Download changes of the vaginal biocenosis in cervical dysplasic lesions

ORIGINAL ARTICLES
CHANGES OF THE VAGINAL BIOCENOSIS IN CERVICAL
DYSPLASIC LESIONS
Elena Bernad1, Doru-Mihai Anastasiu1, Razvan Ilina2, Delia Hutanu3, Marius
Virjan1, Cosmin Brisan4
REZUMAT
Introducere: Este bine cunoscut faptul că disturbarea biocenozei vaginale prin prezenţa unor factori infecţioşi poate duce la modificări celulare locale,
care pot preceda dezvoltarea unor leziuni cervicale. Material şi metode: Am realizat un studiu în cadrul Clinicii Universitare de Obstetrică şi Ginecologie
„Bega” din Timişoara, în perioada 01.07.2008-15.03.2009, pe un număr de 113 paciente cu rezultate patologice la testul Babeș-Papanicolaou. Am
urmărit de asemenea prezenţa altor factori infecţioşi în afară de virusul papilloma uman. S-au recoltat probe pentru examen bacteriologic şi cultură de
la nivel vaginal. Rezultate: Într-un procent ridicat a fost identificată o disturbare a biocenozei vaginale, prin prezenţa unor factori infecţioşi - 41,6%
dintre paciente având un rezultat pozitiv la examenul bacteriologic sau la cultură, 71% prezentând infecţie cu HPV. Mai mult, în grupul cu leziuni HSIL
s-a observat un procent crescut de probe pozitive pentru floră bacteriană (63,6% din pacientele cu HSIL au prezentat examen bacteriologic sau culturi
pozitive). Cea mai mică incidenţă în alterarea ecosistemului vaginal a fost observată la pacienţi cu citologie ASCUS (23,5%). Concluzii: Având în vedere
asocierea proceselor pre-neoplazice cu inflamaţiile genitale cronice, un control regulat care ar evidenţia şi trata infecţiile vaginale ar fi foarte important
pentru a preveni leziunile canceroase.
Cuvinte cheie: biocenoza vaginală, vaginita, citologie Babes-Papanicolaou, leziuni precanceroase
ABSTRACT
Introduction: It is well known that disturbing vaginal biocenosis throught the presence of infectious factors can be blamed for the celular changes
which precede the development of the lesional cervix. Material and methods: We have performed a study at the Bega University Clinic of Obstetrics
and Gynecology, from Timisoara, between 01.07.2008-15.03.2009, on a number of 113 pacients with pathological results of the Babes-Papanicolaou
cytology; we were also interested in the involvement of other infectious factors aside the human papilloma virus. Vaginal discharge samples were taken
for bacterial tests and cultures. Results: In a large procentage of cases a clear disturbance of the vaginal biocenosis was observed, due to a variety of
infectious factors – 41.6% of the pacients included in the cohort had positive bacteriological tests or cultures, 71% HPV infection from the investigated
pacients. Furthermore, in the cohort with HSIL cytology we saw the greatest percentage of positive samples for pathological bacterial flora (63.6% of the
pacients with HSIL cytology had a positive bacteriological test or culture), the lowest incidence of vaginal ecosistem alteration was observed in the pacients
with ASCUS cytology (23.5%). Conclusions: Due to the association of pre-neoplasic processes with chronic genital inflamations, a regular examination
with the discovery and treatment of vaginal infections is of utmost importance for the prevention of cancerous lesions.
Key Words: vaginal biocenosis, vaginitis, Babes-Papanicolaou cytology, precancerous lesion
INTRODUCTION
Cervical cancer is an important public health
problem worldwide, considering that this condition
is the second cause of mortality through cancer in
women, following breast cancer, causing yearly over a
quarter of a million deaths globally.1,2
Department of Obstetrics/Gynecology, 2 Department of Surgical Oncology, Victor
Babes University of Medicine and Pharmacy, Timisoara, 3 Biology Department,
West University, Timisoara, 4 Clinical Emergency County Hospital, Timisoara
1
Correspondence to:
Ass. Prof. Elena Bernad, Bega Hospital of Obstetrics and Gynecology, 12 V.
Babes Str., Timisoara, Tel. +40-745-395220.
Email: [email protected]
Received for publication: Feb. 01, 2010. Revised: Apr. 20, 2011.
_____________________________
54
TMJ 2011, Vol. 61, No. 1 - 2 In developed countries, where the screening
programs using the cytological examination
Babes-Papanicolaou are correctly and completely
implemented, the detection and treatment of
asymptomatic precancerous lesions, which are curable
in almost 100% of the cases, is possible.3 Still, five
of six women with cervical cancer live in developed
countries and 80% of them are diagnosed in a late
stage of the disease.4
The persistent infection with approximately 15
carcinogen genotypes of human papillomavirus
(HPV) determine practically all the cases of cervical
neoplasia, as well as precancerous lesions and in situ
carcinoma.5
The vaginal ecosystem is a complex and dynamic
system of microorganisms which interact with local
factors to maintain the balance of this environment.
The endogenous microflora comprises a variety of
microorganisms of which we can mention aerobic,
anaerobic and optionally anaerobic ones. Of these,
Doderlein bacilli (Lactobacillus acidophilus) represent
a special category in the vaginal ecosystem, having a
role in maintaining the acidity that characterizes the
vaginal secretion in physiological conditions and plays
a vital role in avoiding the colonization of the vagina
with pathogenic organisms, including those which
produce bacterial vaginitis and candidiasis, infections
of the urinary tract or sexually transmitted diseases.
Aside lactobacilli, in the normal vaginal secretion there
are also a number of aerobic bacteria of the diphteroid
type, Streptococcus, Staphylococcus or anaerobic
(Peptococcus, Peptostreptococcus, Bacteroides speciae),
as well as mycolpasma and isolated strains of Candida.
Any change of the local conditions of the vagina (pH
modification, temperature, hormonal impregnation
etc.) can lead to colonization with possible or really
pathogenic germs and the appearance of vulvo-vaginal
infections.
It is also known that altering the vaginal
biocenosis, through the presence of infectious factors
at this level, is involved in the initiation of some
cellular changes at local level, which in time can lead
to a lesional cervix; there are studies which show a
high incidence of cervical dysplasia in patients with
bacterial vaginitis.7- 9
MATERIALS AND METHODS
- Low-risk HPV strains – 6, 11, 40, 43, 44, 54, 70;
- Possible high-risk HPV – 26, 53, 66;
- Other types of HPV – 69, 71 and 74.
Also there were samples where the viral DNA
was amplified, but the genotype involved could not be
detected.
RESULTS
The cyto-bacteriological examination performed
through the Babes-Papanicolau cytology has provided
the following results: out of a total of 113 samples, 74
have been classifies LSIL according to the Bethesda
classification (analogous to CIN I and cellular changes
associated with HPV), 22 samples matched the HSIL
changes, analogous to CIN II and CIN III, and 17
samples have been classified as ASCUS - atypical
squamous cells. (Fig. 1)
HSIL; 22; 19%
ASCUS; 17; 15%
LSIL; 74; 66%
This paper presents a retrospective study done on
a cohort of 113 patients between the ages of 18 and 79
years, which presented to the Bega Clinical Hospital of
Obstetrics and Gynecology, Timisoara, in the interval
01.07.2008-15.03.2009. The study included patients
which had pathological cytologies, and for which we
Figure 1. Distribution of the results obtained at the cytobacteriological test in
have monitored the association of infectious factors
the studies cohort.
aside the infection with HPV, through bacteriological
samples and cultures from the vaginal secretion. Fig 1. Distribution
TheofHPV
DNAobtained
resultsathave
shown the following:
the results
the cytobacteriological
test in the studies c
All the patients included in the study had a Babes30 samples have been negative and 74 samples (71%)
Papanicolaou cytology done, and samples were taken
have been positive for the infection with different
from the vaginal secretion for HPV DNA testing, as
HPV genotypes. Among the positive samples, in 15
well as for other cultures and bacterial tests in order to
cases we detected the simultaneous infection with
detect the possible associated infectious factors.
multiple strains of HPV.
For the detection of the HPV infection the
The detection of other infectious factors
samples have been taken through brushing the cervix
associated with the HPV infection, possibly involved
and have been tested for HPV DNA, through the
in the appearance of the lesional cervix, has been done
polymerization chain reaction (PCR), for the following
through the sampling of cultures and bacteriological
HPVgenotypes:
tests from the vaginal secretion. In a large number of
- High-risk HPV strains – 16, 18, 31, 33, 35, 39,
cases we have found the disturbance of the vaginal
45, 51, 52, 56, 58, 59, 68, 73, 82 – of these , strains 16,
biocenosis with a variety of infectious factors. From
18, 31 and 45 have a very high oncogenic risk;
the total number of 113 bacteriological tests, in 27
_____________________________
Elena Bernad et al 55
cases we have found the replacement of the normal
vaginal flora formed by lactobacilli Doderlein, with a
pathogenic flora represented by Gardnerella vaginalis,
species of Candida, Trichomonas vaginalis or
Lepthotrix vaginalis.
Thus, as presented in Table 1, where we have
represented the distribution of results of the
bacteriological exams of the vaginal secretion, the
most frequent infection was with Gardnerella vaginalis
(12 cases), followed by vaginal candidosis (present
in 8 cases) and the associated infection with both
Gardnerella vaginalis and Candida albicans which was
detected in 3 cases.
Table 2. The distribution of results obtained in the cultures taken from the
vaginal secretion.
Positive cultures
36
Staphylococcus aureus
8
Escherichia coli
6
Enterococcus faecalis
5
Klebsiella
4
Enterococcus spp
3
Associations between different bacterial species
3
Other bacterial species
7
Negative cultures
77
Table 1. The results of the bacteriological testing of the vaginal secretion.
Positive bacteriological tests
27 (23.9%)
Gardnerella vaginalis
12 (10.6%)
Candida
8 (7.1%)
The corroboration
the data obtained
in obtained
the
Table 2. Theofdistribution
of results
in the
tests performed to detect the pathogenic microbial
3 (2.6%)
Gardnerella vaginalis associated with Candida
flora of the
inferior vaginal tract, with the those
secretion.
from
the
cyto-diagnostic
examination of the cervix,
2 (1.7%)
Trichomonas vaginalis
has shown the frequent association of the alteration
2 (1.7%)
Lepthotrix vaginalis
of the vaginal ecosystem with modification of
Negative bacteriological tests
86 (76.1%)
the Babes-Papanicolaou cytology classified HSIL
and an association in a lower percentage with LSIL
modifications or ASCUS. In other words, the presence
The cultures taken from the vaginal secretion have
of the infectious factors in the vaginal secretion was
Table 1. of
The
the bacteriological
testing of
vaginal
shown the alteration
theresults
vaginalofbiocenosis
with a
detected
in the
14 of
the 22secretion
patients with HSIL at the
wide variety of infectious agents, of which the most
cyto-diagnostic examination, 29 patients with LSIL
frequently involved microorganisms were: Escherichia
and only 4 patients with a cytology classified ASCUS.
coli, Streptococcus beta haemolyticus, Klebsiella,
(Fig. 2)
Enterococcus faecalis, Staphylococcus aureus,
Enterococcus spp. and Enterobacter aerogenes.
Of the 113 cultures sampled, in 36 cases several
pathogenically species developed, and 77 cultures have
been negative. In Table 2 is presented the distribution
of results obtained from the cultures sampled from
the vaginal secretion.
We need to mention the fact that five
patients with a positive culture had associated
the presence of Gardnerella vaginalis, detected
Figure 2. Percentual distribution of the presence of the infectious factors,
at the bacteriological test, and three patients with
according to the cyto-diagnostic result.
Fig
2.
Procentual
distribution of the presence of the infectious factors according to the cytoa positive culture has associated the presence of
candidosis. Also it is very important to observe that diagnostic
DISCUSSIONS
result
for 31 patients with positive bacteriological test or
culture, no lactobacilli Doderlein were detected at
Cervical intraepithelial neoplasia (CIN) and
the bacteriological test. The study of the presence
cervical cancer represent a major cause of morbidity
of infectious factors at the level of the vaginal tract
and mortality for women and was several times
has shown the alteration of the vaginal biocenosis
suggested that bacterial vaginitis can play an important
in 47 of the patients in the cohort, in 13 cases
part in the development of CIN.10 It has been proven
there were associations between different bacterial
that the presence of HPV infection in the cells of the
species.
cervix is a major factor in the initiation of the cervical
_____________________________
56
TMJ 2011, Vol. 61, No. 1 - 2 dysplasia processes, but not all pacients infected with
HPV develop cervical lesions, this indicating that
there are additional factors which together lead to
the initiation of displasic transformation processes.11
Moreover there are studies which have shown a greater
incidence of displasic changes of the cervix among the
pacients with an alteration of the vaginal ecosistem.12,13
Bacterial vaginitis is a polimicrobial syndrome
that affects the inferior genital tract, characterized
by the replacement of the normal vaginal flora
that comprises lactobacilli, with pathogenic flora
mostly Gardnerella vaginalis, Mycoplasma hominis,
Ureaplasma urealyticum, certain species of Mobiluncus
or Prevotella, Chlamydia as well as other anaerobic
organisms. Moreover, some Gardnerella vaginalis
biotypes are more frequently associated with bacterial
vaginitis with clinical manifestation.14,15 In our study,
the presence of the infectious factors in the inferior
genital tract was detected in greater procentage of
cases (41.6% of the pacients included in the cohort
had positive bacteriological tests or cultures), this fact
proving the possible involvement of the alteration
of the vaginal biocenosis, together with the HPV
infection, in the development of the lesional cervix.
Furthermore, in the cohort with HSIL cytology we
saw the greatest percentage of positive samples for
pathological bacterial flora (63.6% of the pacients
with HSIL cytology had a positive bacteriological test
or culture), the lowest incidence of vaginal ecosistem
alteration was observed in the pacients with ASCUS
cytology (23.5%). Of the 74 pacients with a LSIL
cytology, 29 cases have shown the presence of the
infectious factors throught the tests done (39.2%).
The most frequent microorganisms involved in the
alteration of the vaginal biocenosis at the pacients in
our cohort were: Gardnerella vaginalis (15 patients),
Candida (11 patients), Staphylococcus aureus (8
patients), Escherichia coli (6 patients). What is more,
13 patients in the cohort (11.5%) have presented
an alteration of the vaginal ecosistem through the
association of two or more pathogenic agents.
Finally, it would be important to emphasize the
fact that in what prevention and early detection of
the premalignant and malignant cervical pathology
are concerned, all women should have cytologies done
every six months or at least once a year. Furthermore,
the periodical colposcopic examination of the cervix
should also be performed.
CONCLUSIONS
The increased incidence of infectious factors
in patients with pathological results of the cytology
shows that the statements, according to which these
factors have an important part in the initiation of the
displasic processes of the cervix together with HPV,
have a real basis. Nevertheless, in the future, studies
are needed to verify if this association exists, as well
as research that helps in understanding the intimate
mechanisms which initiate the dysplasic process.
Until then, though, due to the frequent association
of the pre-neoplasic associations with chronic vaginal
inflammations, we deem extremely important the
regular check-up with the detection and treatment of
the genital infections, as a method of prevention of
the appearance of the cancerous lesions.
ACKNOWLEDGEMENTS
This work has been supported by Romanian
National Authority for Scientific Research, The
National Plan for Research, Development and
Inovation for the period 2007-2013, Programme III CAPACITIES, grant no: 54/2008.
REFERENCES
1.
Speck LM, Tyring SK. Vaccines for the prevention of human
Papillomavirus infections. Skin Therapy Lett 2006;11(6):1-3.
2.Zonfrillo NJ, Hackley B. The quadrivalent human papillomavirus
vaccine: Potential factors in effectiveness. Journal of Midwifery &
Women’s Health 2008;53(3):188-94.
3.Murray CJL, Lopez AD. Global health statistics. Boston, Harvard School
of Public Health, WHO and World Bank, 1996.
4.Stewart BW, Kleihues P. Cancers of the female reproductive tract. In
WHO Cancer Report Lyon, France. IARC 2003;215-22.
5.Castle PE. Invited commentary: Is monitoring of human Papillomavirus
infection for viral persistence ready for use in cervical cancer
screening? Am J Epidemiol 2008;168(2):138-44.
6.Faro S. Vaginitis: diagnosis and management. Menopausal Study. Int J
Fertil 1996;41(2):115-23.
7.Boyle CM, Uthayakumar S, Hlaket J, et al. Does bacterial vaginosis cause
cervical neoplasia? Proceedings of the 29th Annual Meeting of the
Society of Gynecologic Oncologists, February 7–11, 1998;108.
8.Guijon F, Paraskevas M, Rand F, et al. Vaginal microbial flora as a cofactor
in the pathogenesis of uterine cervical intraepithelial neoplasia. Int J
Gynaecol Obstet 1992;37(3):185-91.
9.Platz-Christensen JJ, Sundstrom E, Larsson P. Bacterial vaginosis
and cervical intraepithelial neoplasia. Acta Obstet Gynecol Scand
1994;73:586-8.
10.Sweet RL. Gynecologic conditions and bacterial vaginosis: Implications
for the non-pregnant patient. Infect Dis Obstet Gynecol 2000;8(34):184-90.
11.Bedell MA, Jones KH, Laimins LA. The E6–E7 region of human
papillomavirus type 18 is sufficient for transformation of NIH 3T3
and Rat-1 cells. J Virol 1987;61(11):3635-40.
12.McDougall JK. Cofactors in the progression of HPV-associated
tumors. In: Giraldo G, Salvatore M, Chieco-Bianchi L, Beth-Giraldo
E, Editors. Advanced technologies in research. Diagnosis and
treatment of AIDS in oncology, Basel: Karger. 1994;46:150-164.
13.Bosch FX, Manos MM, Munoz N, et al. International biological study
on cervical cancer (IBSCC study group). Prevalence of human
papillomavirus in cervical cancer: a worldwide perspective. J Natl
Cancer Inst 1995;87(11):796-802.
_____________________________
Elena Bernad et al 57
14.Aroutcheva AA, Simoes JA, Behbakht K, et al. Gardnerella vaginalis
isolated from patients with bacterial vaginosis and from patients with
healthy vaginal ecosystems. Clin Infectious Diseases 2001;33:1022–7.
_____________________________
58
TMJ 2011, Vol. 61, No. 1 - 2 15.Koumans EH, Kendrick JS. CDC Bacterial Vaginosis Working Group.
Preventing adverse sequelae of bacterial vaginosis: a public health
program and research agenda. Sex Transm Dis 2001;28:292-7.