We have studied 498 women seeking for cervical Papanicolaou examination and attending three public health centers of Durango City, Mexico. One hundred and sixty six women from each health center were included in the study. Health centers were a University outpatient clinic (Institute for Scientific Research), a hospital of the Instituto Mexicano del Seguro Social (IMSS), and a hospital of the State Health Office (Secretaria de Salud). All 498 participants were enrolled consecutively from July to December 2003.
Socio-demographic data including age, birth place, residence place, marital status, occupation, age at start of active sexual activity, number of sexual partners, use of condom, and history of smoking from all 498 women studied were obtained.
Cervical papanicolaou cytology
A cervical smear was obtained from each participant by using Ayre spatula and cytology brush. Papanicolaou smears were evaluated according to the Bethesda diagnosis criteria .
HPV DNA PCR and HPV genotyping
A second cervical specimen was obtained with the aid of a cervical brush for HPV DNA PCR and HPV genotyping. DNA extraction was performed by using DNAzol (Invitrogen Inc. Carlsbad. CA, USA). With this method, good quality DNA was obtained inferred by the MW>20 kb for the genomic human DNA analyzed by means of 1% agarose electrophoresis, stained with ethidium bromide. In addition, the latter was confirmed by obtaining positive results for beta globin in all samples. HPV DNA PCR was carried out by using MY09/11 primers as described elsewhere . The MY-PCR system has shown a sensitivity of 90% in samples containing multiple HPV types . Concentrations of Mg++, primers, Taq polimerase, dNTP, tetramethylene sulfoxide, and DNA in each 50 μl reaction were 2 mM, 0.2 μM, 2 units, 0.2 mM, 2%, and 2 μg. DNA concentration used was determined based on preliminary analysis of DNA concentrations. Positive results were obtained with as low DNA concentration as 5 ngr. Thirty nine cycles of 94°C for one minute (denaturation), 55°C for two minutes (annealing), and 72°C for two and a half minutes (extension) were performed. PCR products were run in 2% agarose electrophoresis, stained with ethidium bromide, and visualized with the aid of UV light. Positive HPV DNA PCR samples were further analyzed for HPV genotyping. The presence of 3 genotypes were explored, namely HPV genotypes 16, 18 and 33. HPV genotyping PCR was carried out by using specific primers (Takara Mirus Bio Corp. Madison WI, USA) for amplification of the sequence containing E6 region of HPV 16, 18 and 33. The sequences were: forward common, 5'AAGGGCGTAACCGAAATCGGT3'; reverse 16, 5'GTTTGCAGCTCTGTGCATA3'; 18, 5'GTGTTCAGTTCCGTGCACA3', 33, 5'GTCTCCAATGCTTGGCACA3'. The amplified products correspond to 140 bp for HPV 16 and 18, and 141 bp for HPV 33. Cycling temperatures for HPV genotyping were as follows: forty five cycles of 95°C for one minute (denaturation), 57°C for one minute (annealing), and 72°C for one minute (extension).
This study was approved by the institutional ethical committee. The purpose and procedures of the study were explained to all participants, and a written informed consent was obtained from all of them.
Results were analyzed with the aid of the software Epi Info 6. To assess the association between the characteristics of the subjects and the infection, the crude odds ratio with a 95% exact confidence interval was used. In addition, comparison of the frequencies between groups was performed by the χ2 test. A level of P < 0.05 was considered significant.