Summary

Bacterial vaginosis (BV) is a vaginal infection that occurs when the balance of bacteria in the vagina is altered. It is
a common condition affecting millions of women. Although the syndrome is curable with standard drugs such as
metronidazole and clindamycin, relapse rates are high. Many patients are asymptomatic and recurrence is difficult to
differentiate from treatment failure. The infection can have gynaecological and obstetric complications. In addition,
there is an association with the transmission of sexually transmitted infections (STIs) including HIV/AIDS. This
review focuses on the epidemiology, aetiology, diagnosis, complications and treatment of BV, with emphasis on the
role of non-antimicrobial treatment options. Firstly, the lowering of the vaginal pH is discussed as one possible
treatment option. An overview is given of the use of acetic and lactic acid gels, boric acid suppositories, as well as
studies that reported on the use of douches and tampons. Thereafter, the role of Lactobacillus (probiotic)
supplementation as treatment is discussed. Literature sources recommend that more research on BV be conducted.
Although standard pharmacological therapy is effective, there are limited treatment options available. Recent
research indicating the presence of a structured polymicrobial Gardnerella vaginalis biofilm attached to the
endometrium may have major implications for future research into the pathogenesis and treatment of BV.

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 Chapter 1

Introduction

Bacterial vaginosis is a condition caused by an overgrowth of normal vaginal flora. Most

commonly, this presents clinically with increased vaginal discharge that has a fish-like odor. The

discharge itself is typically thin and either gray or white.(Greenbaum et al., 2019). After being

diagnosed with bacterial vaginosis, women have an increased risk of acquiring other sexually

transmitted infections (STI), and pregnant women have an increased risk of early delivery (Russo

et al., 2019)

Bacterial vaginosis (BV) is the most prevalent cause of abnormal vaginal discharge in women of

childbearing age. Those severely affected experience an offensive fishy-smelling discharge

which recurs frequently, often around the time of menstruation. Others may have BV transiently

and asymptomatically. It usually responds to treatment with antibiotics but can relapse rapidly,

and reported rates of relapse are more than 50% within 3–6 months. There is need for

alternatives to antibiotics and to find a way to prevent relapse. Some recent studies imply that it

is sexually transmitted, with more pathogenic strains of Gardnerella being identified. BV is a risk

factor for adverse pregnancy outcomes, including second-trimester miscarriage, spontaneous

preterm birth, and post-Caesarean section endometritis. It is also consistently associated with

acquisition of sexually transmitted infections (STIs), including HIV.

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 Chapter 2

Literature Review

2.1 Background Information of Bacterial Vaginosis

Bacterial vaginosis (BV), or nonspecific vaginitis, was named because bacteria are the etiologic

agents and an associated inflammatory response is lacking. Many studies have demonstrated the

relation of Gardnerella vaginalis with other bacteria in causing BV, such as Lactobacillus,

Prevotella, and anaerobes, including Mobiluncus, Bacteroides, Peptostreptococcus,

Fusobacterium, Veillonella, and Eubacterium. Mycoplasma hominis, Urea plasma

urealyticum,Streptococcus viridans, and Atopobium vaginae have also been associated with BV

(Brocklehurst et al., 2013).

Gardnerella vaginalis is a facultatively anaerobic gram-variable rod. It has been demonstrated to

cause a wide variety of infections; however, it is most commonly recognized for its role as one of

the organisms responsible for bacterial vaginosis (Schuyler et al., 2015).

2.2 Signs and Symptoms of Bacterial Vaginosis

Typical symptoms of BV include the following: Vaginal odor (the most common, and often

initial, symptom of BV); often recognized only after sexual intercourse, Mildly to moderately

increased vaginal discharge, Vulvar irritation (less common), and Dysuria or dyspareunia

(rare).Risk factors that may predispose patients to BV include the following: Recent antibiotic

use, Decreased estrogen production of the host, Wearing an intrauterine device (IUD),

Douching, Sexual activity that could lead to transmission (eg, having a new sexual partner or a

recent increase in the number of sexual partners)

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Physical findings in BV may include the following: Gray, thin, and homogeneous vaginal

discharge, which adheres to the vaginal mucosa, increased light reflex of the vaginal walls, but

typically with little or no evidence of inflammation, Normal-appearing labia, introitus, cervix,

and cervical discharge, in some case, evidence of cervicitis

2.3 Pathophysiology of Bacterial Vaginosis

In BV, the vaginal flora becomes altered through known and unknown mechanisms, causing an

increase in the local pH. This may result from a reduction in the hydrogen peroxide–producing

lactobacilli. Lactobacilli are large rod-shaped organisms that help maintain the acidic pH of

healthy vaginas and inhibit other anaerobic microorganisms through elaboration of hydrogen

peroxide. Normally, lactobacilli are found in high concentrations in the healthy vagina. In BV,

the lactobacilli population is reduced greatly, while populations of various anaerobes and G.

vaginalis are increased.

Gardnerella Vaginalis forms a biofilm in the vagina (Patterson et al., 2007). Some studies show

that this biofilm may be resistant to some forms of medical treatment. This predominant G.

vaginalis biofilm has been shown to survive in hydrogen peroxide (H 2O2), lactic acid, and high

levels of antibiotics. When the biofilm was subjected in the laboratory to enzymatic dissolution,

susceptibility to H2O2 and lactic acid were restored (Patterson et al., 2007). These findings may

lead to future development of novel therapies involving enzymatic degradation of biofilms. No

such products are currently on the market (Patterson et al., 2007).

Although BV is not considered a sexually transmitted disease, sexual activity has been linked to

development of this infection. Observations in support of this include the following: (1)

incidence of BV increases with an increase in the number of recent and lifetime sexual partners,

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(2) a new sexual partner can be related to BV, and (3) male partners of women with BV may

have urethral colonization by the same organism, but the male is asymptomatic. Evidence that

does not support an exclusive sexually transmitted role of BV is its occurrence in virginal

females and its colonization of the rectum in virginal boys and girls.

More recent studies indicated that BV is associated with changes in select soluble immune

mediators, an increase in HIV target cells, and a reduction in endogenous antimicrobial activity,

which may contribute to the increased risk of HIV acquisition. (Meriwether et al., 2015;

Harrison, 2016).

2.4 Aetiology of Bacterial Vaginosis

The aetiology of BV is poorly understood and remains a subject for debate. BV can arise and

remit spontaneously or develop into a chronic or recurrent disease (Donders, 2010). There are no

proven individual predisposing factors exclusive to BV (Henn et al., 2005). Risk factors that

have been associated with BV include having multiple sex partners, a new male sex partner, sex

with a woman, early age at first intercourse, frequent vaginal douching, use of vaginal foreign

bodies or perfumed soaps, cigarette smoking and lack of vaginal Lactobacilli (Cherpes et al.,

2008). Although BV has never been proven to be sexually transmitted, it has an epidemiological

profile consistent with that of a sexually transmitted infection (STI) (Henn et al., 2005), although

it is better described as a SED. It is more common among women who have an STI or who use

intrauterine devices (Fethers et al., 2008; Wilson et al., 2007). Women who have never had

sexual intercourse may also be affected.BV may sometimes affect women after menopause. The

decrease in oestrogen levels in perimenopausal andpostmenopausal women has been linked to an

abnormal vaginal flora of 35 and 70%, respectively when comparedto the normal flora (Wilson

et al., 2007). It has also been shown that amenorrhoea lowers the risk of BV as the absence of

5
blood maintains vaginal pH, low and stable around pH 4.5. Subclinical iron deficiency (anaemia)

is a strong predictor of BV in pregnant women (Verstraelen et al., 2005), especially in

developing countries. A longitudinal study published in 2006 showed a link between

psychosocial stress and BV independent of other risk factors (Verstraelen et al., 2005).It is

generally acknowledged that vaginal Lactobacilliplay an essential role in maintaining an

environment that limits the growth of pathogenic microorganisms in the vagina (Mania-

Pramanik et al., 2009). It has been suggested that the presence of oestrogen and Lactobacillusare

needed to achieve an optimal vaginal pH of 4.0 to 4.5(Melvin et al., 2008; Suresh et al., 2009).

After puberty under the influence of oestrogen, glycogen is deposited in the vaginal epithelial

cells, which is metabolised by vaginal epithelial cells to glucose (Suresh et al.,

2009).Lactobacilli produce lactic acid from glucose, keeping the vagina at an acidic pH (Suresh

et al., 2009). Some species of Lactobacilli produce hydrogen peroxide which is toxic to various

microorganisms (Suresh et al., 2009).

Bacterial vaginosis is therefore characterized by an alteration of the normal acidic Lactobacilli-

predominant vaginal ecosystem to a vaginal milieu dominated by mixed anaerobic bacteria flora

with an accompanying increase in pH (Geva et al., 2006). The complex aetiology of BV of a

continuum of changes in the vaginal flora, rather than a single pathogen infection (Morris et al.,

2001), includes a log10-fold indigenous Lactobacillus-predominant vaginal microflora. The

development of a more anaerobic environment inhibits the growth of Lactobacilli. Srinivasan

and Fredricks (2008) give a complete overview on the vaginal flora in BV from a

microbiological and molecular perspective. It is unknown whether the loss of Lactobacilli

precedes the BV infection or is a result of the infection (ManiaPramanik et al., 2009).

Furthermore, it is not known whether the change in flora is not the result of an as yet unidentified

6
aetiological factor, suggesting that the altered flora is actually a downstream event of BV

(Nansel et al., 2006). The overgrowth by the facultative anaerobes is associated with an increase

in protease production especially carboxypeptidase which leads to the breakdown of peptides to

amines which in an environment of higher pH can become volatile. Due to the flexible nature of

the disease process, the host response in BV should be considered, although most work has been

performed on the changes in micro-bial flora. It was initially believed that inflammation is absent

during BV (Morris et al., 2001) but it has since been shown that median levels of IL-1β, TNF-α,

IL-6 and Il-8 in intermediate stages(between normal flora and BV) and BV are similar but

significantly higher than in normal flora (Hedges et al., 2006). A subtle but significant inverse

relationship has also been noted between IL-1β and the presence of Lactobacilli in the vagina.

Recurrences frequently occur after treatment. Recurrent BV is generally defined as three or more

proven (clinically by Amsel’s criteria or microscopically) episodes of BV in 12 months (Amsel

et al., 1983; Wilson, 2004; Hay 2009). It is postulated that BV recurs around the time of

menstruation (Henn et al., 2005) when oestrogen levels are low and vaginal pH is higher than

normal (Wilson et al., 2007). With treatment, the cure rates are 80 to 90% at one week, but

recurrences are reported in 15 to 30% within three months (Wilson et al., 2005; Wilson, 2004).

Women with recurrent BV appear to have a lower initial cure rate (Wilson et al., 2005). In a

study of women with recurrent BV, complete clinical and microscopic cure occurred in only

23% of episodes following treatment, raised vaginal pH was present in 65%, positive amine

whiff test in 15% and abnormal Gram-stained flora in 24% (Larsson, 1993). Women who

developed early recurrence tended to still complain of an abnormal discharge or, if

asymptomatic, continued to have significant abnormalities of vaginal flora (Cook et al.,

1992).Recent literature on BV epidemiology has largely focused on the presence of Gardnerella

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vaginalis, which is now recognised as a key pathogen in BV (Verstraelen et al., 2010). Recent

research (Swidsinski et al., 2013) has also indicated that the pathogenic effects of BV are not

confined to the lower genital tract. BV is strongly associated with late foetal loss (Oakeshott et

al., 2002) and preterm birth (Leitich and Kiss, 2007), possibly due to an ascending genital tract

infection pathway, though the precise mechanisms are not clear. Substantially higher rates of BV

have been documented in infertile patients (Wilson et al., 2002). According to Salah et al.,

(2013) BV is strongly implicated in female infertility and is probably an underestimated cause of

unexplained infertility. An increased risk of early pregnancy loss associated with BV has also

been found (Ralph et al., 1999). The microbiological correlate of BV has been shown to involve

a dense, highly structured polymicrobial biofilm,primarily consisting of G. vaginalis, strongly

adhering to the vaginal epithelium (Swidsinski et al., 2005). This may explain the recurrent

nature of this condition (Swidsinski et al., 2008), since this has also been shown for other

biofilm-associated infections.

2.5 Epidemiology of Bacterial Vaginosis

BV is the most common vaginal infection among women in their reproductive years (Donders,

2010; Morris et al., 2001). BV is also the most common cause of vaginal discharge and

malodour (Mania-Pramanik et al., 2009).Generally, it is estimated that 1 in 3 women will

develop the condition at some point in their lives. Its prevalence ranges between 4.9 and 36% in

developed countries (Henn et al., 2005). An increased risk for the development of BV has been

shown with surgery and pregnancy where it is estimated that 15 to 20% of pregnant women have

BV (Alfonsi et al., 2004). Other studies have reported the prevalence of BV among non-pregnant

women to range from 15 to 30%, and have reported that up to 50% of pregnant women have

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been found to have BV (Laxmi et al., 2012). In a recent study by (Nelson et al., 2013) among

urban, primarily African-American Pregnant women, 74% were identified with Nugent score

BV.

The prevalence of BV varies around the world (Kenyon et al., 2013) conducted a systematic

review on the global epidemiology of BV. The BV prevalence varies considerably between

ethnic groups in North America, South America, Europe, the Middle East and Asia. Although

BV prevalence is, in general, highest in parts of Africa and lowest in much of Asia and Europe,

some populations in Africa have very low BV prevalence and some in Asia and Europe have

high rates. If these findings are considered, it can be concluded that RTI has a varying degree of

prevalence rate among people of different communities which might be due to various factors

such as socio-demographic characteristics, sexual practices and hygiene behaviour.BV is often

linked to sexual behaviour, and the epidemiological profile of BV mirrors that of established

sexually transmitted infections (STIs) (Verstraelen et al., 2010). There is, however, not

conclusive evidence whether BV pathogenesis involves sexual transmission of pathogenic micro-

organisms from men to women. Gardnerella vaginalis carriage and BV occurs rarely with

children, but has been observed among adolescents (even sexually non-experienced girls),

contradicting that sexual transmission is a necessary prerequisite to disease acquisition

(Verstraelen et al., 2010). Although male-to-female transmission cannot be ruled out, there is

little evidence that BV acts as an STD. BV is therefore rather considered as a sexually enhanced

disease (SED), with frequency of intercourse being a critical factor (Verstraelen et al., 2010)

2.5.1 Race-, Sex-, and Age-related Demographics

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Some studies have shown that bacterial vaginosis appears to occur more commonly among

African American women than non-Hispanic white women (Payne et al., 2010). The reasons for

this are not entirely clear.

Gardnerella vaginalis colonization and/or infection predominantly occurs in women. Men rarely

develop infections with Gardnerella vaginalis; however, the urethras of men whose sexual

partners have symptoms of BV are frequently colonized with the same strain of Gardnerella

vaginalis. A recent study by Bradshaw et al., 2016, found that Gardnerella vaginalis is not

associated with nongonococcal urethritis.

Gardnerella vaginalis infections typically occur in women of reproductive age. Studies have

documented G vaginalis colonization in prepubertal and/or virginal girls and boys and cases of

BV occurring in prepubertal and/or virginal girls.

2.5.2 Prevalence

For clinicians, BV is a common vaginal condition characterized by at least three of the following

four Amsel criteria: 1) thin, gray/white discharge; 2) malodorous “fishy” discharge upon adding

10 % potassium hydroxide; 3) high vaginal pH (>4.5), and 4) identification of vaginal epithelial

cells heavily coated with bacteria (i.e.,“clue cells”) (Amsel et al., 1983). For research purposes,

BV is commonly diagnosed by a Gram’s stain-based evaluation of vaginal bacterial morphotypes

using the Nugent score (≥7 indicates BV) (Nugent et al., 1991). It is assumed that BV is

characterized by the replacement of normal vaginal lactobacilli with that of anaerobic

microorganisms (e.g., G vaginalis, Prevotella, Peptostreptococcus, and Bacteriodes spp). This

replacement causes an imbalance in the vaginal microflora, which is the pathophysiologic

10
process responsible for the discharge. It is estimated that 20–30 % of women with vaginal

discharge have BV, although the prevalence can be as high as 50–60 % in some high-risk sexual

behavior populations (Rubins & Gross, 2011).

A 2013 systematic review reported that BV prevalence varies between and within countries

worldwide (Kenyon et al., 2013). Women from South and East Africa have higher rates of BV

(68 % in Mozambique, 51 % in Lesotho, 44 % in Kenya, 37 % in Gambia) compared to women

from West Africa (7 % in Burkina Faso) (Nugent et al., 1991). In Norway (24 %), Turkey (23

%), and Poland (19 %), women have moderately high BV rates. Women from Southeast Asia,

Australia, New Zealand, and Indonesia have rates of BV that are typically greater than 30 %.

Women in Latin America and the Caribbean have lower rates of BV, except in rural and

antenatal populations in Jamaica and Peru (rates of −40 %). In the US, BV is a common

condition among women, with prevalence varying by race/ethnicity: African-American (51 %),

Hispanic (32 %), and whites (23 %). Similarly, in Canada, aboriginal and indigenous women

have high BV rates (33 %). Among female military recruits to the US Marine Corps between

1999 and 2000, the prevalence of BV was 27 % (Yen et al., 2003). According to this study,

Native American (34 %), African-American (32 %), and Hispanic (30 %) female recruits had the

highest BV burden. Data from the US Defense Medical Surveillance System indicates that

between 2004 and 2013, 149,666 (15,000 cases per year) BV cases were reported in women in

the US military. Of these, 45 % occurred among U.S. Army personnel (unpublished data). A

recent ecological study conducted by Kenyon and Colebunders among males in 11 countries

(Central African Republic, Brazil, Cote d’Ivoire, Kenya, Lesotho, Philippines, Singapore, Sri

Lanka, Thailand, Zambia, and Tanzania) reported a moderate correlation between the number of

partners (as measured by the question, “Do you now have one or more than one spouse/regular

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partner?”) and BV prevalence (R2 = 0.57). Recent studies conducted among pregnant, HIV-

positive and infertile women have also reported high BV prevalences. Among pregnant women

in northeastern Nigeria and Ethiopia, the prevalence of BV was 17 and 19 %, respectively

(Ibrahim et al., 2014); among human immunodeficiency virus (HIV) positive women, a BV

prevalence of 48 % has been described in India (Lallar et al., 2015), whereas among infertile

women in Qom and Iran the prevalence of BV was found to be 70 %. Information on the burden

of BV in Eastern Europe is limited. However, in Bulgaria, by polymerase chain reaction (PCR)

and Gram’s staining methods, the BV prevalence was found to be 56 and 57 %, respectively. It

has also been reported that the prevalence of BV is high among women with tubal factor

infertility in Nigeria. In recent years, BV among women who have sex with women (WSW) has

received additional research attention. Between 1995 and 2014, five studies have reported high

prevalence estimates (~25 to ~50 %) among WSW. Although there is no specific anatomic or

physiologic reason to explain this high prevalence, it has been hypothesized that vaginal fluid

exchange may represent an efficient mode of transmission, much as occurs with penile-vaginal

sex. Researchers believe that WSW are also unique high-risk population for the study of BV

pathogenesis. Moreover, there is evidence that sexual relationships and behaviors have a strong

influence on BV acquisition. These findings would support the hypothesis that BV is sexually

transmitted.

2.6 Chlamydia and Gonorrhea

Genital Chlamydia trachomatis (CT) is a common bacterial STI worldwide that is spread

through oral, anal, or vaginal sex in both women and men (Vasilevsky et al., 2014). Based on US

National Health and Nutrition Examination Survey (NHANES) data collected from 2007 to

2012, the prevalence of chlamydia has been estimated to be 1.7 % among adults 14–39 years old

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(Torrone et al., 2014). Data from NHANES also indicates that African-Americans (5.2 %),

people with two or more lifetime sexual partners (3.2 %), divorced/widowed/separated

participants (3.0 %), and participants 20–24 years of age (2.9 %) have higher infection rates. In

the US, it is estimated that 2.8 million cases of chlamydia are reported each year .According to

the Armed Forces Health Surveillance Center (AFHSC), from 2000 to 2012, the human

papillomavirus (HPV, 304,021 cases), chlamydia (198,274 cases), and gonorrhea (41,713 cases)

were the three most frequently diagnosed STIs among US military personnel on active duty

status . One epidemiologic characteristic of a Chlamydia infection is that it is usually

asymptomatic (~75 % in women, ~50 % in men). In women, common chlamydia symptoms

include abdominal pain, abnormal vaginal discharge, intermenstrual bleeding, painful

intercourse, burning while urinating, vaginal bleeding after intercourse, and a yellow discharge

with a strong odor. In men, the most common chlamydia symptom is urethritis, followed by a

burning sensation during urination, and itching of the skin of the penis. Younger age is the main

risk factor associated with chlamydia among women, and 60–70 % of chlamydia infections are

reported among adolescents and young adults <25 years of age (Vasilevsky et al., 2014). Other

risk factors associated with infection are: 1) smoking; 2) substance use; 3) preceding HPV

infection; 4) number of lifetime sexual partners; 5) sex with new partners; 6) lack of condom use,

and 7) having a sex partner with a CT infection, cervicitis, and/or prior history of chlamydiaor

other STI (Bebear and Barbeyrac, 2009). In women, chlamydia causes cervicitis, urethritis, and

endometritis. Untreated cervicitis can cause pelvic inflammatory disease (PID), ectopic

pregnancy, infertility, and chronic pelvic pain. In men, untreated chlamydia may cause tender or

swollen testicles and a decline in sperm mobility and concentration, which are both associated

with infertility. Neisseria gonorrhoeae (NG) is a bacterium that causes gonorrhea, a curable and

13
very contagious infection transmitted through genital and anal sex and less frequently through

oral sex. It constitutes the second most common STI worldwide accounting for ~100 million of

the estimated ~500 million new cases of curable STIs worldwide annually. In the US,

approximately one million cases were reported at the disease’s peak in 1975, with decreasing

incidence to 350,000 cases by the year 2000. The principal factor in gonorrhea’s decline was the

widespread use of penicillin as a first line antimicrobial in the mid-1970s. In 2013, the national

incidence of gonorrhea was 106.1 cases per 100,000 persons. Of the 333,004 cases reported that

year, 93 % occurred in persons aged 15–44 years. Gonorrhea is also commonly reported among

active duty personnel in the US Armed Forces, with stable incidence rates since 2001 (~200

cases per 100,000 persons-years). In men, gonorrhea most commonly presents as acute urethritis.

Asymptomatic infection rates may be as high as 75 %, in much the same manner as chlamydia

infections are for women. In women, the bacteria initially infect the endocervical canal causing

gonococcal cervicitis; however, gonorrhea is asymptomatic in up to 70–90 % of women,

providing an important reservoir of infection (Walker and sweet, 201). When symptomatic,

common signs and symptoms in women are vaginal discharge (green or yellow color) with an

unpleasant odor, together with bleeding during sexual intercourse, painful urination, or itching.

For both men and women, the strongest risk factor associated with a NG infection is young age

(15–24 years). Other risk factors/indicators include: 1) African American or Hispanic

race/ethnicity; 2) illicit drug use; 3) casual sex partners; 4) presence of other STI pathogens (e.g.,

chlamydia); 5) history of STIs; 6) lack of barrier contraception, and 7) inconsistent condom use.

Biological, behavioral, and socio-cultural factors influence the risk of gonorrhea transmission in

adolescents and young adult populations .First, compared to older women, younger women have

larger areas of cervical ectropion and thus are biologically more susceptible to infection. Second,

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sexually active younger populations engage in more risky sexual behaviors (e.g., multiple sexual

partners) compared to the general population, increasing the risk of infection. Finally, at the

community level, a gender imbalance represents a factor for transmission; in areas with more

women than men, young women are vulnerable to subtle coercion to engage in high-risk sexual

behaviors. In the past decade, a new generation of non-culture tests called nucleic acid

amplification tests (NAATs), which are both highly sensitive and specific, have revolutionized

diagnosis of gonorrhea and chlamydia infections. Two valuable characteristics of NAAT

‘testing’ are the use of less invasive samples, including self-collected specimens such as urine

samples, and the possibility of detecting both gonorrhea and chlamydia using the same specimen.

2.7 Bacterial Vaginosis and Associations with HIV, Chlamydia, and Gonorrhea

In 1995, Cohen reported a positive relationship between BV and HIV in CSWs from Chiang

Mai, Thailand. Since then, this association has been reported in pregnant and postnatal women in

Malawi, women in South Africa, and CSWs in Kenyan, among others. A systematic review of 23

cohort studies estimated that BV increases the risk of HIV by 60 %. Although causality remains

unclear, a high pH in the vaginal environment (>4.5) may allow for the adherence and survival of

HIV, increasing the risk of HIV infection. Other studies also indicate that BV is a risk for both

ulcerative (e.g.,herpes simplex virus type 2 (HSV-2), syphilis and non-ulcerative STIs (e.g.,

gonorrhea, chlamydia).

Joesoef in 1996 was the first to report an association of BV with chlamydia (two-fold increase)

and gonorrhea (six-fold increase) among pregnant women with BV in Indonesia and suggested

BV as a potential marker for these two common bacterial STIs (Joesoef et al., 1996). One year

later, Keane conducted a case control study among 51 couples and observed a strong association

15
between chlamydia and BV (odds ratio = 5.4) (Keane et al., 1997). That same year, a large

Swedish cross-sectional study involving 1101 women found that high-risk sexual behaviors, such

as a high number of lifetime sexual partners, a history of anal sex, having multiple partners in the

last month, and a history of sexual abuse and rape were similar among both BV and chlamydia

patients. In 1999, Martin found that absence of vaginal lactobacilli increased the risk of

gonorrhea (hazard ratio = 1.7), but not chlamydia. Wiesenfeld reported a strong relationship

between BV and both CT and NG infections following exposure to male partners with urethritis;

this study was conducted among 255 non-pregnant women 15–30 years of age. In this study,

women with BV were 4.1 times more likely to test positive for NG and 3.4 times more likely to

test positive for CT compared to women without BV. Among 1179 African-American women

who were followed for 3 years with visits every 6 and 12 months between 1999 and 2001, Ness

et al., found that BV was associated with concurrent NG and CT infections at baseline but not

subsequently. However, the authors conducted a trial of NG/CT treatment during follow-up and

this might have affected their estimates. According to a secondary data analysis of 535 women at

high-risk for STI, Allsoworth observed that BV severity, as measured by a high Nugent score (8–

10), was associated with incident STIs (NG, CT and Trichomonas vaginalis) and with severe BV

cases experiencing a 2-fold increased risk for STIs compared to women with normal vaginal

flora Findings from two cohort studies associating BV with a CT or NG infection were published

in 2010 and 2012. Both studies were conducted among nonpregnant women. According to

Brotman’s study, which was a secondary analysis of 3620 non-pregnant women 15–44 years of

age enrolled in the Longitudinal Study of Vaginal Flora in Birmingham, Alabama from 1999 to

2002, women with BV (measured as a Nugent score of 7–10) had a 1.8- and 1.9-fold increased

risk for gonorrhea and chlamydia, respectively. The second cohort study conducted by Gallo re-

16
analyzed data from a large condom intervention study among 1159 women who attended public

STI clinics between 1995 and 1998 in Birmingham and Huntsville, Alabama, and used

generalized estimating equations methods to account for multiple individual visits (6 monthly

follow-up visits). The authors reported that women with an incident BV episode at a prior visit

were at a 1.6 times the risk of having chlamydia or gonorrhea at a subsequent follow-up visit. In

addition, Gallo found a relationship between these two bacterial STIs and BV; namely,

chlamydia or gonorrhea increased by 2.4 times the risk of having BV at a subsequent visit. This

represents the first report of a temporal relationship working in both directions between BV and

chlamydia/ gonorrhea. Recently, two studies have reported a significant association between BV

and these STIs among women in Durban, South Africa and among female sex workers in

Uganda, Considered together, multiple studies report BV as a risk factor or at least an important

contributor in subsequent gonorrhea or chlamydia infection. It is important to note that the

observation that BV increases the risk of chlamydia or gonorrhea was mainly seen in high-risk

women (e.g., CSWs, women attending STI clinics, and/or women at risk for unplanned

pregnancies). Additionally, the two recent cohort publications are based on secondary data

analyses of data collected in the late 1990s to early 2000s, and their findings must be interpreted

with caution considering the changes in the epidemiology of chlamydia in the US over the past

decade. We believe that additional research with increased sample sizes and using modern

epidemiological and statistical methods in other young sexually active women affected by these

bacterial STIs are warranted. For instance, women serving in the US Armed Forces can provide

additional data-based evidence about the role of BV as a risk factor or indicator for CT and NG

infection. In addition, molecular studies to determine risk factors and adverse outcomes

associated with the subtypes of BV in different risk behaviors groups are also required. Data

17
from these studies may provide additional evidence as to the epidemiology of this leading

disorder among women of reproductive age.

2.8 Complications of Bacterial Vaginosis

More important than symptoms are complications associated with BV. These appear to be related

to an increased risk of susceptibility to STIs including infection with Chlamydia trachomatis,

Neisseria gonorrhoeae, HSV-1 and -2, and an increased risk of HIV acquisition, and to an

adverse outcome of pregnancy (Geva et al., 2006). BV has been shown to increase the risk of

gynaecological and obstetric complications such as preterm labour and delivery,

chorioamnionitis, post-caesarean endometritis, post-abortion pelvic inflammatory disease and

cervicitis. Several groups had found that bacterial vaginal flora has an impact on these

complications (Johnson et al., 1985), while other studies disproved some of these findings. The

leading hypothesis concerning these associations is that absence of protective Lactobacilli

increases biological susceptibility of acquiring an STI upon exposure (Alfonsi et al., 2004).

However, the temporal nature of the association between BV and acquisition of STIs remains an

ongoing discussion. Although there is evidence favouring the plausibility that BV also incurs an

elevated risk for HPV acquisition (Truter and Graz, 2013), this also remains a matter of debate.

Since the 1970’s BV has been associated with pelvic inflammatory disease in the absence of

Chlamydia or Neisseria gonorrhea (Morris et al., 2001). Finally, there is also a potential link

between BV and an increased risk of HIV infection (Mania-Pramanik et al., 2009).A Cochrane

study (McDonald et al., 2011) found that the administration of antibiotics during pregnancy for

overgrowth of abnormal bacteria in the birth canal does not reduce the risk of babies being

preterm. A more recent Cochrane review confirmed this finding that antibiotic treatment can

18
eradicate bacterial vaginosis in pregnancy, but that the overall risk of preterm birth was not

significantly reduced (Brockhurst et al., 2013).Furthermore, it has been shown that BV increases

the risk of miscarriage between 13 and 24 weeks (Donders, 2010), the risk of babies being

preterm and a 40% elevated risk of low birth weight (Morris et al., 2001).

2.9 Diagnosis of Bacterial Vaginosis

At least 50% of women with BV have no symptoms (Henn et al., 2005) and there is a debate on

whether this form of BV should be considered a disease (Nansel et al., 2006). In the other half,

BV most often manifests clinically as a thin homogenous vaginal discharge, a pH of more than

4.5, presence of “clue cells” and an amine odour (after addition of 10% of KOH). Few or no

Lactobacilli are usually found through microscopy in the vaginal fluid (Larsson, 1992). Several

methods are currently in use for the diagnosis of BV (Cook et al., 1992). Amsel criteria have

been used in most studies as the gold standard.

Clue cells are vaginal squamous epithelial cells with coccobacilli-shaped bacteria densely

adhered to them and obscuring their borders and making these appear indistinct rather than

clearly defined (Truter and Graz, 2013).

Furthermore, there is a significant lack of polymorphonuclear lymphocytes characterised by < 1

PMN per squamous epithelial cell. The sensitivity and specificity of > 20% clue cells in the

diagnosis of BV is 81 and 99%, respectively and clue cells are said to be the single most reliable

predictor of BV (Henn et al., 2005). Other methods rely only on microscopically confirmed

criteria of a Gram-stained smear. The Nugent scoring system classifies vaginal smears into

normal flora, intermediate flora or BV infection according to the number of bacterial

19
morphotypes counted per field of vision. In this scale, a score of 0 to 10 is generated. This

method is time consuming and requires trained staff, but it has high inter observer reliability. The

scores are as follows (Nugent et al., 1992):

1. 0 to 3 is considered negative for BV.

2. 4 to 6 is considered intermediate.

3. 7+ is considered indicative of BV.

A simpler scoring system applied to Gram-stained smears is described by Hay and Ison whereby

only the correlation between the different morphotypes is examined, rather than the exact

number per field of vision (Hay et al., 1994):

1. Grade 1(Normal):Lactobacillus morphotypes predominate.

2. Grade 2 (Intermediate): Mixed flora with some Lactobacilli present, but Gardnerella or

Mobiluncus, morphotypes also present.

3. Grade 3 (Bacterial Vaginosis): Predominantly

The Amsel criteria have subjective components (macroscopic judgement of the vaginal

discharge and reliance on examiner olfaction) and require access to a microscope. The Nugent

and Hay/Ison methods both require a microscope and specially trained staff for Gram-staining

and bacteria counts.Vaginal pH testing alone is highly sensitive, but it is not specific for BV

(Henn et al., 2005; Charonis et al., 2006;Thulkar et al., 2010). Various commercial tests to

diagnose BV are in use (Henn et al., 2005). Molecular techniques have been used to characterise

the normal and BV associated flora but to date are not used in routine diagnosis (Donders, 2010).

2.10 Syndromic Approach to STI

20
WHO introduced a syndromic approach: simple, client friendly, cost effective, applicable in the

community at the primary care level.

STIs managed based on the clinical presentation, and treatment can be given without the

laboratory test.

The main infective agents are grouped according to clinical syndromes they cause and patients

are treated for all the important causes of a syndrome, using combinations of antimicrobials.

2.11 WHO Syndromic Approach

1. Vaginal discharge

2. Urethral discharge

3. Genital ulcer

4. Lower abdominal pain

5. Scrotal swelling

6. Inguinal bubo

7. Neonatal conjunctivitis

2.12 Treatment and Recurrence

The recommended treatments for BV, according to the 2015 Centers for Disease Control and

Prevention Sexually Transmitted Disease guidelines, are: (1) oral metronidazole, 500 mg twice

daily for 7 days; (2) metronidazole, 0.75% gel intravaginally at bedtime for 5 days; or (3)

clindamycin cream, 2% intravaginally at bedtime for 7 days. Alternative regimens include

tinidazole, 2g daily for 2 days; tinidazole, 1g daily for 5 days; clindamycin, 300 mg twice daily

for 7 days; or clindamycin ovules, 100 mg intravaginally at bedtime for 3 days.9 Reported cure

21
rates for an episode of acute BV vary but have been estimated to be between 70% and 80%.

Unfortunately, more than 50% of BV cases will recur at least once within the following 12

months (Bradshaw and Sobe, 2018). Because the etiology of BV is still not entirely understood,

identifying the cause of recurrent cases is challenging. Reinfection may play a role in explaining

recurrent BV, but treatment failure is a more likely contributor. There are several theories that try

to explain recurrence and persistent symptoms. The existence of a biofilm in the vagina is one

such theory and is the subject of ongoing research. Biofilms occur when microorganisms adhere

to surfaces. G vaginalis, one of the primary organisms associated with BV, is now known to

adhere to the vaginal epithelium, which creates a sticky biofilm on the vaginal wall. It is thought

that this film may limit penetration of the antibiotics intended to eradicate bacterial growth. The

film may also serve as a support scaffolding that allows adherence of other organisms to the

biofilm, enhancing colonization of the vagina with various bacterial species. (Machado and

Cerca, 2015). Although BV is considered a polymicrobial condition, G vaginalis is the most

prominent organism. It is the focus of ongoing microbiological studies because of its unique

qualities that can affect virulence and resistance. One of the areas of current interest regarding G

vaginalis is which genotypes of G vaginalis produce sialidase and which do not. It is believed

that sialidase-producing genotypes are more likely to be associated with biofilm development

and subsequently may increase the risk of resistant and recurrent infections.

In 2016, Schuyler et al published a study that helped broaden our understanding of G vaginalis

resistance to metronidazole. Four previously identified findings revealed that 2 of the 4 known G

vaginalis clades were metronidazole resistant. The 2 remaining clades, which showed less

resistance, were more closely related in phylogenetic characteristics. The authors proposed that

women with BV may be colonized with multiple G vaginalis clades and that this inherent

22
sensitivity or resistance to metronidazole may impact treatment outcomes and the likelihood of

recurrence. It has been recommended that further research in this area be undertaken to translate

this finding into the clinical setting and provide clinicians with tests to distinguish between BV

clades that are more likely to fail treatment due to innate resistance (Schuyler et al., 2016).

23
Treatment of Recurrence

The recommended treatment for recurrent BV is metronidazole, 0.75% gel twice weekly for 4 to

6 months, after appropriate treatment of the initial episode of BV with metronidazole or

clindamycin. This method has been shown to reduce the rates of BV recurrence by more than

50%. Some data support the use of boric acid intravaginally to reacidify the vagina and help

create an environment that encourages Lactobacillus and a healthy flora, resulting in a decrease

of BV recurrence. With this regimen, metronidazole is prescribed for the usual 7-day course

along with vaginal boric acid, 600 mg once daily at bedtime for 21 days. The patient is seen

immediately after completion of this regimen and reassessed. If in remission, the patient begins a

twice-weekly metronidazole regimen for 4 to 6 months (Workowski and Bolan, 2015). Probiotics

have been evaluated as an adjunct treatment of acute BV infection and to deter recurrence.

Although individual studies have reported positive outcomes with the use of probiotics, evidence

to support the use of probiotics for treatment or prevention of this condition is not yet available.

There has also been research evaluating the use of presumptive treatment for BV at monthly

intervals to reduce BV recurrences, the results of which have demonstrated some success (Balkus

et al., 2017). A more novel treatment approach is the use of thermoplastic polyurethane-based

intravaginal rings, similar in concept to the NuvaRing which is being investigated as a potential

method of delivering antimicrobials and lactic acid to the vaginal flora for prevention of

recurrent BV. There is, however, some discussion that intravaginal rings can be prone to biofilm

formation and colonization by bacteria, which would be a potential limitation of this type of

treatment, and further studies are needed to evaluate the feasibility of this modality. If

mechanical properties related to drug release and delivery can be optimized and there is an

24
adequate method to address the possibility of biofilm formation, intravaginal rings could become

a convenient treatment option for BV.

2.13 Non-Antibiotic Treatment Options For Bacterial Vaginosis

Various studies have looked at non-antibiotic treatment options for BV if standard antimicrobial

treatment is not available. These studies can be grouped into two categories, namely:

1. Lowering the vaginal pH; and

2. Treatment with Lactobacilli.

2.13.1 Role of pH in the Treatment of Bactrial Vaginosis

Lactobacillus-dominant flora is associated with a vaginal pH in the range of 3.6 to 4.5 (normal

vaginal pH in women of reproductive age). As raised vaginal pH facilitates the proliferation of

the colonising BV-associated bacteria (Alfonsie et al., 2004; Cherpes et al., 2008), one

therapeutic option in the management of recurrent BV is therefore to keep the vaginal pH at 4.5

or less, in order to prevent overgrowth of bacteria until the normal Lactobacilli are re-

established. The different methods that have been or are used to lower vaginal pH are shown in

2.13.2 Role Of Probiotics In The Treatment Of Bacterial Vaginosis

Treatment of BV using recommended antibiotics is often associated with failure and high rates of

recurrence. This led to the concept of replacing the depleted Lactobacilli using probiotic strains

25
as a treatment approach (Senok et al., 2009). If a decrease in the population of Lactobacilli

appears to be the first event leading to BV and relapses are often associated with failure to

restore a healthy, Lactobacillus-dominant vaginal flora, then the administration of Lactobacilli

might contribute to the treatment of BV (Morris et al., 2001). For decades, some women have

used L. acidophilus in yogurt or supplements to treat BV. Two RCTs investigating the efficacy

of Lactobacillus species for recurrent BV were conducted. The first RCT was a cross-over study

comparing the ingestion of yoghurt containing live L. acidophilus with pasteurised yoghurt in 46

women with recurrent BV, recurrent candidiasis or both (Hemmerling et al., 2007). Among

women with recurrent BV, episodes of BV were signi-ficantly reduced in those ingesting live

yoghurt (from 60% at the start of treatment to 25% after 1 month) compared with those ingesting

pasteurised yoghurt (from 60 to 50%; p = 0.004). However, these results must be interpreted with

caution because the dropout rate was high and only seven women completed the entire treatment

protocol.Drago et al., (2007) investigated the effect of a L. acidophilus-strain-based douche in

restoring a normal vaginal flora. They conducted an open label pilot evaluation of 40 women

with BV. The Nugent score decreased significantly from BV or an intermediate flora toward a

normal flora during treatment, and remained low during the follow-up period for almost all of the

patients, indicating BV in 52.5 and in 7.5% of the patients before treatment and at follow-up,

respectively. After treatment, significant decreases in vaginal pH were observed, and the odour

test became negative in all the patients. The study concluded that the treatment of BV with a

vaginal douche containing a strain of L. acidophilus contributed to the restoration of a normal

vaginal environment. Another RCT compared the use of vaginal capsules containing a mixture of

L. gasseri and L. rhamnosus with placebo vaginal capsules (Larsson et al., 2008). After initial

treatment with clindamycin 2% intravaginal cream, 100 women with BV were randomised to

26
receive vaginal gelatine capsules containing either freeze-dried Lactobacilli or identical placebo.

At the end of the study, 65% of the Lactobacilli-treated women had no recurrence of BV,

compared with 46% of the placebo-treated women. The difference between the groups in time

from cure to relapse was statistically significant (p = 0.027) in favour of the Lactobacilli

treatment. A further study (Shalev et al., 1996) investigated the effectiveness of vaginal

administration of L.rhamnosus after conventional metronidazole therapy. However,

randomisation in this study was not blinded, and there was no comparator arm. Some experts

claim that dairy Lactobacillus is not the strain that normally lives in the vagina. This is why dairy

Lactobacillus does not work for the treatment of BV. But researchers have found that two

different types of Lactobacillus - L. crispatus and L. jensenii are most commonly found in a

healthy vaginal environment. Research is now focusing on using these types of Lactobacilli in

capsules. A Cochrane review investigating the evidence for the use of probiotic preparations

either alone or in conjunction with antibiotics for the treatment of BV did not find probiotics

useful. Current research does not provide conclusive evidence that probiotics are superior to or

enhance the effectiveness of antibiotics in the treatment of BV. In addition, there is insufficient

evidence to recommend the use of probiotics either before, during or after antibiotic treatment as

a means of ensuring successful treatment or reduce recurrence (Marcone et al., 2008; Andreeva

et al., 2002; Milani et al., 2003). Larger, welldesigned randomized controlled trials with

standardized methodologies are needed to confirm the benefits of probiotics in the treatment of

BV.

27
 Chapter 3

Conclusion

Bacterial vaginosis (BV) is a commonly-occurring condition of the female sexual tract, mostly

associated with the overgrowth of pathogenic, anaerobic bacteria in the presence of a suitable

biofilm, an adverse change in the vaginal pH, or a lack of H 2O2-producing Lactobacilli in the

vagina. Symptomatic BV warrants proper antibiotic treatment, either locally or systemically,

even in pregnant women and breastfeeding mothers. Effective treatment will reduce the

likelihood of complications and provide much needed relief of the unpleasant symptoms

associated with this condition. These signs form part of the typical clinical appearance of patients

with bacterial vaginosis. It has been suggested by many studies with significantly increasing

evidence, that G. vaginalis is the main pathogen that causes BV. In addition, it has recently been

supported that the development of a biofilm may be a required component of this process of

developing a gradual overgrowth of stable anaerobic vaginal flora. A cohesive form of G.

vaginalis adheres to the vaginal epithelium and then forms the framework to which other species

attach. G. vaginalis accounts for 90% of the bacteria in the biofilm of the microbiota on the

epithelial surfaces of vaginal biopsy specimens, while Atopobium vaginae made up most of the

remainder.

28
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