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Journal of Clinical Microbiology, January 2000, p. 408-411, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Additional Human Papillomavirus Types Detected by the Hybrid
Capture Tube Test among Samples from Women with Cytological and
Colposcopical Atypia
József
Kónya,1,*
György
Veress,1
Attila
Juhász,1
Krisztina
Szarka,1
Tamás
Sápy,2
Zoltán
Hernádi,2 and
Lajos
Gergely1
Department of
Microbiology1 and Department of
Obstetrics and Gynecology,2 University
Medical School of Debrecen, Debrecen, Hungary
Received 3 May 1999/Returned for modification 21 July 1999/Accepted 8 October 1999
 |
ABSTRACT |
The type specificity of the human papillomavirus (HPV) Hybrid
Capture Tube (HCT) test was evaluated by using typing with PCR (MY09-MY11)-restriction fragment length polymorphism (RFLP) and sequencing. All samples HCT test positive for only low-risk HPV (n = 15) or only high-risk HPV (n = 102) were confirmed, whereas 9 of 12 HCT test double-positive samples
contained only high-risk HPV types as determined by PCR-RFLP. Several
high-risk HPV types (HPV-53, -58, -62, -66, -CP8304, and -MM4) not
included in the HCT test were indeed detected, indicating a broader
detection range with retained distinction between low-risk and
high-risk HPV types.
| |
TEXT |
Infection of the female genitalia by
oncogenic human papillomavirus (HPV) types is the major cause of
precancerous squamous intraepithelial lesions (SIL) of the cervix uteri
(10). Invasive cancer is generally preventable by organized
cervical screening programs (17). Persistent infection with
oncogenic HPV types and a high viral load confer an increased risk for
persistent or progressing SIL (9). Infections in women above
30 years of age and infections with cancer-associated HPV types tend to persist longer, and women with persistent HPV infections are at particularly increased risk for cervical cancer (8). Thus, additional testing for concomitant HPV infections may improve screening
efficacy (4, 5, 15).
The commercially available Hybrid Capture Tube (HCT) test (Digene
Diagnostics, Inc., Beltsville, Md.) is designed to detect the genomes
of 14 HPV types by hybridization to type-specific RNA probes
(11). The probes are routinely used in two cocktails, the
first of which (designated A) contains probes specific to low-risk HPV
types (types 6, 11, 42, 43, and 44) causing benign proliferation of the
genitalia and the second of which (designated B) consists of probes
specific to high-risk, oncogenic types (types 16, 18, 31, 33, 35, 45, 51, 52, and 56) associated with high-grade SIL and invasive cervical
cancer. The clinical utility of the HCT test is well established; it
can diagnose cervical dysplasia cases with good sensitivity and
specificity (3, 6, 7, 13, 14, 20). However, the number of
newly identified HPV types is still growing and the hybridization
method of the HCT test raises the possibility of cross-hybridization to
other types. Also, the instructions included with the HCT test mention
cross-reaction with two essentially extragenital types (HPV-13 and
-30). We evaluated whether cross-hybridization may influence the
specific distinction between low-risk and high-risk types.
Clinical samples.
Clinical samples were taken from women
referred to gynecologic outpatient clinics because of cytological or
colposcopical abnormalities (cervical screening in Hungary routinely
includes colposcopy). Exfoliated cells from the cervix uteri were
collected with a Digene specimen collection kit and stored at 
20°C
until processed. Papillomavirus DNA was detected with the Digene HCT test. In the first step of the HCT test protocol, the specimen is
alkali denatured. A 150-µl aliquot of the denatured specimen is
hybridized to each probe, while the remaining part can be stored at
20°C and retested later. Storage and tests were performed in
accordance with the manufacturer's instructions.
PCR amplification.
Of 570 consecutive cervical samples, 145 were positive by the HCT test and were further processed for PCR
amplification. DNA was recovered from a 300-µl aliquot of the frozen,
stored, alkali-denatured specimen. After thawing, 750 µl of ethanol
(96%) and 60 µl of glacial acetic acid were added. The precipitate
was centrifuged for 15 min at 4°C and 12,000 × g and
redissolved in 200 µl of Tris (10 mM, pH 7.5)-EDTA (0.1 mM) buffer.
After the DNA recovery step, 16 specimens were excluded from
HPV-specific PCR testing because they could not be amplified with the
human 
-globin primers pCO3 and pCO4 (18). Ten microliters
of each of the remaining 129 samples was subjected to PCR amplification
with the consensus primers MY09 and MY11 with 40 cycles in 50-µl
volumes (12). A 2-µl aliquot of each PCR mixture was
visualized by silver staining after nondenaturing polyacrylamide (5%;
acrylamide/bisacrylamide ratio, 50:1) gel electrophoresis (PAGE).
RFLP typing of PCR products.
The enzymes AluI,
BamHI, DdeI, HaeIII, HinfI,
and PstI were used for restriction fragment length
polymorphism (RFLP) typing. The RFLP patterns of the MY09-MY11
fragments of the different HPV types are reported elsewhere
(2), with the exception of AluI digestion.
AluI digests the MY09-MY11 fragments of the different types
as follows: HPV30, 281, 86, and 73 bp; HPV32, 271, 109, and 69 bp;
HPV39, 277 and 178 bp; HPV42, 328, 82, and 39 bp; HPV51, 289, 117, and
39 bp; HPV52, 244 and 206 bp; HPV53, 290, 124, and 34 bp; HPV54, 172, 167, and 113 bp; HPV56, 325, 73, and 51 bp; HPV58, 266, 184, and 61 bp;
HPV61, 198, 131, and 127 bp; HPV66, 196, 94, and 88 bp; HPV67, 266 and
183 bp; HPV68, 349 and 107 bp; HPV69, 331 and 124 bp; HPV70, 296 and
159 bp; HPV72, 331, 86, and 39 bp; HPV73, 280, 110, and 68 bp;
HPV-CP141, 296, 124, and 35 bp; HPV-CP4173, 331, 86, and 39 bp;
HPV-CP8061, 247, 166, and 39 bp; HPV-CP8304, 358 and 94 bp; HPV-IS039,
253, 163, and 39 bp; HPV-L1AE1, 276, 104, and 35 bp; HPV-L1AE2, 253, 143, and 19 bp; HPV-LVX100, 331, 86, and 39 bp; HPV-LVX160, 296, 124, and 35 bp; HPV-LVX82, 361 and 91 bp; HPV-MM4, 253, 163, and 39 bp; HPV-MM7, 361 and 91 bp; HPV-MM9, 280, 110, and 68 bp.
The restriction fragments of single PCR products were electrophoresed
in 2% agarose gels, whereas those of multiple infections were
separated by PAGE (see above). We constructed a score table for rapid
evaluation of RFLP. The RFLP patterns of the different HPV types were
coded by a 1-2-4 system (Table 1). Coding
was based on whether the typed MY09-MY11 fragment was cleaved by the different restriction enzymes or not. For instance, HPV33 was cleaved
by DdeI, HinfI, and PstI but not by
AluI, BamHI, and HaeIII. Its score was
therefore 46 (i.e., 1(AluI) × 0 + 2(BamHI) × 0 + 4(DdeI) × 1 = 4 and
1(HaeIII) × 0 + 2(HinfI) × 1 + 4(PstI) × 1 = 6). Most HPV types had
unique scores, while for the HPV types with the same scores, we
selected the restriction enzymes that produced the largest difference
in fragment size. For instance, HPV-18 and HPV-45 both have a score of
64 but can be distinguished due to the different sizes of their
DdeI fragments (Table 1).
DNA sequencing.
PCR amplimers of rare, unexpected HPV types
were purified with a QIAquick Gel Extraction kit (Qiagen, Hilden,
Germany), cloned in a Promega T-Easy vector (Promega Corp., Madison,
Wis.), and sequenced with a Pharmacia T7 sequencing kit (Pharmacia
Biotech AB, Uppsala, Sweden) using primers MY09 and MY11 and direct
[35S]dATP incorporation.
Results and discussion.
The HCT test results of the samples
subjected to PCR-RFLP typing were the following: 15 low-risk HPV
positive (A), 102 high-risk HPV positive (B), and 12 HCT test double
positive (AB); i.e., the latter samples hybridized to both the low-risk
and high-risk probes. The PCR detection method suited two requirements.
(i) It did not alter the HCT test protocol, since the DNA for PCR amplification was recovered from the specimens after the HCT test results had been obtained. (ii) Hybridization and PCR were performed on
the same specimens, allowing reliable comparison of the methods (3, 16). Separate sampling for each method might have biased the intermethod comparisons because different amounts of infected cells, virus, etc. might have been obtained.
By nondenaturing PAGE, PCR products with very similar lengths (449 to
458 bp) have different electrophoretic mobilities due
to the difference
between their nucleotide sequences. Thus, multiple
infections could be
detected as well as single infections (Fig.
1a). For typing of the PCR products, RFLP
(
2) was the method
selected because it is independent of
hybridization. Its combination
with the 1-2-4 evaluation system (Table
1) eliminated the need
for precise restriction fragment length
determination, while its
combination with nondenaturing PAGE enabled us
to identify all
of the coinfecting HPV types in multiple infections
(Fig.
1b and
c). With this method, we identified a triple infection as
well,
which was confirmed 1 month later in another sample from the same
patient.
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FIG. 1.
(a) Different PAGE mobilities of MY09-MY11 fragments.
Marker bands are 500, 400, and 330 bp long. (b) RFLP of a single
infection (#195) in 2% agarose gel with an HaeIII-digested
 X174 marker. (c) RFLP of a double infection (#205) revealed by PAGE
and silver staining. (b and c) Arrows indicate restriction enzyme
cleavage. Identified types: HPV-33 (#195), HPV-52 (#196), HPV-16
(#197), HPV-56 (#198), HPV-6 (#201), HPV-16/33 (#205), and HPV-18
(#212).
|
|
The HCT test results of the samples that reacted exclusively with
either the low-risk (A) or the high-risk (B) probe mixture
were
completely concordant with the PCR-RFLP typing results (Table
2). Among the 102 high-risk (B)-positive
HCT test samples, 13
had multiple infections with all of the
coinfecting types belonging
to the high-risk group. In addition, we
detected single infections
with HPV-53 (two samples), HPV-58 (two
samples), HPV-66 (three
samples), HPV-MM4, and HPV-CP8304, which also
belong to the high-risk
group but are not included in the high-risk HCT
test probe mixture.
These types probably cross-hybridized with some of
the high-risk
probes. Among the additional types, there was HPV-58,
which has
a type-specific probe in the new-generation Hybrid Capture II
plate test. Although we do not know the detection limit of the
HCT test
for additional types, profound infections by them are
probably not
missed even by the first-generation test. Detection
of additional
types, HPV-53, -66, -67, -73, -CP6108, and -CP8061,
by Hybrid Capture
II test has recently been reported (
16). At
least two of
these types, HPV-53 and -66, also hybridized with
the HCT test
high-risk probes.
In the HCT test double-positive samples (AB), PCR-RFLP could detect
both low-risk and high-risk types only in two specimens,
while all but
one of the remaining samples contained high-risk
types (Table
3). The lack of simultaneous detection of
low- and
high-risk HPV types in these specimens is unlikely due to
methodological
failure, since the sensitive silver staining could
readily detect
the multiple PCR bands even if there was a 100-fold
difference
between the copy numbers of the competing target sequences
(data
not shown). Thus, we considered the PCR-RFLP typing method
reliable
and retested these samples with the HCT test, which is useful
for exclusion of false-positive HCT test signals (
19). As 12
of 13 specimens consistently hybridized to both low- and high-risk
probes, we investigated the significance of the amplitude of the
hybridization signal, which was expressed as the ratio of sample
luminescence to the mean luminescence of three positive controls
run in
each test series (
6). A high-risk (B) signal exceeding
the
low-risk (A) signal indicated an infection dominated by high-risk
types, whereas in all three samples containing low-risk HPV, the
A
signal was stronger (Table
3). Interestingly, an additional
HPV type
(HPV-62) was present in one of the specimens with a dominant
low-risk
(A) signal.
For the additional high-risk types (HPV-53, -58, -62, -66, -CP8304, and
-MM4), the RFLP typing results were confirmed by sequencing.
The
MY09-MY11 amplimers were cloned, sequenced, and aligned (
1)
with the corresponding reference HPV types. The sequencing of
the first
four clones was performed in two directions. Since the
major parts of
the sequences obtained from the two directions
overlapped, we performed
unidirectional sequencing of the rest
of the clones. The extent of
homology to the reference sequences
was 96 to 100%. Since the
amplifications were done with
Taq polymerase,
a few
nucleotide differences between the clones and the reference
sequences
might have been due to amplification errors. This did
not influence the
typing and confirmation of the PCR-RFLP results,
however.
The HCT test provides a sufficient diagnostic aid by distinguishing
between lesions with low-risk and high-risk HPV types.
From this point,
HCT test double-positive patients should be grouped
together with
high-risk HPV positives. Indeed, there is a strong
association between
SIL and high-risk HPV types whether or not
low-risk types are also
present (
19). According to this interpretation,
we found a
contradictory PCR-RFLP result only in one HCT double-positive
sample,
where the high-risk type was not detected by PCR-RFLP.
The overall type
specificity of the HCT test was thus 128 of 129
(99.2%). In
conclusion, the HCT test has a broader detection range
than assigned,
which increases its diagnostic value without influencing
the specific
distinction between low-risk and high-risk HPV
types.
| |
ACKNOWLEDGMENTS |
This work was supported by a Hungarian State Eötvös
Fellowship (J. Kónya), by a National Scientific Grant (OTKA
T-023864), and by a research grant (ETT T-10304/96) of the Health
Council from the Ministry of Welfare.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University Medical School of Debrecen, P.O. Box 17, H-4012 Debrecen, Hungary. Phone and Fax: 36 52 417-565. E-mail:
konya{at}mibio.dote.hu.
| |
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Journal of Clinical Microbiology, January 2000, p. 408-411, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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