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The diagnostics and prevalence of genital human papillomavirus (HPV) infection in Hungary
European Journal of Obstetrics & Gynecology and Reproductive Biology 100 (2002) 231–236
The diagnostics and prevalence of genital human papillomavirus (HPV) infection in Hungary Laszlo Kornyaa, Imre Csehb, Judit Deakc, Mihaly Bakd, Vilmos Fulopb,* a
b
Peterfy Hospital (PH), Budapest, Hungary Department of Obstetrics and Gynecology, Haynal Imre University of Health Sciences (HIETE), Budapest, Hungary c Central Clinical Microbiological Laboratory, Szent Gyo¨rgyi Albert University (SZOTE), Szeged, Hungary d Hungarian Oncological Institute (OOI), Budapest, Hungary Received 6 December 1999; accepted 2 July 2001
Abstract Objective: To show the prevalence and determine the type of human papillomavirus (HPV) in healthy women of reproductive age in Hungary. Study Design: We determined HPV nucleic acid using the Digene Hybrid Capture HPV-DNA assay from endocervical swabs of 1121 volunteer women of reproductive age. With the help of the hybridization antibody capture test we determined 14 HPV types (low risk, intermediate and high risk). Results: HPV prevalence was 17.5% considering the whole material. At the Szeged center 27.6% of the women screened were HPV positive, whereas at the three centers in Budapest, HPV prevalence did not exceed 15% in either of them. With a cytological examination out of 1100 cases, 117 (10.6%) were found to be HPV infected. The virus infection could be shown out of 1018 non-malignant cytologies in 60 (5.9%) cases and from 82 epithelial lesions 57 (69.5%) were infected. The cytological and molecular HPV diagnoses showed a significant relation to each other (P < 0:001). The cytological method showed HPV infections with a low degree of efficiency (sensitivity: 23.8%). On the other hand, the specificity (92.2%) is an acceptable method for the real negativity of the light microscopic HPV infection. Conclusions: These facts mean regarding the detection of HPV-DNA genoms that HPV positive cytological reports are false negative and in dysplasias are false positive. Since in HPV infected women the development of CIN is a great risk, it is advisable to carry out the HPV determination and typing in the so-called ‘‘endangered’’ groups. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human papillomavirus; Cervical intraepithelial neoplasia; HPV prevalence; HPV-DNA hybrid capture assay; Cervix cytological classification (Bethesda)
1. Introduction The human papillomavirus (HPV) is sexually transmitted and its lesion formed is considered sexually transmitted disease (STD) [1,2]. The experiences in epidemiology and molecular diagnostics supported the idea that the sexually transmitted HPVs play a leading role in the ethiology of cervical carcinoma. Meisels proposed first that cervical intraepithelial neoplasia (CIN) may be the consequence of papillomavirus infection [3]. Zur Hausen reported in the same year that HPV epithelialis or fibroepitheliasis induces proliferation [4]. HPV virus has more than 70 versions and more than 20 of them can be detected in human genitals. The virus types are classified into three categories: 1. high risk HPV types: 16, 18, 45, 56, etc.; 2. intermediate risk types: 31, 33, 35, 51, 58; 3. low malignancy types: 6, 11, 42, 43, 44. *
Corresponding author. Tel.: þ36-1-239-86-24; fax: þ36-1-239-86-24.
On the basis of clinical manifestation three types of the infection can be distinguished (clinical, subclinical, latent). In its latent phase there are no symptoms or morphological changes and a diagnosis can be established only with the help of the HPV-DNA hybridization technique. In the subclinical form usually both condyloma acuminatum or micropapillomatosis are manifested. The epidemiological status of STDs shows various pictures in different countries of the world and even in different populations of a certain country. It is possible that these differences in HPV infections are valid for different home populations. Only sporadic data were available in Hungary so far in connection with the prevalence of HPV. Therefore, the aim of this study was to screen the proportion of HPV infections furthermore determine the risks factors of the infections. Additionally, this study has also focused on the comparison of the results originated from cervix cytological examinations and HPV-DNA hybrid capture method.
0301-2115/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 1 ) 0 0 4 7 4 - 2
232
L. Kornya et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 100 (2002) 231–236
2. Materials and methods HPV-DNA screening were performed for women in reproductive age at Department of Obstetrics and Gynecology, Haynal Imre University of Health Sciences (HIETE); Department of Obstetrics and Gynecology, Szent Gyo¨ rgyi Albert University (SZOTE); Gynecological Department, Hungarian Oncological Institute (OOI) and Department of Gynecology and Obstetrics, Peterfy Hospital. The SZOTE was the only rural center while all other three centers were from our capital, Budapest. Of the 1121 women, 1100 were evaluated.
labeled with alkaline phosphatase, specific to the DNA:RNA hybrids. The substrate of the alkaline phosphatase conjugate is suitable for signal amplified chemiluminescence measuring. The light emission relative light unit (RLU) produced by the splitting of the substrate can be measured in units in luminometres. The degree of light emission indicates the presence or lack of HPV-DNA in the sample. The value lower than the cut-off value indicates the lack of HPV-DNA, while the RLU at the cut-off value or above it indicates the presence of HPV-DNA. The measured RLU values refer to the amount of HPV-DNA which is present in the sample. The degree of HPV infection can be characterized with one to three crosses [9].
2.1. Epidemiological studies 2.3. Cytological examination We put down the data on separate forms of every volunteer participant after giving them detailed information and after their signing consent. We asked them questions about factors influencing HPV status: age, education (<8 grades, 8 grades of elementary school, secondary school — matriculation, higher education), starting time of sexual life, number of sexual partners (one partner only, more than one partner), method of contraception, previous abortions, number and date of births, gynecological complaints (yes or no: dysuria, vaginal discharge, itching, burning, dipareunia, etc.), and smoking history (yes or no; mild smoker: <10 cigarettes per day, medium smoker: 10–20 cigarettes per day, heavy smoker: >20 cigarettes per day). All results were compared with statistical analysis, respectively. Cytological, colposcopic and gynecological examinations were performed in each cases. Sampling needed for the HPV-DNA determination was done after cleaning the cervix with a swab. A sample was taken with a manufactured cotton tampon suitable for the transport nutrient solution. The cervical swab was placed into the tube containing the transport medium solution was kept at þ48C and taken to the Central Clinical Microbiological Laboratory of SZOTE where HPV was detected and its type was defined. 2.2. The principle of the method To perform the molecular diagnostic method the necessary samples were collected and transported according to the description of the manufacturer’s instruction of the kit (DIGENETM HPV) [5]. The Digene Hybrid Capture HPVDNA assay is a liquid hybridizing test which applies a signal amplifying chemiluminescence detection method for distinguishing 14 HPV types belonging to the low [5–7] intermediate and high risk groups [8]. The heat treated clinical samples containing the HPVDNA get hybridized with the specific cocktail containing HPV-RNA which comes with the kit. The DNA:RNA hybrids produced adhere to the wall of the test tube covered with anti-DNA:RNA monoclonal antibody (produced against the DNA:RNA hybrid). The hybrids on the wall in the next step get linked with the antibody or conjugate
Samples for cytology were taken prior to the HPV sampling. The Bethesda classification was used for cytology evaluation [7,10,11]. 2.4. Statistical analysis The comparison of data originated from HPV nucleic acid hybridizing method with the cytological results were done by using the t and chi-square tests. Data analysis was done with the SPSS program. In the cases of sensitivity, specificity, positive (PPV) and negative (NPV) predictive value and in the mistaken positive and negative cases we considered the hybrid capture method as a standard. We compared the methods with the help of the chi-square test. We indicated significant deviation in case of P < 0:05 [12]. 2.5. Demography Examinations were carried out in four centers. From the 1100 cases 193 were positive, which means a prevalence of 17.5%. The distribution of HPV infected in the different centers studied is very interesting. While 27.4% of the women screened at the Szeged (SZOTE) countryside center proved to be HPV positive, in none of the three Budapest centers HPV prevalence was higher than 15.0% (chi-square 31.5, P < 0:001). The high HPV prevalence in the countryside (Szeged) can be detected in both the low (45 cases ¼ 23:3%) and the high risk (116 cases ¼ 60:2%) HPV carrier category, in 32 (16.5%) cases low þ high risk types occurred together.
3. Results 3.1. Epidemiology The average age of HPV positive women was lower (28:47:9) than that of HPV negative women (32:69:7) (P < 0:001) (Table 1).
L. Kornya et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 100 (2002) 231–236
233
Table 1 The role of risk factors in the development of cervix carcinoma Number of women studied: 1100
HPV infected: 193
Average age S.D.
28.4 7.9
Education <8 grade 8 grade Matriculation Higher education
27 47 69 44
Starting time of sexual life (average age S.D.)
17.4 1.7
Number of sexual partners 1 >1 Method of contraception Pills Not used
HPV negative: 907 32.6 9.7 98 224 293 259 18.1 2.3
P-value 0.0001 NS
0.01
41 87
235 254
0.01
60 129
296 599
NS
Number of previous abortions 1 2 >2
12 2 0
65 29 9
NS
Number of births 1 2 >2
33 30 9
225 242 64
NS
Pregnancies in case history Yes No
111 62
631 198
0.001
Number of pregnancies 1 >1
115 58
385 444
0.01
Smoking history Yes No
75 118
218 689
0.01
Social situation Good Average Bad
74 109 6
410 455 30
NS
Lasting sexual relationship Yes No
51 180
299 570
0.001
The proportion of HPV positive and HPV negative women with the advancement of age was shifted to the advantage of women free from HPV infection. While in the age group bellow 20 years we could detect HPV-DNA in 32.1% of the participating women, in the age group of 20 and 30 years old the number of HPV infected decreased to a greater extent and only 10.4% of the women above 42 years were HPV infected. We have found an inverse proportion between social situation, education and HPV prevalence. In case of a low level of education (8 or fewer grades) the proportion of HPV infected was higher (19%), while among women with more education the number of HPV infected is lower (17%) (Table 1). Among those in bad financial situation (the average income per man-year in the family is not above
the minimal subsistence level of Hungary: 2,88,000 HUF) the proportion of HPV carriers is also higher than among those who considered their social situation good (the average income per man-year in the family is more than 5,75,000 HUF — data were obtained from the Central Statistical Office of Hungary in 2000) (Table 1). There was no significant difference with regard to education. We also recorded the beginning of sexual life and the number of sexual partners (we did not always get an answer for these questions). The relatively favorable influence of monogamy can be detected (Table 1). While in case of lasting sexual relationships (the respondent lives in a faithful, loving long standing relationship with her partner) the proportion of HPV infected was 14.5% (n ¼ 350), there were much more HPV positive cases 24.0% (n ¼ 750)
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Table 2 Comparison of HPV positive cytological diagnoses with hybrid capture DNA resultsa Bethesda diagnoses
Cervix cytological diagnoses
Negative
1018
Cytological diagnoses HPVþ 60 (49 þ 11)
Ascus-Agus
23
11 (7 þ 4)
Low grade SIL
52
39 (16 þ 23)
High grade SIL
7
7 (4 þ 3)
Total a
1100
False positive 49 Equal w. capture 11 False negative 87 False positive 7 Equal w. capture 4 False negative 3 False positive 16 Equal w. capture 23 False negative 0 False positive 4 Equal w. capture 3 False negative 0
Hybrid capture HPV-DNSþ þ147
þ14
þ29
þ3
þ193
117
L 39 H 91 M 17 L7 H5 M2 L7 H 19 M3 L0 H3 M0
871
9
23
4
907
L: low risk type; H: high risk type; M: moderate risk type.
among those with a higher number of sexual partners (the respondent has six or more sexual partners per month). Significant differences were recorded with regard to the increasing number of sexual partners and HPV infections (P < 0:001) (Table 1). We also found a higher proportion of HPV positive cases among smokers (n ¼ 293) (25.6%) while among non-smokers it was only 14.6% (n ¼ 807); the difference is significant (P < 0:01) (Table 1). We also examined the occurrence of HPV during pregnancy. It seems that also pregnancy may be a factor influencing HPV positivity (111 infected out of 742 pregnant — 14.5% versus 62/260 not pregnant — 23.8%). Significant differences were found between those who have already been pregnant and who were not pregnant yet (P < 0:001) (Table 1). When the number of pregnancies increased the number of HPV infections gradually decreased. 3.2. Correlation between cervical cytology and HPV The cytology was negative in 1018 cases. Ascus-Agus occurred in 23 cases, low grade SIL in 52, and high grade SIL in 7 cases. Carcinoma did not occur. The difference between the distribution of HPV infections in Szeged and in Budapest also deserves attention. With a cytological examination, from 328 cases in Szeged 17.7% (58 cases) were HPV positive and from 772 samples in Budapest 7.9% (61 cases) were positive. On the other hand, molecular HPV-DNA expression in Szeged and Budapest HPV infections in 27.4% (90 cases) and in 13.3% (103 cases), respectively. We have summarized the results of Bethesda diagnoses and of hybrid capture HPV-DNA hybridizations in Table 2. HPV infection had the following distribution on the basis of cytological examinations: from the 1100 cases we detected morphological changes associated with HPV with the help of cytological examination in 117 (10.6%) cases. Virus infection could be detected in 60 cases (5.9%) from the
1018 cases where there was a negative cytological result and in 57 (11 þ 39 þ 7) (69.5%) cases from the 82 (23 þ 52 þ 7) cases of SIL lesions. HPV infection was most frequent with low grade SIL cases (slight dysplasia, CIN I) (39 positive cytological results from 52 cases). On the other hand, HPV-DNA method proved to be positive in 193 cases (17.5%). The 117 positive HPV cytological diagnoses were in agreement with the results of molecular biological HPV identification in 41 (11 þ 4 þ 23 þ 3) cases (35%). The number of false positive cases was 76 (49 þ 7 þ 16 þ 4) (65%). Regarding the 59 cases of pathological (low (52) and high grade (7) SIL) lesions we detected HPV infection with the cytological method in 46 (39 þ 7) cases and with nucleic acid hybridization in 32 cases (29 þ 3). In the negative or intact swabs the positive HPV cytological cases are false negative compared to the molecular HPV-DNA positive cases (60 versus 147; false negative: 87), but in dysplasias they are overvalued (46 versus 32; false negative: 0) (Table 2). We found out that cytological and hybrid capture HPV identifications are in significant connection with each other (chi-square 45.9, P < 0:001) (Table 2). The cytological method indicates HPV positive infections with a low level of efficiency.
4. Comment The international literature gives account of a 2–48% occurrence of human papillomavirus, difference may be shown even in the different regions of the country. It is an internationally accepted fact that HPV is sexually transmitted and the different lesions formed are considered STD. The differences in the incidence of HPV are also brought into connection with the different sexual licentiousness [6,13–17]. We found an interesting difference in the geographical distribution of HPV infections. We detected a much higher
L. Kornya et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 100 (2002) 231–236
proportion of HPV infection among woman screened in the countryside, than among those of the capital. The reason for this might not be the use of oral contraceptives, so much more as we did not find significant difference in HPV infection between oral contraceptive users and non-users. However, this geographical distribution may have another explanation. Szeged where the SZOTE is located is a bordering town, the southern gate of Hungary. There is a big transit-commuting traffic through the town between the Balkan countries and western Europe (the commuters (or transient guests) are consisted of mainly Turkish and Yugoslav workers). Therefore, the sexual looseness and activity is much higher in Szeged than in those regions of Budapest which are represented by the three capital centers. It is quite possible that significantly more women with six or more sexual partners per month were included in the study in our rural center than in the capital ones. In our study the average age of the patients was 32 S:D: 9:5 years. Significant difference was found between the ages of HPV infected and non infected women. Our study has shown that the HPV infection occurs mostly in the age group of 20–22 and its tendency decreases in advanced ages. Our results justify that further factors promoting HPV infection are smoking, age and social situation. Based upon the studies performed in four Hungarian centers significant difference was found with regard to the beginning of sexual life. Significant difference was found in respect to smoking between the HPV infected and non infected Hungarian women (P < 0:001). Based upon our study significant difference was found between the women with one sexual partner and those with several sexual partners (P < 0:01). Significant difference was found between the HPV infected women who conduct lasting sexual relationships and those who have unstable changing relationships. Significant difference was also found between women who changed their partners lately and those who have permanent partners. Similarly, significant difference was detected between married women (15.1%; 550/83) and the unmarried ones (30.7%; 345/ 106). It is an important result of our study that there is a significant connection between the cytological and hybrid capture determinants. To sum it up we can declare that the identification of cervical HPV infection can be accepted adequately only on the basis of results of molecular biological processes. Therefore, the Hybrid Capture HPV-DNA assay has to be the gold standard. Cervix cytological examination is a reliable method for detecting both the pathological lesions of the epithelium and the dysplasia of the cervix. The suspicion of HPV infection with negative (P1–P2) swabs and low grade SIL (P3) indicates a control examination after treatment. In case of a persistent HPV infection molecular biological HPV virus type identification is recommended. In case of a high grade SIL (CIN II, P3 and CIN III and in situ P4) the performance of conization is necessary apart from the HPV infection [9,18–20].
235
The further correct cooperation of gynecologists, pathologists and virologists is the pledge of the radical decrease of cervix cancer morbidity and mortality in Hungary as well. According to the studies in case of women infected with HPV the danger of the formation of CIN is 10 times higher than in case of women without the infection. HPV screening test is important also from a virological point of view, first of all in the risk groups. HPV identification is also advisable in all the other cases, when neither the clinical examination nor the colposcopic image show an inflammation but there is a P3 cytological diagnosis. The result of HPV detection may be decisive in such a case: in case of the identification of an oncogenic HPV type cytological repetition and the clearing up treatment applied so far have to be avoided and an immediate histological examination is justified. As in the case of HPV infected women there is a higher risk of the development of CIN, it is advisable to perform HPV detection and type identification in the ‘‘high risk’’ group. The molecular virological result may have prognostic and therapeutic consequences as well.
Acknowledgements The authors are very grateful to Professor Ross S. Berkowitz, MD for his revision of this manuscript. The authors are also beholden to Tamas Pulay, MD (OOI), Janos Szolosi, MD (SZOTE) and their colleagues for taking samples and to Mr. Tibor Nyari for his statistical work, Judit Jakab, Judit Jarmai, and Andrea Thurner for their organizational and technical works in helping of editorial efforts.
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[9] Ferenczy A, Franco E, Arseneau J, Wright TC, Richart RM. Diagnostic performance of hybrid capture human papillomavirus deoxyribonucleic acid assay combined with liquid-based cytological study. Am J Obstet Gynecol 1996;175:651–6. [10] Broder S. The Bethesda system for reporting cervical/vaginal cytologic diagnoses — report of the 1991 Bethesda Workshop. JAMA 1992;267:1892–6. [11] Lundberg GD. The 1988 Bethesda system for reporting cervical/ vaginal cytological diagnosis. JAMA 1989;262:931–4. [12] Fleiss JL. Statistical and WHO International Agency for research on cancer. In: Berslow NE, Day NE, editors. Statistical methods in cancer research, vol. 1. Lyon, 1980. p. 1–19. [13] Petry KU, Bohmer G, Linge G, Llaubitz M, Maschek H. Die HPVDNA hybridiserung ermoglicht eine Differenzierung zervicaler Lasionen bei PAP III Zytobefunden. Geburthsh u Frauenheilk 1996;56:509–12. [14] Melkert WJP, Hopman E, van der Brule JCA, et al. Prevalence of HPV in cytomorphologically normal cervical smears, as determined
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by the polymerize chain reaction, is age-dependent. Int J Cancer 1993;53:919–23. Munoz N, Kato I, Bosch FX, et al. Risk factors for HPV-DNA detection in middle-aged women. Sex Transm Dis 1996;23:504–10. Bosch FX, Munoz N, de Sanjose S, et al. Risk factors for cervical cancer in Columbia and Spain. Int J Cancer 1992;52:750–58. Yuong KT, McNicol P, Beauvais J. Factors associated with human papillomavirus infection detected by polymerize chain reaction among urban Canadian aboriginal and non-aboriginal women. Sex Transm Dis 1997;24:293–8. Goff AB, Muntz HG, Bell AD, Wertheim I, Rice LW. Human papillomavirus typing in patients with Papanikolaou smears showing squamous atypia. Gynecol Oncol 1993;48:384–8. Bodo M. Lecture notes for specialized assistants in cytology. ESZKTI, 1991 [in Hungarian]. Schneider A, Meinhardt D, de Villiers EM, Gissmann L. Sensitivity of the cytologic diagnosis of cervical condyloma in comparison with HPV-DNA hybridization studies. Diagn Cytopathol 1987;3:250–5.
The diagnostics and prevalence of genital human papillomavirus (HPV) infection in Hungary Laszlo Kornyaa, Imre Csehb, Judit Deakc, Mihaly Bakd, Vilmos Fulopb,* a
b
Peterfy Hospital (PH), Budapest, Hungary Department of Obstetrics and Gynecology, Haynal Imre University of Health Sciences (HIETE), Budapest, Hungary c Central Clinical Microbiological Laboratory, Szent Gyo¨rgyi Albert University (SZOTE), Szeged, Hungary d Hungarian Oncological Institute (OOI), Budapest, Hungary Received 6 December 1999; accepted 2 July 2001
Abstract Objective: To show the prevalence and determine the type of human papillomavirus (HPV) in healthy women of reproductive age in Hungary. Study Design: We determined HPV nucleic acid using the Digene Hybrid Capture HPV-DNA assay from endocervical swabs of 1121 volunteer women of reproductive age. With the help of the hybridization antibody capture test we determined 14 HPV types (low risk, intermediate and high risk). Results: HPV prevalence was 17.5% considering the whole material. At the Szeged center 27.6% of the women screened were HPV positive, whereas at the three centers in Budapest, HPV prevalence did not exceed 15% in either of them. With a cytological examination out of 1100 cases, 117 (10.6%) were found to be HPV infected. The virus infection could be shown out of 1018 non-malignant cytologies in 60 (5.9%) cases and from 82 epithelial lesions 57 (69.5%) were infected. The cytological and molecular HPV diagnoses showed a significant relation to each other (P < 0:001). The cytological method showed HPV infections with a low degree of efficiency (sensitivity: 23.8%). On the other hand, the specificity (92.2%) is an acceptable method for the real negativity of the light microscopic HPV infection. Conclusions: These facts mean regarding the detection of HPV-DNA genoms that HPV positive cytological reports are false negative and in dysplasias are false positive. Since in HPV infected women the development of CIN is a great risk, it is advisable to carry out the HPV determination and typing in the so-called ‘‘endangered’’ groups. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Human papillomavirus; Cervical intraepithelial neoplasia; HPV prevalence; HPV-DNA hybrid capture assay; Cervix cytological classification (Bethesda)
1. Introduction The human papillomavirus (HPV) is sexually transmitted and its lesion formed is considered sexually transmitted disease (STD) [1,2]. The experiences in epidemiology and molecular diagnostics supported the idea that the sexually transmitted HPVs play a leading role in the ethiology of cervical carcinoma. Meisels proposed first that cervical intraepithelial neoplasia (CIN) may be the consequence of papillomavirus infection [3]. Zur Hausen reported in the same year that HPV epithelialis or fibroepitheliasis induces proliferation [4]. HPV virus has more than 70 versions and more than 20 of them can be detected in human genitals. The virus types are classified into three categories: 1. high risk HPV types: 16, 18, 45, 56, etc.; 2. intermediate risk types: 31, 33, 35, 51, 58; 3. low malignancy types: 6, 11, 42, 43, 44. *
Corresponding author. Tel.: þ36-1-239-86-24; fax: þ36-1-239-86-24.
On the basis of clinical manifestation three types of the infection can be distinguished (clinical, subclinical, latent). In its latent phase there are no symptoms or morphological changes and a diagnosis can be established only with the help of the HPV-DNA hybridization technique. In the subclinical form usually both condyloma acuminatum or micropapillomatosis are manifested. The epidemiological status of STDs shows various pictures in different countries of the world and even in different populations of a certain country. It is possible that these differences in HPV infections are valid for different home populations. Only sporadic data were available in Hungary so far in connection with the prevalence of HPV. Therefore, the aim of this study was to screen the proportion of HPV infections furthermore determine the risks factors of the infections. Additionally, this study has also focused on the comparison of the results originated from cervix cytological examinations and HPV-DNA hybrid capture method.
0301-2115/02/$ – see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 1 - 2 1 1 5 ( 0 1 ) 0 0 4 7 4 - 2
232
L. Kornya et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 100 (2002) 231–236
2. Materials and methods HPV-DNA screening were performed for women in reproductive age at Department of Obstetrics and Gynecology, Haynal Imre University of Health Sciences (HIETE); Department of Obstetrics and Gynecology, Szent Gyo¨ rgyi Albert University (SZOTE); Gynecological Department, Hungarian Oncological Institute (OOI) and Department of Gynecology and Obstetrics, Peterfy Hospital. The SZOTE was the only rural center while all other three centers were from our capital, Budapest. Of the 1121 women, 1100 were evaluated.
labeled with alkaline phosphatase, specific to the DNA:RNA hybrids. The substrate of the alkaline phosphatase conjugate is suitable for signal amplified chemiluminescence measuring. The light emission relative light unit (RLU) produced by the splitting of the substrate can be measured in units in luminometres. The degree of light emission indicates the presence or lack of HPV-DNA in the sample. The value lower than the cut-off value indicates the lack of HPV-DNA, while the RLU at the cut-off value or above it indicates the presence of HPV-DNA. The measured RLU values refer to the amount of HPV-DNA which is present in the sample. The degree of HPV infection can be characterized with one to three crosses [9].
2.1. Epidemiological studies 2.3. Cytological examination We put down the data on separate forms of every volunteer participant after giving them detailed information and after their signing consent. We asked them questions about factors influencing HPV status: age, education (<8 grades, 8 grades of elementary school, secondary school — matriculation, higher education), starting time of sexual life, number of sexual partners (one partner only, more than one partner), method of contraception, previous abortions, number and date of births, gynecological complaints (yes or no: dysuria, vaginal discharge, itching, burning, dipareunia, etc.), and smoking history (yes or no; mild smoker: <10 cigarettes per day, medium smoker: 10–20 cigarettes per day, heavy smoker: >20 cigarettes per day). All results were compared with statistical analysis, respectively. Cytological, colposcopic and gynecological examinations were performed in each cases. Sampling needed for the HPV-DNA determination was done after cleaning the cervix with a swab. A sample was taken with a manufactured cotton tampon suitable for the transport nutrient solution. The cervical swab was placed into the tube containing the transport medium solution was kept at þ48C and taken to the Central Clinical Microbiological Laboratory of SZOTE where HPV was detected and its type was defined. 2.2. The principle of the method To perform the molecular diagnostic method the necessary samples were collected and transported according to the description of the manufacturer’s instruction of the kit (DIGENETM HPV) [5]. The Digene Hybrid Capture HPVDNA assay is a liquid hybridizing test which applies a signal amplifying chemiluminescence detection method for distinguishing 14 HPV types belonging to the low [5–7] intermediate and high risk groups [8]. The heat treated clinical samples containing the HPVDNA get hybridized with the specific cocktail containing HPV-RNA which comes with the kit. The DNA:RNA hybrids produced adhere to the wall of the test tube covered with anti-DNA:RNA monoclonal antibody (produced against the DNA:RNA hybrid). The hybrids on the wall in the next step get linked with the antibody or conjugate
Samples for cytology were taken prior to the HPV sampling. The Bethesda classification was used for cytology evaluation [7,10,11]. 2.4. Statistical analysis The comparison of data originated from HPV nucleic acid hybridizing method with the cytological results were done by using the t and chi-square tests. Data analysis was done with the SPSS program. In the cases of sensitivity, specificity, positive (PPV) and negative (NPV) predictive value and in the mistaken positive and negative cases we considered the hybrid capture method as a standard. We compared the methods with the help of the chi-square test. We indicated significant deviation in case of P < 0:05 [12]. 2.5. Demography Examinations were carried out in four centers. From the 1100 cases 193 were positive, which means a prevalence of 17.5%. The distribution of HPV infected in the different centers studied is very interesting. While 27.4% of the women screened at the Szeged (SZOTE) countryside center proved to be HPV positive, in none of the three Budapest centers HPV prevalence was higher than 15.0% (chi-square 31.5, P < 0:001). The high HPV prevalence in the countryside (Szeged) can be detected in both the low (45 cases ¼ 23:3%) and the high risk (116 cases ¼ 60:2%) HPV carrier category, in 32 (16.5%) cases low þ high risk types occurred together.
3. Results 3.1. Epidemiology The average age of HPV positive women was lower (28:47:9) than that of HPV negative women (32:69:7) (P < 0:001) (Table 1).
L. Kornya et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 100 (2002) 231–236
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Table 1 The role of risk factors in the development of cervix carcinoma Number of women studied: 1100
HPV infected: 193
Average age S.D.
28.4 7.9
Education <8 grade 8 grade Matriculation Higher education
27 47 69 44
Starting time of sexual life (average age S.D.)
17.4 1.7
Number of sexual partners 1 >1 Method of contraception Pills Not used
HPV negative: 907 32.6 9.7 98 224 293 259 18.1 2.3
P-value 0.0001 NS
0.01
41 87
235 254
0.01
60 129
296 599
NS
Number of previous abortions 1 2 >2
12 2 0
65 29 9
NS
Number of births 1 2 >2
33 30 9
225 242 64
NS
Pregnancies in case history Yes No
111 62
631 198
0.001
Number of pregnancies 1 >1
115 58
385 444
0.01
Smoking history Yes No
75 118
218 689
0.01
Social situation Good Average Bad
74 109 6
410 455 30
NS
Lasting sexual relationship Yes No
51 180
299 570
0.001
The proportion of HPV positive and HPV negative women with the advancement of age was shifted to the advantage of women free from HPV infection. While in the age group bellow 20 years we could detect HPV-DNA in 32.1% of the participating women, in the age group of 20 and 30 years old the number of HPV infected decreased to a greater extent and only 10.4% of the women above 42 years were HPV infected. We have found an inverse proportion between social situation, education and HPV prevalence. In case of a low level of education (8 or fewer grades) the proportion of HPV infected was higher (19%), while among women with more education the number of HPV infected is lower (17%) (Table 1). Among those in bad financial situation (the average income per man-year in the family is not above
the minimal subsistence level of Hungary: 2,88,000 HUF) the proportion of HPV carriers is also higher than among those who considered their social situation good (the average income per man-year in the family is more than 5,75,000 HUF — data were obtained from the Central Statistical Office of Hungary in 2000) (Table 1). There was no significant difference with regard to education. We also recorded the beginning of sexual life and the number of sexual partners (we did not always get an answer for these questions). The relatively favorable influence of monogamy can be detected (Table 1). While in case of lasting sexual relationships (the respondent lives in a faithful, loving long standing relationship with her partner) the proportion of HPV infected was 14.5% (n ¼ 350), there were much more HPV positive cases 24.0% (n ¼ 750)
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Table 2 Comparison of HPV positive cytological diagnoses with hybrid capture DNA resultsa Bethesda diagnoses
Cervix cytological diagnoses
Negative
1018
Cytological diagnoses HPVþ 60 (49 þ 11)
Ascus-Agus
23
11 (7 þ 4)
Low grade SIL
52
39 (16 þ 23)
High grade SIL
7
7 (4 þ 3)
Total a
1100
False positive 49 Equal w. capture 11 False negative 87 False positive 7 Equal w. capture 4 False negative 3 False positive 16 Equal w. capture 23 False negative 0 False positive 4 Equal w. capture 3 False negative 0
Hybrid capture HPV-DNSþ þ147
þ14
þ29
þ3
þ193
117
L 39 H 91 M 17 L7 H5 M2 L7 H 19 M3 L0 H3 M0
871
9
23
4
907
L: low risk type; H: high risk type; M: moderate risk type.
among those with a higher number of sexual partners (the respondent has six or more sexual partners per month). Significant differences were recorded with regard to the increasing number of sexual partners and HPV infections (P < 0:001) (Table 1). We also found a higher proportion of HPV positive cases among smokers (n ¼ 293) (25.6%) while among non-smokers it was only 14.6% (n ¼ 807); the difference is significant (P < 0:01) (Table 1). We also examined the occurrence of HPV during pregnancy. It seems that also pregnancy may be a factor influencing HPV positivity (111 infected out of 742 pregnant — 14.5% versus 62/260 not pregnant — 23.8%). Significant differences were found between those who have already been pregnant and who were not pregnant yet (P < 0:001) (Table 1). When the number of pregnancies increased the number of HPV infections gradually decreased. 3.2. Correlation between cervical cytology and HPV The cytology was negative in 1018 cases. Ascus-Agus occurred in 23 cases, low grade SIL in 52, and high grade SIL in 7 cases. Carcinoma did not occur. The difference between the distribution of HPV infections in Szeged and in Budapest also deserves attention. With a cytological examination, from 328 cases in Szeged 17.7% (58 cases) were HPV positive and from 772 samples in Budapest 7.9% (61 cases) were positive. On the other hand, molecular HPV-DNA expression in Szeged and Budapest HPV infections in 27.4% (90 cases) and in 13.3% (103 cases), respectively. We have summarized the results of Bethesda diagnoses and of hybrid capture HPV-DNA hybridizations in Table 2. HPV infection had the following distribution on the basis of cytological examinations: from the 1100 cases we detected morphological changes associated with HPV with the help of cytological examination in 117 (10.6%) cases. Virus infection could be detected in 60 cases (5.9%) from the
1018 cases where there was a negative cytological result and in 57 (11 þ 39 þ 7) (69.5%) cases from the 82 (23 þ 52 þ 7) cases of SIL lesions. HPV infection was most frequent with low grade SIL cases (slight dysplasia, CIN I) (39 positive cytological results from 52 cases). On the other hand, HPV-DNA method proved to be positive in 193 cases (17.5%). The 117 positive HPV cytological diagnoses were in agreement with the results of molecular biological HPV identification in 41 (11 þ 4 þ 23 þ 3) cases (35%). The number of false positive cases was 76 (49 þ 7 þ 16 þ 4) (65%). Regarding the 59 cases of pathological (low (52) and high grade (7) SIL) lesions we detected HPV infection with the cytological method in 46 (39 þ 7) cases and with nucleic acid hybridization in 32 cases (29 þ 3). In the negative or intact swabs the positive HPV cytological cases are false negative compared to the molecular HPV-DNA positive cases (60 versus 147; false negative: 87), but in dysplasias they are overvalued (46 versus 32; false negative: 0) (Table 2). We found out that cytological and hybrid capture HPV identifications are in significant connection with each other (chi-square 45.9, P < 0:001) (Table 2). The cytological method indicates HPV positive infections with a low level of efficiency.
4. Comment The international literature gives account of a 2–48% occurrence of human papillomavirus, difference may be shown even in the different regions of the country. It is an internationally accepted fact that HPV is sexually transmitted and the different lesions formed are considered STD. The differences in the incidence of HPV are also brought into connection with the different sexual licentiousness [6,13–17]. We found an interesting difference in the geographical distribution of HPV infections. We detected a much higher
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proportion of HPV infection among woman screened in the countryside, than among those of the capital. The reason for this might not be the use of oral contraceptives, so much more as we did not find significant difference in HPV infection between oral contraceptive users and non-users. However, this geographical distribution may have another explanation. Szeged where the SZOTE is located is a bordering town, the southern gate of Hungary. There is a big transit-commuting traffic through the town between the Balkan countries and western Europe (the commuters (or transient guests) are consisted of mainly Turkish and Yugoslav workers). Therefore, the sexual looseness and activity is much higher in Szeged than in those regions of Budapest which are represented by the three capital centers. It is quite possible that significantly more women with six or more sexual partners per month were included in the study in our rural center than in the capital ones. In our study the average age of the patients was 32 S:D: 9:5 years. Significant difference was found between the ages of HPV infected and non infected women. Our study has shown that the HPV infection occurs mostly in the age group of 20–22 and its tendency decreases in advanced ages. Our results justify that further factors promoting HPV infection are smoking, age and social situation. Based upon the studies performed in four Hungarian centers significant difference was found with regard to the beginning of sexual life. Significant difference was found in respect to smoking between the HPV infected and non infected Hungarian women (P < 0:001). Based upon our study significant difference was found between the women with one sexual partner and those with several sexual partners (P < 0:01). Significant difference was found between the HPV infected women who conduct lasting sexual relationships and those who have unstable changing relationships. Significant difference was also found between women who changed their partners lately and those who have permanent partners. Similarly, significant difference was detected between married women (15.1%; 550/83) and the unmarried ones (30.7%; 345/ 106). It is an important result of our study that there is a significant connection between the cytological and hybrid capture determinants. To sum it up we can declare that the identification of cervical HPV infection can be accepted adequately only on the basis of results of molecular biological processes. Therefore, the Hybrid Capture HPV-DNA assay has to be the gold standard. Cervix cytological examination is a reliable method for detecting both the pathological lesions of the epithelium and the dysplasia of the cervix. The suspicion of HPV infection with negative (P1–P2) swabs and low grade SIL (P3) indicates a control examination after treatment. In case of a persistent HPV infection molecular biological HPV virus type identification is recommended. In case of a high grade SIL (CIN II, P3 and CIN III and in situ P4) the performance of conization is necessary apart from the HPV infection [9,18–20].
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The further correct cooperation of gynecologists, pathologists and virologists is the pledge of the radical decrease of cervix cancer morbidity and mortality in Hungary as well. According to the studies in case of women infected with HPV the danger of the formation of CIN is 10 times higher than in case of women without the infection. HPV screening test is important also from a virological point of view, first of all in the risk groups. HPV identification is also advisable in all the other cases, when neither the clinical examination nor the colposcopic image show an inflammation but there is a P3 cytological diagnosis. The result of HPV detection may be decisive in such a case: in case of the identification of an oncogenic HPV type cytological repetition and the clearing up treatment applied so far have to be avoided and an immediate histological examination is justified. As in the case of HPV infected women there is a higher risk of the development of CIN, it is advisable to perform HPV detection and type identification in the ‘‘high risk’’ group. The molecular virological result may have prognostic and therapeutic consequences as well.
Acknowledgements The authors are very grateful to Professor Ross S. Berkowitz, MD for his revision of this manuscript. The authors are also beholden to Tamas Pulay, MD (OOI), Janos Szolosi, MD (SZOTE) and their colleagues for taking samples and to Mr. Tibor Nyari for his statistical work, Judit Jakab, Judit Jarmai, and Andrea Thurner for their organizational and technical works in helping of editorial efforts.
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