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Human papillomavirus DNA testing by PCR-ELISA and hybrid capture II from a single cytological specimen: concordance and correlation with cytological results
Journal of Clinical Virology 25 (2002) 177– 185 www.elsevier.com/locate/jcv
Human papillomavirus DNA testing by PCR-ELISA and hybrid capture II from a single cytological specimen: concordance and correlation with cytological results Simona Venturoli a, Monica Cricca a, Francesca Bonvicini a, Francesco Giosa b, Francesco Renato Pulvirenti c, Claudio Galli c, Monica Musiani a, Marialuisa Zerbini a,* a
Department of Clinical and Experimental Medicine, Section of Microbiology, Uni6ersity of Bologna, Policlinico S. Orsola, 6ia Massarenti 9, 40138 Bologna, Italy b Department of Oncology and Hemathology, S. Orsola Hospital, Bologna, Italy c Scientific Affairs, Abbott Di6isione Diagnostici, Rome, Italy Received 23 October 2001; accepted 20 January 2002
Abstract Background and objecti6es: A persistent infection by high-risk HPV is now considered as the major cause of cervical carcinoma. The use of a single cytological specimen for HPV DNA testing by two different molecular methods was analyzed and validated. Study design: HPV DNA testing by PCR-ELISA and hybrid capture II HPV test (HC-II), was investigated on 317 cytological samples obtained from Italian women. Two hundred twenty-seven women were referred to virological lab for HPV DNA testing during cytological routine screening and 90 during a cytological and virological follow-up after a conization or hysterectomy. Results: Overall, the concordance between the two assays was high (K =0.87). Compared with PCR-ELISA, the HC-II showed a sensitivity of 91.7% and a specificity of 95.4%. Although the analytical sensitivity of the PCR-ELISA was higher, the performance of the two tests did not differ in recognizing HPV DNA positive patients with either low or high-grade squamous intraepithelial lesions (LSIL or HSIL). HPV DNA positivity was directly correlated with the severity of cytological diagnosis (PB 0.005). Conclusions: In view of the comparable results obtained with the two assays and of the ease of use, and higher throughput of HC-II, it seems advisable, with a single cytological specimen, to employ the HC-II test as a first-line assay, either for screening or diagnosis, and to perform reflex PCR on positive samples, if typing of prevalent high risk HPVs is needed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: HPV DNA; HC-II; PCR
1. Introduction * Corresponding author. Tel.: +39-051-341-652; fax: + 39051-341-632. E-mail address: [email protected] (M. Zerbini).
A persistent infection by high-risk types of human papillomavirus (HR-HPV) is now considered
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as the major cause of cervical carcinoma (Myers et al., 2000; Walboomers et al., 1999; Wallin et al., 1999; Ylitalo et al., 2000). While HPV infections are most common in younger women, among whom prevalences as high as 30– 50% have been reported, the persistence of both low- and high-risk types for more than 1 year is infrequent (Franco et al., 1999; Ho et al., 1998; Myers et al., 2000). The high positivity rates and the short duration of infection have so far hampered the implementation of screening programs based on HPV DNA testing, due to the huge number of infected women who will not develop HPV-related pathologies. However, since the sensitivity of Pap smear testing for detecting high-grade lesions is not higher than 80– 85% even under the best conditions (Clavel et al., 1999; Cuzick et al., 2000; Schiffman et al., 2000), some recent studies have suggested that HPV DNA screening in association with Pap testing may substantially reduce the rate of false negatives, i.e. patients with a normal cytological smear and high-grade cervical intraepithelial neoplasia (CIN) in the cervix (Clavel et al., 1999; Poljack et al., 1999). On the other hand, HPV testing has been proven both useful and cost-effective in the evaluation of borderline cytological results (Cox et al., 1995; Ferris et al., 1998; Manos et al., 1999). The major obstacle towards the adoption of HPV DNA testing in clinical routine has for many years been the lack of standardized, easy-to-use assays. The majority of clinical studies have been carried out by molecular biology assays based on polymerase chain reactions (PCR). Such methods are highly sensitive but may suffer from inhibition by substances contained either in the sample or in the transport buffer, causing false-negative results, and require trained and skilled personnel and dedicated laboratory equipment in order to avoid contamination and false-positive results. In recent years, the availability of a direct hybridization test with chemiluminescent signal amplification (HC) has provided a simple and attractive alternative to the more complex PCR assays. This method has been employed on a variety of clinical samples (Peyton et al., 1998) and does not require the stringent contamination
control of target amplification procedures. The aim of the study here described was: (i) to establish if both molecular methods may be run starting from a single sample obtained either with different tools for cervical scrapes or transport media and (ii) to compare the efficiency of the newest version of the HC assay (HC-II) (Ferris et al., 1998; Poljack et al., 1999) with a PCR method (PCR-ELISA) that had previously been described and validated (Venturoli et al., 1998; Zerbini et al., 2001). Additionally, we looked for a correlation between the two HPV DNA tests and Pap test results and for the best possible utilization of both molecular techniques in clinical microbiology practice.
2. Materials and methods
2.1. Clinical specimens HPV DNA testing was performed by PCRELISA and HC-II. Cytological specimens for PCR-ELISA were collected by Dracon-tipped swab and suspended in 10 ml of phosphate buffered saline solution (PBS). Cytological specimens for HC-II were obtained by cytobrush and dipped in 1 ml of HC-II transport medium (Digene Cervical Sampler). In order to evaluate the suitability of PCRELISA and HC-II for HPV DNA testing using different tools for cervical scrapes and transport media, two successive cytological specimens were obtained from the ecto-endocervix and posterior fornix of each of 24 consecutive women enrolled for a routine Pap smear testing. PCR-ELISA was performed on cervical cells suspended in PBS and aliquoted, and on cytological specimens collected in HC-II transport medium. The latter were analyzed both directly and after removal of the transport medium by centrifugation and resuspension in PBS. HC-II was used both directly on specimens collected in HC-II transport medium and on specimens collected in PBS, centrifuged and resuspended in HC-II transport medium. After the preliminary analysis to examine different cervical specimen collection methods, the
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24 samples used for method validation and 293 further cervical specimens, all collected by cytobrush in HC-II transport medium, were analyzed for HPV DNA detection by HC-II and the results compared with the data obtained by PCR-ELISA. The statistical analysis was performed on all 317 cervical specimens. Informed consent for the study was obtained from all the 317 women enrolled in the study (mean age 38.9 years, median 38 years, range 17 to 74 years), 227 of whom had been referred to the virological lab for HPV DNA testing during a routine cytological screening, while 90 were tested during a cytological and virological follow-up after conization (85 cases) or hysterectomy (five cases) for a previously CIN or cervical cancer. The cytological analysis of the 317 cervical specimens was done by Pap test performed by staff physicians at the Department of Oncology and Hemathology, S. Orsola Hospital, Bologna (Italy) using ectocervical and endocervical specimens. Specimens for Pap test were evaluated by the standard practices and results were accessed from cytology records. Cytological atypias, according to the Bethesda system, consisted of: atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade SIL (HSIL). Experienced staff physicians performed colposcopy with biopsy. Examinations were conducted without knowledge of patients’ Pap or HPV results. In the histological diagnosis cervical lesions were grouped as negative, cervical intraepithelial neoplasias (CIN 1, 2/3) and invasive carcinoma (IC).
2.2. PCR-ELISA for HPV DNA 2.2.1. Sample preparation Cervical specimens collected in PBS were centrifuged; aliquots of 100 000 cells from each cytological specimen were protease digested by incubation at 55 °C for 2 h in 200 ml of digestion buffer (50 mM KCl, 10 mM Tris– HCl pH 8.3, 0.05% Tween 20 and 400 mg/ml of Proteinase K), followed by heat inactivation at 95 °C for 10 min. Cervical cells collected by HC-II transport medium were counted and a volume containing
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100 000 cells was protease digested. Alternatively, an equal volume of the same sample was centrifuged and 100 000 cells were resuspended in 200 ml of digestion buffer and processed as above. A 10 ml volume of each digested sample was then processed by PCR-ELISA. DNA preparation of control samples was performed as previously described (Venturoli et al., 1998).
2.2.2. PCR-ELISA method The consensus primer pair MY09 and MY11 (Resnick et al., 1990) was used in PCR amplification. Oligonucleotide probes specific for HPVs 6, 11, 16, 18, 31, 33, 45 were separately utilized in different hybridization reactions. As amplification control, primers GH20 and PC04 (Resnick et al., 1990) recognizing ß globin gene sequence, were used in each PCR reaction and amplified products were detected by PC03 probe in a separate hybridization reaction. All the hybridization probes used in the PCR-ELISA were 5% labeled with biotin. DNA amplification and digoxigenin-labeling was performed in 50 ml of PCR reaction volume containing 10 ml of each digested sample; amplified products were detected by a hybrid capture-immunoassay in microtiter wells (Venturoli et al., 1998). Specificity and sensitivity controls and the cutoff of the PCR-ELISA method were performed as previously described (Venturoli et al., 1998). Results were expressed as net absorbance after the subtraction of absorbance of the buffer blank and index value was calculated as OD sample/OD cut-off; an index value \1 was considered as positive. 2.2.3. Agarose gel electrophoresis All the amplified samples were also analyzed by agarose gel electrophoresis. Ten microliters of amplified products were visualized through 2% agarose gel with ethidium bromide staining. 2.3. Hybrid Capture II HPV Cervical specimens in HC-II transport medium were processed by the commercially available HCII System (Digene). The Digene Hybrid Capture II HPV is a signal amplification method based on
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liquid hybridization of HPV DNA with two different sets of RNA probes aimed at the recognition of 13 high risk (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and 5 low risk (6, 11, 42, 43 and 44) HPV types, respectively, followed by capture of DNA/RNA hybrids on a solid phase by monoclonal antibodies and detection by enhanced chemiluminescence. The results are expressed by comparing the relative light units (RLU) from clinical samples with a positive control (PC): a RLU/PC ratio of 1 or higher is considered as a positive result. This assay has a nominal sensitivity of 0.2 –1 pg/ml, equivalent to 1000–5000 genome copies of HPV.
2.4. HPV DNA testing algorithm Each sample was initially tested by both molecular assays. Samples yielding discrepant results were retested by both PCR-ELISA and HC-II. The specimens that resulted as high negative (0.8– 0.99 RLU/PC ratio) or weak positive (1– 5 RLU/PC ratio) by HC-II were retested by this method, regardless of PCR-ELISA results, in order to check the assay reproducibility around the cut-off. Only repeat testing results were considered for assay comparison and HPV prevalence.
2.5. Statistical analysis Sensitivity and specificity were assessed by 2× 2 contingency tables; chi-square ( 2) or Fisher’s exact test and chi-square for linear trend were performed in order to evaluate the percentage differences between the two assays and to state the significance of different HPV DNA positivity rates among classes of Pap test results, respectively. Kappa coefficient with 95% confidence limits was also performed to evaluate the correlation between PCR-ELISA and HC-II assays.
3. Results
3.1. Methods 6alidation The validation of PCR-ELISA and HC-II on samples stored in the transport buffer specific for
the other method was carried out on 24 samples. All the samples collected by the Digene system and processed by PCR-ELISA without removing the transport medium gave a negative result: this was due to PCR inhibition, since beta-globin amplification, the internal amplification control (IC), was negative as well. Centrifugation and PCR testing of the pellets resuspended in PBS buffer guaranteed an efficient removal of inhibitors: all samples became positive for the IC and gave a 100% concordance (12 positives, 12 negatives) with the PCR-ELISA performed on the duplicate specimens collected in PBS. The HC-II method did not show any influence from different transport media, as a 100% concordance was observed on duplicate specimens with similar RLU/PC values on positive specimens (range: − 46 to + 73%). Sample analysis performed by gel electrophoresis was concordant with results obtained by PCR-ELISA. The endpoint analytical sensitivity of the two molecular assays was checked by analyzing serial dilutions of a lysate of HeLa cells containing the HPV 18 DNA genome. The number of HPV genome copies presents in HeLa cell lysate was determined in comparison with serial dilutions, from 106 to 100 genome copies, of plasmid DNA containing L1 HPV region (Amplimedical SpA, Divisione Bioline, Italy). The HCII assay was able to detect about 1000 genome copies, as compared with the 50–100 genome copies detected by PCR-ELISA.
3.2. Population groups and cytology results Out of the 317 women enrolled in the study, 227 were referred to the virological lab for HPV DNA testing during routine cytological screening, while 90 were tested during a cytological and virological follow-up after a conization (85 cases) or hysterectomy (five cases) for a previously diagnosed CIN or cervical cancer. In the screening population almost half of the Pap smears (94/227: 41.4%) were classified as normal, 30 (13.2%) ASCUS, 40 (17.6%) LSIL and 63 (27.8%) HSIL. Conversely, followedup women had a normal cytology in 74/90 cases (82.2%), 6 were ASCUS (6.7%), 2 LSIL (2.2%) and 8 HSIL (8.9%). The median ages were 38 and 39 years in the two groups and 38 years overall: median ages and ranges among groups with differ-
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ent cytological results were, respectively, 39 years (22–74) for normal smears, 37 years (28– 72) for ASCUS, 37 years (19– 59) for LSIL and 37 years (17–65) for HSIL.
3.3. HPV DNA test results and comparison The comparative analysis of the 317 samples after repeat testing of discordants showed a concordance of 94.0% (298/317) between PCRELISA and HC-II (K =0.87; c.i. 95%= 0.82 – Table 1 Comparison between PCR-ELISA and HC-II on 317 samples HPV-DNA test
PCR-ELISA +
PCR-ELISA −
HC-II + HC-II −
111 10
9 187
The overall concordance was 94.0% (k= 0.87) and HC-II sensitivity and specificity vs. PCR-ELISA were 91.7 and 95.4%.
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0.93). Overall, 111 samples were positive and 187 were negative by both assays, while 9 and 10, respectively, were positive only by HC-II and only by PCR-ELISA (Table 1). In comparison with PCR-ELISA, the HC-II assay showed a sensitivity of 91.7% and a specificity of 95.4%. These percentages were higher among routine patients (94.5 and 96.3%) than in followed-up women (83.3 and 93.3%), and the difference in sensitivity between the two groups was significant (PB 0.05). Out of the 121 HPV DNA samples positive by PCR-ELISA, 77 specimens were positive by electrophoresis and typeable by hybridization (ELISA) (type 16 in 34, type 31 in 18, types 16/31 in 3, type 33 in 7, type 45 in 7, type 18 in 4, type 6 in 4) and 44 positive by electrophoresis but non typeable by hybridization (Fig. 1A). Of the 120 positive HC-II specimens, 84 reacted only with the high-risk probes (70.0%) and 6 only with the low-risk probes (5.0%), while in the remaining 30 (25.0%) the infection appeared to be sustained by both HR and LR types of HPV. This could not be confirmed by PCR because viral typing by this method was available only for 5 high-risk and 2 low-risk types, whereas HC-II covers 13 and 5 types, respectively. The relative frequency of HPV DNA positivity for HR, LR or both by HC-II is reported in Fig. 1B.
3.4. Retesting by HC-II
Fig. 1. HPV typing by PCR-ELISA (A) and HC-II (B); relative frequency on HPV-DNA positive cervical specimens. HPV DNA samples not typeable (HPV NT).
Sixty-five specimens were retested by HC-II because of initial discrepancy with PCR (39 samples) or for low RLU/PC values (26 samples). The initial result was reproducible in 40 (61.5%) of cases (Table 2), and the percentage of reproducible results was significantly higher (88.5 vs. 43.6%; PB 0.01) among samples with low RLU/PC values and concordance with PCR-ELISA. The mean RLU/PC ratio for non confirmed specimens was 2 (range: 1.03–5.02). A low-level positivity for HR types was quite reproducible (70.0% of repeated reactivity). Conversely, the concurrent presence of HR and LR types in the same specimen with a loss signal was unreliable,
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Table 2 Repeat testing by HC-II HPV on 65 specimens yielding discordant results with PCR-ELISA or a low RLU/PC ratio value (B5) Reason for repeat testing
Number of tested samples
Number of confirmed results
% of confirmed results
Discordance with PCR-ELISA Low RLU/PC ratio value (B5) but concordant with PCR-ELISA Total
39 26
17 23
43.6 88.5
65
40
61.5
The difference in the percentage of confirmed results between the two groups was highly significant (PB0.01). Table 3 Cytology results (Bethesda system) and HPV DNA detection by PCR-ELISA and HC-II in 317 Pap smear samples Pap smear result Normal (routine) Normal (FU) ASCUS LSIL HSIL Total
Number of specimens 94 74 36 42 71 317
Positive by HC-II and PCR-ELISA (%) 17 (18.1) 18 7 20 49 111
(24.3) (19.4) (47.6) (69.0) (35.0)
PCR-ELISA positive/HC-II HC-II positive/PCR-ELISA negtive (%) negative (%) 1 (1.1) 4 1 0 4 10
(5.4) (0.3) (–) (5.6) (3.2)
2 (2.1) 4 1 1 1 9
(5.4) (0.3) (2.4) (1.4) (2.8)
FU, follow-up specimens after conization.
since none of the 13 samples showing this pattern was confirmed upon retesting. The finding of low level reacting for HR types by HC-II was not related to specific types as identified by PCRELISA.
3.5. HPV positi6ity and cytology results The number of samples positive for HPV DNA according to cytology classes is summarized in Table 3, while the positivity rates are indicated in Fig. 2. HPV positivity showed a linear trend, as it increased from 21.3% in samples with no abnormality to 76.1% in HSIL specimens. The correlation between HPV positivity and cytological grading was statistically significant (P B0.005). Histological data were available for 120 patients: a CIN 2–3 was evidenced in 77 of them (64.2%), and 59 of the 77 (76.6%) were positive for HPV DNA on the cytological sample. It is of note that a CIN 3 was found in five HPV-positive routine samples with either a normal cytology (three cases) or an ASCUS (two cases).
4. Discussion HPV testing is mostly required as a triage strategy after cytological screening for cervical cancer, and in some instances a limited experience of combined screening by Pap smear and HPV testing has been proposed. In Italy, following the recent set up of regional screening programs for cervical cancer screening, an increasing workload for the reference laboratories as well as for territory-based hospitals is envisaged. Assay standardization has become an issue, in view of the utilization of HPV DNA results in patient management and frequent referral of patients to different clinical settings from the ones in which initial visits and testing are made. From a laboratory standpoint, the suitability of performing both molecular assays from the same sample preparation material is very important and can provide a valid tool for the physician to set up the algorithm for appropriate management. In our experience, the results obtained by the second version of the Hybrid Capture direct hybridiza-
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tion assay were very similar to those given by a well-established PCR system, although HC-II is not as sensitive as the PCR-ELISA we have developed and validated. An overall agreement with PCR results in 94.0% of cases was seen: this is in agreement with the findings of Peyton et al. (1998), who observed a substantial identity of results both on scrapes and tissue specimens. An extensive comparison of HC-II and PCR has also been reported in the ALTS study (Koutsky et al., 2000) on more than 200 women with LSIL, with a 92.0% concordance: similar results have been reported both on cytological and histological samples by several authors (Clavel et al., 1998; Cuzick et al., 1999). It is of note that the category of patients in which the lowest concordance observed was among women with normal cytology tested during a follow-up after conization for CIN 3: 8 out of 74 samples (10.8%) were positive only by PCR (4) or HC-II (4), whereas the discordance rates among women with normal cytology during a routine screening and in patients with abnormal cytology results were 3.2 and 5.4% (P = 0.048 and not significant, respectively). Moreover, the positivity rate for HPV DNA was higher in the follow-up group with normal cytology (35.1%) than among women of the routine screening group with a normal Pap test result (21.3%). This is probably due to the persistence of the infection in
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the first months after the surgical intervention in the former group, and a decreasing viral load may at least in part explain the higher discordance in follow-up samples, because as fewer sites of viral infection remain, the probability of finding a residual HPV infection in the exfoliated cells is reduced. HPV testing as a follow-up strategy after conization has in fact been recommended, since the persistence of HR HPV for more than 1 year has been found to bear an increased risk for the new development of CIN 3, diagnosed in 45.0% of women with those results (Nagai et al., 2000). Thus, in follow-up testing HPV positivity could be assessed either by a combination of the two assays or by HC-II alone followed by repeat testing. This approach will guarantee the sensitivity that is needed if viral clearance is set as the outcome measure of surgical intervention. In many previous studies HPV positivity has shown a strong correlation with the degree of abnormal cytological results (Clavel et al., 1999; Cox et al., 1995; Ferris et al., 1998; Manos et al., 1999; Nobbenhuis et al., 1999), and also with histological severity. Our results are in accordance with these observations, although it is noteworthy that in our experience the HPV prevalence in women with normal cytological smear (21.3%) was higher than previously reported (Cox et al.,
Fig. 2. HPV DNA prevalence according to cytological Pap smear results on 317 cervical samples. HPV DNA positivity was established based on the concordance of HC-II and PCR-ELISA results or on repeat positivity by either of the two methods.
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1995; Schiffman et al., 2000). We have no definite explanation for this finding. The women in our study were not particularly young, with a median age of 38 years and only 5% aged less than 30 years, but 28 of the 94 women with normal cytology (29.8%) were positive for HIV-1, and HIV infection is correlated with high HPV positivity rates and HPV persistence (Myers et al., 2000). Since the performance characteristics of the two assays are very similar but the complexity is different, PCR-ELISA being more demanding, the optimal strategy for routine HPV DNA testing might be to take a single cytological specimen during Pap smear testing (either conventional or liquid-based) that is than divided into two aliquots. Initial testing should be done by Hybrid Capture II, which is less time-consuming and suited for testing batches of different sizes. Specimens that are positive by HC-II can then be further assayed by PCR, if typing of prevalent HR HPVs is needed. PCR testing can also be centralized in specialty labs, to which relevant aliquots may be shipped.
Acknowledgements This work was partially supported by MURST, Target Project on ‘Biotechnology’ CNR and the University of Bologna Funds for Selected Research Topics.
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Human papillomavirus DNA testing by PCR-ELISA and hybrid capture II from a single cytological specimen: concordance and correlation with cytological results Simona Venturoli a, Monica Cricca a, Francesca Bonvicini a, Francesco Giosa b, Francesco Renato Pulvirenti c, Claudio Galli c, Monica Musiani a, Marialuisa Zerbini a,* a
Department of Clinical and Experimental Medicine, Section of Microbiology, Uni6ersity of Bologna, Policlinico S. Orsola, 6ia Massarenti 9, 40138 Bologna, Italy b Department of Oncology and Hemathology, S. Orsola Hospital, Bologna, Italy c Scientific Affairs, Abbott Di6isione Diagnostici, Rome, Italy Received 23 October 2001; accepted 20 January 2002
Abstract Background and objecti6es: A persistent infection by high-risk HPV is now considered as the major cause of cervical carcinoma. The use of a single cytological specimen for HPV DNA testing by two different molecular methods was analyzed and validated. Study design: HPV DNA testing by PCR-ELISA and hybrid capture II HPV test (HC-II), was investigated on 317 cytological samples obtained from Italian women. Two hundred twenty-seven women were referred to virological lab for HPV DNA testing during cytological routine screening and 90 during a cytological and virological follow-up after a conization or hysterectomy. Results: Overall, the concordance between the two assays was high (K =0.87). Compared with PCR-ELISA, the HC-II showed a sensitivity of 91.7% and a specificity of 95.4%. Although the analytical sensitivity of the PCR-ELISA was higher, the performance of the two tests did not differ in recognizing HPV DNA positive patients with either low or high-grade squamous intraepithelial lesions (LSIL or HSIL). HPV DNA positivity was directly correlated with the severity of cytological diagnosis (PB 0.005). Conclusions: In view of the comparable results obtained with the two assays and of the ease of use, and higher throughput of HC-II, it seems advisable, with a single cytological specimen, to employ the HC-II test as a first-line assay, either for screening or diagnosis, and to perform reflex PCR on positive samples, if typing of prevalent high risk HPVs is needed. © 2002 Elsevier Science B.V. All rights reserved. Keywords: HPV DNA; HC-II; PCR
1. Introduction * Corresponding author. Tel.: +39-051-341-652; fax: + 39051-341-632. E-mail address: [email protected] (M. Zerbini).
A persistent infection by high-risk types of human papillomavirus (HR-HPV) is now considered
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as the major cause of cervical carcinoma (Myers et al., 2000; Walboomers et al., 1999; Wallin et al., 1999; Ylitalo et al., 2000). While HPV infections are most common in younger women, among whom prevalences as high as 30– 50% have been reported, the persistence of both low- and high-risk types for more than 1 year is infrequent (Franco et al., 1999; Ho et al., 1998; Myers et al., 2000). The high positivity rates and the short duration of infection have so far hampered the implementation of screening programs based on HPV DNA testing, due to the huge number of infected women who will not develop HPV-related pathologies. However, since the sensitivity of Pap smear testing for detecting high-grade lesions is not higher than 80– 85% even under the best conditions (Clavel et al., 1999; Cuzick et al., 2000; Schiffman et al., 2000), some recent studies have suggested that HPV DNA screening in association with Pap testing may substantially reduce the rate of false negatives, i.e. patients with a normal cytological smear and high-grade cervical intraepithelial neoplasia (CIN) in the cervix (Clavel et al., 1999; Poljack et al., 1999). On the other hand, HPV testing has been proven both useful and cost-effective in the evaluation of borderline cytological results (Cox et al., 1995; Ferris et al., 1998; Manos et al., 1999). The major obstacle towards the adoption of HPV DNA testing in clinical routine has for many years been the lack of standardized, easy-to-use assays. The majority of clinical studies have been carried out by molecular biology assays based on polymerase chain reactions (PCR). Such methods are highly sensitive but may suffer from inhibition by substances contained either in the sample or in the transport buffer, causing false-negative results, and require trained and skilled personnel and dedicated laboratory equipment in order to avoid contamination and false-positive results. In recent years, the availability of a direct hybridization test with chemiluminescent signal amplification (HC) has provided a simple and attractive alternative to the more complex PCR assays. This method has been employed on a variety of clinical samples (Peyton et al., 1998) and does not require the stringent contamination
control of target amplification procedures. The aim of the study here described was: (i) to establish if both molecular methods may be run starting from a single sample obtained either with different tools for cervical scrapes or transport media and (ii) to compare the efficiency of the newest version of the HC assay (HC-II) (Ferris et al., 1998; Poljack et al., 1999) with a PCR method (PCR-ELISA) that had previously been described and validated (Venturoli et al., 1998; Zerbini et al., 2001). Additionally, we looked for a correlation between the two HPV DNA tests and Pap test results and for the best possible utilization of both molecular techniques in clinical microbiology practice.
2. Materials and methods
2.1. Clinical specimens HPV DNA testing was performed by PCRELISA and HC-II. Cytological specimens for PCR-ELISA were collected by Dracon-tipped swab and suspended in 10 ml of phosphate buffered saline solution (PBS). Cytological specimens for HC-II were obtained by cytobrush and dipped in 1 ml of HC-II transport medium (Digene Cervical Sampler). In order to evaluate the suitability of PCRELISA and HC-II for HPV DNA testing using different tools for cervical scrapes and transport media, two successive cytological specimens were obtained from the ecto-endocervix and posterior fornix of each of 24 consecutive women enrolled for a routine Pap smear testing. PCR-ELISA was performed on cervical cells suspended in PBS and aliquoted, and on cytological specimens collected in HC-II transport medium. The latter were analyzed both directly and after removal of the transport medium by centrifugation and resuspension in PBS. HC-II was used both directly on specimens collected in HC-II transport medium and on specimens collected in PBS, centrifuged and resuspended in HC-II transport medium. After the preliminary analysis to examine different cervical specimen collection methods, the
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24 samples used for method validation and 293 further cervical specimens, all collected by cytobrush in HC-II transport medium, were analyzed for HPV DNA detection by HC-II and the results compared with the data obtained by PCR-ELISA. The statistical analysis was performed on all 317 cervical specimens. Informed consent for the study was obtained from all the 317 women enrolled in the study (mean age 38.9 years, median 38 years, range 17 to 74 years), 227 of whom had been referred to the virological lab for HPV DNA testing during a routine cytological screening, while 90 were tested during a cytological and virological follow-up after conization (85 cases) or hysterectomy (five cases) for a previously CIN or cervical cancer. The cytological analysis of the 317 cervical specimens was done by Pap test performed by staff physicians at the Department of Oncology and Hemathology, S. Orsola Hospital, Bologna (Italy) using ectocervical and endocervical specimens. Specimens for Pap test were evaluated by the standard practices and results were accessed from cytology records. Cytological atypias, according to the Bethesda system, consisted of: atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesions (LSIL) and high grade SIL (HSIL). Experienced staff physicians performed colposcopy with biopsy. Examinations were conducted without knowledge of patients’ Pap or HPV results. In the histological diagnosis cervical lesions were grouped as negative, cervical intraepithelial neoplasias (CIN 1, 2/3) and invasive carcinoma (IC).
2.2. PCR-ELISA for HPV DNA 2.2.1. Sample preparation Cervical specimens collected in PBS were centrifuged; aliquots of 100 000 cells from each cytological specimen were protease digested by incubation at 55 °C for 2 h in 200 ml of digestion buffer (50 mM KCl, 10 mM Tris– HCl pH 8.3, 0.05% Tween 20 and 400 mg/ml of Proteinase K), followed by heat inactivation at 95 °C for 10 min. Cervical cells collected by HC-II transport medium were counted and a volume containing
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100 000 cells was protease digested. Alternatively, an equal volume of the same sample was centrifuged and 100 000 cells were resuspended in 200 ml of digestion buffer and processed as above. A 10 ml volume of each digested sample was then processed by PCR-ELISA. DNA preparation of control samples was performed as previously described (Venturoli et al., 1998).
2.2.2. PCR-ELISA method The consensus primer pair MY09 and MY11 (Resnick et al., 1990) was used in PCR amplification. Oligonucleotide probes specific for HPVs 6, 11, 16, 18, 31, 33, 45 were separately utilized in different hybridization reactions. As amplification control, primers GH20 and PC04 (Resnick et al., 1990) recognizing ß globin gene sequence, were used in each PCR reaction and amplified products were detected by PC03 probe in a separate hybridization reaction. All the hybridization probes used in the PCR-ELISA were 5% labeled with biotin. DNA amplification and digoxigenin-labeling was performed in 50 ml of PCR reaction volume containing 10 ml of each digested sample; amplified products were detected by a hybrid capture-immunoassay in microtiter wells (Venturoli et al., 1998). Specificity and sensitivity controls and the cutoff of the PCR-ELISA method were performed as previously described (Venturoli et al., 1998). Results were expressed as net absorbance after the subtraction of absorbance of the buffer blank and index value was calculated as OD sample/OD cut-off; an index value \1 was considered as positive. 2.2.3. Agarose gel electrophoresis All the amplified samples were also analyzed by agarose gel electrophoresis. Ten microliters of amplified products were visualized through 2% agarose gel with ethidium bromide staining. 2.3. Hybrid Capture II HPV Cervical specimens in HC-II transport medium were processed by the commercially available HCII System (Digene). The Digene Hybrid Capture II HPV is a signal amplification method based on
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liquid hybridization of HPV DNA with two different sets of RNA probes aimed at the recognition of 13 high risk (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and 5 low risk (6, 11, 42, 43 and 44) HPV types, respectively, followed by capture of DNA/RNA hybrids on a solid phase by monoclonal antibodies and detection by enhanced chemiluminescence. The results are expressed by comparing the relative light units (RLU) from clinical samples with a positive control (PC): a RLU/PC ratio of 1 or higher is considered as a positive result. This assay has a nominal sensitivity of 0.2 –1 pg/ml, equivalent to 1000–5000 genome copies of HPV.
2.4. HPV DNA testing algorithm Each sample was initially tested by both molecular assays. Samples yielding discrepant results were retested by both PCR-ELISA and HC-II. The specimens that resulted as high negative (0.8– 0.99 RLU/PC ratio) or weak positive (1– 5 RLU/PC ratio) by HC-II were retested by this method, regardless of PCR-ELISA results, in order to check the assay reproducibility around the cut-off. Only repeat testing results were considered for assay comparison and HPV prevalence.
2.5. Statistical analysis Sensitivity and specificity were assessed by 2× 2 contingency tables; chi-square ( 2) or Fisher’s exact test and chi-square for linear trend were performed in order to evaluate the percentage differences between the two assays and to state the significance of different HPV DNA positivity rates among classes of Pap test results, respectively. Kappa coefficient with 95% confidence limits was also performed to evaluate the correlation between PCR-ELISA and HC-II assays.
3. Results
3.1. Methods 6alidation The validation of PCR-ELISA and HC-II on samples stored in the transport buffer specific for
the other method was carried out on 24 samples. All the samples collected by the Digene system and processed by PCR-ELISA without removing the transport medium gave a negative result: this was due to PCR inhibition, since beta-globin amplification, the internal amplification control (IC), was negative as well. Centrifugation and PCR testing of the pellets resuspended in PBS buffer guaranteed an efficient removal of inhibitors: all samples became positive for the IC and gave a 100% concordance (12 positives, 12 negatives) with the PCR-ELISA performed on the duplicate specimens collected in PBS. The HC-II method did not show any influence from different transport media, as a 100% concordance was observed on duplicate specimens with similar RLU/PC values on positive specimens (range: − 46 to + 73%). Sample analysis performed by gel electrophoresis was concordant with results obtained by PCR-ELISA. The endpoint analytical sensitivity of the two molecular assays was checked by analyzing serial dilutions of a lysate of HeLa cells containing the HPV 18 DNA genome. The number of HPV genome copies presents in HeLa cell lysate was determined in comparison with serial dilutions, from 106 to 100 genome copies, of plasmid DNA containing L1 HPV region (Amplimedical SpA, Divisione Bioline, Italy). The HCII assay was able to detect about 1000 genome copies, as compared with the 50–100 genome copies detected by PCR-ELISA.
3.2. Population groups and cytology results Out of the 317 women enrolled in the study, 227 were referred to the virological lab for HPV DNA testing during routine cytological screening, while 90 were tested during a cytological and virological follow-up after a conization (85 cases) or hysterectomy (five cases) for a previously diagnosed CIN or cervical cancer. In the screening population almost half of the Pap smears (94/227: 41.4%) were classified as normal, 30 (13.2%) ASCUS, 40 (17.6%) LSIL and 63 (27.8%) HSIL. Conversely, followedup women had a normal cytology in 74/90 cases (82.2%), 6 were ASCUS (6.7%), 2 LSIL (2.2%) and 8 HSIL (8.9%). The median ages were 38 and 39 years in the two groups and 38 years overall: median ages and ranges among groups with differ-
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ent cytological results were, respectively, 39 years (22–74) for normal smears, 37 years (28– 72) for ASCUS, 37 years (19– 59) for LSIL and 37 years (17–65) for HSIL.
3.3. HPV DNA test results and comparison The comparative analysis of the 317 samples after repeat testing of discordants showed a concordance of 94.0% (298/317) between PCRELISA and HC-II (K =0.87; c.i. 95%= 0.82 – Table 1 Comparison between PCR-ELISA and HC-II on 317 samples HPV-DNA test
PCR-ELISA +
PCR-ELISA −
HC-II + HC-II −
111 10
9 187
The overall concordance was 94.0% (k= 0.87) and HC-II sensitivity and specificity vs. PCR-ELISA were 91.7 and 95.4%.
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0.93). Overall, 111 samples were positive and 187 were negative by both assays, while 9 and 10, respectively, were positive only by HC-II and only by PCR-ELISA (Table 1). In comparison with PCR-ELISA, the HC-II assay showed a sensitivity of 91.7% and a specificity of 95.4%. These percentages were higher among routine patients (94.5 and 96.3%) than in followed-up women (83.3 and 93.3%), and the difference in sensitivity between the two groups was significant (PB 0.05). Out of the 121 HPV DNA samples positive by PCR-ELISA, 77 specimens were positive by electrophoresis and typeable by hybridization (ELISA) (type 16 in 34, type 31 in 18, types 16/31 in 3, type 33 in 7, type 45 in 7, type 18 in 4, type 6 in 4) and 44 positive by electrophoresis but non typeable by hybridization (Fig. 1A). Of the 120 positive HC-II specimens, 84 reacted only with the high-risk probes (70.0%) and 6 only with the low-risk probes (5.0%), while in the remaining 30 (25.0%) the infection appeared to be sustained by both HR and LR types of HPV. This could not be confirmed by PCR because viral typing by this method was available only for 5 high-risk and 2 low-risk types, whereas HC-II covers 13 and 5 types, respectively. The relative frequency of HPV DNA positivity for HR, LR or both by HC-II is reported in Fig. 1B.
3.4. Retesting by HC-II
Fig. 1. HPV typing by PCR-ELISA (A) and HC-II (B); relative frequency on HPV-DNA positive cervical specimens. HPV DNA samples not typeable (HPV NT).
Sixty-five specimens were retested by HC-II because of initial discrepancy with PCR (39 samples) or for low RLU/PC values (26 samples). The initial result was reproducible in 40 (61.5%) of cases (Table 2), and the percentage of reproducible results was significantly higher (88.5 vs. 43.6%; PB 0.01) among samples with low RLU/PC values and concordance with PCR-ELISA. The mean RLU/PC ratio for non confirmed specimens was 2 (range: 1.03–5.02). A low-level positivity for HR types was quite reproducible (70.0% of repeated reactivity). Conversely, the concurrent presence of HR and LR types in the same specimen with a loss signal was unreliable,
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Table 2 Repeat testing by HC-II HPV on 65 specimens yielding discordant results with PCR-ELISA or a low RLU/PC ratio value (B5) Reason for repeat testing
Number of tested samples
Number of confirmed results
% of confirmed results
Discordance with PCR-ELISA Low RLU/PC ratio value (B5) but concordant with PCR-ELISA Total
39 26
17 23
43.6 88.5
65
40
61.5
The difference in the percentage of confirmed results between the two groups was highly significant (PB0.01). Table 3 Cytology results (Bethesda system) and HPV DNA detection by PCR-ELISA and HC-II in 317 Pap smear samples Pap smear result Normal (routine) Normal (FU) ASCUS LSIL HSIL Total
Number of specimens 94 74 36 42 71 317
Positive by HC-II and PCR-ELISA (%) 17 (18.1) 18 7 20 49 111
(24.3) (19.4) (47.6) (69.0) (35.0)
PCR-ELISA positive/HC-II HC-II positive/PCR-ELISA negtive (%) negative (%) 1 (1.1) 4 1 0 4 10
(5.4) (0.3) (–) (5.6) (3.2)
2 (2.1) 4 1 1 1 9
(5.4) (0.3) (2.4) (1.4) (2.8)
FU, follow-up specimens after conization.
since none of the 13 samples showing this pattern was confirmed upon retesting. The finding of low level reacting for HR types by HC-II was not related to specific types as identified by PCRELISA.
3.5. HPV positi6ity and cytology results The number of samples positive for HPV DNA according to cytology classes is summarized in Table 3, while the positivity rates are indicated in Fig. 2. HPV positivity showed a linear trend, as it increased from 21.3% in samples with no abnormality to 76.1% in HSIL specimens. The correlation between HPV positivity and cytological grading was statistically significant (P B0.005). Histological data were available for 120 patients: a CIN 2–3 was evidenced in 77 of them (64.2%), and 59 of the 77 (76.6%) were positive for HPV DNA on the cytological sample. It is of note that a CIN 3 was found in five HPV-positive routine samples with either a normal cytology (three cases) or an ASCUS (two cases).
4. Discussion HPV testing is mostly required as a triage strategy after cytological screening for cervical cancer, and in some instances a limited experience of combined screening by Pap smear and HPV testing has been proposed. In Italy, following the recent set up of regional screening programs for cervical cancer screening, an increasing workload for the reference laboratories as well as for territory-based hospitals is envisaged. Assay standardization has become an issue, in view of the utilization of HPV DNA results in patient management and frequent referral of patients to different clinical settings from the ones in which initial visits and testing are made. From a laboratory standpoint, the suitability of performing both molecular assays from the same sample preparation material is very important and can provide a valid tool for the physician to set up the algorithm for appropriate management. In our experience, the results obtained by the second version of the Hybrid Capture direct hybridiza-
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tion assay were very similar to those given by a well-established PCR system, although HC-II is not as sensitive as the PCR-ELISA we have developed and validated. An overall agreement with PCR results in 94.0% of cases was seen: this is in agreement with the findings of Peyton et al. (1998), who observed a substantial identity of results both on scrapes and tissue specimens. An extensive comparison of HC-II and PCR has also been reported in the ALTS study (Koutsky et al., 2000) on more than 200 women with LSIL, with a 92.0% concordance: similar results have been reported both on cytological and histological samples by several authors (Clavel et al., 1998; Cuzick et al., 1999). It is of note that the category of patients in which the lowest concordance observed was among women with normal cytology tested during a follow-up after conization for CIN 3: 8 out of 74 samples (10.8%) were positive only by PCR (4) or HC-II (4), whereas the discordance rates among women with normal cytology during a routine screening and in patients with abnormal cytology results were 3.2 and 5.4% (P = 0.048 and not significant, respectively). Moreover, the positivity rate for HPV DNA was higher in the follow-up group with normal cytology (35.1%) than among women of the routine screening group with a normal Pap test result (21.3%). This is probably due to the persistence of the infection in
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the first months after the surgical intervention in the former group, and a decreasing viral load may at least in part explain the higher discordance in follow-up samples, because as fewer sites of viral infection remain, the probability of finding a residual HPV infection in the exfoliated cells is reduced. HPV testing as a follow-up strategy after conization has in fact been recommended, since the persistence of HR HPV for more than 1 year has been found to bear an increased risk for the new development of CIN 3, diagnosed in 45.0% of women with those results (Nagai et al., 2000). Thus, in follow-up testing HPV positivity could be assessed either by a combination of the two assays or by HC-II alone followed by repeat testing. This approach will guarantee the sensitivity that is needed if viral clearance is set as the outcome measure of surgical intervention. In many previous studies HPV positivity has shown a strong correlation with the degree of abnormal cytological results (Clavel et al., 1999; Cox et al., 1995; Ferris et al., 1998; Manos et al., 1999; Nobbenhuis et al., 1999), and also with histological severity. Our results are in accordance with these observations, although it is noteworthy that in our experience the HPV prevalence in women with normal cytological smear (21.3%) was higher than previously reported (Cox et al.,
Fig. 2. HPV DNA prevalence according to cytological Pap smear results on 317 cervical samples. HPV DNA positivity was established based on the concordance of HC-II and PCR-ELISA results or on repeat positivity by either of the two methods.
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1995; Schiffman et al., 2000). We have no definite explanation for this finding. The women in our study were not particularly young, with a median age of 38 years and only 5% aged less than 30 years, but 28 of the 94 women with normal cytology (29.8%) were positive for HIV-1, and HIV infection is correlated with high HPV positivity rates and HPV persistence (Myers et al., 2000). Since the performance characteristics of the two assays are very similar but the complexity is different, PCR-ELISA being more demanding, the optimal strategy for routine HPV DNA testing might be to take a single cytological specimen during Pap smear testing (either conventional or liquid-based) that is than divided into two aliquots. Initial testing should be done by Hybrid Capture II, which is less time-consuming and suited for testing batches of different sizes. Specimens that are positive by HC-II can then be further assayed by PCR, if typing of prevalent HR HPVs is needed. PCR testing can also be centralized in specialty labs, to which relevant aliquots may be shipped.
Acknowledgements This work was partially supported by MURST, Target Project on ‘Biotechnology’ CNR and the University of Bologna Funds for Selected Research Topics.
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