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Comparison between Gram stain and culture for the
characterization of vaginal microflora
Definition of a distinct grade that resembles
grade I microflora and revised categorization of
grade I microflora
Rita Verhelst,1 Hans Verstraelen,2 Geert Claeys,1
Gerda Verschraegen,1 Leen Van Simaey,1 Catharine
De Ganck,1 Ellen De Backer,1 Marleen Temmerman,2
Mario Vaneechoutte1
1 Department Clinical chemistry, Microbiology and
Immunology, 2 Department of Obstetrics
Gynaecology, Ghent University Hospital, Belgium
Introduction The microbiological diagnosis of
bacterial vaginosis (BV) is usually made using
Nugents criteria. This method scores the Gram
stain of a vaginal smear in a standardized manner
by quantification of some of the cell types
present designated as Lactobacillus,
Gardnerella vaginalis, Bacteroides and Mobiluncus
morphotypes. However, the Nugent scoring system
conflates women with potentially very different
vaginal microflora in a single category. Since
the method requires considerable time and skill,
simpler versions have been described which assess
the categories in a more qualitative manner.
Recent developments in our knowledge of the
vaginal microflora such as the realisation that
different Lactobacillus species produce different
amounts of hydrogen peroxide and that these
differences might influence pregnancy outcome and
such as the assessment of a strong association of
the metronidazole resistant fastidious anaerobic
coccobacillus Atopobium vaginae with bacterial
vaginosis urge to refine these Gram stain
interpretation criteria in an effort to increase
the agreement between Gram stain and the true
composition of the vaginal microflora. The more
accurate allocation of subjects according to
vaginal microflora status, as assessed by Gram
stain, may enhance the validity of studies on the
etiology of bacterial vaginosis, help better
understand the reason for its recurrence in some
women and its relation to preterm birth. Here we
report our findings obtained by studying a total
of 515 vaginal samples by Gram stain, by
DNA-based techniques - like cloning and
sequencing of amplified 16S rRNA-genes and
species specific PCR for Gardnerella vaginalis
and A. vaginae and by culture in combination with
tDNA-PCR, which allows the rapid identification
of large numbers of cultured isolates, including
isolates from different Lactobacillus species.
Based on these findings, we propose refined
criteria to categorize the status of the
microflora of vaginal smears.
3. The colonisation resistance conferred differs
between Lactobacillus species Overall the
frequency of isolation of all Lactobacillus
species together did not diminish significantly
from grade I to grade III samples in our
population, since lactobacilli were cultured from
96.9 of grade Ia, 94.5 of grade Ib, 96.7 of
grade Iab, 78.9 of grade I-like, 93.5 of grade
II, 59.1 of grade III and 62.5 of grade IV
samples. This is in agreement with previous
reports 32, 34. However, we observed a clear
difference with regard to the Lactobacillus
species frequency distribution for the different
grades. While L. crispatus, known as a strongly
H2O2-producing species 7,8, was cultured from
87.0 of grade Ia specimens, it was absent in
grade III specimens and only present in 2.8 of
grade I-like specimens. In contrast, L. iners,
reported as a weakly H2O2-producing species
7,8, was present in only 3.7 of Ia specimens
but in 39.6 of grade Ib and 31.8 of grade III
specimens. Whether it is the hydrogen peroxide
production by L. crispatus that confers
colonisation resistance remains a matter of
debate, since a correlation between the presence
of hydrogen peroxide production and the type of
vaginal microflora was found by some 35, though
not by others 36. Possibly other species
specific characteristics, present in L.
crispatus, but absent in species like L. gasseri
and L. iners, confer colonisation resistance. It
has also been hypothesized that the onset of
perturbation leading to BV may be due to
competition between Lactobacillus species 36, a
situation possibly reflected by grade Iab
specimens. 4. Grade I-likes a separate category
of vaginal microflora status A number of samples
were initially difficult to score because the
predominant cell types could not be categorized
as Lactobacillus, Gardnerella, Bacteroides-Prevote
lla or Mobiluncus cell types. These samples were
considered as belonging to a separate category
because of the presence of gram-positive rods,
either quite small and short or otherwise
irregularly shaped with clubbing, curved edges
and irregular staining and often arranged like
Chinese letters (diphtheroid cell types). Since
it is likely that most microbiologists would
score this cell type as Lactobacillus-like and
that therefore it would be scored in most cases
as grade I, we designated it as grade I-like.
Culture and species specific PCR confirmed that
indeed these samples represent a separate kind of
vaginal microflora. This is reflected by the
increased species diversity of 0.83, which is
much higher than that for grades Ia, Ib and Iab
(0.15 - 0.30) and which is comparable to that of
grade II (0.76), but even more so by the virtual
absence of L. crispatus (cultured from only one
of 36 samples) as well as of G. vaginalis and A.
vaginae (cultured from respectively 1 and 0
samples) and the presence of Bifidobacterium spp.
in 19 of 36 samples, a much higher prevalence
than in samples from all other grades. This was
confirmed by cloning of two grade I-like samples,
which contained only Lactobacillus delbrueckii,
L. gasseri and Bifidobacterium breve. Importantly,
since Gram stain based categorizing can result
in the interpretation of grade I-like samples as
genuine grade I samples (whereof their
designation), this class of samples may
jeopardize and probably has done so in the past
- the interpretation of the results of clinical
studies.
2. Grade Ia and Iab Agreement with the presence
of L. crispatus From this comparison it became
obvious that it is possible to recognize the
presence of L. crispatus by means of Gram stain,
since this species was cultured in 81.9 of the
grade Ia samples and 76.7 of the grade Iab
samples. Nevertheless, L. crispatus was not
cultured from 21 of the 162 grade Ia samples.
This may be explained by the fact that L.
crispatus is not as easily cultured as other
lactobacilli. Indeed, L. crispatus colonies were
quite often observed as satellites of other
bacteria and in some cases no growth at all was
observed in samples with numerous L.
crispatus-like lactobacilli on Gram stain. Using
non culture dependent t-RFLP-analysis (data not
presented) the Ia samples negative for L.
crispatus culture were tested and 16 were
positive for L. crispatus, bringing the agreement
between Gram stain grading as grade Ia and the
presence of L. crispatus to 96.9. Similarly,
when taking into account t-RFLP-analysis results,
the agreement between categorization as grade Iab
and t-RFLP-analysis positive for L. crispatus was
92.9 whereas L. crispatus was detected by
t-RFLP-analysis only in 27.3, 20.0, 22.5 and
0 for grades Ib, I-like, II and III,
respectively. These results indicate that - for a
trained microbiologist - it is possible to
recognize the L. crispatus bacteria upon cell
morphology, a finding that is of importance since
this species is clearly associated with healthy
microflora, and since we have strong evidence
that L. crispatus also better ensures stable
healthy microflora than other lactobacilli (data
to be presented elsewhere). Samples were scored
as grade Ib when no L. crispatus cell types were
observed, but other Lactobacillus cell types were
predominant. The agreement with culture results
was high only 13.2 contained L. crispatus upon
culture, whereas L. gasseri, L. iners, L.
jensenii and L. vaginalis were present in
respectively 32.4, 39.6, 24.2 and 12.6 of the
grade Ib samples. These were clearly grade I
samples since BV-associated organisms were mostly
absent.

• Methods
• Study design A total of 515 vaginal swabs were
  collected by sampling 197 unselected pregnant
  women attending our out-patient clinic between
  January 2003 and July 2004. Patients were
  scheduled for consecutive vaginal microflora
  status assessment through three Gram-stained
  vaginal smears during pregnancy (respectively
  197, 171 and 147 first, second and third
  trimester samples were collected).
• Grading of slides Gram stained smears from
  vaginal swabs were all scored by one clinical
  microbiologist (GC) according to Ison Hay
  criteria samples were categorized as
• grade I when only Lactobacillus cell types
  (large Gram positive rods) were present,
• grade II (intermediate) when both Lactobacillus
  and Gardnerella or Bacteroides-Prevotella cell
  types were present,
• grade III (BV) when Lactobacillus cell types
  were absent and only Gardnerella,
  Bacteroides-Prevotella or Mobiluncus cell types
  were present and
• grade IV when Gram positive cocci were
  predominantly present.
• Further subdivision of grade I samples into
  categories Ia, Iab and Ib and the description of
  a separate category, designated grade I-like, is
  presented in the Results section.
• Culture and identification of cultured isolates
  by tDNA-PCR, DNA extraction of vaginal swab
  samples, species specific PCR for Atopobium
  vaginae and Gardnerella vaginalis and cloning of
  amplified mixtures of 16S rDNA are described in
  Verhelst et al. 2004.

• Conclusions
• - Our characterization of the vaginal microflora
  by means of detailed Gram stain interpretation
  and by culture in combination with genotypic
  identification helps to refine our understanding
  of normal and disturbed vaginal microflora.
• Our results show that L. crispatus can be
  recognized as such on Gram stain.
• We established the existence of a separate
  additional category, characterized by the absence
  of L. crispatus and the abundance of
  bifidobacteria
• The association of Atopobium vaginae with
  bacterial vaginosis could be confirmed.
• These findings could help us to unravel
• i) why only some women are prone to BV,
• ii) why only half of the women suffering from BV
  are symptomatic,
• iii) why only some women are prone to recurrent
  BV,
• iv) why only some women with bacterial vaginosis
  are prone to preterm birth and
• v) why only some women with preterm labour
  respond to tocolytics and antibiotics whereas
  others do not.

• Results
• 1. Detailed observation of the Gram stained
  vaginal smears in combination with specific PCR
  and tDNA-PCR based identification of cultured
  isolates led to
• A. Subdivision of grade I samples.
• Grade I samples were characterized as
• Grade Ia when only Lactobacillus crispatus cell
  types (plump, mostly short rods) were present
• Grade Ib when only other Lactobacillus cell types
  were present (smaller or more elongated and less
  stained than in Ia smears)
• Grade Iab when both L. crispatus and other
  lactobacilli were present
• B. Recognition of a separate category, designated
  grade I-like.
• Grade I-like samples were scored when
• Gram-positive rods, either quite small or short
  or otherwise irregularly shaped with clubbing ,
  curved edges and irregular staining and often
  arranged like Chinese letters (diphteroid cell
  types) were present.

Literature Verhelst R, Verstraelen H, Claeys G,
Verschraegen G, Van Simaey L, De Ganck C, De
Backer E, Temmerman M, Vaneechoutte M. Comparison
between Gram stain and culture for the
characterization of vaginal microflora
Definition of a distinct grade that resembles
grade I microflora and revised categorization of
grade I microflora. Submitted to BMC
Microbiol. Verhelst R, Verstraelen H, Claeys G,
Verschraegen G, Van Simaey L, De Ganck C,
Temmerman M, Vaneechoutte M. Cloning of 16S rRNA
genes amplified from normal and disturbed vaginal
microflora points to a strong association between
Atopobium vaginae, Gardnerella vaginalis and
bacterial vaginosis. BMC Microbiol 2004, 4 16.
Nugent RP, Krohn MA, Hillier SL Reliability of
diagnosing bacterial vaginosis is improved by a
standardized method of gram stain interpretation.
J Clin Microbiol 1991, 29 297-301. Ison CA, Hay
PE Validation of a simplified grading of Gram
stained vaginal smears for use in genitourinary
medicine clinics. Sex Transm Infect 2002, 78
413-415.
Acknowledgements This work was supported through
a research grant by the Flemish Government and by
the Marguerite-Marie Delacroix Foundation.
Results 1. Detailed observation of the Gram
stained vaginal smears in combination with
specific PCR and tDNA-PCR based identification of
cultured isolates led to subdivision of grade I
samples and the recognition of a separate
category, designated grade I-like Grade I
specimens were characterized as grade Ia when
only Lactobacillus crispatus cell types (plump,
mostly short rods) were present (Figure 1a - 1b),
as grade Ib when only other Lactobacillus cell
types were present (smaller or more elongated and
less stained than in Ia smears)(Figure 1c 1d)
and as grade Iab when both L. crispatus and other
lactobacilli were present (Figure 1e 1f).
Furthermore a number of samples were designated
as grade I-like because of the presence of
gram-positive rods, either quite small and short
or otherwise irregularly shaped with clubbing,
curved edges and irregular staining and often
arranged like Chinese letters (diphtheroid cell
types) (Figure 1g 1i). To corroborate that
grade I-like samples indeed represent a separate
class, cloning was carried out for two samples
that had been categorized as grade I-like. For
completeness, figures 1j 1k represent grade II
vaginal smears and figures 1l 1m represent
grade III vaginal smears.