Bacterial vaginosis is conducive to the persistence of HPV infection
Y-l Guo md*, K You md*, J Qiao md PhD*, Y-m Zhao PhD^ and L Geng md PhD*
*Department of Obstetrics and Gynecology; ^Research Center for Clinical Epidemiology, Peking University Third Hospital, Beijing 100191, China
Summary: We investigated the effects of bacterial vaginosis (BV) on the outcomes of high-risk human papillomavirus infection (HR-HPV). BV was diagnosed on Papanicolaou-stained cytology slides of 707 HPV-positive patients. HR-HPV DNA expression was analysed using the Hybrid Capture II (HC-II) assay. Ofthe 707 HR-HPV-positive female patients, 298 (42.1%) exhibited clearance of HR-HPV. The remaining 409 patients had persistent HR-HPV infection. The persistent HR-HPV group and the clearing group had similar rates of BV at the beginning of the study. At the end of the study, the persistent HR-HPV group had a BV prevalence of 11.2% while the clearing group had a significant lower BV prevalence of 5.0%. A decreased clearance of HPV was found in women with current BV, compared with women without BV. Furthermore, the natural history of HPV was not affected by the HPV viral load or the BV prevalence at the beginning of the study (P > 0.05). Bacterial vaginosis appears conducive to the persistence of HPV infection.
Keywords: human papillomavirus, HPV, bacterial vaginosis, BV, persistence, high-risk HPV, follow-up
INTRODUCTION
A large number of studies have shown that the persistent infection of the high-risk human papillomavirus (HR-HPV) is the major pathological cause for cervical cancer and pre cancerous lesions,1-3 although in most cases the infection would disappear naturally.4 However, it has been difficult to predict whether HPV would undergo spontaneous regression or result in tumour formation. At present, one common strategy is to monitor patients with HPV and cervical smear diagnosed ASC-US (atypical squamous cells of undetermined significance) or LSIL (low-grade squamous intraepithelial lesion).
The anaerobic bacteria associated with bacterial vaginosis (BV) and their metabolites may destroy the innate defenses of the vaginal environment. Moreover, BV is associated with the incidence of many sexually transmitted infections (STIs), such as Chlamydia trachomatis, Neisseria gonorrhoeae, herpes simplex virus (HSV) and HIV.5-7 It has been suggested that HPV-positive women have higher rates of BV8,9 and women with BV may be more susceptible to HPV infection.10 However, it has also been suggested that HPV infection usually occurs prior to or at the same time as BV;11 hence, the relationship between HPV and BV remains controversial. These observations raised interesting questions as to whether BV can influence the outcomes of HR-HPV infection. In the present study, we aimed to address the question about whether BV is conducive to the persistence of HPV infection.
BV can be diagnosed by Nugent's scoring system, Amsel's clinical criteria, modified Amsel's criteria and the presence of clue cells.12 As Papanicolaou-stained vaginal smears (Pap smears) used for cervical cancer screening are routinely
archived they could provide very useful and accessible material for BV diagnosis.7,13,14 Thus in our study cervical Pap smears were used for the diagnosis of BV.
MATERIALS AND METHODS
Patients
Female patients admitted to the Department of Obstetrics and Gynecology, Peking University Third Hospital between April 2005 and July 2011 were given a cervical cytological examin ation and HR-HPV test. In the 6-12 months prior to this study, the patients were diagnosed as HPV-positive with ASC-US or LSIL. All patients included in this study either were normal or had a colposcopic biopsy diagnosed cervical intraepithelial neoplasia (CIN) 1.
The informed consent form was approved by Peking University Health Science Center Institutional Reviewing Board (IRB00001052-06058).
The cervical microscopy slides were prepared using the PrepStain system (BD Diagnostic Systems, Franklin Lakes, NJ, USA) and the Bethesda 2011 System (TBS) was used for cytolo- gical diagnosis.15 The HR-HPV DNA test was performed using the Hybrid Capture II system (Digene, Gaithersburg, MD, USA) with an HR-HPV DNA kit (Digene). The positive result was determined by a >1.0 ratio of relative light units (RLU) between the test group and the positive control (PC) group (1 pg/mL HPV16 DNA). Cervical cytology smear testing was repeated twice for each patient. The BV test was positive with a clue cell positive rate of >20% in the Papanicolaou-stained microscopy slide; below that cut-off point, the BV test was deemed negative.
According to the HR-HPV testing results at the beginning and end of the study, the patients were divided into two groups: the HPV clearing group and the persistent HPV group. According to the HR-HPV testing results at the beginning of the study, all HPV-positive patients were divided into a low viral load group (1-9.99 RLU/PC) and a high viral load group (>10 RLU/PC). According to the patients' age, they were divided into a young patient group (<30 years old) and an older patient group (>30 years old). According to the BV test at the beginning and end of the study, the patients were divided into four groups: the continuously BV-positive group, the BV-clearing group, the BV-positive conversion group (BV was found at the current visit but not at the preceding visit), and the continuously BV-negative group.
Statistical analysis
Data were analysed using the chi-squared analysis and the Binary Logistic Regression method in SPSS 13.0 software (SPSS Inc, Chicago, IL, USA). Statistically significant difference was determined as P < 0.05.
RESULTS
The 707 female patients who qualified for this study had an average age of 39.1 years (range: 19-71), and an average follow-up of 9.5 months. Among them, 298 (42.1%) exhibited clearance of HR-HPV and the remaining 409 patients had per sistent HPV infection.
The persistent HR-HPV group and the clearing group did not show any significant difference in the prevalence of BV at the beginning of the study (x2 = 3.667, P = 0.056). At the end of the study, the persistent HR-HPV group exhibited a BV preva lence of 11.2% while the clearing group had a significantly lower BV prevalence of 5.0% (x2 = 8.442, P = 0.004; Table 1).
The chi-squared test and logistic regression analysis were per formed to analyse the age and HR-HPV load at the beginning of the study, and the relationship between HR-HPV outcome and BV prevalence at the beginning and the end of the study (Table 2). The results showed that more women cleared HPV in the young patient group than the older group (P < 0.05). More women cleared HPV when BV was not present at the end of the study (P < 0.05). Furthermore, the clearance of HPV was not affected by the HPV viral load or BV prevalence at the beginning of the study (P > 0.05).
Using the chi-squared test, we analysed the correlation of BV prevalence and HPV outcomes. We found that the BV-positive conversion group exhibited the lowest rate of HPV clearance (23.3%), followed by the continuously BV-positive group (27.8%). The HPV clearance rate in the continuously BV-negative group was the highest (x2 = 11.525, P = 0.009； Table 3).
Table 1 Correlation between HR-HPV outcomes and BV test results
| BV-positive |
Persistent
HPV group(n=409)
|
HPV-clearing
group (n=298)
|
X2
|
p
|
|
Beginning of study
|
53
|
13.0%
|
25
|
8.4%
|
3.667
|
0.056
|
|
End of study
|
46
|
11.2%
|
15
|
5.0%
|
8.442
|
0.004
|
HR-HPV, high-risk human papillomavirus; BV, bacterial vaginosis
Table 2 Relationship of patient age, HPV load, BV-positiverate and HR-HPV clearing
|
|
n
|
Number that cleared HPV
|
%
|
Chisquared analysis
|
Binary logistic regression
|
|
X2
|
p
|
Wald
|
p
|
|
Age
|
|
<30 yeaes
|
139
|
72
|
51.8
|
6.606
|
0.010
|
6.512
|
0.011
|
|
≥30 years
|
568
|
226
|
39.8
|
|
|
HPV load
|
|
<10RLU/PC
|
116
|
58
|
50.0
|
6.507
|
0.061
|
3.192
|
0.074
|
|
≥10RLU/PC
|
591
|
240
|
40.6
|
|
|
BV test at the beginning of the study
|
|
Negative
|
629
|
273
|
43.4
|
3.667
|
0.056
|
2.227
|
0.136
|
|
Positive
|
78
|
25
|
32.1
|
|
|
BV test at the end of the study
|
|
Negative
|
646
|
283
|
4.38
|
8.442
|
0.004
|
5.619
|
0.018
|
|
Positive
|
61
|
15
|
24.6
|
|
HR-HPV, high-risk human papillomavirus; BV, bacterial vaginosis; RLU, relative light
unit; PC, positive control
Table 3 Correlation between BV test and HPV outcome
|
BV
test group
|
n
|
HPV clearing
|
X2
|
p
|
|
n
|
%
|
|
1.Positive conversion
|
43
|
10
|
23.3
|
11.525
|
0.009
|
|
2.Continuously positive
|
18
|
5
|
27.8
|
|
|
3.BV clearing
|
60
|
20
|
33.3
|
|
4.Continuously negative
|
586
|
263
|
44.9
|
HPV, human papillomavirus; BV, bacterial vaginosis
Both BV and HPV occur frequently in sexually active women, but their relationship remains controversial. BV is associated with an increased risk of detection of HPV, and HPV infection is associated with an increased risk of BV.10 On one hand, HPV infection can damage the vaginal epithelium and degrade the cervical mucus, which in turn can facilitate development of BV. On the other hand, BV is associated with major changes in the vaginal environment.
It has been suggested that lactic acid bacteria can function to resist cervical intraepithelial neoplasia.18 HPV is a common vaginal pathogen in women with a short duration of infection under normal conditions. Persistent infection with HPV may be related to vaginal flora imbalance, especially the reduction of lactic acid bacteria. Korshunov et al.18 studied the distri bution of vaginal flora in HPV-positive patients with CIN and found that the occurrence of CIN was closely related to the reduction of lactic acid bacteria. Women with BV possess Lactobacillus-poor flora, which may increase the production of mucin-degrading enzymes (e.g. sialidases), change the cervical mucosa barrier, reduce the production of H2O2, and affect the production of cytokines.19-21 All these mechanisms could con tribute to the survival of other sexually transmitted agents. Moreover, BV may increase susceptibility to cervical HPV infec tion by facilitating the adherence, invasion and incorporation of HPV oncogenes into the genome of cells of the transformation zone, thus providing biological plausibility for an increased risk or reactivation of an HPV infection. Cervical cancer results from the long-term persistent HPV infection. Patients with BV have frequent vaginal flora imbalance, of which the amount of lactic acid bacteria was reduced while Gardnerella or mixed anaerobic bacteria flourish to produce harmful metab- olites.8 With other cancer-causing factors (e.g. HPV and human cytomegalovirus infection), the vaginal flora imbalance can eventually induce the occurrence of cervical cancer.
Our analysis revealed that BV found at the current visit but not at the preceding visit was predictive of the maintenance of HPV, while the persistently BV-negative women were more able to clear HPV, indicating that the vaginal flora status may affect the outcome of HPV infection.
Our results showed evidence of a positive association between these two common types of infections. A body of evidence has suggested that BV is associated with HPV infec- tion.8 A previous study revealed that HPV infection generally occurs first, or at the same time as BV.11 In the HIV Epidemiology Research Study, it was suggested that BV is associated with increased odds for prevalent and incident HPV as well as delayed clearance among women.16 Although our results could not determine the occurrence order of HPV and BV, we found that those women that were initially BV negative and became positive later were likely to maintain HPV, indicating that a persistent HPV infection predisposes a woman to BV.
Discacciati et al.9 found that the prevalence rate of BV was 33% for patients with CIN, but was only 12% for normal patients. In the present study, the HR-HPV-positive patients showed a low BV prevalence of 8.4-13.0% (Table 1). This dis crepancy could be caused by either different BV diagnostic methods,14 or different population composition.
It is believed that it is not necessary to treat BV patients without symptoms,22 or HPV patients without apparent symp toms of cervical lesions and cervical cancer. Clinically, at least 50% of BV patients do not show obvious BV symptoms,23 and hundreds of HPV-positive patients are not treated to promote HPV clearance.24 Our study suggests that a healthy vaginal environment slows down HPV persistence. It has also been suggested that BV promotes the acquisition of HPV and is associated with the delayed clearance of HPV.16 It is impor tant to develop simple, economic and effective approaches to promote HPV clearance, such as by influencing the vaginal flora by providing lifestyle advice to women infected with HPV. Lactic acid bacteria have been found to be able to have anti-infective and antitumor effects.25,26 McNicol et al.27 suggested that the presence of vaginal lactobacilli could sig- ni^icantly reduce the HPV-positive rate. Hence, it may be appli cable to treat dual BV/HPV-positive patients with Lactobacillus drugs or possibly personal hygiene education.
It remains elusive whether BV and HPV are related simply because both occur frequently in sexually active women or whether there is a biological interaction between the two infec tions. Moreover, it is unknown why the high-risk HPV infection is cancerous in some women whereas in others it is eradicated. Individual differences in immunological defence may be one explanation.28 Prevention of HPV infection through vaccination or behaviour changes may reduce the incidence of cervical cancer among young women, but for those older women with HR-HPV infection, enhancing their immune competence may be more important. BV is a common and treatable vaginal infection that causes major changes in the local environ ment, leading to degradation of innate protective mechanisms against infection and disease. Furthermore, changes in the cervico-vaginal milieu resulting from co-infections may affect the clearance of the HPV infection.
Our results suggest that BV prior to HPV infection makes women more likely to maintain HPV. Therefore, it is important to treat vaginal pathogen infections (e.g. BV) and restore healthy vaginal flora for HR-HPV-positive women.
ACKNOWLEDGEMENTS
We sincerely thank those patients who participated in this study.
REFERENCES
- Koshiol J, Lindsay L, Pimenta JM, Poole C, Jenkins D, Smith JS. Persistent human papillomavirus infection and cervical neoplasia: a systematic review and meta-analysis. Am J Epidemiol 2008;168:123 - 37
- Dalstein V, Riethmuller D, Prt^tet JL, et al. Persistent and load of high-risk HPV are predictors for development of high-grade cervical lesions: a longitudinal Freach cohort study. Int J Cancer 2003;106:396-403
- Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:12-9
- Castellsague X. Natural history and epidemiology of HPV infection and cervical cancer. Gynecol Oncol 2008;110(Suppl. 2):S4-7
- Cherpes TL, Hillier SL, Meyn LA, et al. A delicate balance: risk factors for acquisition of bacterial vaginosis include sexual activity, absence of hydrogen peroxide-producing lactobacilli, black race, and positive herpes simplex virus type 2 serology. Sex Transm Dis 2008;35:78-83
- Wiesenfeld HC, Hillier SL, Krohn MA, et al. Bacterial vaginosis is a strong predictor of Neisseria gonorrhoeae and Chlamydia trachomatis infection.
Clin Infect Dis 2003;369:663-8
- Dols JA, Reid G, Kort R, et al. PCR-based identification of eight lactobacillus species and 18 hr-HPV genotypes in fixed cervical samples of South African women at risk of HIV and BV. Diagn Cytopathol 2011;40:472-77
- Gillet E, Meys JF, Verstraelen H, et al. Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a meta-analysis. BMC Infect Dis 2011;11:10-8
- Discacciati MG, Simoes JA, Lopes ES, et al. Is bacterial vaginosis associated with squamous intraepithelial lesion of the uterine cervix? Diagn Cytopathol 2006;34:323-5
- Watts DH, Fazarri M, Minkoff H, et al. Effects of bacterial vaginosis and other genital infections on the natural history of human papillomavirus infection in HIV-infected and HIV-uninfected women. J Infect Dis 2005;191:1129-39
- Mao C, Hughes JP, Kiviat N, et al. Clinical findings among women with genital human papillomavirus infection. Am J Obstet Gynecol 2003;188:677-84
- Discacciati MG, Simoes JA, Amaral RG, et al. Presence of 20% or more clue cells: an accurate criterion for the diagnosis of bacterial vaginosis in Papanicolaou cervical smears. Diagn Cytopathol 2006;34:272-6
- Eriksson K, Forsum U, Bj0rnerem A, et al. Validation of the use of Pap-stained vaginal smears for diagnosis of bacterial vaginosis. APMIS 2007;115:809-13
- Dols JAM, Smit PW, Kort R, et al. Microarray-based identification of clinically relevant vaginal bacteria in relation to bacterial vaginosis. Am J Obstet Gynecol 2011;204:305.e1-7
- Solomon D, Davey D, Kurman R, et al. The 2001 Bethesda System: terminology for reporting results of cervical cytology. JAMA 2002;287:2114-9
- King CC, Jamieson DJ, Wiener J, et al. Bacterial vaginosis and the natural history of human papillomavirus. Infect Dis Obstet Gynecol 2011;ID 319460
- Morris M, Nicoll A, Simms I, Wilson J, Catchpole M. Bacterial vaginosis: a public health review. Br J Obstet Gynecol 2001;108:439-50
- Korshunov VM, Kafarskaia LI, Bagirova MSh, Minkina GN, Manukhin IB, Bossart W. The effect of Solco Trichovac on the vaginal microflora of patients with a papillomavirus infection associated with a cervical intraepithelial neoplasm. Zh Mikrobiol Epidemiol Immunobiol 1994;5:13-17
- Hillier SL. The vaginal microbial ecosystem and resistance to HIV. AIDS Res Hum Retroviruses 1998;14(Suppl. 1):S17-21
- Cauci S, Guaschino S, Driussi S, et al. Correlation of local interleukin-8 with immunoglobulin A against Gardnerella vaginalis hemolysin and with prolidase and sialidase levels in women with bacterial vaginosis. J Infect Dis 2002;185:1614-20
- Olmsted SS, Meyn LA, Rohan LC, et al. Glycosidase and proteinase activity of anaerobic gram-negative bacteria isolated from women with bacterial vaginosis. Sex Transm Dis 2003;30:257-61
- Centers for Disease Control and Prevention. Sexually transmitted diseases treatment guidelines, 2010. MMWR Morb Mortal Wkly Rep 2010;59:1-110
- Verstraelen H, Verhelst R. Bacterial vaginosis: an update on diagnosis and treatment. Expert Rev Anti Infect Ther 2009;7:1109-24
- Wright TC Jr, Massad LS, Dunton CJ, Spitzer M, Wilkinson EJ, Solomon D. 2006 consensus guidelines for the management of women with cervical intraepithelial neoplasia or adenocarcinoma in situ. Am J Obstet Gynecol 2007;197:340-5
- Femia AP, Luceri C, Dolara P, et al. Antitumorigenic activity of the prebiotic inulin enriched with oligo fructose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lact is on azoxymethane-induced colon carcinogenesis in rats. Carcinogenesis 2002;23:1953 - 60
- Seow SW, Rahmat JN, Mohamed AA, Mahendran R, Lee YK, Bay BH. Lactobacil lus species is more cytotoxic to human bladder cancer
cells than Mycobacterium bovis (bacillus Calmette-Guerin).
J Urol 2002;168:2236 -9
- McNicol P, Paraskevas M, Guijon F. Variability of polymerase chain reaction-based detection of human papillomavirus DNA is associated with the composition of vaginal microbial flora.
Med Virol 1994;43:194-200
- Huh WK. Human papillomavirus infection: a concise review of natural history. Obstet Gynecol 2009;114:139-43
(Accepted 21 February 2012)
