We included in the study 160 adult women who were enrolled in a cervical cancer screening program and attending gynecologic services at different hospitals in the city of Bogota. Informed consent was signed by all participants, who voluntarily agreed taking part in the study for a period of eight months. Inclusion criteria were: women with history of borderline, mild or moderate dysplastic cytology (the criteria used for the cytologic diagnoses were those of The Bethesda System 2001) who were then referred to histopathology analysis, with the outcome of ‘normal’ or ‘abnormal’, in which case the lesions were classified according to the CIN grading system by the pathologist. Exclusion criteria were: pregnancy, previous history of biopsy-confirmed CIN (any grade) or HPV infection, as well as having received antineoplastic therapy, immunotherapy or HPV vaccination. All participants included in the study underwent subsequent exploratory colposcopy with collection of cervical scraping smears and biopsy samples at the site where the abnormal lesion was localized for HPV genotyping and histopathology analysis, respectively. According to the outcome of such analysis, the patients were diagnosed following the CIN grading system (CIN grades I, II and III), CC or as “negative pathology”. The histopathology findings were reviewed by a second pathologist, who was not blinded to the results. Those cases in which the diagnosis made by the two pathologists did not match were excluded from the study.

The cervical scrapings were collected at at the lesion site by colposcopy using cyto-brushes, which were immediately placed in sterile tubes containing 1 ml of nucleic acid-free phosphate buffer saline (PBS) supplemented with 0.05% thimerosal. All samples were subsequently treated with proteinase K (Bioline^®) to a final concentration of 20 ug/ml, and incubated overnight at 37°C. Then, the proteinase K was inactivated at 95°C for 5 minutes, and the samples were centrifuged at 10.000 x g for 5 minutes and stored at -20°C until processed for DNA isolation. Cervical biopsies, taken at the site where the lesion was identified, were fixed with formalin and paraffin-embedded for histopathology analysis.

DNA was isolated from the samples treated with proteinase K in the previous section by a two-step phenol extraction and one-step chloroform-isoamyl alcohol extraction followed by ethanol precipitation. DNA pellets were resuspended in 0.2 ml of 10 mM Tris (pH 8.5) buffer and stored at -20°C until further analysis.

Quality of DNA was confirmed by electrophoresis and by PCR with primers PC03/PC04, which amplify a 110 bp fragment of the ß-globin gene [17Arias Y, Carrillo E, Aristizábal F. Plasma DNA restoration for PCR applications J Clin Pathol 2007; 60: 952-4.]. The reaction mixture contained 1X PCR buffer, 3.5 mM MgCI2, dNTP mix with 200 µM of each, PC03 and PC04 at 1 µM each, 1.25 U of Taq polymerase (Promega^®) and distilled water to a total reaction volume of 25 µL. The reactions were run on the My Cycler^TM Thermal Cycler (BioRad) using the following conditions: 4 minutes at 94 º C, followed by 40 cycles of amplification 94°C for 1 minute, 55ºC for 1 minute and 72ºC for 1 minute, and extension end at 72°C for 4 minutes. Lymphocyte DNA was used as positive control, and distilled water as negative control. The PCR products were visualized by gel electrophoresis on 1% agarose gels stained with ethidium bromide to identify the amplified 110pb band. The electrophoresis was run for one hour at 100 volts in TBE buffer using a BioRad Power PAC 3000 and visualized with a gel scanner with Quantity One software from BioRad.

For all samples positive for the ß-globin amplification, GP PCR was performed using the primers GP5+/5'-biotinylated GP6+ as described elsewhere [15Van Den Brule A, Pol R, Fransen-Daalmeijer N, Schouls L, Meijer C, Snijders P. GP5+/GP6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes J Clin Microbiol 2002; 40: 779-87.]. The reaction mixture contained 10 µL of sample, 1X PCR buffer, 3.5 mM MgCI2, dNTPs mix (200 µM of each deoxynucleoside triphosphate), GP5+ and 5'-biotinylated GP6+ primers at 1 µM each, 1.25 U of Taq polymerase (Promega^®) and sterile distilled water to a total reaction volume of 50 µL. Amplification was carried out in a thermocycler MyCycler^TM (Biorad) and PCR conditions were: one step of denaturation at 94°C for 4 minutes, followed by 40 amplification cycles each with the following steps: denaturation at 94°C for 1 minute, annealing at 40°C for 2 minutes and elongation at 72°C for 1½ minutes. A final elongation step was prolonged by 4 minutes. As positive control we used DNA extracted from the cervical cancer cell lines HeLa and SiHa kindly provided by the laboratory of Pharmacogenetics from the National University of Colombia.

Detection was performed using an Immunetics Minibloter^® 45. This system uses membranes with carboxyl groups that are activated with 16% (w/v) EDAC (1-ethyl-3-(-3-dimethylamininopropyl) carbodiimide) to covalently bind oligonucleotides coupled to amino groups, which detect 37 types of HPV types as reported previously by Van den Brule [15Van Den Brule A, Pol R, Fransen-Daalmeijer N, Schouls L, Meijer C, Snijders P. GP5+/GP6+ PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes J Clin Microbiol 2002; 40: 779-87.]. Subsequently, the membrane is rotated 90 degrees, for hybridization with denatured PCR products at 42ºC for 45 minutes, followed by two washes at 52 º C for 10 minutes with 2X SSPE (1X SSPE contains 0.18 M NaCl, 10 mM NaH2PO4 and 1 mM EDTA pH 7.4, Invitrogen) and 0.5% SDS. Then, the membrane was incubated with streptavidin-peroxidase conjugate (Roche^®) (diluted 1:4000) for 45 minutes at 42°C, followed by two washes in 2X SSPE and 0.5% SDS for 10 minutes at 42 º C and two washes with 2X SSPE for 5 minutes at room temperature. Detection of specific hybridization was performed using ECL (GE Healthcare UK, Amersham) followed by exposure overnight of autoradiography film (Amersham Hyperfilm ™ ECL). The film was developed using a Medical Film Processor Model SRX-101 Konica Minolta Medical & Graphic, Inc.

Multiplex BSGP5+/GP6+ PCR-MPG was carried out with primers reported previously by Schmitt et al., 2008 [11Schmitt M, Dondog B, Waterboer T, Pawlita M. Homogeneous amplification of genital human alpha papillomaviruses by PCR using novel broad-spectrum GP5+ and GP6+ primers J Clin Microbiol 2008; 46: 1050-9.]. The assay used nine sense primers BSGP5+ at 200 nM and 3 antisense primers 5'-biotinylated-BSGP6+ at 400 nM to amplify a ~150 bp fragment of the viral L1 ORF, as well as two additional primers (MS3/MS10) at 300 nM to amplify the ß-globin gene, which was used as DNA integrity control [18Dunbar SA, Vander Zee CA, Oliver KG, Karem KL, Jacobson JW. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the luminex LabMAP system J Microbiol Methods 2003; 53: 245-52.]. The multiplex Luminex hybridization assay enabled the detection of 27 mucosal HPV-genotypes: i) high risk (HR)-HPV types: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68; ii) possibly HR-HPV types: 26, 53, 67, 70, 73, 82; low risk (LR)-HPV types: 6, 11, 30, 43, 44 and 69. In addition, the β-globin product was amplified in each sample.

Oligonucleotide probes with an amino group at the 5' end for each of the 27 HPV types were coupled to carboxylated beads using the carbodiimide procedure described by Schmitt et al., 2006 [16Schmitt M, Bravo I, Snijders P, Gissmann L, Pawlita M, Waterboer T. Bead-Based Multiplex Genotyping of Human Papillomaviruses J Clin Microbiol 2006; 44: 504-12.]. For each combination of probes and beads we used 2.5 million carboxylated beads in 25 µL of 0.1 M2-(N-morpholino) ethanesulfonic acid (MES), pH 4.5, with 200 µg of N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide (EDC) and 400 pmol of probe, the mixture was incubated in the dark under stirring for 30 minutes, the addition of EDC and incubation was repeated once, then the beads were washed with 0.5 ml of 0.2 g/L of Tween 20 and once with 0.5 ml of 1.0 g/L SDS. Finally, they were stored at 4 °C in TE solution. Hybridization was performed in 96 well plates with 10 µL of PCR product in 33 µL of hybridization solution (0.15 M of tetramethylammonium chloride (TMAC), 75 mM Tris-HCl, 6 mM EDTA, 1.5 g/L Sarkosyl, pH 8.0) to which 2000 bead-coupled probes were added. This mixture was incubated at 95°C for 10 minutes and immediately placed on ice for 2 minutes, then it was placed at 41°C for 30 minutes under stirring, then the samples were transferred to a wash plate with filter bottom (Millipore, Bedford, MA) pre-equilibrated with blocking solution (PBS with 0.2 g/L Tween-20 and 2.0 M TMAC), and washed with blocking solution to eliminate non-hybridized DNA in a vacuum wash station (Millipore). Subsequently, biotinylated PCR products were stained with streptavidin-R-phycoerythrin conjugate (Molecular Probes) diluted 1:1600 in 2.0 M TMAC, 75 mM Tris-HCl, 6 mM EDTA, 1.5 g/L Sarkosyl, 1.0 g/L casein, pH 8.0 and incubated 20 minutes at room temperature with shaking. After washing three times with blocking solution, the beads were analyzed in a Luminex 100 reader (Luminex Corp.), which uses two lasers, one that recognizes the bead set by the internal bead color and another to quantify the reporter fluorescence on the bead. The results were expressed in median fluorescence intensities (MIF) of ≥100 beads per sample [11Schmitt M, Dondog B, Waterboer T, Pawlita M. Homogeneous amplification of genital human alpha papillomaviruses by PCR using novel broad-spectrum GP5+ and GP6+ primers J Clin Microbiol 2008; 46: 1050-9., 16Schmitt M, Bravo I, Snijders P, Gissmann L, Pawlita M, Waterboer T. Bead-Based Multiplex Genotyping of Human Papillomaviruses J Clin Microbiol 2006; 44: 504-12., 18Dunbar SA, Vander Zee CA, Oliver KG, Karem KL, Jacobson JW. Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the luminex LabMAP system J Microbiol Methods 2003; 53: 245-52., 19Louie KS, de Sanjose S, Diaz M, et al. Early age at first sexual intercourse and early pregnancy are risk factors for cervical cancer in developing countries Br J Cancer 2009; 100: 1191-7.].

As control, plasmid standards from the WHO panel II [20Eklund C, Zhou T, Dillner J. for the WHO Human Papillomavirus Laboratory Network. Global proficiency study of human papillomavirus genotyping J Clin Microbiol 2010; 48: 4147-55.] with 100 and 1,000 copies for 16 distinct HPV types per 100ng human placental DNA were amplified and served to estimate the HPV viral load. MFI values obtained with 1,000 HPV genome copies of the respective HPV types per 100 ng of human placental DNA served as cutoff for defining low (<cutoff) and high viral load (>cutoff) groups.

The demographic data was analyzed using Statistix 9 software. The genotyping results were analyzed using Excel^®. For each probe, MFI values in reactions with no PCR product added to the hybridization mixture were considered background values. Reactions with net MFI values above 5 were defined as positive reactions. Net MFI values were computed by subtraction of 1.1 times the median background value. For all probes, this cutoff value was above the mean background plus three times the standard deviation.. P values of <0.05 were considered statistically significant.

Inclusion and exclusion criteria for selection of the study population are defined in the Methods section. All participants had a history of abnormal cervical cytology (atypical squamous cells, any grade, according to The Bethesda System 2001) and underwent subsequent colposcopy and histopathology analysis, which led to their classification into one of the five groups: negative pathology, CIN I, CIN II, CIN III and CC. The Table 1 resumes the socio-demographic and anamnestic characteristics of the study population distributed by the grade of the lesions found in the histopathologic analysis. Nearly 51% of the participants in the study were under the age of 34 years, with ~52% belonging to socioeconomic levels 1 or 2, classified according to the criteria of the Secretary for District Planning and the National Administration Department for Statistics (DANE, Decreto 304, 2008). Nearly half of the population (50.6%) had their first sexual intercourse under the age of 18, and about 70% had 1-2 sexual partners. The majority of women participating in the study had children (85.6%), and their first delivery was in most cases (87.6%) previous to the age of 24. A large number (56.9%) never used contraceptive methods. About 63% had no family history of cancer. A family history of CC was infrequent in all types of CIN lesions (Table 1) and even absent in the CC patients. However, a family history of other types of cancer was found in a significant number of women in all groups with a maximum in CIN II (43.8%).

Table 1

Characteristics of the Study Population

Table 2

Histopathologic Diagnosis and Incident Rates of HPV Types 16 and 18 in the Study Population

Table 3

Prevalence of High-Risk (HR), Putative High-Risk (pHR) and Low-Risk (LR) HPV Types in Cervical Smears Taken from Women with Negative Histopathologic Diagnosis and in Patients with CIN and CC

Table 4

Frequencies of HR, pHR and LR-HPV Single and Multiple Infections Among the Different Histopathologic Groups within the Study Population

Table 5

Prevalence by Age of HR, pHR and LR HPV Types (Single and Multiple Infections)

Table 6

Viral Load Values Arranged into Three Groups (<50, 50-500 and >500 Copies), Determined by PCR in the Cervical Smears of the Study Population Distributed by Histopathologic Diagnosis

The distribution of cervical cancer and its precursor lesions among the study population is summarized in Table 2. Of the 160 study participants 41 (25.6%) did not show any detectable cervical lesions in the histopathology analysis. The remainder 119 cases were diagnosed of cervical lesions at different stages: 56 (35%) with low-grade cervical lesions (CIN I), 49 (30.6%) with high-grade cervical lesions (CIN II and CIN III) and 14 women (8.8%) were diagnosed of CC (either in situ or invasive carcinoma).

In a previous study, Wright and colleagues estimated the sensitivity of the cytology screening to be about 60.7% [21Wright TC, Denny L, Kuhn L, Pollack A, Lorincz A. HPV DNA testing of self-collected vaginal samples compared with cytologic screening to detect cervical cancer JAMA 2000; 283: 81-6.], with a trend to increase with higher degree of dysplasia. In our study, the sensitivity of the Pap smear was determined to be 69,6% for CIN I, 40% for CIN II, 63% for CIN III and 78,6% for CC.

The majority of CIN and CC cases are linked to infection by the so-called high-risk HPVs, a subset of oncogenic HPV types with mucosal tropism [6Kisseljov FL, Kisseljova NP, Kobzeva VK, et al. State of human papilloma virus DNA in cervical carcinomas Mol Biol 2001; 35: 399-404., 7Cid-Arregui A. Therapeutic vaccines against human papillomavirus and cervical cancer Open Virol J 2009; 3: 67-83.]. We used both multiplex PCR and Luminex xMAP technology as well as reverse line blot (RLB) analysis for detection and genotyping of the HPV types 16 and 18, the most prevalent in CC, in DNA isolated from cervical smears of all the patients participating in the study. As expected, these two viral types were found at high frequencies in all five histopathologic groups. With the Luminex xMAP technique HPV16 and HPV18 were detected in separate or combined infections in 80.5% of women with negative histopathology diagnosis, 83.9% of low-grade lesions (CIN I), 91.8% of high-grade lesions (CIN II and CIN III) and 100% of CC (Table 2, middle column). In contrast, the RLB analysis detected the presence of these virus types at markedly lower frequencies in all groups (Table 2, right column), suggesting that, in our hands, this technique is less sensitive than the Luminex xMAP method. Furthermore, in those samples positive for HPV by both methods there was a 67.2% coincidence in the HPV type(s) detected (p<0.05). Therefore, Luminex xMAP was chosen to detect up to 27 mucosotropic HPV types in cervical scrapings of all the women included in the study. Table 3 summarizes the prevalence of the HPV types that were detected in the five histopathologic groups of our study population.

Using Luminex xMAP one or more HPV types were demonstrated in the cervical smears of all 160 participants in the study. Table 3 shows the 27 HPV types that were detected. Of these, 16 were high-risk (HR), 2 possibly high-risk (pHR) and 9 low-risk types commonly found in genital warts and low-grade cervical lesions [22Muñoz N, Bosch FX, de San Jose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer N Engl J Med 2003; 348: 518-27.]. The data in Table 3 reflects the HPV types detected either as unique or double/multiple infections. HPV16 was by far the most prevalent virus (overall prevalence 84.4%), ranging from 75.6% in the negative pathology group to 100 in CC. The second most common virus type was HPV18 (overall prevalence 84.4%), with 53.6% in the negative pathology group and over 60% in all CIN groups, the lowest prevalence being in the CC group (28.5%). Among the remainder HR-HPV types, only five were found in CC: HPV45, 52, 56, 68 and 69, although all HR-HPV types detected in our study were also present in the negative pathology and/or the CIN groups. Of the two pHR-HPV types detected (HPV53 and 66) only HPV53 was found in CC, albeit HPV66 was present in all other groups with a relatively high prevalence (about 10-15%). Among the LR-HPV types only four types were found in CC (HPV6, 42, 69 and 70) of which HPV6 showed the highest prevalence (21.4%).

The accuracy of the Luminex genotyping was further confirmed by analyzing the expression of early genes (E6 and E7) of the most prevalent HR-HPVs detected with this technique (HPV types 16, 18, 31, 33, 45, 51, 52, 56 and 58). To this end, we isolated total RNA from biopsy material of the patients positive for these virus types and, after reverse transcription and DNase I treatment, analyzed them by quantitative real time PCR (QRT-PCR) using specific primers for E7 or E6/E7 transcripts. The results validated those obtained with Luminex, since all samples identified as positive for the specific virus types by Luminex amplified their respective transcripts by QRT-PCR (data not shown).

The Table 4 shows the frequencies of infection with HR, pHR and LR-HPVs found in the five histopathologic groups as single or multiple events. Overall, the frequency of multiple infections (129/160 cases, 80.6%) was about 4-fold higher than that of single infections (31/160, 19.4%), a trend that was conserved in the five groups of patients. HR-HPV types were the most prevalent in all cases of infection with a unique virus, the highest prevalence being in the CC group (28.6%). In contrast, single infections with LR-HPVs were rare and were not observed at stages above CIN II. There was just one case of single infection by pHR-HPV in the negative pathology group. In the samples infected with more than one HPV type, the combination of two or more HR-HPVs was the most frequent (70/160, 43.7%), followed by HR+LR infections (43/160, 26.9%). The combination of HR+pHR was rare (16/160, 10%) (Table 4). Within the CC group, HR+LR-HPV infections were more frequent (5/14, 35.7%) than infections with multiple HR-HPV types (4/14, 28.6%). However, such differences were not statistically significant (p>0.1, one way ANOVA).

Analysis of the prevalence of HR-HPVs by age showed an overwhelming presence of these viruses in all age groups, with over 97% of women above the age of 25 being positive (Table 5). Among the HR-HPVs, HPV16 and 18 were found in 80-90% of cases, as single or multiple infections. The LR-HPVs showed much lower frequency rates, with a maximum in the group less than 25 years old (42.1%). Likewise, pHR-HPVs had their maximal prevalence in the same age group, but with a lower frequency rate (23.7%).

The viral load was estimated in the DNA isolated from the cervical scrapings by calculating the HPV copy number against standards. As can be appreciated in the Table 6, the samples infected with HR-HPVs, especially HPV types 16, 18, but also types 31, 33, 52 and 58, had an overall tendency towards a stronger viral load, which correlated with the degree of dysplasia. However the differences seen were not statistically significant. For the two most prevalent HR-HPV types, 16 and 18, the highest copy numbers were found in the group diagnosed of CIN II in which about 80% had more than 50 viral copies. Interestingly, the viral load of these two viruses decreased in CIN III and CC (p<0.05). In contrast, most samples infected with LR-HPVs contained less than 50 viral copies, with the exception of those infected with HPV6, a significant number of which carried higher numbers of copies, particularly in CIN I and II lesions (p<0.05). Of the two pHR-HPVs that were detected, HPV53 was in the range below 50 copies, while HPV66 showed a tendency to higher viral load, largely in the CIN I group, but the difference was not statistically significant.