Efficiency of MY09/11 consensus PCR in the detection of multiple HPV infections Fatih S¸ahiner, Ayhan Kubar, Ramazan G¨umral, Medine Ardıc¸, Nuri Yi˘git, Kenan S¸ener, Murat Dede, Mehmet Yapar PII: DOI: Reference:
S0732-8893(14)00210-7 doi: 10.1016/j.diagmicrobio.2014.03.030 DMB 13621
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
21 November 2013 8 February 2014 6 March 2014
Please cite this article as: S¸ahiner Fatih, Kubar Ayhan, G¨ umral Ramazan, Ardı¸c Medine, Yi˘git Nuri, S ¸ ener Kenan, Dede Murat, Yapar Mehmet, Efficiency of MY09/11 consensus PCR in the detection of multiple HPV infections, Diagnostic Microbiology and Infectious Disease (2014), doi: 10.1016/j.diagmicrobio.2014.03.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
REVISED Important note: The parts highlighted yellow color: newly added or changed The parts highlighted pink color will be deleted
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Running title: MY09/11 PCR efficiency in multiple infections
T
Title: Efficiency of MY09/11 consensus PCR in the detection of multiple HPV infections
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SC
Fatih Şahiner, Md, Department of Medical Virology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3481, Fax: +90 304 34 02.
Ayhan Kubar, Prof, Department of Medical Virology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3414
PT
ED
Ramazan Gümral, Md, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3433
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Medine Ardıç, Ankara Atatürk Training and Research Hospital, Yıldırım Beyazıt University, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 2904
AC
Nuri Yiğit, Md, Department of Medical Pathology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3734
Kenan Şener, Md, Department of Medical Virology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3420
Murat Dede, Md, Department of Obstetrics and Gynecology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 5811
Mehmet Yapar, PhD, Md, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3436
1
ACCEPTED MANUSCRIPT ABSTRACT Human papillomavirus (HPV) DNA testing has become an important component of cervical cancer screening programs. In this study, we aimed to evaluate the efficiency of
RI P
T
MY09/11 consensus PCR for the detection of multiple HPV infections. For this purpose, MY09/11 PCR was compared to an original TaqMan-based type-specific real-time PCR assay,
SC
which can detect 20 different HPV types. Of the 654 samples, 34.1% (223/654) were HPV DNA positive according to at least one method. The relative sensitivities of MY09/11 PCR and
MA NU
type-specific PCR were 80.7% (180/223) and 97.8% (218/223), respectively. In all, 352 different HPV isolates (66 low-risk and 286 high-risk or probable high-risk types) were identified in 218 samples, but 5 samples, which were positive by consensus PCR only, could not be genotyped. The distribution of the 286 high-risk or probable high-risk HPVs were as
ED
follows: 24.5% HPV-16, 8.4% HPV-52, 7.7% HPV-51, 6.3% HPV-39, 6.3% HPV-82, 5.6%
PT
HPV-35, 5.6% HPV-58, 5.6% HPV-66, 5.2% HPV-18, 5.2% HPV-68, and 19.6% the other 8 types. A single HPV type was detected in 57.3% (125/218) of the genotyped samples, and
CE
multiple HPV types were found in the remaining 42.7% (93/218). The false negative rates of MY09/11 PCR were found to be 17.4% in single infections, 23.3% in multiple infections, and
AC
34.6% in multiple infections that contained three or more HPV types, with the condition that the low-risk types HPV-6 and HPV-11 be considered as a monotype. These data suggest that broad-range PCR assays may lead to significant data loss and that type-specific PCR assays can provide accurate and reliable results during cervical cancer screening.
Key words: Cervical smear; genotyping; type distribution; mismatches.
2
ACCEPTED MANUSCRIPT INTRODUCTION To date, more than 200 human papillomavirus (HPV) types have been identified, and all of them exhibit a specific tropism for the squamous epithelium (Conway and Meyers,
RI P
T
2009). HPV types have been classified as high-risk or low-risk based on their oncogenic potential (Muñoz et al., 2003). The low-risk types cause benign lesions, such as genital warts
SC
and low-grade squamous intraepithelial lesions (LSILs), whereas high-risk HPV types can cause a wide variety of illnesses, including benign lesions, high-grade squamous intraepithelial
MA NU
lesions (HSILs), and cancerous lesions (de Villiers et al., 2004; Muñoz et al., 2003). Cervical cancer screening tests, such as the Pap smear and HPV DNA tests, have become increasingly important because cervical cancer is a preventable disease if patients participate in regular screening programs. Today, there are a number of techniques available for the detection of
ED
HPV DNA in clinical samples, and several factors and challenges affect the choice of methods
PT
for detection and genotyping of HPVs. The most important of these are as follows: (i) A number of different HPV types (more than 40) can cause genital infections (zur Hausen, 1996);
CE
(ii) Multiple sequence variations are often observed even in the same HPV genotype (Nindl et al., 1999); (iii) The detection sensitivity of the current methods is variable depending on the
AC
HPV genotypes and the viral DNA copy numbers in the samples examined (Gravitt et al., 2000; Qu et al., 1997); (iv) HPV genotyping is essential for adequate classification of patients into low-risk or high-risk groups and for following up with patients because the natural course of HPV infection is highly variable among different genotypes. However, the capability for detection of multiple infections and the ability to perform genotyping differ among the methods (Gravitt and Viscidi, 2004; Molijn et al., 2005; Sabol et al., 2008); (v) Finally, different degrees of target-DNA degradation may occur for some HPV genotypes during the integration of viral DNA into the host genome. By considering all these factors, a number of diagnostic techniques have been developed for the molecular diagnosis of HPV infections. Each of these methods has their own unique strong and weak features (Molijn et al., 2005). 3
ACCEPTED MANUSCRIPT The consensus polymerase chain reaction (PCR) assays (MY09/11, GP5+/6+, PGMY09/11, SPF-10) are designed to detect most genital HPV types in a single PCR reaction. MY09/11 consensus PCR, which targets a 450 bp conserved sequence in the HPV L1 gene, is
T
one of the most widely used consensus PCR assays (Depuydt et al., 2007), and it is
RI P
theoretically predicted that this method can detect over 40 different genital HPV types using degenerate primers (Gravitt and Viscidi, 2004). Another diagnostic approach is the type-
SC
specific real-time PCR assay, which is a highly sensitive method that is able to perform
MA NU
genotyping and viral DNA quantifying simultaneously in clinical samples (Depuydt et al., 2007; Şahiner et al., 2012). The L1 and E6-E7 gene regions are often preferred targets for the type-specific detection of HPV genotypes by real-time PCR assays (Depuydt et al., 2007; Dictor and Warenholt, 2011; Lee et al., 2011; Şahiner et al., 2012). The main aim of this study
ED
was to evaluate the effectiveness of MY09/11 consensus PCR and thus to draw attention to the
CE
and associated lesions.
PT
importance of the choice of method used for the diagnosis and follow-up of HPV infections
AC
MATERIAL AND METHOD Study group: The study was performed upon approval by the local ethical committee and after informed consent was obtained from the study participants. In all, 654 women who visited our gynecology outpatient clinic were included in this study. Two samples were collected from each patient simultaneously; one was sent to our laboratory for HPV DNA analysis, and the other was sent to the pathology laboratory for cytological examination. Repeat samples from the same patients were excluded from the study. Cytological examination: Exfoliated cervical cells were obtained using a liquid-based collection system (BD SurePath™, BD Diagnostics, USA) and interpreted according to the modified Bethesda system (Solomon et al., 2002).
4
ACCEPTED MANUSCRIPT DNA isolation: Genomic DNA templates were extracted using the standard phenolchloroform-isoamyl alcohol method from the smear samples (Sambrook et al., 1989). Briefly, cervical smear samples were suspended in 500 μl of TE buffer (10 mM Trishydrochloride, 1
T
mM EDTA, pH 8), and homogenized by vigorous mixing on a vortex. A 100 μl of mixed
RI P
specimen was placed in 10 μl of protease solution (65 mg/ml) (Sigma-Aldrich Corp, St. Louis, MO, USA) and 250 μl of K buffer for 60 min at 45°C. Following centrifugation at 10,000 g for
SC
10 min at 12°C, DNA was extracted from the supernatant using a mixture of 250 μl alkali
MA NU
phenol and 250 μl chloroform-isoamyl alcohol (24 : 1), and then precipitated using 500 μl isopropyl alcohol. DNA was washed in 75% ethyl alcohol at 10,000 g for 5 min at 4°C, airdried at 37°C, and dissolved in 100 μl distilled water (Şahiner et al, 2014). Consensus PCR reactions: All samples were tested by a previously described method using
ED
MY09/11 primers (Bauer et al., 1992) (MY09: 5'-cgtccmarrggawactgatc-3') (MY11: 5'-
PT
gcmcagggwcataayaatgg-3'). After completion of 40 cycles of this SYBR green-based real-time PCR, melting-curve data were obtained by continuous fluorescence acquisition from 55 to
CE
95°C with a thermal transition rate of 0.1 °C/s. The melting temperature of MY09/11 PCR products was determined as 82±1.5°C by optimization studies for our laboratory conditions
AC
(Şahiner et al, 2014).
Type-specific real-time PCR reactions: All smear samples were tested for the presence of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 using previously described TaqMan-based type-specific real-time PCR assay (Şahiner et al., 2012, Şahiner et al., 2014). The genotype spectrum of these methods was expanded to include all high-risk HPVs (Table 1). All type-specific primers and TaqMan probes using in this study were designed to target the L1 gene region. The reactions were carried out separately for each different HPV type. The reaction mixture was prepared as follows: 1.25 U Hot Start Taq DNA polymerase (Bioron, Germany), 10 pmol of each primer, 2.5 pmol TaqMan probe, 2.5 mM MgCl2, and 0.2 mM dNTP mix. PCR amplifications were conducted after the addition of 5 μl 5
ACCEPTED MANUSCRIPT of the sample containing the template DNA in a final volume of 25 µL. The PCR amplification cycles were as follows: initial denaturation and at hot-start Taq DNA polymerase activation at 95°C for 10 min, followed by 40 amplification cycles at 95°C for 15 sec and at 60°C for 1 min
T
(annealing-extension step). The TaqMan probes were labeled with fluorescent reporter dyes
RI P
(FAM; 6-carboxy fluorescein and JOE; 6-carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein) at the 5' end and with a black hole quencher (BHQ) as the non-fluorescent quencher at the 3' end.
SC
The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an
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internal control, and the PCR mixture without template DNA was used as a negative control in all PCR reactions. The L1 amplicons of each of the 20 HPV genotypes and the GAPDH amplicon were cloned into plasmid vectors using a TOPO TA Cloning System (Invitrogen, USA), and the detection sensitivities of the PCR assays were analyzed using serial plasmid
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dilutions (108-101 copies/ml). All PCR reactions were performed on an ABI Prism 7500
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Sequence Detection system (Applied Biosystems, USA). Genomic analyses (using databases and software): The primers and probes were designed
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using the OligoYap 4.0 software program (Yapar et al., 2005) and confirmed with a new software program developed in our laboratory for molecular biology research (OligoBee 1.0).
AC
All primer and probe sequences were analyzed with the GenBank BLAST database for specificity and were synthesized (MWG-Biotech, Ebersberg, Germany). The mismatches between MY09/11 primers and target gene regions and the nucleotide sequence similarities of MY09/11 amplicons of 20 different HPV types were analyzed using the NCBI Nucleotide Blast program and the OligoBee 1.0 software application. Agarose gel electrophoresis (AGE): PCR products were viewed by gel electrophoresis to differentiate HPV-6 and HPV-11 because the same probe was used for both HPV types in typespecific PCR reactions. Agarose gels were prepared for electrophoresis at 2.0%. Amplicons were run in the agarose gel in a 0.5X TAE buffer at 120 volts, maintained at a constant current for 35 min, and viewed and photographed using the Gel Doc 2000 gel documentation system 6
ACCEPTED MANUSCRIPT (Bio-Rad) under UV light. A 50-bp DNA ladder was used to determine the amplicon sizes (Invitrogen, USA). Statistical analysis: The unweighted kappa statistic was calculated to adjust for chance
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agreement between MY09/11 PCR and type-specific PCR (Coutlée et al., 2006; Landis and Koch, 1977). The chi-square (2) or Fisher's exact test was used to evaluate the statistical
SC
significance of the difference between groups. The statistical analysis was conducted using the Statistical Package for the Social Sciences (SSPS) 16.0 software (SPSS, Inc., Chicago, IL,
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USA).
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RESULTS
The study group consisted of 654 patients with a mean age of 37.4 years (±8.74), a
PT
median age of 37 years, and a distribution range of 17-81 years. Of the 654 samples, 34.1%
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(223/654) were HPV DNA positive, as indicated by at least one of the two methods, and the remaining 65.9% (431/654) were negative according to both methods. Excellent agreement
AC
(92.6% and kappa: 0.827) was found between the results of the two methods. The relative sensitivities of MY09/11 PCR and the type-specific PCR were 80.7% (180/223) and 97.8% (218/223), respectively (both tests were performed for all samples, and if either was positive, the result was recorded as positive). Genotyping was successfully carried out in 218 samples by type-specific PCR, but 5 samples were positive by consensus PCR only and could not be genotyped by type-specific PCR. A single HPV type was detected in 57.3% (125/218) of the genotyped samples, and multiple HPV types were identified in the remaining 42.7% (93/218). Two types were detected in 64: 3 types in 25, 4 types in 2, 5 types in 1, and 11 types in 1 (types 6, 11, 16, 39, 51, 52, 53, 59, 66, 68, and 82) of the multiple-infected patients. In total, 352 different isolates (66 low-risk 7
ACCEPTED MANUSCRIPT and 286 high-risk or probable high-risk HPV) were identified in 218 samples by type-specific PCR. The distribution of the 286 high-risk or probable high-risk HPVs was as follows: 24.5% HPV-16, 8.4% HPV-52, 7.7% HPV-51, 6.3% HPV-39, 6.3% HPV-82, 5.6% HPV-35, 5.6%
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HPV-58, 5.6% HPV-66, 5.2% HPV-18, 5.2% HPV-68, 4.9% HPV-53, 4.5% HPV-31, 2.8%
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HPV-59, 2.4% HPV-45, 1.7% HPV-33, 1.7% HPV-56, 0.7% HPV-26, and 0.7% HPV-73. Unidentified isolates and the low-risk isolates HPV-6 and HPV-11 were not included in this
SC
percentage. The type distribution of all HPVs is shown in Fig. 1. By using plasmid dilutions,
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the detection sensitivities of type-specific PCR assays for all HPV types were determined as 10
ED
copies/reaction.
MY09/11 PCR could not detect the presence of HPV DNA in 19.3% (43/223) of
PT
positive samples. The false negative rates for MY09/11 PCR in single and multiple infections were 18.4% (23/125) and 21.5% (20/93), respectively. Although missing rates for multiple
CE
infections were higher than they were for single infections, there was no statistically significant
AC
difference between groups (p: 0.569). However, when HPV-6 and HPV-11, which have highly similar sequences for MY09/11 primer binding sites and MY09/11 PCR amplicons (Table 4), were considered as a monotype and our data were reevaluated, the false negative rates were 17.4% (23/132) in single infections, 23.3% (20/86) in multiple infections, and 34.6% (9/26) in multiple infections that contained three or more HPV types. In the last situation, there was no statistically significant difference between single and multiple infections (p: 0.290), but a statistically important difference was found between single and multiple infections that contained three or more HPV types (p: 0.046). Cytological abnormalities were detected in 20.9% (137/654) of all samples, and the abnormal cytology rates for single and multiple infections were 43.2% (54/125) and 54.8% (51/93), respectively (odds ratio: 1.27) (Table 2). In addition, the HSIL rates for single and 8
ACCEPTED MANUSCRIPT
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multiple infections were 8.8% (11/125) and 12.9% (12/93), respectively (odds ratio: 1.47).
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According to retrospective data collected from patient records at our institution, cervical/endocervical biopsy was performed in 12 of the 43 patients who were missed by
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findings were reported for the other seven (Table 3).
SC
MY09/11 PCR. While the biopsy results were normal for five patients, different pathological
We classified the HPV types investigated in this study into three groups according to
ED
the number of mismatches between the MY09/11 primers and the target gene regions.
PT
1. Highly matched group; any, or having only one mismatch: HPV types 6, 11, 18, 33, and 58.
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2. Middle-matched group; having two to four mismatches: HPV types 16, 26, 31, 35, 39, 45, 53, 59, 66, and 73.
AC
3. Low-matched group; having six to seven mismatches: HPV types 51, 52, 56, 68, and 82. In single infections, the missing rates for MY09/11 PCR in the highly matched group and the low-matched group were 3.03% (1/33) and 41.7% (10/24), respectively (p:
S0732-8893(14)00210-7 doi: 10.1016/j.diagmicrobio.2014.03.030 DMB 13621
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
21 November 2013 8 February 2014 6 March 2014
Please cite this article as: S¸ahiner Fatih, Kubar Ayhan, G¨ umral Ramazan, Ardı¸c Medine, Yi˘git Nuri, S ¸ ener Kenan, Dede Murat, Yapar Mehmet, Efficiency of MY09/11 consensus PCR in the detection of multiple HPV infections, Diagnostic Microbiology and Infectious Disease (2014), doi: 10.1016/j.diagmicrobio.2014.03.030
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
REVISED Important note: The parts highlighted yellow color: newly added or changed The parts highlighted pink color will be deleted
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Running title: MY09/11 PCR efficiency in multiple infections
T
Title: Efficiency of MY09/11 consensus PCR in the detection of multiple HPV infections
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SC
Fatih Şahiner, Md, Department of Medical Virology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3481, Fax: +90 304 34 02.
Ayhan Kubar, Prof, Department of Medical Virology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3414
PT
ED
Ramazan Gümral, Md, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3433
CE
Medine Ardıç, Ankara Atatürk Training and Research Hospital, Yıldırım Beyazıt University, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 2904
AC
Nuri Yiğit, Md, Department of Medical Pathology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3734
Kenan Şener, Md, Department of Medical Virology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3420
Murat Dede, Md, Department of Obstetrics and Gynecology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 5811
Mehmet Yapar, PhD, Md, Department of Medical Microbiology, Gulhane Military Medical Academy, Ankara, Turkey. (e-mail: [email protected]). Phone: +90 312 304 3436
1
ACCEPTED MANUSCRIPT ABSTRACT Human papillomavirus (HPV) DNA testing has become an important component of cervical cancer screening programs. In this study, we aimed to evaluate the efficiency of
RI P
T
MY09/11 consensus PCR for the detection of multiple HPV infections. For this purpose, MY09/11 PCR was compared to an original TaqMan-based type-specific real-time PCR assay,
SC
which can detect 20 different HPV types. Of the 654 samples, 34.1% (223/654) were HPV DNA positive according to at least one method. The relative sensitivities of MY09/11 PCR and
MA NU
type-specific PCR were 80.7% (180/223) and 97.8% (218/223), respectively. In all, 352 different HPV isolates (66 low-risk and 286 high-risk or probable high-risk types) were identified in 218 samples, but 5 samples, which were positive by consensus PCR only, could not be genotyped. The distribution of the 286 high-risk or probable high-risk HPVs were as
ED
follows: 24.5% HPV-16, 8.4% HPV-52, 7.7% HPV-51, 6.3% HPV-39, 6.3% HPV-82, 5.6%
PT
HPV-35, 5.6% HPV-58, 5.6% HPV-66, 5.2% HPV-18, 5.2% HPV-68, and 19.6% the other 8 types. A single HPV type was detected in 57.3% (125/218) of the genotyped samples, and
CE
multiple HPV types were found in the remaining 42.7% (93/218). The false negative rates of MY09/11 PCR were found to be 17.4% in single infections, 23.3% in multiple infections, and
AC
34.6% in multiple infections that contained three or more HPV types, with the condition that the low-risk types HPV-6 and HPV-11 be considered as a monotype. These data suggest that broad-range PCR assays may lead to significant data loss and that type-specific PCR assays can provide accurate and reliable results during cervical cancer screening.
Key words: Cervical smear; genotyping; type distribution; mismatches.
2
ACCEPTED MANUSCRIPT INTRODUCTION To date, more than 200 human papillomavirus (HPV) types have been identified, and all of them exhibit a specific tropism for the squamous epithelium (Conway and Meyers,
RI P
T
2009). HPV types have been classified as high-risk or low-risk based on their oncogenic potential (Muñoz et al., 2003). The low-risk types cause benign lesions, such as genital warts
SC
and low-grade squamous intraepithelial lesions (LSILs), whereas high-risk HPV types can cause a wide variety of illnesses, including benign lesions, high-grade squamous intraepithelial
MA NU
lesions (HSILs), and cancerous lesions (de Villiers et al., 2004; Muñoz et al., 2003). Cervical cancer screening tests, such as the Pap smear and HPV DNA tests, have become increasingly important because cervical cancer is a preventable disease if patients participate in regular screening programs. Today, there are a number of techniques available for the detection of
ED
HPV DNA in clinical samples, and several factors and challenges affect the choice of methods
PT
for detection and genotyping of HPVs. The most important of these are as follows: (i) A number of different HPV types (more than 40) can cause genital infections (zur Hausen, 1996);
CE
(ii) Multiple sequence variations are often observed even in the same HPV genotype (Nindl et al., 1999); (iii) The detection sensitivity of the current methods is variable depending on the
AC
HPV genotypes and the viral DNA copy numbers in the samples examined (Gravitt et al., 2000; Qu et al., 1997); (iv) HPV genotyping is essential for adequate classification of patients into low-risk or high-risk groups and for following up with patients because the natural course of HPV infection is highly variable among different genotypes. However, the capability for detection of multiple infections and the ability to perform genotyping differ among the methods (Gravitt and Viscidi, 2004; Molijn et al., 2005; Sabol et al., 2008); (v) Finally, different degrees of target-DNA degradation may occur for some HPV genotypes during the integration of viral DNA into the host genome. By considering all these factors, a number of diagnostic techniques have been developed for the molecular diagnosis of HPV infections. Each of these methods has their own unique strong and weak features (Molijn et al., 2005). 3
ACCEPTED MANUSCRIPT The consensus polymerase chain reaction (PCR) assays (MY09/11, GP5+/6+, PGMY09/11, SPF-10) are designed to detect most genital HPV types in a single PCR reaction. MY09/11 consensus PCR, which targets a 450 bp conserved sequence in the HPV L1 gene, is
T
one of the most widely used consensus PCR assays (Depuydt et al., 2007), and it is
RI P
theoretically predicted that this method can detect over 40 different genital HPV types using degenerate primers (Gravitt and Viscidi, 2004). Another diagnostic approach is the type-
SC
specific real-time PCR assay, which is a highly sensitive method that is able to perform
MA NU
genotyping and viral DNA quantifying simultaneously in clinical samples (Depuydt et al., 2007; Şahiner et al., 2012). The L1 and E6-E7 gene regions are often preferred targets for the type-specific detection of HPV genotypes by real-time PCR assays (Depuydt et al., 2007; Dictor and Warenholt, 2011; Lee et al., 2011; Şahiner et al., 2012). The main aim of this study
ED
was to evaluate the effectiveness of MY09/11 consensus PCR and thus to draw attention to the
CE
and associated lesions.
PT
importance of the choice of method used for the diagnosis and follow-up of HPV infections
AC
MATERIAL AND METHOD Study group: The study was performed upon approval by the local ethical committee and after informed consent was obtained from the study participants. In all, 654 women who visited our gynecology outpatient clinic were included in this study. Two samples were collected from each patient simultaneously; one was sent to our laboratory for HPV DNA analysis, and the other was sent to the pathology laboratory for cytological examination. Repeat samples from the same patients were excluded from the study. Cytological examination: Exfoliated cervical cells were obtained using a liquid-based collection system (BD SurePath™, BD Diagnostics, USA) and interpreted according to the modified Bethesda system (Solomon et al., 2002).
4
ACCEPTED MANUSCRIPT DNA isolation: Genomic DNA templates were extracted using the standard phenolchloroform-isoamyl alcohol method from the smear samples (Sambrook et al., 1989). Briefly, cervical smear samples were suspended in 500 μl of TE buffer (10 mM Trishydrochloride, 1
T
mM EDTA, pH 8), and homogenized by vigorous mixing on a vortex. A 100 μl of mixed
RI P
specimen was placed in 10 μl of protease solution (65 mg/ml) (Sigma-Aldrich Corp, St. Louis, MO, USA) and 250 μl of K buffer for 60 min at 45°C. Following centrifugation at 10,000 g for
SC
10 min at 12°C, DNA was extracted from the supernatant using a mixture of 250 μl alkali
MA NU
phenol and 250 μl chloroform-isoamyl alcohol (24 : 1), and then precipitated using 500 μl isopropyl alcohol. DNA was washed in 75% ethyl alcohol at 10,000 g for 5 min at 4°C, airdried at 37°C, and dissolved in 100 μl distilled water (Şahiner et al, 2014). Consensus PCR reactions: All samples were tested by a previously described method using
ED
MY09/11 primers (Bauer et al., 1992) (MY09: 5'-cgtccmarrggawactgatc-3') (MY11: 5'-
PT
gcmcagggwcataayaatgg-3'). After completion of 40 cycles of this SYBR green-based real-time PCR, melting-curve data were obtained by continuous fluorescence acquisition from 55 to
CE
95°C with a thermal transition rate of 0.1 °C/s. The melting temperature of MY09/11 PCR products was determined as 82±1.5°C by optimization studies for our laboratory conditions
AC
(Şahiner et al, 2014).
Type-specific real-time PCR reactions: All smear samples were tested for the presence of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82 using previously described TaqMan-based type-specific real-time PCR assay (Şahiner et al., 2012, Şahiner et al., 2014). The genotype spectrum of these methods was expanded to include all high-risk HPVs (Table 1). All type-specific primers and TaqMan probes using in this study were designed to target the L1 gene region. The reactions were carried out separately for each different HPV type. The reaction mixture was prepared as follows: 1.25 U Hot Start Taq DNA polymerase (Bioron, Germany), 10 pmol of each primer, 2.5 pmol TaqMan probe, 2.5 mM MgCl2, and 0.2 mM dNTP mix. PCR amplifications were conducted after the addition of 5 μl 5
ACCEPTED MANUSCRIPT of the sample containing the template DNA in a final volume of 25 µL. The PCR amplification cycles were as follows: initial denaturation and at hot-start Taq DNA polymerase activation at 95°C for 10 min, followed by 40 amplification cycles at 95°C for 15 sec and at 60°C for 1 min
T
(annealing-extension step). The TaqMan probes were labeled with fluorescent reporter dyes
RI P
(FAM; 6-carboxy fluorescein and JOE; 6-carboxy-4',5'-dichloro-2',7'-dimethoxy-fluorescein) at the 5' end and with a black hole quencher (BHQ) as the non-fluorescent quencher at the 3' end.
SC
The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an
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internal control, and the PCR mixture without template DNA was used as a negative control in all PCR reactions. The L1 amplicons of each of the 20 HPV genotypes and the GAPDH amplicon were cloned into plasmid vectors using a TOPO TA Cloning System (Invitrogen, USA), and the detection sensitivities of the PCR assays were analyzed using serial plasmid
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dilutions (108-101 copies/ml). All PCR reactions were performed on an ABI Prism 7500
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Sequence Detection system (Applied Biosystems, USA). Genomic analyses (using databases and software): The primers and probes were designed
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using the OligoYap 4.0 software program (Yapar et al., 2005) and confirmed with a new software program developed in our laboratory for molecular biology research (OligoBee 1.0).
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All primer and probe sequences were analyzed with the GenBank BLAST database for specificity and were synthesized (MWG-Biotech, Ebersberg, Germany). The mismatches between MY09/11 primers and target gene regions and the nucleotide sequence similarities of MY09/11 amplicons of 20 different HPV types were analyzed using the NCBI Nucleotide Blast program and the OligoBee 1.0 software application. Agarose gel electrophoresis (AGE): PCR products were viewed by gel electrophoresis to differentiate HPV-6 and HPV-11 because the same probe was used for both HPV types in typespecific PCR reactions. Agarose gels were prepared for electrophoresis at 2.0%. Amplicons were run in the agarose gel in a 0.5X TAE buffer at 120 volts, maintained at a constant current for 35 min, and viewed and photographed using the Gel Doc 2000 gel documentation system 6
ACCEPTED MANUSCRIPT (Bio-Rad) under UV light. A 50-bp DNA ladder was used to determine the amplicon sizes (Invitrogen, USA). Statistical analysis: The unweighted kappa statistic was calculated to adjust for chance
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agreement between MY09/11 PCR and type-specific PCR (Coutlée et al., 2006; Landis and Koch, 1977). The chi-square (2) or Fisher's exact test was used to evaluate the statistical
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significance of the difference between groups. The statistical analysis was conducted using the Statistical Package for the Social Sciences (SSPS) 16.0 software (SPSS, Inc., Chicago, IL,
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USA).
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RESULTS
The study group consisted of 654 patients with a mean age of 37.4 years (±8.74), a
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median age of 37 years, and a distribution range of 17-81 years. Of the 654 samples, 34.1%
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(223/654) were HPV DNA positive, as indicated by at least one of the two methods, and the remaining 65.9% (431/654) were negative according to both methods. Excellent agreement
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(92.6% and kappa: 0.827) was found between the results of the two methods. The relative sensitivities of MY09/11 PCR and the type-specific PCR were 80.7% (180/223) and 97.8% (218/223), respectively (both tests were performed for all samples, and if either was positive, the result was recorded as positive). Genotyping was successfully carried out in 218 samples by type-specific PCR, but 5 samples were positive by consensus PCR only and could not be genotyped by type-specific PCR. A single HPV type was detected in 57.3% (125/218) of the genotyped samples, and multiple HPV types were identified in the remaining 42.7% (93/218). Two types were detected in 64: 3 types in 25, 4 types in 2, 5 types in 1, and 11 types in 1 (types 6, 11, 16, 39, 51, 52, 53, 59, 66, 68, and 82) of the multiple-infected patients. In total, 352 different isolates (66 low-risk 7
ACCEPTED MANUSCRIPT and 286 high-risk or probable high-risk HPV) were identified in 218 samples by type-specific PCR. The distribution of the 286 high-risk or probable high-risk HPVs was as follows: 24.5% HPV-16, 8.4% HPV-52, 7.7% HPV-51, 6.3% HPV-39, 6.3% HPV-82, 5.6% HPV-35, 5.6%
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HPV-58, 5.6% HPV-66, 5.2% HPV-18, 5.2% HPV-68, 4.9% HPV-53, 4.5% HPV-31, 2.8%
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HPV-59, 2.4% HPV-45, 1.7% HPV-33, 1.7% HPV-56, 0.7% HPV-26, and 0.7% HPV-73. Unidentified isolates and the low-risk isolates HPV-6 and HPV-11 were not included in this
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percentage. The type distribution of all HPVs is shown in Fig. 1. By using plasmid dilutions,
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the detection sensitivities of type-specific PCR assays for all HPV types were determined as 10
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copies/reaction.
MY09/11 PCR could not detect the presence of HPV DNA in 19.3% (43/223) of
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positive samples. The false negative rates for MY09/11 PCR in single and multiple infections were 18.4% (23/125) and 21.5% (20/93), respectively. Although missing rates for multiple
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infections were higher than they were for single infections, there was no statistically significant
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difference between groups (p: 0.569). However, when HPV-6 and HPV-11, which have highly similar sequences for MY09/11 primer binding sites and MY09/11 PCR amplicons (Table 4), were considered as a monotype and our data were reevaluated, the false negative rates were 17.4% (23/132) in single infections, 23.3% (20/86) in multiple infections, and 34.6% (9/26) in multiple infections that contained three or more HPV types. In the last situation, there was no statistically significant difference between single and multiple infections (p: 0.290), but a statistically important difference was found between single and multiple infections that contained three or more HPV types (p: 0.046). Cytological abnormalities were detected in 20.9% (137/654) of all samples, and the abnormal cytology rates for single and multiple infections were 43.2% (54/125) and 54.8% (51/93), respectively (odds ratio: 1.27) (Table 2). In addition, the HSIL rates for single and 8
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multiple infections were 8.8% (11/125) and 12.9% (12/93), respectively (odds ratio: 1.47).
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According to retrospective data collected from patient records at our institution, cervical/endocervical biopsy was performed in 12 of the 43 patients who were missed by
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findings were reported for the other seven (Table 3).
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MY09/11 PCR. While the biopsy results were normal for five patients, different pathological
We classified the HPV types investigated in this study into three groups according to
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the number of mismatches between the MY09/11 primers and the target gene regions.
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1. Highly matched group; any, or having only one mismatch: HPV types 6, 11, 18, 33, and 58.
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2. Middle-matched group; having two to four mismatches: HPV types 16, 26, 31, 35, 39, 45, 53, 59, 66, and 73.
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3. Low-matched group; having six to seven mismatches: HPV types 51, 52, 56, 68, and 82. In single infections, the missing rates for MY09/11 PCR in the highly matched group and the low-matched group were 3.03% (1/33) and 41.7% (10/24), respectively (p:
