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Technical Advance |
From the Delft Diagnostic Laboratory,*
the Department of
Pathology,
Diagnostic Center SSDZ, and the
Department of Gynecology,¶
and the Departments
of Virology
and
Gynecology,§
Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As HPV cannot be cultured in vitro and serological assays are still ineffective, diagnosis of HPV injury is based on the use of molecular tools. Direct dot-spot detection and in situ hybridization assays have been described,12,13 but these methods are tedious and appear to lack sensitivity and specificity. DNA amplification methods, such as the polymerase chain reaction (PCR), permit more sensitive detection of the viral DNA. Besides type-specific PCR primers for individual HPV genotypes,14,15 several universal PCR primer sets have been developed, including My11/My09,16 OBI/II,17 CPI/CPII,18 and GP5+/6+.19 At present, these HPV primer sets are widely used for routine diagnosis of HPV infections. However, it has been shown that none of these general primer sets permits adequate detection of the still expanding spectrum of anogenital HPV genotypes.20,21 Moreover, as the sensitivity of these universal primer sets is limited, their use will underestimate the true prevalence of HPV.14,20,21,22
The aim of the present study was to develop a set of HPV PCR primers for highly sensitive detection of all known mucosal HPV types. The assay comprised a novel set of primers for broad-spectrum detection of HPV DNA by short-fragment PCR and a microtiter-format assay for detection of HPV-derived PCR products by a mixture of probes. The sensitivity and specificity of this novel HPV detection system were analyzed and compared with type-specific as well as with other general HPV PCR primer sets. Finally, the performance of the method was evaluated by testing clinical samples from various patient populations, including 1) cervical scrapes, mainly classified as normal cytology from patients previously treated for CIN lesions, 2) mild, moderate, and severe dyskaryotic smears, and 3) formalin-fixed, paraffin-embedded cervical carcinoma samples.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study comprised three different groups of patients. Group 1 comprised 534 cervical scrapes obtained from consecutive women that were treated in the gynecology outpatient clinic of a community hospital in Delft, The Netherlands. Patients had a history of treatment for dysplasia by loop excision of the transformation zone (LETZ). Cervical scrapes were obtained with a Cervex brush (Rovers, Oss, The Netherlands) and were resuspended and transported in 1.5 ml of phosphate-buffered saline (PBS), pH 7.2, at room temperature. Smear preparations were examined and classified.
Group 2 consisted of 304 cervical scrapes of a total of 320 scrapes from women that were tested for the presence of HPV during a community screening program between 1988 and 1993 in The Netherlands.23 The surveyed patients had either one cervical scrape showing severe dyskaryosis (n = 153) or two scrapes showing mild or moderate dyskaryosis (n = 151) with a maximum interval of 1 year. Of the unavailable scrapes, 10 samples were severely dyskaryotic and 6 were mild or moderate dyskaryotic.
Group 3 comprised 184 patients with cervical carcinoma. The cervical biopsies were obtained from women visiting the Russian Cancer Center in Moscow between 1988 and 1994.24 The specimens had been fixed in formalin, embedded in paraffin, histologically examined after hematoxylin and eosin staining, and classified as squamous cell carcinoma (n = 132), adenocarcinoma (n = 31), or adenosquamous carcinoma (n = 21).
DNA Isolation
Cervical scrapes were resuspended in 1.5 ml of PBS pH 7.2. To prepare DNA for PCR, the cell suspension was vigorously shaken, and 120 µl was treated by adding 40 µl of proteinase K (200 µg/ml) in 3% Triton X-100 for 1 hour at 37°C. The proteinase was inactivated by incubation at 95°C for 10 minutes. Subsequently, 10 µl of the solution was used in a PCR reaction.
In group 3, DNA was isolated from formalin-fixed, paraffin-embedded tissue sections by a modified version of the method as described by Claas et al.25 Briefly, a 10-µm section was collected in a 1.5-ml tube and deparaffinized by adding 500 µl of xylol. After gentle shaking for 2 minutes and centrifuging for 5 minutes at 12,000 rpm, the pellet was retreated with 500 µl of xylol. The pellet was then washed twice with 500 µl of 96% ethanol and once with 500 µl of acetone. Subsequently, the pellet was air dried, dissolved in 200 µl of 5 mmol/L TrisHCl, pH 9.0, containing 1 mg/ml proteinase K, and incubated overnight at 37°C. Finally, the proteinase K was activated by incubation at 95°C for 10 minutes, and the supernatant was used directly for PCR.
Plasmids
Plasmids containing HPV genomic DNA were kindly provided by Dr. E.-M. de Villiers, Heidelberg, Germany (HPV genotypes 6b, 11, 13, 16, 18, 40, 45, 51, and 53), Dr. R. Ostrow, Minneapolis, MN (HPV genotype 26), Dr. A. Lorincz, Silver Springs, MD (HPV genotypes 31, 35, 43, 44, 56, 61, and 64), Dr. T. Matsukura, Tokyo, Japan (HPV genotypes 58, 59, 62, 67, and 69), and Dr. G. Orth, Paris, France (HPV genotypes 30, 33, 34, 39, 42, 52, 54, 55, 66, 68, 70, and 74). HPV genotypes were classified according to the Los Alamos Database (available at http://hpv-web.lanl.gov).
PCR
The SPF primers developed in this study are located in the L1 open
reading frame and listed in Table 1
. SPF
was performed in a final reaction volume of 100 µl, containing 10
µl of the isolated DNA, 10 mmol/L TrisHCl, pH 9.0, 50 mmol/L KCl, 2.5
mmol/L MgCl2, 0.1% Triton X-100, 0.01% gelatin, 200
mmol/L each deoxynucleoside triphosphate, 20 pmol of forward and
reverse primers, and 0.25 U of SuperTaq (Sphaero Q, Cambridge, UK). PCR
conditions were as follows: preheating for 1 minute at 94°C was
followed by 40 cycles of 1 minute at 94°C, 1 minute at 45°C, and 1
minute at 72°C and a final extension of 5 minutes at 72°C. Each PCR
experiment was performed with positive and several negative PCR
controls.
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HPV DNA Detection in Microtiter Plates
Amplimers, synthesized by biotinylated PCR primers, were detected
by hybridization to a mixture of HPV-specific probes (Table 1)
. Ten
microliters of PCR product was diluted in 100 µl of hybridization
buffer (150 mmol/L NaCl, 15 mmol/L sodium citrate, pH 7.0, 0.1% Tween
20) and incubated at 42°C for 30 minutes in streptavidin-coated
microtiter plates. Noncaptured materials were removed by three washes
with hybridization buffer. The double-stranded captured PCR products
were denatured by addition of 100 µl of denaturation solution (100
mmol/L NaOH) and incubated for 5 minutes at room temperature, followed
by three washes with hybridization buffer. A mixture of digoxigenin
(DIG)-labeled HPV-specific probes (Table 1)
were diluted in
hybridization buffer and added to the well and incubated at 42°C for
45 minutes. Wells were washed three times, and anti-DIG alkaline
phosphatase conjugate was added and incubated at 42°C for 15 minutes.
After five washes, substrate was added and incubated at room
temperature for 15 minutes. The reaction was stopped by adding 100 µl
of 0.5 mmol/L H2SO4. Optical densities (OD)
were determined at 450 nm in a microtiter plate reader. Samples were
considered positive if the OD450 was 2.5 times higher than
the negative PCR control. In each run, negative controls as well as
positive and borderline positive controls were tested together with the
clinical samples.
Southern Blot Hybridization
Southern blot hybridization was performed according to standard procedures.26 Briefly, 20 µl of the PCR product (GP5+/6+ and My11/09) was electrophoresed on a 2% Tris-borate/ethylenediaminetetra-acetate (TBE) agarose gel. The small (65-bp) SPF1/2 amplimers were separated on a 3% agarose gel. Subsequently, amplimers were transferred onto a nylon membrane (Hybond N+, Amersham, Little Chalfont, UK) by vacuum blotting in the presence of 0.4 N NaOH. The Southern blots were hybridized with a 32P-labeled oligonucleotide probe for 16 hours at 42°C in a solution containing 5X SSC (1X SSC contains 150 mmol/L NaCl and 15 mmol/L sodium citrate), 5X Denhardt's (1X Denhardt's contains 0.02% bovine serum albumin, 0.02% polyvinyl pyrrolidone, and 0.02% Ficoll), 0.5% SDS, 75 mmol/L EDTA, and 0.1 mg/ml denatured herring sperm DNA. Subsequently, the blots were washed twice in 2X SSC/0.1% SDS at 42°C for 15 minutes. Autoradiography was performed with intensifying screens for 3.5 hours at -70°C, using Kodak X-Omat AR film.
Sequence Analysis
SPF1/2 amplimers were excised from a 2% low-melting-point Tris-acetate/ethylenediaminetetra-acetate (TAE)-agarose gel and directly analyzed by the AmpliCycle sequencing kit (Perkin Elmer, Norwalk, CT) using one of the SPF primers. The concentration of dideoxyribonucleosides in the termination mixtures was increased to permit adequate sequencing of the short 65-bp PCR products. Sequence products were separated and detected by an automated sequencing system (Alf-Express, Pharmacia, Uppsala, Sweden). As the software of the sequencing unit was not suitable for interpretation of very short sequences, the profiles were read manually. Sequences were analyzed with the PC-Gene software (Intelligenetics, Mountain View, CA) and compared with HPV sequences of known types using the computer program basic local alignment search tool (BLAST).27
Statistical Analyses
For statistical analyses 
2
and Fisher's exact test
were used to compare the prevalence of a dichotomous variable between
two groups. P values <0.05 were considered to be
statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The L1 region sequences from 39 HPV genotypes were obtained from
Genbank. Alignment of these sequences identified a relatively well
conserved part in the L1 region, located between nucleotides 6582 and
6646, as shown in Figure 1
(all numbers
are according to the prototype PPH16 sequence of HPV type 16; Genbank
accession number K02718). Based on this sequence alignment, target
regions for forward and reverse primers were chosen that were
relatively well conserved and designated as SPF1 (nt 6582-6601) and
SPF2 (nt 6624-6646), respectively. Four primers (SPF1A to -D) were
designed as forward PCR primers, aimed at the SPF1 region. Similarly,
two reverse primers (SPF2B and -D) were aimed at the SPF2 region. These
six primers (Table 1)
were used in equimolar quantities for universal
HPV DNA amplification. The expected amplimer size was only 65 bp (nt
6582-6646), including a 22 bp interprimer fragment, flanked by the SPF1
and SPF2 priming regions. The SPF primer target regions are similar to
the ones used by the sense primers My11 and GP5+.16,19
In
the SPF system, the later target region is used for antisense priming.
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, the sequences of the interprimer region, located
between SPF1 and SPF2, are heterogeneous. This sequence variation is
sufficient to permit recognition of each of the different HPV
genotypes, except for types 68 and 73, which have identical interprimer
regions.
For universal detection of HPV DNA amplimers from a broad range of
genotypes, a total of nine probes (Table 1)
were designed that
permitted hybridization to all anogenital HPV genotypes. These probes
were used in a microtiter-based amplimer detection assay to determine
the presence of amplified HPV DNA after SPF-mediated PCR.
Specificity and Sensitivity of the SPF1/2 Primers
To assess the efficacy of the novel broad-spectrum primer set,
plasmids containing partial or complete HPV genomic sequences from 34
different genotypes (as listed in Materials and Methods) were analyzed
by the SPF system. Amplimers of the expected size of 65 bp were
obtained from all plasmids, as determined by gel electrophoresis (data
not shown). Sequences of the 22-bp interprimer regions were completely
concordant with the published L1 region sequence of each HPV genotype
as shown in Figure 1
. Subsequently, the SPF1/2 amplimers were tested in
the microtiter plate hybridization assay. To test the detection of HPV
genotypes 73, MM4, and MM7, clinical material was used. Hybridization
with the mixture of the nine universal probes yielded positive signals
for all of the 37 amplimers. These results showed that the
SPF1/2-mediated PCR products of all tested HPV genotypes could be
detected by the mixture of HPV DNA detection probes.
To determine the sensitivity of the novel primer set, series of 10-fold
dilutions of plasmids containing genomic DNA from HPV types 16, 35, and
45 were tested by PCR and Southern blot hybridization. The sensitivity
of the SPF1/2 primers was compared with those of the primer sets
My11/My09 and GP5+/GP6+ (Figure 2)
. For
HPV type 16, the three PCR primer sets demonstrated the same
sensitivity. For HPV type 35, the My11/09 primer set was at least
100-fold less sensitive than SPF1/2 and GP5+/6+. Primer sets GP5+/6+
and My11/09 reached equal sensitivity for type 45, whereas SPF1/2 was
10 times more sensitive. These results showed the high sensitivity of
the novel SPF1/2 primers for these HPV genotypes.
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The clinical performance of the novel HPV DNA detection system was
investigated by analysis of materials from three different patient
groups. Group 1 comprised a total of 534 cervical scrapes that were
obtained during follow-up from women after treatment for cervical
intraepithelial neoplasia (CIN). Results of the cytological
examinations are summarized in Table 2
.
The great majority (83.7%) of the follow-up smears did not show
cytological abnormalities. All cervical scrapes were tested for the
presence of HPV DNA with the primer sets SPF1/2 and GP5+/6+. The PCR
results are also shown in Table 2
. Overall, a total of 160 (30.0%) of
the 534 samples were found to be positive for the SPF1/2 primers, which
is significantly higher than the 113 (21.2%) positive samples detected
by GP5+/6+ PCR (P < 0.001). All 113
GP5+/6+-positive samples were also positive with the SPF1/2 primer set.
PCR results of the 47 cases that were initially positive by the SPF1/2
primers, but negative by the GP5+/6+ primer set, were all confirmed by
repeating the PCR with both systems. The HPV DNA detection rate in
scrapes with a normal cytology (n = 447) was
significantly higher by SPF1/2-mediated PCR as compared with GP5+/6+
PCR (P < 0.001). The detection rates among
scrapes with atypical squamous cells of undetermined significance
(ASCUS) or mild to moderate dyskaryotic lesions were not significantly
different. The numbers of cases where no cytological analysis was
possible or with severe dyskaryosis or carcinoma were too low to
compare the efficacy of both primer sets in this group.
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. The distinction between high-risk and
low-risk HPV genotypes was according to zur Hausen.8
Among
the 34 SPF-positive cases in group 1 with mild or moderate dyskaryotic
lesions, 79.4% and 17.6% were classified as high risk and low risk,
respectively. The prevalence of sequences that did not exactly match
any of the known HPV genotype sequences (designated as unknown in Table 3
) among cases with normal cytology or ASCUS (20.5%) was significantly
higher than among the mild or moderate dyskaryotic cases (3.0%). In
this analysis, cases with no cytological diagnosis possible, severe
dyskaryosis, or carcinoma were excluded due to low numbers.
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. A total of 299 (98.4%) of the 304 cases were
positive by SPF-mediated PCR, and only five scrapes (four times mild or
moderate dyskaryosis and one with severe dyskaryosis) were found to be
HPV DNA negative. Again, the amplimers were directly sequenced, and the
presence of HPV-specific sequences from various HPV genotypes was
confirmed in all cases. A total of 140 (95.2%) and 5 (3.4%) of the
147 SPF-positive mild or moderate dyskaryotic cases were classified as
high-risk and low-risk HPV genotypes, respectively. Among the 152
SPF-positive severe dyskaryotic cases, the prevalence of high-risk
genotypes was 98.7% (Table 3)
.
The presence of HPV in cervical carcinoma was assessed by SPF analysis
of the third group, comprising 184 formalin-fixed, paraffin-embedded
cervical cancer biopsies obtained from Russian women. The SPF1/2 primer
set yielded positive results in 100% of cases (184/184). Amplimers
were analyzed by direct sequencing, and the presence of HPV-specific
sequences was confirmed in all cases. Among the 184 carcinomas, 177
(96.2%) and 1 (0.5%) were classified into high-risk and low-risk
groups, respectively. The low-risk type sequence was associated with
HPV genotype 55.8
The remaining six yielded sequences that
did not exactly match with known HPV genotypes (Table 3)
.
Taken together, a total of 634 sequences of the 22-bp SPF interprimer
regions, obtained from patient groups 1, 2, and 3 were compared with
corresponding L1-region sequences of the different HPV genotypes, as
shown in Table 3
. A total of 35 (5.5%) of the 634 sequences did not
exactly match with any of the known HPV genotype L1 sequences. These
were classified as unknown and require more extensive sequence
analysis. The great majority of these 35 unrecognized sequences were
found among scrapes with normal cytology or ASCUS in group 1 patients.
Also, sequences, matching low-risk HPV genotypes were found almost
exclusively among scrapes with normal cytology or ASCUS and appeared to
be virtually absent in the dyskaryotic or carcinoma cases from groups 2
and 3.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As HPV cannot be cultured in vitro and serological assays are still not sensitive enough, diagnosis of HPV infection is currently based on detection of the viral DNA genome. The existence of various HPV genotypes with heterogeneous DNA genomes requires either type-specific or broad-spectrum detection of HPV DNA. Type-specific PCR detection would involve a large number of separate PCR assays for each individual clinical sample and is therefore impractical for routine diagnosis. Several broad-spectrum PCR primer sets have been described for detection of multiple HPV types by a single PCR assay.16-18,29 However, none of the currently available universal primer sets is able to effectively detect all anogenital HPV genotypes,21,30 and recent studies have shown that the overall prevalence of HPV can be underestimated considerably if only a single DNA detection method is used.14,20,30,31
Development of a Novel HPV DNA Detection System
Alignment of a large number of L1-region sequences permitted the design of a novel set of broad-spectrum primers. The amplimer size of SPF1/2 was only 65 bp, whereas the amplimer sizes of GP5+/6+ and My11/09 were 150 and 450 bp, respectively. In general, the sensitivity of DNA detection by PCR is inversely related to the size of the amplimer, as the kinetics of the PCR reaction favor smaller amplimers. The importance of small amplimers has also been shown for the detection of the hepatitis C virus.32 Moreover, as the efficacy of PCR also depends on the quality of the DNA extracted from the clinical specimen, detection with small amplimers will be advantageous, especially when using formalin-fixed, paraffin-embedded materials.14,33 Taken together, the small amplimer size may explain the high sensitivity of the SPF assay.
The other universal primer sets located in the L1 open reading frame
either use degenerated primers (My11/09)16
or consensus
primers (GP5+/6+).19
The novel SPF system employs a mixture
of defined primers for the SPF1 and SPF2 target regions. These regions
are also used for My11 and GP5+ priming, respectively. As the
heterogeneity of the primer target sites is limited, this mixture of
primers may be more effective than consensus or degenerated primers.
The 3' terminus of a primer target region is crucial for the efficacy
of the PCR primer. From the sequence alignment (Figure 1)
, it is clear
that the 3' ends of SPF1 and SPF2 primers are both highly conserved
among all HPV genotypes.
The SPF1/2 primers permitted effective detection of genomic DNA of 34 different anogenital HPV genotypes from plasmid clones. In addition, the SPF1/2 primers also allowed detection of cutaneous HPV genotypes 3, 4, 5, 8, 27, 32, 37, 65, and 71 (data not shown). Sequences of the 22-bp interprimer region were completely concordant with the published sequence for each of the HPV genotypes. This confirms the effective detection of a broad spectrum of HPV genotypes by the novel SPF assay.
For several HPV genotypes, the sensitivity of the SPF primer set was investigated. Results indicated that the SPF-mediated PCR was more or equally sensitive than GP5+/6+ and My11/09 for HPV 16, 35, and 45. My11/09 is less sensitive for HPV types 35 and 45, whereas GP5+/6+ is less sensitive for type 45 as compared with SPF1/2. These results indicated the high sensitivity of SPF1/2, whereas other general primer sets may be less sensitive for specific genotypes, as has been reported earlier.21
Clinical Evaluation of the SPF System
The performance of the SPF1/2 primers was evaluated by testing a large number of clinical specimens, representing clinically different patient categories.
Among group 1 patients, with the majority of cases classified as normal cytology, the HPV detection rate using the SPF1/2 primers was significantly higher than with the GP5+/6+ primers. Semiquantitative studies have shown that the viral load is associated with the cytological grade of abnormality, with the lowest level of HPV DNA in scrapes with normal cytology and ASCUS.30 These results suggest that the SPF assay is able to detect low concentrations of HPV DNA in clinical samples and confirms the higher sensitivity of the SPF primer set as compared with GP5+/6+ primers for a broad range of HPV genotypes.
The detection rate of the SPF1/2 primer set among the second group of patients with scrapes classified as dyskaryotic was close to 100%. Materials from this group had been analyzed by the GP5/6 primer set earlier,23 with a detection rate of only 63.6% and 82.6% among mild or moderate dyskaryotic and severe dyskaryotic cases, respectively. Again this confirmed the high sensitivity of the SPF1/2 primers. These samples have not been tested with the adapted GP5+/6+ primers, which should have a higher sensitivity than GP5/6.19 Among scrapes classified as mild or moderate dyskaryotic, the HPV DNA detection rate, as determined by various universal PCR primer sets, ranged from 71% to 76%.30,34,35 Similarly, among scrapes classified as severe dyskaryotic, the HPV DNA detection rate ranged from 86% to 94%.30,35 The HPV DNA detection rate of the SPF1/2 primer set was considerably higher in both groups, indicating the high sensitivity of this novel method.
Among cervical carcinoma specimens, HPV DNA has been generally found in approximately 70% of the cases using direct DNA detection by Southern blot analysis or type-specific PCR.36,37 Using a combination of multiple universal PCR primers, HPV DNA can be detected in up to 90% of the carcinomas.20,29 There are several explanations for the failure to detect HPV DNA in all cervical carcinomas. It has been postulated that truly HPV-DNA-negative cervical carcinomas exist, but the prevalence is considered to be very low.38 Linear integration of the circular HPV DNA genome into the chromosomal DNA of the host cell may affect the PCR target region, although the preferred cleavage integration site is located in the E2 region.39 Uneven distribution of HPV in the carcinoma tissue may lead to sampling errors, yielding false negative PCR results. Most likely, however, the sensitivity of the current PCR systems may be too limited to detect very low concentrations of HPV DNA in 100% of the 184 formalin-fixed, paraffin-embedded cervical cancer biopsies presented in patient group 3. The high efficacy of the SPF primer set was also shown by comparison with results of other general HPV primer sets in these materials. Among 30 samples that remained negative by HPV-16, -18, -31, and -33 type-specific PCR and positive by the SPF assay, HPV DNA was detected in only three cases by the general primer sets My11/My09 and/or GP5+/6+ (data not shown). This confirmed the high HPV DNA detection rate of the SPF primer set in formalin-fixed, paraffin-embedded cervical carcinomas, most likely due to the very small amplimer size.
Association between HPV Genotypes and Severity of Morphological Changes
The relative risk association of the different HPV types is still disputed. Based on the observed distribution of genotypes in low- and high-grade lesions and carcinomas, HPV genotypes have been classified into high-risk, intermediate-risk, or low-risk groups.40 However, detection and identification of HPV types in various clinical materials strongly depends on the methods used. Direct hybridization methods have a relatively low detection level whereas universal PCR primer sets might have differential sensitivity for different HPV DNA genotypes.21 Therefore, the data obtained so far from epidemiological studies may be incomplete and not completely accurate. The general primer sets My11/09 and GP5+/6+ are both aimed at the L1 region, which is one of the more conserved parts of the HPV genome. Classification of HPV genotypes as well as identification of novel genotypes is based on phylogenetic analysis of the 450-bp PCR fragment generated by My11/My09 primers.8 Direct sequencing analysis of SPF amplimers obtained from reference HPV genomic clones confirmed the distinct heterogeneity of the 22-bp interprimer sequence. Consistent sequence variation in this region can be used to recognize different HPV genotypes. However, formal genotyping analysis of HPV DNA, based exclusively on sequence variation of the SPF1/2 amplimer, needs to be confirmed by phylogenetic analysis. Preliminary data from more extensive sequence analysis of the L1 region indicate that genotype recognition in the SPF1/2 fragment is highly reliable (data not shown). Comparison of the HPV genotype distribution, based on analysis of the 22-bp SPF interprimer sequences, revealed some interesting differences between patient groups with different degrees of cytological abnormalities. Among group 1 patients with normal cytology or ASCUS, the prevalence of low-risk HPV genotypes was approximately 24%. By contrast, in patients belonging to groups 2 and 3 with dyskaryosis or carcinoma, these low-risk genotypes were virtually absent, and all cases contained high-risk genotypes. A single carcinoma case appeared to contain HPV genotype 55. Detection of this potentially low-risk type by direct sequencing could be caused by a mixed infection with a high-risk type, but HPV genotype 55 has been observed in carcinoma earlier.9 Also, the number of 22-bp interprimer sequences that did not exactly match any of the known HPV genotypes, was significantly higher among patients with normal cytology or ASCUS than among patients with at least dyskaryosis. These sequences may either represent novel HPV genotypes or variants of known HPV genotypes. This will be the subject of additional, more extensive phylogenetic studies. Overall, the frequency distribution of HPV genotypes appeared to be consistent with the hypothesis that patients can be initially infected with a broad range of HPV genotypes, but only those infected with potentially high-risk HPV genotypes are at risk for carcinoma.
In conclusion, the present study describes a novel PCR primer set for the detection of a broad spectrum of HPV genotypes. At present, the SPF1/2 primers have been shown to efficiently amplify sequences from at least 43 different HPV genotypes. These results indicated that the SPF1/2 primer set permits highly sensitive detection of a broad range of HPV genotypes. The sequence variation in the amplified fragment probably permits consistent identification of HPV genotypes. PCR mediated by the SPF1/2 primer set seems to be highly sensitive and may facilitate future studies on the clinical importance of HPV infections.
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Acknowledgements |
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Footnotes |
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Accepted for publication August 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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P. E. Gravitt, L. J. van Doorn, W. Quint, M. Schiffman, A. Hildesheim, A. G. Glass, B. B. Rush, J. Hellman, M. E. Sherman, R. D. Burk, et al. Human Papillomavirus (HPV) Genotyping Using Paired Exfoliated Cervicovaginal Cells and Paraffin-Embedded Tissues To Highlight Difficulties in Attributing HPV Types to Specific Lesions J. Clin. Microbiol., October 1, 2007; 45(10): 3245 - 3250. [Abstract] [Full Text] [PDF]
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G. M. Clifford, H.-R. Shin, J.-K. Oh, T. Waterboer, Y.-H. Ju, S. Vaccarella, W. Quint, M. Pawlita, and S. Franceschi Serologic Response to Oncogenic Human Papillomavirus Types in Male and Female University Students in Busan, South Korea Cancer Epidemiol. Biomarkers Prev., September 1, 2007; 16(9): 1874 - 1879. [Abstract] [Full Text] [PDF]
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M. P. Stevens, S. M. Garland, E. Rudland, J. Tan, M. A. Quinn, and S. N. Tabrizi Comparison of the Digene Hybrid Capture 2 Assay and Roche AMPLICOR and LINEAR ARRAY Human Papillomavirus (HPV) Tests in Detecting High-Risk HPV Genotypes in Specimens from Women with Previous Abnormal Pap Smear Results J. Clin. Microbiol., July 1, 2007; 45(7): 2130 - 2137. [Abstract] [Full Text] [PDF]
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M. Safaeian, R. Herrero, A. Hildesheim, W. Quint, E. Freer, L.-J. Van Doorn, C. Porras, S. Silva, P. Gonzalez, M. C. Bratti, et al. Comparison of the SPF10-LiPA System to the Hybrid Capture 2 Assay for Detection of Carcinogenic Human Papillomavirus Genotypes among 5,683 Young Women in Guanacaste, Costa Rica J. Clin. Microbiol., May 1, 2007; 45(5): 1447 - 1454. [Abstract] [Full Text] [PDF]
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C. Payan, A. Ducancelle, M. H. Aboubaker, J. Caer, M. Tapia, A. Chauvin, D. Peyronnet, E. Le Hen, Z. Arab, M.-C. Legrand, et al. Human Papillomavirus Quantification in Urine and Cervical Samples by Using the Mx4000 and LightCycler General Real-Time PCR Systems J. Clin. Microbiol., March 1, 2007; 45(3): 897 - 901. [Abstract] [Full Text] [PDF]
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V. Fontaine, C. Mascaux, C. Weyn, A. Bernis, N. Celio, P. Lefevre, L. Kaufman, and C. Garbar Evaluation of Combined General Primer-Mediated PCR Sequencing and Type-Specific PCR Strategies for Determination of Human Papillomavirus Genotypes in Cervical Cell Specimens J. Clin. Microbiol., March 1, 2007; 45(3): 928 - 934. [Abstract] [Full Text] [PDF]
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L. Giovannelli, M. C. Migliore, G. Capra, M. P. Caleca, C. Bellavia, A. Perino, E. Viviano, D. Matranga, and P. Ammatuna Penile, Urethral, and Seminal Sampling for Diagnosis of Human Papillomavirus Infection in Men J. Clin. Microbiol., January 1, 2007; 45(1): 248 - 251. [Abstract] [Full Text] [PDF]
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D. van Hamont, M. A. P. C. van Ham, J. M. J. E. Bakkers, L. F. A. G. Massuger, and W. J. G. Melchers Evaluation of the SPF10-INNO LiPA Human Papillomavirus (HPV) Genotyping Test and the Roche Linear Array HPV Genotyping Test. J. Clin. Microbiol., September 1, 2006; 44(9): 3122 - 3129. [Abstract] [Full Text] [PDF]
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L.-J. van Doorn, A. Molijn, B. Kleter, W. Quint, and B. Colau Highly effective detection of human papillomavirus 16 and 18 DNA by a testing algorithm combining broad-spectrum and type-specific PCR. J. Clin. Microbiol., September 1, 2006; 44(9): 3292 - 3298. [Abstract] [Full Text] [PDF]
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M. P. Stevens, E. Rudland, S. M. Garland, and S. N. Tabrizi Assessment of MagNA Pure LC Extraction System for Detection of Human Papillomavirus (HPV) DNA in PreservCyt Samples by the Roche AMPLICOR and LINEAR ARRAY HPV Tests. J. Clin. Microbiol., July 1, 2006; 44(7): 2428 - 2433. [Abstract] [Full Text] [PDF]
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