Study of Leucorrhea Cases by Gram Stain and Aerobic Bacterial Culture

20 Pages·2017·0.68 MB·English

Int.J.Curr.Microbiol.App.Sci (2018) 7(2): 1484-1503
International Journal of Current Microbiology and Applied Sciences
ISSN: 2319-7706 Volume 7 Number 02 (2018)

Journal homepage: http://www.ijcmas.com

Original Research Article https://doi.org/10.20546/ijcmas.2018.702.180

Study of Leucorrhea Cases by Gram Stain and Aerobic Bacterial Culture

B. Sumangala, Akshatha B. Sangannavar, N.S. Sahana Shetty* and
Kavya Shree M. Arun

Department of Microbiology, Mandya Institute of Medical Sciences, Mandya, India

*Corresponding author

A B S T R A C T

Vaginitis is infl ammation of cervical mucosa. Women who present with vaginal symptoms
often complain of an abnormal discharge and possibly other symptoms such as an

offensive odour or itching. The most common cause for vaginitis are Trichomoniasis,
Candida Vagini tis (Moniliasis) and Bacterial Vaginosis. Vaginal discharge may be blood-
stained or otherwise. Pathologically significant vaginal discharge can be white, cream,
Key words yellow or green ish discharge. Women coming to the outpatient department of Obstetrics
and Gynaecolo gy Department with complaints of white discharge per vagina were
Bacte rial vaginosis,
Gram staining, approached, ex plained about the purpose of the study and informed consent was taken. A
non-lubricated Sim‟s speculum was inserted into the vagina after keeping the patient in
Nuge nt‟s score,
Vagin itis dorsal position. An anterior vaginal wall retractor was used to retract the anterior vaginal
wall. A high va ginal swab was taken using a sterile swab supplied by the Department of
A rtic le Info Microbiology. A smear was prepared for Gram‟s staining and culture plates, both blood

agar and MacConkey‟s agar were inoculated and incubated. Out of the total 70 samples
Accep ted: collected 25(36 %) were positive for bacterial vaginosis and 45(64%) were negative. Cases
15 January 2018

Avail able Online: of co-infection of bacterial vaginosis and Staphylococcus aureus (A gram positive cocci)
10 Fe bruary 2018 and also Gram Negative bacilli (Escherichia coli, Klebsiella pneumonia, Gram negative
non-fermenters were isolated. Direct microscopic examination of clinical material is often

used in the diag nosis of bacterial vaginosis. In this study, we have evaluated the Gram‟s
stain, standard clinical and microbiological (culture) criteria. In the present study the
incidence of BV was 36%. This correlates with other Indian studies performed by (Bhalla
et al., 2007) wh ich gave a result of 32.8% incidence of bacterial vaginosis
Introduction Vaginosis. Vaginal discharge may be blood-
stained or otherwise. Pathologically
Vaginitis is inflammation of cervical mucosa. significant vaginal discharge can be white,
Women who present with vaginal symptoms cream, yellow or greenish discharge.
often complain of an abnormal discharge and
possibly other symptoms such as an offensive Discharge caused by infection is
odour or itching. The most common cause for mucopurulent or frankly purulent; its colour
vaginitis are Trichomoniasis, Candida therefore varies from cream to yellow or
Vaginitis (Moniliasis) and Bacterial green. It is often offensive, especially when
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coliform bacilli are present as primary or with N. Gonorrhoeae, Chlamydia trachomatis
secondary invaders. Its chief microscopic and Herpes Simplex virus-2, HIV. In the
characteristic is the presence of pus cells. The presence of co-infection the purulent
commonest lesions causing a discharge of this discharge obscures BV (Ison and Hay. 2002).
kind are as follows:
Bacterial vaginosis is polymicrobial in
Vulvovaginitis: This may be due to infection
etiology, usually Gardnerella vaginalis
with the gonococcus, Trichomonas vaginalis,
associated with other bacteria such as
Candida albicans or bacterial vaginosis in the
Lactobacillus, Prevotella, and anaerobes,
adult, and with non-specific organisms in
including Mobiluncus, Bacteroides,
childhood and old age. Cervicitis gonococcal,
Peptostreptococcus, Fusobacterium,
chlamydial, anaerobic or puerperal or senile.
Veillonella and Eubacterium. Mycoplasma
Endometritis, puerperal or senile. Secondary
hominis, Ureaplasma urealyticum,
infection of wounds, abrasions (including
Streptococcus viridans and Atopobiumvaginae
those caused by foreign bodies), burns,
have also been associated with BV. Hence
chemical injuries and neoplasms, sited in any
culture of Gardnerella vaginalis alone and
part of the genital tract (Robert et al., 1991;
non-culture tests cannot be considered as
Martinez-Martinez et al., 2014).
definitive diagnosis of BV. Molecular

Herman Gardner and Dukes in the year 1955 methods are also not useful since it is
described foul smelling discharge in women as polymicrobial. Recurrence and treatment
non-specific vaginitis, which is now termed as failure is fairly common unless it is diagnosed
Bacterial vaginosis. It was named so because and treated with an oral agent and local
bacteria are the etiologic agents and an application for prolonged period. Screening
associated inflammatory response is lacking. It and treatment of BV is necessary before
is an alteration of normal vaginal bacterial gynaecologic surgery and induction of
flora that results in the loss of hydrogen abortion, to prevent pelvic inflammatory
peroxide-producing lactobacilli and an disease (PID). In recurrent BV both sexual
overgrowth of predominantly anaerobic partners need to be screened and treated
bacteria. Lactobacilli are usually absent. It is (Carol et al., 1983).
not known what triggers the disturbance of
normal vaginal flora. It has been postulated Bacterial vaginosis is diagnosed popularly by
that repeated alkalinisation of the vagina, Amsel‟s clinical criteria and in the laboratory
which occurs with frequent sexual intercourse by Nugent‟s criteria based on Gram‟s stain.
or use of douches, plays a role. Most of the
patients are asymptomatic. Perivaginal Bacterial vaginosis is diagnosed on the basis
irritation, dysuria, dyspareunia are rare. of the following findings:
Abdominal discomfort is complained by few
patients. Women with BV are at increased risk A fishy vaginal odour, which is particularly
for pelvic inflammatory disease (PID), noticeable following coitus, and vaginal
postabortal PID, postoperative cuff infections discharge are present. b. Vaginal secretions
after hysterectomy and abnormal cervical are gray and thinly coat the vaginal walls. c.
cytology. BV in pregnancy is known to be The pH of these secretions is higher than 4.5
associated with adverse outcome like pre- (usually 4.7 to 5.7) d. Microscopy of the
mature rupture of the membranes, preterm vaginal secretions reveals an increased
labour and delivery, chorioamnionitis, and number of clue cells, and leucocytes are
postcaesare anendometritis. Women with BV conspicuously absent. e. The addition of KOH
are at increased risk of developing infection to the vaginal secretions (the “whiff” test)
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releases a fishy, amine-like odour. BV is the been prepared after a detailed analysis of the
most common cause of vaginal symptoms results of the experiment.
among women of reproductive age. The
percentage of women affected at any given We hereby propose to use the above
time varies between 5% and 70%. mentioned technique as it is rapid,
economical, accurate, reliable.
Of the diagnostic methods currently available,
assessment of clinical signs is subtle and Microbiologically, bacterial vaginosis is
detection of the signs is very dependent on the characterized by a shift in the vaginal flora
acuity of the clinician performing the test. In from the dominant flora of Lactobacillus spp.
all cases of vaginitis it is difficult to clinically to a mixed vaginal flora that includes G.
rule out all the causes. Definitive diagnosis is vaginalis, Bacteroides spp., Mobiluncus spp.
not possible based on signs and symptoms. and Mycoplasma hominis. As bacterial
vaginosis is a clinical syndrome which has
Empirical therapy based on clinical findings been associated with a group of genital micro-
leads to- organisms rather than a single etiologic agent,
it has been defined primarily by certain
1. Drug resistance 2.Inappropriate therapy 3. clinical signs. Laboratory methods for
Financial burden to the patient diagnosis of bacterial vaginosis have included
culture of Gardnerella vaginalis, direct Gram
Gram stained vaginal smears are the least stain of vaginal secretions, biochemical tests
expensive, require the least time to perform for metabolic by-products of vaginal bacteria
and are more widely available than other (gas chromatography) and more recently, the
laboratory methods which can be preserved prolineaminopeptidase test (Robert et al.,
for later reference. However, this is the most 1991).
interpretive of the laboratory methods. One
study has shown that the Gram stain Robert. P. Nugent and co-workers proposed a
interpretation for diagnosis of bacterial new scoring system that uses the most reliable
vaginosis has high intracenter reliability. morphotypes from the vaginal smear. The
scoring system (0 to 10) was described as a
Grading of the Gram stained smears of vaginal weighted combination of the following
fluid reflects both the change in vaginal morphotypes – lactobacilli, Gardnerella
ecology and the strong microbial associations. vaginalis or Bacteroides (small Gram variable
rods or Gram negative rods) and curved
For performing Gram‟s stain, vaginal swabs Gram-variable rods as tabulated in Materials
have been collected from all patients attending and methods of this article. The new scoring
the obstetrics and gynaecology outpatient system is most successful in tackling inter-
department with complaints of excessive center variability (Robert et al., 1991).
white discharge per vagina and analysed over
the prescribed period of 2 months. Significant A comparative study of various existing
and relevant history has also been collected to diagnostic methods for bacterial vaginosis
correlate the clinical signs and symptoms and were evaluated by W. Martinez et al., The
the laboratory analysis results. Culture of the three most commonly used methods for
procured samples has also been performed for diagnosis of bacterial vaginosis are – Amsel‟s,
further evaluation in case of any doubt Nugent‟s and Claeys‟ methods. The diagnosis
regarding the causative organism. A holistic of bacterial vaginosis by the three methods
study has been performed and the report has showed moderate concordance. Amsel‟s
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criteria – Based on clinical evidence and culture, particularly in a third world setting
characteristics of vaginal discharge. Nugent‟s with fewer or no laboratory facilities. This
Score – The standard method and is used in study highlights the presence of clue cells as a
epidemiological and research studies, based on diagnostic criteria for labelling patients as
the amount of different bacterial morphotypes bacterial vaginosis positive (Rotimi et al.,
present in vaginal ecosystem. Claeys‟ criteria 1991).
– Based on the rate of Lactobacilli and
bacterial vaginosis- associated bacterial Comparison of methods for diagnosing
morphotypes. Claeys‟ criteria is most Bacterial Vaginosis was performed by
effective, Nugent‟s method helps to make a Krohn,et. Al to determine the laboratory
subdiagnosis of the disease and Amsel‟s method that best predicted bacterial vaginosis.
method contains criteria with poor sensitivity The methods compared were – Gardnerella
and specificity (Martinez-Martinez et al., vaginalis culture, Gramstained vaginal smears
2014). and gas-liquid chromatography.

Ison and Hay in the year 2002, used Amsel‟s It was concluded by the above study that the
composite criteria to grade Gram-stained order of –
smears, which is still regarded as „Gold
standard‟. This study states that, of the Sensitivity: Gardnerella vaginalis culture >
different scoring methods, the Amsel‟s criteria Gas-liquid chromatography = Gram-stained
can be used as an alternative method of smears
diagnosis when there is a lack of time or
expertise for assessment of Gram-stained Specificity: Gram – stained vaginal smears >
smears (Ison and Hay, 2002). Gas-liquid chromatography = Gardnerella
vaginalis culture (Marijane et al., 1989).
Spiegel, Amsel and Holmes considered three
diagnostic techniques for thethe diagnosis of Culture of Gardnerella vaginalis is a
bacterial vaginosis – Clinical examination, challenge to microbiologists as isolating a
Microbiological examination (culture for pure strain is extremely tedious. Because of
Gardnerella vaginalis) and Gram‟s staining. the relative difficulty of isolating
They suggest after their study that Gram-
stained smear method is the easiest way for Gardnerella vaginalis which was attemped by
diagnosing bacterial vaginosis by clinicians Ison and Dawson, it was concluded that a
and laboratorians (Carol et al., 1983). presumptive diagnosis is often made on the
microscopy of wet and Gram-stained
In the years, diagnosis of bacterial vaginosis preparations of vaginal discharge, looking for
has seen frequent upgrading of methods, from clue cells and short Gram Negative bacilli
clinical presentation to gas-liquid (Ison et al., 1982).
chromatography (GLC) and thin-layer
chromatography. According to the study Bacterial vaginosis plays are role in patients
conducted by Rotimi and coworkers with HIV. A study performed by Dora Warren
estimating the number of Gardnerella and et al., reports an increased risk of bacterial
Lactobacillus morphotypes in a Gram-stained vaginosis in women with HIV infection. They
smear of vaginal discharge, facilitates the also state that candida is protective against
diagnosis of bacterial vaginosis for clinicians bacterial vaginosis (Dora Warren et al., 2001).
with minimum need for confirmation by Research performed in the Indian setup by
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Hemalatha et al., concluded that determination department of Obstetrics and Gynaecology
of vaginal pH for the diagnosis of bacterial Department with complaints of white
vaginosis is relatively sensitive but less discharge per vagina were approached,
specific. They suggest use of pH glove and pH explained about the purpose of the study and
strip as suitable for screening women with informed consent was taken. A non-lubricated
bacterial vaginosis on out-patient basis Sim‟s speculum was inserted into the vagina
(Hemalatha et al., 2013). after keeping the patient in dorsal position. An
anterior vaginal wall retractor was used to
Prevalence of bacterial vaginosis among retract the anterior vaginal wall. A high
women in Delhi was studied by P. Bhalla and vaginal swab was taken using a sterile swab
co-workers showed an association between supplied by the Department of Microbiology.
bacterial vaginosis and other laboratory A smear was prepared for Gram‟s staining and
confirmed sexually transmitted infections culture plates, both blood agar and
(STI‟s). They also stated in their study that all Macconkey‟s agar were inoculated and
women with Vaginaltrichomoniasis were incubated.
found to have bacterial vaginosis (Bhalla et
al., 2007). Grading - Nugent‟s method is based on
counting bacterial morphotypes present on
The main aim and objectives of this study to Gram stain of vaginal discharge.
identify bacterial vaginosis in women
presenting with white discharge per vagina. Grade I (normal flora), Lactobacillus
And also to test the efficacy of Gram‟s stain as morphotype only. Grade II (intermediate
an outpatient investigation in the diagnosis of flora), reduced Lactobacillus morphotype with
bacterial vaginosis. mixed bacterial morphotypes.  Grade III
(BV), mixed bacterial morphotypes with few
Inclusion Criteria: All married women or absent Lactobacillus morphotypes. Grade
presenting to the out patient department of IV, epithelial cells covered with Gram positive
obstetrics and gynaecology with complaints of cocci only.
white discharge per vagina.
Score Lactobacillus morphotypes
Exclusion Criteria: Un-married and pregnant
women. Gardnerella and Bacteroides spp.
Morphotypes
Materials and Methods
Curved gram-variable rods
Study design: Prospective study. 0 4+ 0 0 1 3+ 1+ 1+ or 2+ 2 2+ 2+ 3+ or 4+ 3
Study setting: Out patient Department of 1+ 3+ 4 0 4+
Obstetrics and Gynaecology in Mandya
The values obtained according to the grading
Institute of Medical Sciences.
are scored as shown in the table. The total sum

is obtained with the sum of partial scores.
Study group: All married women coming to

the obstetrics and gynaecology out patient
Culture was performed in the department of
with complaints of white discharge per vagina.
Microbiology.

Procedure: The study was started after taking The swabs collected were streaked onto Blood
approval of the Ethical Committee of the agar (BA) and Mac-conkey (MA) agar plates.
institution. Women coming to the out patient Post streaking they were incubated at 37◦C for
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24 hours and reported. Plan of Analysis/ Role of Gram negative curved bacilli in
Statistical tools – Data was entered on excel bacterial vaginosis (Mobiluncus spp.)
sheet and analysed by EPI_INFO software.
Gram Negative curved bacilli >10 shows
Results and Discussion about 80% (21 out of 25) of bacterial
vaginosis positive cases.
Incidence of bacterial vaginosis by Nugent’s
criteria Nugent’s score

Out of the total 70 samples collected 25(36%) A Nugent‟s score of >5 shows 100%
were positive for bacterial vaginosis and correlation with bacterial vaginosis
45(64%) were negative (Figure 1).
Conclusion drawn based on growth shown
Incidence of bacterial vaginosis in different in culture media
age groups
The pie chart represents the presence of
Highest incidence was seen in the age group various micro-organisms based on the culture
31 to 40. report.

Significance of epithelial cells in bacterial Sample of normal vaginal study. It can be
vaginosis taken as a scale to compare other infections.

Most of the women who were bacterial A case of co-infection of bacterial vaginosis
vaginosis positive showed an epithelial cell and Staphylococcus aureus (A gram positive
count of <10. cocci). Presence of clue cells indicates that it
is a case of bacterial vaginosis. Other factors
Significance of pus cells in bacterial indicating bacterial vaginosis are Gram
vaginosis variable bacilli (GVB) count of 100 and
presence of 40 Gram Negative bacilli (GNB)
Bacterial vaginosis usually shows a lack of per oil immersion field. Absence of
pus cells in the vagina. lactobacilli is another addition to the list of
factors favouring bacterial vaginosis. Presence
Number of lactobacilli in vaginal flora in a of a significantly high number of pus cells
case of bacterial vaginosis indicates a co-infection which is confirmed to
be of Staphylococcus aureus by culturing and
Number of lactobacilli is NIL in about 94% of shown in Figure 9. Gram Negative bacilli
the bacterial vaginosis positive cases. As the (Escherichia coli, Klebsiella pneumonia, Gram
number of lactobacilli increase the incidence negative non-fermenters) infection presents
of bacterial vaginosis falls drastically. under the microscope as presence of pus cells,
Gram Negative bacilli and absence of clue
Role of gram variable bacilli in bacterial cell. Presence of an almost normal number of
vaginosis lactobacilli is indicative of an absence of
bacterial vaginosis (Figure 10).
Gram variable bacilli are Gardnerella
vaginalis. Presence of >10 organisms per oil Staphylococcal infection encountered in the
immersion field shows 100% result. vagina shows presence of pus cells and

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epithelial cells. There is a considerable material is often used in the diagnosis of
decrease in the number of lactobacilli among bacterial vaginosis.
the vaginal flora (Figure 11).
In this study, we have evaluated the Gram‟s
Bacterial vaginosis with co-infection of stain, standard clinical and microbiological
candida shows a picture similar to that of BV (culture) criteria.
(clue cells) and budding Gram positive yeast
cells with the presence of pseudo-hyphae In the present study the incidence of BV was
under oil immersion field (Figure 12). 36%. This correlates with other Indian studies
performed by (Bhalla et al., 2007) which gave
This is a case of pure bacterial vaginosis a result of 32.8% incidence of bacterial
infection. The values are typical of BV with vaginosis.
minimal lactobacilli in the vagina and
associated rise in the gram variable and Gram A study by (Hemalatha et al., 2013) showed
Negative curved bacilli. Presence of clue cells the mean age of incidence of bacterial
is another classic feature of bacterial vaginosis vaginosis as 29 +/- 4.8. Our results are in
(Figure 13). Candidial infection of the vagina agreement, with maximum incidence of
is another common infection which presents bacterial vaginosis between the age groups 31
under the microscope as presence of to 40. The probable reason for this
pseudohyphae with Grampositive budding coincidence in results can be attributed to the
yeast cells. Absence of clue cells and Gram population chosen which mostly consisted of
variable bacilli differentiates it from BV women belonging to reproductive age group,
(Figure 14). which incidentally is the sexually active
group.
In culture, Staphylococcus aureus was the
predominant organism isolated (9) among As shown in figures 3 and 4 a decrease in the
gram positive organisms and E.coli was number of epithelial cells are consistent with
predominant among gram negative organisms the positive cases of bacterial vaginosis. As
(7). told in (Jeffecoat‟s) about microscopy of
vaginal secretions, presence of leucocytes is
Direct microscopic examination of clinical not significant in BV.

Fig.1 Diagnosis of Bacterial vaginosis by Gram‟s stain

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Fig.2 Incidence of bacterial vaginosis among women of different age groups

Fig.3 Epithelial cells in bacterial vaginosis

Fig.4 Association of pus cells to bacterial vaginosis

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Fig.5 Number of lactobacilli in vaginal flora in a case of bacterial vaginosis

Fig.6 Gram variable bacilli in bacterial vaginosis

Fig.7 Gram Negative curved bacilli in bacterial vaginosis

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Fig.8 Relationship between bacterial vaginosis and Nugent‟s score

Fig.9 Pie chart representing the presence of various micro-organisms based on culture report

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