The International Journal of Biological Markers, Vol. 24 no. 4, pp.

215-222 REVIEW
© 2009 Wichtig Editore

Molecular detection methods of human papillomavirus (HPV)

Apostolos Zaravinos, Ioannis N. Mammas, George Sourvinos, Demetrios A. Spandidos

Department of Clinical Virology, School of Medicine, University of Crete, Heraklion, Crete - Greece

ABSTRACT: Human papillomavirus (HPV) testing can identify women at risk of cervical cancer. Currently, molecular detection
methods are the gold standard for identification of HPV. The three categories of molecular assays that are available are based
on the detection of HPV DNA and include (1) non-amplified hybridization assays, such as Southern transfer hybridization
(STH), dot blot hybridization (DB) and in situ hybridization (ISH); (2) signal amplified hybridization assays, such as hybrid
capture assays (HC2); (3) target amplification assays, such as polymerase chain reaction (PCR) and in situ PCR. STH requires
large amounts of DNA, is laborious and not reproducible, while ISH has only moderate sensitivity for HPV. The sensitivity
of the HC2 assay is similar to that of PCR-based assays, with high sensitivity being achieved by signal rather than target
amplification. PCR-based detection is both highly sensitive and specific. Since PCR can be performed on very small amounts
of DNA, it is ideal for use on specimens with low DNA content. In the future, with the advance of technology, viral DNA
extraction and amplification systems will become more rapid, more sensitive, and more automated. (Int J Biol Markers 2009;
24: 215-22)

Key words: HPV, Molecular detection, PCR, Hybridization

INTRODUCTION 45, 51, 52, 56, 58, 59, 68, 73 and 82. HPV-16 and 18 are
considered to be the most frequent HPV types worldwide
Human papillomavirus (HPV) infection has a global and are responsible for approximately 70% of all cervical
distribution and has been identified as the leading cancer cases (6, 7). Low-risk HPVs have been associated
etiologic agent for cervical cancer and its precursors in with benign warts of oral and urogenital epithelium in
adulthood (1). Different HPV types can cause a wide adults as well as children, and are only rarely found in
range of infections, including common warts, genital malignant tumors. Different HPV types vary in tissue
warts, recurrent respiratory papillomatosis, low-grade distribution, oncogenic potential and association with
and high-grade squamous intraepithelial lesions (SILs), anatomically and histologically distinct diseases.
and cervical cancer. Cervical cancer remains the second HPVs are double-stranded DNA viruses that comprise
most common cancer among women worldwide, with a remarkably heterogeneous family of more than 130
an estimated 493,000 new cases and 274,000 deaths in types (4, 8). HPV is a small virus of 55 nm in diameter,
2002. Cervical cancer is most prevalent in developing which consists of the viral genomic DNA and its coat. The
countries, where 80% of the cases occur, and it accounts viral genome is double-stranded circular DNA of nearly
for at least 15% of all female cancers. 8000 base pairs and encodes 8 proteins: E1, E2, E4, E5,
HPVs can be classified into cutaneous and mucosal E6, E7, L1 and L2. The early proteins E5, E6 and E7 are
types (2). Cutaneous types infect the squamous epithelium involved in cell proliferation and survival, with E6 and E7
of the skin and produce common, plantar and flat warts, playing a key role in HPV-associated carcinogenesis. The
which usually occur on the hands, face and feet. Specific early proteins E1, E2 and E4 are involved in the control
cutaneous types are also detected in Epidermodysplasia of viral gene transcription and viral DNA replication. The
verruciformis, a rare familial disorder which is related coat of the virus is made up of 2 proteins, the major one
to the development of large cutaneous warts that can being L1 and the minor component L2. The coat proteins
progress to skin cancer (3). Mucosal types infect the assemble into structures known as capsomeres and 72 of
mucous membranes and can cause cervical neoplasia in these come together to form the spherical coat.
adults as well as anogenital warts in both children and It is generally accepted that HPV E6 and E7 function as
adults. the dominant oncoproteins of high-risk HPVs by altering
Mucosal HPVs are classified into high-risk and low- the function of critical cellular proteins. Expression of the
risk types. High-risk HPV types have been implicated in E6 and E7 proteins as a consequence of viral integration
the development of SILs and their progression to cervical is paramount to the establishment and maintenance of
cancer (4, 5). To date, 15 HPV types have been classified the tumorigenic state. In addition, expression of E6 and
as high risk and these include HPV-16, 18, 31, 33, 35, 39, E7 increases the genomic instability of the host cell, thus

0393-6155/215-08$25.00/0

215-222_JBM_774_zaravinos.indd 215 26-01-2010 12:30:59

 HPV molecular detection methods

accelerating malignant progression (9). E6 and E7 target can be divided into: (1) target amplification assays/PCR,
important cellular growth regulatory circuits including (2) direct hybridization assays, and (3) signal amplified
the p53 protein and the retinoblastoma tumor suppressor hybridization assays.
protein Rb, respectively. HPV E6 has been shown to
interact with and enhance the degradation of p53, which
plays an important role in cell cycle control and apoptosis TARGET AMPLIFICATION ASSAYS/PCR
in response to DNA damage, while HPV E7 disables
the function of Rb. During the last decade, it has been Polymerase chain reaction (PCR) is the most widely
demonstrated that HPV E6 and E7 interact with both host used method for amplification of nucleic acids. PCR-based
cell targeting and a plethora of key host cellular proteins detection of HPV is both highly sensitive and specific.
that are involved in apoptosis and malignant cellular The chemistry of this assay relies on a thermostable
transformation (10). DNA polymerase which recognizes and extends a pair of
HPV testing was recently introduced into clinical oligonucleotide primers that flank the region of interest.
practice with the aim of identifying women at risk of Finally, the viral DNA is sufficiently amplified in vitro
cervical cancer (Tab. I). HPV testing is recommended to generate an adequate amount of target, which is then
in the triage of women with atypical squamous cells of directly visualized on an agarose gel. Theoretically, PCR is
undetermined significance (ASCUS) (11). The use of HPV able to detect 1 copy of a target sequence in a given sample.
testing in the follow-up of women after local treatment In practice, the sensitivity of the PCR-based method is
of cervical intraepithelial neoplasia (CIN) is also strongly about 10-100 HPV viral genomes in a background of 100
supported by clinical evidence (12). Screening prior to ng cellular DNA. Since PCR can be performed on very
vaccination could identify women who have already small amounts of DNA (10-100 ng), it is ideal for use on
been exposed to HPV and thus have reduced benefit from specimens with low DNA content.
their vaccination. However, financial barriers prevent the Generally, HPV detection by PCR can be performed
prescreening of all women by HPV testing. HPV testing has either by type-specific primers, designed to exclusively
also been proposed as a useful tool for primary cervical amplify a single HPV genotype, or by consensus/general
screening and the management of women with low-grade PCR primer pairs, designed to amplify a broad spectrum
SILs. However, recent evidence is insufficient to support of HPV genotypes. Unfortunately, the use of multiple
HPV testing instead of conventional cytology (13-15). It type-specific PCR reactions in order to detect HPV-DNA
is likely that after the introduction of vaccination against in a single sample has the disadvantage of being costly
HPV, the role of HPV testing for triage for low-grade SILs and time-consuming, and the primers usually have to be
and primary cervical screening will be re-evaluated. confirmed by others (16-18). The use of general primers is
Several molecular assays are available for the detection much more convenient. Usually, general primers identify a
of HPV infection in tissue and exfoliated cell samples, and conserved region among different HPV genotypes, such as
they present different sensitivities and specificities. They the L1 (19) or E1 regions (20). However, most laboratories
utilize consensus primers directed to the conserved L1
TABLE I - R
 ECOMMENDATIONS OF THE USE OF HPV TESTING IN
region.
CLINICAL PRACTICE There are numerous consensus PCR primers that can
be used. The GP5+/6+ pair is aimed at the L1 conserved
1. Primary cervical screening region, but it is 100% complementary to just a few HPV
genotypes. The mismatching produced between the
- HPV testing and Pap smear co-testing increases sensitivity of primary
cervical screening
consensus PCR primers and the various HPV genotypes
is overcome by setting a low annealing temperature at
- HPV testing is not recommended to replace Pap smear as an initial screen the PCR reaction (21-23). Other consensus primers are
2. Cervical screening of women with low-grade SIL
a mixture of many different oligonucleotides and do not
have to be used at a lower annealing temperature. One
- HPV testing can increase the sensitivity of Pap smear to detect “high- example of these primers is the MY09/11 set (24-27).
risk” women with low-grade SIL who require colposcopy Moreover, a combination of various non-degenerate
3. Post-treatment follow-up of SIL primers which target the identical location of the viral
genome can be applied. Combined primers usually contain
- HPV testing can detect residual disease following treatment of SIL ear- inosine, which matches with any nucleotide. Primer sets
lier that Pap smear belonging to this category have the advantage of being
4. Cervical screening prior to vaccination highly reproducible; moreover, PCR can be performed at
optimal annealing temperatures. PGMY, SPF10 (27), LCR/
- HPV testing prior to vaccination is not recommended E7 (28), as well as a combination of the MY11 and GP6+
SIL, squamous intraepithelial lesion primers are examples of combined primers (29).

216

215-222_JBM_774_zaravinos.indd 216 26-01-2010 12:30:59

 Zaravinos et al

Another point of consideration is the size of the the DNA sequence. On the other hand, the frequency
amplicon to be produced after the PCR. Generally, the of multiple HPV infections can be underestimated when
efficiency of a PCR reaction is inversely proportional to DNA sequences that represent a minority in the total PCR
the size of the amplicon. Various treatments of the clinical product remain undetected (42). One such example is the
samples, before or during the DNA extraction procedure, study by Levi et al (43), which compared the sequence
usually result in low quality of the DNA and/or DNA analysis of SPF10 PCR products with reverse hybridization
degradation. Therefore, the PCR primers that produce in 166 HPV-positive cervical scrapes. Well-matched HPV
a small amplicon tend to be more efficient than those genotypes were found in all samples. With the former
responsible for larger amplicons (28, 30, 31). assay, multiple types were detected in only 2% of the
When the PCR is complete, the amplicon sequence cases, whereas the latter assay was capable of detecting
can be examined in various ways. One involves the multiple genotypes in 25% of the samples.
digestion of the amplicon with specific restriction Multiple HPV genotypes are found in up to 35%
endonucleases, an approach known as HPV restriction of HPV-positive patients with advanced cytological
fragment length polymorphism (RFLP), followed by agarose disorders and more than 50% of HIV-infected patients
gel electrophoresis analysis. However, RFLP often results (43, 44), whereas multiple genotypes are less prevalent
in a number of fragments which are difficult to interpret. in carcinoma patients (42). The genotype from an HPV
This becomes more evident when multiple infections are sequence can be deduced through alignment with a set of
present (31-37). known HPV sequences, using the BLAST software (45) of
PCR products can also be detected with a mixture a genome database (e.g., http://www.ncbi.nlm.nih.gov).
of type-specific probes, for example in an enzyme The complete genomes of various papillomaviruses have
immunoassay (EIA) (23). One good example is the HPV been fully sequenced. In order to designate a new type,
oligonucleotide microarray (HPVDNAChip, Biomedlab the L1, E6 and E7 ORFs must differ by more than 10%
Co.), which contains 22 type-specific probes; 15 of the from the closest type known. Differences of 2-10% lead
high-risk group (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, to the definition of a new subtype, whereas differences of
59, 66, 68 and 69) and 7 of the low-risk group (6, 11, 34, less than 2% define intratype variants (46).
40, 42, 43 and 44). Briefly, the PCR product is hybridized Real-time PCR techniques have been developed to
onto the chip, and after a washing step, hybridized signals quantify HPV-DNA in clinical samples (47-49). Real-
are visualized with a DNA chip scanner. The sensitivity time PCR has the following advantages: 1) it is capable
of this assay has been reported to reach 94.9%, enabling of detecting the viral load; 2) with the use of different
this application to be considered a diagnostic tool with fluorochromes which emit fluorescence as the PCR reaction
significant advantages since it can discriminate the HPV proceeds, the reactions can be performed in multiplex,
genotype and identify multiple infections (38, 39). Ideally, thus amplifying simultaneously different nucleic acid
a larger number of HPV type-specific oligonucleotides targets; 3) using a 7-log dynamic range to extrapolate viral
could be spotted on the chip, although this method load/concentration in the standard curve, it is possible to
requires the presence of expensive equipment and may detect nucleic acids even at very small concentrations,
not be suitable for many laboratories. A similar assay which would not be detected by conventional PCR (47,
was recently released by Gen-Probe Inc., called the 48). Finally, real-time PCR is extremely reproducible,
APTIMA(R) HPV Assay. This assay distinguishes 14 high- rapid and pertinent in a clinical setting.
risk HPV genotypes in an amplified HPV nucleic acid. In Novel real-time PCR methods have recently been
particular, it identifies the E6 and E7 mRNA sequences, released and are capable of being used as high-throughput
produced at higher levels when HPV infection progresses screening tools. One such example is the GenoID real-
toward cervical cancer (40). time PCR assay, the amplification of which is based on
Sequencing reactions of double-stranded PCR the L1 region of HPV. The assay is designated to detect the
products can be performed directly with the GP6+ primer, non-integrated copies of HPV. The assay’s calibrators are
using commercially available kits such as the BigDye designed to detect ~10,000 copies/reaction (~100 infected
Terminator cycle sequencing kit (Applied Biosystems, CA, cells). Amplification is balanced over the genotypes,
USA). Subsequently, the DNA sequences obtained from which is important to achieve optimal clinical sensitivity.
the patient samples can be compared to the GenBank Detection of high-risk HPVs (16, 18, 26, 31, 33, 35, 39, 45,
sequences by using the BLAST program at the National 51, 52, 56, 58, 59, 66 and 68), low-risk HPVs (6, 11, 42,
Center for Biotechnology Information website. Thus, the 43 and 44/55), and internal controls is carried out in the
sequencing reaction can now be applied in routine clinical same reaction tube using 3 different color-compensated
analysis (41). Unfortunately, in many patients the presence dye channels (50). However, the exclusive optimization
of multiple HPV genotypes, visualized as multiple peaks of this assay for the LightCycler 2.0 instrument (Roche)
at the sequencing electrophoregram, is very common can be regarded as a weakness. Unlike other HPV tests,
and represents a major obstacle in the determination of the newly released CE-marked Abbott RealTime High Risk

217

215-222_JBM_774_zaravinos.indd 217 26-01-2010 12:30:59

 HPV molecular detection methods

HPV assay can detect the 14 highest-risk HPV genotypes DIRECT HYBRIDIZATION ASSAYS
and, in the same procedure, identify women infected with
the HPV-16 and HPV-18 genotypes. The assay can rapidly Southern blot hybridization (SBH) and in situ
identify HPV-infected patients at risk of cervical cancer by hybridization (ISH) can be used, but they have serious
combining 2 diagnostic tools in one test: HPV high-risk defects such as low sensitivity, delay, and the need for
screening and viral genotyping. possibly large amounts of highly purified DNA (59). Of
Apart from TaqMan oligo-probe technologies, SYBR the direct probe methods, ISH is the least specific for HPV
Green-based real-time PCR assays utilizing the GP5+/6+ detection (60-62) (72% for condylomatous lesions and
primers have also been used for HPV quantification with 30% for invasive cancer cells). Recently, new ISH assays
high specificity and sensitivity, whereas the results showed have emerged, showing better results. The INFORM HPV
excellent concordance with the enzyme immunoassay- 3 (Ventana Medical Systems) test can detect 13 types of
reverse line blot and sequencing assays (51). oncogenic HPV (16, 18, 31, 33, 35, 45, 51, 52, 56, 58,
It seems that the importance of HPV viral load detection 59, 68 and 70). This assay was shown to correlate with
with real-time PCR remains vague so far, since viral load PCR-based assays. Despite the favorable correlation,
values are an average summed over many infected and however, the assay detected significantly fewer HPV-
uninfected cells. The production of a high HPV viral load positive cases among patients with cervical carcinomas
can be observed during severe disease rather than being than PCR assays (63). Another novel ISH assay, the HPV-
the cause thereof (52). CARD assay, was shown to have high analytical sensitivity
Although the majority of HPV detection strategies and a high signal-to-noise ratio, to allow quantification of
are DNA based, the detection of the expression of HPV HPV-infected epithelial cells, and to distinguish between
oncogenes may have significant clinical value. This can be HPV physical states (64).
performed by reverse-transcription real-time PCR. Several
groups have correlated the HPV-16 and HPV-18 E7 transcript
levels with the severity of cervical lesions (53, 54). SIGNAL AMPLIFIED HYBRIDIZATION ASSAYS
Moreover, Cattani et al (55) detected E6/E7 RNA
transcripts in 18.2% of HPV DNA-positive patients with The most reliable signal amplified hybridization assay
normal cytology. The rate of detection increased gradually is the FDA-approved Hybrid Capture 2 (HC2) test (65).
with the grade of the observed lesions, suggesting that This method is based on 13 RNA probes specific for the
testing for HPV E6/E7 transcripts is a useful tool for corresponding number of high-risk HPV types (HPV-16, 18,
screening and patient management, providing more 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). The assay’s
accurate predictions of risk than DNA testing. sensitivity is excellent since it can detect HPV-16 DNA of
The PreTect HPV-Proofer (NorChip AS, Klokkarstua, a concentration down to 1 pg/mL, which corresponds to
Norway) is a commercially available RNA-based HPV ~105 viral gene copies. Nevertheless, there has been some
assay that incorporates the NASBA amplification of E6/ crossreactivity of the various RNA probes with HPV types
E7 mRNA transcripts prior to type-specific detection via not represented in the probe mix (66). However, it has
molecular beacons for the following HPV types: 16, 18, been proven that the HC2 assay scores significantly more
31, 33 and 45 (56, 57). However, large-scale prospective positive samples than ISH and GP5+/6+ PCR (67).
studies need to be performed in order to better determine Hybridization of the biotin-labeled PCR products to
the clinical value of this assay. oligonucleotide probes in streptavidin-coated microtiter
Finally, a protocol for the amplification of papillomavirus plates has been shown to increase the efficiency of
oncogene transcripts (APOT) from cervical specimens has diagnostic assays (23, 28, 68). One such assay is the Roche
been proposed that allows the distinction of episomal from Molecular Systems Amplicor HPV MWP assay, which is
integrated HPV mRNAs (58). In most cervical carcinomas, capable of detecting 13 high-risk genotypes by a broad-
HPV genomes are integrated into host cell chromosomes spectrum PCR in the L1 region, producing an amplicon
in order that transcribed mRNAs can encompass viral of ~170 bp. The sensitivity of this method appears to be
and cellular sequences. In contrast, in early preneoplastic superior to that of the HC2 assay for the detection of the
lesions, HPV genomes persist as episomes, and derived same high-risk HPV types. Moreover, the high throughput
transcripts contain exclusively viral sequences. Thus, of the microtiter format gives the Amplicor HPV MWP
detection of integrated-derived HPV transcripts in cervical assay a further advantage, making it appropriate for the
swabs or biopsy specimens by the APOT assay points to distinction between HPV-positive and HPV-negative DNA
advanced dysplasia or invasive cervical cancer, where loss samples.
of the regulatory E2 protein on integration results in the However, since both the HC2 and Amplicor tests only
upregulation of the oncogenes E6 and E7. However, since differentiate between infection with 1 of 13 high-risk HPV
the assay is based on RT-PCR protocols it has not readily genotypes and no high-risk HPV infection, neither allows
been adapted for use in routine diagnostic testing. for the individual identification of specific genotypes, nor

218

215-222_JBM_774_zaravinos.indd 218 26-01-2010 12:31:00

 Zaravinos et al

do they identify multiple genotypes possibly involved line blot assays, using the PGMY (24, 84-87) and GP5+/6+
in infection. This is an unfortunate weakness, as recent (88) primer sets, respectively. HPV DNA microarrays are
studies have provided evidence for a difference in based on the same principle (89).
oncogenic potential between the different high-risk HPVs
(69), arguing for the importance of HPV genotyping in
screening and triage (70-73). CONCLUSIONS
Furthermore, the CLART HPV 2 system is based
on a low-density microarray that detects infections Molecular assays are the gold standard for HPV
and coinfections of up to 35 of the most relevant HPV identification. If one would like to pinpoint each method’s
genotypes; 21 high risk (16, 18, 26, 31, 33, 35, 39, 43, 45, specific characteristics, the extremely high sensitivity
51, 52, 53, 56, 58, 59, 66, 68, 70, 73, 85 and 89) and 12 and specificity of PCR should be mentioned, along with
low risk (6, 11, 40, 42, 44, 54, 61, 62, 71, 81, 83 and 84). the need for only small amounts of DNA template. The
The diagnostic sensitivity and specificity of this system HC2 assay is equally sensitive, with the difference that
can reach 98.2% and 100%, respectively (74). its sensitivity is achieved by signal rather than target
The simultaneous hybridization of a PCR product to amplification. Southern blot hybridization on the other
multiple oligonucleotide probes can be performed by hand requires large amounts of DNA template, is difficult
reverse hybridization. The first step of this method consists to apply in routine practice and not always reproducible,
of the immobilization of the probes on a solid phase, whereas in situ hybridization is not always as sensitive as
then the PCR product is added in the liquid phase and the PCR and HC2 methods.
this is followed by the detection of the signal produced. Further investigation is required to clarify the role
The most frequently used reverse hybridization assays are of molecular HPV testing in current primary cervical
the line probe assay, line blot assay (Roche Molecular screening programs. In the future, the introduction of a
Systems, Alameda, CA, USA) (75-78), and linear array fast, cheap and reliable test for the molecular detection
(Roche Molecular Systems). These methods are judged of HPV in clinical practice is expected to improve the
advantageous in their ability to rapidly genotype HPVs Pap-test sensitivity for the early diagnosis of cervical HPV
present in samples with high sensitivity and specificity (24, infection. With the advancement of technology, viral DNA
79-82). Castle et al (83) compared the linear array with extraction and amplification systems will become more
the line probe assay and found that the former was more rapid, more sensitive, and even more automated.
analytically sensitive, resulting in increased detection of
individual genotypes and of multigenotype infection. The
findings translated into a more clinically sensitive but less Conflict of interest statement: None declared.
specific test for CIN3 or worse in a population of women
enrolled in a low-grade SIL triage study (ALTS) because of Address for correspondence:
an ASCUS Pap-test result. Professor Demetrios A. Spandidos
Reverse hybridization assays are especially useful for Department of Clinical Virology
detecting type-specific infections and multiple genotypes. School of Medicine, University of Crete
Alternative reverse hybridization assays for the detection Heraklion, Crete, Greece
of HPV and its genotyping are the line blot and reverse e-mail: [email protected]

REFERENCES 5. Peto J, Gilham C, Deacon J, et al. Cervical HPV infection

and neoplasia in a large population-based prospective study:
1. Clifford GM, Smith JS, Plummer M, et al. Human the Manchester cohort. Br J Cancer 2004; 91: 942-53.
papillomavirus types in invasive cervical cancer worldwide: 6. Munoz N, Bosch FX, de Sanjose S, et al. Epidemiologic
a meta-analysis. Br J Cancer 2003; 88: 63-73. classification of human papillomavirus types associated
2. Swygart C. Human papillomavirus: disease and laboratory with cervical cancer. N Engl J Med 2003; 348: 518-27.
diagnosis. Br J Biomed Sci 1997; 54: 299-303. 7. Munoz N, Bosch FX, Castellsague X, et al. Against which
3. Majewski S, Jablonska S. Epidermodysplasia verruciformis human papillomavirus types shall we vaccinate and
as a model of human papillomavirus induced genetic cancer screen? The international perspective. Int J Cancer 2004;
of the skin. Arch Dermatol 1995; 131: 1312-8. 111: 278-85.
4. zur Hausen H. Papillomaviruses and cancer: from basic 8. Sanclemente G, Gill DK. Human papillomavirus molecular
studies to clinical application. Nat Rev Cancer 2002; 2: biology and pathogenesis. J Eur Acad Dermatol Venereol
342-50. 2002; 16: 231-40.

219

215-222_JBM_774_zaravinos.indd 219 26-01-2010 12:31:00

 HPV molecular detection methods

9. Fehrmann F, Laimins LA. Human papillomaviruses: targeting of 27 human papillomavirus types by using L1 consensus
differentiating epithelial cells for malignant transformation. PCR products by a single-hybridization, reverse line blot
Oncogene 2003; 22: 5201-7. detection method. J Clin Microbiol 1998; 36: 3020-7.
10. Mammas I, Sourvinos G, Giannoudis A, Spandidos DA. 25. Coutlee F, Hankins C, Lapointe N. Comparison between
Human papilloma virus (HPV) and host cellular interactions. vaginal tampon and cervicovaginal lavage specimen
Pathol Oncol Res 2008; 14: 345-54. collection for detection of human papillomavirus DNA by
11. The Atypical Squamous Cells of Undetermined Significance/ the polymerase chain reaction. The Canadian Women’s HIV
Low-Grade Squamous Intraepithelial Lesions Triage Study Study Group. J Med Virol 1997; 51: 42-7.
(ALTS) Group. Human papillomavirus testing for triage of 26. Gravitt PE, Peyton CL, Alessi TQ, et al. Improved amplification
women with cytologic evidence of low-grade squamous of genital human papillomaviruses. J Clin Microbiol 2000;
intraepithelial lesions: baseline data from a randomized 38: 357-61.
trial. J Natl Cancer Inst 2000; 92: 397-02. 27. Kleter B, van Doorn LJ, ter Schegget J, et al. Novel short-
12. Paraskevaidis E, Arbyn M, Sotiriadis A, et al. The role of HPV fragment PCR assay for highly sensitive broad-spectrum
DNA testing in the follow-up period after treatment for CIN: detection of anogenital human papillomaviruses. Am J
a systematic review of the literature. Cancer Treat Rev 2004; Pathol 1998; 153: 1731-9.
30: 205-11. 28. Sasagawa T, Minemoto Y, Basha W, et al. A new PCR-based
13. Davies P, Arbyn M, Dillner J, et al. A report on the assay amplifies the E6-E7 genes of most mucosal human
current status of European research on the use of human papillomaviruses (HPV). Virus Res 2000; 67: 127-39.
papillomavirus testing for primary cervical cancer screening. 29. Menzo S, Ciavattini A, Bagnarelli P, et al. Molecular
Int J Cancer 2006; 118: 791-6. epidemiology and pathogenic potential of underdiagnosed
14. Kitchener HC, Almonte M, Wheeler P, et al. HPV testing human papillomavirus types. BMC Microbiol 2008; 8: 112.
in routine cervical screening: cross sectional data from the 30. Meyer T, Arndt R, Stockfleth E, et al. Strategy for typing
ARTISTIC trial. Br J Cancer 2006; 95: 56-61. human papillomaviruses by RFLP analysis of PCR products
15. Naucler P, Ryd W, Törnberg S, et al. Human papillomavirus and subsequent hybridization with a generic probe.
and Papanicolaou tests to screen for cervical cancer. N Engl Biotechniques 1995; 19: 632-9.
J Med 2007; 357: 1589-97. 31. Kado S, Kawamata Y, Shino Y, et al. Detection of human
16. Dokianakis DN, Sourvinos G, Sakkas S, et al. Detection of papillomaviruses in cervical neoplasias using multiple sets
HPV and ras gene mutations in cervical smears from female of generic polymerase chain reaction primers. Gynecol
genital lesions. Oncol Rep 1998; 5: 1195-8. Oncol 2001; 81: 47-52.
17. Sourvinos G, Rizos E, Spandidos DA. p53 Codon 72 32. Lungu O, Wright TC Jr, Silverstein S. Typing of human
polymorphism is linked to the development and not the papillomaviruses by polymerase chain reaction amplification
progression of benign and malignant laryngeal tumours. with L1 consensus primers and RFLP analysis. Mol Cell
Oral Oncol 2001; 37: 572-8. Probes 1992; 6: 145-52.
18. Mammas IN, Zafiropoulos A, Sifakis S, et al. Human 33. Wang TS, Chou HF, Liu WM, Choo KB. Semiautomated
papillomavirus (HPV) typing in relation to ras oncogene typing of human papillomaviruses by restriction fragment
mRNA expression in HPV-associated human squamous length polymorphism analysis of fluorescence-labeled PCR
cervical neoplasia. Int J Biol Markers 2005; 20: 257-63. fragments. J Med Virol 1999; 59: 536-40.
19. Hildesheim A, Schiffman MH, Gravitt PE, et al. Persistence 34. Laconi S, Greco M, Pellegrini-Bettoli P, et al. One-step
of type-specific human papillomavirus infection among detection and genotyping of human papillomavirus in
cytologically normal women. J Infect Dis 1994; 169: 235-40. cervical samples by reverse hybridization. Diagn Mol Pathol
20. Tieben LM, ter Schegget J, Minnaar RP, et al. Detection of 2001; 10: 200-6.
cutaneous and genital HPV types in clinical samples by 35. Jacobs MV, Snijders PJ, Voorhorst FJ, et al. Reliable high
PCR using consensus primers. J Virol Methods 1993; 42: risk HPV DNA testing by polymerase chain reaction: an
265-79. intermethod and intramethod comparison. J Clin Pathol
21. de Roda Husman AM, Walboomers JM, Hopman E, et al. 1999; 52: 498-503.
HPV prevalence in cytomorphologically normal cervical 36. Park TC, Kim CJ, Koh YM, et al. Human papillomavirus
scrapes of pregnant women as determined by PCR: the age- genotyping by the DNA chip in the cervical neoplasia DNA.
related pattern. J Med Virol 1995; 46: 97-102. Cell Biol 2004; 23: 119-25.
22. de Roda Husman AM, Walboomers JM, van den Brule AJ, 37. Grce M, Husnjak K, Skerlev M, Lipozencić J, Pavelić K.
et al. The use of general primers GP5 and GP6 elongated Detection and typing of human papillomaviruses by
at their 30 ends with adjacent highly conserved sequences means of polymerase chain reaction and fragment length
improves human papillomavirus detection by PCR. J Gen polymorphism in male genital lesions. Anticancer Res
Virol 1995; 76: 1057-62. 2000; 20: 2097-102.
23. Jacobs MV, Snijders PJ, van den Brule AJ, et al. A general 38. Kim CJ, Jeong JK, Park M, et al. HPV oligonucleotide
primer GP5+/GP6(+)-mediated PCR enzyme immunoassay microarray-based detection of HPV genotypes in cervical
method for rapid detection of 14 high-risk and 6 low-risk neoplastic lesions. Gynecol Oncol 2003; 89: 210-7.
human papillomavirus genotypes in cervical scrapings. J 39. Seo SS, Song YS, Kim JW, et al. Good correlation of HPV
Clin Microbiol 1997; 35: 791-5. DNA test between selfcollected vaginal and clinician-
24. Gravitt PE, Peyton CL, Apple RJ, Wheeler CM. Genotyping collected cervical samples by the oligonucleotide

220

215-222_JBM_774_zaravinos.indd 220 26-01-2010 12:31:00

 Zaravinos et al

microarray. Gynecol Oncol 2006; 102: 67-73. Cancer 2004; 90: 1407-13.
40. Szarewski A, Ambroisine L, Cadman L, et al. Comparison of 57. Lie AK, Risberg B, Delabie J, et al. DNA versus RNA based
predictors for high-grade cervical intraepithelial neoplasia methods for HPV testing in screening evaluation of Hybrid
in women with abnormal smears. Cancer Epidemiol Capture II and pre-tect HPV proofer in Norway 2004.
Biomarkers Prev 2008; 17: 3033-42. In: Proceedings of the 21st International Papillomavirus
41. Arens M. Clinically relevant sequence-based genotyping of Conference; 2004; 55.
HBV, HCV, CMV, and HIV. J Clin Virol 2001; 22: 11-29. 58. Klaes R, Friedrich T, Spitkovsky D, et al. Overexpression
42. Kleter B, van Doorn LJ, Schrauwen L, et al. Development of p16(INK4A) as a specific marker for dysplastic and
and clinical evaluation of a highly sensitive PCR-reverse neoplastic epithelial cells of the cervix uteri. Cancer Res
hybridization line probe assay for detection and identification 1999; 59: 6132-6.
of anogenital human papillomavirus. J Clin Microbiol 1999; 59. Melchers WJ, Herbrink P, Walboomers JM, et al.
37: 2508-17. Optimization of human papillomavirus genotype detection
43. Levi JE, Kleter B, Quint WGV, et al. High prevalence of in cervical scrapes by a modified filter in situ hybridization
human papillomavirus infections and high frequency of test. J Clin Microbiol 1989; 27: 106-10.
multiple genotypes in HIV-infected women in Brazil. J Clin 60. Ausubel FM. Short protocols in molecular biology:
Microbiol 2002; 40: 3341-5. A compendium of methods from current protocols in
44. Gonçalves MA, Massad E, Burattini MN, Villa L. Relationship molecular biology. 4th ed. New York, NY: Wiley, 1999.
between human papillomavirus (HPV) genotyping and 61. Sambrook J, Russell DW. Molecular cloning: a laboratory
genital neoplasia in HIV-positive patients of Santos City, Sao manual. 3rd ed. Cold Spring Harbor, NY: Cold Spring
Paulo. Brazil Int J STD Aids 1999; 10: 803-7. Harbor Laboratory Press, 2001.
45. Altschul SF, Gish W, Miller W, et al. Basic local alignment 62. Caussy D, Orr W, Daya AD, et al. Evaluation of methods
search tool. J Mol Biol 1990; 215: 403-10. for detecting human papillomavirus deoxyribonucleotide
46. de Villiers EM, Fauquet C, Broker TR, et al. Classification of sequences in clinical specimens. J Clin Microbiol 1988; 26:
papillomaviruses. Virology 2004; 324: 17-27. 236-43.
47. Gibson UE, Heid CA, Williams PM. A novel method for real 63. Guo M, Gong Y, Deavers M, et al. Evaluation of a
time quantitative RT-PCR. Genome Res 1996; 6: 995-01. commercialized in situ hybridization assay for detecting
48. Heid CA, Stevens J, Livak KJ, Williams PM. Real time human papillomavirus DNA in tissue specimens from
quantitative PCR. Genome Res 1996; 6: 986-94. patients with cervical intraepithelial neoplasia and cervical
49. Roberts I, Ng G, Foster N, et al. Critical evaluation of HPV16 carcinoma. J Clin Microbiol 2008; 46: 274-80.
gene copy number quantification by SYBR green PCR. BMC 64. Algeciras-Schimnich A, Policht F, Sitailo S, et al. Evaluation
Biotechnol 2008; 8: 57. of quantity and staining pattern of human papillomavirus
50. Takács T, Jeney C, Kovács L, et al. Molecular beacon-based (HPV)-infected epithelial cells in thin-layer cervical
real-time PCR method for detection of 15 high-risk and 5 specimens using optimized HPV-CARD assay. Cancer 2007;
low-risk HPV types. J Virol Methods 2008; 149: 153-62. 111: 330-8.
51. de Araujo MR, De Marco L, Santos CF, et al. GP5+/6+ 65. Clavel C, Masure M, Putaud I, et al. Hybrid capture II, a
SYBR Green methodology for simultaneous screening and new sensitive test for human papillomavirus detection.
quantification of human papillomavirus. J Clin Virol 2009; Comparison with hybrid capture I and PCR results in
45: 90-5. cervical lesions. J Clin Pathol 1998; 51: 737-40.
52. Hart KW, Williams OM, Thelwell N, et al. Novel method 66. Castle PE, Schiffman M, Burk RD, et al. Restricted
for detection, typing, and quantification of human crossreactivity of hybrid capture 2 with nononcogenic
papillomaviruses in clinical samples. J Clin Microbiol 2001; human papillomavirus types. Cancer Epidemiol Biomarkers
39: 3204-12. Prev 2002; 11: 1394-9.
53. Lamarcq L, Deeds J, Ginzinger D, et al. Measurements of 67. Hesselink AT, van den Brule AJ, Brink AA, et al. Comparison
human papillomavirus transcripts by real time quantitative of hybrid capture 2 with in situ hybridization for the detection
reverse transcription-polymerase chain reaction in samples of high-risk human papillomavirus in liquid-based cervical
collected for cervical cancer screening. J Mol Diagn 2002; samples. Cancer 2004; 102: 11-8.
4: 97-102. 68. Kornegay JR, Shepard AP, Hankins C, et al. Nonisotopic
54. Wang-Johanning F, Lu DW, Wang Y, et al. Quantitation detection of human papillomavirus DNA in clinical
of human papillomavirus 16 E6 and E7 DNA and RNA in specimens using a consensus PCR and a generic probe mix
residual material from ThinPrep papanicolaou tests using in an enzyme-linke immunosorbent assay format. J Clin
realtime polymerase chain reaction analysis. Cancer 2002; Microbiol 2001; 39: 3530-6.
94: 2199-10. 69. Castle PE, Solomon D, Schiffman M, Wheeler CM. Human
55. Cattani P, Siddu A, D’Onghia S, et al. RNA (E6/E7) versus papillomavirus type 16 infections and 2-year absolute risk
DNA (E6/E7) assays for risk evaluation in women infected of cervical precancer in women with equivocal or mild
with human papillomavirus (HPV). J Clin Microbiol 2009; cytologic abnormalities. J Natl Cancer Inst 2005; 97:
47: 2136-41. 1066-71.
56. Kraus I, Molden T, Erno LE, et al. Human papillomavirus 70. Nobbenhuis MA, Walboomers JM, Helmerhorst TJ, et
oncogenic expression in the dysplastic portio; an al. Relation of human papillomavirus status to cervical
investigation of biopsies from 190 cervical cones. Br J lesions and consequences for cervical cancer screening: a

221

215-222_JBM_774_zaravinos.indd 221 26-01-2010 12:31:00

 HPV molecular detection methods

prospective study. Lancet 1999; 354: 20-5. 81. Gravitt PE, Peyton CL, Alessi TQ, et al. Improved amplification
71. Snijders PJ, van den Brule AJ, Meijer CJ. The clinical of genital human papillomaviruses. J Clin Microbiol 2000;
relevance of human papillomavirus testing: relationship 38: 357-61.
between analytical and clinical sensitivity. J Pathol 2003; 82. van Doorn LJ, Quint W, Kleter B, et al. Genotyping of human
201: 1-6. papillomavirus in liquid cytology cervical specimens by the
72. Cuschieri KS, Whitley MJ, Cubie HA. Human papillomavirus PGMY line blot assay and the SPF(10) line probe assay. J
type specific DNA and RNA persistence–implications for Clin Microbiol 2002; 40: 979-83.
cervical disease progression and monitoring. J Med Virol 83. Castle PE, Gravitt PE, Solomon D, et al. Comparison of
2004; 73: 65-70. linear array and line blot assay for detection of human
73. Nobbenhuis MA, Helmerhorst TJ, van den Brule AJ, et al. papillomavirus and diagnosis of cervical precancer and
Cytological regression and clearance of high-risk human cancer in the atypical squamous cell of undetermined
papillomavirus in women with an abnormal cervical smear. significance and low-grade squamous intraepithelial lesion
Lancet 2001; 358: 1782-3. triage study. J Clin Microbiol 2008; 46: 109-17.
74. Perez C, Velasco J, Alonso J, et al. Comparison of the performance 84. Coutlee F, Gravitt PE, Richardson H, et al. The Canadian
of two HPV DNA genotyping methods from paraffin-embedded Women’s HIV study group. Non-isotopic detection and
cervical tissues. 23rd International Papillomavirus Conference typing of human papillomavirus DNA in genital samples by
and Clinical Workshop, Prague, 2006. the line blot assay. J Clin Microbiol 1999; 37: 1852-7.
75. Giuliano AR, Papenfuss M, Abrahamsen M, et al. Human 85. Gravitt PE, Lacey JV, Brinton LA, et al. Evaluation of
papillomavirus infection at the United States-Mexico border: self-collected cervicovaginal cell samples for human
implications for cervical cancer prevention and control. papillomavirus testing by polymerase chain reaction. Cancer
Cancer Epidemiol Biomarkers Prev 2001; 10: 1129-36. Epidemiol Biomarkers Prev 2001; 10: 95-100.
76. Richardson H, Abrahamowicz M, Tellier PP, et al. Modifiable 86. Vernon SD, Unger ER, Williams D. Comparison of human
risk factors associated with clearance of type-specific papillomavirus detection and typing by cycle sequencing,
cervical human papillomavirus infections in a cohort of line blotting, and hybrid capture. J Clin Microbiol 2000; 38:
university students. Cancer Epidemiol Biomarkers Prev 651-5.
2005; 14: 1149-56. 87. Lazcano-Ponce E, Herrero R, Munoz N, et al. Epidemiology
77. Richardson H, Kelsall G, Tellier P, et al. The natural history of HPV infection among Mexican women with normal
of type specific human papillomavirus infections in female cervical cytology. Int J Cancer 2001; 91: 412-20.
university students. Cancer Epidemiol Biomarkers Prev 88. van den Brule AJ, Pol R, Fransen-Daalmeijer N, et al. GP5+/6+
2003; 12: 485-90. PCR followed by reverse line blot analysis enables rapid and
78. Schiffman M, Wheeler CM, Dasgupta A, et al. A comparison high-throughput identification of human papillomavirus
of a prototype PCR assay and hybrid capture 2 for detection genotypes. J Clin Microbiol 2002; 40: 779-87.
of carcinogenic human papillomavirus DNA in women 89. Klaassen CH, Prinsen CF, de Valk HA, et al. DNA microarray
with equivocal or mildly abnormal Papanicolaou smears. format for detection and subtyping of human papillomavirus.
Am J Clin Pathol 2005; 124: 722-32. J Clin Microbiol 2004; 42: 2152-60.
79. Melchers WJ, Bakkers JM, Wang J, et al. Short fragment
polymerase chain reaction reverse hybridization line probe
assay to detect and genotype a broad-spectrum of human
papillomavirus types: clinical evaluation and follow-up. Am
J Pathol 1999; 155: 1473-8.
80. Quint WG, Scholte G, van Doorn LJ, et al. Comparative
analysis of human papillomavirus infections in cervical
scrapes and biopsy specimens by general SPF10 PCR and Received: August 19, 2009
HPV genotyping. J Pathol 2001; 194: 154-8. Accepted: October 14, 2009

222

215-222_JBM_774_zaravinos.indd 222 26-01-2010 12:31:00