Amplificación de genes tempranos del virus del papiloma humano para la detección de nueve genotipos en mujeres venezolanas.

Resumen. La genotipificación de virus del papiloma humano (VPH) por métodos moleculares puede proveer información valiosa para el monitoreo y seguimiento de la infección cervical. Se estudiaron muestras cervicales obtenidas a partir de 250 mujeres, en quienes se realizó, simultáneamente, evaluación citológica y colposcópica. Un ensayo de PCR, en formato Nested-múltiple para las amplificación de la región temparana E6/E7, fue realizado para la detección y genotipificación viral. La infección por VPH se detectó en 26,0% de las muestras, de las cuales, un 98,46% fue positivo para al menos uno de los genotipos probados; los VPH de alto riesgo se identificaron en un 98,44%. Los genotipos más frecuentes fueron VPH18 con un 76,92%, y VPH16 con un 36,92%, ya sea como infecciones individuales o múltiples. En cuanto a la edad, estas infecciones fueron más frecuentes en mujeres menores de 35 años, con un 64,7%. Los resultados citológicos mostraron que 16,92% (11/65) de las muestras cervicales tenían cambios sugestivos de infección por VPH; mientras que la evaluación colposcópica fue sugestiva de la infección por VPH en 47,69% (31/65) de las muestras ADN-VPH positivas. Se determinó una elevada frecuencia de los genotipos de VPH de alto riesgo, particularmente VPH18, solo o en infecciones múltiples. Los hallazgos colposcópicos mostraron un elevado valor predictivo para el diagnóstico de la infección por VPH. Los resultados reflejan que más del 50% de las pacientes VPH positivas tenían colposcopía y/o citología normal, evidenciando la importancia de incluir las pruebas de detección e identificación de VPH en la evaluación ginecológica de rutina.

Palabras clave:  VP H; genotipos; cáncer cervical.

Recibido: 10-12-2012 Aceptado: 17-10-2013

INTRODUCTION 

Cervical carcinoma (CC) represents the second worldwide most frequent cancer in women, being the leading cause of cancer deaths among women in Latin America and the Caribbean (1). In Venezuela, CC is the second cause of mortality in women whose ages vary from 15 to 75 years old (2). In Mérida state, western Venezuela, morbidity and mortality frequency of CC has shown to be as high as in the rest of the country (3, 4). It is very well established today that infection with specific high-risk HPV genotypes are necessary, but not sufficient, to virtually cause all CC, and to be most likely responsible for other anogenital neoplasm and oral squamous cell carcinomas; thus, high-risk HPV genotypes emerge as one of the most important infectious carcinogens in humans (5). 

Clinical trials based on the natural history of human papillomavirus (HPV) infection have demonstrated that most infections are usually asymptomatic and spontaneously cleared by the immune system, or produce transient minor lesions (6, 7). Nevertheless, a little fraction of untreated viral infections may persist and progress to different stages of what it’s known as cervical intraepithelial neoplasia (CIN), a cancer precursor. Without intervention, a considerable fraction of women with CIN 3 can develop cancer, in about one to two decades after the initial infection. The DNA from high-risk HPV genotypes can be detected in virtually all cases of invasive cancer (8, 9). Therefore, women infected with high-risk HPV have a higher risk for the development of CC than those infected with low-risk HPV or women not infected (6, 10). 

CC screening based on the Papanicolaou (Pap) smear test has been credited with a significant reduction in the incidence of this disease (10). Nevertheless, studies performed up to date, have concluded that cervical cytology is moderately accurate and that the average sensitivity to detect CC or precancerous lesions was considerably lower than generally believed; in addition, its sensitivity has shown to be highly variable among different countries (11). 

Epidemiological and clinical studies had detected high-risk HPV between 95.0-99.0% of CC, which 70.0% of them are due to HPV16 and HPV18, representing major risk factors related with the progression of cervical lesion to CIN and/or CC (8, 12). Considering the central role played by infection with high-risk HPV genotypes in the etiology of CC, DNA testing for identification of these genotypes has been introduced for early diagnosis (13). Because HPV testing in combination with cytological evaluation, has proven to have higher sensitivity than cytological test alone for detection of CIN 3 and CC, the International Agency for Research on Cancer considered an acceptable alternative to Pap smears/cervical cytology for CC screening (14). 

HPV genotyping is also a powerful tool as a primary evaluation test for viral infection and determination of carcinogenic HPV genotypes persistence, which could be used for specific persistent high-risk-HPV infection (13,15). Moreover, HPV genotyping is needed to predict how prophylactic vaccination of young women could affect the secondary prevention of cervical cancer (16). The aim of this study was to detect and genotype HPV, from cervical samples of Venezuelan women, along with the analysis of colposcopy and cytology examination. 

MATERIALS AND METHODS 

Study population 

Cervical samples were obtained from 250 sexually active women (15 to 69 years-old), who attended a gynecology outpatient clinic at the Department of Obstetrics & Gynecology (Gyn/OB)-Los Andes University Hospital Autonomic Institute (IAHULA) in Mérida State, western Venezuela, between August 2008 and August 2011. 

For the development of the research, randomly selected women were included, attending gynecology when undergoing routine screening. Likewise, the study excluded all women who, by the time of the interview and sampling, had any genital bleeding because of normal menstruation, metrorrhagia or menorrhagia, and women who showed changes in the cervix caused by surgical procedures that prevented proper sampling. Individuals and parent/guardian consent were obtained from each participant following the Helsinki and WHO ethics principles in human research (17). This study was also conducted in compliance with the local institutional ethical board. 

Practitioners were instructed to obtain the sample from the cervix transformation zoneusing a DNA collection device (Digene® Corporation, Gaithersburg, MD, USA). Samples were stored at 4°C and transported to the Microbiology and Public Health Laboratory, University of Los Andes of Mérida, Venezuela (ULA) within the same day of sample collection. Simultaneously, Pap-smear sampling and colposcopy were performed. 

Colposcopy 

All study participants underwent a colposcopic examination of the cervix, vagina, and vulva by the Gyn/OB specialists. Practitioners examined the cervix by inserting an unlubricated bivalve vaginal speculum, with the help of a halogen focus lamp; immediately, lugol iodine solution was applied to the cervix under direct vision. Lesions in the transformation zone (TZ) were assessed by applying 5.0% acetic acid and iodine solution under ×8 to ×12 magnification. An international nomenclature (IFCPC) was used to classify the colposcopic patterns (18).

Cytology 

Pap-smear analysis of cervical cells was carried out by medical pathologists (Department of Pathology, Medicine School, ULA), adopting the conventional Bethesda terminology/classification (19, 20). Negative and benign changes were kept as originally categorized. Atypical changes were classified as atypical squamous cells of undetermined significance (ASC-US) and atypical squamous cells, cannot exclude HSIL (ASC-H). Mild dysplasia was classified as low-grade intraepithelial lesions (LSIL) of squamous or glandular type; moderate to severe dysplasia were classified as high-grade intraepithelial lesions (HSIL) of the squamous or glandular type.

DNA-HPV Specimen processing

Cells kept in the DNA collection device were centrifuged and two aliquots of 700 µL each were used for DNA isolation in silicon-based column technology using the QIAamp DNA Mini Kit (QIAGEN®, Hilden, Germany). Purified DNA was eluted in 100 µL of buffer in accordance with the manufacturer recommendations. DNA-quantification was performed at 260nm wavelength (UV1101/1101T, Biotech, Cambridge, UK) and purified DNA was stored at –20°C until processing.

Amplification of early genome regions (E6/E7) by PCR 

DNA extraction processes and all pre- and post-PCR procedures were carried out in separate rooms and cabinets under the most stringent conditions. Buffer and blank controls were always included for the extraction protocol to obtain sufficient number of negative controls in order to monitor contamination. 100 ng of DNA were used for each PCR assay in a final volume of 25 µL. 

A Nested-PCR-Multiplex assay was used for amplification of conserved early region from HPV genome. The first PCR amplified a segment of a 630bp-length of E6/E7 gene. These regions were amplified using the consensus sequences GP-E6 3F/5B/6B (21), and 10 pmol of primers PC04/GH20, with an initial denaturation step of 15 min at 95°C, followed by 40 cycles (1 min of denaturation at 94°C, annealing at 55°C/1 min and an extension step at 72°C/1 min) with a final extension step of 72°C/10 min program. A commercially available positive control was used in each PCR assay (HPV-4011, Maxim Biotech, USA). 

In the second PCR, which allows the genotyping of HPV in a multiplex format, 1 microliter of the first PCR product was used as template. Amplicon sizes varied from 151 to 457bp for HPV16-18-31-45 (Coctail I) and 33-6/11-58-52 and 56 (Coctail II) (21). Commercially available positive controls were used in each PCR assay: HPV-C001 (HPV16/18), HPV-4011 (HPV18), HPV-4009-33 (HPV33) HPV-4012-11 (HPV11) (Maxim Biotech, USA). These reactions were performed in a program of 35 cycles (94°C/30 second, 56° C/30 second and 72°C/45 second) with a final extension step of 72°C/4 min; adapted after Sotlar et al. (21). 

All PCRs were performed on an ABI 2400 instrument (Applied Biosystems) and amplification products were visualized on 2.0% agarose gels containing 10 µL of ethidium bromide/100 mL agarose, under UV light (UV transilluminator, Vilber Lourmat, France).

Statistical analysis 

Patient information was collected in a formulary designed for this objective. Data base and statistical analyses were performed using EPI Info 2008, version 3.5.1. A descriptive analysis of the variables was firstly carried out. The distribution of cervical results was determined by evaluating the proportion of normal and abnormal results. Chi-Squared test and P value were used to assess HPV DNA infection rate in each age group and to establish relationships between HPV DNA infection and colposcopic and/or cytological atypia. Any difference was considered statistically significant when P<0.05. 

RESULTS 

Cervical samples from 250 women, between 15 and 69 years of age (the median age of women was 33.74 years with standard deviation of 10.45 years), were tested for HPV infection. Viral detection was performed by PCR assay for E6/E7 regions. HPV genotypes were identified by a Nested-PCR-Multiplex assay for amplification of early viral genome region; in Fig. 1 is showed PCR amplification products of HPV E6/E7 region, then visualized on 2.0% agarose gels. Taken together, 26.0% (65/250) of samples was positive for HPV; from which, 98.46% (64/65) were positive for at least one of the genotypes assayed. The distribution of the HPV genotypes in the 65 HPV positive samples is summarized in Fig. 2: HPV18 (50/64) along with HPV16 (24/64) were the most frequent genotypes found in this study; 1 sample was negative when evaluating genotypes. It should be noted that the high-risk HPV genotypes were identified in 98.44% (63/64) positive samples.

Twenty seven patients (41.53%) were infected with one single HPV genotype, while 56.92% (37/65) showed multiple infection with two or more HPV genotypes, being the combination of HPV18/6/11 (14/65) the most frequent. Fig. 3 shows the distribution of HPV infection by groups of age; the prevalence of HPV infections was higher (60.0%), in women under 35 years old than in older women participating in the present study; statistical analysis did not show any significant association between the age of patients and HPV detection (p=0.2635; square-Chi Test).

Table I shows the varying degrees of dysplasia and the histological findings during colposcopy examination versus the HPV detection and genotyping. Colposcopy evaluation was suggestive of HPV infection in 47.69% (31/65) positive samples by HPV testing, while the remaining 52.31% HPV positive samples showed normal colposcopic findings. HPV18 was the most frequent genotype identified in women with positive colposcopy, both as individual (9/27) and multiple infection (12/37). There was not any significant association between Schiller test and the most frequent HPV genotypes: HPV18 (p=0.8182; square-Chi Test) and HPV16 (p=0.4080; square-Chi Test).

TABLE I

CORRELATION BETWEEN RESULTS OF THE COLPOSCOPIC DIAGNOSIS AND HPV GENOTYPES IDENTIFIED IN CERVICAL SAMPLES OF WOMEN ATTENDING THE GYN/OB OUTPATIENT PUBLIC CLINIC [LOS ANDES UNIVERSITY HOSPITAL], MERIDA STATE, VENEZUELA 

^¥HPV diagnosis by PCR-E6/E7 (GP E6/E7).

8.0% (20/250) of Pap smears showed cytological changes suggesting HPV infection, from which 95.0% (19/20) was described as LSIL. In samples from patients positive for HPV DNA, Pap-smear examination showed that 10 patients had LSIL (Table II), from which 7 were positive for HPV18. Statistical analysis did not show any significant association between the identification of HPV18 and the cytological findings associated to HPV infection (p=0.3598; square-Chi Test). Overall, colposcopy findings showed the best predicted value for diagnosis of HPV infection compared to Pap-smear examination.

TABLE II

CORRELATION BETWEEN THE RESULTS OF THE CYTOLOGICAL DIAGNOSIS AND HPV GENOTYPES IDENTIFIED IN CERVICAL SAMPLES OF WOMEN ATTENDING THE GYN/OB OUTPATIENT PUBLIC CLINIC [LOS ANDES UNIVERSITY HOSPITAL], MERIDA STATE, VENEZUELA 

^¥HPV diagnosis by PCR-E6/E7 (GP E6/E7). ^—ASC-H: Atypical Squamous Cells cannot exclude High-grade Intraepithelial Lesion (HSIL); ^¥LSIL: Low-grade Intraepithelial Lesion. 

DISCUSSION

This study was designed to investigate the presence of HPV infection and to determine the respective genotype from cervix with or without lesions in women attending an outpatient clinic in Mérida, western Venezuela. The overall percentage of HPV positivity was 26.0%, higher than that found in the general population, which has been estimated between 9.0 and 13.0% worldwide (8). Regarding other reports from Venezuela, the findings of this study are consistent with that conducted by Reigosa et al. (22) in Carabobo state, in which 58 asymptomatic women for cervical pathology studied, 35.50% were HPV positive. However, our results differ from reports of Caracas (23-25) and Sucre (26), in women with diagnosis of impaired cervical cytology or histopathology, which showed higher percentages (between 43.0-98.70%) of HPV infection prevalence.

The high percentage of HPV positive samples found in our study may be due to the nature of genital HPV infections, which are mainly transmitted through sex, an event that promotes its high transmission in sexually active women and men. It must be considered that most of these infections are transient, and therefore could be eliminated by the immune system in a relatively short period of time without apparent clinical consequences (5).

It is important to emphasize the elevated frequency of high-risk HPV genotypes identified in this study, mainly HPV18 and/or HPV16. This distribution is consistent with previously published data in Merida, Venezuela, in which the predominance of HPV18 infections over other genotypes was evidenced (3); similarly, two recent studies developed in samples from patients diagnosed with cervical cancer have reported higher frequency of HPV18 over other genotypes identified (27, 28). On the other hand, the results of this study differ from reports previously published around the world, in which HPV16 has been consistently more identified. This variation can be explained by the fact that HPV genotypes reported in different regions worldwide may vary both in type and relative incidence (15, 29-31). 

Investigations have shown that HPV16 and HPV18 infections preferentially progress to CC more readily than those due to other high-risk genotypes (8-9). Thereby, HPV testing have demonstrated to be a strong predictor of risk in the development of cervical neoplasia, supporting the hypothesis by which HPV testing effectively stratifies patients according to cancer risk (32). Moreover, HPV16 and 18 have been the most frequent genotypes identified in cervical smears from women harboring CC (8). 

Among HPV-positive samples identified in this study, multiple high-risk genotype-infections were more frequent than single infections, showing the relevance of HPV genotype testing in patients with a positive report for HPV detection. Multiple HPV infection increases the probability of harboring a highly oncogenic HPV genotype (7). These co-infections seem to have a higher occurrence than expected by chance, suggesting a higher likelyhood of synergy amongst the co-infecting HPV genotypes (33). 

HPV16/18 was the second more frequent combination in the present investigation. Previous publications concerning the etiologic role to HPV genotypes in neoplasic lesions and CC, have showed an elevated risk of progression of incident HPV16/18 infections to CIN and then to CC (34, 35). Similarly, case-control studies have yielded more important relative risks of invasive CC of 100 to 500 among women with these genotypes (8). 

Epidemiologic studies have shown that sexually active young women at early ages are at greater risk of acquiring HPV (36, 37). The present study showed a prevalence of HPV infections higher in women whose ages were less than 35 years than in older women. This is a very relevant finding considering that HPV16 and HPV18 positive invasive cancers are more prevalent in younger women than invasive cancers related to other carcinogenic non-HPV16/18 genotypes. Even if HPV infections are very common among young women, and although they usually clear within one or two years (38), infections associated to cervical changes have been reported in 80.0% of women under 25 years of age, 66.0% in the age group of 26 to 35 years, and 51.0% in the group of 36 to 45 years old (37). In this study, the HPV prevalence declined in an age-related fashion; similar findings were presented in an epidemiological study of global data on age-specific prevalence of HPV infection overall, by Smith et al. (38), who observed a substantial decrease in HPV prevalence in older women. 

Colposcopy evaluation of the population studied here was suggestive for HPV infection in a higher range than Pap smears, showing a better predicted value for diagnosis of HPV infection. In a meta-analysis developed by Mitchell et al. (39), a mean sensitivity of 96.0% and a mean specificity of 48.0% for colposcopy to detect histological abnormalities, including CIN 1-3 and cancer were found.

It has been recognized that among women who are HPV positive but cytological negative, about 60.0% become HPV negative within six months. However, even in the presence of negative cytology, older women who are HPV positive have a greater risk of developing CIN 3 within 10 years, compared with younger women (21.0 vs 13.6%, respectively) (10). In this study, HPV18 was the most frequent genotype identified in patients with LSIL, the majority of these women were younger than 35 years old. It has been suggested that women with LSIL, positive for HPV16 and HPV18, have a higher risk to progress to CC than those positives for other HPV genotypes; together, HPV16 and HPV18 account for 35.0% of HPV positive LSIL but nearly 70.0% of worldwide CC (30); so that the specificity of HPV detection tests can be enhanced by setting an age limit at 35 years in cases of LSIL (40). 

The variation of HPV distribution in LSIL and cancer, suggests that the distinction of high-risk HPV genotyping has the potential to improve the management of LSIL in clinical practice (29, 41). Thus, performing HPV testing and cytology screening simultaneously should improve the detection of prevalent disease; additionally, if Pap testing is negative in women infected with HPV, then there is future low risk for cervical neoplasia, so the screening interval for women with negative tests could be lengthened, and the subgroup of patients with positive results could be targeted for more frequent surveillance (32, 42). 

In conclusion, the results obtained in this study showed a high frequency of HPV infection with specific HPV oncogenic-risk genotypes in women from Mérida state. This should be a key point when considering the implementation of the vaccination system against HPV infection,. Colposcopy should be considered a priority in women’s gynecological routine examination, together with HPV testing; as well as prevention plans extended to men, aiming the education of sexual and reproductive health items in order to modify sexual practice and to avoid major risk factors associated to HPV infection. 

ACKNOWLEDGMENTS

This study was supported by grants FONACIT-G-2005000408; Proyecto Misión Ciencia LUZPROLAB N° 2007001088 and CDCHTA-ULA N° M-945-08-07-A and M-946-08-07-C.

The authors thank, Dr. Pierina Petrosino and Dr. Asmiria Sotolongo from the Pathology Laboratory, Dept. de Anatomía Patológica, ULA, Mérida. The authors also appreciate the professional services of the gynecologists who gave time for this project [Dr. Nazira Monsalve and Dr. Manuel Santos from IAHULA].

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