Journal of Virological Methods 114 (2003) 135–144

Real-time PCR assays using internal controls for quantitation

of HPV-16 and ␤-globin DNA in cervicovaginal lavages
Jonas Lefevre a,b , Catherine Hankins c,d , Karina Pourreaux d ,
Hélène Voyer a,b , François Coutlée a,b,c,e,∗
The Canadian Women’s HIV Study Group1
a Laboratoire de Virologie Moléculaire, Centre de Recherche du Centre Hospitalier de l’Université de Montréal, Montréal, Qué., Canada
b Département de Microbiologie et Immunologie, Université de Montréal, Montréal, Qué., Canada
c Joint Departments of Epidemiology, Biostatistics and Occupational Health, and Department of Oncology, McGill University, Montréal, Que., Canada
d Direction de la Santé Publique de Montréal-Centre, Institut National de la Santé Publique du Québec, Montréal, Qué., Canada
e Département de Microbiologie et Infectiologie, Hôpital Notre-Dame du Centre Hospitalier de l’Université de Montréal,

1560 Sherbrooke est, Montréal, Qué., Canada H2L 4M1

Received 31 March 2003; received in revised form 21 August 2003; accepted 4 September 2003

Abstract

High-risk human papillomavirus 16 (HPV-16) DNA viral load has been measured with real-time PCR assays by amplifying HPV-16 and
a human gene. However, these assays have not used internal controls (ICs) to screen for the presence of inhibitors contained in samples. To
quantitate HPV-16 DNA and cell content with real-time PCR, ICs for HPV-16 DNA and ␤-globin were synthesised and used to control for
inhibition. The assays were sensitive and linear over 5 logs. Good reproducibility was achieved with inter-run coefficients of variation of 23%
(102 HPV-16 copies), 12% (104 HPV-16 copies), 17% (274 ␤-globin DNA copies) and 7% (27 400 ␤-globin DNA copies). Samples containing
56 800 000, 306 000, 18 000, and 4070 HPV-16 copies/␮g of cellular DNA were tested blindly and estimated to contain 48 800 000, 479 000,
20 300, and 6620 HPV-16 copies/␮g of DNA (mean ratio of measured to expected viral load of 1.27 ± 0.32). Inhibition of amplification of
HPV-16 and ␤-globin ICs by six samples known to contain PCR inhibitors was variable: four inhibited both ICs while two inhibited only the
HPV-16 IC. The use of internal controls with real-time PCR for HPV-16 quantitation allows to screen for the presence of inhibitors that do
not affect equally primer-driven genomic amplification.
© 2003 Elsevier B.V. All rights reserved.

Keywords: PCR; Real-time PCR; Cervical cancer; HPV; Internal controls

1. Introduction

∗ Corresponding author. Tel.: +1-514-890-8000/25162;
fax: +1-514-412-7512.
E-mail address: francois.coutlee@ssss.gouv.qc.ca (F. Coutlée).
1 The Canadian Women’s HIV Study Group includes the follow-
ing investigators from across Canada—Principal investigator: Catherine
Hankins. Halifax: Janet Conners∗ , Rob Grimshaw, David Haase, Lynn
Johnston∗ , Wally Schlech∗ , Arlo Yuzicappi-Fayant∗ . Hamilton: Stephen
Landis, Fiona Smaill∗ , Marek Smieja. Kingston: Wendy Wobeser∗ , Peter
Ford. London: Ole Hammerberg, Ted Ralph, Bill Thompson∗ . Montréal:
François Coutlée∗ , Julian Falutz, Alex Ferenczy∗ , Marina Klein∗ , Louise
Labrecque, Richard Lalonde, Grégoire Noël∗ , Chantal Perron∗ , Jean-Pierre
Routy, and Emil Toma. Ottawa: Claire Touchie, . Québec: Louise Coté,
Hélène Senay, Sylvie Trottier∗ . Saskatoon: Kurt Williams∗ . Sherbrooke:
Louise Dion, Alain Piché. Sudbury: Roger Sandre. Toronto: Louise
Binder∗ , Donna Keystone, Anne Phillips, Anita Rachlis∗ , Irving Salit,
Cheryl Wagner, Sharon Walmsley∗ . Vancouver: Paula Braitstein∗ , David
Infection with high-risk human papillomavirus (HPV)
types is a very strong determinant for the presence of
squamous intraepithelial lesions and invasive cancer of the
uterine cervix (Bosch et al., 2002). The association be-
tween HPV viral load measured by Hybrid Capture and the
presence or grade of squamous intraepithelial lesions was
demonstrated by some but not all cross-sectional studies
(Sun et al., 1995; Nindl et al., 1998; Clavel et al., 1998a,b).
Some studies reported that high-grade squamous intraep-
ithelial lesions contained higher HPV DNA copy numbers,
Burdge∗ , Marianne Harris, Deborah Money∗ , Julio Montaner∗ . (∗ Are
co-investigators).

0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2003.09.003
136 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144
although there was an important overlap between low-grade
and high-grade squamous intraepithelial lesions (Hall et al.,
1996; Cox et al., 1995; Sigurdsson et al., 1996). Higher
levels of HPV DNA measured with semi-quantitative PCR
assays were related to the presence or grade of squamous
intraepithelial lesions (Ho et al., 1995; Schneider et al.,
1997; Ho et al., 1998; Cuzick et al., 1994).
Few truly quantitative PCR assays have been developed
for measurement of HPV viral load. There is great interest
in using validated quantitative HPV assays in epidemiolog-
ical studies to better define the evolution of HPV infection
and lesion progression. Several real-time PCR assays have
been developed to measure HPV-16 DNA load by adjust-
ing the signal obtained for HPV-16 DNA with the amount
of cellular DNA calculated from amplification of a human
gene (Swan et al., 1997, 1999; Josefsson et al., 1999, 2000;
Ylitalo et al., 2000; Peitsaro et al., 2002; Nagao et al., 2002;
Beskow and Gyllensten, 2002). In one study, cervical car-
cinoma in situ occurred in women with consistently high
HPV-16 viral load (Ylitalo et al., 2000). HPV16 viral load
also predicted the risk of carcinoma before squamous in-
traepithelial lesions (Josefsson et al., 2000). Increased HPV
DNA viral load could be a candidate marker for the pres-
ence of cervical lesions (Josefsson et al., 2000; Ylitalo et al.,
2000). However, none of the latter assays used internal con-
trols (ICs) to screen for the presence of inhibitors, implying
that modulation of amplification efficiency by inhibitors is
independent of primer sequences (Rosenstrauss et al., 1998).
The goal of the current work was to apply a TaqMan pro-
tocol previously described to a Light Cycler system. ICs for
HPV-16 and ␤-globin were also constructed and used in this
quantitative real-time PCR assay to screen for the presence
of amplification inhibitors. The sensitivity, specificity and
reproducibility of these assays were determined. The pres-
ence of inhibitors in clinical specimens that selectively im-
pede the amplification of HPV-16 but not amplification of
␤-globin DNA is described here.
2. Material and methods
2.1. Cell lines and clinical specimens
The cervical carcinoma cell line HeLa (which contains
40 copies of HPV-18 DNA per cell) was obtained from
the American Type and Culture Collection (Rockville, MD,
USA). Titration curves of internal controls and HPV-16
DNA were obtained by serial ten-fold dilutions in a fixed
amount of 150 ng of human fibroblast DNA in 10 mM
Tris–HCl [pH 8.2], of purified HPV-16 IC DNA or of a
HPV-16 plasmid, that had been kindly provided by Prof. zur
Hausen. Titration curves of human DNA were obtained by
serial dilutions of a stock of human placental DNA D4642
(Sigma, St-Louis, MO) in 10 mM Tris–HCl [pH 8.2].
Eight clinical specimens, that had been shown in previ-
ous studies to contain PCR inhibitors, were selected for this
study (Mayrand et al., 2000; Coutlée et al., 1997a). Two
endocervical swab samples had been tested for Chlamydia
trachomatis (Coutlée et al., 2000; Coutlée et al., 1997a).
One endocervical cytobrush sample had been processed as
described previously (Tran-Thanh et al., 2003). Twenty-five
cervicovaginal lavage samples were also analysed and
had been collected from women recruited in the Canadian
Women’s HIV Study (Hankins et al., 1998, 1999; Coutlée
et al., 1997a). All participants provided written informed
consent to participate. Ethics committees of each partic-
ipating institution had approved the Canadian Women’s
HIV Study protocol. Cervicovaginal lavages were collected,
centrifuged at 2500 rpm for 10 min at 4 ◦ C, resuspended in
500 ␮l of 10 mM Tris–HCl [pH 8.2] and stored frozen at
−70 ◦ C until processed as described previously (Coutlée
et al., 1997a). Cell suspensions were thawed, lysed by addi-
tion of Tween 20 at a final concentration of 0.8% (v/v) and
digested with 250 ␮g/ml of proteinase K for 2 h at 45 ◦ C.
Cell lysates were boiled for 10 min and stored at −70 ◦ C
until tested. Some cervicovaginal lavages were further
processed with the Master Pure Extraction kit (Epicentre,
Madison, WI, USA) according to the manufacturer’s rec-
ommendations, and resuspended in 20 ␮l of TE (Tarkowski
et al., 2001). Cervicovaginal lavages had been screened
for HPV-16 DNA with the L1 consensus HPV primers
MY09/MY11 and primer HMB01, as previously described
(Hildesheim et al., 1994; Coutlée et al., 1997a,b). Amplified
products were spotted onto nylon membranes and hybridised
under stringent conditions with a 32 P-labelled oligonu-
cleotide HPV-16 probe (Bauer et al., 1991; Hildesheim
et al., 1994; Coutlée et al., 1997a).
2.2. Construction and use of the internal controls (ICs)
for HPV-16 and for β-globin
The IC for HPV-16 was generated by amplifying the
pSP65 plasmid with two primers containing pSP65 se-
quences (twenty underlined bases) to which HPV-16 se-
quences of primers U6564 or L7012 (see below) were
appended at the 5 end: primer IC-16-1 5 -CCTTATTG-
GTTACAACGAGCACGAATACACGGAATTCGAGCT-3
(bp1-20 of pSP65) and primer IC-16-2 5 -GCGTCCTA-
AAGGAAACTGATCTATTTGCTCACATGTTCTTTCC-3
(bp 407-426 of pSP65). Amplification of pSP65 with these
primers generated a pSP65 fragment flanked at both ends
by sequences of primers used for HPV-16 amplification.
HPV-16 IC Amplicons had a length similar to that of
HPV-16 (447 bp versus 449 bp). The amplification mixture
contained 2.5 mM MgCl2 , 50 mM KCl, 2.5 units of Am-
pliTaq DNA polymerase (Roche Molecular Diagnostics),
200 ␮M each of dATP, dCTP, dGTP, dTTP, and 50 pmol of
each primer pool. The HPV-16 IC was generated by 40 cy-
cles of amplification with the following cycling parameters:
one cycle at 95 ◦ C for 30 s, 55 ◦ C for 30 s and 72 ◦ C for
30 s, followed by 39 cycles at 94 ◦ C for 30 s, 55 ◦ C for 30 s
and 72 ◦ C for 30 s. The last cycle had an elongation time
 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144
of 5 min. The HPV-16 IC was purified from agarose gels
with QIAquick gel extraction kit protocol (Quiagen Inc.)
and measured in a spectrophotometer. A known amount
of HPV-16 IC was added to each amplification reaction
and was coamplified with HPV-16 DNA sequences. Since
the DNA sequences of the HPV-16 IC located between
the HPV-16 primer sequences were from pSP65, a pSP65
IC-specific probe measured the HPV-16 IC copy number in
a separate reaction (see below).
The IC for ␤-globin was constructed with pSP65 se-
quences using the same strategy with primers IC-glob-1
(5 -GAAGAGCCAAGGACAGGTACGAATACACGGAA-
TTCGAGCT-3 ; bp1-20 of pSP65) and primer IC-glob-2
(5 -CAACTTCATCCACGTTCACCAGGTTTCCCGACT-
GGAAAGC-3 ; bp 209-228 of pSP65), derived from the
␤-globin primer sequences U61992 and L62240 (see below).
The amplification mixture and parameters were identical to
those used for the HPV-16 IC. The ␤-globin and ␤-globin
IC amplicon lengths were 248 and 268 bp, respectively.
HPV-16 and ␤-globin fluorogenic PCR. HPV-16 and
␤-globin DNA were measured in separate reactions in a
Light Cycler PCR and detection system (Roche Molecu-
lar Systems). The real-time PCR assays used hydrolysis
probes described previously, but with some modifications
(Swan et al., 1999). For HPV-16 quantitation, each sam-
ple was tested in duplicate capillaries: one contained the
fluorogenic HPV-16 probe while the other capillary con-
tained the HPV-16 IC fluorogenic probe. For HPV-16
quantitation, the twenty-␮l reaction mixture contained 1x
DNA Master Hybridization Probe Mix containing the Fast-
Start Taq DNA polymerase (Roche Molecular Biochem-
icals), 4.5 mM MgCl2 , 0.3 ␮M of each HPV-16 primer.
HPV-16 primers were U6564 5 -CCTTATTGGTTACAAC-
GAGCAC (nucleotide positions 6564–6585) and L7012
5 -GCGTCCTAAAGGAAACTGATCTA (nucleotide po-
sitions 7012–7034), 500–103 copies of HPV-16 IC and
4 ␮l of processed sample. Each sample was amplified in
the presence of 50 nM of the HPV-16 fluorogenic probe
U6862 (nucleotide positions 6862–6883) labeled with
the reporter dye FAM (6-carboxyfluorescein) (FAM-5 -
CCCCAGGAGGCACACTAGAAGAT-3 -TAMRA) in one
capillary and in the presence of 50 nM of the pSP65 oligonu-
cleotide fluorogenic probe (FAM-5 -CTGCAGCCCAAGC-
TTGGCGTAAT-3-TAMRA) in another capillary. Although
the IC was included in each reaction mixture, the IC probe
was added only to the reaction mixture not containing
the HPV-16 probe since both probes were labeled with the
same dye. For ␤-globin DNA, the same reaction mixture
was used but included primers U61992 and L62240, 50 nM
of ␤-globin probe U62049 labeled with FAM and TAMRA
in one capillary, and the pSP65 oligonucleotide fluorogenic
probe described above in another capillary (Swan et al.,
1999).
After Taq polymerase activation for 7 min at 95 ◦ C, the
following cycling parameters were utilised: 95 ◦ C for 15 s,
60 ◦ C for 5 s and 65 ◦ C for 60 s for a total of 50 cycles in the
137
Light Cycler system. Negative and positive (103 copies of
each target) controls were included. Thus, each sample was
tested four times (quantitation of HPV-16 DNA, ␤-globin
DNA, HPV-16 IC and ␤-globin IC). Samples that showed
inhibition with the ICs as defined in the result section were
further diluted in TE and retested. Samples that still showed
persistent inhibitory activity were processed by the Master
Pure technique as described above.
2.3. Analysis of results and statistical methods
Hydrolysis probe-based real-time PCR assays are based
on the increase in fluorogenic signal when the fluorogenic
probes are degraded by the exonuclease activity of the Fast-
Start Taq DNA polymerase (Swan et al., 1997). FAM can
then diffuse away from the quencher TAMRA located at
the 3 end of the probes, generating a fluorescent signal
that can be detected at the end of each cycle. The level of
fluorescence is proportional to the amount of DNA gener-
ated during each cycle PCR. On-line fluorescence data is
used to calculate the first cycle in which the logarithmic lin-
ear phase of fluorescence can be distinguished from back-
ground. This cycle number is designated the threshold cycle
(Ct) (Ylitalo et al., 2000) (Josefsson et al., 2000). Compar-
ison of Ct value obtained for a sample to that of external
standards from a titration curve, allowed the quantification
of unknown amounts of HPV-16 DNA. The same applied
to the evaluation of ␤-globin quantity in samples. HPV-16
viral load was expressed as a number of HPV copies per ␮g
of human DNA.
The intra-assay coefficient of variation (CV) was deter-
mined by amplifying four times in the same PCR run 200
and 5000 copies of HPV-16 DNA, and human DNA from
54 800 (0.2 ␮g) and 2740 (0.01 ␮g) lysed fibroblast cells. To
determine the inter-assay CV, 100 and 10 000 HPV-16 DNA
copies, as well as 274 and 27 400 ␤-globin DNA copies were
tested on eight different days. The CV was calculated as the
ratio of the standard deviation over the mean, multiplied by
100. One ␮g of human DNA corresponds to 137 000 cells.
HPV-16 IC DNA copy numbers measured in lysates, diluted
samples and purified DNA from the same samples were com-
pared using a two-tailed Student’s t-test. The number of IC
copies measured in samples with inhibition was compared
to the number of ICs measured in TE using a two-tailed
Student’s t-test.
3. Results
3.1. Characteristics of the real-time PCR assays
Internal controls were constructed for two quantitative
real-time PCR assays: one measuring the HPV-16 copy num-
ber and the other measuring the quantity of cellular DNA,
allowing to derive the HPV-16 viral load. We first assessed
the sensitivity for detection of each target and IC. Serial
138 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144

Fig. 1. Quantitation of HPV-16, ␤-globin and their respective internal controls (IC) DNA with real-time PCR assays. Serial dilutions of HPV-16 DNA
(䉱) and of the HPV-16 IC DNA (䊉) contained in a fixed amount of 0.5 ␮g of human DNA, and serial dilutions of ␤-globin DNA (䊏) and of the
␤-globin IC DNA (䉲), were prepared and tested as described in the methods section. Results are expressed as the mean of Ct values ± S.D. of duplicate
testing. No fluorescence was obtained when DNA from 105 human cells was amplified with HPV-16 primers and HPV-16 or the HPV-16 IC fluorogenic
probes, 104 copies of HPV-16 were amplified in presence of HPV-16 primers and the HPV-16 IC probe or when 103 HPV-16 IC copies were amplified
with HPV-16 primers and HPV-16 probe, 104 copies of HPV-16 DNA was amplified with the ␤-globin primers and probe, 103 copies of the ␤-globin
IC were amplified in the presence of the ␤-globin probe, 106 copies of ␤-globin DNA were amplified in the presence of the ␤-globin IC probe. Neither
HPV-16, ␤-globin or ICs reagents reacted with 1 ng of pSP65 plasmid.

dilutions of HPV-16 DNA, HPV-16 IC DNA, human pla-
cental DNA and ␤-globin IC DNA were tested twice on dif-
ferent days (Fig. 1). As mentioned in the figure legend, no
fluorescence was generated when one ␮g of human placental
DNA was amplified with HPV-16 primers and the HPV-16
or HPV-16 IC fluorogenic probes or when HPV-16 was am-
plified with ␤-globin primers in the presence of ␤-globin or
␤-globin IC fluorogenic probes. The assays could detect 10
copies of HPV-16, ␤-globin or ICs DNA. The linear range of
each assay extended from 5–6 logs. Fig. 1 demonstrates that
the Ct and the slopes of the curves for detection of HPV-16
DNA or ␤-globin and their respective ICs were within one
standard deviation of the other curves. The ICs were thus
amplified as efficiently as their targets. Signals generated by
amplification of HPV-16 were lower than those obtained by
amplification of ␤-globin. These results were confirmed by
retesting the same dilutions on different days.
The reproducibility of the various real-time PCR assays
was then evaluated. The average estimates of 200 and 5000
copies of HPV-16 were 265 ± 20 and 5566 ± 174 copies, for
intra-assay CVs of 7.5 and 3.1%, respectively. Human DNA
from 54 800 and 2740 lysed fibroblast cells were estimated
to contain an average of 1918±274 cells (0.014±0.002 ␮g)
and 37 127 ± 685 cells (0.271 ± 0.005 ␮g), for intra-assay
CVs of 15.9 and 7.0%, respectively. For inter-assay CVs,
the mean measured numbers of 100 and 10 000 HPV-16
DNA copies were 104 ± 24 and 9757 ± 1181, for a vari-
ability of 23.4 and 12.1%, respectively. The mean measured
number of ␤-globin DNA copies in samples containing
274 and 27 400 ␤-globin DNA copies were 244 ± 41 and
30 004 ± 2008 copies, for inter-assay CVs of 16.7 and
6.7%, respectively. The variability of the ICs was also as-
sessed by amplifying 103 copies of HPV-16 and ␤-globin
IC DNA separately five times on the same run and once on
five different days: CVs obtained for HPV-16 IC reached
4.2% (1001 ± 42 copies) and 11.5% (1030 ± 119 copies),
and for ␤-globin IC 10.6% (1004 ± 126 copies) and 11.5%
(1023 ± 151 copies), respectively.
A panel of five samples containing known quantities
of HPV-16 plasmid DNA and placental human DNA was
tested blindly with the real-time PCR assays for quantitation
of HPV-16 and ␤-globin DNA. Results of the estimated
HPV-16 viral load in these specimens containing from 4070
to 56 800 000 HPV-16 DNA copies/␮g of cellular DNA are
 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144 139

Table 1
Blind testing of samples containing known amounts of HPV-16 DNA and human DNA
Sample Input HPV-16 DNA viral Measured HPV-16 DNA viral Ratio (measured/input)
load (HPV-16 copies/␮g) load (HPV-16 copies/␮g)
1 56800000 48800000 0.86
2 306000 479000 1.57
3 239000 280000 1.17
4 18000 20300 1.13
5 4070 6620 1.63
Various amounts of HPV-16 DNA were mixed with various quantities of placental human DNA. Samples were tested once blindly with the real-time
PCR assays for HPV-16 and ␤-globin DNA as described in the methods section. The measured number of copies from each target was derived by
comparing the Ct against HPV-16 and ␤-globin standard curves.

presented in Table 1. The ratio between input and measured HPV-16 copies interfered with the amplification of all con-
copy numbers ranged from 0.86 to 1.63, for an average of centrations of HPV-16 IC and 105 HPV-16 copies reduced
1.27 ± 0.32. the reactivity of 102 HPV-16 IC copies (data not shown).
The presence of other HPV types contained in samples Results from two experiments are presented in Table 2 using
could interfere with quantitation of HPV-16 by hybridising 103 copies of HPV-16 IC. In these experiments, 10 copies
with HPV-16 primers and probe, and generate false-positive of HPV-16 DNA were consistently detected even when
results, or by competing with HPV-16 for HPV-16 primers coamplified with 103 copies of internal control. Amounts
and impede amplification efficiency of HPV-16 contained of HPV-16 DNA up to 105 copies did not interfere with the
in the sample tested. To investigate if HPV types other than detection of 103 copies of HPV-16 IC (Table 2). The same
16 could alter the quantitation of HPV-16, 106 copies of results were obtained with ␤-globin DNA and IC (data not
each type 6, 11, 16, 18, 31, 33, 35, 45, 66 cloned into shown). However, the signal obtained by amplification of
plasmids, five ␮l of processed samples that had generated 103 HPV-16 IC copies in the presence of 105 HPV-16 DNA
a strong signal in a consensus L1 PCR assay with one of copies was increased with a decreased Ct value as demon-
the following types 39, 51, 52, 53, 56, or 58, and a lysate strated in Table 2. In some runs, 103 copies of HPV-16 IC
of 25 000 HPV-18-positive HeLa cells were amplified sep- were estimated at 2 × 103 copies. Increases in estimations of
arately with HPV-16 primers and the fluorogenic HPV-16 IC copy number in the presence of non-homologous DNA
probe. They were also amplified separately in another re- was also encountered for ␤-globin IC (data not shown). Be-
action with 103 copies of the HPV-16 IC and the HPV-16 cause of the variable increased amplification efficiency with
IC fluorogenic probe. In these experiments, the mean esti- total DNA content, we choose not to adjust the measured
mates of 103 HPV-16 copies and of 103 HPV-16 IC copies
tested in duplicates were 1016 ± 257 HPV-16 copies and Table 2
1002 ± 70 copies, respectively. Specificity of the assay was Coamplification of various quantities of HPV-16 DNA with 103 copies
excellent since no fluorescence was generated with types of HPV-16 IC
other than 16. The signals generated by the HPV-16 IC 103 No. of input copies HPV-16 DNA HPV-16 IC
copies were not altered by the presence of other HPV types. reactivity (Ct reactivity
HPV-16 DNA HPV-16 IC ± S.D.) (Ct ± S.D.)
However, the presence of 106 copies of HPV-16 interfered
1000000 0 21.66 ± 0.93
significantly with amplification of the HPV-16 IC generat-
1000000 1000 20.89 ± 1.44 No fluorescence
ing signals corresponding to 36 ± 2 copies (P = 0.003, 100000 0 25.96 ± 0.35
Student’s t-test). The latter phenomenon suggested compe- 100000 1000 24.86 ± 0.48 29.09 ± 1.50
tition between HPV-16 and IC and was investigated below. 10000 0 29.36a
10000 1000 28.20a 30.61 ± 1.10
3.2. Optimisation of IC use conditions 1000 0 33.94 ± 0.73
1000 1000 33.04 ± 0.45 30.84 ± 1.10
100 0 37.02 ± 0.25
Although HPV-16 DNA and HPV-16 IC were measured 100 1000 37.09 ± 0.68 30.53 ± 0.42
in separate capillaries, coamplification occurred in both 10 0 40.90 ± 1.35
reactions. Ideally, the IC should not interfere with the quan- 10 1000 40.96 ± 0.78 31.03 ± 1.40
titation of small amounts of HPV-16 or ␤-globin DNA. 1 0 43.68 ± 0.60
Conversely, since the copy number of HPV-16 will vary 1 1000 41.24a 31.17 ± 1.60
0 0 No fluorescence
between specimens, amplification of a high copy number 0 1000 No fluorescence 30.68 ± 1.10
of HPV-16 must not interfere with amplification of the IC.
To investigate the level of competition between HPV-16 or Samples from a titration curve of HPV-16 DNA were mixed with 103
copies of HPV-16 IC and were amplified with the real-time PCR assay
␤-globin DNA amplification and their respective IC, serial
on two different days. Results are from duplicate ± one S.D.
ten-fold dilutions of HPV-16 DNA (1–106 DNA copies) a Samples that were not tested in duplicate. Similar results were ob-
were spiked with 102 to 104 copies of HPV-16 IC. 106 tained with the PCR for ␤-globin and ␤-globin IC.
140 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144

Table 3
Analysis of a panel of serially diluted HPV-16-positive cervicovaginal lavage lysates
Sample Dilution HPV-16 DNA copies ␤-Globin DNA copies HPV-16 viral load CV (%)
(No. of copies) (No. of copies) (No. of copies/␮g)
A Undiluted 9647500 ± 210011 56535 ± 1379 46783468 ± 2158852 22.8
1:10 2387500 ± 235467 8458 ± 1959 80389562 ± 26252743
1:30 623650 ± 27931 2567 ± 169 66627256 ± 1405373
1:103 10046 ± 1844 47 ± 10 61052528 ± 23610256
B 1:10 1253000 ± 67882 9020 ± 1028 38192892 ± 2291313 4.6
1:30 278600 ± 5798 2166 ± 215 35454022 ± 4252050
1:100 111200 ± 566 789 ± 38 38657500 ± 1674387
C Undiluted 4690 ± 1187 24115 ± 3557 54875 ± 21583 2.2
1:10 274 ± 78 1384 ± 254 56632 ± 25796
D Undiluted 10750000 ± 296985 92145 ± 14927 32463411 ± 6142015 18.0
1:10 1096500 ± 112430 7108 ± 677 42253373 ± 307123
1:100 114500 ± 17536 782 ± 366 46691203 ± 27989803
E Undiluted 148 ± 20 2068 ± 95 18943 ± 2620 8.2
1:30 4±5 38 ± 17 21282 ± 24151
F Undiluted 11 ± 8 9682 ± 2175 330 ± 294 6.9
1:30 1±1 808 ± 79 364 ± 515
G Undiluted 92 ± 1 34875 ± 1450 724 ± 41 41.6
1:30 6±9 1157 ± 219 1327 ± 1877
Serial dilutions of cervicovaginal lavage lysates were tested with real-time PCR assays for quantitation of HPV-16 and ␤-globin DNA. Dilutions were
tested on different runs and depended on the initial reactivity of sample. HPV-16 viral load was the number of HPV-16 DNA copies/␮g of cellular DNA
deduced from the number of measured ␤-globin copies. CV: coefficient of variation. Results are from duplicate ± one S.D. For each sample viral load
estimates of each dilution were not significantly different with a Student’s t-test (P > 0.05 for all comparisons).

HPV-16 DNA copies with the reactivity of the HPV-16 IC as
is usually done in quantitative PCR assays with internal con-
trols, but rather screen samples for inhibition with the ICs.
3.3. Real-time PCR assay results for serially diluted
clinical specimens
Several lysates from HPV-16-positive cervicovaginal
lavages were tested in duplicate for HPV-16 and ␤-globin
DNA. Lysates were diluted (1:10 to 1:103 ) in TE and
retested in duplicate (Table 3). The variability of HPV-16
viral load determination was assessed by calculating CVs
from viral loads measured in dilutions for each sample. CVs
ranged from 2.2% for sample C to 42% for sample G. The
latter sample contained a very low copy number of HPV-16
DNA, explaining the important differences between viral
load estimations. The calculated HPV-16 viral load from
dilutions always came within two-folds of the viral load
measured in the undiluted sample. According to these re-
sults, HPV-16 viral load estimates from diluted samples
because of the presence of inhibitors should be adequate.
Sixteen undiluted samples were tested in duplicate ini-
tially and then after purification with the Master Pure tech-
nique on a different day. The difference between the HPV-16

Table 4
HPV-16 DNA viral load measures in cell lysates and purified DNA from 16 samples
Sample HPV-16 DNA copies/␮g of cellular DNA HPV-16 viral load on P value
purified DNA/lysate
Cell lysate Purified DNA

1–7 0 0 ND ND
8 13521 ± 5007 18114 ± 4808 1.33 0.59
9 1688 ± 2387 1224 ± 157 0.73 0.81
10 28587 ± 9367 31218 ± 3993 1.09 0.75
11 349748 ± 210820 214326 ± 20946 0.61 0.46
12 123426 ± 23337 131316 ± 5564 1.06 0.69
13 908185 ± 139590 865492 ± 8087 0.95 0.71
14 19 ± 26 59 ± 12 3.10 0.19
15 155922 ± 53979 354409 ± 169192 2.27 0.255
16 159493 ± 48918 337338 ± 23186 2.12 0.04a
HPV-16 DNA viral load was measured on undiluted lysates and after DNA purification by Master Pure on 16 samples. ND is for not done. Results are from
duplicate ± one S.D. The ratio is calculated by dividing the HPV-16 viral load obtained on purified DNA and that obtained on the undiluted cell lysate.
a Significant results obtained with a Student’s t-test.
 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144
Table 5
Effect of samples containing PCR inhibitors on the amplification efficiency of 500 copies of HPV-16 and ␤-globin internal controls
Sample no. Sample tested
1 Genital swab sample
2 Genital cytobrush sample
3 Cervicovaginal lavage
4 Cervicovaginal lavage
5 Cytobrush sample
6 Bloody sample
7 Genital swab sample
8 ICs in water
9 No IC
Five hundred copies of HPV-16 and ␤-globin ICs mixed with processed samples that had demonstrated inhibition of HPV amplification in previous
studies were amplified in separate reactions. Results are from duplicates ± one S.D.
viral load estimated on lysates and purified DNA was sig-
nificant for sample 16 (Table 4). A very high ratio be-
tween HPV-16 viral load estimated from purified DNA and
from lysate was obtained with sample 14 which contained a
very low viral load (59 ± 12 versus 19 ± 26 HPV-16 DNA
copies/␮g of cellular DNA, P = 0.187). The ratios of viral
load estimated on purified DNA and on lysate were usually
within two-folds (range 0.61–2.27, mean of 1.47 ± 0.84).
3.4. Effect of inhibitors on HPV-16 and β-globin
quantitation
In vitro experiments were conducted to study the effect
of sample inhibition on amplification of the HPV-16 and
␤-globin ICs. Aliquots of samples previously suspected of
containing inhibitors were mixed with 500 copies of both
ICs in separate reactions in order to assess whether amplifi-
cation of ICs with ␤-globin and HPV-16 primers was simi-
larly impeded by the presence of PCR inhibitors (Table 5).
Genital sample #1 had been shown to contain inhibitors on
the basis of Chlamydia trachomatis IC in the Amplicor test
(Coutlée et al., 2000). Cervicovaginal lavage samples 3 and
4 as well as cytobrush sample #2 had been shown previ-
ously to contain inhibitors on the basis of ␤-globin detection
(Tran-Thanh et al., 2003; Coutlée et al., 1997a). For sam-
ple 5, a cytobrush was left in TE buffer for several hours
and then retrieved. Sample 6 was a supernatant of blood
collected with heparin. The ICs were also amplified with
a genital sample that had successfully been amplified for
␤-globin (specimen 7). As shown in Table 5, four samples
showed inhibition for both ICs. For two samples, inhibition
for the HPV-16 IC was demonstrated in the absence of inhi-
bition for the ␤-globin IC. The latter results were confirmed
on repeat testing a different day. Sample #1 demonstrated
strong inhibition for HPV-16 IC but ␤-globin amplification
was enhanced.
To discriminate between PCR inhibition and quenching of
fluorescent signal from fluorogenic probes, capillaries were
spun in a microfuge after completion of PCR and 10 ␮l of
reaction mixture was migrated by electrophoresis on a 2%
agarose gel stained with ethidium bromide. IC amplicons
141
Measured HPV-16 IC copies Measured ␤-globin IC copies
0±0 1781 ± 35
49 214
135 325
250 235
1±1 692 ± 141
97 ± 93 258 ± 164
632 ± 134 526 ± 101
504 ± 73 503 ± 60
No fluorescence No fluorescence
could not be demonstrated in samples that showed inhibition
(data not shown). Each IC was amplified with sample 6
in the presence of SYBERGREEN. The signal generated
corresponded to 16 ± 22 copies and 181 ± 55 copies of
HPV-16 and ␤-globin ICs, respectively. Spiking experiments
with HPV-16 or ␤-globin DNA could not be performed since
some samples already contained human or HPV DNA.
4. Discussion
The use of ICs in real-time PCR assays for HPV-16 viral
load determination is described in this work. Several groups
have utilised real-time PCR assays for HPV DNA viral load
determinations (Josefsson et al., 1999, 2000; Swan et al.,
1997; Ylitalo et al., 2000; Peitsaro et al., 2002; Nagao et al.,
2002; Beskow and Gyllensten, 2002). Real-time PCR as-
says provide accurate measurements of the initial copy num-
ber of target DNA in samples by continuously measuring
the increments of fluorescence released during the reaction
(Loeffler et al., 2000). This monitoring is impossible with
conventional PCR. Although the assay devised by Swan and
colleagues is very sensitive (Swan et al., 1999), some of the
HPV-16 positive samples tested here scored negative in the
real-time PCR assay for HPV-16 DNA in the absence of in-
hibitors. A greater volume of lysate was however tested in
the conventional PCR assay.
Real-time PCR assays for HPV-16 and ␤-globin were
shown to be specific and reproducible. The specificity of
the real-time PCR assay for HPV-16 had been assessed in
the original description of the assay for a limited number of
HPV types (Swan et al., 1997). In the original description of
the assay, cross-reactivity was not encountered with types
31, 33, 45, 51, 52 but was demonstrated with types 18, 35
and 56. A panel of HPV types contained in plasmids and
samples did not generate false-positive result. In the initial
evaluation of this TaqMan protocol, amplifications were
performed in a 9600 thermal cycler and then read in a fluo-
rometer. The LightCycler combines amplification by rapid
thermocycling with glass capillaries and online fluorescence
detection (Nitsche et al., 1999). It may be that a higher
142 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144
degree of stringency was achieved using this instrument,
although we did not compare these instruments in parallel.
Excellent CVs of at most 23% were obtained. Intra-run
CVs were lower than inter-run CVs. CVs were greater
when the amount of HPV-16 or ␤-globin DNA was smaller.
Reproducibility was also evaluated by comparing viral
load results obtained after lysate dilution or DNA purifica-
tion to those measured on undiluted lysates. Results were
similar considering errors with sample manipulation and
that the viral load determination results of the ratio of
two measures. These experiments and the blind testing of
samples suggest that two-fold or greater differences in vi-
ral load between samples could be demonstrated with this
assay.
Few truly quantitative PCR assays have been developed
for HPV quantitation. The low-stringency PCR assay uses a
non-stringent amplification reaction with primers GP5/GP6
that consistently generates a specific band from human DNA
and permits calculation of the ratio of signals of HPV DNA
and human DNA (Caballero et al., 1995). This assay can-
not be applied on samples containing multiple types. More-
over, the correlation between HPV viral load and grade of
lesions may be dependent of HPV type (Swan et al., 1999).
Real-time PCR assays for HPV DNA viral load determina-
tions measure the human DNA in samples to adjust HPV re-
sults for cellular content (Josefsson et al., 1999; Swan et al.,
1997; Josefsson et al., 2000; Ylitalo et al., 2000). Such cor-
rection is essential if bias in viral load measurements is to
be avoided, since more cellular material can be obtained
from sampling high-grade cervical lesions, thus potentially
generating artificially higher HPV viral loads (Swan et al.,
1999).
ICs have been described for several infectious agents but
not for HPV (Yang et al., 1993; Coutlée et al., 1998; Boeckh
and Boivin, 1998). Coamplification of HPV DNA and HPV
IC, which use the same primer binding sites but have a
different intervening sequence, is the best means to mea-
sure the impact of inhibitors or competition by other HPV
types on HPV DNA amplification (Coutlée et al., 1997c;
Boivin et al., 1997; Wang et al., 1989a; Wang and Mark,
1989b). The size of the IC amplicons was similar to that
of the target DNA. Any conditions that modify the ampli-
fication efficiency of the target DNA will affect the rate
of amplification of the IC. The reduced amplification effi-
ciency can thus be measured by comparing the estimated
copy number of IC to the actual number of IC copies in-
troduced into the reaction mixture. Real-time PCR assays
for HPV quantitation did not use an internal control (IC)
to specifically control for PCR inhibitors, although inhibi-
tion could be partly screened using ␤-globin amplification
(Coutlée et al., 2000). To measure precisely the HPV-16
viral load in cervicovaginal lavages, two quantitative PCR
assays were done separately to avoid competition between
targets, one for HPV-type-specific DNA quantitation and
one for ␤-globin quantitation (Josefsson et al., 1999). The
amount of IC introduced in each reaction was also opti-
mized to avoid competition with HPV-16 DNA quantita-
tion. Internal controls can screen for the presence of ampli-
fication inhibition (Rosenstrauss et al., 1998; Wang et al.,
1989a; Wang and Mark, 1989b). We have demonstrated that
␤-globin amplification cannot replace the use of ICs for de-
tection of the presence of inhibitors (Coutlée et al., 2000). In
the latter study, more than half of urethral samples contain-
ing inhibitors scored positive for ␤-globin. This discrepancy
could be explained by a higher number of ␤-globin DNA
molecules compared with the low IC copy numbers usually
introduced in quantitative PCR tests. Variations in HPV viral
load caused by inhibitors could go unnoticed with ␤-globin
PCR amplification alone, thus underestimating HPV viral
load amplification. ICs can also be utilised in PCR to nor-
malise the signal obtained for quantitation of microbial DNA
(Mulder et al., 1994). However, the increased signal gener-
ated by ICs in the presence of non-homologous DNA still
remains unexplained. ICs were thus utilised only to screen
for inhibitors.
Inhibitors could affect to different degrees the efficiency
of amplification of different primer pairs for the same DNA
template (Coutlée et al., 1991). In another report, the rate of
inhibition of lysates was evaluated with ICs for ␤-globin,
herpes simplex virus and varicella–zoster virus (Bezold
et al., 2000). Inhibition rates for each of these ICs were
23, 16 and 25%, respectively. Not all primer sequences are
equally sensitive to inhibitors. Thus investigators should
not rely solely on ␤-globin amplification to control for in-
hibitors affecting amplification of other viral targets. Find-
ings from the current work support this conclusion, since
some inhibitors, as yet unidentified, were impeding prefer-
entially HPV-16 amplification, resulting in an underestima-
tion of HPV-16 DNA copies contained in these samples.
We also found one sample for which the HPV-16 IC was
inhibited while the amplification of the ␤-globin IC was
enhanced. Amplification facilitators such as bovine serum
albumin, DNA binding T4 gene gp32, betaine and others,
have been described for both conventional and real-time
PCR (Abu and Radstrom, 2000). Proteinase inhibitors are
also known as facilitators of PCR amplification (Abu and
Radstrom, 2000). Measurement of HPV-16 viral load can
be influenced greatly by these opposite effects of samples
on ␤-globin and HPV-16 amplification.
Quantitative PCR assays will increase our knowledge
of HPV-induced carcinogenesis. Results from this work
suggest that samples should be first screened for inhibitors
against all targets being quantitated. Samples should be
first screened for inhibitors. Only samples or diluted sam-
ples without inhibitory activity against HPV and ␤-globin
DNA amplification can be tested. These ICs will now al-
low us to investigate the progression of HPV-16 viral load
during infection and better define the relationship between
viral load and squamous intraepithelial lesions. Investi-
gators using real-time PCR for viral quantitation should
determine if inhibitors similarly affect primer sequences
used.
 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144
Acknowledgements
We would like to thank Mme Diane Gaudreault and
Mme Diane Bronsard for processing genital samples. This
study was supported by CANFAR. The Canadian Institutes
of Health Research supports The Canadian Women’s HIV
Study. F.C. is a chercheur national supported by the Fonds
de la Recherche en Santé du Québec and by le Réseau
FRSQ-SIDA. HPV-16 plasmid was kindly provided by
Prof. zur Hausen.
References
Abu, A., Radstrom, P., 2000. Effects of amplification facilitators on di-
agnostic PCR in the presence of blood, feces, and meat. J. Clin.
Microbiol. 38, 4463–4470.
Bauer, H.M., Ting, Y., Greer, C.E., Chambers, J.C., Tashiro, C.J., Chimera,
J., Reingold, A., Manos, M.M., 1991. Genital human papillomavirus
infection in female university students as determined by a PCR-based
method. J. Am. Med. Assoc. 265, 472–477.
Beskow, A.H., Gyllensten, U.B., 2002. Host genetic control of HPV 16
titer in carcinoma in situ of the cervix uteri. Int. J. Cancer 101, 526–
531.
Bezold, G., Volkenandt, M., Gottlober, P., Peter, R.U., 2000. Detection
of herpes simplex virus and varicella-zoster virus in clinical swabs:
frequent inhibition of PCR as determined by internal controls. Mol.
Diagn. 5, 279–284.
Boeckh, M., Boivin, G., 1998. Quantitation of cytomegalovirus: method-
ologic aspects and clinical applications. Clin. Microbiol. Rev. 11,
533–554.
Boivin, G., Handfield, J., Murray, G., Toma, E., Lalonde, R., Lazar, J.G.,
Bergeron, M.G., 1997. Quantitation of cytomegalovirus (CMV) DNA
in leukocytes of human immunodeficiency virus-infected subjects with
and without CMV disease by using PCR and the SHARP Signal
Detection System. J. Clin. Microbiol. 35, 525–526.
Bosch, F.X., Lorincz, A., Munoz, N., Meijer, C.J.L.M., Shah, K.V., 2002.
The causal relation between human papillomavirus and cervical cancer.
J. Clin. Pathol. 55, 244–265.
Caballero, O.L., Villa, L.L., Simpson, J.G., 1995. Low stringency-PCR
(LS-PCR) allows entirely internally standardized DNA quantitation.
Nucl. Acids Res. 23, 192–193.
Clavel, C., Bory, J.P., Rihet, S., Masure, M., Duval-Binninger, I., Putaud,
I., Lorenzato, M., Quereux, C., Birembaut, P., 1998a. Comparative
analysis of human papillomavirus detection by Hybrid Capture assay
and routine cytologic screening to detect high-grade cervical lesions.
Int. J. Cancer 75, 525–528.
Clavel, C., Masure, M., Putaud, I., Thomas, K., Bory, J.P., Gabriel,
R., Quereux, B.P., 1998b. Hybrid capture II, a new sensitive test
for human papillomavirus detection. Comparison with Hybrid Cap-
ture I and PCR results in cervical lesions. J. Clin. Pathol. 51, 737–
740.
Coutlée, F., St-Antoine, P., Olivier, C., Kessous, A., Voyer, H., Berrada, F.,
Begin, P., Giroux, L., Viscidi, R.P., 1991. Inhibitors of Taq polymerase:
a potential cause for discordant results for detection of HIV-1 DNA
with PCR. J. Infect. Dis. 164, 817–818.
Coutlée, F., et al., 1997a. Comparison betwen vaginal tampon and cervi-
covaginal lavage specimens collection for detection of human papil-
lomavirus DNA by the polymerase chain reaction. J. Med. Virol. 51,
42–47.
Coutlée, F., Trottier, A.M., Ghattas, G., Leduc, R., Toma, E., Sanche, G.,
Rodrigues, I., Turmel, B., Allaire, G., Ghadirian, P., 1997b. Risk factors
for oral human papillomavirus in adults infected and not infected with
human immunodeficiency virus. Sex. Transm. Dis. 24, 23–31.
143
Coutlée, F., Mayrand, M.H., Provencher, D., Franco, E., 1997c. The future
of HPV testing in clinical laboratories and applied virology research.
Clin. Diagn. Virol. 8, 123–141.
Coutlée, F., Saintlouis, G., Voyer, H., Daloze, P., Ghadirian, P., 1998. My-
coplasma fermentans DNA is infrequently detected in urine specimens
from renal transplant recipients. Mol. Cell. Probes 12, 201–206.
Coutlée, F., de Ladurantaye, M., Tremblay, C., Vincelette, J., Labrecque,
L., Roger, M., 2000. An important proportion of genital samples sub-
mitted for Chlamydia trachomatis detection by PCR contain small
amounts of cellular DNA as measured by beta-globin gene amplifica-
tion. J. Clin. Microbiol. 38, 2512–2515.
Cox, J.T., Lorincz, A.T., Schiffman, M.H., Sherman, M.E., Cullen, A.,
Kurman, R.J., 1995. Human papillomavirus testing by hybrid capture
appears to be useful in triaging women with a cytologic diagnosis of
atypical squamous cells of undetermined significance. Am. J. Obstet.
Gynecol. 172, 946–954.
Cuzick, J., Terry, G., Ho, L., Hollingworth, T., Anderson, M., 1994.
Type-specific human papillomavirus DNA in abnormal smears as a
predictor of high-grade cervical intraepithelial neoplasia. Br. J. Cancer
69, 167–171.
Hall, S., Lorincz, A.T., Shah, F., Sherman, M.E., Abbas, F., Paull, G.,
Kurman, R.J., Shah, K., 1996. Human papillomavirus DNA detection
in cervical specimens by hybrid capture: correlation with cytologic
and histologic diagnoses of squamous intraepithelial lesions of the
cervix. Gynecol. Oncol. 62, 353–359.
Hankins, C., Coutlée, F., Lapointe, N., Simard, P., Tran, T., Samson, J.,
Hum, L.,. The Canadian Women’s HIV study Group 1999. Prevalence
of risk factors associated with human papillomavirus infection in
women living with HIV. Can. Med. Assoc. J. 160, 185–191.
Hankins, C., Lapointe, N., Walmsley, S., 1998. Participation in clinical
trials among women living with HIV in Canada. Can. Med. Assoc. J.
159, 1359–1365.
Hildesheim, A., Schiffman, M.H., Gravitt, P.E., Glass, A.G., Greer, C.E.,
Zhang, T., Scott, D.R., Rush, B.B., Lawler, P., Sherman, M.E., Kur-
man, R.J., Manos, M.M., 1994. Persistence of type-specific human
papillomavirus infection among cytologically normal women. J. In-
fect. Dis. 169, 235–240.
Ho, G.Y.F., Burk, R.D., Klein, S., Kadish, A.S., Chang, C.J., Palan, P.,
Basu, J., Tachezy, R., Lewis, R., Romney, S., 1995. Persistant genital
human papillomavirus infection as a risk factor for persistent cervical
dysplasia. J. Nat. Cancer Inst. 87, 1365–1371.
Ho, G.Y.F., Palan, P.R., Basu, J., Romney, S.L., Kadish, A.S., Mikhail, M.,
Wassertheilsmoller, S., Runowicz, C., Burk, R.D., 1998. Viral charac-
teristics of human papillomavirus infection and antioxidant levels as
risk factors for cervical dysplasia. Int. J. Cancer 78, 594–599.
Josefsson, A., Livak, K., Gyllensten, U., 1999. Detection and quantitation
of human papillomavirus by using the fluorescent 5 exonuclease assay.
J. Clin. Microbiol. 37, 490–496.
Josefsson, A.M., Magnusson, P.K.E., Ylitalo, N., Sorensen, P.,
Qwarforth-Tubbin, P., Anderson, P.K., Melbye, M., adami, H.O., yl-
lensten, U.B., 2000. Viral load of human papilloma virus 16 as a
determinant for development of cervical carcinoma in situ: a nested
case–control study. Lancet 355, 2189–2193.
Loeffler, J., Henke, N., Hebart, H., Schmidt, D., Hagmeyer, L., Schu-
macher, U., Einsele, H., 2000. Quantification of fungal DNA by using
fluorescence resonance energy transfer and the Light Cycler System.
J. Clin. Microbiol. 38, 586–590.
Mayrand, M.H., Coutlée, F., Hankins, C., Lapointe, N., Forest, P., De
Ladurantaye, M., Roger, M., 2000. Detection of human papillomavirus
type 16 DNA in consecutive genital samples does not always represent
persistent infection as determined by molecular variant analysis. J.
Clin. Microbiol. 38, 3388–3393.
Mulder, J., McKinney, N., Christopherson, C., Sninsky, J.J., Greenfield,
L., Kwok, S., 1994. Rapid and simple PCR assay for aquantitation of
human immunodeficiency virus type 1 RNA in plasma: application to
acute retroviral infection. J. Clin. Microbiol. 32, 292–300.
144 J. Lefevre et al. / Journal of Virological Methods 114 (2003) 135–144
Nagao, S., Yoshinouchi, M., Miyagi, Y., Hongo, A., Kodama, J., Itoh, S.,
Kudo, T., 2002. Rapid and sensitive detection of physical status of
human papillomavirus type 16 DNA by quantitative real-time PCR.
J. Clin. Microbiol. 40, 863–867.
Nindl, I., Lorincz, A., Mielzynska, I., Petry, U., Baur, S., Kirchmayr, R.,
Michels, W., Schneider, A., 1998. Human papillomavirus detection
in cervical intraepithelial neoplasia by the second generation hybrid
capture microplate test, comparing two different cervical specimen
collection methods. Clin. Diagn. Virol. 10, 49–56.
Nitsche, A., Steuer, N., Schmidt, C.A., Landt, O., Siegert, W., 1999. Dif-
ferent real-time PCR formats compared for the quantitative detection
of human cytomegalovirus DNA. Clin. Chem. 45, 1932–1937.
Peitsaro, P., Johansson, B., Syrjanen, S., 2002. Integrated human papil-
lomavirus type 16 is frequently found in cervical cancer precursors
as demonstrated by a novel quantitative real-time PCR technique. J.
Clin. Microbiol. 40, 886–891.
Rosenstrauss, M., Wang, Z., Chang, S.-Y., Debonville, D., Spadoro, J.P.,
1998. An internal control for routine diagnostic PCR: design properties
and effect on clinical performance. J. Clin. Microbiol. 36, 197.
Schneider, A., Zahm, D.M., Greinke, C., Kirchmayr, R., Nindl, I., 1997.
Different detectability of high-risk HPV in smears from incident and
prevalent high-grade squamous intraepithelial lesions of the cervix.
Gynecol. Oncol. 65, 399–404.
Sigurdsson, K., Arnadottir, T., Snorradottir, M., Benediktsdottir, K., Sae-
mundsson, H., 1996. Human papillomavirus (HPV) in an Icelandic
population: correlation of HPV DNA to cytologic and histopathologic
lesions and evaluation of treatment strategies. Int. J. Gynecol. Cancer
6, 175–182.
Sun, X.W., Ferenczy, A., Johnson, D., Koulos, J.P., Lungu, O., Richart,
R.M., Wright Jr., T.C., 1995. Evaluation of the hybrid capture human
papillomavirus deoxyribonucleic acid detection test. Am. J. Obstet.
Gynecol. 173, 1432–1437.
Swan, D.C., Tucker, R.A., Holloway, B.P., Icenogle, J.P., 1997. A sen-
sitive, type-specific, fluorogenic probe assay for detection of human
papillomavirus DNA. J. Clin. Microbiol. 35, 886–891.
Swan, D.C., Tucker, R.A., Tortolero-Luna, G., Mitchell, M.F., Wideroff,
L., Unger, E.R., Nisenbaum, R.A., Reeves, W.C., Icenogle, J.P., 1999.
Human papillomavirus (HPV) DNA copy number is dependent on
grade of cervical disease and HPV type. J. Clin. Microbiol. 37, 1030–
1034.
Tarkowski, T.A., Rajeevan, M.S., Lee, D.R., Unger, E.R., 2001. Improved
detection of viral RNA isolated from liquid-based cytology samples.
Mol. Diagn. 6, 125–130.
Tran-Thanh, D., Provencher, D., Koushik, A., Duarte-Franco, E.,
Kessous-Elbaz, A., Drouin, P., Wheeler, C., Dubuc-Lissoir, J., Gau-
thier, P., Allaire, G., Vauclair, R., DiPaolo, J., Gravitt, P., Franco,
E.L., Coutlée, F., 2003. Herpes simplex virus type II (HSV-2) is not
a cofactor to human papillomavirus in cancer of the uterine cervix.
Am. J. Obstet. Gynecol., in Press.
Wang, A.M., Doyle, M.V., Mark, D.F., 1989a. Quantitation of mRNA
by the polymerase chain reaction. Proc. Natl. Acad. Sci. U.S.A. 86,
9717–9721.
Wang A.M., Mark D.F., 1989b. Quantitative PCR. In: Innis, M.A.,
Gelfand, D.H., Sninski, J.J., White, T.J. (Eds.), PCR Protocols: A
Guide to Methods and Applications. Academic press, New York,
pp. 70–75.
Yang, B., Yolken, R.H., Viscidi, R.P., 1993. Quantitative polymerase chain
reaction by monitoring enzymatic activity of DNA polymerase. Anal.
Biochem. 208, 110–116.
Ylitalo, N., Sorensen, P., Josefsson, A.M., Magnusson, P.K.E., Anderson,
P.K., Ponten, J., Adami, H.O., Gyllensten, U.B., Melbye, M., 2000.
Consistent high viral load of human papillomavirus 16 and risk of
cervical carcinoma in situ: a nested case–control study. Lancet 355,
2194–2198.