Short Fragment Polymerase Chain Reaction Reverse Hybridization Line Probe Assay to Detect and Genotype a Broad Spectrum of Human Papillomavirus Types
1999; Elsevier BV; Volume: 155; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65462-4
ISSN1525-2191
AutoresWillem J. G. Melchers, Judith M. J. E. Bakkers, Jinhua Wang, Peter C.M. de Wilde, H. Boonstra, Wim Quint, Antonius G.J.M. Hanselaar,
Tópico(s)Molecular Biology Techniques and Applications
ResumoThe purpose of this study was to detect and genotype 16 different human papilloma virus (HPV) types simultaneously using a short fragment polymerase chain reaction (SPF) hybridization line probe assay (LiPA). 152 women who were referred to the gynecologist because of abnormal cervical smear underwent colposcopic examination and repeat cervical smear. In addition, the cervical scrapes were analyzed for the presence of HPV by a novel general HPV polymerase chain reaction assay followed by a single reaction genotyping assay allowing for a simultaneous detection and identification of 16 different HPV types. HPV DNA was detected in 38% of normal follow-up cervical scrapes, 51% of scrapes with atypical squamous cells of undetermined significance, 78% of scrapes with mild dysplasia (low grade squamous intraepithelial lesions), 86% of scrapes with moderate dysplasia (high grade squamous intraepithelial lesions), and in 88% of scrapes with severe dysplasia and carcinoma in situ. One case of invasive squamous cell carcinoma was positive for HPV 16. Overall, a single HPV type was detected in 56% of HPV positive scrapes, with HPV 16 being the most common and accounting for 45% of all single infections. Forty-four percent of the positive scrapes contained multiple HPV types, of which double infections prevailed. Follow-up results proved the reproducibility and reliability of SPF HPV LiPA. In conclusion, we have used and evaluated the SPF-HPV-LiPA system for the detection and genotyping of HPV infections. The combined detection-typing method proved to be sensitive, specific, simple, and fast, making mass screening of cervical scrapes accessible for routine practice and facilitating individual patient management. The purpose of this study was to detect and genotype 16 different human papilloma virus (HPV) types simultaneously using a short fragment polymerase chain reaction (SPF) hybridization line probe assay (LiPA). 152 women who were referred to the gynecologist because of abnormal cervical smear underwent colposcopic examination and repeat cervical smear. In addition, the cervical scrapes were analyzed for the presence of HPV by a novel general HPV polymerase chain reaction assay followed by a single reaction genotyping assay allowing for a simultaneous detection and identification of 16 different HPV types. HPV DNA was detected in 38% of normal follow-up cervical scrapes, 51% of scrapes with atypical squamous cells of undetermined significance, 78% of scrapes with mild dysplasia (low grade squamous intraepithelial lesions), 86% of scrapes with moderate dysplasia (high grade squamous intraepithelial lesions), and in 88% of scrapes with severe dysplasia and carcinoma in situ. One case of invasive squamous cell carcinoma was positive for HPV 16. Overall, a single HPV type was detected in 56% of HPV positive scrapes, with HPV 16 being the most common and accounting for 45% of all single infections. Forty-four percent of the positive scrapes contained multiple HPV types, of which double infections prevailed. Follow-up results proved the reproducibility and reliability of SPF HPV LiPA. In conclusion, we have used and evaluated the SPF-HPV-LiPA system for the detection and genotyping of HPV infections. The combined detection-typing method proved to be sensitive, specific, simple, and fast, making mass screening of cervical scrapes accessible for routine practice and facilitating individual patient management. Worldwide, cervical cancer is the second most common cancer affecting women. Although screening programs to identify precursor lesions of cervical cancer have reduced significantly the mortality and morbidity of this disease, 500,000 new cases of invasive cancer of the cervix are diagnosed annually.1Parkin DM Pisani P Ferlay J Estimates of the worldwide incidence of eighteen major cancers in 1985.Int J Cancer. 1993; 54: 594-606Crossref PubMed Scopus (1582) Google Scholar Epidemiological and molecular studies over the past two decades have demonstrated convincingly that certain types of human papillomaviruses (HPVs) are etiologically related to the development of most cases of cervical cancer.2Bosch FX Manos MM Munoz N Sherman M Jansen AM Peto J Schiffman MH Moreno V Kurman R Shah KV the IBSCC Study Group Prevalence of human papillomavirus in cervical cancer: a worldwide perspective.J Natl Cancer Inst. 1995; 87: 796-802Crossref PubMed Scopus (2976) Google Scholar, 3Koutsky LA Holmes KK Critchlow CW Stevens CE Paavonen J Beckmann AM DeRouen TA Galloway DA Vernon D Kiviat NB A cohort study of the risk of cervical intraepithelial neoplasia grade 2 and 3 in relation to papillomavirus infection.N Engl J Med. 1992; 327: 272-278Crossref Scopus (859) Google Scholar, 4Schiffman MH Bauer H Hoover R Glass AG Cadell DM Rush BB Scott DR Sherman ME Kurman RJ Wacholder S Stanton CK Manos MM Epidemiologic evidence showing that HPV infection causes most cervical intraepithelial neoplasia.J Natl Cancer Inst. 1993; 85: 958-964Crossref PubMed Scopus (1088) Google Scholar To date, more than 85 types of HPV types have been identified, of which 30 different HPVs have been found to infect the genital mucosa.5Chan SY Delius H Halpern AL Bernard HU Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy.J Virol. 1995; 69: 3074-3083Crossref PubMed Google Scholar Several HPV types, such as HPVs 16, 18, 31, 33, and 35 have been implicated in cervical carcinogenesis,6zur Hausen H Papillomavirus infections—a major cause of human cancers.Biochim Biophys Acta. 1996; 1288: F55-F78Crossref PubMed Scopus (1468) Google Scholar whereas other types, such as HPVs 6 and 11, are frequently detected in benign lesions such as condylomata acuminata. Women infected with high risk HPV types are considered to be at a higher risk for the development of cervical cancer than those who are not infected with HPV or are infected with low risk HPV types.7Cuzick J Szarewski A Terry G Ho L Hanby A Moddox P Anderson M Kocjan G Steele ST Guillebaud J Human papillomavirus testing in primary cervical screening.Lancet. 1995; 345: 1533-1536PubMed Scopus (312) Google Scholar, 8Remmink AJ Walboomers JMM Helmerhorts TJM Voorhorts FJ Roozendaal L Risse EKJ Meijer CJLM Kenemans P The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: natural history up to 36 months.Int J Cancer. 1995; 61: 306-311Crossref PubMed Scopus (342) Google Scholar Several studies have shown the potential relevance of HPV testing in cervical cancer screening program and management of patients with atypical squamous cells of undetermined significance.7Cuzick J Szarewski A Terry G Ho L Hanby A Moddox P Anderson M Kocjan G Steele ST Guillebaud J Human papillomavirus testing in primary cervical screening.Lancet. 1995; 345: 1533-1536PubMed Scopus (312) Google Scholar, 9Bollen LJ Tjong-A-Hung SP van der Velden J Brouwer K Mol BW ten Kate FJ ter Schegget J Human papillomavirus deoxyribonucleic acid detection in mildly or moderately dysplastic smears: a possible method for selecting patients for colposcopy.Am J Obstet Gynecol. 1997; 177: 548-553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 10Cox JT Lorincz AT Schiffman MH Sherman ME Cullen A Kurman RJ Human papillomavirus testing by hybrid capture appears to be useful in triaging women with cytological diagnosis of atypical squamous cells of undetermined significance.Am J Obstet Gynecol. 1995; 172: 946-954Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 11Cuzick J Terry G Ho L Hollingsworth T Anderson H Human papillomavirus type 16 DNA in cervical smears as a predictor of high-grade cervical cancer.Lancet. 1992; 339: 959-960Abstract PubMed Scopus (143) Google Scholar, 12Sherman ME Schiffman MH Lorincz AT Manos MM Scott DR Kurman RJ Kiviat NB Stoler M Glass AG Rush BB Toward objective quality assurance in cervical cytopathology: correlation of cytopathologic diagnosis with detection of high-risk human papillomavirus types.Am J Clin Pathol. 1994; 2: 182-187Google Scholar, 13Walboomers JMM De Roda Husman AM Snijders PJF Stel HV Risse EKJ Helmerhorst ThJM Voorhorst FJ Meijer CJLM Human papillomavirus in false negative archival cervical smears: implications for screening for cervical cancer.J Clin Pathol. 1995; 48: 728-732Crossref PubMed Scopus (71) Google Scholar Incorporation of HPV tests into screening programs might identify women at risk for developing invasive cervical cancer. Furthermore, absence of high risk HPV in the cervical smear would permit less aggressive management of women with mild or equivocal cytological abnormalities. General polymerase chain reaction (PCR) assays with a broad spectrum specificity for HPV are widely used for the detection of the virus in clinical specimens.9Bollen LJ Tjong-A-Hung SP van der Velden J Brouwer K Mol BW ten Kate FJ ter Schegget J Human papillomavirus deoxyribonucleic acid detection in mildly or moderately dysplastic smears: a possible method for selecting patients for colposcopy.Am J Obstet Gynecol. 1997; 177: 548-553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 14De Roda Husman AM Walboomers JMM van den Brule AJ Meijer CJLM Snijders PJF The use of general primers GP5 and GP6 elongated at their 3′-end with adjacent highly conserved sequences improves human papillomavirus detection by PCR.J Gen Virol. 1995; 76: 1057-1062Crossref PubMed Scopus (1112) Google Scholar, 15Hildesheim A Schiffman MH Gravitt PE Glass AG Greer CE Zhang T Scott DR Rush BB Lawler P Sherman ME Kurman RJ Manos MM Persistence of type-specific human papillomavirus infections among cytologically normal women.J Infect Dis. 1994; 169: 235-240Crossref PubMed Scopus (622) Google Scholar, 16Melchers WJG Claas ECJ Quint WGV The use of the polymerase chain reaction to study the relationship between human papillomavirus infections and cervical cancer.Eur J Clin Microbiol Infect Dis. 1991; 10: 714-727Crossref PubMed Scopus (24) Google Scholar However, additional typing by hybridization or type-specific PCR assays is required to identify the individual HPV type. This is laborious and time-consuming. In addition, for many of the newly described HPV types, specific primers are not available. For these reasons, it is important to pursue further development of genotyping systems applicable for mass screening of cervical scrapes. In this report we describe the practical application of a reverse hybridization line probe assay (HPV-LiPA), recently developed for genotyping of a broad spectrum of HPV types in a single assay.17Kleter B van Doorn L-J Schrauwen L Molijn A Sastrowijoto S ter Schegget J Lindeman J ter Harmsel B Burger M Quint W Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.J Clin Microbiol. 1999; 37: 2508-2517PubMed Google Scholar Combined with a novel general (SPF) PCR assay, amplifying a fragment of only 65 bp, this SPF-HPV-LiPA permits a highly sensitive, simultaneous detection of 16 individual HPV types in a single reaction scheme.18Kleter B van Doorn L-J ter Schegget J Schrauwen L van Krimpen C Burger M ter Harmsel B Quint W Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol. 1998; 153: 1731-1739Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar Cervical scrapes obtained from women who were referred to the gynecologist because of an abnormal result of their cervical smear were analyzed by SPF-HPV-LiPA to determine its practical usefulness in detection and identification of HPV in large numbers of clinical samples. Cervical scrapes were obtained from 152 women who participated in the Cervical Cancer Screening Program and who were referred by general practitioners to gynecologists for colposcopic examination after two successive cervical scrapes with atypical squamous cells of undetermined significance (ASCUS) or one smear with mild dysplasia (LSIL), moderate dysplasia, severe dysplasia, or carcinoma in situ (the latter three according to the Bethesda classification as HSIL). The visit to the gynecologist took place from 2 weeks to 3 months after the last cytological examination. On referral to the Department of Gynecology, during colposcopic examination, before the application of acetic acid/Lugol solution, two cervical scrapes were taken. One slide was prepared for a routine cytological diagnosis. The residual material from the first scrape and the entire material from the second scrape were collected into ethanol carbowax solution, centrifuged, and embedded in agar (Kerstens HMJ, Robben JCM, De Wilde PCM, Hanselaar AGJM, submitted for publication). The slides were cytomorphologically classified according to the standard Dutch cytological classification system and linked to the Bethesda classification as indicated. To study the predictive value of HPV infections for short and long term progression of intraepithelial lesions, a protocol for follow-up of patients is included in the overall study design. Because of the high regression rate, the Dutch management policy for women with ASCUS, mild dysplasia (LSIL), and moderate dysplasia (HSIL) is to keep these patients under strict follow-up surveillance and not to treat these women directly for their cervical disorder. In the current study, we were able to examine repeat cytological scrapes and perform repeat HPV analysis in a small group of women after a 3-month follow-up period. SPF-HPV-LiPA tests were performed in a blind fashion without a knowledge of the results of cytological examination or previous HPV testing. A single cell section of every specimen 3 μm thick was put into a reaction tube and incubated overnight at 56°C in 200 μl of 10 mmol/L Tris-HCl with 1 mmol/L EDTA, 0.2% Tween 20, and proteinase K (0.3 mg/ml). Proteinase K was inactivated by 10 minutes of incubation at 100°C. The sample was centrifuged for 10 minutes at 11,000 rpm and 10 μl was directly used for PCR analysis. A water blank control was processed with each batch of 10 samples. Broad-spectrum HPV DNA amplification was performed using a recently developed short PCR fragment (SPF-PCR) assay.18Kleter B van Doorn L-J ter Schegget J Schrauwen L van Krimpen C Burger M ter Harmsel B Quint W Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol. 1998; 153: 1731-1739Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar All HPV tests were performed blinded to the cytological results and other clinical data. SPF-PCR system amplifies a 65-bp fragment of the L1 open reading frame allowing for detection of at least 43 different HPV types.18Kleter B van Doorn L-J ter Schegget J Schrauwen L van Krimpen C Burger M ter Harmsel B Quint W Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol. 1998; 153: 1731-1739Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar SPF-PCR was performed in a final reaction volume of 50 μl containing 10 μl of the isolated DNA sample, 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 2.0 mmol/L MgCl2, 0.1% Triton X-100, 0.01% gelatin, 200 μmol/L each of deoxynucleoside triphosphate, 15 pmol each of the forward and reverse primers tagged with a biotin at the 5′ end, and 1.5 units of AmpliTaq gold (Perkin Elmer). The mixture was overlaid with two drops of mineral oil and incubated for 9 minutes at 94°C followed by 40 cycles of 30 seconds at 94°C, 45 seconds at 45°C, 45 seconds at 72°C, and a final extension of 5 minutes at 72°C. Each experiment was performed with separate positive and negative PCR controls. A total 15 μl of each PCR product was electrophoresed in a 3% agarose gel and was stained with ethidium bromide. To avoid contamination by PCR products, different steps such as sample preparation and the amplification reaction were performed in strictly separated rooms. A poly(dT) tail was enzymatically added to the 3′ end of each of 16 oligonucleotide specific for 16 different HPV types, namely HPVs 6, 11, 16, 18, 31, 33, 35, 40, 42, 43, 44, 45, 51, 52, 56, and 58. The tailed probes were applied as horizontal lines to the membrane strips. A biotinylated poly(dT) control for conjugate was applied. The HPV-LiPA genotyping assay was performed as described previously.17Kleter B van Doorn L-J Schrauwen L Molijn A Sastrowijoto S ter Schegget J Lindeman J ter Harmsel B Burger M Quint W Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.J Clin Microbiol. 1999; 37: 2508-2517PubMed Google Scholar Briefly, equal volumes (10 μl each) of the biotinylated products of PCR and the denaturation solution (400 mmol/L NaOH, 10 mmol/L EDTA. were mixed in test troughs and incubated at room temperature for 5 minutes, after which 1 ml of the prewarmed (37°C) hybridization solution (3X SSC (1X SSC is 0.15 mol/L NaCl plus 0.015 mol/L sodium citrate), 0.1% sodium dodecyl sulfate) was added, followed by the addition of one strip per trough. Hybridization was performed for 1 hour at 50 ± 0.5°C in a closed water bath with back-and-forth shaking. The strips were washed twice with 1 ml of wash solution (3X SSC, 0.1% sodium dodecyl sulfate) at room temperature for 20 seconds and once at 50°C for 30 minutes. Following this stringent washing, strips were rinsed twice with 1 ml of a standard rinse solution.17Kleter B van Doorn L-J Schrauwen L Molijn A Sastrowijoto S ter Schegget J Lindeman J ter Harmsel B Burger M Quint W Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.J Clin Microbiol. 1999; 37: 2508-2517PubMed Google Scholar Strips were incubated on a rotating platform with an alkaline phosphatase-labeled streptavidin conjugate diluted in a standard conjugate solution at 20 to 25°C for 30 minutes. Strips were then washed twice with 1 ml of rinse solution and once with standard substrate buffer, and color development was initiated by addition of 5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium to 1 ml of substrate buffer.17Kleter B van Doorn L-J Schrauwen L Molijn A Sastrowijoto S ter Schegget J Lindeman J ter Harmsel B Burger M Quint W Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.J Clin Microbiol. 1999; 37: 2508-2517PubMed Google Scholar After 30 minutes of incubation at room temperature, the color reaction was stopped by aspiration of the substrate buffer and addition of distilled water. After drying, the strips were visually interpreted using a grid. In 4 scrapes only the conjugate control line on the HPV-LiPA-strip showed reaction indicating presence of HPV DNA; however, no reaction with any of the 16 HPV type specific probes was observed. To identify the HPV type involved the SPF PCR amplimers were cloned and sequenced. PCR products were ligated into pCR2.1 vector immediately after amplification using the Original TA Cloning Kit (Invitrogen Corporation) and purified using Wizard plus Minipreps DNA purification systems (Promega) according to the manufacturer's instructions. The PCR fragments were sequenced using the Ampli Cycling sequencing kit (Perkin Elmer) and M13 forward primer for the pCR2.1 vector. All four samples were found to be positive for HPV 66. The results of cytological examination correlated with the results of HPV DNA detection as shown in Table 1. In 39 of the women the follow-up smear was negative. However, in 38. of these negative scrapes HPV DNA was detected. HPV DNA was also detected in 24 of 47 (51%) cervical scrapes reported as ASCUS, in 21 of 27 (78%) scrapes with mild dysplasia (LSIL), in 19 of 22 (86%) scrapes with moderate dysplasia, and in 14 of 16 (88%. scrapes with severe dysplasia and carcinoma in situ. One scrape with invasive squamous cell carcinoma was positive for HPV 16.Table 1HPV Occurrence in Cervical Scrapes from Women Combined with Cytological Classification ResultsHPV typeClassificationNo.HPV positive611161831333540424344455152565866*66, HPV 66, typed by sequence analysis.D†D, double infection.T‡T, triple infection.>T§>T, more than triple infection.Normal3938%1141521ASCUS4751%23211212532Mild dysplasia2778%14111922Moderate dysplasia2286%17121421Severe dysplasia and CIS1688%631121Squamous cell carcinoma1100%1Total152585424313112221425106* 66, HPV 66, typed by sequence analysis.† D, double infection.‡ T, triple infection.§ >T, more than triple infection. Open table in a new tab The results of HPV genotyping and type distribution are summarized in Table 1. An example of the results of HPV-LiPA genotyping assay is shown in Figure 1. As seen in Figure 1, interpretation is very easy and multiple infections can readily be identified. In 53 of 94 HPV positive scrapes (56%) a single HPV type was detected. HPVs 6, 11, 16, 18, 31, and 33 accounted for 75% of the single infections, with HPV 16 being the most common and accounting for 45% of all single infections. A remarkable finding was the high prevalence of multiple HPV types in cervical scrapes. Forty-one of 94 HPV positive cervical scrapes contained two or more HPV types, accounting for 44% of the HPV positive scrapes (Table 1). The distribution of the individual HPV types within the multiple infections was random (statistical analysis not shown) and any combination of HPV types could be found. Multiple HPV infection was more common in smears diagnosed as LSIL. The ratio of single versus multiple HPV type infection in normal, and ASCUS smears was similar, 46:54 and 58:42, respectively. The ratio in smears with LSIL was 38:62, and a reverse ratio 69:31 in smears with HSIL was observed. Up to six different HPV types were detected in a single sample. Double infections were most common, accounting for 61. of the multiple infections. Infections with three HPV types were detected in 24% of the scrapes, four HPV types in 7%, five HPV types in 5%, and six HPV types in 2% in the scrapes. The distribution of high risk HPV types (16, 18, 31, 33, 35, 45, 51, 52, 56, and 58) and low risk HPV types (6, 11, 40, 42, 43, and 44. in the scrapes is shown in Figure 2. Prevalence of high risk HPV types was increasing with the severity of cervical smear abnormality. The ratio of high risk to low risk HPV types in normal scrapes was 1.9:1, in ASCUS 2.9:1, in LSIL18.5:1, and HSIL 28:1. Seven percent of all multiple infections contained low risk HPV types only, 37% contained high risk HPV types only, and 56% contained a mixture of both low and high risk HPV types. The ratio of low to high to low/high risk multiple HPV infections in normal scrapes was 1:1:6.3, in ASCUS 1:1:3.5, in LSIL 0:1:1.2, and in HSIL 0:1:0.5. The results of the second cytological examination and repeat HPV analysis in a group of 12 patients after 3 months of follow-up is shown in Table 2. SPF-HPV-LiPA testing for the presence of HPV showed a complete correlation with the results obtained during the first sampling. In 4 cases with ASCUS diagnosis and negative first HPV testing result, the follow-up with second HPV testing was also negative. In one case of ASCUS, positive for HPV 16, the follow-up HPV testing was also positive for HPV 16 indicating a persistent infection. Cytological examination revealed LSIL. In one case of LSIL, positive for HPV 31, the second sampling revealed the presence of an extra type, HPV 18, suggesting additional exposure.Table 2SPF-HPV-LiPA Follow-UpFirst samplingSecond samplingCytological diagnosisHPV typeCytological diagnosisHPV typeASCUSNegativeASCUSNegativeASCUSNegativeASCUSNegativeASCUSNegativeASCUSNegativeASCUSNegativeASCUSNegativeASCUS16Mild dysplasia16Mild dysplasia31Mild dysplasia18,31Mild dysplasia16Mild dysplasia16Mild dysplasia52Mild dysplasia52Mild dysplasia6,16,31Mild dysplasia6,16,31Moderate dysplasia52Moderate dysplasia52Severe dysplasia18Severe dysplasia18Severe dysplasia18Severe dysplasia18 Open table in a new tab Cytological screening programs together with clinical and public awareness have resulted in a remarkable decrease in the incidence of cervical cancer and the mortality due to this disease.19Koss LG The Papanicolaou test for cervical cancer detection: a triumph and a tragedy.JAMA. 1989; 261: 737-743Crossref PubMed Scopus (655) Google Scholar, 20Oleson F A case-control study of cervical cytology before diagnosis of cervical cancer in Denmark.Int J Epidemiol. 1988; 17: 501-508Crossref PubMed Scopus (35) Google Scholar, 21Pontén J Adami HO Bergström R Dillner J Friberg LG Gustafsson L Miller AB Parkin DM Sparén P Trichopoulos D Strategies for global control of cervical cancer.Int J Cancer. 1995; 60: 1-26Crossref PubMed Scopus (239) Google Scholar However, substantial overtreatment of noninvasive intraepithelial lesions is the consequence of early surgical intervention.22Nasiell K Nasiell M Vaclavinkova V Behavior of moderate cervical dysplasia during long term follow-up.Obstet Gynecol. 1983; 61: 609-614PubMed Google Scholar, 23Nasiell K Roger V Nasiell M Behavior of mild cervical dysplasia during long term follow-up.Obstet Gynecol. 1986; 67: 665-669Crossref PubMed Scopus (335) Google Scholar It is well established that only 1% of LSIL and about 12% of HSIL progress to invasive cervical cancer. As the markers of progression of squamous intraepithelial lesions are not established, identification of high risk HPV types in cervical scrapes would help to identify patients with increased risk for development of cervical cancer. Worldwide at least 99% of the cervical carcinomas are HPV DNA positive.2Bosch FX Manos MM Munoz N Sherman M Jansen AM Peto J Schiffman MH Moreno V Kurman R Shah KV the IBSCC Study Group Prevalence of human papillomavirus in cervical cancer: a worldwide perspective.J Natl Cancer Inst. 1995; 87: 796-802Crossref PubMed Scopus (2976) Google Scholar, 18Kleter B van Doorn L-J ter Schegget J Schrauwen L van Krimpen C Burger M ter Harmsel B Quint W Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol. 1998; 153: 1731-1739Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar, 24Walboomers JMM De Roda Husman AM van den Brule AJC Snijders PJF Meijer CJLM Detection of genital human papillomavirus infections: critical review of methods and prevalence studies in relation to cervical cancer. Human Papillomavirus and Cervical Cancer: Biology and Immunology.in: Stern PL Stanley MA Oxford University Press, Oxford1994: 41-71Google Scholar, 25Ngelangel C Munoz N Bosch FX Limson GM Festin MR Deacon J Jacobs MV Santamaria M Meijer CJLM Walboomers JMM Causes of cervical cancer in the Philippines: a case-control study.J Natl Cancer Inst. 1998; 90: 43-49Crossref PubMed Scopus (155) Google Scholar, 26Chichareaon S Herrero R Munoz N Bosch FX Jacobs MV Deacon J Santamaria M Chongsuvivatwong V Meijer CJLM Walboomers JMM Risk factors for cervical cancer in Thailand: a case-control study.J Natl Cancer Inst. 1998; 90: 50-57Crossref PubMed Scopus (174) Google Scholar In addition to HPV 16 and 18, 13 other HPV types were detected as a single HPV type in the cervical carcinomas. These HPV types have been identified as high risk HPVs. Infection with high or low risk HPV type cannot be determined on the basis of cytological examination.27Hirschowitz L Raffle AE Mackenzie EFD Hughes AO Long term follow-up of women with borderline cervical smear result: effects of age and viral infection on progression to high grade dyskaryosis.Br Med J. 1992; 304: 1209-1212Crossref PubMed Scopus (44) Google Scholar, 28Jonsson M Karlsson R Evander M Gustavsson A Rylander E Wadell G Acetowhitening of the cervix and vulva as a predictor of subclinical human papillomavirus infection: sensitivity and specificity in a population-based study.Obstet Gynecol. 1997; 90: 744-747Crossref PubMed Scopus (23) Google Scholar, 29Walker EM Dodgson J Duncan ID Does mild atypia on a cervical smear warrant further investigation?.Lancet. 1986; ii: 672-673Abstract Scopus (88) Google Scholar The importance of high risk HPV in the pathogenesis of cervical neoplasia suggests that detection of an infection with high risk HPV would be useful in patient management.30Kjaer SK van den Brule AJC Bock JE Poll PA Engholm G Sherman ME Walboomers JMM Meijer CJLM Determinants for genital human papillomavirus (HPV) infection in 1000 randomly chosen young Danish women with normal Pap smear: are there different risk profiles for high risk and nonhigh risk HPV types?.Cancer Epidemiol Biomarkers Prev. 1997; 6: 799-805PubMed Google Scholar Much effort has been taken to develop HPV detection and typing assays in the last two decades, which led to the development of broad-spectrum HPV PCR assays allowing for detection of at least 34 anogenital HPV types in a single reaction.9Bollen LJ Tjong-A-Hung SP van der Velden J Brouwer K Mol BW ten Kate FJ ter Schegget J Human papillomavirus deoxyribonucleic acid detection in mildly or moderately dysplastic smears: a possible method for selecting patients for colposcopy.Am J Obstet Gynecol. 1997; 177: 548-553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 14De Roda Husman AM Walboomers JMM van den Brule AJ Meijer CJLM Snijders PJF The use of general primers GP5 and GP6 elongated at their 3′-end with adjacent highly conserved sequences improves human papillomavirus detection by PCR.J Gen Virol. 1995; 76: 1057-1062Crossref PubMed Scopus (1112) Google Scholar, 15Hildesheim A Schiffman MH Gravitt PE Glass AG Greer CE Zhang T Scott DR Rush BB Lawler P Sherman ME Kurman RJ Manos MM Persistence of type-specific human papillomavirus infections among cytologically normal women.J Infect Dis. 1994; 169: 235-240Crossref PubMed Scopus (622) Google Scholar, 16Melchers WJG Claas ECJ Quint WGV The use of the polymerase chain reaction to study the relationship between human papillomavirus infections and cervical cancer.Eur J Clin Microbiol Infect Dis. 1991; 10: 714-727Crossref PubMed Scopus (24) Google Scholar In the present study, we have used a novel, ultrasensitive SPF HPV general PCR assay. The SPF assay was shown to be more sensitive than other assays using general primer sets, My11/09 and GP5+/6+.18Kleter B van Doorn L-J ter Schegget J Schrauwen L van Krimpen C Burger M ter Harmsel B Quint W Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol. 1998; 153: 1731-1739Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar The other advantage of the SPF HPV general PCR assay is the combination with the reverse hybridization line probe assay permitting the simultaneous detection of 16 different HPV types individually in a single, quick assay. When this study was prepared for publication the HPV-LiPA has been further extended for genotyping of 25 different HPV types, including the 16 probes described in this report using a slight modification of the primer content.17Kleter B van Doorn L-J Schrauwen L Molijn A Sastrowijoto S ter Schegget J Lindeman J ter Harmsel B Burger M Quint W Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.J Clin Microbiol. 1999; 37: 2508-2517PubMed Google Scholar Recently, a phylogenetic tree based on sequence homology was developed in which HPV types were classified in groups of high risk (HPVs 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58) and low risk (HPVs 6, 11, 40, 42, 43, and 44).5Chan SY Delius H Halpern AL Bernard HU Analysis of genomic sequences of 95 papillomavirus types: uniting typing, phylogeny, and taxonomy.J Virol. 1995; 69: 3074-3083Crossref PubMed Google Scholar The classification of HPV type as high risk was further substantiated by follow-up studies showing that the persistent infection with high risk HPV correlated strongly with progression of cervical intraepithelial neoplasia.8Remmink AJ Walboomers JMM Helmerhorts TJM Voorhorts FJ Roozendaal L Risse EKJ Meijer CJLM Kenemans P The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: natural history up to 36 months.Int J Cancer. 1995; 61: 306-311Crossref PubMed Scopus (342) Google Scholar, 15Hildesheim A Schiffman MH Gravitt PE Glass AG Greer CE Zhang T Scott DR Rush BB Lawler P Sherman ME Kurman RJ Manos MM Persistence of type-specific human papillomavirus infections among cytologically normal women.J Infect Dis. 1994; 169: 235-240Crossref PubMed Scopus (622) Google Scholar, 27Hirschowitz L Raffle AE Mackenzie EFD Hughes AO Long term follow-up of women with borderline cervical smear result: effects of age and viral infection on progression to high grade dyskaryosis.Br Med J. 1992; 304: 1209-1212Crossref PubMed Scopus (44) Google Scholar, 31Ranst van M Kaplan JB Burk RD Phylogenetic classification of human papillomaviruses: correlation with clinical manifestation.J Gen Virol. 1992; 73: 2653-2660Crossref PubMed Scopus (192) Google Scholar In the current study, 13 cases with single or multiple infection of low risk HPV was detected and 77 cases with single or multiple infection of high risk HPVs or multiple infection of combined high risk and low risk HPV was detected. The detection of high risk HPVs increased with the severity of the lesion, from 38% in ASCUS up to 84% of the smears with HSIL. This finding illustrates that HPV infection, especially high risk HPV infection, is important for progression of cervical dysplasia. We were able to perform a repeat HPV testing on a cohort of 12 women after a 3-month follow-up. The results of the second HPV testing correlated completely with the results of the first HPV testing indicating that, SPF-HPV-LiPA is a highly sensitive, specific, and reproducible assay. We found a high incidence of HPV in women with normal cervical smears (38%) or with atypical squamous cells of undetermined significance in their cervical smears (51%) compared to other data obtained in the Dutch population or European women.16Melchers WJG Claas ECJ Quint WGV The use of the polymerase chain reaction to study the relationship between human papillomavirus infections and cervical cancer.Eur J Clin Microbiol Infect Dis. 1991; 10: 714-727Crossref PubMed Scopus (24) Google Scholar This reflects both the higher sensitivity of the system compared to other general HPV detection systems as described previously, as well as (and probably mainly) patient selection bias.9Bollen LJ Tjong-A-Hung SP van der Velden J Brouwer K Mol BW ten Kate FJ ter Schegget J Human papillomavirus deoxyribonucleic acid detection in mildly or moderately dysplastic smears: a possible method for selecting patients for colposcopy.Am J Obstet Gynecol. 1997; 177: 548-553Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 32Qu W Jiang G Cruz Y Chang CJ Ho GY Klein RS Burk RD PCR detection of human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems.J Clin Microbiol. 1997; 35: 1304-1310PubMed Google Scholar Women were incorporated into the study who were referred to the gynecologist after at least two successive cervical smears containing cells consistent with atypical squamous cells of undetermined significance. Indeed, this apparent difference in prevalence rate was equalized in the higher grade lesions in which the prevalence of HPV was not different from those described in general. We found multiple HPV infections in 27% of the total scrapes analyzed, which accounted for 44% of the total amount of HPV positive scrapes. This prevalence is much higher than previously described in the Dutch population or in general.8Remmink AJ Walboomers JMM Helmerhorts TJM Voorhorts FJ Roozendaal L Risse EKJ Meijer CJLM Kenemans P The presence of persistent high-risk HPV genotypes in dysplastic cervical lesions is associated with progressive disease: natural history up to 36 months.Int J Cancer. 1995; 61: 306-311Crossref PubMed Scopus (342) Google Scholar, 14De Roda Husman AM Walboomers JMM van den Brule AJ Meijer CJLM Snijders PJF The use of general primers GP5 and GP6 elongated at their 3′-end with adjacent highly conserved sequences improves human papillomavirus detection by PCR.J Gen Virol. 1995; 76: 1057-1062Crossref PubMed Scopus (1112) Google Scholar, 16Melchers WJG Claas ECJ Quint WGV The use of the polymerase chain reaction to study the relationship between human papillomavirus infections and cervical cancer.Eur J Clin Microbiol Infect Dis. 1991; 10: 714-727Crossref PubMed Scopus (24) Google Scholar, 24Walboomers JMM De Roda Husman AM van den Brule AJC Snijders PJF Meijer CJLM Detection of genital human papillomavirus infections: critical review of methods and prevalence studies in relation to cervical cancer. Human Papillomavirus and Cervical Cancer: Biology and Immunology.in: Stern PL Stanley MA Oxford University Press, Oxford1994: 41-71Google Scholar The difference may be due to the high sensitivity of the method used in the current study. The major advantage of the SPF-HPV-LiPA is the possibility to discriminate very easily whether the amplification has resulted in heterogeneous PCR clusters, which cannot be differentiated by conventional hybridization methods used for genotyping general PCR products. The results indicate that the incidence of multiple infections has been highly underestimated. Kleter et al17Kleter B van Doorn L-J Schrauwen L Molijn A Sastrowijoto S ter Schegget J Lindeman J ter Harmsel B Burger M Quint W Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus.J Clin Microbiol. 1999; 37: 2508-2517PubMed Google Scholar reported an incidence of multiple HPV infections in 34% of cervical scrapes obtained from women with mild or moderate dysplasia using the SPF-HPV-LiPA. Very recently, a similar reverse line blot detection method has been described by Gravitt et al33Gravitt PE Peyton CL Apple RJ Wheeler CM Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.J Clin Microbiol. 1998; 36: 3020-3027PubMed Google Scholar in which 27 HPV types could be identified in a single blot system. Although this genotyping system appears to have similar specificity as the SPF-HPV-LiPA, Gravitt et al33Gravitt PE Peyton CL Apple RJ Wheeler CM Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.J Clin Microbiol. 1998; 36: 3020-3027PubMed Google Scholar found only 8.5% of multiple infections among all HPV positive specimens. The main difference between the HPV-LiPA and the reverse line blot detection method, as described by Gravitt et al,33Gravitt PE Peyton CL Apple RJ Wheeler CM Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.J Clin Microbiol. 1998; 36: 3020-3027PubMed Google Scholar is the primary step of PCR amplification. Gravitt et al33Gravitt PE Peyton CL Apple RJ Wheeler CM Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method.J Clin Microbiol. 1998; 36: 3020-3027PubMed Google Scholar used the My11/09 general PCR as initial step which proved to be less sensitive than the SPF HPV general PCR, especially for HPV types 16, 35, and 45, which may have influenced the rate of detection of multiple infections,18Kleter B van Doorn L-J ter Schegget J Schrauwen L van Krimpen C Burger M ter Harmsel B Quint W Novel short-fragment PCR assay for highly sensitive broad-spectrum detection of anogenital human papillomaviruses.Am J Pathol. 1998; 153: 1731-1739Abstract Full Text Full Text PDF PubMed Scopus (452) Google Scholar although a difference in patient selection may also have affected the prevalence of multiple infections. In conclusion, the SPF-HPV-LiPA system for the detection and genotyping of HPV infections proved to be a sensitive, specific, and simple assay. This combined detection-genotyping assay makes mass screening of cervical scrapes in principle accessible for routine clinical practice and research applications.
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