Abnormal Vaginal Flora in Low-Risk Pregnant Women Cared for by a Public Health Service : Prevalence and Association with Symptoms and Findings from Gynecological Exams

Corresponding Author: Cristina Maria Garcia de Lima Parada Universidade Estadual Paulista “Júlio de Mesquita Filho”. Faculdade de Medicina de Botucatu. Departamento de Enfermagem Campus Universitário de Rubião Júnior, s/n Bairro Rubião Júnior CEP: 18618-970 Botucatu, SP, Brasil E-mail: cparada@fmb.unesp.br Abnormal Vaginal Flora in Low-Risk Pregnant Women Cared for by a Public Health Service: Prevalence and Association with Symptoms and Findings from Gynecological Exams


Introduction
Bacterial vaginosis (BV) is a relevant issue during pregnancy because it is associated with a higher risk of late miscarriage, infection of the amniotic cavity, premature rupture of membranes, preterm labor, prematurity, and infant low birth weight (1) .The mechanism through which these obstetrical complications occur is not yet totally clarified, though it is known that BV consists of changes in vaginal flora, which produces endotoxins, making some women more vulnerable to inflammatory responses with the production of cytokines and prostaglandins that trigger labor.Microorganisms might ascend and invade the chorioamniotic membranes, decidua and amniotic fluid.Even the possibility of protease production by the microorganism that compose BV is possible, which would participate in the pathogenesis of the premature rupture of membranes (2) .
It is also known that some bacterial species that colonize the Lower Genital Tract (LGT), especially those associated with BV, release sialidase and prolidase.Sialidase are enzymes that cleave sialic acid from glycoproteins, among them the IgA, mucins and cell receptors and thus is associated with evasion of innate and acquired immunity, due to degradation of cervical IgA and changes in the cellular membrane receptors (3) .Prolidase are proteolytic enzymes that degrade the extracellular matrix, encourage cell infiltration and thus contribute to breaking down the protective mucosal barrier (4) .Women in the second trimester of pregnancy, with higher activities of sialidase and prolidase, accompanied by increased vaginal pH, are at a higher risk of premature delivery because of the synergistic interrelationship among the virulence factors produced by bacteria in the altered vaginal microbiota with a consequent risk of an adverse pregnancy outcome (5) .
The metabolism resulting from the proliferation of these bacteria promote increased production of aromatic amines, the putrescine and cadaverine, which evaporate and induce a bad genital odor (6) .Another frequent symptom is a grayish and fluid vaginal discharge with small bubbles (7) .
The Lactobacillus SP is a gram-positive bacillus that produces components such as lactic acid, bacteriocins and hydrogen, and which has properties that protect the vaginal flora.These components determine an acidic vaginal pH, lower than 4.5, inhibiting the growth of pathogenic bacteria.The vaginal flora is also composed of other commensal microorganisms, which in certain situations can become pathogenic (8) .
We must take into account that under normal conditions, progesterone increases the number of intermediate epithelial cells, resulting in a higher availability of glycogen and decreased vaginal pH.Even though these factors favor the presence of lactobacilli, they are also associated with the development of Candida sp (9) .
Vulvovaginal candidiasis is caused by several species of Candida sp, while Candida albicans is considered a commensal of the vaginal flora.The high levels of vaginal glycogen, and local heat and humidity, are conducive to fungus activation, especially in the second trimester of pregnancy.
Its most common symptoms are itching, urinary discomfort, perineal irritation, and whitish discharge in plates, while between 25% and 40% of women are asymptomatic (10) .Although it is highly prevalent, it is not related to relevant perinatal complications (11) .
Vaginal trichomoniasis is an important sexually transmitted disease, caused by Trichomonas Vaginalis (TV), an agent that mainly infects the LGT squamous epithelium.It is an anaerobic organism that grows well in the absence of oxygen, in pH between 5.0 and 5.7 (12) .
Trichomoniasis symptoms depend on individual conditions, aggressiveness and the number of parasites and vary in intensity depending on the type of infection, which can be classified as acute or chronic.In acute cases, there is the classical sign of abundant, foamy, yellowish mucopurulent discharge (13) .This infection is related to various complications such as infertility, increased risk of HIV transmission, and undesirable pregnancy intercurrences such as the premature rupture of membranes, premature labor, and low birth weight newborns (12) .
Even though cytolytic vaginosis is not an infectious pathology, it is a condition frequently diagnosed as vaginal candidiasis because of its similar symptoms.Complaints reported by patients include whitish discharge, vaginal and vulvar itching, dyspareunia, dysuria, and perineal burning, markedly in the luteal phase of the menstrual cycle.These symptoms would be determined by the elimination of irritating substances from the cytoplasm of intermediate cells, arising from the cytolysis process from the action of lactobacilli.Diagnosis should include microscopic examination of samples directly from vaginal content through Gram's method, to exclude the possibility of infection by Candida sp, observing a significant increase of the number of lactobacilli, generally adhered to epithelial cells, which are also in a larger number, while leucocytes, when present, are rare (14) .

Aerobic vaginitis is associated with aerobic microorganisms, especially Streptococcus agalactiae
and Escherichia coli, while its characteristics are different from those observed in bacterial vaginosis.Aerobic vaginitis raises an important inflammatory response that can cause complications during pregnancy such as ascendant chorioamnionitis, premature rupture of membranes and premature labor (15) .Therefore, diagnosing and treating changes in the vaginal flora and their respective associations is especially relevant during pregnancy.Despite this relevance, health services frequently do not have the means to provide etiological treatment.Aiming to seek scientific evidence to base clinical practice during prenatal care, an important area of nursing practice (16)(17) , this study's www.eerp.usp.br/rlaeRev. Latino-Am.Enfermagem 2010 Sep-Oct;18(5):919-27.
general objective is to identify the prevalence of vaginal flora alterations in low-risk pregnant women based on gold-standard tests and association with reported symptoms and gynecological exams.

Method
This quantitative, descriptive and cross-sectional study was carried out in Botucatu with approximately 120,000 inhabitants, located in the central region of the state of São Paulo, Brazil.The city has public primary health care delivered through three polyclinics, three health centers and two school center units and another eight Family Health Units with 10 teams.In addition to providing primary care within their scope areas, the polyclinics are also referral centers in pediatrics, gynecology and obstetrics, and general practice for the Family Health units and Health centers.
Based on a prevalence of altered vaginal flora of 20% with confidence interval of 95% and a margin of error of 5%, the sample minimum size was determined by: 1.96 0.20x0.800.05 With a total of 1,006 pregnant women cared for in the various health units in 2005, a stratified sample was obtained by unit, considering the coefficient: Half way through the study, the prevalence found was used to recalculate the sample size.As the value obtained was lower than that initially computed, we opted for keeping the original computation.
A total of 289 pregnant women cared for during the prenatal period in the primary care public service in Botucatu, of any pregnancy stage, regardless of gynecological complaint, with at least 72 hours of sexual abstinence and/or vaginal procedures and absence of antibiotherapy in the 30 days prior to the collection were included in the study.

Data Collection
Data were collected from October 2006 to March 2008 by one of the authors.To characterize the studied women, socio-demographic information and personal and obstetrical antecedents were obtained.
Patients included in the study were submitted to speculum examination using a bi-valve vaginal Collins speculum, sterilized and without any lubricating.
After this procedure, the macroscopic characteristics of the vaginal content were noted, followed by a pH measurement with Merk  tape graduated between four and seven, pressed against the middle third of the lateral vaginal wall for one minute, then proceeding to the reading according to the manufacturer's instructions.
Then, the material of the lateral vaginal wall was collected with a sterile swab and that content smeared onto glass slides.After this procedure, two drops of 10% KOH were added to the swab with vaginal content to perform a Whiff test (18)(19) .

Microscopy of vaginal content was performed using
Gram's method.Collection of vaginal content for testing for TV was performed with an Ayre spatula and cultured in Diamond's medium (18)(19) .
The characteristics of vaginal content, a foul odor, vaginal itching, dyspareunia and bleeding after sexual intercourse were considered symptoms and signs when reported by the women (18)(19) .

Diagnosis of abnormal vaginal flora
The proportion between Lactobacillus SP and other microorganisms determine the type of vaginal microbiota: Flora I indicates there is a predominance of lactobacilli and diminished ancillary flora; Flora II is intermediary, with a diminished number of lactobacilli coexisting with other bacteria; and Flora II or bacterial vaginitis, occurs with marked diminished or absence of lactobacilli and predominance of bacterial morphotypes (20) .
The diagnosis of Candidiasis was based on the presence of blastoconidia or pseudohyphae and neutrophils in the microscopy exam of the vaginal content stained by Gram's method.The diagnosis of aerobic (15) and cytolytic (14) vaginitis was also performed according to criteria previously mentioned.
Abnormal vaginal flora was defined as the absence

Studied variables
The

Data analysis
A database were created in the Excel and analyzed using the EpiInfo software.One of the researchers input the entire data and data consistency was checked through verification and comparison of the distribution of frequencies in associated questions; errors were corrected.Statistical analysis was performed through the χ 2 test, whose level of significance was fixed at α=0.05, with computation of respective odds ratio and confidence interval (CI=95%).Whenever relevant, correction of Yates was performed.

Ethical Procedures
This

Results
The median age of the 289 pregnant women studied was 25 years (14-43).Most of the women reported they had a partner (79.2%), nine of more years of schooling (55.4%), no paid job (56.1%) and 42.2% of them did not have children.
The prevalence of abnormal vaginal flora was 49.5% and the most frequent ones were BV, VC and intermediate flora: 20.7%, 11.8% and 11.1%, respectively, not considering associations.Mixed flora totaled 3.4% of the cases (Table 1).3).

Discussion
This study enabled the identification of the prevalence of abnormal vaginal flora in low-risk pregnant women, regardless of the reported complaint, using gold-standard exams with a sample of pregnant women from a medium sized city in the interior of São Paulo, Brazil.
Taking into account the general prevalence (49.5%), the results reveal a high rate of abnormal vaginal flora, which is in agreement with other recent studies involving pregnant women (18)(19) .BV was the most frequent alteration in the flora, with an isolated prevalence of 20.7% and 23.4% considering associations.These or predominance of lactobacilli (BV and intermediate vaginal flora) or a positive culture for Candida sp or TV through microscopy.Mixed flora was defined as positive for BV and VC, intermediate flora and TV and BV and trichomoniasis.All the slides with vaginal content were evaluated by an experienced examiner from the Universidade Estadual Paulista (UNESP) Medical Faculty with relative blinding concerning clinical data.Gondo DCAF, Duarte MTC, Silva MG, Parada CMGL.

Table 1 -
Occurrence of abnormal vaginal flora in the

Table 2 -
Relationship between reported signs and symptoms and abnormal vaginal flora (n=289).Botucatu, SP,

Table 5 -
Relationship between reported signs and symptoms, data from the gynecological exam and candidiasis (n=289).Botucatu, SP, Brazil 2008 * Cases of association with bacterial vaginosis were excluded † Yates correction.