Enhanced detection and typing of human papillomavirus (HPV) DNA in anogenital samples with PGMY primers and the Linear array HPV genotyping test.
ABSTRACT The Roche PGMY primer-based research prototype line blot assay (PGMY-LB) is a convenient tool in epidemiological studies for the detection and typing of human papillomavirus (HPV) DNA. This assay has been optimized and is being commercialized as the Linear Array HPV genotyping test (LA-HPV). We assessed the agreement between LA-HPV and PGMY-LB for detection and typing of 37 HPV genotypes in 528 anogenital samples (236 anal, 146 physician-collected cervical, and 146 self-collected cervicovaginal swabs) obtained from human immunodeficiency virus-seropositive individuals (236 men and 146 women). HPV DNA was detected in 433 (82.0%) and 458 (86.7%) samples with PGMY-LB and LA-HPV (P = 0.047), respectively, for an excellent agreement of 93.8% (kappa = 0.76). Of the 17,094 HPV typing results, 16,562 (1,743 positive and 14,819 negative results) were concordant between tests (agreement = 96.9%; kappa = 0.76). The mean agreement between tests for each type was 96.4% +/- 2.4% (95% confidence interval [CI], 95.6% to 97.2%; range, 86% to 100%), for an excellent mean kappa value of 0.85 +/- 0.10 (95% CI, 0.82 to 0.87). However, detection rates for most HPV types were greater with LA-HPV. The mean number of types per sample detected by LA-HPV (4.2 +/- 3.4; 95% CI, 3.9 to 4.5; median, 3.0) was greater than that for PGMY-LB (3.4 +/- 3.0; 95% CI, 3.1 to 3.6; median, 2.0) (P < 0.001). The number of types detected in excess by LA-HPV in anal samples correlated with the number of types per sample (r = 0.49 +/- 0.06; P = 0.001) but not with patient age (r = 0.03 +/- 0.06; P = 0.57), CD4 cell counts (r = 0.06 +/- 0.06; P = 0.13), or the grade of anal disease (r = -0.11 +/- 0.06; P = 0.07). LA-HPV compared favorably with PGMY-LB but yielded higher detection rates for newer and well-known HPV types.
- SourceAvailable from: mcgill.ca[Show abstract] [Hide abstract]
ABSTRACT: HIV-seropositive men having sex with other men (MSM) are at increased risk for anal Human Papillomavirus (HPV) infection, anal intraepithelial neoplasia (AIN) and anal cancer. Because most studies on the subject were cross-sectional, longitudinal investigations were needed to improve our understanding of anal carcinogenesis. Objective: To explore the longitudinal dynamics of anal HPV infection and predictors of progression to AIN 2/3 in HIV-infected MSM. Methods: Data from a prospective cohort of HIV-infected MSM in Montreal (the HIPVIRG cohort) were analysed for this thesis. Results: Incidence and clearance rates of anal HPV infection were calculated for each HPV type. The strongest predictor of progression to AIN 2/3 was a low nadir CD4 counts. We also found that a longer time on highly active antiretroviral therapy (HAART) was protective against progression. Conclusion: Results from this thesis contributes important knowledge to research of anal HPV and AIN. Les hommes infectés par le VIH ayant des relations avec des hommes (HARSAH) ont un risque accru d'être infectés par le virus du papillome humain (VPH) au niveau anal et de développer une néoplasie intra épithéliale (AIN) ou un cancer anal. Puisque la majorité des études publiées sur le sujet étaient transversales, des études longitudinales étaient désirées. Objectif : Explorer la dynamique longitudinale de l'infection anale par le VPH et identifier des prédicteurs de la progression vers des lésions AIN de haut-grade. Méthodes: Les données d'une étude de cohorte prospective chez des HARSAH infectés par le VIH (la cohorte HIPVIRG) ont été analysées pour ce mémoire. Résultats: Les taux d'incidence et de clairance du VPH anal ont été calculés pour chaque type de VPH. Un nadir de CD4 très bas a été identifié comme un prédicteur de progression vers un AIN 2 ou 3 alors que la durée de prise des antirétroviraux est protectrice. Conclusion: Les résultats de ce mémoire contribuent des apports de connaissances importants dans la recherche sur le VPH et les lésions AIN.
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ABSTRACT: Cervical cancer, the third most common cancer that affects women worldwide, is caused by the human papillomavirus (HPV) and is treatable when detected at an early stage. To date, more than 100 different HPV types have been described, and the development of simple, low-cost, and accurate methods to distinguish HPV genotypes is highly warranted. In this study, an HPV genotyping assay based on polymerase chain reaction (PCR) was evaluated. This method involved the use of MY09/11 primers followed by restriction fragment length polymorphism (RFLP) analysis with the restriction enzymes HpyCH4V and NlaIII. Cervical specimens preserved using CytoRich Blue fluid were collected from 1,134 female volunteers for HPV detection, and 1,111 valid samples were amplified using PCR. The PCR method was sensitive enough to detect 25 copies of HPV18, and three copies of HPV16. Out of 202 PCR-positive samples, HPV genotypes were determined in 189 samples (93.6%) by this RFLP method. Results were then evaluated further by capillary sequencing method. Concordant results between the two tests were as high as 96.0%. Thirteen samples, which tested negative with RFLP, were verified as non-specific amplifications with PCR. In conclusion, this PCR-RFLP method using restriction enzymes HpyCH4V and NlaIII is simple, non-labor intensive, and is applicable for the inexpensive determination of HPV genotypes in clinical samples. J. Med. Virol. 85:1229-1234, 2013. © 2013 Wiley Periodicals, Inc.Journal of Medical Virology 07/2013; 85(7):1229-34. · 2.37 Impact Factor
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ABSTRACT: Human papillomaviruses (HPVs) have been recognized as etiologic factors in cervical carcinoma and several other anogenital cancers in females and males. HPV are classified as low risk (LR), probable high risk and high risk (HR) on the basis of their oncogenic potential. HPV genotypes, which are crucial for diagnosis and relationship with carcinogenesis, have been determined by several genotyping methods. In this study, two genotyping methods were compared: direct sequencing and INNO-LiPA. In total, 2,494 cervical specimens were tested and 27.2 % of these were found to be HPV DNA positive with 24.5% showing normal cytology. Specimens were divided into four groups according to their pathological cytology as normal, LSIL, HSIL and cancer and 134 specimens were selected for HPV genotyping by both methods. HPV genotyping results showed 87.5% positive correlation. With 17 specimens, the results were discordant, 12 specimens showed different genotypes. Others had genotypes that could not be typed by the INNO-LiPA method. Neither did direct sequencing in 3 different regions yield unequivocal results. Both genotyping methods have advantages and disadvantages. Consequently, the method most suitable for the study objective, budget and predominance of HPV genotype in any given area should be selected.Asian Pacific journal of cancer prevention: APJCP 01/2011; 12(4):989-94. · 1.50 Impact Factor
JOURNAL OF CLINICAL MICROBIOLOGY, June 2006, p. 1998–2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 6
Enhanced Detection and Typing of Human Papillomavirus (HPV)
DNA in Anogenital Samples with PGMY Primers and
the Linear Array HPV Genotyping Test
Franc ¸ois Coutle ´e,1,2,3* Danielle Rouleau,1,2Patrick Petignat,1Georges Ghattas,1Janet R. Kornegay,4
Peter Schlag,4Sean Boyle,4Catherine Hankins,3,5Sylvie Ve ´zina,6Pierre Cote ´,7John Macleod,3
He ´le `ne Voyer,1Pierre Forest,1Sharon Walmsley,8The Canadian Women’s HIV Study Group,† and
Laboratoire de Virologie Mole ´culaire du Centre de Recherche, De ´partements de Microbiologie et Infectiologie, Obste ´trique-Gyne ´cologie et
Gastro-ente ´rologie, Ho ˆpital Notre-Dame du Centre Hospitalier de l’Universite ´ de Montre ´al, Montre ´al, Que ´bec, Canada1; De ´partement de
Microbiologie et Immunologie, Universite ´ de Montre ´al, Montre ´al, Que ´bec, Canada2; Division of Cancer Epidemiology and Department of
Medicine, McGill University, Montreal, Quebec, Canada3; Roche Molecular Systems, Alameda, California4; Direction de la Sante ´ Publique
de Montre ´al-Centre, Institut National de Sante ´ Publique du Que ´bec, Montre ´al, Que ´bec, Canada5; Clinique Me ´dicale l’Actuel,
Montre ´al, Que ´bec, Canada6; Clinique Me ´dicale du Quartier-Latin, Montre ´al, Que ´bec, Canada7; and Division of
Clinical Investigation and Human Physiology, Toronto General Research Institute,
Toronto General Hospital, University of Toronto, Toronto, Canada8
Received 17 January 2006/Returned for modification 7 March 2006/Accepted 17 March 2006
The Roche PGMY primer-based research prototype line blot assay (PGMY-LB) is a convenient tool in epidemi-
ological studies for the detection and typing of human papillomavirus (HPV) DNA. This assay has been optimized
and is being commercialized as the Linear Array HPV genotyping test (LA-HPV). We assessed the agreement
between LA-HPV and PGMY-LB for detection and typing of 37 HPV genotypes in 528 anogenital samples (236 anal,
146 physician-collected cervical, and 146 self-collected cervicovaginal swabs) obtained from human immunodefi-
ciency virus-seropositive individuals (236 men and 146 women). HPV DNA was detected in 433 (82.0%) and 458
(86.7%) samples with PGMY-LB and LA-HPV (P ? 0.047), respectively, for an excellent agreement of 93.8%
(kappa ? 0.76). Of the 17,094 HPV typing results, 16,562 (1,743 positive and 14,819 negative results) were concor-
dant between tests (agreement ? 96.9%; kappa ? 0.76). The mean agreement between tests for each type was
96.4% ? 2.4% (95% confidence interval [CI], 95.6% to 97.2%; range, 86% to 100%), for an excellent mean kappa
value of 0.85 ? 0.10 (95% CI, 0.82 to 0.87). However, detection rates for most HPV types were greater with
LA-HPV. The mean number of types per sample detected by LA-HPV (4.2 ? 3.4; 95% CI, 3.9 to 4.5; median,
3.0) was greater than that for PGMY-LB (3.4 ? 3.0; 95% CI, 3.1 to 3.6; median, 2.0) (P < 0.001). The number
of types detected in excess by LA-HPV in anal samples correlated with the number of types per sample (r ?
0.49 ? 0.06; P ? 0.001) but not with patient age (r ? 0.03 ? 0.06; P ? 0.57), CD4 cell counts (r ? 0.06 ? 0.06;
P ? 0.13), or the grade of anal disease (r ? ?0.11 ? 0.06; P ? 0.07). LA-HPV compared favorably with
PGMY-LB but yielded higher detection rates for newer and well-known HPV types.
Infection by human papillomavirus (HPV) causes squamous
intraepithelial lesions and invasive cancer of the uterine cervix
and anus (3). HPV testing relies on the detection and analysis
of viral DNA. Epidemiological studies and vaccine clinical
trials require reliable and reproducible identification and
genotyping of genital HPV infections. Since only a fraction of
the 40 HPV genotypes infecting the anogenital tract are asso-
ciated with malignant lesions, the detection method has to
identify types individually. Specific genotyping also provides
information on mixed HPV infections (26). Type-specific PCR
assays are impractical for epidemiological studies because of
the multiplicity of relevant genotypes infecting the anogenital
tract. Consensus PCR assays that target conserved regions of
the HPV genome have been devised to amplify all relevant
genital types in one reaction, with analysis of amplicons by
direct sequencing, restriction fragment length polymorphism
analysis, or type-specific hybridization.
The most common PCR methods use the consensus primer
set MY09/MY11/HMB01 (20, 25), GP5?/GP6? (9, 21), PGMY09/
PGMY11 (13, 34), or SPF10 (30, 34). Convenient assays for
detection and typing of HPV have been developed for all of
these primer sets. HPV amplicons generated by PGMY or MY
primers can easily be detected and typed by a nonisotopic
* Corresponding author. Mailing address: De ´partement de Micro-
biologie et Infectiologie, Ho ˆpital Notre-Dame du Centre Hospitalier
de l’Universite ´ de Montre ´al, 1560 Sherbrooke est, Montre ´al, Que ´bec
H2L 4M1, Canada. Phone: (514) 890-8000, ext. 25162. Fax: (514)
412-7512. E-mail: firstname.lastname@example.org.
† The Canadian Women’s HIV Study Group includes the following
members: in Halifax, Janet Conners, Rob Grimshaw, David Haase,
Lynn Johnston, Wally Schlech, and Arlo Yuzicappi-Fayant; in Hamil-
ton, Stephen Landis and Fiona Smaill; in London, Tom Austin, Ole
Hammerberg, and Ted Ralph; in Montre ´al, Franc ¸ois Coutle ´e, Julian
Falutz, Alex Ferenczy, Catherine Hankins, Marina Klein, Louise La-
brecque, Richard Lalonde, John Macleod, Gre ´goire Noe ¨l, Chantal
Perron, Jean-Pierre Routy, and Emil Toma; in Ottawa, Claire Touchie
and Garry Victor; in Que ´bec, Louise Cote ´, He ´le `ne Senay, and Sylvie
Trottier; in Saskatoon, Kurt Williams; in Sherbrooke, Alain Piche ´; in
Sudbury, Roger Sandre; in Toronto, Louise Binder, Donna Keystone,
Anne Phillips, Anita Rachlis, Irving Salit, Cheryl Wagner, and Sharon
Walmsley; and in Vancouver, Paula Braitstein, David Burdge, Mari-
anne Harris, Deborah Money, and Julio Montaner.
reverse hybridization assay, the line blot assay (14). In previous
studies, the reverse line blot assay combined with PCR using
the MY09-MY11 or PGMY09-PGMY11 primers compared
favorably to a dot blot assay using type-specific radiolabeled
oligonucleotide probes for HPV typing of PCR-amplified
products (6, 7, 23).
Consensus L1 PGMY09/11 primers were reported to im-
prove the sensitivity for HPV detection over that of the MY09/
MY11/HMB01 primers (6, 12, 13, 23). The research prototype
version of the assay, the PGMY line blot assay (PGMY-LB),
has been further developed and is now commercially available
from Roche Molecular Systems under the designation Linear
Array HPV genotyping test (LA-HPV). The reagents used for
LA-HPV are now standardized and produced under quality-
controlled conditions. Amplification profiles and reagents have
been optimized to increase the sensitivity and reproducibility,
mainly by avoiding competition during coamplification of
?-globin and HPV DNA (J. Kornegay, personal communica-
tion). The use of standardized protocols for HPV detection
and typing increases the reproducibility of results and should
facilitate the comparison of results between studies (22).
The aim of the present study was to compare results ob-
tained with PGMY-LB and LA-HPV for 528 anogenital spec-
imens collected from 236 men and 146 women for HPV DNA
detection and typing. The potential to separately identify types
in multiple-type infections, a frequent occurrence in human
immunodeficiency virus (HIV)-seropositive hosts, was also ex-
plored. The rates of detection of the 37 genotypes were com-
pared between assays. This information is useful in view of the
wider use of LA-HPV by numerous research groups as well as
its potential application in diagnostic laboratories for HPV
MATERIALS AND METHODS
Cell lines and clinical specimens. The cervical carcinoma cell line HeLa
(which contains 40 copies of HPV type 18 [HPV-18] DNA per cell) was obtained
from the American Type Culture Collection (Rockville, Md.). Overall, 528 gen-
ital specimens were obtained from 382 participants (146 women and 236 men) in
two different cohort studies on the natural history of anogenital HPV infection
during the course of HIV infection. Each participant in both studies gave written
informed consent for HPV testing and completed a standardized questionnaire.
Both studies were approved by the local research ethics committees of partici-
pating institutions. Male subjects were participants in a cohort study of the
natural history of anal intraepithelial neoplasia (AIN). Anal swabs obtained at
the inclusion visit for the cohort were collected from men and agitated in 1.5 ml
of Preservcyt (Cytyc Corporation, Boxborough, MA) (33). After centrifugation
at 13,000 ? g for 15 min at 22°C, the supernatant was discarded, and the cell
pellet was left to dry and resuspended in 300 ?l of 20 mM Tris buffer, pH 8.3 (31,
33). Female subjects were from The Canadian Women’s HIV Study, conducted
across Canada to evaluate relationships between HPV and HIV infection and
cervical disease (18, 28). Self-collected cervicovaginal (n ? 146) and physician-
collected cervical (n ? 146) swabs were obtained from the same women to
evaluate self-sampling for HPV detection, as described previously (28). After
being sampled, each swab was agitated in a tube containing 1 ml of 10 mM Tris,
pH 8.3, with 0.1 mM EDTA and then discarded. Purification of DNA was done
with a Master Pure extraction kit (Epicenter, Madison, WI) for all samples (17).
Five microliters of processed sample diluted in 45 ?l of sterile water was tested
in each PCR assay, since 50 ?l of processed sample collected in Preservcyt is
tested in the commercial LA-HPV protocol. All samples yielded a ?-globin
amplicon when amplified individually with 10 pmol each of the PC04 and GH20
primers, as previously described, and migrated in an ethidium bromide-stained
agarose gel, confirming the presence of amplifiable DNA (2, 8).
LA-HPV. PCR was performed in a final reaction volume of 100 ?l with 50 ?l
of kit working master mix containing MgCl2, KCl, AmpliTaq Gold DNA poly-
merase, uracil-N-glycosylase, dATP, dCTP, dGTP, dUTP, dTTP, and biotin-
ylated PGMY primers and ?-globin primers GH20 and PC04. The mixture was
incubated in a TC 9700 thermal cycler set at maximum ramp speed for 2 min at
50°C and 9 min at 95°C, followed by 40 cycles of 30 s at 95°C, 1 min at 55°C, and
1 min at 72°C, with a final extension at 72°C (ramp rate set at 50%) for 5 min.
Biotinylated amplicons were denatured in 0.4 N NaOH and hybridized to an
immobilized probe array containing probes for 37 HPV genotypes according to
the protocol provided by Roche Molecular Systems. Positive hybridization reac-
tions were detected by streptavidin-horseradish peroxidase-mediated color pre-
cipitation on the membrane at the probe line. The probe for detection of
HPV-52 amplicons on the array was a cross-reactive probe that also hybridized
with types 33, 35, and 58. Samples positive with the HPV-52 cross-reactive probe
and containing at least one of these types were also tested with a real-time PCR
assay specific for type 52 validated in the laboratory of F. Coutle ´e (unpublished
data; see below). Only samples reactive in the HPV-52 real-time PCR assay were
considered HPV-52 positive.
PGMY-LB. HPV DNA was amplified with the L1 consensus HPV PGMY09/
PGMY11 primer set, as previously described (13). The amplification mixture
contained 4 mM MgCl2, 50 mM KCl, 7.5 units of AmpliTaq Gold DNA poly-
merase (Perkin-Elmer, Foster City, CA), a 200 ?M concentration (each) of
dATP, dCTP, and dGTP, 600 ?M of dUTP, 100 pmol of each biotinylated
PGMY primer, and 2.5 pmol each of the 5?-biotinylated ?-globin primers GH20
and PC04. HPV was amplified in a TC9600 thermal cycler at 95°C for 9 min,
followed by denaturation for 1 min at 95°C, annealing for 1 min at 55°C, and
extension at 72°C for 1 min for a total of 40 cycles. Amplification was followed
by a 5-minute terminal extension step at 72°C. HPV genotyping was performed
with a reverse line blot detection system as previously described (14). PCR
products were denatured in 0.4 N NaOH and hybridized to an immobilized probe
array containing probes for 37 mucosotropic HPV genotypes. The array was
similar to that used with LA-HPV but contained an HPV-52-specific probe.
Positive hybridization was detected by streptavidin-horseradish peroxidase-me-
diated color precipitation on the membrane at the probe line.
Real-time PCR assay for HPV-16. Each 20-?l reaction mixture contained 10
mM Tris-HCl, pH 8.0, 50 mM KCl, a 200 ?M concentration (each) of dATP,
dGTP, and dCTP, 400 ?M dUTP, 0.05 ?M of TaqMan probe HPV 16-TM, 0.3
pmol each of primers HPV 16-U and HPV 16-L (targeting the E6 gene), 3.0 mM
MgCl2, and 5 units of AmpliTaq Gold DNA polymerase (15, 16). Capillaries
were placed in a Light Cycler system (Roche Molecular Systems) and amplified
at 95°C for 10 min, followed by 50 cycles at 95°C for 15 s and 55°C for 30 s. Ten
copies of an HPV-16-expressing plasmid in 500 ng of cellular DNA served as a
weak positive control.
Real-time PCR assay for HPV-52. Each 20-?l reaction mixture contained 10
mM Tris-HCl, pH 8.0, 50 mM KCl, a 200 ?M concentration (each) of dATP,
dGTP, and dCTP, 400 ?M dUTP, 0.05 ?M of TaqMan probe 52-TM (CGTG
CAGGGTCCGGGGTC), 0.3 pmol each of primers 52JA-3 (GAACACAGTG
TAGCTAACGCACG) and 52JA-4 (GCATGACGTTACACTTGGGTCA)
(targeting the E6 gene), 2.0 mM MgCl2, and 5 units of AmpliTaq Gold DNA
polymerase. The primers had been used for PCR-sequencing experiments in
previous work (1). Capillaries were placed in a Light Cycler system and amplified
at 95°C for 10 min, followed by 50 cycles at 95°C for 15 s and 60°C for 60 s. Ten
copies of an HPV-52-expressing plasmid in 500 ng of cellular DNA served as a
weak positive control. This assay was shown to be sensitive and specific for
HPV-52 DNA detection (F. Coutle ´e, unpublished data).
Statistical methods. The crude percent agreement between both detection
methods was the percentage of samples with identical results in both meth-
ods. Percentages of agreement for overall positivity (HPV DNA-positive
samples), for positivity for high-risk HPV types as a group (types 16, 18, 26,
31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, and 82), and for positivity for
each HPV type were calculated. Low-risk types were types 6, 11, 40, 42, 54,
61, 70, 72, 81, and CP6108, and types with unknown risk were types 55, 62, 64,
67, 69, 71, 73, 83, 84, and IS39 (HPV-82 subtype). The unweighted kappa
statistic was calculated to adjust for chance agreement between HPV detec-
tion methods (11). In general, kappa values above 0.75 indicate excellent
agreement, values between 0.40 and 0.75 indicate fair to good agreement, and
values below 0.40 represent poor agreement beyond chance. The mean num-
ber of types detected per sample by each PCR test was compared using the
sign test, since the frequencies of types per sample were not normally dis-
tributed. Two-sided McNemar’s chi-square analysis for matched pair data was
performed to analyze contingency tables comparing both PCR tests. Propor-
tions were compared with the Z statistic.
VOL. 44, 2006DETECTION AND TYPING OF HPV DNA WITH PCR 1999
A total of 528 anogenital samples from 378 HIV-seroposi-
tive participants were tested in one laboratory using the
PGMY-LB and LA-HPV systems. The characteristics of these
378 participants are shown in Table 1. The human ?-globin
gene was detected with the high-concentration probe in 528
(100%) and 527 (99.8%) samples and with the low-concentra-
tion probe in 497 (94.1%) and 466 (88.3%) samples by LA-
HPV and PGMY-LB, respectively (P ? 0.001). All 528 sam-
ples had tested positive for ?-globin after amplification with 10
pmol of GH20 and PC04 and gel electrophoresis of the am-
plicons. Of the 31 ?-globin-negative samples with the low
probe concentration in LA-HPV, 22 (71.0%) were HPV pos-
itive. Of the 62 ?-globin-negative samples with the low probe
concentration in PGMY-LB, 44 (71.0%) were HPV positive. A
total of 12 samples tested negative with the low probe concen-
tration for ?-globin in both assays. The number of types de-
tected per specimen was greater for ?-globin-positive than
?-globin-negative samples in LA-HPV (median and mean, 4.0
and 4.3 ? 3.4 versus 2.0 and 2.0 ? 2.3; P ? 0.001) or
PGMY-LB (median and mean, 3.0 and 3.5 ? 3.0 versus 2.0 and
2.2 ? 2.2; P ? 0.001).
Results from both assays were first compared for HPV DNA
detection, irrespective of type(s). As shown in Table 2, HPV
DNA was detected in 433 (82.0%) and 458 (86.7%) of 528
samples by PGMY- LB and LA-HPV, respectively (P ? 0.047).
There was high agreement for HPV DNA by both methods for
495 (93.8%) samples (kappa ? 0.76) (Table 2). Similar results
were obtained when only the 447 ?-globin-positive samples in
both assays were considered in the comparison: 373 were pos-
itive and 47 were negative for HPV in both assays (concor-
dance, 94.0%; kappa, 0.76; 95% confidence interval [CI], 0.69
to 0.84). Ninety-nine samples reacting with the HPV-52 cross-
reactive probe and containing HPV-33, HPV-35, or HPV-58
DNA were retested with the HPV-52-specific real-time PCR
assay. Sixty-three were shown to contain HPV-52 DNA.
We then compared typing results for 37 genotypes for the
462 specimens found to be HPV positive with at least one of
the PCR tests, for a total of 17,094 type-specific results. Over-
all, 1,743 positive results for the same type and 14,819 negative
results were obtained in both PCR tests, while 492 positive
typing results were obtained only with LA-HPV and 40 were
obtained only with PGMY-LB. The overall agreement was
96.9% (16,562 of 17,094 results), with a kappa value of 0.76
(95% CI, 0.68 to 0.85).
Since these agreement calculations are influenced by the
high rates of negative typing results in both tests, we compared
the specific identification of individual genotypes by each of
the two methods for all samples (Fig. 1). The mean agreement
reached 97% ? 2.5% (95% CI, 95.8% to 98.5%) and ranged
from 93.2% to 100%. The mean kappa value reached 0.86 ?
0.07 (95% CI, 0.83 to 0.88). Kappa values below 0.75 were
obtained only for types 66 and 53. Since histological diagnosis
was available only for men, we then compared agreements
between tests, considering the AIN grades for the eight most
TABLE 1. Clinical characteristics of 378 HIV-seropositive participants
Value (no. of subjects [%], unless
CWHIS cohort HIPVIRG cohort
Age (median yrs [range]) 32.5 (18.0–68.3) 43.4 (26.8–66.5)
Age categories (yrs)
Mean CD4 cell count ? SD 507 ? 318443 ? 261
CD4 count categories
Not currently smoking
Median no. of lifetime sexual
Cytology smear results
AIN grade 1
AIN grades 2 and 3
aASCUS, atypical squamous cells of undetermined significance; LSIL, low-
grade squamous intraepithelial lesions; HSIL, high-grade squamous intraepithe-
lial lesions; AIN, anal intraepithelial neoplasia.
bNA, not available.
cMedian of two to five male sexual partners per year.
TABLE 2. Comparison of PGMY-LB and LA-HPV assays for
detection of HPV DNAs from 37 genotypes in 528
No. (%) of samples with indicated resulta
PGMY-LB positive PGMY-LB negative
Total 433 (82.0)95 (18.0) 528 (100)
aAbsolute agreement, 93.8%; kappa ? 0.76 (95% CI, 0.68 to 0.85).
2000 COUTLE´E ET AL. J. CLIN. MICROBIOL.
frequent types (types 16, 18, 53, 58, 6, 42, 61, and 84). The AIN
grade did not modify agreement between tests, as the mean
agreement for men without disease was 94.6% ? 5.4% (kappa,
0.83 ? 0.13), that for men with AIN grade 1 was 94.3% ? 2.7%
(kappa, 0.84 ? 0.07), and that for men with AIN grades 2 and
3 was 94.6% ? 3.4% (kappa, 0.86 ? 0.11). These differences
were not significant (P ? 0.10 for each comparison). HPV-67
was the only type for which PGMY-LB detected a greater
number of samples than LA-HPV, with 25 samples being pos-
itive in both tests, 1 testing positive in LA-HPV only, and 3
testing positive in PGMY-LB only.
For each type but HPV-64 and HPV-71, at least one speci-
men was positive only by LA-HPV. HPV-16 was the type for
which we obtained the lowest specificity with respect to the
results obtained with PGMY-LB, measured at 92.1% (95% CI,
89.0% to 94.3%). The mean specificity obtained for all types
reached 95.5% ? 6.3% (95% CI, 93.5% to 97.4%). For HPV-
16, 113 samples were positive in both tests, 33 were positive by
LA-HPV only, none were positive by PGMY-LB only, and 382
were negative in both tests. All samples had been tested ini-
tially before this evaluation, using PGMY-LB, but without
coamplification of ?-globin (6): 10 (30.3%) of the 33 LA-HPV-
positive, PGMY-LB-negative samples for HPV-16 had tested
positive with PGMY-LB without coamplification. Additional test-
ing was performed in duplicate on these 33 samples which were
Thirty (90.9%) of these samples tested positive for HPV-16.
We then determined if the use of LA-HPV over PGMY-LB
could improve the type-specific detection rate for HPV infec-
tions (Fig. 2a, b, and c). The most frequent HPV type was
HPV-16. For all but two types (67 and 71), the rate of detection
was higher with LA-HPV. This difference reached statistical
significance for types 16, 51, 52, 53, 66, and 61 as well as the
high-risk types as a group (P ? 0.05). More than one HPV type
per sample was detected in 223 (94.5%) of 236 anal swab
samples and 167 (57.2%) of 292 cervical or cervicovaginal swab
samples (P ? 0.001). The median numbers of HPV types per
specimen detected in anal swab samples and cervical or cervi-
covaginal swabs with PGMY-LB were 5 (range, 0 to 18) and 1
(range, 0 to 12), respectively. Figure 3 demonstrates a strong
correlation between the numbers of types per sample detected
with LA-HPV and PGMY-LB. However, a greater number of
types per sample was identified with LA-HPV (mean, 4.2 ?
3.4; 95% CI, 3.9 to 4.5; median, 3.0; range, 0 to 17) than with
PGMY-LB (mean, 3.4 ? 3.0; 95% CI, 3.1 to 3.6; median, 3.0;
range, 0 to 15) (P ? 0.001). Figure 4 shows the distribution of
the numbers of types identified per sample in excess with
LA-HPV. Although nearly half of the samples contained the
same number of types with both assays, the distribution of
differences shows a shift towards a greater number of types per
sample detected by LA-HPV.
We then investigated the determinants of detecting a greater
number of types per sample with LA-HPV for the anal cohort
study (n ? 236), since histology data had been obtained only
for these individuals. The number of types per sample found in
excess with LA-HPV correlated with the total number of types
per sample (r ? 0.49 ? 0.06; P ? 0.001) but not with age (r ?
0.03 ? 0.06; P ? 0.57), blood T-lymphocyte CD4 counts (r ?
0.06 ? 0.06; P ? 0.13), or AIN grade (r ? ?0.11 ? 0.06;
P ? 0.07). LA-HPV detected 0.66 ? 1.04 (95% CI, 0.46 to
0.87) type in excess compared to PGMY-LB in samples con-
taining five types of fewer and 1.41 ? 1.42 (95% CI, 1.17 to
1.66) types in excess for samples containing more than five
types (P ? 0.001). A greater number of types per sample was
also associated with having more than two types detected by
LA-HPV over PGMY-LB (odds ratio, 1.4; 95% CI, 1.2 to 1.6),
FIG. 1. Agreement between results obtained for 528 anogenital samples with LA-HPV and PGMY-LB. Kappa coefficients are presented, with
VOL. 44, 2006DETECTION AND TYPING OF HPV DNA WITH PCR 2001
controlling for age, AIN grade, and blood T-lymphocyte CD4
count by logistic regression.
The precise typing of HPV isolates is essential for the perfor-
mance of epidemiological studies. It is an essential tool for mea-
suring the impact of HPV vaccination on the risk of acquisition of
individual HPV types. The oncogenic potential of some rare or
new types also needs to be assessed. Persistent infections by high-
risk HPV types are relevant to identifying women at greater risk
of progressing to cervical cancer. The transmission of HPV be-
tween sexual partners as well as determinants of disease progres-
sion is still under study in large-scale cohort studies.
FIG. 2. Detection rates for HPV types by LA-HPV and PGMY-LB. (a) High-risk types; (b) low-risk types; (c) types with unknown risk. Rates
of detection were compared, and P values are shown at the bottom of each graph near the axis. Bars represent 95% CI.
2002COUTLE´E ET AL.J. CLIN. MICROBIOL.
In this evaluation, results obtained with LA-HPV were com-
pared with those generated with PGMY-LB, a prototype assay
that uses the same primers. In previous studies, PGMY-LB was
shown to be a convenient, sensitive, and reproducible alterna-
tive to PCR assays based on radiolabeled probes (6, 7). Cycle
sequencing has been reported to detect some HPV types better
than PGMY-LB but did not perform as well on samples con-
taining several HPV types (35). Moreover, PGMY-LB gener-
ally detected more HPV-positive samples than did cycle se-
quencing (35). One study reported that PGMY-LB performed
similarly to the SPF10 Line Probe assay, a PCR-based system
that also uses reverse hybridization for genotyping (34). An
international proficiency study had also shown the reliability of
PGMY-LB (22). We carried out the present study to assess the
relative gain in diagnostic yield of using the commercial LA-
HPV assay instead of PGMY-LB for the detection and typing
of HPV DNA in 528 anogenital samples from HIV-seroposi-
Based on these analyses, we found substantial agreement
between the assays for the detection of HPV DNA. Our eval-
uation also demonstrated very good agreement between assays
for consideration of each of the 37 genotypes individually. Our
results indicate that HPV detection and typing can be signifi-
cantly improved to various degrees for most genital types with
the use of LA-HPV, including the most frequent genotype,
HPV-16. Most samples positive for HPV-16 only with LA-
HPV also tested positive with an HPV-16-specific real-time
PCR assay, demonstrating a greater sensitivity of LA-HPV
than of PGMY-LB. More HPV types per specimen were also
identified with LA-HPV, especially with samples that con-
tained over five genotypes.
The 93% to 100% agreement between the LA-HPV and
PGMY-LB systems, whether considering all results or those
for each of the 37 genotypes, is greater than the levels of
agreement reported in the first evaluations of PGMY primers
and in other studies comparing two consensus L1 PCR systems
(4, 12, 13, 19, 27, 29, 32, 35). Since both PGMY systems use the
same primer sets, a greater level of agreement was expected.
Moreover, type-specific probes were identical between the two
assays, except those for type 52. The high prevalence of HPV
infection and the presence of multiple-type infections in our
population allowed us to derive levels of agreement with rel-
atively narrow 95% confidence intervals for most types studied.
However, four types (HPV-82, -64, and -71 and IS39) were
detected in ?10 samples each. Since all individuals recruited
FIG. 3. Correlation between numbers of types detected in each
sample by PGMY-LB and LA-HPV. The dashed lines represent the
95% CI of the regression line.
VOL. 44, 2006DETECTION AND TYPING OF HPV DNA WITH PCR2003
for the anal cohort study had high-resolution anoscopies, we
could assess if the correlation between assays was dependent
on histological diagnosis. For this limited subset of samples,
correlation was similar, irrespective of the grade of disease.
Several reasons could explain the increased ability of LA-
HPV to detect HPVs compared with PGMY-LB. The oligo-
nucleotide probes and consensus PGMY primers were identi-
cal for both systems, making it less likely that the excess
detection rate for LA-HPV was due to nonspecific hybridiza-
tion or amplification. Moreover, when samples positive for
HPV-16 with LA-HPV only were further investigated with a
real-time PCR assay, most were confirmed as positive for
HPV-16 DNA. In contrast to PGMY-LB, the LA-HPV proto-
col uses standardized reagents that are quality controlled and
cycling parameters that are optimized, along with an optimal
concentration of ?-globin primers to minimize competition
due to coamplification of ?-globin and HPV DNAs. Interest-
ingly, previous runs using PGMY-LB without coamplification
of ?-globin on the same samples confirmed the results ob-
tained with LA-HPV for HPV-16 DNA in a substantial pro-
portion of samples.
Coamplification of HPV with ?-globin could reduce the
level of sensitivity of consensus L1 PCR assays for HPV de-
tection, especially for multiple-type infections (7, 35). More-
over, all samples had tested positive initially for ?-globin in a
PCR assay amplifying ?-globin separately from HPV DNA
sequences (6, 7). Samples that tested negative for ?-globin in at
least one PGMY assay contained HPV DNA. More samples
were positive for ?-globin with LA-HPV. Since ?-globin is
coamplified with HPV in PGMY assays, these assays could also
be more sensitive to the effects of inhibitors than an assay
testing only for ?-globin sequences with a higher primer con-
centration (5). This hypothesis could be investigated further
with internal controls for ?-globin. Sample degradation could
also have occurred since the initial testing for ?-globin. Nev-
ertheless, more samples were considered adequate by testing
positive for ?-globin with LA-HPV. Optimized amplification
parameters could also have facilitated HPV typing for multi-
ple-type infections, a frequent occurrence in samples from
Detection and genotyping of HPVs become more complex
in samples containing multiple genotypes because of competi-
tion for reagents during amplification and the discrimination of
types amplified by PCR. The correlation between the number
of types contained in a sample and the number of additional
types detected with LA-HPV suggests that less competition
during amplification was encountered with LA-HPV than with
One limitation of the current study is the use of specimens
from HIV-seropositive individuals. HIV-infected individuals
have a high prevalence of HPV infection and are also infected
more frequently with more than one HPV type. The test panel
thus cannot be representative of the prevalence of HPV in a
random population. On the other hand, our decision to use
only samples from HIV-infected individuals afforded the op-
portunity to scrutinize assay performance under the more ex-
treme conditions seen with specimens containing several HPV
types. HIV-seropositive individuals are not only prone to ac-
quiring multiple-type infections but also tend to harbor higher
HPV viral loads (24). Another consideration may be that the
FIG. 4. Differences in numbers of types detected per sample by LA-HPV and PGMY-LB. The number of types detected with PGMY-LB was
subtracted from the number of types detected with LA-HPV for each sample. The percentage of the total number of samples is shown above each
2004COUTLE´E ET AL. J. CLIN. MICROBIOL.
specimens analyzed were stored for several months, and there
could be some specimen degradation over time. The same
sample preparations were evaluated in each assay concur-
rently, and degradation was thus not an issue here.
In conclusion, LA-HPV will be of great value for epidemi-
ological studies and clinical trials to monitor HPV infection at
the genotype level in genital samples. Since high-risk HPV
persistence is the most important predictor of cervical cancer,
genotyping assays could prove useful for assessing viral persis-
tence, but further clinical studies are required before precise
recommendations can be made. Eventually, women with per-
sistent infections caused by the same types could be monitored
more closely (10, 26). There was good agreement between
these two assays for HPV DNA detection and HPV typing.
Still, LA-HPV had a superior ability to detect HPV DNA and
individual types compared with PGMY-LB. In light of the
present results, studies currently using PGMY-LB will have to
assess the opportunity to use LA-HPV because of the greater
sensitivity of LA-HPV. Nevertheless, although there are some
detection gains from using the LA-HPV commercial assay, the
agreement with the prototype PGMY-LB assay is excellent,
and therefore previous epidemiological studies using the pro-
totype reagents are unlikely to suffer from severe misclassifi-
cation bias. Further evaluations should be performed with
We thank Jean-Marc Tre ´panier and Serge Cote ´ for maintenance of
the database for the HIPVIRG study and for sampling of men and
Diane Gaudreault and Diane Bronsard for processing genital samples.
This work was supported by Roche Molecular Systems, which sup-
plied reagents for PGMY assays, and by the Re ´seau FRSQ-SIDA
Maladies Infectieuses. The National Cancer Institute of Canada sup-
ports the HIPVIRG cohort. The Canadian Institutes for Health Re-
search and Health and Welfare Canada supported the Canadian
Women’s HIV Study. F.C. is a clinical research scholar supported by
the Fonds de Recherche en Sante ´ du Que ´bec. E.F. holds a Distin-
guished Scientist Award from the Canadian Institutes for Health Re-
search. S.W. holds career scientist salary support from the Ontario
HIV Treatment Network.
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