Abstract
Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin–labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.
Keywords: High-risk human papillomavirus (HR-HPV), FFPE cervical samples, Cervical cancer, Microplate hybridization, Fluorometric assay
Introduction
During the clinical course of human papillomavirus (HPV) infection, nearly 90% of people infected with high- or low-risk HPV clear the infection within almost 2 years [1, 2]. However, persistent infection with high-risk HPV (HR-HPV), especially types 16 and 18 in minority of the individuals, may lead to cervical intraepithelial neoplasia (CIN), which is the precursor lesion of cervical cancer [3]. On the other hand, CIN is considered to progress in severity over time, passing from grade I (CIN1 or mild epithelial dysplasia), over grade II (CIN2 or moderate dysplasia) to grade III (CIN3 or severe dysplasia), and then to invasive carcinoma [4–6].
Although most CINs spontaneously regress, early diagnosis of HR-HPV type infection is of great value for monitoring and preventing cervical cancer.
Cervical biopsy through colposcopy procedure for histopathological analysis along with DNA examination in detecting HR-HPV genotypes as an indicator of lesion severity is essential [7–9].
In histopathological approaches, cervical tissue is used to provide valuable information on the diagnostic along with resolving the discrepancy in patients with cervical intraepithelial neoplasia. Nevertheless, HPV detection and genotyping in purified DNA extracted from the cervical tissue are the other major aspect for using tissues to improve patient’s care management [10–12].
Based on cell morphology, Pap smear test, a method to screen abnormities, has a limited application in detecting cervical cancer and its precursors (CINs), due to its low sensitivity, high dependency on sampling, and tissue preservation, and “it cannot detect type HPV” [13–15].
At present, several HPV DNA testing assays based on nucleic acid hybridization, signal-amplification, and nucleic-acid amplification are available for cervical tissue specimens, each with its strengths and drawbacks. Amongst the hybridization methods, microplate colorimetric hybridization assay (MCHA) is a method to identify HR-HPV based on the amplification of a fragment within the L1 gene region by consensus primers GP5+/6+, followed by colorimetric hybridization using type-specific probes coated on microplates [16]. Although, it has been shown that MCHA is in good agreement with PapilloCheck®; a DNA array–based diagnostic tool for the simultaneous detection and genotyping of 18 HR-HPV and 6 low-risk HPV genotypes, sensitivity, and specificity of this assay remained to be determined.
Microplate fluorometric hybridization assays (MFHAs), similar to MCHA, are more sensitive than colorimetric assays, and they can detect more analytes than colorimetric assays. However, they widen the dynamic range of the assay by allowing very high readings. Using Alexa Fluor 488, streptavidin comprises biotin-binding protein (streptavidin) covalently attached to a fluorescent label (Alexa Fluor® dye); hence, the aim of this study was to compare the specificity and sensitivity of colorimetric and fluorometric microplate hybridization assays for detection and genotyping of HR-HPV 16 and 18 DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissue specimens of patients within different grades of CIN and cervical cancer.
Brightness, fluorescent photo-stability, high solubility in aqueous solutions, and availability in multiple colors ideal for microplate assays are the important features of the streptavidin-fluorophore conjugate used in this experiment.
Materials and methods
Source of plasmids DNA
DNA plasmids containing the entire genomes of HPV16 (ATCC 45113D) and HPV18 (ATCC 45152D) were purchased from American Type Culture Collection (ATCC, Manassas, USA) and used as the control to develop efficient hybridization methods.
Cervical tissue specimens
In order to evaluate the performance of two in-house microplate hybridization detection methods, 60 archival formalin-fixed-paraffin-embedded (FFPE) cervical tissue samples were included in this study. Based on histopathological diagnosis and the World Health Organization (WHO) nomenclature and criteria, the specimens were divided into CIN grade 1 (n = 23), CIN 2 (n = 17), CIN 3 (n = 9), and cervical cancer (n = 11). All the clinical specimens were obtained from the pathology department archive with local Ethics Committee approval at the Namazi Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. Clinical data were also obtained from patients’ records by obtaining their permission. The mean age of the patients was 38.5 years (SD = 10.6, range = 20–63).
Sample preparation and DNA extraction
For the purification of total genomic DNA from formalin-fixed-paraffin-embedded tissues, 8 paraffin sections of 5–10-μm thick in a 1.5-mL microcentrifuge tube were used. The tissue sections were deparaffinized in xylene, followed by two 100% ethanol washes to remove residual xylene, and QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) was used for DNA extraction according to the manufacturer’s protocol.
Microplate hybridization method
Amplification of HPV DNA using consensus primers
HPV DNA sequence detection was carried out by nested PCR assay using MY09/11 as the outer primers [17] and GP5 +/6 + as inner primers [18]. To develop microplate hybridization assay, a 150-bp PCR product of the HPV L1 gene was labeled using 5′-biotinylated forward set of inner primers [19]. As an internal control and indicator of PCR assay efficiency, β-globin gene was also amplified using specific primers, PC03 (50-ACACAACTGTGTTCACTAGC-30) and PC04 (50-CAACTTCATCCACGTTCACC-30) generating a 110-bp PCR product.
The PCR products were electrophoresed on 1.5% agarose gels and stained with ethidium bromide.
Probe design
Using Allele ID software (version 7.5), type-specific probes were designed to distinguish between HPV-16 and HPV-18 genotypes. To evaluate the specificity of the selected oligonucleotide probes, the sequences were compared with those in the GenBank databases (www.ncbi.nlm.nih.gov/blast) by using the BLAST program. Oligonucleotide probes were synthesized with a 12-carbon (C12) amine (NH2) containing group on the 5′ end, to ensure that the attached oligo is raised off the microplate well surface, and to facilitate that the interaction with the target and reporter molecules needs to be captured in the assay. As a control probe, HPV-16 type–specific biotinylated probe with the same sequence as the test probe was designed to determine if the captured probes were able to couple with microplate surface.
Colorimetric microplate assay
Corning PureCoat amine 96-well plate (Corning Life Science, USA) was used to develop microplate hybridization assay. In order to attach 5′-amine-labeled capture probe on the surface of microplate wells, 200 μL of 0.01% glutaraldehyde as a crosslinker was added to each well and incubated at 4 °C for 1 h. 2 pmol/100 μL of each probe was then added to each well and incubated at 4 °C overnight.
Following three-times washing with phosphate buffered saline (PBS, pH 7.4), the wells were blocked with 300 μL of 5% bovine serum albumin (BSA) and incubated at room temperature for 2 h. The blocked wells were then washed three times with 100 μL of pre-heated (47 °C) hybridization buffer containing 20X SSC, 50X Denhardt’s solution, 10% SDS, 0.5 M EDTA (pH 8.0), and 10 mg/mL salmon sperm DNA.
Biotin-labeled PCR products were denatured in 20 μL of denaturation solution (2 M NaOH) at room temperature for 15 min, and then hybridized by adding to each well of one of the type-specific probes in 100-μL hybridization buffer and incubated for 60 min at 47 °C.
After the hybridization, the wells were washed three times with pre-warmed (50 °C) washing buffer containing 0.5X SSC and 0.05% Tween 20 and incubated for 15 min at 50 °C. The plates were washed with PBS, and 100 μL streptavidin-peroxidase conjugate was added to each well and incubated for 30 min at 37 °C. Plates washed 3 times with PBS at room temperature and 75 μL TMB chromogenic substrate solution was added to each well and incubated at room temperature for 30 min to develop color. The reaction was terminated by adding 75 μL of sulfuric acid stop solution as the color is forming reaction. The colorimetric reaction was measured by a spectrophotometer at an OD of 450 nm (reference filter 630 nm).
Fluorometric microplate assay
In this approach, the reactions were carried out under conditions similar to those used in the colorimetric microplate assay, except the streptavidin conjugated to Alexa Fluor 488 which was used to further increase the stability and consistency of the color formation. In this assay, instead of the streptavidin-peroxidase conjugate, 100 μL of 2.5 μg/mL of Alexa Fluor 488-conjugated streptavidin was added to each well and incubated for 30 min at 37 °C. The plates were washed three times with PBS, and then, 100 μL PBS was added for fluorometric measurements. The reaction was carried out in the darkness, and the intensity of fluorescent was measured by a Fluorescence Microplate Reader (FluStar Omega, BMG, Germany) at 495 nm of excitation and 519 nm of emission.
Type-specific PCR
Microplate hybridization assays were further confirmed by type-specific nested PCR using specific primers to detect HPV16 and HPV18 DNA as described before [20–23].
Determination of microplates positive/negative cut-off value
The “cut-off” point between positive and negative FFPE samples for HPV 16 and HPV 18 DNA detection by microplate hybridization assays was determined by calculating the mean + 3SD optical density (OD) of 20 HPV-negative samples for each assay.
Sensitivity and specificity assessments
The threshold sensitivity of microplate hybridization assays for HPV 16 and HPV 18 detection was determined using 10-fold serial dilution (1012–100 copies/reaction) of HPV DNA plasmids.
The specificity of type-specific primers for HPV types 16 and 18 detection was performed using BLAST search of the GenBank nucleotide database for the primers’ sequences to verify genotype specificity. In addition, PCR and microplate hybridization assays were performed with each type-specific primers and probes on control plasmid DNA types 16 and 18. Cross hybridization was carried out in triple, for each HPV 16 and 18 separately, in 10 runs, through each colorimetric and fluorometric assay separately.
Statistical analysis
The results were analyzed by SPSS 22 software (SPSS Inc., Chicago, USA). The agreement between the high-risk HPV identification methods was evaluated using kappa value. P values lower than 0.05 were considered to be statistically significant. Diagnostic measures were also used such as sensitivity, specificity, and precision/accuracy.
Results
Determination of microplate hybridization cut-off values
The “cut-off” optical density values (OD 450/630 nm) between positive and negative for HPV types 16 and 18 DNA detection by colorimetric assay in FFPE samples were found to be 0.207 and 0.202, respectively. Furthermore, these values (OD 495/519 nm) for HPV types 16 and 18 detection by fluorometric assay were 997 and 900.
Sensitivity of the microplate hybridization assays
When evaluated by microplate colorimetric hybridization assay (MCHA), the detection limit of the assay was found to be 50 and 30 copies of HPV DNA 16 and 18/reaction, respectively. However, the limit sensitivity of the HPV DNA types 16 and 18 by microplate fluorometric hybridization assay (MFHA) was found to be 5 and 3 copies/reaction.
Specificity of the oligonucleotide probes
The signal generated from both MCHA and MFHA indicated that the oligonucleotide probes for the detection and distinguishing of HPV 16 and 18 had good specificity (Figs.1 and 2).
Fig. 1.
Specificity of the HPV 16 and 18 probes for detection in MCHA
Fig. 2.
Specificity of the HPV 16 and 18 probes for detection in MFHA
Evaluation of archival clinical samples
From the total of 60 FFPE cervical samples, HPV DNA types 16 and 18 were found in 31 (51.7%) and 1 (1.6%) samples, respectively. HPV 16 and 18 co-infections were detected in 25 (41.7%) of the cases. However, 3 (5%) FFPE samples were tested negative for HPV DNA (Table 1). β-globin amplification showed that all samples had good-quality DNA and no inhibitors that would block a PCR reaction.
Table 1.
HPV types 16 and 18 prevalence in patients with different types of cervical dysplasia
| Stage | HPV 16 and 18 co-infections | HPV 16 | HPV 18 | Negative for HPV DNA |
|---|---|---|---|---|
| Cervical cancer | 8/11 (72.8%) | 2/11(18.1%) | 1/11(9.1%) | 0/11 (0%) |
| CIN1 | 2/23 (8.8%) | 18/23 (78.2%) | 0/23 (0%) | 3/23 (13%) |
| CIN2 | 7/17 (41.2%) | 10/17 (58.8%) | 0/17 (0%) | 0/17(0%) |
| CIN3 | 8/9 (88.9%) | 1/9 (11.1%) | 0/9 (0%) | 0/9 (0%) |
| Total | 25/60 (41.7%) | 31/60 (51.7%) | 1/60 (1.6%) | 3/60 (5%) |
CIN, cervical intraepithelial neoplasia
Analysis of three different PCR-based assays for the detection of HPV DNA
We analyzed and compared the sensitivity and specificity of three different PCR-based detection methods using plasmid DNA containing HPV genotypes 16 and 18 as a template. Validations of these methods were carried out on DNA extracted from FFPE cervical samples, representing different grades of HPV-associated lesions and cervical samples (Table 2).
Table 2.
HPV types 16 and 18 overall detection via three different methods
| Detection method | HPV 16 and 18 co-infections | HPV 16 | HPV 18 | Negative for HPV DNA |
|---|---|---|---|---|
| TSnPCR | 25/60 (41.7%) | 31/60 (51.6%) | 1/60 (1.7%) | 3/60 (5%) |
| MCHA | 19/60 (30%) | 35/60 (56.7%) | 2/60 (33.3%) | 4/60 (6.7%) |
| MFHA | 26/60 (41.7%) | 30/60 (50%) | 1/60 (1.7%) | 3/60 (5%) |
TSnPCR, type-specific PCR assay; MCHA, microplate colorimetric hybridization assay; MCFH, microplate fluorometric hybridization assay. (For MCHA and MFHA, amplified PCR by HPV GP5+/6+ primers were used)
Assay comparison
TSnPCR Vs. MCHA
Cohen’s Kappa coefficient was carried out to assess the level of agreement between the two detection methods. First, TSnPCR and MCHA were compared for the degree of HPV DNA 16 detection in 60 FFPE cervical samples from patients with CIN lesions and cervical cancer, and 20 embedded specimens of the normal cervix. Overall, both assays were able to detect 54 (90%) of HPV 16 DNA in samples from the patients. TSnPCR rated two samples as positive for HPV 16 DNA, while they were negative by MCHA. However, of the 20 normal samples, none of them were positive with the assays.
There was a very good agreement between the two methods’ judgments, κ = 0.942 (95% CI, 0.862 to 1.000), p < 0.0005 (Table 3). The sensitivity and specificity of MCHA method for HPV 16 detection were calculated as 96.43% (95% CI, 87.69 to 99.56%), and 100.00% (95% CI, 85.75 to 100.00%), respectively (Table 3).
Table 3.
Comparison between the results obtained for the identification of HPV 16 and 18, with TSnPCR and with MCHA
| Probe | Positive results (n = 80) (%) | Compared results (n = 80) | ||||||
|---|---|---|---|---|---|---|---|---|
| MCHA | TSnPCR | MCHA+/TSnPCR+ | MCHA+/TSnPCR− | MCHA−/TSnPCR+ | MCHA−/TSnPCR− | Kappa (%) (95% CI) | p value | |
| HPV 16 | 54 (67.5%) | 56 (70%) | 54 | 0 | 2 | 24 | 94.2 (86.1–100) | < 0.0005 |
| HPV 18 | 21 (26.3%) | 26 (32.5%) | 21 | 0 | 5 | 24 | 85 (72.4–97.5) | < 0.0005 |
MCHA, microplate colorimetric hybridization assay; TSnPCR, type-specific nested PCR; + positive result; − negative result
With respect to the concordance of the two assays for HPV 18 DNA detection, they were able to detect 22 HPV DNA 18 in patients’ samples, while 53 samples were negative for HPV 18 DNA. However, TSnPCR rated five samples as positive for HPV 18, while MCHA rated them as negative. There was a very good agreement between the two methods’ judgments, κ = 0.850 (95% CI, 0.724 to 0.975), p < 0.0005 (Table 3). The sensitivity and specificity of MCHA method for HPV 18 detection were calculated as 81.48% (95% CI, 61.92 to 93.70%) and 100.00% (95% CI, 93.28 to 100.00%), respectively.
TSnPCR Vs. MFHA
A comparison of the results obtained from DNA cervical samples tested with TSnPCR and MFHA is summarized in Table 4. Kappa value (k) showed 100% agreement for both HPV 16 and HPV 18 (κ = 1.000, p < 0.0005). The sensitivity and specificity of MFHA method for HPV 16 detection were calculated as 100.00% (95% CI, 93.62 to 100.00%) and 100.00% (95% CI, 85.75 to 100.00%), respectively. The sensitivity and specificity of MFHA method for HPV 18 detection were calculated as 100.00% (95% CI, 87.23 to 100.00%) and 100.00% (95% CI, 93.28 to 100.00%), respectively.
Table 4.
Comparison of the results obtained for the identification of HPV 16 and 18, with TSnPCR and with MFHA
| Probe | Positive results (n = 80) (%) | Compared results (n = 80) | ||||||
|---|---|---|---|---|---|---|---|---|
| MFHA | TSnPCR | MFHA+/TSnPCR+ | MFHA+/TSnPCR− | MFHA−/TSnPCR+ | MFHA−/TSnPCR− | Kappa (%) (95% CI) | p value | |
| HPV 16 | 56 (70%) | 56 (70%) | 56 | 0 | 0 | 24 | 100 | < 0.0005 |
| HPV 18 | 26 (32.5%) | 26 (32.5%) | 26 | 0 | 0 | 24 | 100 | < 0.0005 |
MFHA, microplate fluorometric hybridization assay; TSNPCR, type-specific nested PCR; + positive result; − negative result
Discussion
The accurate identification of human papillomavirus genotypes in FFPE cervical tissue from patients with cervical neoplasia is important for monitoring the progress of the disease and cervical cancer prevention approaches. Here, microplate colorimetric and fluorometric hybridization assays were designed with the aim of achieving equal or higher sensitivity in comparison with type-specific nested PCR assay for HPV genotypes 16 and 18 detection and typing in FFPE cervical tissue samples of patients with different CIN grades and cervical cancer.
Microplate hybridization method permits handling of 96 samples at the same time and using automatic pipetting and washing devices. Hence, to achieve the optimal microplate hybridization condition for HPV DNA detection and typing, several hybridization assay parameters were optimized. For the optimization and validation of microplate assay, control probe with the same nucleotide sequence homology of HPV-16, but 3′biotin labeled, was used through entire assay procedures. Different hybridization buffers were used to optimize DNA probe immobilization in amine coated microplate. Using buffer containing formamide, no or very low hybridization reactions were obtained. However, replacing the buffer with Denhardt’s solution containing BSA, Ficoll, Polyvinylpyrrolidone (PVP), and Salmon sperm removed high background signals but increased the hybridization reaction.
The performance of microplate colorimetric hybridization assay has already been evaluated on DNA extracted from cervical scrap [16]. When compared with PapilloCheck®, the agreement between both methods was reported 69.4% for HPV 16 and 82.4% for HPV 18 with the higher sensitivity of MCHA than Papillocheck®. In the present study, using the same primers, but different specific probes for HPV types 16 and 18 detection, we compared the sensitivity and specificity of MCHA with type-specific nested PCR. In our study, the combination of HPV general primers GP 5+/6+ and specific probes provided a good sensitivity and specificity that were in agreements with TSnPCR detection (κ = 1.000, p < 0.0005).
Nevertheless, in comparison with TSnPCR assay, MCHA had lower sensitivity and was not able to detect the two genotypes well. While 56 FFPE cervical samples were positive for HPV type 16 by TSnPCR assay, MCHA was not able to detect two positive HPV 16 samples, which are false-negative results of this method. However, the MCHA is shown to have equal specificity to TSnPCR assay.
When clinical samples were further tested for HPV DNA type 18, similar results were obtained for MCHA assay in comparison with TSnPCR assay that indicates the lower sensitivity of MCHA. One possible explanation could be a relatively low copy number of HPV DNA extracted from the cervical samples amplified by type-specific nested PCR, but not amplified by general PCR as a simple pre-screening PCR using GP 5+/6+ primers. Therefore, the microplate colorimetric assay needs to be improved for more accurate detection of both types in FFPE cervical samples.
Fluorometric assays that are very similar to colorimetric assays are more sensitive than colorimetric assays and they are able to detect more cases than colorimetric assays [24].
We performed fluorometric hybridization assay under similar conditions to those used in the colorimetric microplate assay, except the streptavidin conjugated to Alexa Fluor 488 was used to further increase the stability and consistency of the color formation. When the fluorometric assay results compared with type-specific nested PCR, a significant agreement of Kappa value (k) for detection of both HPV 16 and HPV 18 genotypes (κ = 1.000, p < 0.0005) was obtained.
The sensitivity and specificity of two assays were the same, indicating that the fluorometric hybridization assay is more sensitive than a colorimetric assay for HPV DNA types 16 and 18 detection and typing extracted from FFPE cervical samples.
Even though colorimetric assay is less sensitive than type-specific nested PCR and fluorometric assay, the technique remains promising for HPV DNA detection in FFPE cervical samples, and there is still room for improving sensitivity and specificity of the assay to be applied in clinical diagnosis.
Considering cost-effectiveness, the feasibility of instrumentation, simple, and easy to use the technique, colorimetric assays can be easily used for diagnosis purposes. However, the disadvantage of this assay is its lower sensitivity in comparison with the other two assays in this study which can be improved.
From the epidemiological point of view, the percentage of HPV16 and 18 positive samples and the rate of co-infection of types 16 and 18 in this study can be discussed. All 60 samples in this study were taken from patients that categorized in one of the CIN grades or cancer grade. From a total of 60 FFPE cervical samples, HPV DNA types 16 and 18 were found in 31(51.7%) and 1 (1.6%) samples, respectively. HPV 16 and 18 co-infections were detected in 25 (41.7%) of the cases. However, co-infection up to 76% has been reported [25]. The prevalence and pattern of co-infection differ depending on the geographic area of study as well.
In conclusion, in terms of specificity, both colorimetric and fluorometric microplate assays designed here for HPV types 16 and 18 detection and typing were in agreement with type-specific nested PCR assay. However, the flurometric assay was more sensitive compared with the colorimetric assay. Type-specific PCR assay only allows identification of individual genotypes, while the microplate hybridization assays can be adjusted for simultaneous detection of more genotypes in an assay run. Hybridization assays can be applied quantitatively and automatically as well. Colorimetric assay has a limited dynamic range in comparison with fluorometric assay. So, fluorometric assay would be a better candidate for quantitative detection of HPV in samples. This assay can be developed for detection and typing of all HPV types in different samples such as liquid-based Pap smear, fresh tissue, and FFPE samples.
Acknowledgments
The authors wish to thank Mr. H. Argasi at the Research Consolation Center (RCC) at Shiraz University of Medical Sciences for his invaluable assistance in editing this manuscript.
This work is based on a thesis in Virology by Negin Nikouyan (Project No. 93-7242), supported by Shiraz University of Medical Sciences, Shiraz, Iran.
Abbreviations
- MCHA
microplate colorimetric hybridization assay
- MFHA
microplate fluorometric hybridization assay
- TSNPCR
type-specific nested PCR
Compliance with ethical standards
All the clinical specimens were obtained from the pathology department archive with local Ethics Committee approval at the Namazi Hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. A statement of ethical approval is required to appear before the references for studies involving human or animal subjects.
Conflict of interest
The authors declare that they have no conflict of interest.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Plummer M, Schiffman M, Castle PE, Maucort-Boulch D, Wheeler CM. A 2-year prospective study of human papillomavirus persistence among women with a cytological diagnosis of atypical squamous cells of undetermined significance or low-grade squamous intraepithelial lesion. J Infect Dis. 2007;195(11):1582–1589. doi: 10.1086/516784. [DOI] [PubMed] [Google Scholar]
- 2.Rodriguez AC, Schiffman M, Herrero R, Hildesheim A, Bratti C, Sherman ME, Solomon D, Guillen D, Alfaro M, Morales J, Hutchinson M, Katki H, Cheung L, Wacholder S, Burk RD. Longitudinal study of human papillomavirus persistence and cervical intraepithelial neoplasia grade 2/3: critical role of duration of infection. J Natl Cancer Inst. 2010;102(5):315–324. doi: 10.1093/jnci/djq001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Li J, Zhou C, Zhou H, et al. The association between methylated CDKN2A and cervical carcinogenesis, and its diagnostic value in cervical cancer: a meta-analysis. Ther Clin Risk Manag. 2016;12:1249–1260. doi: 10.2147/tcrm.s108094. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Jaisamrarn U, Castellsague X, Garland SM, et al. Natural history of progression of HPV infection to cervical lesion or clearance: analysis of the control arm of the large, randomised PATRICIA study. PLoS One. 2013;8(11):e79260. doi: 10.1371/journal.pone.0079260. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mizushima T, Asai-Sato M, Akimoto K, Nagashima Y, Taguri M, Sasaki K, Nakaya MA, Asano R, Tokinaga A, Kiyono T, Hirahara F, Ohno S, Miyagi E. Aberrant expression of the cell polarity regulator aPKClambda/iota is associated with disease progression in cervical intraepithelial neoplasia (CIN): a possible marker for predicting CIN prognosis. Int J Gynecol Pathol. 2016;35(2):106–117. doi: 10.1097/pgp.0000000000000228. [DOI] [PubMed] [Google Scholar]
- 6.Mitra A, MacIntyre DA, Lee YS, et al. (2015) Cervical intraepithelial neoplasia disease progression is associated with increased vaginal microbiome diversity ;5:16865. doi:10.1038/srep16865https://www.nature.com/articles/srep16865#supplementary-information [DOI] [PMC free article] [PubMed]
- 7.Stoler MH, Schiffman M. Interobserver reproducibility of cervical cytologic and histologic interpretations: realistic estimates from the ASCUS-LSIL Triage Study. Jama. 2001;285(11):1500–1505. doi: 10.1001/jama.285.11.1500. [DOI] [PubMed] [Google Scholar]
- 8.Sasieni PD, Cuzick J, Lynch-Farmery E. Estimating the efficacy of screening by auditing smear histories of women with and without cervical cancer. The national co-ordinating network for cervical screening working group. Br J Cancer. 1996;73(8):1001–1005. doi: 10.1038/bjc.1996.196. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Renshaw AA, Davey DD, Birdsong GG, Walsh M, Styer PE, Mody DR, Colgan TJ, College of American Pathologists Comparison Program in Cervicovaginal Cytology Precision in gynecologic cytologic interpretation: a study from the College of American Pathologists Interlaboratory Comparison Program in Cervicovaginal Cytology. Arch Pathol Lab Med. 2003;127(11):1413–1420. doi: 10.1043/1543-2165(2003)127<1413:pigcia>2.0.co;2. [DOI] [PubMed] [Google Scholar]
- 10.Castro FA, Koshiol J, Quint W, Wheeler CM, Gillison ML, Vaughan LM, Kleter B, van Doorn LJ, Chaturvedi AK, Hildesheim A, Schiffman M, Wang SS, Zuna RE, Walker JL, Dunn ST, Wentzensen N. Detection of HPV DNA in paraffin-embedded cervical samples: a comparison of four genotyping methods. BMC Infect Dis. 2015;15:544. doi: 10.1186/s12879-015-1281-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Baldassarri R, Aronberg R, Levi AW, Yarbrough WG, Kowalski D, Chhieng D. Detection and genotype of high-risk human papillomavirus in fine-needle aspirates of patients with metastatic squamous cell carcinoma is helpful in determining tumor origin. Am J Clin Pathol. 2015;143(5):694–700. doi: 10.1309/ajcpcza4pszcfhq4. [DOI] [PubMed] [Google Scholar]
- 12.Lagheden C, Eklund C, Kleppe SN, Unger ER, Dillner J, Sundstrom K. Validation of a standardized extraction method for formalin-fixed paraffin-embedded tissue samples. J Clin Virol. 2016;80:36–39. doi: 10.1016/j.jcv.2016.04.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Dijkstra MG, Snijders PJF, Arbyn M, Rijkaart DC, Berkhof J, Meijer CJLM. Cervical cancer screening: on the way to a shift from cytology to full molecular screening. Ann Oncol. 2014;25(5):927–935. doi: 10.1093/annonc/mdt538. [DOI] [PubMed] [Google Scholar]
- 14.Pileggi C, Flotta D, Bianco A, Nobile CG, Pavia M. Is HPV DNA testing specificity comparable to that of cytological testing in primary cervical cancer screening? Results of a meta-analysis of randomized controlled trials. Int J Cancer. 2014;135(1):166–177. doi: 10.1002/ijc.28640. [DOI] [PubMed] [Google Scholar]
- 15.Kim G, Cho H, Lee D, Park S, Lee J, Wang HY, Kim S, Park KH, Lee H. Comparison of FFPE histological versus LBP cytological samples for HPV detection and typing in cervical cancer. Exp Mol Pathol. 2017;102(2):321–326. doi: 10.1016/j.yexmp.2017.02.015. [DOI] [PubMed] [Google Scholar]
- 16.Barcellos RB, Almeida SE, Sperhacke RD, et al. Evaluation of a novel microplate colorimetric hybridization genotyping assay for human papillomavirus. J Virol Methods. 2011;177(1):38–43. doi: 10.1016/j.jviromet.2011.06.010. [DOI] [PubMed] [Google Scholar]
- 17.Manos MM, Ting Y, Wright DK, Lewis J, Broker TR, Wolinsky SM. The use of polymerase chain reaction amplification for the detection of genital human papillomaviruses. Cancer Cells. 1989;7:209–214. [Google Scholar]
- 18.Jacobs MV, de Roda Husman AM, van den Brule AJ, Snijders PJ, Meijer CJ, Walboomers JM. Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes. J Clin Microbiol. 1995;33(4):901–905. doi: 10.1128/JCM.33.4.901-905.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ. The use of general primers GP5 and GP6 elongated at their 3′ ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol. 1995;76(Pt 4):1057–1062. doi: 10.1099/0022-1317-76-4-1057. [DOI] [PubMed] [Google Scholar]
- 20.Tornesello ML, Duraturo ML, Salatiello I, Buonaguro L, Losito S, Botti G, Stellato G, Greggi S, Piccoli R, Pilotti S, Stefanon B, de Palo G, Franceschi S, Buonaguro FM. Analysis of human papillomavirus type-16 variants in Italian women with cervical intraepithelial neoplasia and cervical cancer. J Med Virol. 2004;74(1):117–126. doi: 10.1002/jmv.20154. [DOI] [PubMed] [Google Scholar]
- 21.Biedermann K, Dandachi N, Trattner M, Vogl G, Doppelmayr H, More E, Staudach A, Dietze O, Hauser-Kronberger C. Comparison of real-time PCR signal-amplified in situ hybridization and conventional PCR for detection and quantification of human papillomavirus in archival cervical cancer tissue. J Clin Microbiol. 2004;42(8):3758–3765. doi: 10.1128/jcm.42.8.3758-3765.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, Vollmer N, Menton S, Menton M, Dietz K, Wallwiener D, Kandolf R, Bultmann B. Detection and typing of human papillomavirus by E6 nested multiplex PCR. J Clin Microbiol. 2004;42(7):3176–3184. doi: 10.1128/jcm.42.7.3176-3184.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Farhadi A, Behzad-Behbahani A, Geramizadeh B, Sekawi Z, Rahsaz M, Sharifzadeh S. High-risk human papillomavirus infection in different histological subtypes of renal cell carcinoma. J Med Virol. 2014;86(7):1134–1144. doi: 10.1002/jmv.23945. [DOI] [PubMed] [Google Scholar]
- 24.Bopp SK, Lettieri T. Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line. BMC Pharmacol. 2008;8:8. doi: 10.1186/1471-2210-8-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Gallegos-Bolaños J, Rivera-Domínguez JA, Presno-Bernal JM, Cervantes-Villagrana RD. High prevalence of co-infection between human papillomavirus (HPV) 51 and 52 in Mexican population. BMC Cancer. 2017;17(1):531. doi: 10.1186/s12885-017-3519-7. [DOI] [PMC free article] [PubMed] [Google Scholar]