What are the laboratory investigations of vaginitis? (explain in detail)

Bacterial Vaginosis (BV) is the most common vaginal infection in the world, constituting a public health problem due to its association with obstetric and gynecological pathology and the significant risk of acquiring sexually transmitted infections (STIs). This infection is caused by a large number of anaerobic bacteria with a predominance of Gardnerella vaginalis, discovered in 1953 by Leopold in isolates of vaginal secretions from women with cervicitis. In 1955, Gardner and Dukes visualized this bacterium in vaginal secretions from women with nonspecific vaginitis and named it Haemophilus vaginalis. Because it was initially isolated only on blood agar, it was associated with the nutritional requirements of Haemophilus. Later, Gadnerella vaginalis was related to other genera of Gram positive bacilli such as Corynebacterium, Butyribacterium and even Lactobacillus, to finally classify it in the new genus Gardnerella, with a single species G. vaginalis.

It is the most frequent type of vaginal infection in women who are of reproductive age between 15 and 44 years; it represents one third of all vulvo vaginal infections. It affects 10% of the general population and 30% or more, according to specific population groups. In 50% of cases, Bacterial Vaginosis is asymptomatic, hence the importance of knowing its frequency, identifying and treating this condition, to avoid future complications such as pelvic inflammatory disease (PID), the increase in infections after gynecological surgery , the risk of miscarriage, preterm delivery, postpartum endometritis, and low birth weight. In economic terms, it generates more than 10 million medical consultations a year around the world, with a minimum estimated cost of one billion dollars. 6 Microbiologically, it is characterized by a change from the normal, predominantly aerobic vaginal bacterial flora (lactobacilli) to a mixed anaerobic flora that includes Gram negative organisms such as Gardnerella vaginalis, Mobiluncus Prevotella, Bacteroides species, Fusobacterium, Veillonella and Gram positive bacilli such as Propionibacterium, Eubacterium and Bifidobacterium, among others.

Given the high prevalence of bacterial vaginosis and the high percentage of affected asymptomatic women, the objective of this work is to update on the clinical aspects and laboratory diagnosis of bacterial vaginosis.

A descriptive review was carried out, which provided basic concepts on the topic Bacterial vaginosis. Information was provided on the vaginal ecosystem, the etiology of BV, associated risk factors, obstetric-gynecological complications, clinical manifestations and laboratory diagnosis, through electronic databases such as Medline, SciELO, Lilacs, Pubmed, that allowed locating the primary sources that contain original publications of scientific studies and recent information on the topic addressed.

The bibliographic review was carried out with the works published in the last 5 years with the exception of those articles that, due to their importance and relevance, had to be documented regardless of their publication date. The collected documentation was analyzed and classified according to the subject and in this way it was possible to structure the descriptive review of the subject, which sought to give a quick and subjective look at bacterial vaginosis.

Historical review

The history of Bacterial Vaginosis, dating back more than 40 years, has been characterized by multiple changes in the name of the bacteria most commonly isolated in this syndrome and what is related to the names of the disease and its diagnosis. In 1982, Krönig for the first time observed by means of Gram staining in vaginal secretions of women with atypical discharges, the characteristic Gram-negative coccobacilli compatible with G. vaginalis, a bacterial morphology totally different from that usually found at that time (Trichomonas and Candida spp.) . Krönig attributed the disorder to anaerobic streptococci. 8 For several years, different researchers came up with various names for this bacterium and finally in 1980, Greenwood and Pickett, came up with the now known name Gardnerella vaginalis. The disease was initially named with the term * vaginitis *. Until 1954, any "vaginal discharge" that was not caused by Neisseria gonorrhoeae, Trichomonas or Candida, was called nonspecific vaginitis, an aspect that evolved until 1984; After reviewing all the clinical and microbiological data collected so far, Weström et al., demonstrated that the disease is characterized by a very abnormal amount of anaerobic and aerobic bacteria, therefore they proposed the adjective bacterial; since the disease did not produce a typical inflammatory response with the presence of neutrophil polymorphous nuclear leukocytes, the term "vaginitis" was considered incorrect and they raised the name * vaginosis *. Since then, this pathology has been called Bacterial Vaginosis. Currently, this pathology is considered a polymicrobial infection with a predominance of facultative and strict anaerobic bacteria.

The normal vaginal flora includes a group of saprophytic bacteria that fulfill a defense function of the vaginal ecosystem. Within the facultative aerobic or anaerobic bacteria, there are the Döderlein or Lactobacilli bacilli. Other aerobic bacteria, mainly diphtheroids and streptococcal species, Staphylococcus epidermidis, and Escherichia coli are also found in the vaginal ecosystem. Anaerobic bacteria have been detected in 80% of women, mainly Gardnerella vaginalis, Mobiluncus, and Bacteroides among other species. This pathology is described as a heterogeneous polymicrobial syndrome, characterized by the absence or depletion of Lactobacillus spp. preferably the producers of hydrogen peroxide and lactic acid, which generates an increase in the number and diversity of anaerobic or facultative bacteria. such as Mobiluncus spp, Bacteroides spp, Prevotella spp, Peptostreptococcus spp, Fusobacterium, Veillonella, Mycoplasma hominis, Peptococcus spp and Ureaplasma urealyticum, In addition to Gardnerella vaginalis, which due to its frequency has become a public health problem due to complications gynecological that it entails.

The etiology of Bacterial Vaginosis is not very clear. Sexual transmission has been implicated because this disease is rarely found in women who have not experienced sexual intercourse. Additionally, a high percentage of this infectious disease has been documented in monogamous homosexual women and has been associated with new sexual partners, oral sex, and lack of condom use. The acquisition of G. vaginalis and Bacterial Vaginosis occurs rarely in girls, but it is common among adolescents even though they have not yet had sexual intercourse, which contradicts that the disease requires sexual transmission. The acquisition of G. vaginalis increases with sexual contact when there is penetration; however, some other types of non-penetrative sexual contact such as oral sex and masturbation have also been associated, indicating that sexual contact per se is involved in several of its forms and not just the coital relationship.

Associated risk factors

Many studies have described an association between this syndrome and the use of the intrauterine device (IUD) as a contraceptive method (MAC). Likewise, a statistically significant relationship has been established between tobacco consumption and BV; It has been found that the risk of acquiring it would be proportional to the number of cigarettes smoked daily and that the accumulation of various cigarette chemicals in the cervical mucus would directly alter the vaginal microbiota, and would produce local immunosuppression. There is controversy among the authors that BV is a sexually transmitted infection (STI), since it can be found in sexually inactive women. Risk factors associated with this syndrome are known to include smoking, alcohol consumption, condom use, hormonal contraceptives, low educational levels, and age of first sexual intercourse. Other factors favor the appearance of this pathology: pregnancy, use of estrogens, oral contraceptives, systemic antibiotics, having multiple sex partners, in addition to a new monogamous sexual relationship; the retention of tampons, contraceptive devices (IUDs), diaphragms or sponges in addition to the use of broad-spectrum antibiotics because these can destroy bacteria in the normal flora of the vagina promoting infection. Uncontrolled diabetes, immunosuppression, HIV infections, wearing tight pants, douching are also important causes of BV. Additionally, other predisposing factors such as inadequate personal hygiene, multiple sexual partners, irritation or allergy to chemicals such as detergents, fabric softener, vaginal deodorants have been reported.

Gynecological-obstetric complications

Bacterial vaginosis has been associated with chronic endometritis, pelvic inflammatory disease (PID), and vaginal cellulitis after invasive procedures, such as endometrial biopsy, hysteroscopy, hysterosalpingography, IUD insertion, caesarean section, and uterine curettage. During pregnancy, BV is associated with adverse outcomes, including premature rupture of membranes, preterm delivery, intra-amniotic infection, and postpartum endometritis. This syndrome has other important reproductive and gynecological risks, such as its association with cervical intraepithelial neoplasia, chorioamnionitis and infections after gynecological surgery. In addition, it is associated with pelvic inflammatory processes and various longitudinal studies suggest that it increases the susceptibility to acquire gonorrhea, Chlamydia trachomatis, trichomoniasis, human immunodeficiency virus (HIV) and herpes simplex 2 (HSV-2). It has also been associated with abnormal uterine bleeding and with the decrease in the success of in vitro fertilization procedures and with an increased risk of developing cystitis. Although it does not affect conception, it increases the risk of abortion in the first trimester in women undergoing in vitro fertilization. The fact that a minority of pregnant women with BV has adverse consequences is not clear, but among the influencing factors is the response of the host, especially low IgA values ??against hemolysin (produces pores in amniotic cells) of G. vaginalis, TNF and high values ??of sialidase or protease, produced by bacteria associated more specifically with Bacterial Vaginosis.

Clinical manifestations

About 50% of patients with bacterial vaginosis are asymptomatic. In symptomatic cases, stench (referred to as a fishy odor) and vulvar itching. Less frequent are irritative symptoms such as vaginal burning, dysuria and dyspareunia. The stench can be increased during menstruation periods and during unprotected sexual intercourse, since the alkalinity of the blood and semen favor the release of volatile amines. In BV, the pH is usually above 4.5. The clinical manifestations are variable: increase in vaginal discharge of a grayish or whitish color, with a milky consistency. Itching, burning, pain sensations are also reported, which can be confused with other causes of vaginitis. Usually there are no signs of inflammation and the cervix appears normal.

Laboratory diagnostics

Because it is a polymicrobial infection, the diagnosis of Bacterial Vaginosis is based on 4 clinical criteria that reflect the pathophysiology of BV proposed by Amsel and colleagues at the 1983 International Symposium on Vaginosis in Stockholm. The criteria accepted as diagnostic indicators to show the presence of the disease are:

- pH higher than 4.5.

- Fine discharge, adherent and homogeneous white.

- Positive amine test in the presence of 10% KHO.

- presence in 10% to 20% of indicator, guide or clue cells in the microscopic examination in saline preparation. The presence of at least three of these criteria are diagnostic parameters of this infection.

Secondly, there is the Nugent Method, for the demonstration of the imbalance in the vaginal microbiota, through the quantification of four bacterial morphotypes in the direct examination of the vaginal discharge stained with Gram: long Gram positive rods compatible with Lactobacillus spp; Gram-variable or Gram-negative coccobacilli corresponding to Gardnerella vaginalis, Gram-negative bacilli type Bacteroides spp. and Gram negative curved rods representing Mobiluncus spp. The interpretation of the results is numerical based on the score obtained by adding the crosses of the morphotypes of the bacteria present in the vaginal discharge: 7 or more points are a diagnosis of bacterial vaginosis, from 4 to 6 points is considered intermediate and from 0 to 3 is considered normal.

As for cultures, they are not always reliable due to the fact that it is a poly-microbial infection, therefore, they are not recommended due to the lack of specificity. It is important to remember that G. vaginalis has been shown to grow in 100% of cultures in women with bacterial Vvaginosis, but it has also been grown in more than 70% of asymptomatic women. Although commercial laboratories now offer PCR modalities for the diagnosis of Bacterial Vaginosis using various criteria, there is no clear evidence of clinical superiority of these expensive tests over the Amsel and Nugent criteria, nor have they been shown to be helpful in guiding analysis. therapy.

As numerous studies have shown, the sensitivity and specificity of the Amsel criteria compared to the Nugent parameters for the diagnosis of Bacterial Vaginosis are relatively low and unreliable. For example, the pH is increased in Bacterial Vaginosis and also when Trichomonas vaginalis is present, the day of the cycle the woman is on, sexual activity, estrogen insufficiency, pregnancy and the use of vaginal douches; therefore, this criterion is not reliable for the diagnosis of Bacterial Vaginosis. Second, some patients with BV may have epithelial cells that are not indicator cells, or the guide cells are not seen, probably because some women have a chronic condition with inflammation and ulceration of the cervix and consequently there is production of type A immunoglobulins , which blocks the injury of bacteria to the cell through interaction with surface proteins, while other biotypes register in the table a high activity of enzymes that causes a decrease in immunoglobulins and, therefore, the immune response of the host, which then reduces the sensitivity and specificity of this clinical criterion. Finally, the amine test only predicts the diagnosis of BV exactly in 94% of patients, since it can also be found in women with trichomoniasis and, although it constitutes the least sensitive parameter of Amsel, it is one of the most specific. Furthermore, the positivity of this criterion is very subjective as it depends on the olfactory capacity of the examiner.

In many countries the most widely used and accepted method for the diagnosis of Bacterial Vaginosis is the Nugent scoring system. However, in Colombia only the Amsel criteria are used despite the fact that for the quantification of bacterial morphotypes it requires fewer inputs and consequently less costs for its execution. In addition, with the application of the Nugent scoring system, the use of a speculum is not required to obtain the adequate sample and it is a diagnostic test that has been validated and standardized as a gold test for diagnosis. Bearing in mind that the diagnostic validity of a test may vary depending on the method, the examiner, the technique used, the prevalence of the condition in the population is essential, and the low sensitivity and specificity of some of the Amsel et al. Criteria, All laboratories should carry out the diagnosis of Bacterial Vaginosis using the two diagnostic methods to guarantee a fast, reliable and timely diagnosis of this pathology, taking into account the gynecological-obstetric consequences that it produces.

Treatment

Given that bacterial vaginosis is associated with the significant increase in different anaerobic microorganisms and among them, being the most abundant and implicated in most gynecological-obstetric complications, G. vaginalis, treatment must be established not only topically, but also orally, since G. vaginalis is colonizing in addition to the vagina, other sites such as the Bartolini glands, the perianal region (since it has been isolated very abundantly from rectal samples); of these anatomical areas, it can pass to other regions and produce pathologies such as endometritis, pelvic inflammatory disease, urinary tract infections, among others.38

Metronidazole (oral or topical) and clindamycin (oral or topical) are recommended for BV treatment: Metronidazole in doses of 500mg orally, twice a day for 7 days and / or metronidazole gel 0.75% by route intravaginal, once a day for 5 days. Clindamycin cream 2%, intravaginally at bedtime for 7 days, and / or clindamycin 300mg orally 2 times a day for 7 days. 15,27 Oral treatment of BV is important since once the upper reproductive tract has been reached or other anatomic sites are affected, treatment with vaginal clindamycin or metronidazole would not eradicate this infection. Therefore, a systemic treatment is necessary to treat this type of infection and to try to reduce preterm labor.

All drugs offer equivalent efficacy and can be distinguished from each other according to cost, mode of administration, and adverse events. After treatment, recurrence may occur on one or more occasions in up to 58% of women within a 12-month period. These relapses are most likely due to the formation of biofilms, a type of slime produced by bacteria, which coat certain surfaces, and within which bacteria hide and protect themselves from the effects of antibiotics.

According to the analysis by Verstraelen and Swidsinski, there are recent studies that have found that 90% of women with bacterial vaginosis and 10% without it have a complex polymicrobial biofilm that can be demonstrated in the electron microscopy of vaginal biopsies. With standard antibiotic regimens, the bacterial load may decrease, but the biofilm may not be shed, thus setting the stage for recurrence after treatment.

Muzny and Schwebke, showed that the sexual transmission of Bacterial Vaginosis can be transmitted between female sexual partners. Therefore, bacterial vaginosis and its recurrence could be the result of one or more mechanisms: reinfection through sexual activity, lack of reestablishment of the dominant normal flora of lactobacilli, or persistence of a vaginal biofilm, factors to take into account during treatment to avoid recurrence of this pathology.

Since bacterial Vaginosis is one of the most frequent vaginal infections worldwide, this work provides basic tools for a better understanding of the pathophysiology of this disease and the predisposing factors for its acquisition, taking into account that a high percentage of women who carry this syndrome are asymptomatic. Similarly, by offering information on the laboratory methods for the diagnosis of BV, it is intended that these implement the two stipulated methodologies (Amsel criteria and quantification of bacterial morphotypes by Gram staining), thus improving rapid detection. and reliable of this pathology. In this sense, this work provides updated theoretical conceptualization on the most relevant topics of this disease, so that other researchers or health professionals have solid bases for understanding and updated conceptualization for future research or similar works.

By understanding the importance of the vaginal microbiota and how changes in its composition and functioning can affect women's health, therapeutic approaches today are aimed at restoring the normal vaginal microbiota and decreasing the possibility of reinfection in sexual partners. that may also impact the ability to determine the efficacy of current therapeutic approaches to achieve high and sustained long-term cure rates.

Bacterial Vaginosis is the most common cause of vaginal infection in women of reproductive age, so it is necessary to make a quick and effective diagnosis to avoid the multiple gynecological-obstetric complications of this disease. Most of the studies consulted only apply the Amsel parameters as criteria to diagnose the presence of BV, based on the presence of at least 3 of the 4 established clinical criteria, which are insufficient criteria for an adequate clinical diagnosis of this pathology.

The diagnosis of bacterial vaginosis should be complemented with the Gram stain to apply the Nugent parameters that allow quantifying four bacterial morphotypes: Lactobacillus, Gardnerella, Bacteroides and Mobiluncus. The joint performance of these two methods will allow an accurate diagnosis of this syndrome with a high degree of sensitivity and specificity.

In an attempt to avoid reinfection, consistent condom use should be implemented routinely and in women who have sex with other women, cleaning shared sex toys between uses is recommended.