Swedish women. human papillomavirus infection in ...

Comparison of a one-step and a two-step polymerase chain reaction with degenerate general primers in a population-based study of human papillomavirus infection in young Swedish women. M Evander, K Edlund, E Bodén, A Gustafsson, M Jonsson, R Karlsson, E Rylander and G Wadell J. Clin. Microbiol. 1992, 30(4):987.

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JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1992, p. 987-992 0095-1137/92/040987-06$02.00/0 Copyright ©) 1992, American Society for Microbiology

Vol. 30, No. 4

Comparison of a One-Step and a Two-Step Polymerase Chain Reaction with Degenerate General Primers in a Population-Based Study of Human Papillomavirus Infection in Young Swedish Women MAGNUS EVANDER,1* KARIN EDLUND,' ELISABETH BODEN,2 AKE GUSTAFSSON,l MONICA JONSSON,3 ROGER KARLSSON,3 EVA RYLANDER,2 AND GORAN WADELL' Departments of Virology, 1 Obstetrics and Gynecology, 2 and Family Medicine, 3 University of Umeac, S-901 85 Umea, Sweden Received 15 July 1991/Accepted 26 December 1991

Human papillomaviruses (HPVs) are double-stranded DNA viruses that cause various proliferative diseases in the infected epithelium (18). Over 60 different types have been isolated from human tissue. Different types of HPV are associated with specific lesions. Several types, HPV types 6, 11, 16, 18, 31, 33 to 35, 39, 40, 42 to 45, and 51 to 59, infect the genital tract (4). HPV types 16, 18, 31, and 33 are mainly associated with malignant lesions of the cervix (2, 6, 12, 14), while HPV types 6 and 11 are generally limited to low-grade lesions which rarely progress to malignancy (3, 14). In large cancer-screening programs, about 2 to 3% of Papanicolaou (Pap) smears from essentially asymptomatic women show abnormal cytologies (16). Nearly all squamous cell abnormalities in the Pap smears (koilocytosis, cervical intraepithelial neoplasia type I, higher-grade lesions) appear to be associated with HPV infections (24). Koutsky et al. (11) have estimated that genital tract HPV infections are prevalent in approximately 10% of the men and women in the 15- to 49-year age group in the United States and that a majority of these infections are subclinical. They further suggest that the true prevalence may be significantly higher because of the lower sensitivity of the commonly used HPV detection methods (dot blot, Southern blot, and filter in situ hybridization). During the last few years, studies based on polymerase chain reaction (PCR) technology have been performed in which type-specific as well as general HPV primers have been used. HPV DNA was demonstrated in 5 to 49% of cytologically normal women (1, 17, 25-28). HPV DNA sequences were present in 80 to 100% of patients with

*

cervical cancers and in 60 to 90% of patients with high-grade cervical intraepithelial neoplasia. HPV type 16 was the most frequently present type, but unknown HPVs have been detected by PCR in a portion (maximum of 10 to 15%) of the cases (24). We determined the prevalence of HPV infection in a population-based study of young Swedish women by use of a general primer-based PCR. We also compared the sensitivity of a one-step general primer-based PCR with that of a two-step general primer-based PCR.

MATERLALS AND METHODS

Population and specimen collection. All women aged 19, 21, 23, and 25 years who were inhabitants of a primary health care area in Umea, Sweden, according to the public record, were asked to participate in the study during the period September 1989 to September 1990. Among the women asked to participate, 70 could not be reached. Cervical scrapes from 602 women were taken by the same registered midwife; 590 of the scrapes were analyzed for presence of HPV DNA. At the same time, a second cell smear was taken from 558 of the women for cytological evaluation. Cytology. A total of 558 Pap smears were taken for cytological evaluation. They were evaluated at the Department of Cytology, University Hospital of Umea, and abnormal results were categorized according to the Bethesda system.

DNA preparation. Cervical cells were collected by scraping a cotton-tipped swab over the entire surface of the portio vaginalis. Subsequently, the swab was suspended in a plastic tube with 1.5 ml of STE (0.1 M NaCl, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA) and centrifuged to pellet the cells. The

Corresponding author. 987


The prevalence of human papillomavirus (HPV) infection in cervical cell scrapes from young women was determined by polymerase chain reaction (PCR) by using general primer pairs localized within the Li region. With a one-step general PCR, 5.9%Yo (35 of 590) of young women in a population-based study were found to contain HPV DNA. The proportion of HPV-positive women increased with age, from 1.4% (1 of 69) among women aged 19 years to 9.2% (13 of 142) among women aged 25 years. Among the cervical scrapes from women with normal cytology, 5.6% (30 of 539) harbored HPV DNA. A total of 5 of 19 (26.3%) of the women with pathological signs were positive for HPV DNA. By a two-step PCR, using nested general primers, 20.3% (118 of 581) of all women were shown to contain HPV DNA. The proportion of HPV-positive women also increased with age, from 17.4% (12 of 69) among women aged 19 years to 31.9%o (43 of 135) among women aged 25 years, when the two-step PCR was used. Some 19.2% (102 of 530) of cervical scrapes from women with normal cytology contained HPV DNA. Among the women with pathological signs, 16 of 19 (84.2%) were positive for HPV DNA. The HPV DNA-positive specimens were demonstrated to contain HPV type 6, 11, 16, 18, 31, 33, 35, 39, 40, 45, 55, or 56. The most prevalent HPV types were 6 (2.0%o) and 16 (2.7%). More than one type was found in 16 specimens. Sixty HPV-positive samples could not be typed.

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DNA was prepared as described previously (8). For amplification, 20 RI of the solution was used directly. PCR. Amplification of HPV DNA was carried out in three ways. First, a consensus primer pair, MY11-MY09, which spans nucleotides 6722 to 7170 in HPV type 6 and the

PCR. Detection and hybridization. For detection, 40% of the amplified DNA (20 p.l) was separated on a 2.0% NuSieve GTG plus 1.0% SeaKem ME agarose gel (FMC Bioproducts, Rockland, Maine) by electrophoresis and stained with ethidium bromide. Two 32P-end-labeled internal oligonucleotide probes, MY1019 and MY18 (15), were used for hybridization of the MY11-MY09 one-step amplification products from 223 of the samples. For HPV typing, 10% of the amplified DNA (5 p.l) was bound to a nylon filter by slot blotting by using a Minifold II Slot-Blotter (Schleicher & Schuell GmbH, Dassel, Germa-

ny). The entire genomes of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 45, 54, 55, 56, and 58 (kindly provided by E.-M. de Villiers, A. Irincz, T. Matsukura, G. Orth, K. Shah, and H. zur Hausen) were radioactively labeled and used as probes for hybridization. The nylon filters were prehybridized and hybridized in a solution containing 5 x SSC (0.75 M NaCl plus 0.075 M sodium citrate), 5x Denhardt solution (0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), sonicated salmon sperm DNA (100 p,g/ml), and 50% formamide at 42°C. The filters were then washed at 20°C in 2x SSC-0.1% SDS for 30 min, two times for 15 min each time with 2x SSC-0.1% SDS at 65°C, and finally, two times for 15 min each time with 0.5x SSC-0.1% SDS at 65°C. The filters were exposed to Cronex 4 film (DuPont) for 1 to 3 days by using intensifying screens. RESULTS Cytology. The cytological evaluation of the cell samples revealed that of 558 cell samples, 539 (96.6%) showed normal cytology and 19 (3.4%) had pathological signs. Of these, 12 (2.2%) had cytological patterns of condyloma, 3 (0.5%) had patterns of dysplasia, and 4 (0.7%) had signs of inflammation. One-step PCR with the outer primer pair. To determine the prevalence of HPV infection in the cell samples, we first used a one-step PCR procedure with the MY11-MY09 consensus primers (Fig. 1 and 2A). Among cervical scrapes from women with normal cytology, 30 of 539 (5.6%) were shown to contain HPV DNA. Of the women with pathological signs, 5 of 19 (26.3%) were positive for HPV DNA (Table 1). In total, samples from 590 women were tested by the MY11-MY09 one-step PCR. We detected HPV DNA in 35 (5.9%) of the cervical scrapes (Table 2). The proportion of HPV-positive women increased with age, from 1.4% HPV positive among the women aged 19 years to 9.2% HPV positive among the women aged 25 years (Table 2). The SiHa cell line (which contains one to two copies of HPV type 16) was used to determine the sensitivity of the one-step PCR. By diluting SiHa cell DNA, which was prepared in the same way as the cervical scrapes were, we detected 100 copies of the HPV type 16 genome by ethidium bromide staining. Two-step PCR. We then assembled a nested PCR with general primers to be able to increase the sensitivity of HPV DNA detection (Fig. 1 and 2B). The two-step PCR was used for analysis of all samples except for nine samples that were not available for analysis, that were negative by the MY11MY09 one-step PCR. Among cervical scrapes from women with normal cytology, 102 of 530 (19.2%) were positive for HPV DNA after the two-step PCR. Among the women with pathological signs, specimens from 16 of 19 (84.2%) of them had HPV DNA (Table 1). In addition to the 35 HPV DNA-positive specimens from the MY11-MY09 one-step PCR, we detected 83 HPV-containing specimens by the two-step PCR, giving a total HPV prevalence of 118 of 581 (20.3%) (Table 3). The proportion of HPV-positive women increased with age, also when the two-step PCR was used, from 17.4% among women aged 19 years to 31.9% among the women aged 25 years (Table 3). The two-step PCR was shown to detect 1 to 10 copies of the HPV type 16 genome by using the SiHa cell line as described above. Hybridization. After amplification, 223 of the MY11-MY09 one-step PCR amplimers were hybridized to the oligonucleotide probes MY1019 and MY18 (15). All samples that were negative after ethidium bromide staining (209 of 223) were


corresponding regions of the other genital HPVs (15), was used in a one-step amplification of the DNA prepared from the cervical scrapes (40 cycles) (Fig. 1 and 2A). Second, another general primer pair, GP5-GP6, which spans nucleotides 6764 to 6902 in HPV type 6 and the corresponding regions of other genital HPVs (23), was used in combination with the MY11-MY09 primer pair in a nested general primer two-step amplification of the HPV DNA (20 cycles for MY11-MY09, 30 cycles for GP5-GP6) (Fig. 1 and 2B). Third, the GP5-GP6 primer pair was used in a one-step amplification of the HPV DNA (40 cycles). All primers were synthesized on a Beckman DNA SM automated DNA synthesizer. For a typical one-step amplification reaction, 20 ,ul of DNA prepared from cervical cells was mixed with PCR buffer [16.6 mM (NH4)2SO4, 67 mM Tris-HCl (pH 8.8) at 25°C, 6.7 mM MgCl2, 10 mM 1-mercaptoethanol]; the four deoxynucleotides dATP, dlTP, dCTP, and dGTP at final concentrations of 50 puM each; 12 pmol of either the MY11MY09 or the GP5-GP6 primer pair; and 2 U of Taq DNA polymerase (Amplitaq; Cetus, Berkeley, Calif.) to a final volume of 50 plI. This mixture was overlaid with 2 to 3 drops of mineral oil. Thermal cycling of the amplification mixture (denaturation, annealing, and extension) was performed in a programmable heat block (Techne PHC-2; Teche Ltd., Cambridge, United Kingdom) for a total of 40 cycles. Denaturation was performed at 94°C for 30 s, annealing was performed at 45°C for 30 s, and extension was performed at 72°C for 30 s for all amplifications. For a typical two-step amplification, 0.2 pmol of the MY11-MY09 primer pair was mixed with the PCR buffer, the four deoxynucleotides at a final concentration of 100 puM each, 2 U of Taq DNA polymerase, and 20 pul of the DNA prepared from the cervical scrapes. The mixture was overlaid with mineral oil, and a total of 20 cycles was performed as described above. A total of 20 pmol of the GP5-GP6 primer pair was then added, together with 2 U of Taq DNA polymerase, to the reaction mixture, and 30 additional cycles were performed. All clinical specimens were also amplified with the P-globin primers PCO3 and PCO4 (19) to exclude falsenegative results. Samples that were negative for 3-globin amplification were extracted with phenol and precipitated with ethanol, and a second 13-globin PCR was performed. All 3-globin-positive samples (590 of 602 samples) were amplified with the HPV general primers. All PCRs were performed so that every third sample was a negative control. All such controls were negative. To minimize the risk of contamination, strict precautions were taken during sample collection, preparation of DNA, and

J. CLIN. MICROBIOL.

HPV PREVALENCE DETECTION BY PCR

VOL. 30, 1992 6722

_

6764

6904

7170

MYll GP5

GP6

MY09

-b

989

-04

El

also

negative

after

hybridization.

Of the ethidium bromide-

positive samples, 9 of 14 were also hybridization positive. Detection by ethidium bromide staining revealed 83 specimens that were negative by the MY11-MY09 one-step PCR A-

1

2

-

but that were positive by the two-step PCR. Of these, 36 specimens were hybridized to the oligonucleotide probes. None of the samples was positive after hybridization. One-step PCR with the inner primer pair. We also performed a one-step PCR with the inner primer pair GP5-GP6. We analyzed 62 of the 83 specimens earlier scored as HPV positive by the two-step PCR but HPV negative by the MY11-MY09 one-step PCR. After ethidium bromide staining, 35 specimens were found to be HPV positive. We also analyzed 90 HPV-negative specimens by the two-step PCR with the GP5-GP6 primer pair. All of these specimens were negative. The number of HPV-positive specimens in the GP5-GP6 one-step PCR corresponds to about 14% of HPVpositive specimens in the total population. The GP5-GP6 one-step PCR was shown to detect 10 copies of the HPV type 16 genome by using the SiHa cell line as described above. HPV typing. The HPV types were determined by slot blot hybridization of the HPV DNA-positive amplification products to 14 different genital HPV types. The most prevalent types were HPV type 6, 12 of 590 (2.0%) specimens, and HPV type 16, 16 of 590 (2.7%) specimens (Table 4). We also TABLE 1. Prevalence of HPV infection compared with cytological evaluation of Pap smears determined by MY11-MY09 one- or two-step PCR'

.142 bp

FIG. 2. DNA from cervical scrapes was analyzed by the MY11MY09 one-step PCR (A) and the two-step PCR (B). Lanes 1 to 6, DNA from cervical scrapes. +, cloned HPV type 16 DNA amplified as a positive control; -, negative control containing all PCR reagents except DNA; m, DNA molecular mass standard. In the one-step PCR, all samples were subjected to 40 cycles of amplification with the MY11-MY09 primer pair (15). In the two-step PCR, all samples were subjected to 20 cycles of amplification with the MY11-MY09 primer pair; this was followed by 30 cycles of amplification with the GP5-GP6 primer pair (23).

No.

(%) HPV:

Cytology and PCR method (no. of patients)

Positive

Negative

Normal cytology One-step PCR (539) Two-step PCR (530)

30 (5.6) 102 (19.2)

509 (94.4) 428 (80.8)

Pathological signsb One-step PCR (19) Two-step PCR (19)

5 (26.3) 16 (84.2)

14 (73.7) 3 (15.8)

a All the HPV DNA-positive specimens from the MY11-MYO9 one-step PCR are included as two-step PCR positives. b Cytological patterns of condyloma (n = 12), dysplasia (n = 3), or inflammation (n = 4).


FIG. 1. Locations of the MY11-MYO9 consensus primers (15) and the GP5-GP6 general primers (23) in the Li region of the HPV genome.

990

J. CLIN. MICROBIOL.

EVANDER ET AL.

TABLE 2. MY11-MYO9 one-step amplification for determination of the prevalence of HPV infection in comparison with age No. (%)

Patient age (yr [no. of patients]) 19 21 23 25

1 6 15 13

(69) (158) (221) (142)

Negative

68 152 206 129

(1.4) (3.8) (6.8) (9.2)

(98.6) (96.2) (93.2) (90.8)

555 (94.1)

35 (5.9)

Total (590)

HPV type

HPV:

Positive

detected HPV types 11, 18, 31, 33, 35, 39, 40, 45, 55, and 56. Sixty HPV-positive samples could not be typed. Infection with more than one type was found in 16 specimens (Table 4).

women.

By using the MY11-MY09

consensus

primer pair in

a a nonse-

lected, young female population, we demonstrated HPV DNA in 5.9% of the women. Among the women with normal cytology, 5.6% were shown to be HPV DNA positive. To check the sensitivity of the one-step PCR directly on cytological scrapes, we assembled a two-step PCR. We took advantage of the fact that the GP5-GP6 general primers (23) were positioned within the MY11-MY09 consensus primers and used these two primer pairs to set up a two-step PCR with the primers arranged in a nested fashion. A new PCR system should be evaluated in a clinical setting before it can TABLE 3. Two-step PCR amplification for determination of the prevalence of HPV infection in comparison with agea Patient (yr [no. of

No. (%)

age

patients])

19 (69) 21 (158) 23 (218) 25 (135) Total (581)

Positive

12 24 39 43 118

(17.4) (15.2) (17.9) (31.9) (20.3)

HPV: Negative

57 134 179 92 463

(82.6) (84.8) (82.1) (68.1) (79.7)

a All the HPV DNA-positive specimens from the MY11-MYO9 one-step PCR are included as two-step PCR positives.

No. positive

6 ................................................. 11 ................................................. 16 ................................................. 18 ................................................. 31 ................................................. 33 ................................................. 35 ................................................. 39 ................................................. 40 ........................................... 45 ........................................... 54 ...........................................

12 2 16 7 8 8 4 3 4 4 0

55 ........................................... 56 .................................................

1 4

58 ................................................. Unknown ...........................................

0 60

a Sixteen specimens were demonstrated to contain two or more types: 4 contained HPV types 6 and 16; two contained HPV types 16 and 33; and the 10 remaining specimens contained HPV types 6 and 31, HPV types 6 and 39, HPV types 11 and 16, HPV types 16 and 18, HPV types 16 and 56, HPV types 18 and 45, HPV types 31 and 33, HPV types 33 and 40, HPV types 35 and 56, and HPV types 6, 39, and 45, respectively.

be regarded as an applicable system (20). The nested primerbased PCR was evaluated on samples from the same nonselected female population that were analyzed by the MY11MY09 one-step PCR. The total detection rate of HPV DNA increased from 5.9 to 20.3%. Among the women with normal cytology, the detection rate of HPV increased from 5.6 to 19.2%. It was clear from the MY11-MY09 one-step PCR that HPV prevalence increases with age (Table 2). The frequency of HPV-positive women also increased with age in the samples analyzed by the two-step PCR (Table 3). Demonstration by the two-step PCR of 83 HPV-positive specimens that were negative by the MY11-MY09 one-step PCR may indicate variations in the HPV copy number in the lesions. The two-step PCR was more sensitive than the one-step PCR; i.e., fewer copies of the HPV genome could be detected. The inner primer pair GP5-GP6 was also used for amplification of some of the specimens, which increased the sensitivity of the one-step PCR. However, the two-step PCR was the most sensitive technique. The GP5-GP6 primer pair spans a region of about 140 bp compared with 450 bp for the MY11-MY09 primer pair. The shorter amplification product may increase the efficiency of the PCR. Furthermore, the GP5-GP6 primers do not contain any degenerate bases, which may result in increased amplification efficiency. The HPV type 6 genome was detected in 10 of 35 HPVpositive samples when the MY11-MY09 one-step PCR was used, but only 2 additional HPV type 6-containing cervical scrapes were found by the two-step PCR (data not shown). This could reflect the fact that HPV type 6 was present at high copy numbers in the lesions, while other types may have had a lower genome copy number. It is also possible that some types (unknown types) could not be amplified to a detectable level by using ethidium bromide-stained gels in the one-step PCR. In the two-step system, the amplification products can be visualized on an agarose gel when the second amplification round is performed with the inner primer pair. The conditions for amplification can also vary considerably between different specimens, since the PCR was performed directly on the DNA prepared from cell scrapings. However, all samples that were analyzed with the


DISCUSSION Several approaches have been tested to determine the prevalence of HPV in the population. Southern blot, dot blot, and filter in situ hybridization have been used for the detection of HPV DNA in women attending clinics for routine gynecologic screening (5, 13) or large populationbased studies (10). More recently, PCR has been applied for the demonstration of HPV DNA in clinical specimens. Several selections of type-specific primers for HPV detection are available (7, 17, 21, 30). Primers for the detection of a broad spectrum of HPV types, consensus or general primers, have been presented (8, 9, 15, 23). Degenerate primers for HPV DNA detection, arranged in a nested fashion, have also been developed (29). We chose to use the MY11-MY09 consensus primer pair (15) for detection of HPV DNA, to allow comparison of our results with those of other studies. The MY11-MY09 primer pair is localized within the Li region of the HPV genome. The Li region can be lost at late stages of neoplasia (22), but this is hardly relevant in HPV infections in very young one-step PCR directly on cytological scrapes from

TABLE 4. Determination of the HPV type in the HPV-containing specimens by slot blot hybridizationa

VOL. 30, 1992

ACKNOWLEDGMENTS We are grateful to T. Angstrom of the Department of Cytology, University of Umea, for cytological analysis. This work was supported by a grant from the Labor Market Insurance Company, grant 1547-B90 from the Swedish Cancer Society, and grants from the Research Foundation of the Department of Oncology, University of Umeg. REFERENCES 1. Bauer, H. M., Y. Ting, C. E. Greer, J. C. Chambers, C. J. Tashiro, J. Chimera, A. Reingold, and M. Manos. 1991. Genital human papillomavirus infection in female university students as determined by a PCR-based method. JAMA 265:472-477. 2. Boshart, M., L. Gissmann, H. Ikenberg, A. Kleinheinz, W. Scheurlen, and H. zur Hausen. 1984. A new type of papillomavirus DNA and its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J. 3:1151-1157. 3. Chow, L. T., M. Nasseri, S. M. Wolinsky, and T. Broker. 1987. Human papillomavirus types [sic] 6 and 11 mRNAs from genital

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condylomata. J. Virol. 61:2581-2588. 4. de Villiers, E.-M. 1989. Heterogeneity of the human papillomavirus group. J. Virol. 63:4898-4903. 5. de Villiers, E.-M., D. Wagner, A. Schneider, H. Wesch, H. Miklaw, J. Wahrendorf, U. Papendick, and H. zur Hausen. 1987. Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet ii:703-706. 6. Durst, M., L. Gissmann, H. Ikenberg, and H. zur Hausen. 1983. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc. Natl. Acad. Sci. USA 80:3812-3815. 7. Evander, M., E. Boden, L. Bjersing, E. Rylander, and G. Wadell. 1991. Oligonucleotide primers for DNA amplification of the early regions 1, 6, and 7 from human papillomavirus types 6, 11, 16, 18, 31, and 33. Arch. Virol. 116:221-233. 8. Evander, M., and G. Wadell. 1991. A general primer pair for amplification and detection of genital human papillomavirus types. J. Virol. Methods 31:239-250. 9. Gregoire, L., M. Arella, J. Campione-Piccardo, and W. D. Lancaster. 1989. Amplification of human papillomavirus DNA sequences by using conserved primers. J. Clin. Microbiol. 27:2660-2665. 10. Kjaer, S. K., E.-M. de Villiers, B. J. Haugaard, R. B. Christensen, C. Teisen, K. A. M0ller, P. Poll, H. Jensen, B. F. Vestergaard, E. Lynge, and 0. M. Jensen. 1988. Human papillomavirus, herpes simplex virus and cervical cancer incidence in Greenland and Denmark. A population-based cross-sectional study. Int. J. Cancer 41:518-524. 11. Koutsky, L. A., D. A. Galloway, and K. K. Holmes. 1988. Epidemiology of genital human papillomavirus infection. Epidemiol. Rev. 10:122-163. 12. Lbrincz, A. T., W. D. Lancaster, and G. F. Temple. 1986. Cloning and characterization of the DNA of a new human papillomavirus from a woman with dysplasia of the uterine cervix. J. Virol. 58:225-229. 13. Lirincz, A. T., G. F. Temple, J. A. Patterson, A. B. Jenson, R. J. Kurman, and W. D. Lancaster. 1986. Correlation of cellular atypia and human papillomavirus deoxyribonucleic acid sequences in exfoliated cells of the uterine cervix. Obstet. Gynecol. 68:508-512. 14. MacNab, J. S., J. Walkinshaw, J. Cordiner, and J. B. Clements. 1986. Human papillomavirus in clinically and histologically normal tissue of patients with genital cancer. N. Engl. J. Med. 315:1052-1058. 15. Manos, M. M., Y. Ting, D. K. Wright, A. J. Lewis, T. R. Broker, and S. M. Wolinsky. 1989. Use of polymerase chain reaction amplification for the detection of genital human papillomavirus. Cancer Cells 7:209-214. 16. Meisels, A., C. Morin, and M. Casas-Cordero. 1982. Human papillomavirus infection of the uterine cervix. Int. J. Gynecol. Pathol. 1:75-94. 17. Melchers, W., A. van den Brule, J. Walboomers, M. de Bruin, M. Burger, P. Herbrink, C. Mejer, J. Lindeman, and W. Quint. 1989. Increased detection rate of human papillomavirus in cervical scrapes by the polymerase chain reaction as compared to modified FISH and Southern blot analysis. J. Med. Virol. 27:329-335. 18. Pfister, H. 1984. Biology and biochemistry of papillomaviruses. Rev. Physiol. Biochem. Pharmacol. 99:111-181. 19. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, ahd N. Arnheim. 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354. 20. Schiffman, M. H., H. M. Bauer, A. L. Lorincz, M. M. Manos, J. C. Byrne, A. G. Glass, D. M. Cadell, and P. M. Howley. 1991. Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus DNA. J. Clin. Microbiol. 29:573-577. 21. Shibata, D. K., N. Arnheim, and J. Martin. 1988. Detection of human papillomavirus in paraffin-embedded tissue using polymerase chain reaction. J. Exp. Med. 167:225-230. 22. Shirasawa, H., Y. Tomita, K. Kubota, T. Kasai, S. Sekiya, H. Takamizawa, and B. Simizu. 1988. Transcriptional differences of


HPV-specific primers were positive for the P-globin gene by a one-step PCR. HPV types 6 and 16 were the most commonly found HPV types among the women in this study. In total, we detected 12 different types. We were not able to determine the HPV type among a large proportion of the two-step PCR-positive specimens in comparison with the proportion we determined among the one-step PCR-positive specimens. A higher proportion of unknown HPV types was amplified with the nested primer-based PCR system, and it is also possible that the typing was easier to perform on the larger amplimer (450 bp) from the MY11-MY09 one-step PCR. The increase in the unknown HPV types among the positive specimens after the two-step PCR may indicate nonspecific amplification. However, the ethidium bromidestained amplification product was always of the predicted size. Furthermore, women with antichlamydial antibodies displayed a statistically significant higher presence of HPV than other women did (P < 0.001; data not shown). This correlates with the increase in the number of HPV-positive women with age determined by the two-step PCR. We also analyzed DNA prepared from the HPV-negative cell line A549 originating from a human lung carcinoma. No amplification product of the predicted HPV amplimer size was detected (data not shown). The nature of the amplimers from the unknown HPV types can be verified by sequencing. By use of the two-step general PCR, we determined the HPV prevalence to be 19.2% among young (age range, 19 to 25 years) Swedish women with normal cytology in a population-based study. Some 20.3% of all women were HPV positive, and the prevalence of HPV 16 was 2.7%. In Holland, HPV DNA was found by using the GP6-GP6 general primer pair in 14 to 25% of cervical scrapes from women with normal cytology attending gynecological clinics for a wide range of gynecological complaints (age range, 16 to 60 years) (26, 27). Among female university students from Berkeley, Calif., who underwent a routine gynecological examination, 31% had normal cytology and 46% were HPV positive when the consensus primer pair MY11-MY09 was used (1). Their mean age was 22.9 years. The one-step PCR consensus primer system from the Li region (15) was, in our hands, not sufficiently robust to detect all the HPV DNA-positive cytological scrapes that were positive by use of a two-step general PCR from the Li region. By using the two-step PCR, we found that even in a population-based study of young women, HPV infection of the uterine cervix is common.

HPV PREVALENCE DETECTION BY PCR

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23.

24. 25.

26.

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