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Journal of Clinical Microbiology, March 1999, p. 796-797, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Comparative Evaluation of First- and
Second-Generation Digene Hybrid Capture Assays for Detection of Human
Papillomaviruses Associated with High or Intermediate Risk for
Cervical Cancer
Mario
Poljak,1,*
Anja
Bren
i,1
Katja
Seme,1
Adriana
Vince,2 and
Irena J.
Marin1
Institute of Microbiology and Immunology,
Medical Faculty, Ljubljana, Slovenia,1 and
Dr. Fran Mihaljevi
University Hospital for Infectious
Diseases, Zagreb, Croatia2
Received 2 July 1998/Returned for modification 22 October
1998/Accepted 17 November 1998
 |
ABSTRACT |
In the present study we comparatively evaluated the first- and
second-generation Digene Hybrid Capture assays for detection of human
papillomaviruses (HPV) associated with high or intermediate risk for
cervical cancer in cervical specimens. Concordant results were obtained
with 468 of 483 (96.8%) specimens. All 15 specimens which gave
repeatedly discordant results were positive by the second-generation
test only, and 14 of them tested PCR positive. The enhanced sensitivity
of the second-generation assay is mainly a result of the reformulation
of hybridization reagents and, to a lesser extent, a result of the
addition of new HPV probes.
| |
TEXT |
The strong association between
infection with certain human papillomavirus (HPV) types and cervical
cancer that has emerged in the past 10 years has brought new urgency to
the need for establishing accurate methods to diagnose HPV infection.
Although cytological screening by the Papanicolaou test, or Pap smear,
continues to play a major role in the early diagnosis of cervical
neoplastic lesions, assays that detect HPV DNA in cervical smears may
represent an important source of adjunct information for the purposes
of counselling, selection of therapy, and follow-up (18).
Several HPV DNA detection methods have been described during the last
decade, each of which allows the detection of a wide spectrum of HPV
types, but none has fulfilled all expectations (reviewed in reference
6). Amplification-based methods, mainly PCR, are
currently the most sensitive methods for detection of HPV DNA. They are
ideal instruments for research and epidemiological purposes since they
allow the detection of low-viral-load infections and also minimize the
risk of misclassification of HPV infection status (6).
However, due to frequent contamination problems and consequent
false-positive results and the present unacceptably high costs of
amplification technology, they are not currently readily applicable to
diagnostic laboratories for the routine detection of HPV infection
(6, 18).
In order to overcome these problems, the first-generation Hybrid
Capture System DNA detection test (Digene Diagnostic, Silver Spring,
Md.) for the rapid detection of genital HPV types in clinical settings
was developed a few years ago (13). In this assay, exfoliated cells are treated with alkali-denaturate solution, and the
processed samples are hybridized under high-stringency conditions in a
solution containing two mixtures of single-stranded RNA probes: one for
HPV types 6, 11, 42, 43, and 44, associated with a low risk for
cervical cancer, and one for HPV types 16, 18, 31, 33, 35, 45, 51, 52, and 56, associated with a high or intermediate risk for cervical cancer
(referred to in this work as low-risk and indeterminate- or high-risk
HPV types, respectively). Positive specimens are detected by binding
the hybridization complexes to tubes coated with a monoclonal antibody
to RNA-DNA hybrids. The bound hybrids are detected by the addition of
an alkaline phosphatase-conjugated antibody to RNA-DNA hybrids,
followed by the addition of a chemiluminiscent substrate. The emission
of light is measured as relative light units (RLU) in a luminometer (13). The analytical sensitivity of the assay was estimated as 50,000 copies of HPV 16 DNA (4, 13). Several studies have evaluated the clinical sensitivity and specificity of the
first-generation assay mainly against PCR and histology (2-5, 7,
9, 10, 14-17). However, as expected, the first-generation Hybrid
Capture assay was less sensitive than PCR and other amplification
techniques, but its specificity and positive predictive value were
higher than those of PCR. To improve the sensitivity of the Hybrid
Capture assay, the Digene Corporation has recently modified the
first-generation assay that has been approved by the U.S. Food and Drug
Administration. The improved second-generation assay (Digene HPV Test
Hybrid Capture II) was launched in Europe at the beginning of 1998. According to the manufacturer's statement, in the second-generation
assay analytical sensitivity was increased to 1,000 HPV DNA copies by the reformulation of hybridization reagents and by the addition of new
probes for four high- or intermediate-risk HPV types, 39, 58, 59, and
68. Additionally, microtiter plates replaced the hybridization tubes
used in the first-generation test.
In the present study we comparatively evaluated both generations of
Hybrid Capture assay for detection of high- or intermediate-risk HPV in
a total of 483 random consecutive cervical specimens obtained from the
same number of women with abnormal Papanicolaou screening test results.
All specimens were collected with the Digene Specimen Collection Kit
and tested by both assays in parallel according to the manufacturer's
instructions, as previously described (14). The evaluation
was performed in a double-blinded manner. According to the
manufacturer's interpretation criteria, specimens with RLU/cutoff
value ratios of 
1.0 were considered positive for one or more high- or
intermediate-risk HPV types, and samples with RLU/cutoff value ratios
of <1.0 were considered negative for the HPV types tested.
Concordant HPV DNA results were obtained in 468 of 483 (96.8%)
cervical specimens. Thus, 193 cervical specimens tested negative and
275 specimens tested positive for high- or intermediate-risk HPV types
by both assay versions. In total, 15 cervical specimens gave repeatedly
discordant results, and all were positive by second-generation assay
only. For 12 specimens with discordant results the RLU/cutoff value
ratios obtained in the second-generation assay ranged from 2.8 to 8.9. For the remaining three specimens with discordant results the
RLU/cutoff value ratios obtained were 28.32, 97.23, and 439.29, respectively. To resolve the HPV DNA status for the discordant
specimens, all 15 specimens were additionally tested in duplicate by an
HPV L1 consensus primer MY09/MY11 PCR-based test, which was originally
developed by Manos et al. (8) and modified as previously
reported by our group (11). Altogether, 14 of 15 discordant
specimens tested clearly HPV DNA PCR positive. A 536-bp fragment of the
beta-globin gene was successfully amplified from all 15 discordant
specimens. Seven discordant specimens contained HPV type 16, two
contained HPV type 56, one contained HPV type 18, one contained HPV
type 31, one contained HPV type 35, one contained HPV type 58, and one
contained HPV type 68, as determined by restriction fragment analysis
of the MY09/MY11 PCR products with seven restriction enzymes
(1). Interestingly, in all discordant samples containing HPV
DNA types covered by the hybridization probes included in both assay
generations (HPV types 16, 18, 31, 35, and 56) the RLU/cutoff value
ratios obtained in the second-generation assay were below 28.32 (in 11 of 12 samples they were below 8.9), which would indicate that the low
HPV DNA levels present in these samples produced false-negative results
in the first-generation assay. By contrast, however, both discordant
specimens with relatively high RLU/cutoff values (97.23 and 439.29)
contained HPV DNA types covered by hybridization probes included in the
second-generation assay only (HPV types 58 and 68), suggesting that the
lack of appropriate probes in the first-generation assay hybridization solution produced false-negative results with these specimens. A
specimen obtained from a 36-year old woman with a cervical
intraepithelial neoplasia, grade 1, lesion
which produced repeatedly
positive results by the second-generation assay, giving RLU/cutoff
values from 2.3 to 4.1, and which was negative both by the
first-generation assay and by MY09/MY11 HPV PCRwas additionally
tested with two other HPV consensus primers and four type-specific
primer sets, as described previously (12). All six
additional PCRs gave negative results, suggesting a false-positive
result by the second-generation assay for this sample.
According to the results of our study, the second-generation Digene
Hybrid Capture HPV DNA assay is more sensitive than its first-generation version. The enhanced sensitivity of the
second-generation version is mainly a result of the reformulation of
reagents and, to a lesser extent, a result of the addition of new HPV
probes. The second-generation test is more rapid and easier to perform and thus more appropriate for use in the diagnostic microbiology laboratory for routine detection of HPV infection.
| |
ACKNOWLEDGMENTS |
We thank Robi Kro
elj for his excellent technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Microbiology and Immunology, Medical Faculty, Zaloka 4, 1105 Ljubljana, Slovenia. Phone: 386 61 1403 042. Fax: 386 61 1423 518. E-mail: poljak{at}animus.mf.uni-lj.si.
| |
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Journal of Clinical Microbiology, March 1999, p. 796-797, Vol. 37, No. 3
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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