debut is a risk factor for HPV infection [14,20] . Licensed by the U.S. Food and Drug Administration, the quadrivalent HPV vaccine has been used in clinical trials which indicate that the vaccine has high efficacy in preventing persistent HPV infection, cervical cancer precursor lesions, vaginal and vulvar cancer precursor lesions, and genital warts caused by HPV types 6, 11, 16 or 18 among females who have not already been infected with the respective HPV type. Although routine vaccination is recommended at age 11–12 years, the majority of females aged 13–26 years also can benefit from vaccination [21] . Ideally, the vaccine should be administered before potential exposure to HPV through sexual contact; however, females who might already have been exposed to HPV should be vaccinated. Sexually active females who have not been infected with any of the HPV vaccine types would receive full benefit from vaccination. The prevalence of HR-HPV infection varies with age and geography [3] . Our study showed a “U” shape of age-specific HR-HPV prevalence with the first peak in the ≤20-year age group and the second in the ≥61-year group. After the first peak of prevalence in the ≤20-year group, the curve declined rapidly; this was also described in other studies [22] . The rapid decrease might relate to the spontaneous clearance of infected HPV through human immunity. The risk of women contracting HPV was about 80% during their entire lives [23] . As the immune response is stimulated, a great proportion of primary HPV infections would clear up spontaneously [24] . Only 5–10% of HPV-infected women developed persistent infections, which might result in CIN or even cervical cancer. In our study, the rapid decline of HR-HPV infection prevalence from 45.2% in women aged ≤20 years to 28.5% in women aged 21–30 years might represent the spontaneous clearing up of the virus. But it is pity that due to less follow-up of HR-HPV infection younger patients, we cannot find a precise decreasing ratio to validate it. After the rapid fall, the HPV infection rate increased slowly among the 21–60-year-old and then reached the second peak in women aged 60 years and older. Contrary to our study, several reports concerning age-specific HPV prevalence showed that the curve was still declining on a small slope with a slight increase in women aged 55 (50–60 years and up) [7,25] . Franceschi et al. reported that HPV prevalence peaked below age 25 or 35, and declined with age in Italy, the Netherlands, Spain, Argentina and Korea, and in Lampang, Thailand and Ho Chi Minh City, Vietnam. In Chile, Colombia and Mexico, a second peak of HPV prevalence was detected among older women [26] . Biological mechanisms, including cervical immaturity, inadequate production of protective cervical mucus and increased cervical ectopy, may make younger women more susceptible to HPV infection. As for older women, one possible explanation is that maybe the highest prevalence of cervical cancer is seen in the oldest age group and the highest RLUs will also be seen here. The mean HR-HPV viral load is a “U” shape based on the features of RLU/PC among different age groups also. Although the highest prevalence of HR-HPV infection occurred in the ≤20-year-old group (45.2%), in this age group the RLU/PC was the second highest. The highest RLU/PC was in the ≥61-year-old (560.30 ± 672.87) group in the viral load curve. It may also relate to the persistent high HPV viral load and the occurrence of cervical cancer. Although incidence of cervical cancer in 41–50-year-old women is usually the highest among age groups, the morbidity of cervical cancer in older women is still higher; advanced stage is more common in women older than 70 while there is a lower occurrence of CIN2 or worse in adolescents younger than 20 [27,28] . The viral load of women aged 17–20 years (382.87 RLU/PC) is the second highest among age groups, and is significantly different from that of 40–50-year-old women (286.94 RLU/PC). Whether HPV viral load is correlated with the severity of cervical lesions or not is still uncertain [8,29] and multicenter studies will be applied to enrich the data of the prevalence of HR-HPV in Shanghai, China for further application of HPV detection. References [1] F.X. Bosch M.M. Manos N. Munoz Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group J Natl Cancer Inst 87 11 1995 796 802 [2] F.X. 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