Human Papillomaviruses and genital co-infections in gynaecological outpatients

  • Rosita Verteramo1,

    Affiliated with

    • Alessandra Pierangeli2,

      Affiliated with

      • Emanuela Mancini1,

        Affiliated with

        • Ettore Calzolari1,

          Affiliated with

          • Mauro Bucci2,

            Affiliated with

            • John Osborn3,

              Affiliated with

              • Rosa Nicosia4,

                Affiliated with

                • Fernanda Chiarini4,

                  Affiliated with

                  • Guido Antonelli2 and

                    Affiliated with

                    • Anna Marta Degener2Email author

                      Affiliated with

                      BMC Infectious Diseases20099:16

                      DOI: 10.1186/1471-2334-9-16

                      Received: 12 August 2008

                      Accepted: 12 February 2009

                      Published: 12 February 2009

                      Abstract

                      Background

                      High grade HPV infections and persistence are the strongest risk factors for cervical cancer. Nevertheless other genital microorganisms may be involved in the progression of HPV associated lesions.

                      Methods

                      Cervical samples were collected to search for human Papillomavirus (HPV), bacteria and yeast infections in gynaecologic outpatients. HPV typing was carried out by PCR and sequencing on cervical brush specimens. Chlamydia trachomatis was identified by strand displacement amplification (SDA) and the other microorganisms were detected by conventional methods.

                      Results

                      In this cross-sectional study on 857 enrolled outpatients, statistical analyses revealed a significant association of HPV with C. trachomatis and Ureaplasma urealyticum (at high density) detection, whereas no correlation was found between HPV infection and bacterial vaginosis, Streptococcus agalactiae, yeasts, Trichomonas vaginalis and U. urealyticum. Mycoplasma hominis was isolated only in a few cases both in HPV positive and negative women and no patient was infected with Neisseria gonorrhoeae.

                      Conclusion

                      Although bacterial vaginosis was not significantly associated with HPV, it was more common among the HPV positive women. A significant association between HPV and C. trachomatis was found and interestingly also with U. urealyticum but only at a high colonization rate. These data suggest that it may be important to screen for the simultaneous presence of different microorganisms which may have synergistic pathological effects.

                      Background

                      Infection with high grade Human papillomavirus (HPV) plays a central role in cervical carcinogenesis. Epidemiological and molecular investigations have shown unequivocally that high grade HPV infection is the main causal factor in initiating the progressive transformation that leads to cervical intraepithelial neoplasia and to cervical cancer [1, 2]. Many reports have demonstrated that HPV infections are mostly transitory and that only a small percentage of infections persist and may progress to dysplastic cervical lesions: type-specific HPV persistence is the strongest risk factor for cervical cancer [3]. The reasons for the variable natural history remain poorly understood but it has been generally accepted that different cofactors are involved in the development of dysplasia in HPV infected women. Factors, such as specific HPV genotype, viral load, and co-infection with more HPV types, are likely to be involved in the progression to precancerous cervical lesions [46]. In addition, environmental factors [79] including smoking, long term hormone contraceptives and co-infection with other microorganisms have also been investigated as potentially involved in the disease process. The sexually transmitted agents that have shown some evidence of an association with cervical dysplastic and neoplastic lesion are Chlamydia trachomatis [10] and Herpes simplex virus (HSV) [11, 12], although the specific role of these microorganisms in the pathogenesis of cervical cancer has not been completely clarified.

                      The aim of this cross-sectional study was to evaluate the prevalence of co-infection HPV and other genital microorganisms: Chlamydia trachomatis, Neisseria gonorrhoeae, Streptococcus agalactiae, Mycoplasma spp, Trichomonas vaginalis. Yeasts and bacterial vaginosis were also investigated.

                      Methods

                      Study population

                      Women attending clinics for routine gynaecologic care in the Department of Gynaecology, Perinatology and Child Health of "Sapienza" University in Rome, between 2000 and 2006 were included in this study. Informed consent was obtained from all women and this study was approved by the Ethics Committees and/or Institutional Review Boards of the participating institution. Cervical samples were taken by cytobrush from the ectocervix and from the endocervix for cytological analysis, detection of HPV DNA and bacteriological tests. Cytology was classified according to the Bethesda system, using standard forms and classified as negative, atypical squamous cell of undetermined significance (ASCUS), atypical glandular cell of undetermined significance (AGUS), low-grade intraepithelial lesion (LSIL) or high-grade (HSIL) categories [13]. All the samples were analysed independently by two cyto-technologists and the final diagnosis was based on the consensus of both. Women were eligible for the study if they: were not currently pregnant nor planning to become pregnant for a year; had an intact uterus and no current referral for hysterectomy; reported no use of vaginal medication in the previous 3 days; reported no treatment for cervical disease in the previous 6 months; answered a structured questionnaire on socio-demographic characteristics, sexual behaviour, reproductive history and smoking habits; provided samples adequate for PCR assays and sequencing. According to these criteria, 857 women were enrolled in this cross-sectional study.

                      HPV detection and typing

                      Cervical brush samples collected in Phosphate Saline Buffer (PBS) were centrifuged at low speed and the cell pellets underwent DNA extraction using QIAampTissue kit (Qiagen, Milan-Italy). Human Leukocyte Antigen (HLA)-specific primers were used in polymerase chain reaction (PCR) analysis to assess the suitability of the target DNA (9). A 450 bp fragment from the L1 region of HPV-DNA was amplified using the consensus primer MY09/11 as described elsewhere [14]. Four pairs of degenerated consensus primers (able to detect 36 HPV types) were used concomitantly to amplify the complete E6 gene and part of the E7 gene [15], thus allowing the detection of a broader range of HPV genotypes. The PCR products were detected by ethidium bromide staining after electrophoretic migration through 2% agarose gels.

                      HPV-positive reactions were purified using a QIAquick PCR purification kit (QIAGEN), and an automatic DNA sequencer (Applied Biosystems, Foster City, CA) was used for sequencing utilising the PCR forward primer(s). Sequence homology was determined using BLAST (NCBI of NIH, Bethesda, MD) and Clustal W (EMBL-EBI, Heidelberg-Germany) programs.

                      Detection of microorganisms

                      Streptococcus agalactiae

                      Streptococcus agalactiae (group B) strains were identified from clinical samples by being weakly beta-haemolytic on Columbia CNA Agar A (Oxoid). The suspected colonies were serologically confirmed (Lancefield's classification) [16, 17].

                      Chlamydia trachomatis

                      endocervical swabs were tested for the presence of C. trachomatis using the BD ProbeTec ET Assays (BDPT, Becton Dickinson and Company, Franklin Lakes, NJ) according to the manufacturer's instructions. This method amplifies DNA from C. trachomatis in separate wells and optionally can monitor inhibition of amplification for each specimen using strand displacement amplification (SDA) and detection by fluorescent energy transfer (FRET) probes, producing a method-other-than-acceleration (MOTA) score for each specimen. Specimen processing and BDPT performance followed the manufacturer's instructions. The original algorithm involved retesting specimens with MOTA scores between 2000 and 9999. A negative repeat result (MOTA score <2000) was considered indeterminate.

                      Neisseria gonorrhoeae

                      presumptive identification of N. gonorrhoeae was carried out by growth on media selective for pathogenic Neisseria species (Oxoid) incubated for up to 48 hours in 5–10% CO2 at 35–37°C. Colonies obtained were identified by API NH (bioMerieux) according to manufacturer's instructions.

                      Mycoplasma spp

                      Mycoplasma IST 2 (bioMérieux) was used for the isolation of Mycoplasma hominis and Ureaplasma urealyticum according to the manufacturer's instructions. The diagnostic kit provided information regarding the presence or absence of M. hominis and U. urealyticum and an estimate of the density of each organism (cut-off 104-colour-changing units- CCU/ml) together with its antimicrobial susceptibilities to doxycycline, josamycin, ofloxacin, erythromycin, tetracycline, pristinamycin, azithromycin, clarythromycin, ciprofloxacin.

                      Candida spp

                      cervical-vaginal samples were cultured on Sabouraud dextrose agar (Oxoid) aerobically at 37°C for 24–48 h [16, 17]. The identity of clinical isolates was confirmed by conventional mycological methods [18, 19], such as the germ tube induction test in serum (C. albicans or C. dubliniensis), microscopic morphology, and growth on Oxoid Chromogenic Candida Agar (Oxoid) [18].

                      Non-Candida albicans yeast isolates were further speciated with API 20C AUX or API 32C.

                      Trichomonas vaginalis

                      the vaginal swab was placed in 0.2 ml of sterile physiologic saline for wet mount evaluation and examined microscopically (400×) within 15 min for motile trichomonads.

                      The culture was carried out on Oxoid's Trichomonas Medium n.2 for the recovery of T. vaginalis and incubated aerobically a 37°C. The broth was examined microscopically for motile trichomonads on days 1, 3 and 5 [16, 17].

                      Bacterial vaginosis

                      the presence of bacterial vaginosis was evaluated microscopically on samples collected from the posterior vaginal fornix. Bacterial vaginosis was diagnosed by the presence of three of the clinical and microscopic findings standardized by Amsel et al [20]: vaginal pH greater than 4.5; presence of clue cells; grey homogenous vaginal discharge; positive Whiff-test in which a fishy odour is released after the addition of 10% potassium hydroxide solution to vaginal fluid.

                      Statistical analysis

                      The χ2 test was used in the statistical analysis, while the Odd Ratio was used to measure the strength of the association with HPV. Statistical tests were considered significant if the p value was 0.05 or less. Logistic regression analysis was used to assess the simultaneous effect of more than one variable on the risk of HPV infection and to identify possible confounding factors.

                      Results

                      Study group characteristics

                      Out of 857 enrolled outpatients, two groups of patients were identified on the basis of HPV DNA test positivity. The demographic and behavioural characteristics of the study population (266 HPV positive and 591 HPV negative women) are presented in table 1. The women were aged between 17 and 57 years (mean 32.7 years). As expected, the features significantly associated with HPV-DNA detection in univariate analysis were: young patient's age; age at first intercourse; high number of lifetime sexual partners. Smokers were 29.98% in the total population with a significantly higher percentage in HPV positive group (38.1% vs. 28.0; p = 0.003). HPV-DNA detection was significantly higher in the nullipara women and in those without spontaneous abortion. Only 22.40% of the study participants usually use at least one contraceptive method with no significant differences in HPV positivity.
                      Table 1

                      Characteristics of studied groups: univariate analysis of age, sexual, reproductive and behavioural history.

                       

                      HPV(+)

                      HPV(-)

                      Total

                      HPV(+) %

                      Comparison*

                      95% CI

                      significance

                      TOTAL: frequency

                      266

                      591

                      857

                      31.0%

                         

                      Age

                             

                      mean age (years) (sd)

                      31.1(8.0)

                      33.5(7.5)

                      32.7(7.6)

                       

                      diff. -2.4

                      -1.3 to -3.5

                      t = -4.26,

                      P < 0.00005

                      Frequency:

                             

                         <25 years

                      63

                      72

                      135

                      46.7%

                      OR = 1.00

                        

                         25- years

                      58

                      116

                      174

                      33.3%

                      OR = 0.57

                      0.36 to 0.91

                      P = 0.017

                         30- years

                      68

                      142

                      210

                      32.4%

                      OR = 0.55

                      0.35 to 0.85

                      P = 0.008

                         35- years

                      36

                      144

                      180

                      20.0%

                      OR = 0.29

                      0.17 to 0.47

                      P < 0.00005

                         40+ years

                      41

                      117

                      158

                      25.9%

                      OR = 0.40

                      0.25 to 0.65

                      P = 0.0003

                      Age at 1st intercourse

                             

                      mean age (years)

                      18.6

                      19.6

                      19.3

                       

                      diff. -1.01

                      -0.49 to -1.54

                      t = -3.79,

                      P = 0.0002

                      Frequency:

                             

                         <16 years

                      25

                      42

                      67

                      37.3%

                      OR = 1.00

                        

                         16 years

                      34

                      55

                      89

                      38.2%

                      OR = 1.04

                      0.54 to 2.00

                      P = 0.91

                         17 years

                      44

                      73

                      117

                      37.6%

                      OR = 1.01

                      0.54 to 1.88

                      P = 0.97

                         18 years

                      58

                      115

                      173

                      33.5%

                      OR = 0.85

                      0.47 to 1.52

                      P = 0.58

                         19 years

                      36

                      55

                      91

                      39.6%

                      OR = 1.10

                      0.57 to 2.11

                      P = 0.77

                         20 years

                      21

                      87

                      108

                      19.4%

                      OR = 0.41

                      0.20 to 0.81

                      P = 0.01

                         21 years

                      15

                      24

                      39

                      38.5%

                      OR = 1.05

                      0.47 to 2.37

                      P = 0.91

                         22+ years

                      33

                      140

                      173

                      19.1%

                      OR = 0.40

                      0.21 to 0.74

                      P = 0.004

                      Number of partners

                             

                         mean

                      4.20

                      2.83

                      3.26

                       

                      diff. 1.37

                      0.96 to 1.79

                      t = 6.51,

                      P < 0.00005

                         frequency:

                             

                            1 partner

                      39

                      234

                      273

                      14.3%

                      OR = 1.00

                        

                            2 partners

                      46

                      129

                      175

                      26.3%

                      OR = 2.14

                      1.33 to 3.45

                      P < 0.00005

                            3 partners

                      58

                      86

                      144

                      40.3%

                      OR = 4.05

                      2.52 to 6.51

                      P < 0.00005

                            4 partners

                      41

                      42

                      83

                      49.4%

                      OR = 5.86

                      3.39 to 10.13

                      P < 0.00005

                            5–9 partners

                      54

                      75

                      129

                      41.9%

                      OR = 4.32

                      2.65 to 7.03

                      P < 0.00005

                            10+ partners

                      28

                      25

                      53

                      52.8%

                      OR = 6.72

                      3.55 to 12.71

                      P < 0.00005

                      Parity

                             

                         Nulliparous

                      215

                      432

                       

                      33.2%

                      OR = 1.00

                        

                         Parous

                      51

                      159

                       

                      24.3%

                      OR = 0.64

                      0.45 to 0.92

                      P = 0.0154

                      Spontaneous abortion

                             

                         No

                      238

                      485

                      723

                      32.9%

                      OR = 1.00

                        

                         Yes

                      28

                      106

                      134

                      20.9%

                      OR = 0.54

                      0.35 to 0.84

                      P = 0.0063

                      Induced abortion

                             

                         No

                      227

                      493

                      720

                      31.5%

                      OR = 1.00

                        

                         Yes

                      39

                      98

                      137

                      28.5%

                      OR = 0.86

                      0.58 to1.29

                      P = 0.48

                      Contraceptives

                             

                      never used any type

                      210

                      455

                      665

                      31.5%

                      OR = 1.00

                        

                      have used at least one type

                      56

                      136

                      192

                      29.2%

                      OR = 0.89

                      0.63 to 1.27

                      P = 0.52

                      Smoking

                             

                      never smoked

                      168

                      432

                      600

                      28.0%

                      OR = 1.00

                        

                      ever smoked

                      98

                      159

                      257

                      38.1%

                      1.58

                      1.17 to 2.16

                      P = 0.003

                      *diff. is the difference between two mean values and OR is the odds ratio compared with the first category.

                      HPV genotyping

                      HPV genotypes found in samples from HPV positive women were shown in table 2. Thirty-five different HPV types were identified: HPV16 was the most prevalent (14.50%), followed by HPV58 (9.16%), HPV53 (8.01%) and HPV42 (6.10%); whereas each other genotype was present in less than 5% of HPV positive women. In addition there were 4 cases of "missed typing" (1.5%), probably due to mixed infections with different HPV genotypes. Moreover it should be considered that PCR amplification and sequencing detect precisely the more abundant (and probably clinically relevant) type and underestimate co-infections. According to the reclassification proposed by Munoz et al. (2006) [3], the aggregated percentage (HR and probably HR) of detected HPV was 58.77%, 27.86% for low-risk and 13.35% for unknown-risk HPV types. Of the HPV negative women, 2.3% revealed an abnormal cervical cytology (2% LG-SIL; 0.3% HG-SIL). Among HPV positive women more than 60% had normal cytology, whereas only 3.98% had HG-SIL. LG-SIL and ASCUS were found in 29.8% and 3.1% respectively of HPV positive women.
                      Table 2

                      Genotypes in HPV positive women.

                      Risk category*

                                  

                      HIGH:

                                 

                      total %

                         HPV type

                      16

                      18

                      31

                      33

                      35

                      45

                      51

                      52

                      56

                      58

                      59

                       

                         Frequency

                      38

                      7

                      13

                      8

                      2

                      1

                      5

                      8

                      5

                      24

                      3

                      114 43.51

                      Probably HIGH:

                                  

                         HPV type

                      53

                      66

                      68

                      73

                              

                         frequency

                      21

                      12

                      1

                      6

                             

                      40 15.27

                      LOW:

                                  

                         HPV type

                      6

                      11

                      42

                      43

                      44

                      54

                      61

                      70

                      72

                      81

                      91

                       

                         frequency

                      13

                      6

                      16

                      1

                      2

                      11

                      6

                      10

                      1

                      5

                      2

                      73 27.86

                      UNKNOWN:

                                  

                         HPV type

                      30

                      34

                      62

                      67

                      74

                      84

                      85

                      86

                      87

                         

                         frequency

                      3

                      3

                      1

                      2

                      5

                      7

                      2

                      2

                      10

                        

                      35 13.36

                      *According to Munoz et al. (2006) [3], 11 types were classified as high risk, 4 types as probably high-risk; 11 were classified as low-risk. Nine types were grouped as unknown-risk HPV because of their incomplete characterization due to their rare detection or recent identification.

                      Different microorganisms infection rate

                      In the total population, C. trachomatis was revealed in 8.05% of the samples, U. urealyticum was present in 28.35% and S. agalactiae in 8.86%; T. vaginalis was found only in 1.16%, bacterial vaginosis in 6.30%, and yeast in 13.06%.

                      M. hominis was isolated only in a few cases whereas N. gonorrhoeae was never found.

                      Co-infection HPV-other microorganisms

                      The detection of genital infections in the HPV negative population was not infrequent: in fact S. agalactiae was found in 9.4% of samples, yeasts in 13.5%, bacterial vaginosis in 5.2%, C. trachomatis in 5.4% and U. urealyticum in 24.8%. Generally U. urealyticum is a common commensal of the female lower genital tract and it seems to be an important opportunistic pathogen during pregnancy and in other genital diseases such as cervicitis. Its pathogenic potential seems to be related to the high density in the analysed samples (>10,000 CCU/ml). In HPV negative patients the percentage of U. urealyticum > 10,000 CCU/ml was 5.24% (table 3).
                      Table 3

                      Different microorganisms infection in HPV positive and negative women

                       

                      HPV(+)

                      HPV(-)

                      Total

                      % positive

                      odds ratio

                      95% CI

                      Significance

                      C. trachomatis

                             

                         negative

                      229

                      559

                      788

                      29.0%

                      OR = 1.00

                        

                         Positive

                      37

                      32

                      69

                      53.6%

                      OR = 2.82

                      1.74 to 4.57

                      P < 0.00005

                      U. urealyticum

                             

                         negative

                      170

                      444

                      614

                      27.7%

                      OR = 1.00

                        

                         <10000 CCU/ml

                      61

                      116

                      177

                      34.5%

                      OR = 1.37

                      0.96 to 1.96

                      P = 0.08

                         >10000 CCU/ml

                      35

                      31

                      66

                      53.0%

                      OR = 2.95

                      1.79 to 4.85

                      P < 0.00005

                      S. agalactiae

                             

                         negative

                      246

                      535

                      781

                      31.5%

                      OR = 1.00

                        

                         Positive

                      20

                      56

                      76

                      26.3%

                      OR = 0.78

                      0.46 to 1.32

                      P = 0.35

                      Yeasts

                             

                         negative

                      234

                      511

                      745

                      31.4%

                      OR = 1.00

                        

                         Positive

                      32

                      80

                      112

                      28.6%

                      OR = 0.87

                      0.56 to 1.35

                      P = 0.55

                      Bacterial vaginosis

                             

                         negative

                      243

                      560

                      803

                      30.3%

                      OR = 1.00

                        

                         Positive

                      23

                      31

                      54

                      42.6%

                      OR = 1.71

                      0.98 to 2.98

                      P = 0.06

                      T. vaginalis

                             

                         negative

                      263

                      584

                      847

                      31.1%

                      OR = 1.00

                        

                         Positive

                      3

                      7

                      10

                      30.0%

                      OR = 0.95

                      0.24 to 3.71

                      P = 0.94

                      The number of the specimens in which T. vaginalis was detected was very low in both populations (1.12% in HPV positive women vs. 1.18%).

                      Statistical analyses did not reveal any association between HPV presence and S. agalactiae, yeasts, T. vaginalis and U. urealyticum < 10,000 CCU/ml detection.

                      Although the bacterial vaginosis was diagnosed in a higher percentage among HPV positive women (8.6% vs. 5.2% control group), the difference in the prevalence of bacterial vaginosis was not statistically significant between the 2 groups.

                      A significant association between HPV-DNA presence and concurrent genital infection with C. trachomatis (p < 0.001) was observed (table 3). Interestingly, among HPV positive women co-infected by C. trachomatis 67.56% belonged to the HR-HPV infected patients.

                      Moreover another significant association was found between the HPV positivity and the presence of U. urealyticum > 10,000 CCU/ml (p < 0.001) (table 3).

                      Logistic regression analyses did not substantially change these results.

                      Discussion

                      Our findings confirm that an increased risk of HPV infection is strongly associated with a young age at first intercourse, and with an elevated number of sexual partners as would be expected for a sexually transmitted viral infection.

                      Epidemiological studies have demonstrated the role of other sexually transmitted infectious agents in the pathogenesis of cervical cancer, but knowledge about the specific role of these pathogens in the natural history of HPV infection is limited. There are several mechanisms by which these co-infections may act, such as direct genotoxicity, but probably the most likely biologic mechanism is the induction of cervical inflammation leading genotoxic damage through oxidative metabolites [21]. An association between cervical cancer and C. trachomatis has been established [22]. In fact, C. trachomatis infection may increase susceptibility to HPV causing microabrasions or alterations of epithelial cells thus facilitating the entry of virions. Some reports have suggested that concurrent C. trachomatis infection can reduce host ability to resolve HPV infection: chronic cervical inflammation seems to influence the HPV persistence through a raised production of free radicals and a reduction of host cell-mediated immunity [2, 10]. C. trachomatis infection induces a shift in the immune response and the unresolved infections have been associated with a humoral (T helper cell type 2) immune response, whereas cellular (T helper cell type 1) immune response is important for the clearance of HPV lesions. Therefore, the modulation of cervical immune response by C trachomatis may influence the clearance of HPV lesions [10].

                      Our data showed, in HPV positive patients, particularly in HR HPV positive women, an increased C. trachomatis infection rate; this result may suggest more accurate screening for C. trachomatis in HPV positive patients with an atypical cytology. Since both pathogens are co-variables related to sexual behaviour, probably they synergize in inducing cervical epithelium alterations [3, 23].

                      An increased infection rate of U. urealyticum in HPV positive group was also observed, but a significant association was found only with high density cervical presence. The biological role of U. urealyticum infection in the HPV outcome was not previously clarified, but high level of U. urealyticum seems to be a cofactor in the development of HPV related cervical dysplasia [24]. It is tempting to speculate that the presence of U. urealyticum may play a role both in initiating cellular anomalies and in viral persistence. It was reported that Mycoplasma infections cause in vitro chromosomal changes and cell transformation throughout gradual progressive chromosomal loss and translocations [25]. Robertsonian chromosome translocation has been identified in the presence of Mycoplasma infections [26].

                      Previous researches have produced conflicting results regarding the role of bacterial vaginosis as cofactor in the development of cervical intraepithelial neoplasia [27]. Thus, although bacterial vaginosis may have an influence in acquisition of genital infections, its role into HPV acquisition and persistence remains to be clarified [28]. Moreover, to date the only prospective study on bacterial vaginosis and HPV infection, showed that both infections occur simultaneously [29] and suggested that the presence of HPV may have an influence in the vaginal flora. In our findings, bacterial vaginosis is not statistically associated with HPV infection, even if alterations of vaginal flora tend to be more common among HPV positive than HPV negative women (8.6% vs. 5.2% respectively).

                      This study evidenced also the lack of protective precautions against sexually transmitted infections, both in HPV positive and HPV negative women. Despite the use of condom is not reported to be highly effective in reducing HPV transmission [30, 31], counselling on sexually transmitted diseases (STDs) prevention use should be continued, for instance, to prevent C. trachomatis transmission.

                      Conclusion

                      In this study a statistically significant association between HPV and C. trachomatis was found and interestingly also with U. urealyticum at a high colonization rate. Although bacterial vaginosis was not significantly associated with HPV, it was more common among HPV positive women. These data confirm that screening for genital infections may be important to reveal the simultaneous presence of different sexually transmitted microorganisms. These results suggest and emphasize the value of the screening for genital infections in HPV positive patients in order to decrease the presence of other microorganisms and to reduce the probable synergistic effects of co-infections. Prevention is important not only to avoid other STDs and their sequelae, but also to reduce the influence of concomitant microorganisms on HPV infection.

                      Declarations

                      Acknowledgements

                      This work was partially supported by grant from MIUR-Italy (Ministero dell'Università, Istruzione, Ricerca scientifica).

                      Authors’ Affiliations

                      (1)
                      Department of Gynaecology, Perinatology and Child Health, "Sapienza" University of Rome
                      (2)
                      Department of Experimental Medicine, Section of Virology, "Sapienza" University of Rome
                      (3)
                      Department of Public Health, "Sapienza" University of Rome
                      (4)
                      Department of Public Health, Section of Microbiology, "Sapienza" University of Rome

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                      32. Pre-publication history

                        1. The pre-publication history for this paper can be accessed here:http://​www.​biomedcentral.​com/​1471-2334/​9/​16/​prepub

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                      © Verteramo et al. 2009

                      This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.