Download Wagdy Mougahed Ali Elsayed Amer_chapter 1 ( B .V )

‫بسم هللا الرحمن الرحيم‬
‫وبه نستعين ‪...............‬‬
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History:
Bacterial vaginosis was first reported in 1955 by Gardner and
Dukes, who described the unique clinical signs and symptoms and the
distinctive increased often foul smelling vaginal discharge that was not
associated with any recognized pathogen. They named that condition
"Non- specific vaginitis" in order to distinguish it from other causes of
vaginitis such as trichomonas vaginalis and Candida species.
They also described a new causative organism, which they named
"Haemophilis vaginalis" subsequently named as corynebacterium
vaginale and now known as Gardnerella vaginalis, Gardner and Dukes
change the name to haemophilus vaginalis vaginitis. This term, later
modified to "Gardnerella-associated vaginitis", was used by many
workers until it became clear that anaerobes were also present in this
condition and the term "Anaerobic vaginitis or vaginosis" was favoured.
(Gardner and Dukes, 1955).
Later, a working group reached a consensus that it would be more
appropriate to call this condition “bacterial vaginosis” because of its
association with many bacteria and , vaginosis because of the lack of an
inflammatory response. vaginal bacteriosis has been suggested as a more
correct name, but bacterial vaginosis remains the most widely used and
accepted term (Easmon el al., 1992).
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Epidemiology of bacterial vaginosis:
Bacterial vaginosis (B.V) is the most common cause of vaginal
discharge (Klebanoff et al., 2004) and one of the most prevalent lower
genital tract infections in women of reproductive age (Beigi et al., 2004).
The prevalence of BV ranges from 5-52% in pregnant women
(Govender et al., 1996) and the prevalence is reported to be between
25% and 36% in women attending sexually transmitted infection clinics.
(Weir, 2004). The prevalence in Banha gynecology clinic was 38.5 % .
(Ayman Azazy et al, 1993). The prevalence in Banha gynecology clinic
according to gram stain was 40.4 % among pregnant women , and 36 %
among non pregnant women . ( Mohamed A. Elhadi - et al 2009 )
Bacterial vaginosis is not an "all or nothing" phenomenon . In
some women , it shows relapse and remission course , With apparently
spontaneous onset and resolution. In women with recurrent bacterial
vaginosis, B.V arises most often around the time of menstruation and
resolve spontaneously in midcycle. (Hay et al., 1997).
The incidences of bacterial vaginosis in different populations are
found to be lowest (4%) in asymptomatic college students and highest
(64%) in patients attending a sexually transmitted disease clinic. Various
factors like diagnostic criteria, use of special tests like Gram staining of
clue cells, gas-liquid chromatography screening for asymptomatic
patients and demographic characteristics of the study population might be
responsible for these wide variations in the incidence of bacterial
vaginosis (Thomason et al., 1991).
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Amsel et al., (1983), found that intrauterine devices were highly
associated with bacterial vaginosis in population of university students.
Among patients with bacterial vaginosis, 18.8% were IUD users, among
normal patients only 5.4%.
The number of sexual partners that a woman had during the month
preceding examination was related directly to the difference of bacterial
vaginosis. Estrogen containing contraceptive (COCP) might have a
protective effect on the development of bacterial vaginosis by promoting
lactobacilli predominant vaginal flora (Brunk, 2000).
Royce el al, 1999, investigated bacterial vaginosis by vaginal
swabs taken from pregnant women between 24 and 29 weeks gestation.
Direct film stained with Gram stain was done for the swabs. They found
22.3% of blacks and 8.5% of whites had bacterial vaginosis. Adjustment
of sociodemographic, sexual activity, sexually transmitted diseases,
health behavior, and sexual hygiene did not explain these differences. In
conclusion they observed race/ethnicity differences in vaginal flora
ecology, high in black .
Race/ethnicity differences ultimately play a rule in larger
proportion of preterm deliveries among black women compared to white
women who had never had sex (Meis et al., 2000).
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Risk factors for bacterial vaginosis:
There are several proposed risk factors for B V . Some of which
are still disputed. The trigger for the change from lactobacillus-dominated
flora to BV-associated flora has been linked to many possible factors
including: some sexual, sociodemographic, behavioral, and reproductive
risk factors. They independently associated with BV in pregnancy. Thus,
a single woman, with daily coitus, smoker, with a previous sexually
transmitted disease, or high alcohol consumer was at increased risk for
B V in pregnancy. Daily coitus 4 weeks prior to test was found to be the
strongest risk factor for BV in pregnancy (Thorsen et al., 2006).
1- Sexual activity:
Multiple sexual partners are associated with increased risk for B V
(Peter et el., 1995). Sexually transmitted diseases as gonorrhea,
chlamydial infection, syphilis and trichomoniasis (Martin et al., 1999),
oral receptive sex (Schmid, 1999) and first coitus in young age are
regarded as risk factors for bacterial vaginosis (Nilson et a1., 1997).
2- Contraception:
Two American studies recorded a significant increase risk of bacterial
vaginosis with use of intrauterine devices than non intrauterine devices
contraceptive users. (Skegg, 1999 and Lossick, 1991).
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Some studies found a significant association between bacterial
vaginosis and the use of intrauterine devices compared with oral
contraceptive or barrier contraceptive (Shoubnikova, 1997 and Avants et
al., 1999).
The major risk factor for bacterial vaginosis in contraceptive users
is the use of an intrauterine device as contraception. (Amsel et al., 1983,
Holset et al., 1987, Hansen et al., 1987, and Lefeure et al., 1988).
3- Vaginal douching:
Zhang et al., 2004, found that vaginal douching has a role in
increased rate of B V in women who douched one or more times per
week when compared to those who douched less frequently or not at all.
Scbwebke el al., 2004, studied douching and the prevalence of
B V among predominately black adolescents who douched regularly. B V
was diagnosed in 44.8% of the study population. they found that
douching in the previous week was positively associated with B V and
there was a strong association between douching after menses and the
prevalence of BV. The reason for these associations is not known. It is
possible that douching decreases lactobacilli, thus facilitating the growth
of the bacteria known to cause B V, but findings have been inconsistent.
The douching during the time of unstable flora such as the time
after menses, may lead to the acquisition of B V (Zhang et al., 2004).
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4- Smoking:
Smoking suppresses the immune system so facilitates the infection,
but may simply reflect poor health seeking behavior (Peter et al., 1995).
Smoking has been associated with bacterial vaginosis and sexually
transmitted diseases in UK. The results from an antenatal population in
London gave a percentage population attributable risk of 8.5% for current
smokers against non smokers (Hay et al., 1994).
5- Racial origin:
Racial factors are also implicated as a risk factor for BV (Bailey et
al, 2004). Klebanoff et al., 2004, evaluated more than 1000 women
diagnosed with B V and found it more common in young women who are
African, American and who were smoking. Others have reported that
African and American women have a greater than 2-fold higher rate of
having B V than white women (Fiscell et al., 2004).
Other factors as, menstruation, socioeconomic state, hygiene
habits, use of diaphragms, immunological status, long term use of
antibiotics and corticosteroids, genital pathology and abnormal uterine
bleeding are regarded as risk factors for bacterial vaginosis (Hiller and
Holmes, 1990).
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Microbiology of Bacterial Vaginosis:
Bacterial vaginosis is a polymicrobial condition associated with
significant reduction of the lactobacilli species and replaced by over
growth of aerobic, anaerobic and microaerophilic species, such as,
Gardnerella
vaginalis,
Bacteroids,
Peptostreptococcus
species
Mycoplasma Hominis, Ureaplasma Urealyticum and Mobiluncus species
in very large numbers (Mclean, 2000).
All of these bacteria with possible exception of Mobiluncus species
may also colonize the vagina of normal women although in smaller
numbers (Easman et al., 1992).
(A) Gardenerella vaginalis
In 1955 Gardener and Dukes reported the isolation of an organism
believed to be responsible for non- specific vaginitis. They named it at
first haemophilus vaginalis and reported that this species was present only
in patients with non- specific vaginitis. At 1963 Zineman and Turner
renamed it corynebacterium vaginalis because it sometimes appeared
gram +ve and formed chinese letters. Then Green Wood and Pickett at
1980 showed a lack of genetic relationship between this organism and the
other organisms in the corynebacterium group, thus it was named
Gardenerella vaginalis (referring it to Gardner) (Gale and Hill, 1993).
Morphology:
These are small, non- motile, non-capsulated non-spore forming,
and Gram - ve bacilli. Under some conditions, it may appear (+ve) or
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variable or form metaphosphate (volutin) granules in the presence of
fermentable compounds (Well and Goel, 1981).
It posses an unusual laminated cell wall structure with a possible
glycocalyx which helps adherence to vaginal epithelium. Pilli also exist
on the surface of clinical isolates from vaginae of women with B V
(Boustouller et al., 1987 , Johnson and Davis 1989,).
Biochemical Reactions:
It ferments glucose, maltose, raffinose, sucrose, dextrin, glycogen
and starch with production of acid only. Its ability to hydrolyse starch is
seen in the production of zone of clearing around colonies on the opaque
peptone starch dextrose (P.S.D) agar. It is catalase, oxidas, urease and
indole negative and it does not reduce nitrate or nitrite. It prouces alpha
but not beta glycosidase and it is able to hydrolyse sodium hippurate.
(Michelsen, 1977).
Gardenerella vaginalis is a facultative anaerobe that grows slightly
better anaerobically in humid atmosphere enriched with 5-10% CO2. The
optimum temperature is 35 – 37oc and optimum pH is 6-6.5, it is slowly
growing organism and requires an enriched media with blood, serum and
starch. Colonies appear small < 0.9 mm in diameter, colourless, rounded
and transparent. They exhibit B haemolysis with diffuse edge on human
or rabbit blood, but not horse or sheep blood (Shaw et al., 1981).
Incubation at 37oc for 48 hours in a humidified atmosphere of air
plus 5-10% CO2 or in candle flame extinction Jar is satisfactory and
practical (Totten et al., 1982).
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There are various selective media for Gardenerella vaginalis such
as that of Michelsen, which consist of Columbia agar base with colistin,
nalidixic acid and 1% cornstarch. Ison et al., (1983), develop another
media which consist of human blood agar with gentamycin, nalidexic
acid and amphotericin B. Totten et al., (1982), developed human bilayer
agar media with tween 80 medium or without tween 80 medium, which is
a good selective and differential medium (Totten et al., 1982).
Culture media and colony morphology for Gardenerella Vaginalis
G V:
(A) Peptone Starch Dextrose medium (PSD)
On this medium , after 48 hours of incubation, G V forms small
colonies (0.5-2 mm) in diameter, which are dull white, convex domed,
somewhat conical in shape , with a dense or compact appearance
(Dunkelberg et al., 1970).
(B) Cosman's Blood Agar
Defibrinated rabbit blood (5%) is added to the agar base. This
medium can be used for primary isolation; the colonies are characteristic
(Colonies appear small < 0.9 mm in diameter, colourless, rounded and
transparent. they exhibit B haemalysis with diffuse edge on rabbit blood)
but minute and difficult to be seen by naked eye. Dissecting microscope
is helpful. (Gardner 1980).
(C) Columbia Colistin Nalidlxic Acid (C.N.A) agar
The medium consists of Columbia agar base, 10mg of sodium
colistin and 10mg of nalidixic acid per liter. This medium appears to
contain all of growth requirements for G. vaginalis (Piot et al., 1982).
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(D) Human Blood Agar
It consists of a single layer of Columbia agar bas containing colistin
and nalidixic acid with amphotericin B, plus 5% human blood.
Gardenerella vaginalis colonies appear as small colonies, which produce
B haemolysis after 48 hours of incubation. Beta haemolysis is more
apparent on the bilayer medium. Also the addition of tween 80 or
protease peptone no 3 to both layers further enhance the haemolysis and
thus facilitate the detection of the organism although tween 80 may cause
spontaneous haemolysis during storage or incubation (Totten et al.,
1982).
(E) Colistin, Oxalinic Acid Blood Agar (COBA)
COBA consists of Columbia agar base with 5% defibrinated sheep
blood , 10mg sodium colistinathate per liter and 10mg of oxalinic acid
per liter.
An advantage of COBA is that it inhibits the growth of
staphylococci and coryneform as well as most Gram negative bacteria
(Thompson 1985).
Dawson et al (1982) has used two selective human blood agar both
contained amphotericin B and nalidexic acid. One contained, in addition,
gentamycin and the other contained colistin. Both media inhibited the
growth of yeasts, anaerobic bacteria, proteus species and cell catalase and
oxide negative and gram variable bacilli, which showed haemolysis on
blood agar.
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Sensitivity and resistance:
Gardenerella vaginalis, in general, sensitive to a wide range of
antimicrobial agents including metronidazole, nitrofurantion and
trimethoprim. (Piot et al., 1982).
Most strains are sensitive only to high concentrations of
metronidazole (e.g. 128mg /L). Although strains have been described as
being sensitive to 2-16mg /L,they show less sensitivity than anaerobes
(Pheifer et al., 1978).
Gardenerella vaginalis highly sensitive to H2O2 and thionin basic
fuchsin, crystal violet but relatively resistant to pyronine dye (Piot et al.,
1982b). They are also sensitive to 2% Nacl. They die rapidly so it is
necessary to achieve rapid transport of sample or swabs of the patient to
the laboratory and working cultures must be transferred every 48 hours.
The organism is sensitive to bile and like gram +ve bacteria, it is sensitive
to ampicillin, clindamycin, trimethoprim, penicillin, vancomycin but
these drugs are less effective than metronidazole. The organism is
resistant to colistin, gentamycin, and nalidixic acid and to sulfonamide
and neomycin (Piot et al., 1982).
(B) Facultative lactobacilli:
Facultative Lactobacilli predominate in the healthy vagina.
Lactobacilli are thought to be responsible for maintaining an acid pH in
the vagina by metabolism of the glucose generated by the metabolism of
glycogen (Wylie et al., 1969). Low pH may directly inhibit some
organisms. H2 O2 produced by facultative lactobacilli may also control the
quantity of catalase negative organisms such as anaerobes (Eschenbach
et al., 1989).
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In patient with B V, Lactobacilli are replaced by G V and a mixed
predominanty anaerobic flora. The decrease in Lactobacilli in patients
with B.V and the return of these organisms after therapy have been
documented by using culture (Blackwell et al., 1983; Spiegel et al., 1980)
and microscopic teachniques (Amsel et al., 1983; Spiegel et al., 1983).
In study by Eschenbach et al (1989), species of lactobacilli were
determind, hydrogen peroxide producing Lactobacilli were recovered
from 96% of healthy women and from 6% women with B V.
Another feature, typical of bacterial vaginosis, is the lack of
lactobacilli in vagina, normal usually protective, and it may be the lack or
incomplete restoration of this protection that allow pathogens to ascend.
Little is known about the local immune function and protection in the
vagina and cervix, which surely has an important role in preventing
ascending infection (Alanen A 2004).
(C) Mycoplasma hominis:
Its role as an etiologic agent in B V is unclear. M.hominis has been
associated with B V in pregnant women (Krohn et al., 1995) and in non
pregnant women (Holst et al., 1987).
It is present in 24 to 75% of women with B.V and 13 to 22% of
women without B.V. M.hominis persisted in 50% of women treated with
sulfonamide, 27% of those treated with doxycyclin, 88% of those treated
with ampicillin and 40% of those treated with metronidazole and, by 35
days after therapy, it had recolonized 70% of the metronidazole treated
women.M.hominis was with abnormal gas liquid chromatogram and
presence of diamines whether or not G V was also present. Also serum
antibody level to M.hominis was significantly high in women with B V.
(Paavonen et al., 1983).
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( D ) Anaerobic Bacteria:
Pheifer et al (1978) were the first to suggest that anaerobic bacteria
might be involved in the pathogenesis of B V. They observed that, when
patients with mixed trichomoniasis and signs of B V were treated with
metronidazol, and antimicrobial agent with activity against protozoa and
anaerobes, the signs of B V. were eradicated.
Mobiluncus:
Curved anaerobic rod – shaped bacteria (CARS) were isolated by
Curtis as early as 1913 from women with pathological discharge and
from uterus of patients with puerperal infection.
Mobiluncus is an anaerobic motile rod associated with bacterial
vaginosis. But is also associated with extragenital infection including
breast abscesses, septicaemia and umbilical discharge and in women
presenting by vaginal bleeding. The reservoir of Mobiluncus spp. may be
the intestinal tract (Hallen et al., 1988).
Astrong association of Mobiluncus with B V is evidenced by their
detection in wet smear preparation, Gram stained smears or their isolation
in culture from 40-60% of women with B V.
There are currently two known species of the genus Mobiluncus:
M. Curtisii and M. muliers. Two subspecies of M. curtisii have been
described M. curtisii subsp. Curtisii and M.Curtisii subsp. Holmessii.
(Spiegel and Roberts, 1984).
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DIAGNOSIS OF BACTERIAL VAGINOSIS:
General speaking B V is asymptomatic condition . The two most
widely accepted methods for the diagnosis of bacterial vaginosis are
Amsel's composite criteria (ACC) and Nugent's Gram stain evaluation of
bacterial morphotypes (NBM) (Hilmarsdóttir et al., 2006).
The main symptoms and signs of Bacterial Vaginosis are:
Symptoms:
More than 50% of women with B V are asymptomatic or on the
contrary may present with vaginal bleeding irregularities (Bump et al.,
1984).
Patients with B V are mostly women in the childbearing period,
presenting mainly with increased vaginal discharge which may have fishy
odour more noticed after menstruation or sexual intercourse, and is
usually not associated with itching or irritation. However, the condition is
essentially mild and patients vary in their tolerance of the increased
discharge, which may be present for months or years(Gardner and
Dukes, 1955).
Signs:
Four clinical criteria have been suggested by Amsel et al., (1983) for
diagnosis of B.V of which at least 3 should be present they are:
a) Thin homogenous vaginal discharge grayish .
b) Vaginal pH of >4.5.
c) Characteristic amine or fishy odour when alkali (10% KOH) is
added to the specimen of vaginal secretion. ( whiff test ).
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d) The presence of clue cells on wet mount examination of vaginal
fluid.
A) Thin homogenous discharge:
Amsel et al., (1983) described the vaginal discharge of women
with B.V as being thin (of low viscosity) and homogenous. It has the
consistency of whipped cream and the color may vary from white to gray.
Since the discharge is not harmful to tissues there is no erythema,
ulceration or other signs of tissue injury.The amount of discharge may be
scanty, moderate or profuse (Chowdhury, et al., 1985). It is usually
adherent to vaginal walls (Hillier and Holmes, 1990).
The malodour is often noticed after coitus where the alkaline
seminal fluid has resulted in the release of volatile amines and fatty acids.
Pruritus and burning occur in a minornitory of cases (Friedrich, 1985).
Although a malodorous vaginal discharge described as “fishy”
suggests the presence of B V, it is not reliable enough to be used as the
only criterion for diagnosis. Some women with B V may report abnormal
discharge, but this symptom is also unreliable (Klebanoff et al., 2004).
B) Vaginal discharge with pH >4.5:
A pH greater than 4.5 indicates decrease lactobacilli , and pH may
be elevated in up to 90% of patients with B V. (Hansen et al., 2002).
Gardner and Dukes (1955) analyzed the pH range of vaginal
secretions. They found that in normal individuals , the pH ranged from
3.8 to 4.2 while, in patients with bacterial vaginitis, pH ranged from 5.0
to 5.5.
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The normal vagina of a woman of reproductive age is colonized
with lactobacilli. These lactobacilli produce bacteriocins, hydrogen
peroxide, and lactic acid, all of which are substances that lower the
vaginal pH. The low pH creates a hostile environment for bacteria other
than lactobacilli. If the number of lactobacilli is decreased, the resulting
increase in pH favors an overgrowth of anaerobic and facultative bacteria,
which can predispose to the development of B V (Ness et al., 2004).
Amsel et al (1983) found that a pH of more than 4.5 was the best
to discriminate that normal from abnormal state. Error in pH
measurement may be made by sampling cervical mucus, which has a
higher pH than that of vaginal fluid.
PH is considered the most sensitive but least specific criterion.
Certain antimicrobials can reduce the flora and elevate the pH. Vaginal
pH may be artifactually elevated by recent unprotected coitus because the
pH of semen is more neutral than that of vaginal discharge. Also, cervical
discharge has a relatively elevated pH; contamination of the vaginal pool
with this cervical discharge can result in an erroneous finding, vaginal
douching is the most common cause in Egyptian women. (Larsen, 1993).
C) Amine test(whiff_test):
The whiff test may be positive in up to 70% of B V patients.
(Hansen, et al., 2002).
A whiff test is made directly on the vaginal discharge accumulated
in the examination speculum with the addition of 10% (or 20%)
potassium hydroxide. The bacteria produce amines, which most noses
will find malodorous, but this is quite subjective. Droping the vaginal
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secretion with a 10% potassium hydroxide solution can make a more
objective test . (Wolrath et al., 2002).
The whiff test is considered positive if an odor of rotten fish is
perceived after the application. Trimethylamine, which is produced by
Mobiluncus when it is included in the anaerobic flora distinguishing BV
cases, evaporates as gas in a matter of seconds. The human nose is very
sensitive to the odour of trimethylamine and can register isolated
molecules of this amine. Since the gas evaporates immediately, it is
necessary to whiff to do hand moving or air blowing at the same time
potassium hydroxide is applied. Some find it difficult to perceive the
trimethylamine odor, that is why the test may be considered subjective.
(Wolrath el al., 2002).
Amine test is a specific but a relatively sensitive test (MarquezDavil 1995). 43% of women with B.V and 1% of women without B.V
giving a positive amine test (Eschenbach et al., 1988).
D) Clue cells:
Demonstration of clue cells on a wet mount saline smear examined
with light microscope X400 ( HPF) is the most special criterion for B V.
(Hansen et al., 2002).
Gardner and Dukes (1955) reported that clue cells show varying
degrees of disintegration with many of the cells showing indefinite
outline and the cytoplasm shows characteristic granular appearance. They
attribute this character to G V being uniformly spaced upon the surface
of the cells. Not all cells show only partial involvement.
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Schmidt and Hansen, 2000, stated that the most objective method
of detecting clue cells is to observe the epithelial cells border, which is
relatively obscured by bacteria rather than detection of cytoplasma
granularity, if the vaginal cell border has a serrated appearance and
cannot be identified clearly because of the attachment of large number of
bacteria , a clue cell is present.
Occasionally , a patient with malodorous vaginal discharge, of high
pH, will have no clue cells on wet mount , it has been suggested that
some women produce local IgA which block attachment of bacteria to the
cell wall. In such cases stained slides will show masses of gram –ve to
gram variable coccobacilli which do not adhere to the cell wall. (Pheifer
et al. 1978).
Thomason et aL., (1990) suggested an average of only one clue
cell per 20 high power (x 400) field to be sufficient when examining wet
mount specimens since very few false positive results occurred, while the
number of false positive results were higher during gram stain
examination of the specimen, that is why it is probably necessary to have
5% to 20% clue cells on Gram stain before that specimen is designated
positive for clue cells (Eschenbach et al., 1988). This is not proved by
many authors and there is some controversy over the number or ratio of
clue cells to epithelial cells that must be visualized to make a diagnosis
(Eschenbach et al., 1988).
Clue cells have been associated with B V by several investigators,
although the association has not been confirmed by others (Gardner and
Dukes, 1955 and Dunkelberg et al., 1962).
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The papanicolaou smear also has been evaluated as a diagnostic
test for bacterial vaginosis. One study has shown that papanicolaou smear
is an excellent diagnostic test for bacterial vaginosis. Whereas other
reports have indicated that this is less specific than gram stain or wet
smear diagnosis (John et al., 1997).
In a study conducted by John et al (1997) to determine the
reliability of papanicolaou smear in making the diagnosis of bacterial
vaginosis, with the Gram stain used as the standard, the results show that
the papanicolaou smear has only 55% sensitivity for diagnosis of
bacterial vaginosis. Therefore it is not an adequate screening test for
bacterial vaginosis .(John et al., 1997).
Acridine orange (AO) is a fluochrome dye which differentially
stain the nucleic acids of microorganisms. Its advantage over wet mount
preparation in detecting trichomoniasis (TV) has been shown. In
prospective study conducted by Martens, (1996), AO was superior to
other methods (including wet mount preparation and Gram stain method)
in detecting clue cells among the patient with bacterial vaginosis. The use
of AO in detecting clue cells had a high sensitivity rate of 77.8% with
negative predictive value of 94.1% . (Martens, 1996).
For Acridine oranges staining a high vaginal swab is taken and
examined on a dry mount after acridine orange staining. Then the slides
are examined with a fluorescence microscope. (Martens, 1996),
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Fig (1): Clue cells of G. vaginalis on a saline wet mount (high power) (X 400 )
(Sodhani, 2005).
Fig (2): Clue cells. Squamous epithelial cells cohesively covered by gram-negative to
gram-variable coccobacilli (×1,000) (Sodhani, 2005).
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Fig (3): Clue cell (arrow) as recognized by cytologic review. Dark, velvety coat of
coccobacilli adhere to the squamous epithelial cytoplasm (Pap stain, ×600) (Sodhani,
2005).
Fig (4): Collection of clue cells, epithelial cells covered with bacteria, coccobacilli
morphology (Pap stain, ×600) (Sodhani, 2005).
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Laboratory diagnosis Of Bacterial Vaginosis:
Diagnosis of bacterial vaginasis is usually made clinically
according to criteria set by Amsel et al., (1983). Patients must have at
least three of the following four signs (1) thin homogenous vaginal
discharge (2) vaginal pH more than 4.5 (3) clue cells on wet smear and
(4) presence of amine odor with addition of KoH.
Elevated vaginal pH (5.0 to 6.5) in a normally estrogenized patient
is almost always associated with bacterial vaginosis (John Davis et al.,
1997).
The papanicolaou smear also has been evaluated as a diagnostic
test for bacterial vaginosis. One study has shown that Papanicolaou smear
is an excellent diagnostic test for bacterial vaginosis. Whereas other
reports have indicated that this is less specific than gram stain or wet
smear diagnosis (John et al., 1997).
The possibility of using papanicolaou smear as a screening method
for bacterial vaginosis is attractive because the Papanincolaou smear is
routinely performed and or additional specimens would to be obtained. In
a study conducted by John et al (1997) to determine the reliability of
papanicolaou smear in making the diagnosis of bacterial vaginosis, with
the Gram stain used as the standard, the results show that the
papanicolaou smear has only 55% sensitivity for diagnosis of bacterial
vaginosis. Therefore it is not an adequate screening test for bacterial
vaginosis (John et al., 1997).
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Increased awareness of the problems associated with bacterial
vaginosis is likely to result in an increase in demand for screening of
women at risk. Speculum examination to obtain smear or high vaginal
secretions are uncomfortable, time consuming and need sterile specula.
This combination of disadvantages will interfere with attempts to set up a
screening service. Blind vaginal swabbing is a suitable alternative for
obtaining high vaginal swabbing and preparation of smears for Gram
staining in the way that it will overcome all the disadvantages of sterile
speculum examination. First it is easier and faster to take a vaginal swab
blindly than a swab at speculum examination. Second, women much
preferred have a swab taken blindly than via a speculum examination.
Third there is no evidence of trauma or bleeding using blind swab
technique (John Morgan et al., 1996).
1) Vaginal Cultures:
Vaginal culture for Gardenella vaginalis is often the primary
laboratory test available for the diagnosis of vaginitis. However, the
usefulness of these cultures is doubtful. G vaginalis can be recovered on
laboratory media from 83% to 94% of women having the clinical signs of
bacterial vaginosis, which gives culture for G. vaginal a high degree of
sensitivity (Esehenbach et al., 1988 and Krohn et al., 1989).
Gardenerella vaginalis is also recovered from 36% to 55% of
women without clinical signs of bacteriasl vaginosis. The actual finding
of G. Vaginalis from a routine vaginal culture should not be used to
diagnose bacterial vaginosis unless clinical signs and Gram stain criteria
are also present. Thus a culture positive for G V should not be used as the
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(
basis of directing therapy or for test of cure after treatment, bacuase many
women who are colonized by G V lack any objective signs of bacterial
vaginosis (Sharon and Hillier, 1993).
Krohn et al (1989) evaluated the sensitivity and specificity of
vaginal cultures for anaerobic bacteria (bacteriods and peposterpto
coccus) and Mycoplasma hominis. She found that the presence of these
organisms was a more specific indicator of bacterial vaginosis than was
the presence of G V but that their detection had inadequate
sensitivity.Mobiluncus species, another group of anaerobic bacteria
highly associated with bacterial vaginosis, are very difficult to recover
with culture methods. It should be stressed that the culture of the high
vaginal swabs for gardenella, Mobiluncus, or any other organism, is
unhelpful in the diagnosis of B V as the disorder is due to an imbalance
of the normal organism, without any pathodgens necessarity being
present.
2) Gram stain :
( A ) ordinary Gram stain .
Gram stain of vaginal fluid has been used for Laboratory
confirmation of bacterial vaginosis by Dunkelberg (1995) (Sharon and
Hillier, 1993) .
Gram stain of vaginal secretions is relatively rapid, objective and
inexpensive method for diagnosing bacterial vaginosis by identifying the
characteristic change in the vaginal flora. In bacterial vaginosis there is a
greater than 400-fold increase in anaerobic flora with a diminished
proportion of lactobacilli are easily differentiated from gram negative
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(
organisms, Cocci, Gardenella and other anaerobic bacterial morphotypes
(Spiegel et al., 1983).
(B) Spiegel Gram stain interpretation.
Spiegel et al (1983) modified the Gram stain criteria for bacterial
vaginosis to include those smears with the Gradenella morphotype plus
other bacteria (cocci, fusiform and curved rods) and less than fivelactobacillus morphotype per oil immerision field. They reported
complete agreement between Gram-stained vaginal smears and clinical
signs for the diagnosis of bacterial vaginosis.
Recently, studies evaluating Gram stains interpreted by the criteria
of spiegel have shown that 62% to 97% of women with clinical signs of
bacterial vaginosis have a vaginal Gram smear consistent with a diagnosis
of bacterial vaginosis (Eschenbach et al., 1988 and Krohn et al., 1989).
Table(1) Spiegel et al., (1983) scoring system.
Mohphotypes per field
Evaluation
<1
+
1-5
++
6-30
+++
>30
++++
The patient was diagnosed as having B V if lactobacillus
morphotypes were < 2+, Gardenella morphotypes were 4+, and at least
one other morphotype was present per oil immersion field (Spiegel et al.,
1983).
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(C) Nugent's Gram stains evaluation of bacterial morphotypes.
Bacterial vaginosis can also be diagnosed on the basis of a Gram
stain of vaginal discharge. In bacterial vaginosis, the normal
lactobacillary flora, which manifests as gram-positive rods, is replaced by
large numbers of gram-variable coccobacilli (Gardnerella vaginalis),
which may be seen adhering to the epithelial cells (clue cells) or in
clumps in the vaginal material (Schwebke et al., 1996).
The Nugent score is calculated by assessing for the presence of
large gram-positive rods (Lactobacillus morphotypes; decrease in
Lactobacillus scored as 0 to 4), small gram-variable rods ( G V
morphotypes; scored as 0 to 4), and curved gram-variable rods
(Mobiluncus spp. morphotypes; scored as 0 to 2) and can range from 0 to
10. A score of 7 to 10 is consistent with B V (Beverly et al., 2005).
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Table (2): Standardized scoring method for evaluation of Gramstained smears for the diagnosis of bacterial vaginosis (Nugent et al.,
1991).
Points scored per morphotype
Bacterial morphotypes
0
+1
+2
+3
+4
Large Gram positive rods
4
3
2
1
0
Small Gram-negative/variable
0
1
2
3
4
Curved Gram-variable rods
0
1
1
2
2
- Score of 0-3 points: Normal.
- 1+ = ≤1/1000x.
- Score of 4-6: Intermediate.
- 2 + = 1-5/l000x.
- Score of 7-10: Bacterial vaginosis.
- 3+ = 6-30/l000x
- 4+ = ≥ 30/l000x.
( X ) = number of magnification by microscope.
3) DNA probe for detection of Gardnerella vaginalis:
Gazi et al., 2006, evaluated DNA hybridization as an alternative
to gram stain for the rapid diagnosing of bacterial vaginitis. They
conducted a prospective study on 321 symptomatic women. Vaginal
specimens from each patient were analyzed using both gram stain and
DNA hybridization. The gram stain results and the DNA assays were
recorded by two separate examiners. When the outcomes were compared,
the researchers found: gram stains were positive for B V on 115 patients;
126 stains were negative, including 80 that showed intermediate results,
while the Affirm VPIII showed: 107 positive and 8 negative outcomes.
When the researchers compared the results from the negative and
intermediate gram stains to the outcomes of the DNA assays, an
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additional 15 patients were determined to have vaginitis. These results
produced a sensitivity of 87.7% (107/122) and a specificity of 96%
(191/199), with an overall accuracy of 92.8% versus gram stain, which
was considered the gold standard during this study. Due to these
outcomes, the researchers concluded that DNA hybridization is useful as
a rapid diagnostic tool for the diagnosis of B V in symptomatic women
but it's not cost effective especially in developing countries .
4) Proline amino peptidase assay:
Proline amino peptidase is presumed to be a product of the bacteria
associated with bacterial vaginosis. Mobiluncus and gardnella vaginalis
have been shown to produce proline aminopeptidase. Other genera known
to produce it , include actinomyces, streptococcus and peptostreptococcus
(Judith and Schoonmaker, 1991).
Proline amino peptidase is a marker enzyme that is present in
vaginal secretions. Elevated proline amino peptidase activity has been
identified as a reliable marker enzyme for bacterial vaginosis; it has been
shown to predict accurately women with clinical diagnosis of bacterial
vaginosis. However this assay has significant practical disadvantages, the
most notable of which is the production of carcinogenic endproduct,
Alphanathylmine. A modified assay for this bacterial vagnosis marker
enzyme with L-proline r-nitroanilide, a substrate that does not yields
carcinogenic end product was done (Judith and Schoonmaker, 1991).
The two proline amino peptidase assay substrate, L-Proline Bnaphthylamide and L-proline P-nitroanilide, yield similar results with
respect to a diagnosis of bacterial vaginosis. When compared with the
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results of Gram stain, both yielded similar findings. The proline amino
peptidase assay sensitivity was (91%) to (93%) (Judith and
Schoonmaker, 1991).
Both proline amino peptidase assays offer rapid, easy, and
objective means for the diagnosis of bacterial vaginosis (Judith and
Schoonmaker, 1991).
5) OSOM BV blue:
A newer, easy test for detecting BV is OSOM BV blue
(Genzyme Corporation, Cambridge ). The kit comes with sample swabs
and reagent. The vagina is swabbed and the swab placed into the test tube
with the reagent. The reagent turns blue or green if the sample is positive
for BV (Medcompare, 2006). This is a 10-minute test that detects
elevated sialidase activity in the vaginal fluid. The sialidase is produced
by bacterial pathogens associated with BV, including Gardnella and
Mobiluncus (Medcompare, 2006).
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Complications of Bacterial Vaginosis:
1-Bacterial vaginosis and cervical intraepithelial neoplasia:
Evidence regarding a causal relationship between B V and CIN
has been so far incomplete and conflicting. The possibility exists that B V
is in some way associated with the development of CIN, i.e. as a cofactor
to human papilloma virus. Therefore, B V must be taken in consideration
in future studies on CIN (Callahan et al., 2003).
To determine whether B V is associated with CIN, a
retrospective study was conducted at the Genitourinary Medicine Clinic
at Southlands Hospital, Shoreham- by-Sea, UK. That study has shown
that an increased prevalence of CIN was associated with B V
(Uthayakumar et al., 1998).
The study by Barrington et al., 1997, has shown a statistically
significant association between anaerobic vaginosis that produce amines
and CIN grade 2 and grade 3. It is, therefore, theoretically possible that
nitrosamines may be an important agent in the development of
premalignant disease of the cervix.
It has been suggested that important etiological factor in cervical
cancer may be the relative absence of hydrogen peroxide producing
lactobacilli that may have a protective role in the development of cervical
dysplasia (Blackwell, 1996).
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Neurr and Menton, (1995), demonstrated Gardnerella vaginalis
and mycoplasma in 22% of 216 patients with cervical interaepithelial
neoplasia (CIN) I-III and in 5% of symptom free controls.
It is not possible to say if the association between anaerobic vaginosis
and cervical precancer is causal or casual. (Pavic, 1984),
2-Bacterial vaginosis and obstetric complications:
Bacterial vaginosis is associated with adverse effects in the first,
second and third trimester of pregnancy (Romero et al., 2001).
A- Premature rupture of the membranes (PROM)
PROM occurs in approximately 5 to 10% of all pregnancies, and is
associated with increased risks of perinatal as well as maternal morbidity
and mortality. Recent data suggest that the presence of B V may be
implicated in the occurrence of PROM. Some studies had demonstrated a
7.3 fold risk for preterm PROM for women with bacterial vaginosis.
Bacteria associated with B V produce proteases that may destroy or
weaken collagen (which gives both strength and elasticity to fetal
membranes) and so, causes them to become inflamed and friable and thus
liable to give way under the stress of the strong Braxton-Hicks
contraction or other strains (McGregor et al., 1994).
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B- Preterm labor
Over the past 20 years, there had been a great number of studies,
which had linked B V with the pre-term delivery. These studies had come
from the United Kingdom, United States, Finland, Jakarta, Indonesia and
Australia, and had obtained the same results. The women who had B V
had an increased risk of pre-term delivery (Hay el al., 2001).
Preterm birth rates have increased in the past decade (Hoyert et
al., 2006) and strong epidemiologic evidence has suggested an
association between BV and preterm birth.
Okun et al., 2005, conducted a meta-analysis of 14 randomized
controlled studies to determine whether antibiotic treatment for B V or
trichmons vaginalis during pregnancy decreased the risk of preterm birth
and associated adverse outcomes. The authors concluded that there is no
evidence to support the screening of antibiotic treatment for neither the
general population nor high-risk pregnant women with B V in the second
or third trimesters of pregnancy
.
C- Miscarriage
The women with B V also have an increased risk of pregnancy
loss or miscarriage between 14 and 22 weeks. There are now some
studies which show that B V is significantly associated with pregnancy
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loss earlier during pregnancy prior to the pre-term delivery (Hay el al.,
2001).
D- Chorioamnionitis
There is also little evidence showing that women with B V had a
threefold increased risk of amnionitis even in the second trimester. If
there are no complications until the late second or early third trimester,
there are data showing that women with B V has a two- to threefold
increased risk of histologic chorioamnionitis (Larsson et al., 2005).
Some microbiologic and epidemiologic studies have shown strong
association between B V on one hand, and clinically evident
intraamniotic infection and histologic chorioamnonitis on the other hand.
Clinically, it has been defined on the basis of fever, maternal and/or fetal
tachycardia, uterine tenderness, foul discharge and maternal leukocytosis
(Gibbs, 1993).
E- Post partum endometritis
Postpartum endometritis is the most common obstetric infection. It
affects approximately 1 to 3% of women delivering vaginally and 10 to
30% of those delivering-by caesarian section. Antepartum B V is an
important risk factor for post partum endometritis. The most important
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element of the diagnosis is the development of fever exceeding 38°C,
pain in the lower abdomen, tender uterus, foul-smelling Lochia in
addition to leukocytosis (Arias, 1993).
3-Bacterial vaginosis and complications of gynecologic
surgery:
Pelvic inflammatory disease (PID) was broadly defined as
infection of the female upper genital tract, including endometritis,
salpingitis, oophoritis, pelvic peritonitis, or tuboovarian abscess (Peipert
et al., 1997).
Bacterial vaginosis had been associated with endometritis, PID,
and vaginal cuff cellulites after invasive procedures, including
endometrial biopsy, hysterectomy, hysterosalpingography, placement of
an IUD, cesarean section, and uterine curettage (Larsson et al., 1989).
Studies had found B V to be more common among women with
PID and had isolated organisms associated with B V in upper genital
tract. However, this did not prove the cause, nor did it indicated how
much PID was attributable to B V (Taylor and Robinson, 1997).
Lin et al., 1999, published a study encompassing 175 women
undergoing gynecologic surgery including vaginal hysterectomy,
laparotomy, and total abdominal hysterectomy. It was shown that women
with B V were substantially more likely to develop postoperative
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infections despite either preoperative or perioperative antibiotics
administration.
4-Tubal infertility:
Several UK studies had investigated the association between B V
and tubal infertility after noting that the prevalence of bacterial vaginosis
tended to be higher in women attending infertility clinics than among the
general population (Wilson et al., 2002).
In another observational study Wilson and associates, 2002, found
that one third of women with tubal factor infertility had BV, compared
with 16% where male factor infertility was diagnosed, and around 19%21% with other causes of infertility.
5-Bacterial vaginosis and female genital tract
infections:
Up to 50% of women with cervicitis had B V Numerous studies
had shown an association of BV with mucopurulent endocervicitis (MPC)
(Moi and Paavonen, 1990).
Additional supporting evidence was offered by the observation that
BV is associated with inflammatory changes noted on cervical cytology.
It appeared from this minor study that when you see cervicitis you should
at least consider screening those women for BV (Schwebke et al., 1995).
) 39
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Schwebke et al., 1995, suggested the need to treat coexistent B V
in women with MPC. Failure to do so resulted in an excess rate of
persistent mucopurlent cervcitis (MPC) , 47% in the BV-untreated group
compared with 14% in the B V-treated group.
6-Bacterial vaginosis and Human Immunodefecincy Virus.
(HIV).
Several factors could lead to the acquisition of HIV when the
normal vaginal microflora is disturbed (Hashemi et al., 1999).
firstly, depletion of lactobacilli may limit production of hydrogen
peroxide and other antibacterial activities, which are protective against
potentially pathogenic organisms such as STDs and possibly HIV.
Secondly,
low vaginal pH has been postulated to inhibit CD4
lymphocyte activation and reduce HIV target cells in the vagina.
Therefore, the lack of lactic acid production by lactobacilli could lead to
an elevated pH which may be more conducive to growth and survival of
the virus.
Thirdly,
similar to the experience with several bacteria,
elevated vaginal pH may enhance HIV adherence to vaginal eukaryotic
cells (Taha et al., 1998).
The study from North Carolina showed that in 724 women (from
rural areas) with BV, there was a fourfold increased risk of HIV. These
studies, like many others, confirm that in the population affected with
BV, there is a great risk of HIV (Schwebke, 2003).
) 40
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Some studies, conducted and published in the United States, Africa
and Thailand, have all shown that women having B V have a higher
prevalence of HIV (Al-Harthi et al., 1999).
Taha et al., 1998, showed in the study published 10 years ago that
women having B V were 3.5 times more likely to become HIV positive
by the time of delivery when compared to women without BV. He also
found that women with gonorrhea and syphilis were at increased risk of
HIV seroconversion. However, trichomoniasis was not significantly
associated with becoming HIV seropositive. The risk of BV, syphilis, and
gonorrhea was similar, but because B V was more prevalent, it was more
influential in transmission than these two classical STDs.
7-Urinary tract infection ( UTI )
The most recent study done by Hillebrand et al., 2002, who were
examining 503 pregnant women for BV and UTIs, showed that nineteen
(13.6%) of 140 women who had BV had UTIs compared with 24 (6.6%)
of 363 women without it. B V in pregnancy was associated with an
increased risk for UTIs.
Another study shown that 67 women among one hundred twentynine had bacterial vaginosis and 62 women did not have bacterial
vaginosis. 15 women with bacterial vaginosis (22.4%) had urinary tract
infections, compared with (9.7%) of those without it. Bacterial vaginosis
was associated with an increased risk of urinary tract infections
(Harmanli et al., 2000).
) 41
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Harmanli et al., 2000, concluded that women with B V are at
increased risk for UTIs. Frequency of sexual intercourse may be a risk
factor for both conditions. The authors suggested that screening for one
condition might be prompted by diagnosis of the other.
Urogenital tract infections of bacterial origin have a high incidence
among the world female population at reproductive age. A great
proportion of these diseases, such as vaginosis and B V are often caused
by pathogens that emerge from the intestinal microbes and ascend along
perineum to the vagina and then to the urethra and bladder (Reid et al.,
2006).
) 42
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Treatment of bacterial vaginosis:
General measures:
Patients should be advised to avoid sanitary water vaginal
douching, use of shower gel, and use of antiseptic agents or shampoo in
the bath (Hay, 1999).
Specific measures:
The ideal antimicrobial agent for treatment of B V
should
eradicate bacteroids species, G V, M. hominis and other anaerobes
without inhibiting lactobacilli or causing an overgrowth of other
potentially harmful micro-organisms. (Livengood et al., 1994).
1) Metronidazole: is a 1-B-Hydroxy ethyl-2-Methyl-5-nitroimidazole. .
Its mechanism of action is reflected in a selective toxicity to
anaerobic or microaerophilic orangisms. (Livengood et al., 1994).
Metronidazole is currently the treatment of choice with reported
cure rates of 72% to 100% (Joesoef and Schmid 1995).
The two most common dosages are 500 mg orally twice daily for 5 to 7
days, or 2g as a single dose. (Banks, 1999).
Metronidazole vaginal gel 0.75% is an effective alternative to oral
metronidazole (Hillier, 1993 and Sullivan, 1996).
) 43
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The common side effects of metronidazole are gastrointestinal
upset, in the form of nausea, vomiting, anorexia, a metallic taste,
headache, dizziness, darkening of the urine, and secondary yeast
infections-skin rashes are less common (Banks, 1999).
2)Clindamycin: It is a 7-deoxy, 7-cholorderivative of lincomycin.
It inhibits synthesis of the bacterial protein by binding to 50 S.
ribosomal subunits of sensitive microorganisms (bacteriostatic in usual
concentrations). It is well absorbed orally; most of the drug is
metabolized to inactive sulfoxide form, which is then excreted in the
urine and bile (Jacob, 1992).
A dose of 300 mg oral every 8 hours daily for 7 days produced a
94% cure rate that in comparable with metronidazole (Graveas, 1988).
It is particularly useful in pregnancy, metronidazole treatment
failure and when patients can not tolerate metronidazole. Adverse
reactions include gastrointestinal complains (Banks, 1999).
Local clindamycin cream 5g of 2% administered, intravaginally
twice daily for 5 or 7 days, was followed by 95% cure rate at 1 weeks
assessment after the onset of the treatment (Hillier et al., 1993).
) 44
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Lactobacillus replacement therapy:
Recently, McLean et al (2000) reported that vaginal colonization
by lactobacilli is important for competitive exclusion of bacteria
associated with B V.
For decades, some women have used Lactobacillus acidophilus in
yogurt or supplements to treat bacterial vaginosis. But researchers now
know that dairy lactobacillus does not work for bacterial vaginosis. This
is because dairy lactobacillus is not the kind of bacteria that normally
lives in the vagina. Researchers have found two different types of
lactobacillus—L. crispatusand , L. jensenii—that are most commonly
found in a healthy vaginal environment. Research is now focusing on
using these types of lactobacilli in capsules. ( Kessel KV, et al. 2003).
Clinical research shows certain strains of Lactobacillus might help
treat bacterial vaginosis when applied inside the vagina. Researchers have
found Lactobacillus acidophilus suppositories (Vivag, Pharma Vinci A/S,
Denmark) and vaginal tablets (Gynoflor, Medinova, Switzerland) may be
effective. Researchers also found that vaginal capsules Lactobacillus
gasseri and Lactobacillus rhamnosus, seem to lengthen the time between
infections.( Larsson PG et al 2008 ) .
) 45
(
Methods of application : 1-2 vaginal tablets (Gynoflor, Medinova, Switzerland) daily
containing living Lactobacillus acidophilus (10 million
colon-forming units/tablet) and 0.3 mg estriol for 6 days.
 Intravaginal suppositories containing 100 million to 1 billion
colony forming units of Lactobacillus acidophilus (Vivag,
Pharma Vinci A/S, Denmark) given twice daily for 6 days
has also been used.
 Vaginal capsules containing Lactobacillus gasseri and
Lactobacillus rhamnosus, 100 million to 1 billion colonyforming units of each strain per capsule (EcoVag Vaginal
Capsules,
Bifodan
A/S,
Denmark),
following
usual
treatment, for 10 days in three menstrual cycles following
the infection has also been used.
(Shalev E et al 1996 ) .
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