Concomitant infections with human papillomavirus and various mycoplasma and ureaplsasma species in women with abnormal cervical cytology

· Advances in Medical Sciences · Vol. 56 · 2011 · pp 299-303 · DOI: 10.2478/v10039-011-0028-9 © Medical University of Bialystok, Poland

Concomitant infections with human papillomavirus and various mycoplasma and ureaplasma species in women with abnormal cervical cytology Biernat-Sudolska M1*, Szostek S1, Rojek-Zakrzewska D1, Klimek M3, Kosz-Vnenchak M1,2 1 Department of Virology, Chair of Microbiology, Jagiellonian University Medical College, Krakow, Poland 2 Laboratory of Molecular Genetics and Virology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland 3 Centre of Oncology, Maria Skłodowska-Curie Memorial Institute, Krakow, Poland

* CORRESPONDING AUTHOR: Department of Virology, Chair of Microbiology Jagiellonian University Medical College, 18 Czysta Street, 31-121 Krakow, Poland Phone. +48 12 634-54-00; Fax. +48 12 423-39-24 e-mail: [email protected] (Małgorzata Biernat-Sudolska)

Received 15.09.2010 Accepted 09.05.2011 Advances in Medical Sciences Vol. 56 · 2011 · pp 299-303 DOI: 10.2478/v10039-011-0028-9 © Medical University of Bialystok, Poland

ABSTRACT Purpose: The objective of the present study is to verify possible association between infections with mycoplasmas and ureaplasmas and the presence of HPV infections in women diagnosed with abnormal cervical cytology. Material/Methods: The investigation included 387 non-pregnant women among whom: 62 were diagnosed with ASCUS, 167 with LSIL, 27 with HSIL, 49 with cervical carcinomas, and 82 females with normal cytology . The presence of HPV infection and identification of both ureaplasma and mycoplasma were confirmed by PCR using specific primers. Results: HPV infections were demonstrated in 156 females (40%), with mycoplasmas and/or ureaplasmas were confirmed in 93 cases (24%). In HPV-positive patients, infections with mycoplasmas/ureaplasmas were more frequent, particularly for ureaplasmas (U. urealyticum p=0.004, U. parvum p=0.027). The percentage of females infected with U. urealyticum significantly increased in women diagnosed with cervical carcinoma as compared to controls. The statistical analysis demonstrated that the risk of HPV infection while already infected with any of the four analyzed species of Mycoplasmataceae increased two-fold. With concomitant of U. urealyticum infection, the risk of HPV infection was 4.7-fold greater than in the absence U. urealyticum infection. Conclusion: Since the presence of U.urealyticum associates significantly with the HPV infection, genotyping of the ureaplasma species should be recomended. Key words: HPV, mycoplasma, ureaplasma, squamous intraepithelial lesions

INTRODUCTION Human papillomavirus (HPV) infections strongly associate with cervical carcinogenesis [1]. It appears that a significant stimulating effect on the process of carcinogenesis may be also exerted by other factors, such as concomitant sexually transmitted diseases [2,3]. Microorganisms from the family Mycoplasmataceae (mycoplasmas and ureaplasmas) represent atypical lacking cellular wall bacteria that either closely adhere to epithelial cells of the urinary tract or penetrate these cells.

The presence of different members of the Mycoplasma species (M. hominis - Mh, M. genitalium - Mg) is also associated with various urogenital diseases [4-6]. These microorganisms adhere to various other human cells, penetrate the cells and disturb the normal cell metabolism, and cause DNA damage (progressive chromosomal loss and translocations). These bacteria may also trigger chronic inflammation of the urinary tract. Mycoplasma-mediated oncogenesis has a long latency and requires a chronic persistent infection (close interaction with mammalian cells for long-period of time without causing acute cytopathic effects) [7-10]. Lukic et al. suggested that

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Table 1. Percentage of women infected with HPV and Ureaplasma urealyticum (Uu), Ureaplasma parvum (Up), Mycoplasma hominis (Mh), Mycoplasma genitalium (Mg) and total mycoplasmas and ureaplasmas (M+U) depending on clinical diagnosis. Group

No. of investigated women

Percentage of infected women HPV

Uu

Up

Mh

Mg

M+U

ASCUS

62

32

3

21

2

19

32

LSIL

167

42

4

19

1

4*

47

HSIL

27

59

7

26

0

15

41

Cervical carcinoma

49

92

10

10

0

0

18

Control – normal cytology

82

6

1

13

1

7**

39

Total

387

40

4

18

1

9***

36

* No. of subjects = 52; ** No. of subjects = 28; *** No. of subjects = 218

the presence of U. urealyticum may play a role both in HPV persistence and initiation of viral cellular anomalies [3]. These initial processes may lead to the HPV–dependent malignant transformation. The main goal of the present investigations was to show an association between presence of urogenital mycoplasmas and HPV infection in women with abnormal cytology of various grades.

MATERIAL AND METHODS The investigations included 387 non-pregnant women. The controls consisted of 82 females at the mean age of 35.37±10.01 years, without atypical lesions of the cervix. The remaining 305 women were divided into four groups in keeping with the Bethesda classification system, depending on their cytology results. The groups included 62 women at the mean age of 34.56±10.8 years, in whom cytology demonstrated lesions described as ASCUS (atypical squamous cells of undetermined significance), 167 females at the mean age of 36.9±11.91 years with the diagnosis of low-grade squamous epithelial lesions (LSIL), 27 women at the mean age of 35.95±11.51 years diagnosed as high grade squamous epithelial lesions (HSIL) and 49 females at the mean age of 54.69 ±11.24 years diagnosed as cervical cancer, respectively. Material for examinations consisted of cervical smears collected using a cytobrush and collected to 5 ml of saline solution. The material in saline was frozen and stored until needed for determinations.

CaSki cells infected with HPV 16. The PCR conditions and amplification profile was described previously [14].

Mycoplasmas/ureaplasmas detection The presence of mycoplasmas and ureaplasmas and the quantitative assessment of infections were carried out by cultures using the PPLO medium [15] and BioMerieux tests. Species identification, both for ureaplasmas and for mycoplasmas, was done by PCR using the previously described two pairs of primers specific for the U. urealyticum and U. parvum genes [16]. The primers H1 and H2 for 16S rRNA allowed for detecting M. hominis, while the primers G3A and G3B served for amplification of the M. genitalium adhesin encoding sequence [17]. In view of the small amount of the isolated material available, tests for M. genitalium were performed in 218 females. As the positive control reference strains: U. urealyticum (ATCC 27816), U. parvum (ATCC 27815), M. hominis PG 21 (ATCC 23114) and M. genitalium DNA (ATCC 33530D) were used.

Statistical analysis Intergroup differences were analyzed using the Fisher’s exact test and the 2test. To determine the contribution of mycoplasmic and ureaplasmic infections to progression of cervical pathological lesions and the susceptibility to infections with HPV types of high oncogenic potential the age-adjusted logistic regression analysis was employed. The statistical analysis was performed using the STATA 8.0 software.

Detection of HPV DNA

RESULTS

DNA was isolated using Genomic Mini kit (A&A Biotechnology). Beta-actin PCR analysis was performed to confirm the presence of human DNA in all specimens [11]. The presence of HPV infections was detected by PCR using the consensus primers MY09/MY11 (specific for the viral L1 sequence) [12], followed by the pU-1M/pU-2R primers (specific for the HPV E6/E7 region of high oncogenic potential, i.e. HPV 16, 18, 31, 33, 35, 52, 58) [13]. The negative control was water, and the positive control was provided by

Infections with HPV were demonstrated in 156 females (40%). Infections with mycoplasma and/or ureaplasma were confirmed in 93 women; the group included 4% of subjects infected with U. urealyticum, 18% with U. parvum, 1% with M. hominis and 9% with M. genitalium. In 10 cases, we detected the presence of more than one species belonging to the Mycoplasmataceae. Nine patients were positive for two mycoplasma species; and one demonstrated the presence of all

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Figure 1. Proportion of HPV and Ureaplasma urealyticum infection in controls and study groups.

HPV: human papillomavirus; Uu: Ureaplasma urealyticum; ASCUS: atypical squamous cells of undetermined significance; LSIL: lowgrade squamous epithelial lesions; HSIL: high grade squamous epithelial lesions.

four species, examined. Statistically significant differences were shown in total prevalence rates of mycoplasmic and ureaplasmic infections as depending on clinical diagnosis (p=0.016; Tab.1). Mycoplasmic/ureaplasmic infections were significantly more common in females with lower grade cervical lesions (ASCUS + LSIL) as compared to women diagnosed as higher grade lesions (HSIL + cervical carcinoma; 2 p<0.05). The percentage of infected patients significantly decreased in the group with cervical carcinoma as compared to the controls (2p<0.05). In the 58 women positive for HPV urogenital mycoplasmas coinfections were detected. Among them U. parvum was detected in 35 patients, U.urealyticum in 12, M. genitalium, in 10 and M. hominis in one woman. We also analyzed concomitant infections with HPV and more than one mycoplasma/ureaplasma species. In three patients diagnosed as ASCUS, concurrent presence of HPV, U. urealyticum and M. genitalium was noted. In the group of females with LSIL, HPV infection concomitant with U. urealyticum and M. hominis was observed in one case and co-infection with U. parvum and M. genitalium in another female. In one woman with HSIL and in one with cervical cancer the sequences specific for U. urealyticum and U. parvum were detected. Females infected with HPV were older as compared to patients without such infections (mean age 40±14.29 vs. 37.13±11.52 years; p=0.109). The mean age of women infected with ureaplasmas and/or mycoplasmas was lower than the mean age of non-infected females (35.9±10.49 vs. 40.30±13.97 years; p=0.05). The percentage of patients infected with particular mycoplasma and ureaplasma species and HPV is presented in Tab. 1. In HPV-positive patients, infections with microorganisms from the Mycoplasmataceae family were more frequently confirmed (p=0.03); this was particularly true for ureaplasmas (U. urealyticum - p=0.004, and U. parvum - p=0.027). The prevalence of infections with HPV clearly increased with higher grades of cervical lesions demonstrated in cytology. While analyzing the prevalence rate of various mycoplasma

species in the controls and in particular investigated groups, we noted a growing tendency in the proportion of females infected with U.urealyticum (Fisher’s test, p=0.108; Fig. 1). The percentage significantly increased in women with cervical cancer as compared to the controls (2p<0.05, Tab. 1). Using the logistic regression method, it was demonstrated that both the risk of HPV-positive patients developing U. urealyticum infections and the risk of HPV infection occurring in females with prior U. urealyticum infections was 4.7-fold higher (p<0.008, 95 % CI: 1.496396-14.94592) and was maintained at the same level following the age-adjustment (p=0.05, 95 % CI: 0.973289-1.049262). The method also showed that the risk of a patient acquiring HPV infection while already infected with any of the four analyzed species increased by 2-fold (p<0.001, 95 % CI: 1.362031-3.507517), and the increase amounted to 2.5-fold following adjustment for age and diagnostic group.

DISCUSSION The results of the present study indicate a significant increase in the prevalence of HPV infections with a higher grade of cervical lesions. The observation does not differ from the results obtained by other authors and confirms the role of HPV in cervical cancer, however, the role of microorganisms from the family of Mycoplasmataceae in human cancer is still not commonly accepted, and still requires further investigation. In this respect, mycoplasma infections, especially of a chronic character, induce chromosomal alterations that may lead to transformation of mammalian cells [8,10,18,19]. The association between mycoplasma infections and the possibility of induction of benign neoplastic lesions in prostate cells cultured in vitro was strongly emphasized by Namiki et al. [20]. Huang et al. [9] found mycoplasmas in 55% of gastrointestinal cancers and in 45.5% of other confirmed carcinomas. Sasaki et al. [21], obtained similar results and estimated the percentage of mycoplasmic infections in ovarian cancers as 59.3% and in gastric cancers as 48%. The possible cooperation of ureaplasmas in cervical oncogenesis is rarely addressed in the literature, most likely in view of the accepted status of physiological flora components given to these microorganisms. Nevertheless, in our previous study, we demonstrated that in the presence of ureaplasmas, infections with highly oncogenic HPVs were almost two-fold more frequent (OR=1.79; 95% CI: 0.90-3.53; p=0.093) in women with LSIL-type lesions, and 4-fold higher in ASCUS, as compared to non-ureaplasma infected females (OR=4.12; 95% CI: 1.08-15.7; p=0.038) [22,23]. Therfore, our present study represents a logic continuation of our earlier work related to the role of urogenital mycoplasmas in the development of abnormal cervical lesions, including women with diagnosed HSIL and cervical carcinoma. Here we demonstrate a particularly high association between the prevalence of HPV infections and the presence of

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U.urealyticum. In patients infected with this species, the risk of them developing HPV infection increased 4.7-fold (p<0.001, 95 % CI: 1.362031-3.507517). Lukic et al. and Ekiel et al. also stressed the role of ureaplasmas as a possible cofactor interacting with HPV in the development of precancerous cervical lesions [3,24]. Lukic et al. using Mycoplasma IST 2 kit showed differences in frequency of ureaplasmas depending on cytological diagnosis [3]. Ekiel et al. in their study demonstrated that U.urealyticum was significantly more common in women with high oncogenic HPV infection and occurred more frequently in women with squamous intraepithelial lesions [24]. In our investigation, women with cervical cancer demonstrated a significant increase in the detection rate of U. urealyticum in comparison to women with normal cytology. On the other hand, the prevalence of U. parvum declined in cervical cancer. The frequency of U. urealyticum and U. parvum in the controls was 1% and 13%, respectively. This observation is in accordance with data from the literature on the subject that describe the U. parvum species as predominant with respect to incidence in the genital tract of sexually active women [17,25]. In our study, the percentage was identical for both species reaching 10% among females with cervical cancer. The results prove an evident increase in the prevalence of U. urealyticum and possibly support the association between the species and the process of oncogenesis initiated by HPV. It may probably be related to higher pathogenicity of this species, which was also shown in studies of neonatal respiratory tract infections [26-28]. In patients with cervical cancer, we did not detect infections with the Mycoplasma species (M. hominis, M. genitalium) although such infections were observed in other groups. Thus, it appears that these infections do not play any major role in cervical carcinoma development. Possibly the microorganisms instead play a role in the early phase of HPV infection, i.e. in viral penetration and survival, rather than affecting development of precancerous and cancerous cervical lesions. Pisani et al. nevertheless, emphasized an apparent association between mycoplasma infections and HPV [29]. Our data demonstrate that the prevalence of U. urealyticum in woman with HSIL and cervical cancer is higher in comparison to females with ASCUS and LSIL (p=0.05), and additionally it correlates well with the abnormalities found during cervical smear.

CONCLUSIONS The problem of coactivity of infectious factors in oncogenesis is still unclear and requires further investigations in the field of immunology, cytogenetics and microbiology. Although the role of HPV as an initiating factor in the process of cervical carcinogenesis is unquestionable, it has been suggested that HPV alone may not be sufficient to

promote malignant transformation. Other cofactors interacting with HPV could be involved in such a process. Our results demonstrate that ureaplasmas, especially U. urealyticum are strongly associated with HPV infection. It seems very important to detect and type these microorganisms to rapidly introduce treatment of infected women.

ACKNOWLEDGEMENTS Supported by a grant from the Ministry of Science and Higher Education, Poland NN401219034

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