Clinical Chemistry 54:1 86 –92 (2008)

Molecular Diagnostics and Genetics

High-Throughput Genotyping of Oncogenic Human Papilloma Viruses with MALDI-TOF Mass Spectrometry Anna So¨derlund-Strand,1,2 Joakim Dillner,1,2 and Joyce Carlson3*

BACKGROUND: Human papilloma virus (HPV) is the major cause of cervical cancer. Use of HPV genotyping in cervical screening programs and for monitoring the effectiveness of HPV vaccination programs requires access to economical, high-throughput technology. METHODS:

We used the Sequenom MassARRAY platform to develop a high-throughput mass spectrometric (MS) method for detecting 14 specific oncogenic HPV genotypes in multiplex PCR products. We compared results from 532 cervical cell samples to the comparison method, reverse dot blot hybridization (RDBH).

RESULTS:

The MS method detected all samples found positive by RDBH. In addition, the MS method identified 5 cases of cervical disease (cervical intraepithelial neoplasia of grade I or higher) that RDBH analysis had missed. Discrepancies in specific genotypes were noted in 20 samples, all positive by MS, with an overall concordance of ␬ ⫽ 0.945.

CONCLUSIONS: The MS high-throughput method, with a processing capacity of 10 ⫻ 384 samples within 2 working days and at a consumables cost of about US$2 per sample, performed as well as or better than the comparison method.

© 2007 American Association for Clinical Chemistry

Genotyping of HPV infections is important, because different HPV genotypes confer distinctly different risks for development of cervical disease (5 ). The addition of HPV testing to cytology in primary screening produces a higher sensitivity for the detection of CIN than cytology alone (6, 7 ). A knowledge of the prevalence and associated risks for each specific HPV genotype also has been essential for the development of vaccines against HPV. High-throughput HPV genotyping will be essential not only for cervicalscreening programs but also for monitoring the effectiveness of HPV vaccination programs, i. e., for documenting decreasing prevalence of the HPV types for which vaccines are available and possibly for changing the occurrence of HPV types without vaccines via type replacement or cross-protection (8 ). Although several primarily PCR-based methods have been used to type HPV (9 ), the massive scale of the monitoring and primary-screening efforts likely to be launched in the near future will require more efficient methods. In the present study, we developed a high-throughput multiplex analysis of 14 distinct HPV high-risk genotypes on the Sequenom MassARRAY matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectrometry (MS) system. We compared its performance with that of another method for HPV genotyping, reverse dot blot hybridization (RDBH) of PCR amplimers, as well as with histopathologic diagnoses.

Infection with oncogenic types of human papillomavirus (HPV)4 is the principal cause of invasive cervical cancer and its precursor lesion, cervical intraepithelial neoplasia (CIN) (1, 2 ); HPV DNA is found in almost all cervical cancers (3 ). HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are found in cervical cancer, whereas HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81 are not associated with cervical cancer (4 ).

Materials and Methods

1

Received May 28, 2007; accepted October 10, 2007. Previously published online at DOI: 10.1373/clinchem.2007.092627 4 Nonstandard abbreviations: HPV, human papillomavirus; CIN, cervical intraepithelial neoplasia; RDBH, reverse dot blot hybridization; MS, mass spectrometry; and hME, homogeneous mass extend.

WHO HPV LabNet Global Reference Laboratory and Departments of 2 Medical Microbiology and 3 Clinical Chemistry, Lund University, Malmo¨ University Hospital, Malmo¨, Sweden. * Address correspondence to this author at: Department of Clinical Chemistry, Lund University, Malmo¨ University Hospital, SE-205 02 Malmo¨, Sweden. Fax 46-40-336286; e-mail [email protected].

86

STUDY POPULATION

We obtained samples from 271 patients, ages 23– 81 years (mean, 38 years; median, 36 years), who were referred to the Department of Gynecology, Va¨sterås Hospital, Va¨sterås, Sweden, for colposcopy after an atypical cervical smear, as described (10 ). A sample of

Human Papilloma Virus Genotyping with Mass Spectrometry

Table 1. Sequences of the new primary PCR primers compared with the original GP5ⴙ/6ⴙ primers. Primer

Primer sequencea

RDBH

GP5⫹

5⬘-TTTGTTACTGTGGTAGATACTAC-3⬘

MS

FA

5⬘-acgttggatgTTTGTTACTGTGGTGGATACTAC-3⬘

MS

FB

5⬘-acgttggatgTTTGTTACCGTTGTTGATACTAC-3⬘

MS

FC

5⬘-acgttggatgTTTGTTACTGTGGTGGATACCAC-3⬘

MS

FD

5⬘-acgttggatgTTTGTTACTGTTGTGGATACAAC-3⬘

RDBH

GP6⫹

5⬘-GAAAAATAAACTGTAAATCATATTC-3⬘

Method

a

MS

RG

5⬘-acgttggatgGAAAAATAAACTGTAAATCATATTCCT-3⬘

MS

RH

5⬘-acgttggatgGAAAAATAAATTGTAAATCATACTC-3⬘

MS

RI

5⬘-acgttggatgGAAATATAAATTGTAAATCAAATTC-3⬘

MS

RJ

5⬘-acgttggatgGAAAAATAAACTGTAAATCATATTC-3⬘

The 10-base 5⬘ extension is indicated in underlined lowercase letters. Nucleotides that differ from GP5⫹/6⫹ primers are in bold type and underlined.

cervical cells for cytologic and HPV testing and a colposcopy-guided tissue biopsy sample were obtained from all consenting patients. Patients testing positive by at least 2 of the criteria (initial cytology results ⱖCIN I, biopsy results ⱖCIN I, or abnormal colposcopy results) were treated with loop or laser conization (n ⫽ 199). All patients were invited to follow-up visits at 4 – 6 months and 16 –18 months after treatment, whether they received treatment or not, and 261 new samples of cervical cells were obtained for cytologic and HPV testing, for a total of 532 samples. The study was approved by the ethics review board of Va¨sterås Hospital.

COMPARISON METHOD

GP5⫹/6⫹ consensus primers (11 ) and BGPCO5/ BGPCO3 primers (12 ) were used to amplify HPV DNA and the human ␤-globin gene (HBB), respectively, in separate 50-␮L PCR reaction mixes, each containing 10 ␮L DNA solution as template. The presence of any of the 14 target HPV types or HBB DNA in 5-␮L aliquots of PCR product was detected by enzyme immunoassay (13 ). Samples positive for HPV (n ⫽ 260) in the enzyme immunoassay were further tested by RDBH (10, 14 ) for the specific HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68. MASS SPECTROMETRY

SAMPLE PREPARATION

Cervical cells were suspended in 1 mL 154 mmol/L NaCl and stored at –20 °C. After thawing, the cells were centrifuged at 3 000g for 10 min, and pellets were resuspended in 1 mL 10 mmol/L Tris-HCl, pH 7.4. Before RDBH analysis, 100-␮L aliquots were freeze-thawed and boiled for 10 min to release the DNA. After storage at – 80 °C for 3– 6 years, 40-␮L aliquots of the same 532 DNA extracts were dried in 96well plates at 37 °C overnight and resuspended in 8 ␮L sterile water; 2 ␮L of this suspension was used as template in the primary PCR reaction of the MS method. To evaluate the sensitivities of the 2 methods, we used standard 10-fold dilution series of 1–1 000 plasmids/reaction with type-specific inserts of each target HPV type and 1–1 000 copies/reaction of DNA extracted from SiHa cells (a cervical cancer cell line containing 1 copy of HPV 16 DNA per cell; American Type Culture Collection).

The MS method involves a multiplex primary PCR, in this case with several HPV primers, followed by a homogeneous mass extend (hME) reaction with a single primer of distinct mass that is specific for each genotype. Subsequently, unextended primers demonstrate the absence and specifically extended primers verify the presence and identity of each specific genotype. After laser deionization, the time of flight, which increases with the m/z, is detected for each hME primer. Unless otherwise specified, all procedures were performed with protocols and materials from Sequenom. PRIMER DESIGN

For the primary PCR, we used Oligo 6.0 software (Cambio) to modify the design of GP5⫹/6⫹ consensus primers to provide optimal annealing to the 14 target HPV types (Table 1). We produced new forward primers FA (all 14 HPV types), FB (HPV 16, 39, 45, 51, 56, 59, 66), FC (HPV 18, 31, 33, 52, 58), and FD (HPV 35, 68) and reverse primers RG (HPV 16, 18, 45, 56, 66, 68), RH (HPV 51, 39), RI (HPV 31, 52, 59), and RJ Clinical Chemistry 54:1 (2008) 87

Table 2. Sequences and molecular weights of unextended (UEP) and extended (EP) hME primers. HPV type

a

hME primer sequencea

UEP, Da

Extension

EP, Da

16

TTAAAGTTAGTATTTTTATATGTAGT

8 019

T

8 307

18

TATACTGCTTAAATTTGGTAGCA

7 053

T

7 341

31

TTTAAAATTACTACTTTTAAATGTAGT

8 261

AT

8 863

33

TGTCACTAGTTACTTGT

5 166

G

5 480

35

CTAGAAGACACAGCAGAAC

5 824

ACACAG

7 655

39

TCCTCCACGTGCCTGG

4 809

T

5 097

45

CTACTATACTGCTTAAACTTAGTAG

7 615

G

7 928

51

AACCGCAGCAGTGGC

4 587

AG

5 213

52

TGTGCTTTCCTTTTTAACCTCAG

6 947

CACAT

8 440

56

GTCTAAGGTACTGATTAATTT

6 450

T

6 738

58

TGTACCTTCCTTAGTTACTTCAG

6 956

T

7 244

59

TCCTCCACATGTCTGGCA

5 411

T

5 699

66

GGCGAAGGTATTGATTGATTT

6 531

CACG

7 736

68

CTCAACATGCCTAACATATT

6 020

CCT

6 887

All primer sequences are given from the 5⬘ end.

(HPV 33, 35, 58). The number of mismatches was minimized, and A-C mismatches were avoided. All primers have the 5⬘ 10-base extension recommended by Sequenom to improve annealing stability in multiplex PCR reactions (see Table 1). PCR programs were optimized, and products were viewed after agarose gel electrophoresis. The 14 unique HPV type–specific hME primers are shown in Table 2. PRIMARY PCR

PCR reaction mixes (6 ␮L) contained 2 ␮L DNA template and a final concentration of 0.3 ␮mol/L of each outer primer, 200 ␮mol/L of each deoxynucleoside triphosphate, 10 mmol/L Tris HCl, pH 8.3, 50 mmol/L KCl, 2.5 mmol/L MgCl2, and 0.2 U AmpliTaq Gold DNA polymerase (Roche Molecular Systems). The mixes were robotically pipetted with disposable tips and amplified in 384-well plates. Separate dilutions of plasmids with HPV type–specific inserts, 1 000 copies/␮L of each HPV type, and a pool of all HPV plasmids with 1 000 copies of each type/␮L were used as positive controls. Negative controls were sterile water and 10 ng/␮L of human DNA extracted from pooled human peripheral blood leukocytes. We performed all primary PCR amplifications with a GeneAmp PCR System 9700 (Applied Biosystems) as follows: denaturation at 95 °C for 10 min; 5 cycles of 95 °C for 30 s, 42 °C for 30 s, and 72 °C for 45 s; 45 cycles of 95 °C for 30 s, 64 °C for 30 s, and 72 °C for 45 s; and a final step at 72 °C for 10 min. Primary PCR reaction 88 Clinical Chemistry 54:1 (2008)

mixes were dephosphorylated with shrimp alkaline phosphatase (Sequenom). hME REACTION

The hME reaction mix was added to the dephosphorylated primary PCR reaction mix and included 1 ␮mol/L of each hME primer (Table 2), 0.229 ␮L terminator mix (Sequenom) containing equal amounts of dATP, dCTP, ddGTP, and ddTTP, and 1.25 U Thermo Sequenase (Sequenom) in a final volume of 10 ␮l. The PCR program was 94 °C for 2 min, followed by 99 cycles of 94 °C for 5 s, 42 °C for 10 s, and 72 °C for 5 s. After desalting by the addition of 6 mg Clean Resin (item #08040, Sequenom) to each 384-well plate, we applied 15 nL of each hME product to a 384-spot SpectroChip (Sequenom). MS analysis was performed and interpreted with MassARRAY Typer software (Sequenom). DISCREPANCY BETWEEN METHODS

Seventy-eight samples showed genotypic discrepancies between the RDBH and MS results. For these samples, we purified DNA from 100-␮L aliquots of the original cell suspension in 10 mmol/L Tris-HCl by proteinase K digestion and DNA precipitation (15 ). The dried pellets were dissolved in 20 ␮L sterile water. The presence of human DNA in these samples was demonstrated by real-time PCR analysis on a 7900HT instrument (Applied Biosystems) with primers and probes for the

Human Papilloma Virus Genotyping with Mass Spectrometry

Table 3. HPV status measured by RDBH and MS compared with the histopathologic diagnosis for 253 primary samples or cytologic results for 249 samples at follow-up. Diagnosis missing, n

Normal, na

CIN I, n

CIN II, n

CIN III, n

Cancer, n

RDBH⫹/MS⫹

0

34

36

42

59

3b

RDBH⫺/MS⫺

0

49

14

8

2

0

RDBH⫹/MS⫺

0

0

0

0

0

0

RDBH⫺/MS⫹

0

2

2

0

1

1c

HPV status

Histopathology

Cytology RDBH⫹/MS⫹

7

21

11

5

2

0

RDBH⫺/MS⫺

6

187

5

3

0

0

RDBH⫹/MS⫺

0

0

0

0

0

0

RDBH⫺/MS⫹

0

1

1

0

0

0

a

Including the cytologic diagnosis koilocytosis (“signs of HPV infection”). One patient with vulva cancer (HPV 45), 1 with adenocarcinoma (HPV 18), and 1 with squamous cell carcinoma (HPV 16). c One patient with squamous cell carcinoma (HPV 68). b

human F25 gene [coagulation factor II (thrombin)] (C 8726802; Applied Biosystems). Samples testing negative for human DNA (n ⫽ 26) were considered not evaluable and were excluded. All 52 samples positive for human DNA were reanalyzed at least once by the MS method. Fluctuating results for specific genotypes in a few samples were apparently due to viral dosage effects. Of the 52 samples, 39 had 1 or more HPV genotypes detected with the RDBH method than were detected with the MS method, 8 had 1 or more HPV genotypes detected with the MS method than were detected with the RDBH method, and 5 had 1 discrepant HPV genotype detected with each method before the MS reanalysis. These 52 samples with persisting discrepancies between the RDBH and MS results were reanalyzed by RDBH with the GP5⫹/6⫹ primers. After reanalysis with MS and RDBH, discrepant results remained for 24 samples, of which 10 had been positive for a single HPV type. After a new PCR reaction, these 10 samples were sequenced with the same mix of forward primers as in the primary PCR (Table 1). The presence of HPV found only in the MS method for 6 samples was verified by DNA sequencing. In 4 other cases [none with the diagnosis CIN I or higher (CIN I⫹)], DNA sequencing was inconclusive.

5

Human genes: F2, coagulation factor II (thrombin); and HBB, (hemoglobin, beta).

These 4 samples were considered not evaluable and were excluded, for a final total of 502 samples. STATISTICAL METHODS

Sensitivity, specificity, and positive predictive value were calculated for each method separately regarding the detection of HPV in comparison with histopathologic diagnosis. The concordance between the RDBH and MS methods for the detection of each of the 14 HPV types in all 502 evaluable samples was calculated by means of ␬ statistics (16 ). Results Analysis of the dilution series of plasmids revealed the following detection limits per PCR reaction for the MS method: 1 copy for HPV types 16, 52, 58, 59, and 66; 10 copies for HPV types 18, 33, 35, 45, 51, 56, and 68; and 100 copies for HPV types 31 and 39. The detection limits per PCR reaction for the comparison method (RDBH) were as follows: 1 copy for HPV types 16, 51, 58, and 66; 10 copies for HPV types 18, 31, 33, 39, 45, and 56; 100 copies for HPV types 35, 52, and 59; and 1 000 copies for HPV 68. With CIN II or higher (CIN II⫹) in the histopathologic results as a clinical reference, the sensitivity for the MS method was 91.4% [95% confidence interval (CI), 84.3%–95.6%], the specificity was 46.0% (95% CI, 37.5%–54.7%), and the positive predictive value was 58.9%, whereas the sensitivity of the comparison method was 89.7% (95% CI, Clinical Chemistry 54:1 (2008) 89

Table 4. HPV types detected by RDBH and MS in all samples, with focus on discrepant results.

a b

No. of discrepant samples positive for a single HPV type

Histopathology/cytology results for samples with discrepant results

HPV type

RDBHⴙ, na

MSⴙ, na

No. of discrepant resultsb

16

84

82

2

0

CIN III, CIN I

18

24

22

2

0

CIN III, CIN II

31

19

19

0

0

33

18

20

2

1

CIN III, normal

35

5

5

0

0

39

12

13

1

0

CIN I

45

12

10

2

0

CIN II, CIN I

51

23

26

3

1

CIN III, 2 normal

52

12

14

2

0

2 CIN I

56

22

19

3

0

CIN III, 2 CIN I

58

15

19

4

2

CIN II, CIN I, 2 normal

59

8

7

1

0

CIN II

66

14

12

2

0

2 CIN I

68

0

4

4

4

Cancer, 2 CIN I, normal

Numbers of samples positive by RDBH or MS. A total of 28 discrepant results were found in 20 samples.

82.3%–94.3%), the specificity was 48.9% (95% CI, 40.3%–57.5%), and the positive predictive value was 59.8% (Table 3). CONCORDANCE OF METHODS

The degree of overall concordance (␬) between the MS method and the RDBH method was 0.945. Both methods detected multiple HPV types in the same 40 samples and single HPV types in the same 175 samples. In 2 samples, the MS method detected multiple types, whereas the RDBH method detected single types. In another 3 samples, the RDBH method detected multiple types, whereas the MS method detected single types. In another 8 samples, the MS method detected single types, whereas the RDBH method detected no HPV. We compared the efficiencies of type-specific HPV detection by the 2 methods for all samples. Table 4 shows an overview of the number of type-specific infections detected with the 2 methods. For all 502 samples (with 2 ⫻ 14 ⫻ 502 results), 28 results (0.2%) in 20 samples did not agree between the 2 methods. Fifteen of the 20 discrepant samples were derived from patients with the diagnosis CIN I⫹. The HPV types missed by RDBH were among 10 patients with a histopathologic or cytologic diagnosis of CIN I⫹ and among 5 patients with no CIN, whereas the HPV types missed by MS were among 9 patients with CIN I⫹ (Table 4). In samples with multiple HPV types present, 90 Clinical Chemistry 54:1 (2008)

the 2 methods may have had slightly different abilities to detect certain HPV types (Table 4). The MS method alone detected all cases of cancer (Tables 3 and 4) and detected HPV 68 in 4 samples. The latter result is in contrast to RDBH, which consistently failed to identify this genotype (Table 4). Discussion Compared with the comparison method and most other reported methods, our new MS-based method had both greater clinical sensitivity for detecting HPV in HPV-associated disease (high-grade CIN II⫹) and better or comparable analytical sensitivity for the number of HPV copies that could be detected. The use of robotic pipetting and the MS method enables the analysis of 10 ⫻ 384 samples within 2 working days, with computerized documentation of sample identity and position at all steps, distinct identification of positive or negative signals by the Sequenom software, and current costs for consumables of about US$2 per sample. By comparison, an analysis of 36 samples by RDBH requires 4 days of labor, and the results depend on a subjective interpretation of dot blots. Our evaluation was based on a large clinical sample consecutively enrolled from a population-based screening program, suggesting that our results are generally valid with respect to the method’s usefulness in a screening setting. An additional strength of the study is

Human Papilloma Virus Genotyping with Mass Spectrometry

that we used the detection of HBB or F2 human DNA in each available aliquot to demonstrate the presence of amplifiable DNA. Because the clinical priority to be able to review cytology prevents centrifugation of cervical samples and lysis of all cells to provide a homogeneous DNA solution, the risk for false-negative HPV results due to nonrepresentative aliquots increases with each successive use of a sample. Although there were discrepancies in the genotyping results, all but 8 discrepant samples (all MS positive and RDBH negative) had at least 1 concordant HPV type and therefore would not have been misclassified as HPV negative in a screening situation. The causes for discrepancy include sampling error, DNA degradation during storage, and potential interference or false-positive reporting of weak RDBH dot blots. Plasmid dilution series generally demonstrated lower detection limits for MS than for the comparison method, with the most notable difference being the greater ability of the MS method to detect HPV type 68. According to a pooled international analysis of HPV type–specific prevalence in 3 085 cervical cancer cases (17 ), HPV 68 is the 14th most common type in cervical cancer, more common than HPV 66. The numbers of type-specific HPV infections detected with the 2 methods were almost equal, but the results of the genotyping differed slightly between methods. HPV 33, 52, and 58 (detected more frequently by MS) and HPV 45 (detected more by RDBH) are among the 8 most common types in cervical cancer (18 ). HPV types 16 and 18 account for about 70% of cervical cancers worldwide (18 ). In samples positive for multiple HPV types, these genotypes were each detected weakly in 2 samples by RDBH, whereas MS detected other HPV types, suggesting possible crosshybridization. For one of the samples positive for HPV 18 by RDBH, there was a weak HPV 18 peak (below cutoff) in the MS analysis, suggesting a low viral load in the sample. Eight additional results were also both MS negative and RDBH positive, and all originated from samples that were concordant with respect to at least 1 HPV type. All samples that were negative for all 14 genotypes by MS were also negative by RDBH. In a WHO international collaborative study for the assessment of the performance of various HPV-

detection assays (19 ), a panel of HPV types was analyzed in a number of laboratories, of which half failed to detect 10 000 copies of HPV 31 plasmids. Some laboratories did not detect the same copy numbers of HPV 35 and HPV 52. In the present study, the MS analysis detected 100 copies of HPV 31, 10 copies of HPV 35, and a single copy of HPV 52. The primary PCR primers of the MS method were designed to match some target HPV types better than others. A few primers match their templates perfectly, whereas others contain a minimal number of non–C-A mismatches. Although the detection limit for HPV 31 is slightly higher than for all the other types (except HPV 39) with the MS method, the MS method was generally more sensitive for HPV 31, 35, and 52 than the methods in the WHO study. In general, the number of mismatches is slightly lower with the MS primers than with GP5⫹/6⫹ primers, but, more importantly, the mismatches are preferentially G-T to optimize thermodynamic stability. In summary, we have designed a highly automated, potentially high-throughput MS method for the specific detection of 1–100 copies of 14 different oncogenic HPVs in samples of cervical cells. The performance compares well with the comparison method and with other methods reported in the literature. With access to robotic pipetting and the Sequenom system, the MS method allows large-scale HPV genotyping at a low cost, and this method may be useful for cervical screening and to evaluate the effectiveness of HPV vaccination programs via monitoring the circulation of HPV types in vaccinated populations.

Grant/funding Support: This work was supported by grants from the Swedish Cancer Society, the WHO, and by the EU 6th Framework Grant CCPRB (Cancer Control Using Population-Based Registries and Biobanks), principal investigator Joakim Dillner. The SWEGENE genotyping facility was supported by the Knut and Alice Wallenberg Foundation. Financial Disclosures: None declared. Acknowledgments: We thank Maria Sterner and Liselott Hall for technical assistance and Per Rymark for the enrollment of patients.

References 1. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International Biological Study on Cervical Cancer (IBSCC) Study Group. J Natl Cancer Inst 1995;87:796 – 802. 2. Bosch FX, Munoz N. The viral etiology of cervical cancer. Virus Res 2002;89:183–90. 3. Walboomers JM, Meijer CJ. Do HPV-negative

cervical carcinomas exist? J Pathol 1997;181: 253– 4. 4. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348:518 –27. 5. Schiffman M, Herrero R, Desalle R, Hildesheim A, Wacholder S, Rodriguez AC, et al. The carcinoge-

nicity of human papillomavirus types reflects viral evolution. Virology 2005;337:76 – 84. 6. Bulkmans NW, Rozendaal L, Voorhorst FJ, Snijders PJ, Meijer CJ. Long-term protective effect of high-risk human papillomavirus testing in population-based cervical screening. Br J Cancer 2005; 92:1800 –2. 7. Clavel C, Cucherousset J, Lorenzato M, Caudroy S, Nou JM, Nazeyrollas P, et al. Negative human

Clinical Chemistry 54:1 (2008) 91

8.

9.

10.

11.

papillomavirus testing in normal smears selects a population at low risk for developing high-grade cervical lesions. Br J Cancer 2004;90:1803– 8. Dillner J, Arbyn M, Dillner L. Translational minireview series on vaccines: monitoring of human papillomavirus vaccination. Clin Exp Immunol 2007;148:199 –207. Molijn A, Kleter B, Quint W, van Doorn LJ. Molecular diagnosis of human papillomavirus (HPV) infections. J Clin Virol 2005;32(Suppl 1): S43–51. Soderlund-Strand A, Rymark P, Andersson P, Dillner J, Dillner L. Comparison between the Hybrid Capture II test and a PCR-based human papillomavirus detection method for diagnosis and posttreatment follow-up of cervical intraepithelial neoplasia. J Clin Microbiol 2005;43: 3260 – 6. de Roda Husman AM, Walboomers JM, van den Brule AJ, Meijer CJ, Snijders PJ. The use of general primers GP5 and GP6 elongated at their 3⬘

92 Clinical Chemistry 54:1 (2008)

ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR. J Gen Virol 1995;76(Pt 4):1057– 62. 12. de Roda Husman AM, Snijders PJ, Stel HV, van den Brule AJ, Meijer CJ, Walboomers JM. Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. Br J Cancer 1995;72: 412–7. 13. Jacobs MV, Snijders PJ, van den Brule AJ, Helmerhorst TJ, Meijer CJ, Walboomers JM. A general primer GP5⫹/GP6(⫹)-mediated PCR-enzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings. J Clin Microbiol 1997;35:791–5. 14. Forslund O, Hansson BG, Bjerre B. Typing of human papillomaviruses by consensus polymerase chain reaction and a non-radioactive reverse dot blot hybridization. J Virol Methods 1994;49: 129 –39.

15. Sambrook J, Russell D. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press, 2001:6.4 – 6.11. 16. Landis JR, Koch GG. The measurement of observer agreement for categorical data. Biometrics 1977;33:159 –74. 17. Munoz N, Bosch FX, Castellsague X, Diaz M, de Sanjose S, Hammouda D, et al. Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int J Cancer 2004;111:278 – 85. 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. 19. Quint WG, Pagliusi SR, Lelie N, de Villiers EM, Wheeler CM. Results of the first World Health Organization international collaborative study of detection of human papillomavirus DNA. J Clin Microbiol 2006;44:571–9.