|Year : 2013 | Volume
| Issue : 2 | Page : 138-141
Distribution of human papillomavirus genotypes in women with cervical alterations from north Argentina
GD Deluca1, HM Marín2, NS Blanco1, JA Basiletti3, JV González3, AL Merino1, MA Picconi3
1 Department of Clinical Microbiology, Laboratory of Molecular Application, Faculty of Medicine, Northeast National University, Corrientes, Argentina
2 Department of Virology, Laboratory of Molecular Biology, Institute of Regional Medicine, Northeast National University, Chaco, Argentina
3 Department of Virology, Oncogenic Virus Service, National Reference Laboratory on Human Papillomavirus, National Institute of Infectious Diseases "Carlos G. Malbrán", Buenos Aires, Argentina
|Date of Submission||16-Aug-2012|
|Date of Acceptance||02-May-2013|
|Date of Web Publication||19-Jul-2013|
G D Deluca
Department of Clinical Microbiology, Laboratory of Molecular Application, Faculty of Medicine, Northeast National University, Corrientes
Source of Support: None, Conflict of Interest: None
Background: Cervical cancer remains a major public health problem in northern Argentina, showing the highest mortality rate in the country (approximately 22 cases/100000 women). Objective: The aim of this study was to provide epidemiological data on the prevalence and type distribution of human papillomavirus (HPV) in women with pre-neoplastic lesions before the massive introduction of HPV vaccination in the country. Materials and Methods: Exfoliated cervical cells were collected to screen for HPV using the widely known MY09/11 PCR, followed by the restriction fragment-length polymorphism (RFLP) technique from a total of 714 women with previously diagnosed atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LG-SIL) and high-grade squamous intraepithelial lesion (HG-SIL). Results: Overall HPV prevalence was 48.2% in ASCUS (24 different HPV types detected), 66.5% in LG-SIL (37 HPV types detected) and 82.6% in HG-SIL (16 HPV types detected). HPV-16 was the most prevalent type among all cases. With respect to multiple HPV infections, 9.6% were observed in ASCUS, 14.3% in LG-SIL and 11.4% in HG-SIL. Conclusion: The major strength of our study is the assessment of a large series of women with cytological alterations in this region. The information attained will be useful as a regional baseline for future epidemiological vigilance, in the context of the national HPV vaccination program.
Keywords: ASCUS, cervical infection, HPV, HG-SIL, LG-SIL
|How to cite this article:|
Deluca G D, Marín H M, Blanco N S, Basiletti J A, González J V, Merino A L, Picconi M A. Distribution of human papillomavirus genotypes in women with cervical alterations from north Argentina. Indian J Med Microbiol 2013;31:138-41
|How to cite this URL:|
Deluca G D, Marín H M, Blanco N S, Basiletti J A, González J V, Merino A L, Picconi M A. Distribution of human papillomavirus genotypes in women with cervical alterations from north Argentina. Indian J Med Microbiol [serial online] 2013 [cited 2019 Sep 17];31:138-41. Available from: http://www.ijmm.org/text.asp?2013/31/2/138/115228
| ~ Introduction|| |
Although infection with oncogenic human papillomavirus (HPV) genotypes is frequent among sexually active women, ,, most cases are self-limiting; the development of malignant cervical lesions only occurs in a small proportion of infected women who harbour persistent infections with oncogenic types. ,,
Argentina is characterised by the multi-ethnic composition of its population. This unusual situation explains the existence of large discrepancies in socioeconomic development, cultural characteristics and sexual behaviour among communities from different regions of the country.  The annual mortality rate for cervical cancer (CC) is 7.8/100000 women,  but this rate shows a significant variation among different regions, from 7 cases out of 100000 women in the metropolitan area of Buenos Aires city to 17-22 cases out of 100000 women in northeast Argentina. This study is intended to provide valuable baseline knowledge about the prevalence and type distribution of HPV in pre-neoplastic lesions in northern Argentina prior to the massive introduction of HPV vaccination. 
| ~ Materials and Methods|| |
A total of 714 sexually active women, aged 15-73 years and with previously diagnosed atypical squamous cells with undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LG-SIL) and high-grade squamous intraepithelial lesion (HG-SIL) were randomized and included in this study to analyse HPV genotype distribution. All samples were collected between January 2007 and December 2011 from different gynaecological clinics of Chaco Province (Argentina).
The study was reviewed and approved by the Bioethics and Research Committee of the School hospital 'General José F. de San Martín', Corrientes (Argentina).
Exfoliated cervical cells were collected from all women by scrapping the uterine ecto-endo cervix with a cytobrush. Cells were washed in sterile buffered saline, and the final cellular pellet was kept at −70°C until DNA extraction.
DNA was obtained by treating pellets with 400-700 μl of homogenization solution (2% cetyltrimethylammonium bromide-CTAB, 1.4 M NaCl, 0.2% β-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HCl pH 7.5), extracting the DNA using the chloroform: Isoamyl alcohol (24:1) method and then precipitating with absolute ethanol. Finally, the DNA pellet was dissolved in 50-100 μl of double distilled sterile water. All samples were checked to assess the quality and integrity of the DNA by amplifying a known region of the human ß-globin gene (data not shown). Samples that were negative for the ß-globin test were considered inadequate and discarded.
HPV DNA amplification and typing were conducted using the widely known MY09/11 (Eurofins MWG Operon, USA) PCR followed by the restriction fragment-length polymorphism (RFLP) technique, as previously described. , HPV-positive samples that were difficult to analyse by PCR-RFLP, either due to the presence of nonspecific amplimers or weak amplicon signals on agarose gels, were reanalysed in the Oncogenic Virus Service Lab of the 'Carlos G. Malbrαn' Institute (Buenos Aires, Argentina) by PCR and reverse line blot, as previously described. ,
| ~ Results|| |
A total of 714 ecto-and endo-cervical samples corresponding to 110 ASCUS, 535 LG-SIL and 69 HG-SIL cases were analysed. Overall, HPV prevalence was 48.2% in ASCUS, 66.5% in LSIL and 82.6% in HSIL. In ASCUS cases, it was possible to identify 23 different HPV viral types, including 12 high-risk (HR) genotypes (involved in 73.6% of positive cases), 6 low-risk (LR) types and 5 genotypes of undetermined risk (UR). In the LG-SIL cases, 40 genotypes were detected, including 18 types considered HR (involved in 78.1% of positive cases), 7 of LR and 15 of UR. Finally, 16 HPV types were identified from HSIL cases, including 10 of HR type (involved in 91.2% of positive cases), 2 of LR type and 4 of UR. [Figure 1], [Figure 2] and [Figure 3] shows the individual HPV type frequencies in ASCUS, LSIL and HSIL patients, respectively; in each graphic, the five most prevalent genotypes are also highlighted. As can be seen, the only HPV types that were present among the five most frequent across all the cervical disease grades considered were HPV-16, HPV-53 and HPV-58. [Figure 4] shows HPV prevalence by risk-group as compared with HPV-16.
|Figure 1: Frequency of HPV genotypes in ASCUS samples of women from different communities of Chaco, Argentina. ASCUS: Atypical squamous cells of undetermined signifi cance, Other HPVs: HPV-31, HPV-33, HPV-40 and HPV-61 (1.82% each one); HPV-26, HPV-34, HPV-39, HPV-51, HPV-52, HPV-56, HPV-66, HPV-67, HPV-72, HPV-84, HPV-100 and HPV-106 (0.91% each one)|
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|Figure 2: Frequency of HPV genotypes in LG-SIL samples of women from different communities of Chaco, Argentina. LG-SIL: Low-grade squamous intraepithelial lesion, Other HPVs: HPV-66 (5.42%); HPV-18 and HPV-61 (3.18% each one); HPV-11 (2,99%); HPV-33, HPV-51 and HPV-52 (2.43% each one); HPV-59 (1.68%); HPV-56 (1.31%); HPV-45 and HPV-70 (1.12% each one); HPV-35, HPV-39 and HPV-81 (0.93% each one); HPV-62, HPV-84 and HPV-100 (0.75% each one); HPV-26, HPV-42, HPV-44, HPV-55, HPV-73, HPV-83 and HPV-8304 (0.56% each one); HPV-68, HPV-69, HPV-71 and HPV-106 (0.37% each one); HPV-9, HPV-34, HPV-64, HPV-67, HPV-82, HPV-86 and HPV-8061 (0.19% each one)|
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|Figure 3: Frequency of HPV genotypes in HG-SIL samples of women from different communities of Chaco, Argentina. HG-SIL: High-grade squamous intraepithelial lesion, Other HPVs: HPV-6, HPV-35, HPV-45 and HPV-59 (2.90% each one); HPV-66, HPV-67, HPV-72, HPV-84, HPV-100 and HPV-106 (1.45% each one)|
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|Figure 4: HPV Frequencies of high-risk group and low-risk group as compared with the frequency of HPV-16 alone in women from Chaco, Argentina. ASCUS: Atypical squamous cells of undetermined signifi cance, LG-SIL: Low-grade squamous intraepithelial lesion, HG-SIL: High-grade squamous intraepithelial lesion|
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With respect to multiple HPV infections (cases with 2 to 5 genotypes involved), 10% were observed in ASCUS, 14.8% in LG-SIL and 11.6% in HG-SIL. In HG-SIL, all HPV multiple infections included at least one HR type; in LG-SIL, the HR types were present in 93.7% of the cases.
Finally, [Figure 5] presents the comparative frequencies of negative and positive cases (single and multiple HPV infections separately) for each grade of cervical disease.
|Figure 5: Distribution of single and multiple infections by cervical alteration in women from Chaco, Argentina. ASCUS: Atypical squamous cells of undetermined signifi cance, LG-SIL: Low-grade squamous intraepithelial lesion, HG-SIL: High-grade squamous intraepithelial lesion|
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| ~ Discussion|| |
This is the first study to analyse the northern Argentinean population in order to obtain data on HPV type distribution across ASCUS, LG-SIL and HG-SIL cervical diseases. , The major strength of our present study is the assessment of a large series of women with cytological alterations in this region.  The information attained will be useful as a regional baseline for future epidemiological vigilance in the context of the national HPV vaccination program. 
HPV-16 was found to be the most frequently detected genotype regardless of the cervical alteration considered, , but its prevalence increases notoriously in the context of HG-SIL disease with a HG-SIL/ASCUS ratio of 2.15. As in other studies, this is consistent with the observation that HPV-16 notoriously increases its presence in true pre-neoplastic lesions.  As expected, while the prevalence of the HR-HPV group (excluding HPV-16) increase with increasing severity of cervical disease, we found the prevalence of the LR-HPV group was maintained without much variation. ,, Also, the differences in HR-HPV type distribution between HPV-positive ASCUS and LG-SIL were not as evident as between these lesions and HG-SIL. This suggests that cytological diagnoses of ASCUS or LG-SIL are of limited utility for risk-stratification of HPV-positive women. Similarly, the diversity of HPV-types decreased from 23 to 40 genotypes in ASCUS and LG-SIL to 16 different types in HG-SIL; a similar situation has been presented in other series worldwide. In addition, HPV-53 and HPV-58 were fairly common in HPV-positive women across all the cervical alterations analysed.  This observation is in agreement with previous studies carried out by the same research group that had already noted a high prevalence of these two types in north Argentina. , There is not much explanation for this observation, but several studies around the world reflect that, in certain regions, some HPV genotypes are more frequents for preneoplastic lesions; however, for CC, the HPV genotype distribution is similar worldwide.
The presence of multiple infections was within the expected range of values and without much variation among ASCUS, LG-SIL and HG-SIL (10-15%). , the extent and importance of multiple HR-HPV infections in the progression of CC and its management remain unknown and require more study. 
HPV-16 and HPV-18 were the two types that suffered the greatest increase in frequencies in this study, with a notorious presence in HG-SIL. Although this phenomenon has been presented in almost all series worldwide, the major question that remains unsolved is what will occur with regard to the distribution of the other types of HPV once the vaccine hypothetically eradicates HPV-16 and HPV-18 transmission. There have been several hypotheses on this topic, but more studies like the present one are necessary in different regions of the country to obtain sufficient data for subsequent evaluation.  In conclusion, it would be of interest to observe the distribution of HPV types once the actual vaccination program has broad coverage in order to analyse the real impact of the actual vaccine formula. 
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]