Background
The association of human papillomavirus (HPV) infection with cervical cancer and its precursor lesions is well established [
1,
2]. More than one hundred HPV genotypes have been described, about 40 of which can infect mucosal epithelia [
3], causing mainly transient infections [
4].
Based on the etiological role in the progression to cervical cancer, 15 HPV genotypes have demonstrated strong oncogenic potential and are classified as high-risk HPVs [
5]. Persistent infection with oncogenic types of HPV is considered the main risk factor associated with this disease. The HPV genotypes and prevalence found in different regions of the world vary both in type and incidence, probably as a consequence of the many factors associated with HPV infection [
6]. In a substantial percentage of HPV infections, two or more genotypes may be found. About 20-50% of the infected women carry more than one viral type [
7,
8]. For some groups, co-infection with multiple HPV types correlates with the severity of cervical intraepithelial neoplasia [
9]. In contrast, other authors [
10,
11] have reported that infection with multiple types does not increase the risk of developing or progression of cervical neoplasia. HPV typing has important prognostic and therapeutic value and the recent development of HPV vaccines makes it increasingly more important. It has been suggested that the impact of the vaccines could vary depending on the regional distribution of HPV types and co-infection patterns. To investigate the potential impact of vaccination strategy, it is essential to assess the local distribution of HPV types and the incidence of co-infections. The aim of the present study was to investigate the distribution of HPV subtypes and its relationship with cervical lesions in women in Madrid, Spain.
Results
There were 2,461 samples from women aged 15-75 years analysed for HPV infection from May 2006 to June 2010: 1,399 (56.8%) were negative for HPV and 1,062 (43.2%) were positive. The cytological diagnosis was: 1,656 (67.3%) with normal cytology (NC); 336 (13.7%) with atypical squamous cell of undetermined significance (ASCUS); 387 (15.7%) low-grade squamous intraepithelial lesions (LSILs); and 82 (3.3%) high-grade squamous intraepithelial lesions (HSILs). The percentage of cases positive for HPV infection in the different diagnostic categories was: 20% in NC, 84% in ASCUS, 94% in LSILs, and 96% in HSILs (Table
1).
Table 1
Association between cytological lesions and HPV positivity
NC (n = 1656) | 1322 (79.8%) | 334 (20.2%) |
ASCUS (n = 336) | 52 (15.5%) | 284 (84.5%) |
LSIL (n = 387) | 22 (5.7%) | 365 (94.3%) |
HSIL (n = 82) | 3 (3.7%) | 79 (96.3%) |
The current analysis focused on the 1,062 HPV-positive samples to establish the distribution of HPV subtypes in our area. Cytological diagnosis of these HPV-positive samples included 334 (31%) from women with negative cytology and 728 (69%) with some cytological abnormality. The cytological diagnosis of abnormal cases was as follows: 284 cases (27%) with ASCUS, 365 (34%) with LSILs, and 79 (8%) with HSILs.
Mean age of the patients was 34 ± 10.5 years (range 15-75). Median age of NC patients was 34.5 years, 34.2 years for ASCUS, 32.3 years for LSILs, and 39.6 years for HSILs. The distribution of the cytological diagnoses with regard to age is shown in Table
2. Women older than 32 years had the following cytological distribution: 33% NC, 25% ASCUS, 30% LSIL, and 12% HSIL, whereas in women younger than 33 years, only 4% had HSILs, 39% LSILs, 28% ASCUS and 29% NC.
Table 2
Distribution of cytological diagnosis in HPV-positive samples with regard to age
NC | 33.3% | 28.8% |
ASCUS | 25.5% | 27.8% |
LSIL | 29.7% | 39.6% |
HSIL | 11.5% | 4.0% |
The most common HPV types in the complete series were as follows: 16 (28%), 53 (16%), 31 (12%), 52 (10%), 6 (9%), 18 (8%), 58 (8%), and 66 (8%). The prevalence of other HPV types was: 33 (5%), 39 (5%), 56 (5%), 81 (5%), 42 (4%), 45 (4%), 11 (3%), and 51 (3%). The most common HPV types in cytologically normal women were: 16 (21%), 53 (16%), 6 (10%), 31 (10%) and 52 (8%). HPV18 was found in 6% of NC cases. In abnormal samples, the distribution was as follows (Table
3). ASCUS: 16 (31%), 53 (16%), 31 (11%), 52 (11%) and 6 (10%); LSILs: 16 (26%), 53 (19%), 31 (14%), 18 (11.5%), 52 (11%) and 66 (11%); and HSILs: 16 (51%), 52 (14%), 31 (11%), 33 (10%) and 58 (8%). The three most frequent HPV types were 16, 53 and 31 in NC, ASCUS and LSILs, respectively.
Table 3
Distribution of HPV genotypes found in each cytological diagnosis
NC (n = 334)
| 10.2% (34) | 2.1% (7) | 21% (70) | 6.3% (21) | 10.5% (35) | 4.8% (16) | 5.4% (18) | 4.2% (14) | 3.6% (12) | 1.2% (4) | 8.4% (28) | 16.2% (54) | 2.4% (8) | 7.5% (25) | 6% (20) | 4.8% (16) | 15% (50) |
ASCUS (n = 284)
| 9.9% (28) | 3.9% (11) | 30.6% (87) | 7.7% (22) | 11.3% (32) | 4.2% (12) | 5.3% (15) | 3.2% (9) | 4.9% (14) | 1.1% (3) | 10.9% (31) | 15.8% (45) | 3.2% (9) | 7% (20) | 7% (20) | 6.3% (18) | 13% (37) |
LSIL (n = 365)
| 10.1% (37) | 3.8% (14) | 26.3% (96) | 11.5% (42) | 14.2% (52) | 3.8% (14) | 5.2% (19) | 7.1% (26) | 3.3% (12) | 8.5% (31) | 10.7% (39) | 18.9% (69) | 7.9% (29) | 10.1% (37) | 11.5% (42) | 5.2% (19) | 16.2% (59) |
HSIL (n = 79)
| 1.3% (1) | 0 | 50.6% (40) | 5.1% (4) | 11.4% (9) | 10.1% (8) | 1.3% (1) | 0 | 2.5% (2) | 1.3% (1) | 13.9% (11) | 5.1% (4) | 5.1% (4) | 7.6% (6) | 1.3% (1) | 3.8% (3) | 6.3% (5) |
Single infection (one HPV type) was observed in 696 (65.5%) samples, and 366 (34.5%) patients were infected with multiple HPV types. Double infections (two different genotypes) accounted for 60% of the multiple infections, three genotypes were detected in 26% of these 366 samples, and four or more types in 14%. In women under 33 years old, infections due to more than one HPV type were found in 39.6% of cases, whereas in those older than this, the prevalence was 26.7% (p < 0.01). HPV 16 was the most common genotype detected in co-infections, and was present in 38% of cases, followed by HPV 53 (27%) and HPV 31 (20%). Co-infections were found in 25% of NC, 36% of ASCUS, 45% of LSIL, and 20% of HSIL cases (Table
4). Distribution of cytological lesions in those 366 samples was as follows: 23% NC, 28% ASCUS, 45% LSIL, and 4% HSIL. Single infection in HSIL samples was found in 80% of cases (63 samples), and HPV 16 was detected in 50% of them. Other genotypes found in HSIL single infection were HPV 31 (9.5%), HPV 33 (7.9%), HPV 52 (6.3%), and HPV 58 (4.8%). HPV 18 as single infection in HSILs was found in one case and in three cases as co-infection with HPV 52.
Table 4
Correlation between HPV co-infection and cytological lesions
NC (n = 334) | 251 (75.1%) | 83 (24.9%) |
ASCUS (n = 284) | 183 (64.4%) | 101 (35.6%) |
LSIL (n = 365) | 199 (54, 5%) | 166 (45.5%) |
HSIL (n = 79) | 63 (79.7%) | 16 (20.3%) |
Total
|
696 (65.5%)
|
366 (34.5%)
|
The global results demonstrated that HPV 16 was the most prevalent infection (28%), whereas HPV 18 was found more rarely (8%).
Discussion
Our specimens came from a group of women who attended a cervical pathology unit because of a previous abnormal Pap test result. We selected the HPV-positive cases to investigate the genotype distribution in our area. Several studies have been published concerning genotype distribution in several Spanish regions [
14‐
18], but few data are available about the distribution in Madrid [
19]. The prevalence and distribution of HPV genotypes varies greatly worldwide, and these differences might be related to the complex geographical and biological interplay between different HPV types and host immunogenetic factors [
20].
Consistent with previously published data, genotype HPV 16 was the most common type in our series, and was detected in 28% of samples. With the exception of Eastern Africa, China, Japan and Taiwan [
21‐
23], HPV 16 is the most prevalent type in all parts of the world. Our study showed that HPV 53 was the second most common genotype (16% in the whole series), which is similar to previous Italian [
24,
25] and Spanish [
17,
19,
26] studies, however, HPV 53 is not a frequent genotype in other Mediterranean countries, such as Greece and Turkey [
27,
28]. The epidemiological patterns of HPV type prevalence could differ depending on the region of origin. Another factor that could influence the genotype distribution is the primer system used. The GP5+/6+ primer set is less sensitive for HPV 53 detection, whereas MY09/MY11 is less sensitive for HPV 31 [
29].
Nearly a third of HPV-positive samples in our series were detected in patients with normal cytology. This prevalence is higher than that of other Spanish studies. This might be due to the fact that the women in our study had an abnormal Pap test and attended a specialised gynaecology unit for cervical pathology.
In NC samples, HPV 16 was the most common genotype (21%), followed by HPV 53 (16%). The HPV distribution found in NC cases is similar to that reported by Conesa et al. [
26] from a cohort of women from Eastern Spain. The same distribution was found in ASCUS and LSIL samples with HPV 16 (31% and 26%, respectively) being the most prevalent, followed by HPV 53 (16% and 19%, respectively). The high prevalence of HPV 53 in ASCUS and LSILs is similar to that in other studies from Spain and France.
In HSILs, the most frequent type was HPV 16 (51%), followed by 52 (14%) and 31 (11%). In our series, HPV 53 was not detected in HSILs, suggesting that it is not a high-risk type. An interesting finding of our study was that HPV 52 was the second most common genotype in HSIL cases, whereas its prevalence in NC and LSILs was lower.
Recently, multiple HPV infections have been analysed because, with the development of anti-HPV vaccines not covering all genotypes, the distribution of infection with types not covered by vaccines could be affected. The elimination of one HPV type could affect the natural history of the remaining genotypes. Therefore, obtaining a solid knowledge of genotype HPV distribution is becoming imperative. Multiple types of HPV have been described in up to 50% of NC and LSIL cases in the population in Northwest Spain [
15], in accordance with other studies [
30].
In the present study, multiple infections were detected in 34% of samples, and were more frequent in younger women (< 33 years). This observation is consistent with the results of Mejlhede et al. [
8], and supports the fact that greater sexual activity in younger women may be associated with the transmission of multiple HPV types. The presence of co-infection was more frequent in LSIL cases (45%), followed ASCUS (36%) and NC (25%), whereas a decreased frequency of co-infection was found in HSILs (20%). Similar to other Spanish studies [
15,
26], in our series, ASCUS/LSILs were associated with co-infections, whereas HSILs were associated with single infection (p < 0.01). It is still not clear whether co-infection with several types increases the risk of progression. It seems that, as lesions progress from low to high grade, high-risk oncogenic types may persist, whereas less oncogenic types are eliminated [
26].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PM designed the experiments, performed analysis and interpretation of data, and drafted the manuscript; LK and DG performed genotyping methods; AMLG performed pathological diagnosis; MJMA performed data analysis; and CB performed pathological diagnosis and supervised the study. VA performed statistical treatment of data.
All authors read and approved the final manuscript.