Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent

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Journal of Virological Methods 147 (2008) 333–337

Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent Elisabete A. Campos a , Jos´e Antonio Sim˜oes a,∗ , Silvia H. Rabelo-Santos b , Luis Ot´avio Sarian a , Denise Rocha Pitta a , Jos´e Eduardo Levi c , Sophie Derchain a a

Department of Obstetrics and Gynecology, Universidade Estadual de Campinas (UNICAMP), S˜ao Paulo, Brazil b School of Pharmacy, Federal University of Goi´ as (UFG), Goiˆania, Brazil c Laboratory of Virology, Institute of Tropical Medicine, University of S˜ ao Paulo (USP), S˜ao Paulo, Brazil Received 28 July 2007; received in revised form 18 September 2007; accepted 20 September 2007 Available online 5 November 2007

Abstract The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at −20 ◦ C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human ␤-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully. © 2007 Elsevier B.V. All rights reserved. Keywords: HPV DNA; Universal collection medium; Denaturing reagent; Detection; Storage; Cervical cancer; Genotype

1. Introduction Persistent infection with oncogenic types of human papillomavirus (HPV) has been established as the primary risk factor for the development of cervical cancer. The infection is known to be a prerequisite for the development of neoplastic disease (Walboomers et al., 1999) and almost all cases of invasive cervical cancer are HPV-positive. New molecular techniques for the detection of HPV DNA have become available widely for use in research studies, allowing the identification of many different HPV types, including those considered to be of high oncogenic potential (Castellsagu´e et al., 2006; Clifford et al., 2006; Mu˜noz

∗ Corresponding author at: Caixa Postal: 6181, Cidade Universit´ aria Zeferino Vaz, 13.083-970 Campinas, S˜ao Paulo, Brazil. Tel.: +55 19 3289 2856/3521 9306; fax: +55 19 3289 2440. E-mail address: [email protected] (J.A. Sim˜oes).

0166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2007.09.014

et al., 2003; Mun˜oz et al., 2006). As a result, a large number of studies on the epidemiological and clinical behaviour of the different HPV types have been published. Diagnosing HPV infections requires the retrieval of HPV DNA from cells collected from the infection site. The signalamplified hybridization microplate-based Hybrid Capture 2 (HC2, Digene Corp, Gaithersburg, MD, USA) is the only FDA approved assay for detection of carcinogenic HPV DNA using cytological samples for clinical use and that has sufficient scientific data to support its performance in different clinical settings. Therefore, HC2 has become the standard test for HPV detection in many countries and has been used extensively in large clinical studies during the last decade (Arbyn et al., 2006; Carozzi et al., 2007; Clavel et al., 1999). However, this test does not allow determination of specific HPV types. HPV typing is performed typically using the polymerase chain reaction (PCR), and several commercial assays are available currently or under development (Stevens et al., 2006; Schiffman, 2007).

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As a limiting factor to many retrospective studies, however, accurate identification of HPV genotypes depends on a number of technical details. Most important of these is the quality of the DNA sample, which in turn is affected by the presence of denaturing agents, time of storage and the conditions of storage. Other important factors include the quality of HPV primers and the type of polymerase enzyme (Mesquita et al., 2001; Melo et al., 2005; Nonogaki et al., 2004). Several researchers refrain from using their stored HPV samples because of these technical limitations. HC2 testing is approved for use on cervical specimens collected in PreservCyt (ThinPrep, Cytyc, Boxborough, MA, USA) or in specimen transport medium (STM—Digene Corp, Gaithersburg, MA, USA) (Carozzi et al., 2005). HC2 requires alkalinization of specimens that may render the DNA susceptible to degradation. Previous studies have demonstrated that HPV detection with PCR-based methods is also possible in samples preserved in specimen transport medium (STM) after alkalinization (Poljak et al., 2002; Rabelo-Santos et al., 2005). Poljak et al. (2002) described a method for extracting DNA from samples preserved in STM containing denaturing reagent. These researchers were successful in extracting DNA from 98% of the HC2-positive samples that had been frozen at −70 ◦ C. Following a meticulous extraction protocol, Rabelo-Santos et al. (2005) recovered adequate amounts of DNA in 90% of samples stored for 18 months in STM at −20 ◦ C. STM is an aqueous buffer containing a proprietary aliphatic amine designed as a DNA preservative, however, it is not suitable for use in morphological analysis. More recently, universal collection medium (UCM–Digene Brasil, S˜ao Paulo, Brazil) has been proposed as a collection medium for these samples, possibly to permit both cytological evaluation and HPV testing (Taha et al., 2006). Nonogaki et al. (2004) suggested that residual material from the DNACitoliq system (UCM) adequately preserves HPV DNA for detection by HC2 and PCR, with performance similar to that of STM. It is not known, however, how long DNA is preserved adequately in UCM after being exposed to denaturing reagents to permit a posteriori HPV genotyping. The objective of this study was to evaluate the recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents. Samples stored for different periods of time were used, allowing for the assessment of storage time as factor contributing to DNA deterioration after exposure to the denaturing reagent. 2. Materials and methods The sample consisted of 60 HC2 positive cervical specimens, collected between 2003 and 2005 from women with cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. The specimens were collected from women that had received care at the Department of Obstetrics and Gynaecology, School of Medicine Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil. All samples were stored in UCM and had been frozen at −20 ◦ C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for HC2 testing for the identification of HPV

DNA. The samples were stored for 6, 12 and 24 months (20 samples for each storage time). 2.1. DNA extraction from denatured samples Total DNA was extracted from the samples containing the denaturing reagent according to the protocol used by RabeloSantos et al. (2005). Briefly, a 450 ␮l aliquot of the denatured specimen was transferred to a 1.5 ml Eppendorf tube with 1.0 ml precipitation solution composed of 2 ml of 3M NaAc pH 5.2, 200 ␮g of glycogen (prepared with 10 ␮l of 20 mg/ml glycogen solution) and 100 ml of absolute ethanol. Following vortex mixing, the solution remained overnight in a freezer at −70 ◦ C. On the following day, the solution was centrifuged at 12,000 × g for 15 min at 4 ◦ C. The pellet was washed with 400 ␮l of 70% ethanol and then it was centrifuged at 3000 × g for 15 min. The tube was inverted for 30 min to decant the fluid and to allow the ethanol to evaporate, and the pellet was then diluted in a 200 ␮l 1× Tris–EDTA buffer (Tris 1 mM EDTA 100 ␮M, pH 8.2) and stored at −20 ◦ C prior to HPV detection. 2.2. DNA quantitation The quantitation of DNA and its purity were determined by optical density (OD) using spectrophotometry (DU-70, Beckman, CA). The concentration of DNA in solution was measured by reading absorbance at a wavelength of 260 nm (1 OD = 50 ng). Samples were diluted at 1:100 with sterile water in a 1.5 ml tube and transferred to a 1 ml quartz cuvette. DNA purity was then evaluated by means of the absorbance ratio at 260–280 nm. Ratios in the range of 1.7–2.0 indicated pure DNA concentration. The final DNA concentration present in each sample was determined by the formula OD260 × 50 × factor of dilution = ng/␮l, where OD260 represents the absorbance of the samples at 260 nm. 2.3. β-Globin amplification The samples were submitted to PCR with ␤-globin primers G73 (5 -GAAGAGCCAAGGACAGGTAC-3 ) and G74 (5 CAACTTCATCCACGTTCACC-3 ), resulting in a 268 bp amplicon to exclude false-negative results, which served as an internal control to evaluate the quality and sufficiency of the DNA present in the sample (Greer et al., 1991). Two and a half microliters of DNA were added to a mix containing 0.4 ␮M G73/74 primers, 3.0 mM MgCl2 , 0.25 mM dNTPs and 0.05 units of taq polymerase in a final volume of 25 ␮l. Each run was tested with quality-control samples that had been shown previously to be ␤-globin positive. 2.4. HPV DNA detection HPV DNA was amplified in replicate tubes using the L1 consensus primers PGMY 09 and PGMY 11. These primers amplify a 450 bp fragment of the HPV genome (Gravitt et al., 2000; Perrons et al., 2002). Two and a half ␮l of DNA were added to a PCR mix containing 0.2 mM PGMY 09/11 primers, 4.0 mM MgCl2 , 0.2 mM dNTPs and 0.1 units of taq platinum in a final

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Table 1 DNA extraction and HPV detection in cervical samples collected and stored at −20 ◦ C in universal collection medium (UCM) with denaturing reagent Storage time

Number of samples

DNA extractiona

6 months 12 months 24 months

20 20 20

19 (95%) 19 (95%) 19 (95%)

Total

60

57 (95%)

a b c d

HPV DNA detectionb

HPV DNA genotypingc

pd

19 (100%) 19 (100%) 18 (95%)

10 (52%) 10 (52%) 6 (33%)

– – 0.19

56 (98%)

26 (46%)

␤-Globin amplification. By HC2 (57). In HC2-positive samples(56). Chi-square for trends.

volume of 25 ␮l. During each PCR run, all samples were tested together with one negative control (water) and one positive control (HPV 18-containing cells) (Levi et al., 2002; Rabelo-Santos et al., 2005). 2.5. HPV genotyping by the Roche Linear Array® The Linear Array genotyping assay (Roche Diagnostics, USA) is based on PCR amplification of targed DNA using HPV primers, hybridization of the amplified product to oligonucleotide probes and their detection by a colourimetric reaction. Particularly, the master mix contains primers for the amplification of 450 bp fragment of the L1 region of more than 37 HPV genotypes and 268 bp fragment of the human ␤-globin gene. The detection and genotype determination was performed using the denatured amplified DNA and an array of oligonucleotide probes, located in the polymorphic region of L1 that permit independent identification of individual HPV genotypes. Negative samples were included in each assay to demonstrate the specificity of the test (Giuliani et al., 2006). 3. Results The DNA extracted from the denatured material was recovered successfully in 57 (95%) of the samples studied. DNA failed to be recovered in three samples, one from each storage time evaluated (␤-globin was not identified). HPV DNA (n = 56) was not detected in only one sample that had been stored for 24 months. It is important to highlight that different sets of 20 specimens were evaluated at different time points because the small volume available of each specimen permitted only one measurement on each. Genotyping, however, failed in the case of 9 of 19 specimens (48%) stored for 6 months, 9 of 19 (48%) specimens stored for 12 months and 12 of 18 specimens (67%) stored for 24 months. There was no trend of deterioration over time (p = 0.19) (Table 1). HPV type 16 was the most prevalent in 6 and 24 months samples. The second most prevalent HPV type was 58, present in roughly 25% of the samples, regardless of the storage time (data not shown). 4. Discussion The present study provides clear evidence that recovery of detectable HPV DNA from samples stored in UCM for up to

24 months is feasible. However, genotyping of HPV with the recovered HPV DNA was not successful, because in only 46% of the samples it has been possible to determine the HPV types. Our data suggest, therefore, that the HPV DNA amplifies but the DNA is sufficiently damaged that typing is problematic. It is possible that while the percentage of HPV-positive specimens did not change, the number of HPV types detected among the positives have declined due to the exposure of specimens to the alkali reagent. Linear Array is one of the most promising techniques for HPV genotyping currently available in the market, and proved to be successful in a number of recent studies (Stevens et al., 2007; Woo et al., 2007). However, to our knowledge, this is the first report on an attempt to use the Linear Array in UCMstored HPV samples. It is important to stress that in the present study, however, the DNA extraction procedures performed were not those recommended by Roche for the Linear Array assay. DNA had been already extracted previously and was used for the detection of HPV with standard PCR. The use of the Linear Array was an additional and further step for the study, which was not initially planned, and we did not investigate if Linear Array has limitations. Dunn et al. (2007) warned that alternate HPV extraction techniques might introduce unpredictable variability in the results obtained with Linear Array. These researchers, however, found that a DNA volume smaller than recommended by Roche (10 ␮l instead 50 ␮l) allowed much higher rates of HPV detection. In the present study, 10 ␮l of HPV DNA was used. Nevertheless, it is possible that technique incompatibilities led to a large number of false-negative results in our Linear Array experiments. On the other hand, 16 of the specimens considered negative by the Linear Array experiment, actually presented weak hybridization signals at line 14. According to Roche, line 14 is a cross-reactive probe that hybridizes with HPV genotypes 33, 35, 52 and 58. However, there was no ␤-globin amplification in these strips, suggesting inadequate specimen collection, inadequate processing, and presence of inhibitors or competition with a high titer HPV reagent. Studies have suggested that time-related DNA degradation results in decreased DNA recovery rates due to fragmentation of genomic DNA. In fact, a study of formalin-fixed histology samples found that the age of the sample from which DNA can successfully be recovered is increased if a shorter ␤-globin gene fragment is targeted for amplification (Ben-Ezra et al., 1991). In the present study, the three samples from which it was not

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possible to extract DNA had been stored for 6, 12 and 24 months, suggesting that storage time cannot be the only cause of failure to recover DNA. Because there was no trend over time concerning DNA degradation, it is likely that the damage occurred at a much earlier phase, probably when specimens were exposed to the alkali reagent. In a study carried out by Castle et al. (2003), specimens preserved in PreserveCyt® for varying periods were tested for preservation of HPV DNA, ␤-globin DNA and cell nuclear features. These investigators also found that detection of HPV DNA by the HC2 assay was not affected by storage time. Nevertheless, in approximately 15% of specimens, amplification for any ␤-globin DNA fragment could not be performed after 5 years of storage. However, those specimens were collected and stored at uncontrolled ambient temperatures until the beginning of 1996 when at which time specimens finally began to be stored in a well-controlled temperature environment. Apart from the technical limitations regarding the compatibility of the DNA extraction and the Linear Array, the present study has other minor limitations: the low number of specimens tested for each one of the three storage periods and that the samples tested from the different periods of storage time were not the same. It was noted that the ideal design for the study of possible damage caused to samples stored in denaturing reagent would have been to evaluate the same sample at different storage times. Unfortunately, this was not possible because the remaining volume of the stored samples was only sufficient to carry out one attempt at DNA HPV extraction, since the only available protocol for denatured samples in the institute required 450 ␮l. Previous studies on this topic were carried out only on STM, since this was the only collection medium used in performing the HC2 assay (Poljak et al., 2002; Rabelo-Santos et al., 2005). Currently, there is a trend towards substituting STM by UCM in view of the possible advantages of the latter method, particularly with respect to the possibility that it may also permit cytological evaluation of cervical samples collected in UCM. The data reported in the present study adds another possible advantage for the use of this medium since it appears to be just as good as STM for the recovery of nucleic material after long periods of storage. One of the major advantages of detecting adequate amounts of intact DNA for HPV DNA amplification by PCR in stored HC2-tested samples is the identification of the specific virus genotypes in material stored following large studies. Valuable information regarding the persistence of HPV DNA may eventually be helpful in making decisions with respect to individual treatments, in population and epidemiological programs and also for vaccine development. However, genotyping by the Linear Array was unsuccessful for more than half of the samples stored for up to 2 years in UCM with denaturing reagent in the present study. Nonogaki et al. (2004) compared HC2 and PCR for identifying HPV infections in samples collected in UCM. In that study, concordance between the two detection techniques was good and the authors concluded that the medium adequately preserves HPV DNA. Their study differs substantially from the present study, inasmuch they did not use archived material with denatured reagent, a purification kit was used (GFX Genomic Blood DNA Purification) for the DNA extraction, and amplitaq gold polymerase.

The present study adds further knowledge on the compatibility of conventional DNA extraction with the automated Linear Array amplification procedure, and prompts investigators to start conducting research focused on the improvement of technical ways that can make retrieval of information from stored HPV samples more reliable and cost-effective. The present data should encourage researchers with stored HC2 material to go back to their samples and perform innovative studies focused on specific-site DNA HPV amplification. Valuable and reliable information may be obtained from storage material and may therefore obviate the need to carry out prospective studies that are much more time consuming and expensive. However, because HPV genotyping has not been successful in our sample, it is necessary to acknowledge that technical details remain to be refined. Acknowledgment This study was funded by a research grant from the Fundac¸a˜ o de Amparo a` Pesquisa do Estado de S˜ao Paulo (FAPESP, Grant #05/54482-0). References Arbyn, A., Sasieni, P., Meijer, C.J.L.M., Clavel, C., Koliopoulos, G., Dillner, J., 2006. Clinical applications of HPV testing: a summary of meta-analyses. Vaccine 24S3, 79–89. Ben-Ezra, J., Johnson, D.A., Rossi, J., Cook, N., Wu, A., 1991. Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction. J. Histochem. Cytochem. 39, 351–354. Carozzi, F.M., Del Mistro, A., Confortini, M., Sani, C., Puliti, D., Trevisan, R., De Marco, L., Tos, A.G., Girlando, S., Palma, P.D., Pellegrini, A., Schiboni, M.L., Crucitti, P., Pierotti, P., Vignato, A., Ronco, G., 2005. Reproducibility of HPV DNA testing by Hybrid Capture 2 in a screening setting. Am. J. Clin. Pathol. 124, 716–721. Carozzi, F., Bisanzi, S., Sani, C., Zappa, M., Cecchini, S., Ciatto, S., Confortini, M., 2007. Agreement between the Amplicor human papillomavirus test and the Hybrid Capture 2 assay in detection of high-risk human papillomavirus and diagnosis. J. Clin. Microbiol. 45, 364–369. Castellsagu´e, X., Diaz, M., de Sanjose, S., Mun˜oz, N., Herrero, R., Franceschi, S., 2006. Worldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactores: implications for screening and prevention. J. Natl. Cancer Inst. 98, 303–315. Castle, P.E., Lorincz, A.T., Scott, D.R., Sherman, M.E., Glass, A.G., Rush, B.B., Wacholder, S., Burk, R.D., Manos, M.M., Schussler, J.E., Macomber, P., Schiffman, M., 2003. Comparison between prototype Hybrid Capture 3 and hybrid capture 2 human papillomavirus DNA assay for detection of high grade cervical intraepithelial neoplasia and cancer. J. Clin. Microbiol., 4022–4030. Clavel, C., Masure, M., Bory, J.P., Putaud, I., Mangeonjean, C., Lorenzato, M., Gabriel, R., Quereux, C., Birembaut, P., 1999. Hybrid Capture II-based human papillomavirus detection, a sensitive test to detect in routine highgrade cervical lesions: a preliminary study on 1518 women. Br. J. Cancer 80, 1306–1311. Clifford, G., Franceschi, S., Diaz, M., Mun˜oz, N., Villa, L.L., 2006. HPV type-distribuition in women with and without cervical neoplastic diseases. Vaccine 24S3, 26–34. Dunn, S.T., Allen, R.A., Wang, S., Walker, J., Schiffman, M., 2007. DNA extraction: an understudied and important aspect of HPV genotyping using PCR-based methods. J. Virol. Methods 143, 45–54. Giuliani, L., Coletti, A., Syrj¨anen, K., Favalli, C., Ciotti, M., 2006. Comparison of sequencing and Roche Linear Array® in human papillomavirus (HPV) genotyping. Anticancer Res. 26, 3939–3941.

E.A. Campos et al. / Journal of Virological Methods 147 (2008) 333–337 Gravitt, P.E., Peyton, C.L., Alessi, T.Q., Wheeler, C.M., Coutlee, F., Hildesheim, A., Schiffman, M.H., Scott, D.R., Apple, R.J., 2000. Improved amplification of genital human papillomaviruses. J. Clin. Microbiol. 38, 357–361. Greer, C.E., Peterson, S.L., Kiviat, N.B., Manos, M.M., 1991. PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time. Am. J. Clin. Pathol. 95, 117–124. Levi, J.E., Kleter, B., Quint, W.G., Fink, M.C., Canto, C.L., Matsubara, R., Linhares, I., Segurado, A., Vanderborght, B., Neto, J.E., Van Doorn, L.J., 2002. High prevalence of human papillomavirus (HPV) infections and high frequency of multiple HPV genotypes in human immunodeficiency virusinfected women in Brazil. J. Clin. Microbiol. 40 (9), 3341–3345. Melo, A., Roa, I., Montenegro, S., Capurro, I., Roa, J.C., 2005. Detection of human papillomavirus in cytologic samples or biopsies of the cervix. Rev. Med. Chil. 133, 639–644. Mesquita, R.A., Anzai, E.K., Oliveira, R.N., Nunes, F.D., 2001. Evaluation of three methods of DNA extraction from paraffin-embedded material for the amplification of genomic DNA using PCR. Pesqui. Odontol. Bras. 15, 314–319. Mu˜noz, N., Bosch, F.X., de Sanjose, S., Herrero, R., Castellsague, X., Shah, K.V., Snijders, P.J., Meijer, C.J., International Agency for Research on Cancer Multicenter Cervical Cancer Study Group, 2003. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N. Engl. J. Med. 348, 518–527. Mun˜oz, N., Castellsagu´e, X., de Gonz´alez, A.B., Gissmann, L., 2006. HPV in the etiology of human cancer. Vaccine 24S3, 1–10. Nonogaki, S., Wakamatsu, A., Longatto Filho, A., Pereira, S.M.M., Utagawa, M.L., Alves, V.A.F., Di Loreto, C., Maeda, M.Y.S., Lima, T.P., RoteliMartins, C., Syrj¨anen, 2004. Hybrid Capture II and polymerase chain reaction for identifying HPV infections in samples collected in a new collection medium. Acta Cytol. 48, 514–520. Perrons, C., Kleter, B., Jelley, R., Jalal, H., Quint, W., Tedder, R., 2002. Detection and genotyping of human papillomavirus DNA by SPF10 and MY09/11

337

primers in cervical cells taken from women attending a colposcopy clinic. J. Med. Virol. 67, 246–252. Poljak, M., Marin, I.J., Seme, K., Vince, A., 2002. Hybrid capture II HPV test detects at least 15 human papillomavirus genotypes not included in its current high-risk probe cocktail. J. Clin. Virol. 25 (Suppl. 3), S89–S97. Rabelo-Santos, S.H., Levi, J.E., Derchain, S.F., Sarian, L.O., Zeferino, L.C., Messias, S., Moraes, D.L., Campos, E.A., Syrjanen, K.J., 2005. DNA recovery from hybrid capture II samples stored in specimen transport medium with denaturing reagent, for the detection of human papillomavirus by PCR. J. Virol. Methods 126, 197–201. Schiffman, M., 2007. Integration of papillomavirus vaccination, cytology and human papillomavirus testing. Cancer Cytopathol. 111, 145–153. Stevens, M.P., Rudland, E., Garland, S.M., Tabrizi, S.N., 2006. Assessment of MagNa pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by Roche Amplicor and Linear Array HPV tests. J. Clin. Microbiol. 44, 2428–2433. Stevens, M.P., Garland, S.M., Rudland, E., Tan, J., Quinn, M.A., Tabrizi, S.N., 2007. 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. 45, 2130–2137. Taha, N.S., Focchi, J., Ribalta, J.C., Castelo, A., Lorincz, A., Dˆores, G.B., 2006. Universal collection medium (UCM) is as suitable as the standard transport medium (STM) for Hybrid Capture II (HC-2) assay. J. Clin. Virol. 36, 32–35. Walboomers, J.M., Jacobs, M.V., Manos, M.M., Bosch, F.X., Kummer, J.A., Shah, K.V., Snijders, P.J., Peto, J., Meijer, C.J., Munoz, N., 1999. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J. Pathol. 189, 12–19. Woo, Y.L., Damay, I., Stanley, M., Crawford, R., Sterling, J., 2007. The use of HPV Linear Array for multiple HPV typing on archival frozen tissue and DNA specimens. J. Virol. Methods 142, 226–230.