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JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 1998, p. 3248–3254 0095-1137/98/$04.
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tibodies. Immobilized hybrids are reacted with a monoclonal antibody reagent that is conjugated to alkaline phosphatase, and the complexes are detected via a chemiluminescent substrate reaction. In HCT, a tube luminometer is employed, whereas in HC II, a microplate luminometer reads the light output and displays the assay results as relative light units (RLU). HPV positivity or negativity is based on comparison to a standard positive reference (RLU of a clinical specimen divided by the mean RLU of three positive calibrator references). The HCT Probe B cocktail detects a limited number of high-risk HPV types, including HPVs 16, 18, 31, 33, 35, 45, 51, 52, and 56, and has been reported to have a diagnostic sensitivity similar to that of the Papanicolaou (Pap) smear (6). HC II detects HPV types at an increased sensitivity compared to that of HCT (23). In addition, probes for HPV types 39, 58, 59, and 68 have been added to the HC II Probe B group. One potential advantage of this test is that it may provide a semiquantitative measure of viral load (8, 9, 11, 16, 23, 31, 32). It has been suggested that viral load may lend prognostic and diagnostic value (13, 20). To date, investigators in most reported HCT-based studies have used a Dacron swab to collect cervical cells into 1.0 ml of Specimen Transport Medium (STM; Digene Corporation) (31, 34, 35). Recently, a conical cytology brush has been used to collect cervical cells. This modiﬁcation was made with the expectation that the brush would increase the number of cells obtained and thus provide a specimen with an increased concentration of HPV.
Aliquots for PCR testing were taken prior to HC II analyses of these specimens. Arlington Heights. The membranes were hybridized at 53°C overnight with biotinylated HPV type-speciﬁc oligonucleotide probes for HPVs 6 or 11. 45. Calif. 0. 44. and a widely used PCR assay based on the MY09-MY11HMB01 primer set. MM9 (also called P238a). followed by a second 2-h or overnight exposure. 11. HPV testing of specimens in PC has recently been approved by the U. Five microliters of each specimen was ampliﬁed per 100 l of ampliﬁcation reaction mixture with the MY09-MY11-HMB01 L1 consensus primers and buffer systems (1. until processing. Specimens collected in PC and STM were stored at ambient temperature and 20°C. MM4 (W13B). Unimar. An advantage of using a liquid cytology medium for the collection of cervical specimens is that multiple diagnostic tests can be performed with a single sample. Approximately half of the 208 women had a second cervical specimen collected with a Dacron swab and placed into 1. 73. 58. Amplimers were analyzed by ethidium bromide-stained agarose gel electrophoresis. 26.) thinlayer slides are produced. MM4 (also called W13B).1% Laureth-12) and digested at 56°C for 1 h. Blots were exposed to Kodak X-Omat AR 5 ﬁlm for an initial 10 min. IS39. 16. 51. 59. Immobilized probes were detected with streptavidin-horseradish peroxidase (Vector. has been introduced. The pellet was resuspended in 150 l of digestion solution (10 mM Tris.) into 0. 73 (P238A). Ill. 1 mM EDTA. The resulting data were analyzed manually by two observers and recorded on a database spreadsheet. As HPV test formats and specimen collection devices have continued to evolve. 26.) for the Pap smear.) was used for all reactions.5-ml Eppendorf tube and centrifuging it for 10 min at 13. 34. a 55°C annealing for 1 min. 25). Conn. These three methods are under consideration as HPV sampling strategies for routine clinical use. 39. The remaining women had a second cervical specimen collected with a conical brush (Medical Packaging Corporation. CP6108. AmpliTaq Gold polymerase (Perkin-Elmer. MM7 (P291). 68. Calif. HPV detection in these specimens was evaluated by comparing the ﬁrst-generation HCT.000 g. Camarillo. the GH20-PC04 human consensus -globin primer system was coampliﬁed with the HPV consensus primers. 55. PCR and dot blot-based HPV testing methods. Wilton. Food and Drug Administration. 64. The selected L1 consensus primer PCR-based method detects a broad spectrum of anogenital HPV types that include. 40. 53. To determine specimen adequacy. 52. 61. MM7 (also called P291).). a new technique that employs the collection of cervical cells into PreservCyt (PC) liquid cytology medium. 200 g of proteinase K per ml. Recently. The laboratory was unaware of the clinical statuses and HCT and HC II results of the study subjects. 53.).175 women conducted in Costa Rica (17). 66. and CP8304 (1–3. 33. membranes were washed at 56 to 57°C to remove nonspeciﬁcally bound probe. 14. 33).S. The cell pellet was dried overnight at room temperature. 18. and a 72°C terminal extension for 1 min. the prototype second-generation HC II.5 l per well) with dot blot apparatuses (Bio-Rad Laboratories. Cervical diagnoses were virtually all within normal limits but included a small number of low-grade abnormalities (n 10). 35. and MM8 (P155). Crude DNA extracts were stored at 20°C until ampliﬁcation. 55. The digestion was followed by a 15-min incubation at 95°C to inactivate the proteinase K. Two hundred eight specimens were selected from specimens collected in a prospective population-based natural-history study of 9. Burlingame. HPV testing can be performed on the temporarily stored Pap smear specimen without the cost of a clinical follow-up visit. These two collection methods were alternated between consecutive patients. PCRs were conducted in a Perkin-Elmer model 9600 thermocycler with the following ampliﬁcation proﬁle: an initial 95°C AmpliTaq Gold activation for 9 min.S. 31. Boxborough. 68. All specimens positive for PCR by gel electrophoresis that did not bind to any of the HPV type-speciﬁc oligonucleotide probes were subjected to restriction . 56. new liquid specimen collection media have also become available for routine use in Pap smear screening. MM8 (also called P155). This cohort consisted of women with past histories or no history of squamous intraepithelial lesions. 57. The supernatant was immediately removed and discarded with a plugged Pasteur pipette. 40 cycles of a 95°C denaturation for 1 min. Calif.5 l) were applied as HPV type-speciﬁc controls. Food and Drug Administration (22. 66. 57. The median age of these women was 37 years. residual cells on each broom device were placed into 20 ml of PC liquid cytology medium (Cytyc). 25). Flowchart illustrating the cervical specimen collection and HPV testing strategies. This method has received approval for clinical use from the U. For a cervical diagnosis within the atypical squamous cells of undetermined signiﬁcance category. 52. 62. 33. 70. 35. the crude digests were allowed to reach room temperature and centrifuged brieﬂy. 45. 59. A cervical specimen collected in PC was processed by placing a 1. 42. respectively. 1998 STRATEGIES FOR PCR AND HYBRID CAPTURE HPV TESTING 3249 FIG. Hercules. Following hybridization. CP8061. 18. Following the preparation of a conventional Pap smear.0 ml of STM. Mass. and a 72°C ﬁnal extension for 5 min. Prior to ampliﬁcation. Accordingly. from which ThinPrep (Cytyc. but are not limited to. Initial specimens were collected from all 208 women with a broom device (Cervex Brush. Foster City. Discrepant results were independently arbitrated. Positive and negative PCR controls were included with each ampliﬁcation. 67.VOL. 56. 19. 36. PCR was performed only on the specimens collected in PC. 58. The PCR and dot blot analyses were performed at the University of New Mexico. MATERIALS AND METHODS Study population and collection of specimens. This study compares HPV detection in specimens collected by three different means. A -globin probe was used to assess specimen adequacy. Calif. 51. 54. Probes for HPV types 26 and MM8 and types 40 and 42 were pooled as pairs during hybridization. HPVs 6.) and Enhanced Chemiluminescent Substrate (Amersham. A collection of previously characterized HPV PCR products (3. 54. 39. HPV typing analyses were carried out by dot blot hybridization and with biotinylated HPV type-speciﬁc oligonucleotide probes as previously described (1). PCR products were denatured and applied to replicate nylon membranes (3. 72. 31.6 ml of STM.5-ml aliquot into a 1. Figure 1 summarizes the cervical specimen collection and HPV testing strategies. 1. 16.
2-pg/ml cutoff.0-pg/ml assay cutoff and did not include RNA probes for HPVs 39.7 96.0-pg/ml assay cutoffs. 18.0-pg/ml detection cutoff limits. and 1.0 69.5.67. respectively).2-pg/ml cutoff. 0.2 92.63 (95% CIs 0.0 77. despite randomization. Data analyses. 13.0. 0. 0. A higher HPV positivity was observed with PC specimens from women from whom a second specimen was obtained with a conical brush than with PC specimens from women from whom the second specimen was obtained with a Dacron swab.59 (0.0-pg/ml cutoff. In comparison.0. Initial comparisons of HC II and PCR results included PCR detection of only those HPV types included in the HC II Probe B cocktail.05.59 (95% CIs 0. differences in HPV positivity may actually reﬂect chance differences in the prevalence of HPV among the two groups of women. and 0.69.1. respectively). one-half volume of NaOH-based denaturation reagent was added to each sample.5. HCT used a single ﬁxed 10. Agreement between HC II results obtained from STM and PC specimens was good at the 0. A 2-ml aliquot was removed from each specimen and placed into a microcentrifuge tube and centrifuged at 13. the remainder of the HC II procedure was identical to that applied to the STM specimens. fragment length polymorphism (RFLP) analysis as previously described (3). Cervical specimens collected in STM (0. of specimens HC II assay cutoff (pg/ml) % of specimens that were positive STM HC II PC HC II % Total Agreementa % Positive Kappa value (95% CIs) Collection device(s) Conical brush 105 0.3 93. Agreement was measured by both absolute agreement and Cohen’s kappa statistic ( ).40.78) 0.47 and 0.3 12.5 11. 0.72 (0.5 1.90 (95% CIs 0. 52.5 24.2 13.and 1.53.5% by HC II of specimens in STM at the 1.74) 0.0 pg/ml.34. 56. Proportions were compared with exact P values for the Pearson chi-square test. and the specimens were denatured for 45 min at 65°C. 13.5% by PCR (any HPV type).0-pg/ml assay cutoff.43 and 0.75 (0. HCT versus HC II. and the pellet was resuspended in 50 l of STM. Brieﬂy.2% positive) and the difference was not statistically signiﬁcant.1% by HC II of specimens in PC at the 0. and 68. with a kappa value of 0. and 68.5%.2. The resulting RFLP proﬁles were compared to known HPV type proﬁles.91) 0.1 15.9 13. the decrease in agreement ( 0. and when the test was restricted to the 13 cancer-associated HPV types present in the HC II Probe B cocktail. Dacron swab versus conical brush.0. and 0.2-pg/ml HC II assay cutoffs.46.62.79) for HC II assay cutoff values of 0.2 14. a measure of the agreement between two methods in excess of that due to chance (12). All hybrid capture testing of clinical specimens was performed at Digene Corporation.0. This possible explanation was supported by the results of HC II when it was applied to the PC specimens. All analyses were performed with SAS version 6. However. At the 0. the level of detection of HC II was nearly equivalent to that of the HCT. From this point.5-. MICROBIOL. and tests: 22.5 0.74).00). Hybrid capture-based HPV testing methods.9 13.2 0. and the HPV types were designated.60) was largely attributable to the much greater HPV DNA positivity obtained with the PC specimens (22.3250 PEYTON ET AL.2 0. Statistical signiﬁcance was achieved when the P value of the test was less than 0.61 (0. and 1. RESULTS HPV DNA positivity.03.81) 0. 0.12 software (28).40.2-. 0.99) 0. 58.5 1.0 ml) were processed according to the manufacturer’s HCT or HC II package insert.00) 0. 45. Agreement was actually slightly better when results of HC II from STM specimens were compared to the PCR results from PC specimens (kappa values.85) 0.000 g for 15 min.5 1. In addition.1% in PC versus 13. When HC II was compared to the PCR method with PC specimens.47 (95% CIs 0.04.0% by HC II of specimens in PC at the 1. and 10.0-pg/ml HC II cutoff levels (P 0. The supernatant was carefully aspirated.2-.9% by HC II of specimens in STM at the 0.2-pg/ml HC II assay cutoff. Approximate 95% conﬁdence intervals (CIs) were computed with an asymptotic variance and critical values from the normal distribution. HPV detection was only slightly greater by HC II than by HCT (8.0. the overall HPV DNA positivity was as follows for different media. Results of the HCT and HC II at the 10.2 97.3 19.88 (0. and 150 l was removed for HCT.7 50.79 and 1. % Positive.8 15. HPV DNA detection by HC II was greater for specimens obtained with the conical brush than for specimens obtained with the Dacron swab at 0.5-. Seventy-ﬁve microliters of the denatured specimen was removed for analysis by HC II.3 50.0-.0 94. 1.2-.6 11.5-.0. Since specimens were collected alternately with a conical brush and Dacron swab from consecutive patients. and 7.5.7 22. HC II versus PCR. 59. 0.85 (0. 0. respectively (Table 1). and 0.2 0.91) 0.0 61. 0.0-. Agreement between HC II and PCR for the group of women sampled with a conical brush was greater than for the group sampled with a Dacron swab. and 1. 51. However.0 15. 58.5-. CLIN. Twenty-ﬁve microliters of denaturation reagent was added.0-pg/ml assay cutoffs (Table 1). with kappa values of 0.6 and 1. and 1.9% in STM). Specimens collected into PC were analyzed by HC II as follows.0 0. Percentages of HPV positivity and agreement between HC II results with STM and PC liquid cytology medium specimens No.2 88.0 86. J.0. cutoffs. TABLE 1.2.79 (0.89) Dacron swab 103 Conical brush and Dacron swab 208 a % Total.2% by HCT testing of specimens in STM. percentage of the total number of specimens collected in STM and PC that produced identical results.0 0. the kappa values were 0. and 0. the HPV DNA positivity was 12.71.4 64.0-pg/ml assay cutoff were equivalent for Dacron swab and conical brush specimens and revealed that. Agreement was better for the group of specimens obtained with a conical brush than for those obtained with a Dacron swab at the 1. 33.5 11. The laboratory technicians were unaware of the clinical statuses and HPV PCR results of the study subjects. these differences were not statistically signiﬁcant.60 (0.4 16.0.02.8 81.7 9. and the kappa values were signiﬁcantly different at the 0.2 15.75.32 and 0. the kappa statistics were not significantly different.68 for HC II cutoff levels of 0. HPV positivity by HC II was determined with 0. which was then mixed vigorously and incubated at 65°C for 45 min. percentage of specimens positive in either STM or PC that produced identical results.7 versus 7. 2). The HC II Probe B cocktail contains RNA probes for high-risk HPV types 16. respectively (Fig.62). .1 46.75.7 94. 0. 31. The RFLP results were subsequently conﬁrmed by an independent PCR-based detection method (15).79 and 0.70.0 pg/ml. at this assay cutoff.7 13. 59. The overall HPV DNA positivity was 24. 11.59 (0. 39.1 89.7 50. 35.
52 (0. 36. PCR detected high-risk HPVs in eight specimens that were not detected by HC II in PC specimens. 73.69) 0.6 88. 18.1 15.0 18. 18.0-pg/ml HC II cutoff.43. b c a .. 66.5 24. Only data for the HPV types present in the HC II Probe B cocktail (i. A listing of HPV types detected by L1 consensus PCR and those detected by HC II is shown in Table 3.36. 70.34. 66.0 45.5 91. 11. In addition.73) 0. Overall.8 41. Discrepant results between HC II and PCR were ascertained and conﬁrmed by repeat PCR testing of the PC specimens. agreement decreased substantially.43. 67.0 13. and 73. In the same analysis. These HPV types do not have corresponding probes in the HC II Probe B cocktail. HPVs 16.1 86. CP6108 (27). Agreement between results of HC II and PCR when HPV types other than those in the HC II Probe B cocktail were included HC II cutoff (pg/ml) % of specimens that were positive HC II PCRa % Total Agreementc % Positive Kappa value (95% CIs) Medium HPV types detected by PCR STM HC II Probe B types plus related typesb 0. CP8061.3 44.40.84) 0. 0. 67.2 44. the 13 HPVs in the Probe B group. 67. 0.48 (0.0 0. 0. This study set out to compare different specimen collection methods for HPV testing. 10.67) 0.58.5% of these women. and CP8061 (27) as determined by PCR. HPV types considered genomically related to HC II Probe B types included HPVs 53.56.5 46. HC II identiﬁed one STM specimen with a high-risk HPV type that was not detected by PCR testing. HCT and HC II results for all specimens collected with a conical brush or Dacron swab were combined.3 85.42. 33.VOL.5 1.54 (0.54 (0. percentage of specimens positive by either HC II or PCR that produced identical results. CP6108. When we performed an identical analysis of results of PCR and HC II with STM specimens.5 24.8 92. HC II. 31.1 84. % Total. We compared two TABLE 2.5 61. and 68) are presented. 0.40.84) 0. MM7.39. 21.8 47. 2.9 13.0 0.83) 0. kappa statistics were calculated for HCT and HC II results compared to the PCR results.0 22.56.9 13. Absolute agreement and kappa values decreased slightly when related HPV types were included in the comparison.2 0.9 13. HC II.58 (0.6 44.6 59.56 (0.5 11.0 59.53 (0. 39. 59. HC II detected some infections with HPVs 53.3 18. 66. 35. 51. The results shown in the upper panel were used to derive the values shown in the lower panel. % Positive.6 84. 52. 0.62) 0.5 1. 54.2 0.38.71 (0.6 86. 0.1 15.3 18.5 13. 0.2 0.5 22. and 73. HC II detected four samples positive for high-risk HPV types that were not detected by the PCR test. 0.2 0. HC II and PCR results with PC liquid cytology medium specimens are also shown. and MM8). The upper panel shows the percentages of specimens positive by HCT.5 1.68) 0. 62.5 1.58 (0. DISCUSSION FIG. 0. 26).68) 0. 45.5 11. 32.3 92. It has been proposed that HPV DNA testing might be used as an adjunct in the management of women with minor cytological abnormalities (8. 0. it was important to compare results of HPV testing of specimens in PC liquid cytology medium by wellestablished HPV testing methods and to further characterize the newly available HC II HPV method.0 0.9 13. Agreement between HPV test results of HCT. HPVs 53.70 (0. percentage of the total number of specimens tested by HC II and PCR that produced identical results.0 88.51 (0. In this study. 54. especially with STM samples.67) 0.9 81. and PCR.65) All types PC HC II Probe B types plus related typesb All types The PCR test was done only on PC specimens. 58. 56. 11 specimens that were found to contain high-risk HPV types by PCR were detected by HC II. 0.3 24.8 0. 0.72) 0.70 (0.e.. When we used all HPV types detected by PCR (i. and the additional HPVs 6. At the 1. In the lower panel.7 42.9 47. and PCR. 1998 STRATEGIES FOR PCR AND HYBRID CAPTURE HPV TESTING 3251 We performed further analyses of HC II and PCR results in which we considered additional HPV types phylogenetically related to the 13 HPVs included in the HC II Probe B cocktail (Table 2). 70. cervical HPV DNAs were detected by PCR in 24.e.3 84.
5 0.5 0. the comparison to PCR results with STM specimens at 0. 66 58 58. indicating good agreement. of specimens % of total no.5. the apparent difference between these two sampling methods may be due to a chance difference in subjects. not sequence data. In addition.4 0.5 0.and 1.0-pg/ml cutoff) STM HC II (1. and 0.0-. Although collection with a conical brush appeared to result in higher HPV DNA detection by HC II than collection with a Dacron swab. Therefore. As previously reported (23). However. an HC II cutoff of about 1. MM8 62 66 67 70 73 73. when HCT was compared to HC II at the 1.3252 PEYTON ET AL. the kappa value was only fair (0. There is uncertainty as to which HC II cutoff is best in terms of accuracy and clinical utility. which . reﬂecting an approximately 10-fold increase in sensitivity over that of HCT. The conical brush STM collection method may be an improvement over the Dacron swab STM collection method. illustrating the correspondence of the two hybrid capture HPV tests. 0.5 1. The HC II detection cutoff value represents the relative light unit reading above which samples are considered positive.2-pg/ml HC II cutoff was observed only with PC specimens.0-pg/ml cutoff) STM HC II (10. for specimens collected in PC. 53 32 33. the device used in the second specimen collection should not have affected the initial specimen.5 0.69.4 0. 56 52 53 55 56 56. Some of the disagreement between PCR and HC II results may be attributed to the use of separate PC aliquots that were processed and tested at two laboratory sites.5 3. In contrast. MM8 35 51 51.5 75.0 pg/ml was optimal and that the use of a 0. the samples were collected in a random manner. of specimens positive by: PC HC II (1. Two recent studies reported reasonable correlation between PCR and HCT HPV assay results (7.5 0. CP8061 No HPV DNA detected a 1 7 1 1 2 1 1 1 1 3 1 2 3 1 3 1 1 1 1 1 1 1 5 1 1 2 5 1 157 0. Results by HC II were in excellent agreement with results by HCT at the 10.5 0.47).0-pg/ml cutoff) 6 or 11 16 16. 34).5 1. hybrid capture methods (HCT and HC II) and L1 consensus PCR for HPV DNA testing. as expected. MM8 CP6108 CP8061 MM7 MM7. the agreement progressively decreased.2-pg/ml HC II cutoff introduced a measurable level of false positivity.2-pg/ml HC II cutoff. In other words. HPV types detected by L1 consensus PCR and those detected by HCT and HC II J.5 0. Also. there was no obvious difference in the ages of and clinics attended by the two groups of women for Pap smear diagnosis. 73 31 31.0 2.5 0.4 0.0-pg/ml cutoff.4 0.5 1. TABLE 3.5 1. further analyses of results stratiﬁed by STM sampling device demonstrated a potential problem with subject randomization. MICROBIOL.5 0 6 1 1 1 0 0 1 1 2 1 1 1 0 2 1 0 0 0 1 1 0 0 0 1 1 0 0 4 0 6 1 1 1 1 0 1 1 1 1 2 1 0 1 1 0 0 0 1 1 0 2 0 0 0 0 0 1 0 6 1 1 1 0 0 1 1 1 1 0 1 0 1 1 0 0 0 0 1 0 0 0 0 0 0 0 1 0 6 1 1 1 0 0 0 1 1 1 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 Based on dot blot and RFLP analyses of MY09-MY11 PCR amplimers. a 1. It is anticipated that the increased sensitivity may improve the clinical utility of this second-generation HPV hybrid capture test.2 pg/ml resulted in a kappa value of 0. 39.0-pg/ml HC II cutoffs. PCR HPV type(s)a No. However. at the 0.5 1.0 1. This increase in background at the 0. When the subjects were separated into two groups based on STM collection device and analyses were limited to those specimens collected in PC. the HC II data showed a higher prevalence of HPV and correlated signiﬁcantly better with PCR data from the group of women who had an STM sample collected with a conical brush (data not shown).2-pg/ml assay cutoffs.5 2.5 0.4 0. This is suggested by both the increase in overall HPV DNA detection and the higher correlation of HC II data from STM conical brush specimens with HC II and PCR data from PC specimens. The comparison of results of HC II and PCR with PC specimens demonstrated a good correlation at 0. of specimens (n 208) No. the level of agreement may have been higher if both tests were performed with the same aliquot.0-pg/ml cutoff) STM HCT (10. However. CLIN.4 0. Our data suggested that.5 0.5 0. using specimens collected in both STM and PC. neither of these previous studies used PC.0-pg/ml cutoff can routinely be used in HC II. 54 16. Because the PC specimen was collected prior to the STM specimen.0 0.5-.
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Review: Application of cytology and molecular biology in diagnosing premalignant or malignant oral lesions.

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