- Email: [email protected]
Automation of the linear array HPV genotyping test and its application for routine typing of human papillomaviruses in cervical specimens of women without cytological abnormalities in Switzerland
Journal of Clinical Virology 45 (2009) 23–27
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Automation of the linear array HPV genotyping test and its application for routine typing of human papillomaviruses in cervical specimens of women without cytological abnormalities in Switzerland Marinko Dobec ∗ , Fridolin Bannwart, Franz Kaeppeli, Pascal Cassinotti Medica, Medizinische Laboratorien Dr. F. Kaeppeli, Wolfbachstrasse 17, CH-8024 Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 29 September 2008 Received in revised form 7 March 2009 Accepted 11 March 2009 Keywords: HPV typing Automated linear array HPV test HPV molecular epidemiology
a b s t r a c t Background: There is a need for reliable, automated high throughput HPV detection and genotyping methods for pre- and post-prophylactic vaccine intervention analyses. Objectives: To optimize the linear array (LA) HPV genotyping test (Roche Diagnostics, Rotkreuz) in regard to possible automation steps for the routine laboratory diagnosis of HPV infections and to analyze the HPV genotype distribution in cervical specimens of women without cytological abnormalities in Switzerland. Study design: 680 cervical cell specimens with normal cytology, obtained from women undergoing routine cervical screening by liquid-based Pap smear, were analyzed by the LA HPV genotyping test for HPV-DNA. Results: The automation of the LA HPV genotyping test resulted in a total hands-on time reduction of 255 min (from 480 to 225 min; 53%). Any of 37 HPV genotypes were detected in 117 (17.2%) and high-risk (HR) HPV in 55 (8.1%) of 680 women with normal cytology. The highest prevalence of any HPV (28.1%) and HR-HPV (15.1%) was observed in age-group 21–30 and showed a continuous decrease in older age-groups. The most common HR-HPV genotypes were HPV-16 (12%), HPV-31 (9.4%), HPV-52 (6%), HPV-51 (5.1%), HPV-45 (4.3%), HPV-58 (4.3%) and HPV-59 (4.3%). Conclusions: The optimization and automation of the LA HPV genotyping test makes it suited for high throughput HPV detection and typing. The epidemiological data provides information about distribution of HPV genotypes in women without cytological abnormalities in Switzerland and may be important for determining the future impact of vaccines and potential changes in the country’s epidemiological HPV profile. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Human papillomaviruses (HPV) are considered as the most common known sexually transmitted agents worldwide.1 There are more than 100 recognized HPV genotypes, of which approximately 40 have tropism specifically for the anogenital mucosa.2 Based on their epidemiological association with cervical cancer, anogenital HPV genotypes are divided into high-risk (HR) and low-risk (LR) types.3 Almost all cases of cervical cancer are caused by persistent infection with about 15 genotypes of HR human papillomavirus.4 In light of the introduction of a vaccine against HPV there is a need for reliable, automated high throughput HPV detection and genotyping methods for pre- and post-prophylactic vaccine intervention analyses.5 In addition, population-based data for HPV genotype distribution is prerequisite to assessment of the effect of
∗ Corresponding author. Tel.: +41 44 269 99 99; fax: +41 44 269 99 09. E-mail address: [email protected] (M. Dobec). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.03.005
future vaccination on HPV infections, but these data are limited or missing for many world regions.6 The current specimen processing protocol for the linear array (LA) HPV genotyping test (Roche Diagnostics, Rotkreuz, Switzerland) is based on a manual DNA extraction protocol. This method of DNA preparation is time-consuming and labor-intensive and is prone to potential specimen cross-contamination, particularly when large numbers of specimens are being processed.7 Furthermore, the hybridization step is performed using a water-bath, which is cumbersome and does not meet the requirements of a modern routine diagnostic laboratory. Finally, data analysis, interpretation, transfer to laboratory database and storage are also part of manual procedure. The purpose of our study was to optimize the LA HPV genotyping test in regard to possible automation steps for the routine laboratory diagnosis of HPV infections and to analyze the HPV genotype distribution in cervical specimens of women without cytological abnormalities undergoing routine cervical screening by liquid-based Pap smear in Switzerland.
24
M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27
2. Materials and methods 2.1. Study population and collection of specimens The study comprised 680 cervical specimens obtained from females living in the German speaking area of Switzerland. Selection criterion for these specimens was normal cervical cytology in specimens sent to our laboratory for routine cytological examination by liquid-based Pap smear. Specimens were collected by Cervex brush (Rovers Medical Devices B.V., The Netherlands) and rinsed into ThinPrep vials containing PreservCyt fixative solution (ThinPrep® liquid Pap vial, Cytyc Corporation, Boxborough, MA). Following routine cytological examination, a subsequent HPV detection and genotyping were performed. The age range of women tested for HPV was from 16 to 88 years (mean age, 40 years; median, 38 years). 2.2. Linear array HPV genotyping test The LA HPV genotyping test is a qualitative in vitro test for the detection of human papillomavirus in clinical specimens. The test amplifies target HPV-DNA by the polymerase chain reaction (PCR) followed by nucleic acid hybridization and colorimetric detection and allows the detection of 37 HPV-DNA genotypes (Table 1). 2.3. DNA extraction For automated DNA extraction from cervical cells in PreservCyt Solution, a procedure was specifically developed in our laboratory for the Cobas AmpliPrep instrument (Roche Diagnostics, Rotkreuz, Switzerland) as follows. To 750 l of specimen 240 l of tissue lysis buffer ATL (Qiagen, Hombrechtikon, Switzerland) and 60 l Qiagen proteinase K at >600 mAU/ml (Qiagen, Hombrechtikon, Switzerland) were added. The 1050 l resulting mix was incubated at 56 ◦ C for 30 min prior to extraction with the COBAS AmpliPrep Total Nucleic Acid Isolation Kit TNAI (Roche Diagnostics, Rotkreuz, Switzerland). The extracted DNA was eluted in 75 l of elution buffer. 2.4. PCR amplification of HPV-DNA The LA HPV genotyping test (Roche) uses biotinylated primers to amplify a 450 base pair fragment within the polymorphic L1 region of the HPV genome. A mixture of HPV specific primers allows amplification of DNA from 37 different HPV genotypes (Table 1). In addition, a 268 base pair fragment of the cellular -globin gene was amplified simultaneously in order to assess cellular adequacy as well as extraction and amplification steps. Selective amplification of target nucleic acid from the specimen and carry-over contamination prevention was achieved by the incorporation of dUTP and inclusion of the enzyme AmpErase (uracil-N-glycosylase) in the master-mix. Each 100 l amplimix consisted of 12 l processed specimen, 50 l working master-mix and 38 l molecular biology grade water. PCR amplification was performed with the Applied Biosystems Gold-plated 96-well GeneAmp PCR System 9700 as follows: 2 min Table 1 HPV genotypes detected by the linear array (LA) HPV genotyping test. Carcinogenic risk
HPV genotype
High risk
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 (MM9), 82 (MM4) 26, 53, 66 6, 11, 40, 42, 54, 61, 70, 72, 81, CP6108 55, 62, 64, 67, 69, 71, 83 (MM7), 84 (MM8), IS39
Probably high risk Low risk Indeterminate risk
at 50 ◦ C, 9 min at 95 ◦ C, followed by 40 cycles of 30 s at 95 ◦ C, 1 min at 55 ◦ C and 1 min at 72 ◦ C. After a final extension step lasting 5 min at 72 ◦ C, the HPV and the -globin amplicons were chemically denatured and kept at room temperature (for no longer than 5 h) until hybridization was performed. A positive (HPV genotype 16) and a negative control, provided in the kit, were included in each run. 2.5. Hybridization and detection Following PCR amplification, 75 l aliquots of the denatured HPV and -globin amplicons were transferred to the corresponding well of a typing tray containing hybridization buffer and a single linear array genotyping strip coated with HPV and -globin probe lines. Instead of performing hybridization and genotype detection manually in a water bath, these steps were automated using the Tecan ProfiBlot 48 instrument (Tecan, Maennedorf, Switzerland). The ProfiBlot 48 allows the processing of up to 48 strips per run with a stored program identical to the linear array HPV genotyping test protocol. The biotin-labelled amplicons hybridized to the oligonucleotide probes on the strip forming a blue coloured complex after successive addition of streptavidin-horseradish peroxidase conjugate and substrate. 2.6. Result interpretation Instead of manually reading the strips using the LA HPV genotyping test Reference Guide included in the kit, they were scanned and analyzed using the Linear Array Image Analysis Software LAIAS v.2.3.1 (Roche Diagnostics, Rotkreuz, Switzerland). LAIAS is a Roche proprietary software enabling the scanning, reading and interpreting of LA HPV genotyping tests. HPV data interpreted with the LAIAS software were electronically exported from the LAIAS platform to the laboratory information system (LIS). The use of the LAIAS software obviated manual interpretation of the strips as well as manual entry of the results in the laboratory database. In addition, the digitalized strips, once saved and archived, remain available for later retrieval and backup purposes. 2.7. Statistical analysis For the evaluation of the test results 95% confidence interval (CI) for proportions based on Agresti and Coull was applied.8 3. Results 3.1. LA HPV genotyping test performance The implementation of several automated steps in the LA HPV genotyping resulted in significant reduction of hands-on time. The automation of DNA extraction reduced hands-on time by 140 min (from 200 to 60 min). The introduction of ProfiBlot T48 (automated hybridization and detection system, Tecan, Switzerland) instead of the standard LA manual protocol brought an additional reduction in hands-on time of 75 min (from 120 to 45 min). The Linear Array Image Analysis Software v. 2.3.1 (Roche Molecular Systems) reduced hands-on time for data analysis by an additional 40 min (from 110 to 70 min). In summary, optimizations and automations developed and implemented in our laboratory resulted in total hands-on time reduction of 255 min (from 480 to 225 min; 53%), based on the evaluation of 46 specimens and two controls (Table 2). 3.2. Epidemiological data Any HPV-DNA was detected in 117 (17.2%) and HR-HPV in 55 (8.1%) of 680 women with normal cytology (Table 3). The highest
M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27
25
Table 2 Linear array (LA) HPV genotyping test performance using standard and modified LA protocol for 46 samples. HPV genotyping (for 46 samples)
Standard LA protocol
Modified LA protocol
Hands-on time saving (min)
Hands-on time (min)
Method
Hands-on time (min)
Method
DNA extraction PCR amplification Hybridization and Detection Interpretation Total time (min)
200 50 120 110 480
AmpliLute Thermocycler 9700 Manual incubation Manual reading
60 50 45 70 225
Cobas AmpliPrep Thermocycler 9700 ProfiBlot T48 LAIAS Software
140 0 75 40 255
Table 3 Prevalence of any human papillomavirus (HPV) and high-risk (HR) HPV according to age in cervical specimens of 680 women without cytological abnormalities. Age (years)
≤20 21–30 31–40 41–50 51–60 >60 Total
No. of specimens tested (%)
40 (5.9) 139 (20.4) 165 (24.3) 165 (24.3) 100 (14.7) 71 (10.4) 680 (100)
Any HPV positive
HR-HPV positive
#
Prevalence, % (95% Confidence Interval)
#
Prevalence, % (95% Confidence Interval)
6 39 32 24 14 2
15 (6.7–29.5) 28.1 (21.2–36.1) 19.4 (14.1–26.1) 14.5 (9.9–20.8) 14 (8.4–22.3) 2.8 (0.2–10.3)
3 21 17 8 5 1
7.5 (1.9–20.6) 15.1 (10.0–22.1) 10.3 (6.5–16.0) 4.8 (2.3–9.4) 5 (1.9–11.5) 1.4 (0.1–8.3)
117
17.2 (14.6–20.2)
55
8.1 (6.3–10.4)
Fig. 1. Prevalence of high-risk (HR) human papillomavirus (HPV) and any HPV in cervical specimens of 680 women without cytological abnormalities according to age.
Fig. 2. Prevalence of 15 high-risk (HR) human papillomavirus (HPV) genotypes and low-risk (LR) HPV-6 and HPV-11 in 117 HPV positive samples of 680 women without cytological abnormalities. As HPV-52 positive were interpreted only specimens that hybridize with a cross-reactive 52/33/35/58 probe and that tested negative for HPV-33, HPV-35, and/or HPV-58 DNA. For clinical specimens that tested positive for HPV-33, HPV-35, and/or HPV-58 DNA, co-infection with HPV genotype 52 could not be ruled out.
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M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27
prevalence of any HPV (28.1%) and HR-HPV (15.1%) was observed in age-group 21–30 and showed a continuous decrease in older agegroups (Fig. 1). The seven most common HR-HPV genotypes in HPV positive women with normal cytology were HPV-16 (12%), HPV-31 (9.4%), HPV-52 (6%), HPV-51 (5.1%), HPV-45 (4.3%), HPV-58 (4.3%) and HPV-59 (4.3%). LR-HPV vaccinal genotype 6 was detected in 5 (4.3%) of 117 HPV positive specimens and HPV-11 was not found (Fig. 2). One HPV genotype only was detected in 74 (63.2%) and infection with multiple HPV genotypes was found in 43 (36.8%) of 117 HPV positive specimens [two genotypes were detected in 26 (22.2%), three genotypes in 9 (7.7%), four genotypes in 7 (6%) and five genotypes in 1 (0.9%)].
4. Discussion The LA test is type specific, sensitive and reproducible, but it includes many time-consuming manual steps, from DNA extraction up to result interpretation.7,8–11 The automation of the LA test developed in our laboratory resulted in total hands-on time reduction of 255 min (from 480 to 225 min; 53%). Also, the use of the LAIAS software obviated manual interpretation of the strips as well as manual entry of the results in the laboratory database, thus eliminating potential typing errors. In addition, the digitalized strips, once saved and archived, remain available for later retrieval and backup purposes. This optimization and automation makes the LA test suitable and convenient for high throughput HPV detection and typing. HPV prevalence analysis in cervical specimens of women without cytological abnormalities undergoing routine cervical screening by liquid-based Pap smear in Switzerland showed an overall infection rate of 17.2% which is comparable with studies in Italy and the USA.12,13 Higher HPV prevalence was observed in Germany and in France, but, for these studies samples were not selected randomly and the examined population consisted of hospital patients.14,15 The HR-HPV prevalence was 8.1% and is consistent with previous studies in Switzerland and worldwide.6,13,16,17 HPV positivity was highest among women aged 21–30 years and then decreased at older ages. This agrees with other studies and is most likely related to sexual habit.1,6,12,13 In contrast to some other observations we did not detect a second peak of HPV prevalence in women over 44 years.1 HPV-16 was the most prevalent genotype and is comparable with findings worldwide.18 However, the second most prevalent genotype is HPV-18 in the Western European countries, whereas in our study it is HPV-31.1,6,14,18 These findings have to be interpreted in light of widespread agreement that the vast majority of HPV infections are transient in young women, in that those infections are cleared by the immune system within 1–2 years of exposure without any detectable lesions. With longer HPV persistence of a given genotype, the probability of subsequent clearance over a fixed interval decreases and the risk of precancer diagnosis increases.4 Although HPV infections in women with normal cytology may be transient, the analysis of the HPV prevalence is reflecting the most frequent genotypes circulating in the population studied. Multiple HPV genotype infections (36.8%) were more frequent than in other studies.12,15,19,20 These differences are probably influenced by the sensitivity of the LA HPV genotyping test.10,11 Future studies are necessary to answer how the vaccine will affect other oncogenic strains of HPV and if there will be selective pressure on the remaining strains of HPV.21,22 In summary, the optimization and automation makes the LA test suitable and convenient for high throughput HPV detection and typ-
ing. This new Swiss epidemiological data, providing additional HPV genotype distribution, will be important for determining the impact of vaccines and potential changes in the country’s epidemiological HPV profile. Conflicts of interest No conflicts of interest have been identified. Acknowledgements This study was performed at medica Laboratories, Zurich, Switzerland and our thanks go to Mrs. Liliane Kunz, laboratory technologist from the Laboratory for molecular diagnostics and Mrs. Britt-Marie Boerelius from the Institute of pathology for help in performing tests. We also thank Roche Diagnostics (Switzerland), Roche Molecular Systems and Cytyc (Cytyc Corporation, Boxborough, MA) for the support provided during the study. References ˜ 1. De Sanjosé S, Diaz M, Castellsagué X, Clifford G, Bruni L, Munoz N, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis 2007;7:453–9. 2. Schiffman M, Castle PE. Human papillomavirus: epidemiology and public health. Arch Pathol Lab Med 2003;127:930–4. ˜ 3. Munoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27. 4. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet 2007;370:890–7. 5. FUTURE II Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 2007;356:1915–27. 6. Clifford GM, Gallus S, Herrero R, Munoz N, Snijders PJ, Vaccarella S, et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005;366:991–8. 7. Stevens MP, Rudland E, Garland SM, Tabrizi SN. Assessment of MagNA pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by the Roche AMPLICOR and LINEAR ARRAY HPV tests. J Clin Microbiol 2006;44:2428–33. 8. Agresti A, Coull BA. Approximate is better than “exact” for interval estimation of binomial proportions. Am Stat 1998;52:119–26. 9. Steinau M, Swan DC, Unger ER. Type-specific reproducibility of the Roche linear array HPV genotyping test. J Clin Virol 2008;42:412–4 [epub]. 10. Stevens M, Garland SM, Rudland E, Tan J, Quinn MA, Tabrizi SN. Comparison of the Digene Hybrid Capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal Pap smear results. J Clin Microbiol 2007;45:2130–7. 11. Coutlée F, Rouleau D, Petignat P, Ghattas G, Kornegay JR, Schlag P, et al. Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the Linear array HPV genotyping test. J Clin Microbiol 2006;44:1998–2006. 12. Del Prete R, Di Taranto AM, Lipsi MR, Nirchio V, Antonetti R, Miragliotta G. Prevalence and genotypes identification of human papillomavirus infection in a population of South Italy. J Clin Virol 2008;42:211–4. 13. Dunne EF, Unger ER, Sternberg M, McQuillan G, Swan DC, Patel SS, et al. Prevalence of HPV infection among females in the United States. JAMA 2007;297:813–9. 14. Pannier-Stockman C, Segard C, Bennamar S, Gondry J, Boulanger JC, Sevestre H, et al. Prevalence of HPV genotypes determined by PCR and DNA sequencing in cervical specimens from French women with or without abnormalities. J Clin Virol 2008;42:353–60 [epub]. 15. Speich N, Schmitt C, Bollmann R, Bollmann M. Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing: genotype spectrum of patients from the west German area. J Med Microbiol 2004;53:125–8. 16. Boulanger JC, Sevestre H, Bauville E, Ghighi C, Harlicot JP, Gondry J. Epidemiology of HPV infection. Gynecol Obstet Fertil 2004;32:218–23. 17. Petignat P, Faltin D, Goffin F, Billieux MH, Stucki D, Sporri S, et al. Age-related performance of human papillomavirus testing used as an adjunct to cytology for cervical carcinoma screening in a population with a low incidence of cervical carcinoma. Cancer 2005;105:126–32. 18. Clifford G, Franceschi S, Diaz M, Munoz N, Villa LL. Chapter 3: HPV type distribution in women with and without cervical neoplastic diseases. Vaccine 2006;24(Suppl. 3):S26–34.
M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27 19. Hwang HS, Park M, Lee SY, Kwon KH, Pang MG. Distribution and prevalence of human papillomavirus genotypes in routine pap smear of 2470 Korean women determined by DNA chip. Cancer Epidemiol Biomarkers Prev 2004;13: 2153–6. 20. Sowjanya AP, Jain M, Poli UR, Padma S, Das M, Shah KV, et al. Prevalence and distribution of high-risk human papilloma virus (HPV) types in invasive squa-
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mous cell carcinoma of the cervix and in normal women in Andhra Pradesh, India. BMC Infect Dis 2005;5:116. 21. Haug CJ. Human papillomavirus vaccination—reasons for caution. N Engl J Med 2008;359:861–2. 22. Kim JJ, Goldie SJ. Health and economic implications of HPV vaccination in the United States. N Engl J Med 2008;359:821–32.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Automation of the linear array HPV genotyping test and its application for routine typing of human papillomaviruses in cervical specimens of women without cytological abnormalities in Switzerland Marinko Dobec ∗ , Fridolin Bannwart, Franz Kaeppeli, Pascal Cassinotti Medica, Medizinische Laboratorien Dr. F. Kaeppeli, Wolfbachstrasse 17, CH-8024 Zurich, Switzerland
a r t i c l e
i n f o
Article history: Received 29 September 2008 Received in revised form 7 March 2009 Accepted 11 March 2009 Keywords: HPV typing Automated linear array HPV test HPV molecular epidemiology
a b s t r a c t Background: There is a need for reliable, automated high throughput HPV detection and genotyping methods for pre- and post-prophylactic vaccine intervention analyses. Objectives: To optimize the linear array (LA) HPV genotyping test (Roche Diagnostics, Rotkreuz) in regard to possible automation steps for the routine laboratory diagnosis of HPV infections and to analyze the HPV genotype distribution in cervical specimens of women without cytological abnormalities in Switzerland. Study design: 680 cervical cell specimens with normal cytology, obtained from women undergoing routine cervical screening by liquid-based Pap smear, were analyzed by the LA HPV genotyping test for HPV-DNA. Results: The automation of the LA HPV genotyping test resulted in a total hands-on time reduction of 255 min (from 480 to 225 min; 53%). Any of 37 HPV genotypes were detected in 117 (17.2%) and high-risk (HR) HPV in 55 (8.1%) of 680 women with normal cytology. The highest prevalence of any HPV (28.1%) and HR-HPV (15.1%) was observed in age-group 21–30 and showed a continuous decrease in older age-groups. The most common HR-HPV genotypes were HPV-16 (12%), HPV-31 (9.4%), HPV-52 (6%), HPV-51 (5.1%), HPV-45 (4.3%), HPV-58 (4.3%) and HPV-59 (4.3%). Conclusions: The optimization and automation of the LA HPV genotyping test makes it suited for high throughput HPV detection and typing. The epidemiological data provides information about distribution of HPV genotypes in women without cytological abnormalities in Switzerland and may be important for determining the future impact of vaccines and potential changes in the country’s epidemiological HPV profile. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Human papillomaviruses (HPV) are considered as the most common known sexually transmitted agents worldwide.1 There are more than 100 recognized HPV genotypes, of which approximately 40 have tropism specifically for the anogenital mucosa.2 Based on their epidemiological association with cervical cancer, anogenital HPV genotypes are divided into high-risk (HR) and low-risk (LR) types.3 Almost all cases of cervical cancer are caused by persistent infection with about 15 genotypes of HR human papillomavirus.4 In light of the introduction of a vaccine against HPV there is a need for reliable, automated high throughput HPV detection and genotyping methods for pre- and post-prophylactic vaccine intervention analyses.5 In addition, population-based data for HPV genotype distribution is prerequisite to assessment of the effect of
∗ Corresponding author. Tel.: +41 44 269 99 99; fax: +41 44 269 99 09. E-mail address: [email protected] (M. Dobec). 1386-6532/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2009.03.005
future vaccination on HPV infections, but these data are limited or missing for many world regions.6 The current specimen processing protocol for the linear array (LA) HPV genotyping test (Roche Diagnostics, Rotkreuz, Switzerland) is based on a manual DNA extraction protocol. This method of DNA preparation is time-consuming and labor-intensive and is prone to potential specimen cross-contamination, particularly when large numbers of specimens are being processed.7 Furthermore, the hybridization step is performed using a water-bath, which is cumbersome and does not meet the requirements of a modern routine diagnostic laboratory. Finally, data analysis, interpretation, transfer to laboratory database and storage are also part of manual procedure. The purpose of our study was to optimize the LA HPV genotyping test in regard to possible automation steps for the routine laboratory diagnosis of HPV infections and to analyze the HPV genotype distribution in cervical specimens of women without cytological abnormalities undergoing routine cervical screening by liquid-based Pap smear in Switzerland.
24
M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27
2. Materials and methods 2.1. Study population and collection of specimens The study comprised 680 cervical specimens obtained from females living in the German speaking area of Switzerland. Selection criterion for these specimens was normal cervical cytology in specimens sent to our laboratory for routine cytological examination by liquid-based Pap smear. Specimens were collected by Cervex brush (Rovers Medical Devices B.V., The Netherlands) and rinsed into ThinPrep vials containing PreservCyt fixative solution (ThinPrep® liquid Pap vial, Cytyc Corporation, Boxborough, MA). Following routine cytological examination, a subsequent HPV detection and genotyping were performed. The age range of women tested for HPV was from 16 to 88 years (mean age, 40 years; median, 38 years). 2.2. Linear array HPV genotyping test The LA HPV genotyping test is a qualitative in vitro test for the detection of human papillomavirus in clinical specimens. The test amplifies target HPV-DNA by the polymerase chain reaction (PCR) followed by nucleic acid hybridization and colorimetric detection and allows the detection of 37 HPV-DNA genotypes (Table 1). 2.3. DNA extraction For automated DNA extraction from cervical cells in PreservCyt Solution, a procedure was specifically developed in our laboratory for the Cobas AmpliPrep instrument (Roche Diagnostics, Rotkreuz, Switzerland) as follows. To 750 l of specimen 240 l of tissue lysis buffer ATL (Qiagen, Hombrechtikon, Switzerland) and 60 l Qiagen proteinase K at >600 mAU/ml (Qiagen, Hombrechtikon, Switzerland) were added. The 1050 l resulting mix was incubated at 56 ◦ C for 30 min prior to extraction with the COBAS AmpliPrep Total Nucleic Acid Isolation Kit TNAI (Roche Diagnostics, Rotkreuz, Switzerland). The extracted DNA was eluted in 75 l of elution buffer. 2.4. PCR amplification of HPV-DNA The LA HPV genotyping test (Roche) uses biotinylated primers to amplify a 450 base pair fragment within the polymorphic L1 region of the HPV genome. A mixture of HPV specific primers allows amplification of DNA from 37 different HPV genotypes (Table 1). In addition, a 268 base pair fragment of the cellular -globin gene was amplified simultaneously in order to assess cellular adequacy as well as extraction and amplification steps. Selective amplification of target nucleic acid from the specimen and carry-over contamination prevention was achieved by the incorporation of dUTP and inclusion of the enzyme AmpErase (uracil-N-glycosylase) in the master-mix. Each 100 l amplimix consisted of 12 l processed specimen, 50 l working master-mix and 38 l molecular biology grade water. PCR amplification was performed with the Applied Biosystems Gold-plated 96-well GeneAmp PCR System 9700 as follows: 2 min Table 1 HPV genotypes detected by the linear array (LA) HPV genotyping test. Carcinogenic risk
HPV genotype
High risk
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73 (MM9), 82 (MM4) 26, 53, 66 6, 11, 40, 42, 54, 61, 70, 72, 81, CP6108 55, 62, 64, 67, 69, 71, 83 (MM7), 84 (MM8), IS39
Probably high risk Low risk Indeterminate risk
at 50 ◦ C, 9 min at 95 ◦ C, followed by 40 cycles of 30 s at 95 ◦ C, 1 min at 55 ◦ C and 1 min at 72 ◦ C. After a final extension step lasting 5 min at 72 ◦ C, the HPV and the -globin amplicons were chemically denatured and kept at room temperature (for no longer than 5 h) until hybridization was performed. A positive (HPV genotype 16) and a negative control, provided in the kit, were included in each run. 2.5. Hybridization and detection Following PCR amplification, 75 l aliquots of the denatured HPV and -globin amplicons were transferred to the corresponding well of a typing tray containing hybridization buffer and a single linear array genotyping strip coated with HPV and -globin probe lines. Instead of performing hybridization and genotype detection manually in a water bath, these steps were automated using the Tecan ProfiBlot 48 instrument (Tecan, Maennedorf, Switzerland). The ProfiBlot 48 allows the processing of up to 48 strips per run with a stored program identical to the linear array HPV genotyping test protocol. The biotin-labelled amplicons hybridized to the oligonucleotide probes on the strip forming a blue coloured complex after successive addition of streptavidin-horseradish peroxidase conjugate and substrate. 2.6. Result interpretation Instead of manually reading the strips using the LA HPV genotyping test Reference Guide included in the kit, they were scanned and analyzed using the Linear Array Image Analysis Software LAIAS v.2.3.1 (Roche Diagnostics, Rotkreuz, Switzerland). LAIAS is a Roche proprietary software enabling the scanning, reading and interpreting of LA HPV genotyping tests. HPV data interpreted with the LAIAS software were electronically exported from the LAIAS platform to the laboratory information system (LIS). The use of the LAIAS software obviated manual interpretation of the strips as well as manual entry of the results in the laboratory database. In addition, the digitalized strips, once saved and archived, remain available for later retrieval and backup purposes. 2.7. Statistical analysis For the evaluation of the test results 95% confidence interval (CI) for proportions based on Agresti and Coull was applied.8 3. Results 3.1. LA HPV genotyping test performance The implementation of several automated steps in the LA HPV genotyping resulted in significant reduction of hands-on time. The automation of DNA extraction reduced hands-on time by 140 min (from 200 to 60 min). The introduction of ProfiBlot T48 (automated hybridization and detection system, Tecan, Switzerland) instead of the standard LA manual protocol brought an additional reduction in hands-on time of 75 min (from 120 to 45 min). The Linear Array Image Analysis Software v. 2.3.1 (Roche Molecular Systems) reduced hands-on time for data analysis by an additional 40 min (from 110 to 70 min). In summary, optimizations and automations developed and implemented in our laboratory resulted in total hands-on time reduction of 255 min (from 480 to 225 min; 53%), based on the evaluation of 46 specimens and two controls (Table 2). 3.2. Epidemiological data Any HPV-DNA was detected in 117 (17.2%) and HR-HPV in 55 (8.1%) of 680 women with normal cytology (Table 3). The highest
M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27
25
Table 2 Linear array (LA) HPV genotyping test performance using standard and modified LA protocol for 46 samples. HPV genotyping (for 46 samples)
Standard LA protocol
Modified LA protocol
Hands-on time saving (min)
Hands-on time (min)
Method
Hands-on time (min)
Method
DNA extraction PCR amplification Hybridization and Detection Interpretation Total time (min)
200 50 120 110 480
AmpliLute Thermocycler 9700 Manual incubation Manual reading
60 50 45 70 225
Cobas AmpliPrep Thermocycler 9700 ProfiBlot T48 LAIAS Software
140 0 75 40 255
Table 3 Prevalence of any human papillomavirus (HPV) and high-risk (HR) HPV according to age in cervical specimens of 680 women without cytological abnormalities. Age (years)
≤20 21–30 31–40 41–50 51–60 >60 Total
No. of specimens tested (%)
40 (5.9) 139 (20.4) 165 (24.3) 165 (24.3) 100 (14.7) 71 (10.4) 680 (100)
Any HPV positive
HR-HPV positive
#
Prevalence, % (95% Confidence Interval)
#
Prevalence, % (95% Confidence Interval)
6 39 32 24 14 2
15 (6.7–29.5) 28.1 (21.2–36.1) 19.4 (14.1–26.1) 14.5 (9.9–20.8) 14 (8.4–22.3) 2.8 (0.2–10.3)
3 21 17 8 5 1
7.5 (1.9–20.6) 15.1 (10.0–22.1) 10.3 (6.5–16.0) 4.8 (2.3–9.4) 5 (1.9–11.5) 1.4 (0.1–8.3)
117
17.2 (14.6–20.2)
55
8.1 (6.3–10.4)
Fig. 1. Prevalence of high-risk (HR) human papillomavirus (HPV) and any HPV in cervical specimens of 680 women without cytological abnormalities according to age.
Fig. 2. Prevalence of 15 high-risk (HR) human papillomavirus (HPV) genotypes and low-risk (LR) HPV-6 and HPV-11 in 117 HPV positive samples of 680 women without cytological abnormalities. As HPV-52 positive were interpreted only specimens that hybridize with a cross-reactive 52/33/35/58 probe and that tested negative for HPV-33, HPV-35, and/or HPV-58 DNA. For clinical specimens that tested positive for HPV-33, HPV-35, and/or HPV-58 DNA, co-infection with HPV genotype 52 could not be ruled out.
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M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27
prevalence of any HPV (28.1%) and HR-HPV (15.1%) was observed in age-group 21–30 and showed a continuous decrease in older agegroups (Fig. 1). The seven most common HR-HPV genotypes in HPV positive women with normal cytology were HPV-16 (12%), HPV-31 (9.4%), HPV-52 (6%), HPV-51 (5.1%), HPV-45 (4.3%), HPV-58 (4.3%) and HPV-59 (4.3%). LR-HPV vaccinal genotype 6 was detected in 5 (4.3%) of 117 HPV positive specimens and HPV-11 was not found (Fig. 2). One HPV genotype only was detected in 74 (63.2%) and infection with multiple HPV genotypes was found in 43 (36.8%) of 117 HPV positive specimens [two genotypes were detected in 26 (22.2%), three genotypes in 9 (7.7%), four genotypes in 7 (6%) and five genotypes in 1 (0.9%)].
4. Discussion The LA test is type specific, sensitive and reproducible, but it includes many time-consuming manual steps, from DNA extraction up to result interpretation.7,8–11 The automation of the LA test developed in our laboratory resulted in total hands-on time reduction of 255 min (from 480 to 225 min; 53%). Also, the use of the LAIAS software obviated manual interpretation of the strips as well as manual entry of the results in the laboratory database, thus eliminating potential typing errors. In addition, the digitalized strips, once saved and archived, remain available for later retrieval and backup purposes. This optimization and automation makes the LA test suitable and convenient for high throughput HPV detection and typing. HPV prevalence analysis in cervical specimens of women without cytological abnormalities undergoing routine cervical screening by liquid-based Pap smear in Switzerland showed an overall infection rate of 17.2% which is comparable with studies in Italy and the USA.12,13 Higher HPV prevalence was observed in Germany and in France, but, for these studies samples were not selected randomly and the examined population consisted of hospital patients.14,15 The HR-HPV prevalence was 8.1% and is consistent with previous studies in Switzerland and worldwide.6,13,16,17 HPV positivity was highest among women aged 21–30 years and then decreased at older ages. This agrees with other studies and is most likely related to sexual habit.1,6,12,13 In contrast to some other observations we did not detect a second peak of HPV prevalence in women over 44 years.1 HPV-16 was the most prevalent genotype and is comparable with findings worldwide.18 However, the second most prevalent genotype is HPV-18 in the Western European countries, whereas in our study it is HPV-31.1,6,14,18 These findings have to be interpreted in light of widespread agreement that the vast majority of HPV infections are transient in young women, in that those infections are cleared by the immune system within 1–2 years of exposure without any detectable lesions. With longer HPV persistence of a given genotype, the probability of subsequent clearance over a fixed interval decreases and the risk of precancer diagnosis increases.4 Although HPV infections in women with normal cytology may be transient, the analysis of the HPV prevalence is reflecting the most frequent genotypes circulating in the population studied. Multiple HPV genotype infections (36.8%) were more frequent than in other studies.12,15,19,20 These differences are probably influenced by the sensitivity of the LA HPV genotyping test.10,11 Future studies are necessary to answer how the vaccine will affect other oncogenic strains of HPV and if there will be selective pressure on the remaining strains of HPV.21,22 In summary, the optimization and automation makes the LA test suitable and convenient for high throughput HPV detection and typ-
ing. This new Swiss epidemiological data, providing additional HPV genotype distribution, will be important for determining the impact of vaccines and potential changes in the country’s epidemiological HPV profile. Conflicts of interest No conflicts of interest have been identified. Acknowledgements This study was performed at medica Laboratories, Zurich, Switzerland and our thanks go to Mrs. Liliane Kunz, laboratory technologist from the Laboratory for molecular diagnostics and Mrs. Britt-Marie Boerelius from the Institute of pathology for help in performing tests. We also thank Roche Diagnostics (Switzerland), Roche Molecular Systems and Cytyc (Cytyc Corporation, Boxborough, MA) for the support provided during the study. References ˜ 1. De Sanjosé S, Diaz M, Castellsagué X, Clifford G, Bruni L, Munoz N, et al. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis 2007;7:453–9. 2. Schiffman M, Castle PE. Human papillomavirus: epidemiology and public health. Arch Pathol Lab Med 2003;127:930–4. ˜ 3. Munoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27. 4. Schiffman M, Castle PE, Jeronimo J, Rodriguez AC, Wacholder S. Human papillomavirus and cervical cancer. Lancet 2007;370:890–7. 5. FUTURE II Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 2007;356:1915–27. 6. Clifford GM, Gallus S, Herrero R, Munoz N, Snijders PJ, Vaccarella S, et al. Worldwide distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. Lancet 2005;366:991–8. 7. Stevens MP, Rudland E, Garland SM, Tabrizi SN. Assessment of MagNA pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by the Roche AMPLICOR and LINEAR ARRAY HPV tests. J Clin Microbiol 2006;44:2428–33. 8. Agresti A, Coull BA. Approximate is better than “exact” for interval estimation of binomial proportions. Am Stat 1998;52:119–26. 9. Steinau M, Swan DC, Unger ER. Type-specific reproducibility of the Roche linear array HPV genotyping test. J Clin Virol 2008;42:412–4 [epub]. 10. Stevens M, Garland SM, Rudland E, Tan J, Quinn MA, Tabrizi SN. Comparison of the Digene Hybrid Capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal Pap smear results. J Clin Microbiol 2007;45:2130–7. 11. Coutlée F, Rouleau D, Petignat P, Ghattas G, Kornegay JR, Schlag P, et al. Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the Linear array HPV genotyping test. J Clin Microbiol 2006;44:1998–2006. 12. Del Prete R, Di Taranto AM, Lipsi MR, Nirchio V, Antonetti R, Miragliotta G. Prevalence and genotypes identification of human papillomavirus infection in a population of South Italy. J Clin Virol 2008;42:211–4. 13. Dunne EF, Unger ER, Sternberg M, McQuillan G, Swan DC, Patel SS, et al. Prevalence of HPV infection among females in the United States. JAMA 2007;297:813–9. 14. Pannier-Stockman C, Segard C, Bennamar S, Gondry J, Boulanger JC, Sevestre H, et al. Prevalence of HPV genotypes determined by PCR and DNA sequencing in cervical specimens from French women with or without abnormalities. J Clin Virol 2008;42:353–60 [epub]. 15. Speich N, Schmitt C, Bollmann R, Bollmann M. Human papillomavirus (HPV) study of 2916 cytological samples by PCR and DNA sequencing: genotype spectrum of patients from the west German area. J Med Microbiol 2004;53:125–8. 16. Boulanger JC, Sevestre H, Bauville E, Ghighi C, Harlicot JP, Gondry J. Epidemiology of HPV infection. Gynecol Obstet Fertil 2004;32:218–23. 17. Petignat P, Faltin D, Goffin F, Billieux MH, Stucki D, Sporri S, et al. Age-related performance of human papillomavirus testing used as an adjunct to cytology for cervical carcinoma screening in a population with a low incidence of cervical carcinoma. Cancer 2005;105:126–32. 18. Clifford G, Franceschi S, Diaz M, Munoz N, Villa LL. Chapter 3: HPV type distribution in women with and without cervical neoplastic diseases. Vaccine 2006;24(Suppl. 3):S26–34.
M. Dobec et al. / Journal of Clinical Virology 45 (2009) 23–27 19. Hwang HS, Park M, Lee SY, Kwon KH, Pang MG. Distribution and prevalence of human papillomavirus genotypes in routine pap smear of 2470 Korean women determined by DNA chip. Cancer Epidemiol Biomarkers Prev 2004;13: 2153–6. 20. Sowjanya AP, Jain M, Poli UR, Padma S, Das M, Shah KV, et al. Prevalence and distribution of high-risk human papilloma virus (HPV) types in invasive squa-
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