HPV detection using primers MY 09 / MY 11 and GP 5 + / GP 6 + in patients with cytologic and / or colposcopic changes

First submission on 23/04/12; last submission on 03/12/13; accepted for publication on 16/05/14; published on 20/08/14 1. MSc in Health and Environment-Universidade Tiradentes (UNIT); biomedical Scientist. 2. MSc Health and Environment-Universidade Tiradentes (UNIT); gynecologist. 3. Biomedical scientist. 4. PhD in Theoretical Medicine (Theoretischen Medizin-Doctor rerum medicinalium) – School of Medicine at Rheinisch-Westfalische Technische Hochschule Aachen; AachenGermany; adjunct professor at Universidade Federal de Santa Catarina (UFSC). 5. PhD in Parasitology-Universidade Estadual de Campinas (UNICAMP); permanent professor at UNIT. 6. PhD in Parasitology-Universidade de São Paulo (USP); permanent professor at UNIT. ABStrACt

cancer, as well as its precursors lesions, implying that the virus presence is a cancer-causing agent (7,28) .Walboomers et al. (26) , using the polymerase chain reaction (PCR) and serological tests, showed that HPV is present in 99.7% of patients with invasive cervical cancer.
Molecular diagnosis of HPV infection is important for virus screening, and is mainly based on methods such as: hybrid capture (CH2) (20,23) , in situ hybridization (2) , and PCR (8,21,24) .These techniques have vary widely in terms of sensitivity and specificity, and PCR is the most used today in various areas of molecular diagnostics due to its great ability to detect small fragments of deoxyribonucleic acid (DNA) (4,11) .
The system uses MY09/MY11 and GP5+/GP6+ primers, that amplify the L1 region of viral genome, are more frequently used for HPV detection in clinical and histological studies (12) .These primers are effective for amplifying wide spectrum of HPV genotypes in cells obtained from cervical smears and paraffin-embedded tissues (13,16) .The pair of oligonucleotides MY09/MY11 primers flanks a sequence of approximately 450 pb, while the pair GP5+/ GP6+, a sequence of approximately 150 pb, which is internal to the sequence flanked by MY (22) .The pair MY is synthesized from several degenerate nucleotides in each primer, it is a mixture of 25 primers oligonucleotides able to amplifying > 25 genital HPV types.On the other hand, the pair GP5+/GP6+ consists of a fixed sequence of nucleotide for each oligonucleotide primer.This pair uses low annealing temperature during PCR (18) , and may be used as a single oligonucleotide primer or in PCR "nested", after the MY oligonucleotide primer amplification (9) .This study aimed to investigate HPV infection in women with cytological and/or colposcopic abnormalities, which were seen in a poor city of Alagoas, and also verify the performance of MY09/ MY11 and GP5+/GP6+ primers for HPV detection.

MAtEriAL AnD MEthoD
This is a cross-sectional study, performed during May 2009 to March 2011, from epidemiological data on cytological and/or colposcopic abnormalities cases diagnosed at Clinic of Gynecology of Núcleo de Apoio à Saúde da Família (NASF) in Penedo-Alagoas, city with high poverty (60.62%) (14) .
The study included women had as cytological result: atypical squamous cells of undetermined significance (ASC-US), atypical glandular cells of undetermined significance (AGUS), low-grade intraepithelial lesion (LSIL), high-grade intraepithelial lesion (HSIL), and invasive carcinoma.Patients with colposcopic changes such as acetowhite epithelium (AWE), dotted, mosaic, partial iodine-positive epithelium and iodine-negative epithelium, classified as positive colposcopy, were also included in this study.Patients' data were obtained from medical records.Cervical samples were only collected from patients who signed the Informed Consent.This project was approved by Research Ethics Committee of Universidade Tiradentes, Aracaju-SE.
Cervical smears were collected using Ayre spatula, then placed in Falcon tubes, with 25 ml of PBS buffer solution (phosphatebuffered saline, pH 7.4), and stored at -20ºC until DNA extraction.

DNA extraction
DNA extraction from cervical smears was performed using QIAamp DNA Mini Kit (QiagenLtd, Crawley, UK), according to manufacturer's instructions.Aliquots of 200 μl of samples were digested with 20 µl of K proteinase and 200 μl of AL buffer at 56ºC, for 10 minutes.DNA precipitation was performed by adding 200 μl of ethanol (96%).DNA was eluted in 200 μl of AE buffer and stored at -20ºC until further use.
To determine extracted DNA quality and quantity, each DNA was analyzed by electrophoresis on a 1% agarose gel, stained with blue green loading dye (LGC) (Figure 1).As an intern control of the reaction, samples with DNA were subjected to PCR for human β-globin gene amplification, using PCO3 (5'CTTCTGACACAACTGTGTTCACTAGC3') and PCO4 (5'TCACCACAACTTCATCCACGTTCACC3') oligonucleotides, which flanks a sequence of about 110 pb (Figure 2).Reagents quantities and concentrations were just the same as PCR reaction for HPV, using MY oligonucleotide primer (Table 1).
Samples that were positive for human ß-globin gene were subjected to DNA viral of HPV research.It was excluded from the analysis only negative samples for ß-globin gene, after reanalyzing samples for confirmation.

DNA amplification
Two different pairs of oligonucleotides primers were used in this study to HPV detection: MY09/MY11, GP5+/GP6+ (Figures 3 and 4).PCR reactions for each oligonucleotide primer were carried out separately, as previously described by Bernard et al. (3) and De Roda-Husman et al. (6) .Table 1 shows the oligonucleotides primers used in PCR, as well as their amplification conditions.For each PCR reaction, it was used a positive control (DNA extracted from a condyloma sample) and a negative control (H 2 O).Result was considered positive for DNA-HPV when one of the MY09/MY11 or GP5+/GP6+ oligonucleotides primers testing detected the viral DNA.

Statistical analysis
For data statistical analysis we used chi-squared and/or Fisher's exact test, through SPSS software (Chicago, IL, USA), 16.0 version, considering significant variables those that reached p < 0.05 in the two-tailed test.

rESuLtS
A sample survey of cytological and/or colposcopic abnormalities cases diagnosed during May 2009 and March 2011 was performed at the Clinic of Gynecology of NASF, in the city of Penedo-AL.The average age of the studied population was 36 years (range: 18-58 years).Regarding cytologic results, 21 patients (30%) showed ASC-US; 20 (29%), AGUS; 12 (17%), LSIL; and five (7%), HSIL.Twelve patients (17%) showed positive colposcopy (Figure 5 and Table 2).Only one patient has HSIL and a positive sample to HPV, which demonstrate the effectiveness of PCR as standard molecular method.From the seven patients with LSIL, only three were positive for o HPV.The most likely possibility is that the results for the other four samples were false negative.
Results showed that DNA of HPV was detected in 15% and 30%, using MY-PCR and GP+-PCR, respectively.There was no statistical difference between the two oligonucleotides used in PCRs (Table 3).

DiSCuSSion
PCR is being increasingly used in clinical laboratories to HPV diagnose.For the L1 region, there is consensus in indicating two oligonucleotides primers systems commonly used: MY09/MY11 and GP5+ /GP6+.Both detect a wide range of HPVs.Degenerated MY09/MY11 oligonucleotide primer uses annealing high temperature (55ºC) and can amplify multiple HPV infections.However, GP5+/GP6+ oligonucleotide primer has lower annealing temperature (42ºC), allowing the best amplification of single HPV infections, when compared to multiple infections (18) .
Studies in the literature indicate variables rates of HPV detection.This differences in detecting DNA of HPV suggest a potential difference in the ability to amplify fragments of different sizes and specific types of HPV, depending on the methods used for DNA detection, and may also de due to the types of studied material (smears, frozen material, paraffin material), anatomical localization, population issue, and design of oligonucleotides (5) .rESuMo Introdução: O câncer cervical é uma das doenças mais frequentes entre mulheres, e causa considerável morbidade e mortalidade.

Unitermos: câncer cervical; citologia; colposcopia; HPV; PCR.
In this study we observed a greater number of positive samples using GP5+/GP6+ oligonucleotides, when compared to MY09/ MY11.From 27 samples, eight (30%) were positive for HPV.DNA-HPV was found in 15% and 30%, using MY e GP+ oligonucleotides primers, respectively.From these, four (15%) were detected by both methods, and eight (30%), only by PCR do GP+.However, there was no statistical difference between the two oligonucleotides used in PCRs (p = 0.3261), even with twice detection efficiency using GP+ oligonucleotides primers.According to Poljak et al. (17) and Vince et al. (25) , one cannot rule out the possibility of false negative in PCR due to DNA-HPV low concentration in the clinical sample and the presence of PCR endogenous inhibitors or from sample processing.
In a Zehbe and Wilander study (26) , they compared the sensitivity of these oligonucleotides primers for PCR in cervical biopsies classified as cervical intraepithelial neoplasia (CIN) I to III.The authors demonstrated that the two oligonucleotides primers systems were equally sensitive, with a correlation of 98%.Using GP5+/GP6+, the authors found HPV positivity of 95% with MY09/MY11 of 94%.QU et al. (18) , compared the detection rates of the same oligonucleotides primers used in our experiment (MY09/MY11 and GP5+/GP6+) in cervicovaginal washing sample, they found DNA of HPV in 45,5% and 42,8%, respectively, with no statistical difference between the two oligonucleotides used in PCRs, only in the evaluation of HPV types detected by each pair of oligonucleotide, result similar to ours, which also showed no statistical difference.
Remmerbach et al. (19) , through oral mucosal smears analysis of patients suspected of carcinoma lesions, showed that DNA-HPV was detected in 2.8% of sample using PCR with MY09/MY11 oligonucleotides and 35,8% with GP5+/GP6+ oligonucleotides.These authors also reported that HPV detection with GP5+/GP6+ oligonucleotides was higher than MY09/MY11 oligonucleotides, corroborating the results found in our study.This can be explained by the smaller size of DNA fragment amplified by GP-PCR system.Thus, this oligonucleotide would have greater efficiency in HPV detection in samples with fragmented DNA.These authors also found a greater HPV detection in biopsy samples, when compared to cervical smear sample, which may indicate the difference between our study and the Zehbe and Wilander study (27) .
Iftner and Villa (15) reported that the sensitivity and specificity of PCR method may vary depending mainly on the choice of oligonucleotides primers used, the number of base pairs of the product amplified by PCR, performance of DNA polymerase used in the reaction, DNA-HPV type spectrum amplified, and the ability to detect different types in the infection.Studies in the literature indicate variables HPV detection rates.
The choice of suitables oligonucleotides to clinical and epidemiological uses should consider the source of clinical material, the size of PCR product, range of DNA-HPV types to be amplified, oligonucleotides ability to amplify the HPV multiple types and the availability of sequencers systems or specific probes to identifying the different HPV genotypes (5) .

ConCLuSion
Considering the great intra-and inter-observer variability associated to cytological interpretation of preneoplastic and neoplastic cervical lesions, the use of molecular detection methods, such as PCR technique, becomes an important tool in identifying patients with HPVs responsible for developing cervical neoplasia, thus supporting medical management.This study suggests that more than one type of oligonucleotide primer should be used in clinical samples to increase the sensitivity in HPV detection.

figurE 5 -
figurE 5 -Results of cytological and/or colposcopic reports from cervical samples of patients admitted in the city of Penedo-AL (2009-2011) ASC-US: atypical squamous cells of undetermined significance; AGUS: atypical glandular cells of undetermined significance; LSIL: low-grade squamous intraepithelial lesions; HSIL: high-grade squamous intraepithelial lesions.

tABLE 3 -
Percentage of DNA of HPV detection with oligonucleotide primers pairs *: Pearson's chi-square test.DNA: deoxyribonucleic acid; HPV: human papillomavirus; n: number of collected samples.

tABLE 2 -
Correlation between diagnostic categories to cytologic and colposcopic examinations and HPV molecular detection *: HPV positive means the sum of the two PCR methods used; **: Fisher's exact test.HPV: human papillomavirus; ASC-US: atypical squamous cells of undetermined significance; AGUS: atypical glandular cells of undetermined significance; LSIL low-grade squamous intraepithelial lesions; HSIL: high-grade squamous intraepithelial lesions; PCR: polymerase chain reaction.