Distribution of human papillomavirus genotypes in Paraguayan women according to the severity of the cervical lesion
Abstract
The incidence of cervical cancer in Paraguay is among the highest in the world. This study aimed to determine the distribution of human papillomavirus (HPV) genotypes in Paraguayan women, according to the severity of the cervical lesion. This cross-sectional study included 207 women without a squamous intraepithelial lesion, 164 with low-grade squamous intraepithelial lesions, 74 with high-grade squamous intraepithelial lesions, and 41 with cervical cancer. Type-specific HPV was determined by the polymerase chain reaction with MY9/11 L1 and GP5+/GP6+ L1 primers, followed by restriction fragment length polymorphism and reverse line blotting hybridization, respectively. In total, 12 high-risk and 24 low-risk HPVs types were detected. HPV 16 was the most prevalent, followed by HPV 18 in cervical cancer (14.6%), HPV 31 in high-grade squamous intraepithelial lesions (14.9%), HPVs 58/42 in low-grade squamous intraepithelial lesions (9.1% each), and HPVs 31/58 (2.4% each) in women without squamous intraepithelial lesions. Among 285 positive samples, 24.2% harbored multiple HPV types, being this more prevalent in women with squamous intraepithelial lesions (30.8% in low-grade squamous intraepithelial lesions, 22.5% in high-grade squamous intraepithelial lesions, and 22.0% in cervical cancer) than in women without lesions (9.3%). The higher prevalence of HPV 16 and other high-risk HPVs in women both with and without cervical lesions may explain the high incidence of cervical cancer in Paraguay. This information may be of importance for local decision makers to improve prevention strategies. In addition, these results may be useful as baseline pre-vaccination data for a future virological surveillance in Paraguay. J. Med. Virol. 83:1351–1357, 2011. © 2011 Wiley-Liss, Inc.
INTRODUCTION
Cervical cancer represents the second most common malignancy in women around the world and contributes to 9.8% of all female cancers. In 2008, there were about 529,800 cases and 275,000 deaths worldwide due to cervical cancer. More than 13% of this burden was suffered by women from Latin American countries. Since this cancer can be prevented, these figures are alarming [Ferlay et al., 2010].
Infections with human papillomavirus (HPV) are considered as the main cause of cervical squamous intraepithelial lesions and cervical cancer [Muñoz et al., 2003]. Approximately 40 distinct HPV types are known to infect, and frequently co-infect, the anogenital mucosa; they are grouped into high-risk HPV and low-risk HPV categories, on the basis of their ability to cause cervical cancer, with high-risk HPVs detected in virtually all of the patients with cervical cancer [Muñoz et al., 2003; Evans et al., 2010].
Two vaccines have been licensed worlwide: a bivalent vaccine against HPVs 16 and 18 and a quadrivalent vaccine against HPVs 6, 11, 16, and 18. Many controlled trials have demonstrated that these vaccines have almost complete efficiency for preventing lesions caused by these HPV types [Harper, 2009]. Although HPV 16 is the most prevalent type worldwide, several meta-analyses have shown that the distribution of HPV types varies according to several factors such as the geographic region and the severity of the injury [Smith et al., 2007; de Sanjosé et al., 2007; Bosch et al., 2008]. Thus, monitoring HPV infection endpoints needs to include type-specific data. The World Health Organization has recommended recently that countries should establish the prevalence of the different HPV types, the incidence of cervical abnormalities and pre-cancerous lesions, and the incidence and mortality caused by cervical cancer [Brotherton et al., 2010].
Paraguay occupies the seventh place in incidence of cervical cancer in Latin America and the Caribbean countries, with a rate of 35.0 and a mortality rate of 16.6 × 100,000 women, with 864 new cases and an annual mortality of 407 women [Ferlay et al., 2010]. However, there are only two local studies related to HPV typing, carried out only in women with cervical cancer [Kasamatsu et al., 1996; Rolón et al., 2000]. Therefore, the aim of this study was to determine the distribution of HPV genotypes in cervical samples from Paraguayan women with and without lesions.
MATERIALS AND METHODS
Study Design
The present cross-sectional study included a total of 502 cervical samples (463 cervical exfoliated cells and 39 formalin-fixed paraffin-embedded sections of cervical cancer) from Paraguayan women aged 16–77 years, who attended the Instituto Nacional del Cáncer, Ministerio de Salud Pública y Bienestar Social; Instituto de Previsión Social; Instituto de Investigaciones en Ciencias de la Salud; and the Facultad de Ciencias Médicas, Universidad Nacional de Asunción, from March 2007 to April 2009. The participants were included using the following selection criterion: sexually active, non-pregnant, Paraguayan women, without total histerectomy, medical treatment, or surgery. Based on the cytological diagnosis and applying the Bethesda System (2001), 209 women negative for squamous intraepithelial lesions, 166 with low-grade squamous intraepithelial lesions, 76 with high-grade squamous intraepithelial lesions, and 50 with cervical cancer were also included [Solomon et al., 2002]. All the abnormal results of cytological diagnosis were confirmed histologically.
The sample size to determine the distribution of HPV types according to the severity of the cervical lesion was calculated considering the average proportions of HPV for each group of lesions in women from Latin American countries, which are known to be 80% for low-grade squamous intraepithelial lesions, 89% for high-grade squamous intraepithelial lesions, 97% for cervical cancer, and 14% for women negative for squamous intraepithelial lesions [Kasamatsu et al., 1996; Rolón et al., 2000; Clifford et al., 2005; Smith et al., 2007; de Sanjosé et al., 2007; Bosch et al., 2008]. Finally, a sample size was calculated for each group of lesions according to the table to compare proportions of dichotomous variables, using an α-value of 0.05 (bilateral) and a β-value of 0.20, being 120 women with low-grade squamous intraepithelial lesions, 49 with high-grade squamous intraepithelial lesions, 35 with cervical cancer, and 204 negative for squamous intraepithelial lesions the minimum sample size required for the study [Hulley et al., 2007].
The study protocol was approved by the institutional Ethics Committee of the Instituto de Investigaciones en Ciencias de la Salud, Universidad Nacional de Asunción, in accordance with the ethical standards of the Declaration of Helsinki. All the participants signed an individual informed consent after receiving a detailed explanation of the study. All the data were processed respecting the confidentiality of the participants.
DNA Extraction From Cervical Samples
Cervical cells were collected using a conical brush, which was placed in a tube with a preservative solution; the sample was subsequently stored at −70°C until processing.
Cervical cells were centrifuged at 12,000 × g and washed with 1 ml of PBS and the cell pellets kept at −70°C until processing. For the extraction of genomic DNA, the cell pellet was resuspended in the extraction buffer solution, containing proteinase K (Invitrogen by Life Technologies, Carlsbad, CA) and incubated at 56°C for 1 hr; the enzyme was then inactivated at 95°C for 30 min and the crude digest was used as source of the DNA template for the PCR assays.
Five to ten sections from fixed and paraffin-embedded cervical cancer biopsies were placed in a sterile Eppendorf tube. To avoid contamination between blocks, each sample was cut with a new disposable microtome blade. Later, deparaffinization and extraction was performed according to Sato et al. [2001].
Crude digest samples were tested for the β-globin gene to confirm the presence of a satisfactory template for PCR assay [Saiki et al., 1988].
HPV Detection and Typing
In order to ensure that the testing of the specimens met the highest international standards, a strict protocol which reduced greatly the possibility of contamination was followed.
The classification of HPVs as high-risk types was performed according to Bouvard et al. [2009]; HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 59, 56, 58 were included in this group. All other HPVs types were considered low-risk.
PCR Followed by Restriction Fragment Length Polymorphism
HPV DNA was detected and typed by PCR using MY09/11 primers, combined with restriction enzyme digestion of PCR products (restriction fragment length polymorphism analysis) [Manos et al., 1989; Bernard et al., 1994]. These degenerate consensus primers target a region of approximately 450 bp of the viral L1 gene.
Briefly, aliquots of the PCR products were mixed with 10 U of seven different restriction enzymes (BamHI, HaeIII, HinfI, DdeI, PstI, RsaI, and Sau3AI) in separate reactions [Bernard et al., 1994]. Digestion products were separated by 7% polyacrylamide gel and stained with silver nitrate. In each electrophoretic run, the molecular weight marker Phi-X 174 DNA cut with Hae III (Invitrogen by Life Technologies) was used. Finally, the band patterns obtained were compared with published data [Bernard et al., 1994].
GP5+, 6+ PCR Followed by Reverse Line Blot Hybridization
When the detection by PCR MY09/11 was negative or showed non-specific products that prevented subsequent restriction fragment length polymorphism typing, the samples were analyzed by the GP5+, 6+ PCR- reverse line blot hybridization system as described elsewhere [van den Brule et al., 2002]. Briefly, a PCR using GP5+ and biotin-labeled GP6+ generic HPV primers was performed. These primers target a region of approximately 140 bp in the L1 viral region. The GP-PCR products were typed by reverse line blot hybridization using the type-specific oligoprobes corresponding to 37 HPVs types (HPVs 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 6, 11, 26, 34, 40, 42, 43, 44, 53, 54, 55, 57, 61, 70, 71 [CP8061], 72, 73, 81 [CP8304], 82/MM4, 82/IS39, 83 [MM7], 84 [MM8], and CP6108). Positive reactions were revealed by chemiluminescence using an ECL kit, according to the manufacturer's recommendations (Amersham™ ECL™ Detection Reagents, GE Healthcare, Little Chalfont, UK).
For both PCRs, CaSki and HeLa cell DNAs (which harbor HPVs 16 and 18 sequences, respectively), were used as positive controls and water as negative control (without DNA template).
Statistical Analysis
The data were analyzed using descriptive and analytical statistics. To estimate the distribution of HPV genotypes, single and multiple HPV infections were considered either separately or combined. The prevalence ratios and their corresponding 95% confidence intervals (CIs) were used to compare the relative frequency of the most common HPV types detected in HPV positive women with different degrees of cervical lesions with that of HPV positive women negative for squamous intraepithelial lesions.
RESULTS
A total of 486 (97%) out of the 502 cervical samples collected in the study amplified the β-globin gene, and were thus considered appropriate for the PCR study, leaving 207 women negative for squamous intraepithelial lesions, 164 with low-grade squamous intraepithelial lesions, 74 with high-grade squamous intraepithelial lesions, and 41 with cervical cancer. The demographic characteristics, as well as the reproductive and sexual history of the women included, are shown in Table I.
| Characteristics | Cyto-histological diagnosis (N = 279) | |||
|---|---|---|---|---|
| Low-squamous intraepithelial lesions (N = 164) | High-squamous intraepithelial lesions (N = 74) | Cervical Cancer (N = 41)a | Negative for squamous intraepithelial lesions (N = 207) | |
| Age (year) Median, CI25–75% | 29 (24–37) | 32 (28–43) | 45 (39–56) | 35 (26–44) |
| Place of origin No (%) | ||||
| Asunción's suburbs | 117 (71) | 60 (80) | 38 (98) | 156 (75) |
| Asunción | 47 (29) | 14 (19) | 3 (7) | 51 (25) |
| Education, No (%) | ||||
| Primary | 29 (18) | 26 (35) | 6 (54) | 40 (19) |
| Secondary and higher | 135 (82) | 48 (65) | 5 (45) | 167 (81) |
| Age at 1st sexual intercourse (years), No (%) | ||||
| Median, CI25–75% | 18 (16–21) | 17 (16–20) | — | 19 (17–21) |
| ≤15 | 25 (15) | 18 (24) | 2 (18) | 16 (8) |
| >15 | 139 (85) | 56 (76) | 9 (82) | 191 (92) |
| No sexual partners (%) | ||||
| Median, CI25–75% | 2 (1–3) | 2.5 (1–4) | — | 1 (1–2) |
| <4 | 131 (80) | 54 (73) | 9 (82) | 192 (93) |
| ≥4 | 33 (20) | 20 (27) | 2 (18) | 15 (7) |
| No pregnancies (%) | ||||
| Median, CI25–75% | 2 (1–4) | 3 (2–5) | 6 (4–8) | 3 (2–4) |
| No pregnancies | 57 (35) | 9 (12) | — | 58 (28) |
| pregnancies | 107 (65) | 65 (88) | 41 (100) | 149 (72) |
| No pregnancies (%) | ||||
| <6 | 98 (92) | 52 (80) | 19 (46) | 136 (91) |
| ≥6 | 9 (8) | 13 (20) | 22 (54) | 13 (9) |
| Age at 1st pregnancy, year, No (%) | ||||
| Median, CI25–75% | 20 (19–23) | 20 (17–24) | — | 22 (19–33) |
| ≤19 | 37 (35) | 32 (49) | 7 (64) | 42 (28) |
| >19 | 70 (65) | 33 (51) | 4 (36) | 107 (72) |
| Oral contraceptive use | ||||
| Yes | 62 (38) | 22 (30) | 4 (36) | 60 (29) |
| No | 102 (62) | 52 (70) | 7 (64) | 147 (71) |
| Smoking | ||||
| Yes | 23 (14) | 15 (20) | 3 (27) | 20 (10) |
| No | 141 (86) | 59 (80) | 8 (73) | 187 (90) |
- a It was not possible to obtain data of: the level of education, the age at 1st sexual intercourse, the number of sexual partners, the age at 1st pregnancy, oral contraceptive use, and smoking habit of 30 of the women with cervical cancer.
In total, 13 high-risk HPVs types and 23 low-risk HPVs types were detected. The viral type could not be determined in 3% of the HPV positive cervical samples. HPV 16 was the most prevalent in all women with and without lesions, whereas, the remaining HPV types showed a difference in their distribution according to the cyto-histological diagnosis. In cervical cancer, the high-risk HPVs after HPV16 were HPV 18 (14.6%) and HPV 45 (12.2%), being HPV 16 and/or HPV 18 detected in 82.8% of women with cervical cancer. Considering only single infections, HPVs 16/18 were detected in 71.5% of women with cervical cancer. Among low-risk HPVs cases with single infections, the HPV types detected were HPV 42 (2.4%) and HPV 73 (2.4%). In women with high-grade squamous intraepithelial lesions, the second most prevalent viral type was HPV 31 (14.9%), followed by HPV 33 (12.2%). Infections with one low-risk HPV type (HPV 26, HPV 73, or HPV 89) were found in low frequencies (1.4% each). Finally, in women with low-grade squamous intraepithelial lesions, HPV 16 was followed by HPV 58 (9.1%) and HPV 31 (5.5%) (Table II).
| Variable | Low-squamous intraepithelial lesions | High-squamous intraepithelial lesions | Cervical cancer | Negative for squamous intraepithelial lesions | ||||
|---|---|---|---|---|---|---|---|---|
| Alla | Single typeb | Alla | Single typeb | Alla | Single typeb | Alla | Single typeb | |
| No total | 164 | 120 | 74 | 54 | 41 | 32 | 207 | 202 |
| High-risk HPV, No (%) | ||||||||
| 16 | 32 (19.5) | 21 (17.5) | 31 (41.9) | 22 (40.7) | 28 (68.2) | 19 (59.4) | 9 (4.3) | 8 (4.0) |
| 31 | 9 (5.5) | 5 (4.2) | 11 (14.9) | 8 (14.8) | 2 (4.9) | 2 (6.2) | 5 (2.4) | 5 (2.5) |
| 58 | 15 (9.1) | 8 (6.7) | 4 (5.4) | 4 (7.4) | 2 (4.9) | — | 5 (2.4) | 4 (2.0) |
| 33 | 7 (4.3) | 5 (4.2) | 9 (12.2) | 7 (13) | 1 (2.4) | 1 (3.1) | 1 (0.5) | — |
| 51 | 8 (4.9) | 2 (1.7) | 5 (6.8) | — | — | — | 1 (0.5) | — |
| 45 | 6 (3.6) | 2 (1.7) | — | — | 5 (12.2) | 4 (12.5) | 3 (1.4) | — |
| 18 | 6 (3.6) | 2 (1.7) | 1 (1.4) | — | 6 (14.6) | 4 (12.5) | — | — |
| 56 | 8 (4.9) | 3 (2.5) | 3 (4.1) | 2 (3.7) | — | — | — | — |
| 52 | 4 (2.4) | 2 (1.7) | 7 (9.4) | 2 (3.7) | — | — | 2 (1.0) | 1 (0.5) |
| 39 | 5 (3.0) | 2 (1.7) | — | — | 1 (2.4) | — | — | — |
| 59 | 3 (1.8) | — | 2 (2.7) | — | 1 (2.4) | — | 2 (1.0) | 2 (1.0) |
| 35 | 2 (1.2) | 2 (1.7) | 1 (1.4) | 1 (1.8) | — | — | — | — |
| Low-risk HPV, No(%) | ||||||||
| 42 | 15 (9.1) | 3 (2.5) | — | — | 6 (14.6) | 1 (3.1) | 2 (1.0) | 1 (0.5) |
| 81 | 12 (7.3) | 4 (3.3) | 1 (1.4) | — | 5 (12.2) | — | 4 (1.9) | 2 (2.0) |
| 6 | 9 (5.5) | 4 (3.3) | 2 (2.7) | — | — | — | 1 (0.5) | — |
| 66 | 9 (5.5) | 1 (0.8) | 2 (2.7) | 1 (1.8) | — | — | — | — |
| 53 | 8 (4.9) | 5 (4.2) | — | — | — | — | — | — |
| 11 | 5 (3.0) | 2 (1.7) | 2 (2.7) | — | — | — | 2 (1.0) | 2 (1.0) |
| 73 | 4 (2.4) | 1 (0.8) | 2 (2.7) | 1 (1.8) | 1 (2.4) | 1 (3.1) | — | — |
| 70 | 3 (1.8) | 3 (2.5) | 1 (1.4) | — | — | — | 1 (0.5) | 1 (0.5) |
| 40 | 2 (1.2) | 2 (1.7) | — | 2 (3.7) | — | 1 (3.1) | 1 (0.5) | |
| 26 | 1 (0.6) | — | 2 (2.7) | 1 (1.8) | — | — | 1 (0.5) | 1 (0.5) |
| 89 | 1 (0.6) | — | 1 (1.4) | 1 (1.8) | — | — | 1 (0.5) | 1 (0.5) |
| 55 | 1 (0.6) | — | — | — | 3 (7.3) | — | — | |
| 82 | 1 (0.6) | — | — | — | 1 (2.4) | — | 1 (0.5) | 1 (0.5) |
| HPV X | 17 (10.4) | 7 (5.8) | — | — | — | — | 8 (3.9) | 7 (3.5) |
| Indeterminate viral type | 4 (2.4) | 4 (5.4) | — | 1 (0.5) | ||||
- HPV, human papillomavirus.
- a Prevalence data contain HPV types detected in single and/or multiple infections.
- b The prevalence was calculated excluding the samples with multiple infections or positive for indeterminate viral type.
According to the cyto-histological diagnosis of 285 HPV positive women, a higher prevalence ratio of HPVs16/18 was observed with the severity of the cervical lesion, being a prevalence ratio of 3.96 95% CI (2.18–7.20) detected in women with cervical cancer. Among positive samples, 24.2% (69/285 women) harbored multiple HPV types. Multiple-type infections were more common in HPV positive women with squamous intraepithelial lesions than in those without lesions (Table III).
| HPV type | Low-squamous intraepithelial lesions, All(%)a, Prevalence ratio (95%CI) | High-squamous intraepithelial lesions, All (%)a, Prevalence ratio (95%CI) | Cervical cancer All (%)a, Prevalence ratio (95%CI) | Negative for squamous intraepithelial lesions, All (%)a |
|---|---|---|---|---|
| No Total | 130 | 71 | 41 | 43 |
| 16 | 32 (24.6), 1.18 (0.87–2.27) | 31 (43.7), 2.09 (1.1–3.95) | 28 (68.2), 3.26 (1.76–6.05) | 9 (20.9), |
| 16/18 | 38 (29.2), 1.40 (0.74–2.65) | 32 (45.1), 2.15 (1.14–4.06) | 34 (82.9), 3.96 (2.18–7.20) | 9 (20.9)b |
| Other high-risk types | 67 (51.5), 1.17 (0.80–1.70) | 42 (59.2), 1.34 (0.91–1.97) | 12 (29.3), 0.66 (0.37–1.19) | 19 (44.2) |
| Multiple infections | 40 (30.8), 3.31 (1.26–8.71) | 16 (22.5), 2.42 (0.87–6.77)c | 9 (22.0), 2.36 (0.79–7.07)d | 4 (9.3) |
- HPV, human papillomavirus; CI, confidence interval.
- a Prevalence data contain HPV types detected in single and/or multiple infections.
- b HPV 18 was not detected in women negative for squamous intraepithelial lesions.
- c All women presented multiple infections with at least one type of high-risk HPV.
- d All the multiple infections were accompanied with HPV 16.
DISCUSSION
HPV, the causative agent of cervical cancer, has a major social and public health impact on the female population of Paraguay due to its high morbidity and mortality. The present study provides further information about the distribution of HPV genotypes in Paraguayan women with different degrees of cervical lesions.
In agreement with previous studies, HPV 16 was the most prevalent type [Clifford et al., 2005; Smith et al., 2007; Bosch et al., 2008]. The HPV type remained undetermined in only 3% of the HPV positive cervical samples; these infections could be caused by viral types which are not included among the 37 reverse line blot hybridization probes.
The prevalence of HPV 16 in women negative for squamous intraepithelial lesions (4.3%) was higher than that estimated worldwide (2.6%) and Central and South America (3.1%; 95% CI 2.8–3.4) [de Sanjosé et al., 2007; Bosch et al., 2008]. Given the greater tendency of HPV16 to persist and induce lesions with higher risk of malignant progression than other high-risk HPVs, the predominance of HPV 16 observed in women negative for squamous intraepithelial lesions may partly contribute to the high incidence of cervical cancer in Paraguay [Castle et al., 2005; Kjær et al., 2010; Rodríguez et al., 2010].
The fact that HPV 16 (68.2%) and HPV 18 (14.6%) were detected in approximately 82% of women with cervical cancer is comparable with that found in two studies conducted in Paraguay, in which HPVs 16/18 were detected in 82% and 77.9% of the cases, respectively [Kasamatsu et al., 1996; Rolón et al., 2000]. These rates were higher than those found in a recent meta-analysis published by Li et al. [2011], where the proportion of women with cervical cancer associated with HPVs 16/18 was between 70 and 76% in all world regions. In the present study, considering only single infections, 71.9% of women with cervical cancer harbored HPV 16 (59.4%) or HPV 18 (12.5%). Therefore, this study has public health significance, since it may help to assess the potential impact of HPV vaccines and to confirm that the current vaccine formula would be able to prevent more than 70% of cervical cancer in Paraguayan women [Smith et al., 2007; Bosch et al., 2008]. In this series, HPV 45 was third in prevalence for cervical cancer. This agrees with that found by de Sanjose et al. [2010], who analyzed 3,404 cases of invasive cervical cancer of Central South America in Latin America.
In the present study, HPV 16 was detected in 41.9% of the cases with high-grade squamous intraepithelial lesions, in agreement with the global HPV16 prevalence in high-grade squamous intraepithelial lesions (45.4%), which ranges from 51.8% in Europe to 33.3% in Oceania. The prevalence of this HPV type in Latin America and the Caribbean countries has been observed to be of 39.6% [Bosch et al., 2008]. The following two most common types were HPV 31 (15%) and HPV 33 (12%). In a meta-analysis that included 7,094 women from 38 countries, HPV 31 (8.7%) and HPV 33 (7.3%) were the second and third, respectively, in the worldwide prevalence, with regional variations, and HPV 58 and HPV 18 occupied the second and third place in Latin America and the Caribbean countries [Smith et al., 2007; Bosch et al., 2008]. Therefore, the results of this study are consistent with those observed worldwide, but not with those detected in Latin America and the Caribbean, suggesting that further research is needed at the regional level, and that variations in the distribution of HPV genotypes can occur, especially in cancer precursor lesions.
In low-grade squamous intraepithelial lesions, HPV 16 was also the most frequent type (19.5%), a rate which was within the range detected in a worldwide meta-analysis (16.3% in Africa to 32.6% in Asia) by Clifford et al. [2005]. In the present study, HPVs 58/42 (9.1% each) and HPV 81 (7.3%) were the second and third most important HPV types, while in Clifford's worldwide meta-analysis, the second, third, and fourth places were occupied by HPV 31 (11.5%), HPV 51 (10.6%), and HPV 53 (10.2%), respectively; in South and Central America, the second place was occupied by HPV 33 (11.8%). These differences may be due to the variety of typing methods applied, being many of them unable to identify a large proportion of multiple infections [Clifford et al., 2005]. Therefore, a greater number of studies in South American countries using similar methods are needed to obtain more solid conclusions on the distribution of HPV.
In this study, multiple infections were detected in 24.2% of HPV positive women. Several studies have reported variable frequencies of multiple infections, from less than 10% to above 80%. Remarkably, these disparities seen in earlier works may be due to various factors such as geographic and ethnic parameters and sexual behavior, being the sensitivity of the techniques used for HPV genotyping one of the most important ones [Trottier et al., 2006; Spinillo et al., 2009; Schmitt et al., 2010]. The combination of the MY PCR-restriction fragment length polymorphism and GP5+/6+ PCR-reverse line blot hybridization assays used in the present study is a good alternative since it allows detecting new types of HPV as well as multiple HPV infections [van den Brule et al., 2002].
Multiple-type infections were more common in HPV positive women with squamous intraepithelial lesions than in those without lesions. Several studies have found that multiple infections are more common in women with squamous intraepithelial lesions (50%) than in those negative for squamous intraepithelial lesions (12.1%) [Herrero et al., 2005; Kjaer et al., 2008]. More recently, Quint et al. [2009] have suggested that although there are many viruses that can infect the same anatomical site, only one genotype would be responsible for each lesion.
In summary, this study provides robust information about the molecular epidemiology of HPV in Paraguayan women. It demonstrates a higher prevalence of HPV 16 and other high-risk HPVs types in women both with and without cervical lesions, which could partly explain the high incidence of cervical cancer in Paraguay. Finally, these data can also contribute to the control of the HPV related-diseases, providing relevant information for the implementation of prevention programs, including an appropriate introduction of vaccination and virological surveillance in the vaccine era [WHO, 2009].
Acknowledgements
We thank M. Samudio for her technical assistance in the statistical analysis. We also thank the specialized physicians; E. Segovia, Z. Benítez, M. González, S. Hamuy, and J. Bellasai (from the health centers from Paraguay) for their assistance in collecting biological specimens and all the women who agreed to participate in the study. This work was supported by Virology Section, Faculty of Sciences, University of the Republic (Uruguay); Oncogenic Virus Service, National Institute of Infectious Diseases (INEI) - ANLIS “Dr. Malbrán” (Argentina) and performed in the framework of activities of the PAHO HPV Regional Reference Laboratory (INEI-ANLIS Malbrán, Argentina), within the WHO HPV LabNet.
