|Vaginal microflora, PCR, Lactobacillus, Nugent score.
|One of the most common gynaecological problems is vaginal infections like bacterial vaginosis
(BV), Candida vulvovaginitis, trichomonal infection and aerobic vaginitis (AV). BV is caused
by an overgrowth of Gardnerella vaginalis (G.vaginalis), anaerobes, Mycoplasma hominis
(M.hominis), Ureaplasma urealyticum (U.urealyticum) and clinically diagnosed by Amsel
criteria: presence of thin, grayish homogenous discharge; vaginal pH greater than 4.5; presence
of clue cells, positive whiff test (detection/enhancement of fishy odor on additions of potassium
hydroxide to the vaginal specimen). AV is absence of lactobacilli, presence of cocci or coarse
bacilli, parabasal epithelial cells, vaginal leucocytes. AV is associated with growth of group B
streptococci (GBS), E.coli, Staphylococcus aureus (S.aureus), has different immunological
inflammation reaction and clinical signs – such as red, inflamed vaginal mucosa, yellowish
sticky discharge, high vaginal pH, “not fish-like” odor.1,2 Vaginal infection especially bacterial
vaginosis and aerobic vaginitis can be the reason for such complications as preterm delivery,
horioamnionitis and low birth weight. The studies with antibiotic treatment in high risk preterm
delivery pregnancies have not proven to be effective except clindamycin, but more studies are
|Mostly these infections are asymptomatic, 60% of patients that have bacteria vaginosis (BV) had
no complains.5 It is important to discover all cases of vaginal infections early, because such
deviations before 14th week of delivery can be as risk factor for preterm delivery.3
|There are different diagnostic methods for vaginal infections – pH measure, KOH test, visual
vaginal discharge evaluation, dark field microscopy, Grams staining microscopy, cultivation on
artificial media and polymerase chain reaction (PCR). There is still a great dilemma - which
method is easier, cheaper and more widely available for the specialist. Vaginal pH level is
important diagnostic criteria which can be helpful in asymptomatic cases of BV but how precise
this method is comparing to other diagnostic methods? 3
|Nowadays, molecular diagnostic methods are becoming more popular, because they provide
precise information about species of normal and pathogenic vaginal flora inhabitants. It is not
fully understood how microorganisms determined with molecular methods correlate with basic
daily tests like pH and Nugent score.6 Studies show that vaginal microbial composition is dependent on a variety of factors, including
geographical, for example, the normal female flora in African women is considered as
pathogenic for other region women.7-10 The most common lactobacillus in India is considered
L.reuteri, but in Finland L.crispatus.11,12 There were no study data about which Lactobacillus and
pathogenic microflora species present in the vaginal flora of Latvian women. So it was quite
important to maintain this information and compare it with world data and also find the
correlation between molecular diagnostic methods and Nugent score.13
|The goal of this study was to analyze different vaginal flora types by Grams staining and PCR in
pregnant women during their first trimester of pregnancy with a special emphasis on lactobacilli
and pathogenic bacteria species connected with bacterial vaginosis.
|A total of 65 pregnant Latvian women were enrolled in this study during routine prenatal visits at
SIA "Dzirciema Clinic" Ltd "Aura R" and the "Jugla Medical Center" from August 2012 to
February 2013. Informed consent was obtained from all participants in verbal and written form.
All necessary approvals were received from Riga Stradins University ethical comity. Including
criteria in the study were pregnant women older than 18 years with no serious extragenital
abnormalities in their 6-12 week of pregnancy. Estimated date of delivery was determined from
the last menstrual period and early gestational fetal ultrasonographic measurements. Patients
were divided into 2 groups: group A with normal pH (<4,5) and group B with elevated pH
(≥4,5), pH set to Machery Nagel pH strips (measuring range 3.6-7.0). A sterile speculum was
inserted into the vagina and two specimens of vaginal fluid were obtained by brushing the
posterior vaginal fornix with a swab. A vaginal smear was prepared by rolling a swab onto a
glass slide, which was then air-dried, heat-fixed, and Gram-stained. The smears were then
assessed according to Nugent criteria (Table 3). Nugent score for the diagnosis of BV is ≥ 7 and
it is considered as pathogenic flora, intermediate flora 4-6 and for normal flora ≤ 3. Overall, the
Nugent scoring system for Gram –stained vaginal smears has shown high intracenter and
intercenter reliability and reproducibility, however practitioners are not usually familiar with
performing in-office Gram-stain-based diagnosis, Nugent’s criteria are widely applied in the
absence of standardized pre-analytical and analytical conditions and interpretation, especially of
the so called intermediate flora, is also a matter of concern.1,6,14 Second specimen was placed in
Amies media and immediately transported for molecular investigation.
|Swab from Amies media was placed 2 ml containers and the pellet was digested with proteinase
K at 56°C for 60–90 min and the DNA was extracted and purified with a QIAmp DNA
Investigator Kit (Qiagen, Germantown, MD) in accordance with the manufacturer's instructions,
resulting in 100 μl of DNA solution. PCR mixtures consisted of PCR buffer with 1.5 mM of
MgCl2, 10 pmol of each primer, 2.0 μM of each deoxyribonucleoside triphosphate, 0.1 μl of Taq
DNA polymerase, and 1 μl of template DNA solution in a final volume of 25 μl.
|Sequences and annealing temperatures for the various primer sets are listed in 1st table. All
primers were located in the 16S rDNA region. PCR was carried out for 40 cycles. For the Lactobacillus genus and its four species, the denaturation was performed at 95°C for 15 sec
followed by a 1-min annealing and extension step. For four BV-related bacteria, the denaturation
step was set at 94°C for 30 sec, followed by the annealing step for 40 s, with extension at 72°C
for 1 min for all reactions. A final extension step at 72°C for 7 min was added for all reactions.
Aliquots of 8 μl of the PCR products were electrophoreses in agarose gels and visualized by
ultraviolet transillumination after ethidium bromide staining.
|The statistical analysis was made using SPSS, Chi square test and Pearson correlation was
|There were 65 pregnant women included in the study 45 with pH < 4,5 and 20 with pH ≥ 4,5.
The mean age in both groups were 28 ± 5.2. Minimal age of participant was 18 and maximal–
|Comparing both pH and Nugent score diagnostic methods statistically significant difference was
found (X 2= 6,607; p=0,01), high pH was measured only in 17% of participants, but Nugent
score shoed pathogenic flora in 37% of participants, (diagram 1).
|Based on our data pH test sensitivity compared with Grams staining method was 54% and
|Genus Lactobacillus (any Lactobacillus) was detected by PCR in all women irrespective of
Nugent score, the most common species were L. crispatus, L. jensenii, and L. inners, less
common were L. gasseri, L. plantaris, but least likely was L. rhamnosus and L. reuteri species
which were found only in two patients specimen (diagram 2).
|L. jensenii was detected in normal vaginal flora significantly more frequently (p<0,01) than in
pathogenic flora, but in L. inners detection frequency was no significant difference both in
normal and pathogenic flora.
|Gardnerella vaginalis was detected in both normal and pathogenic group, but in BV and middle
flora by Nugent score and high pH group it was detected more often (85-90% of cases) compared
with normal pH and 0-3 Nugent scores (67-73%). Megasphaera, Leptotrichia were found less
frequent in 0-3 Nugent scores, but Leptotrichia detection in both pH groups did not differ
statistically. An A. vagina was detected mainly in pathogenic flora group.
|The incidence of microorganisms, detected with PCR, in vaginal flora, depending on the
diagnostic method are displayed in 2nd table.
|Performing Pearson correlation test on all summarised data some significant correlations was
discovered. The correlation between Nugent and pH test was moderate (r=0,453; p<0,01) also
there was found moderate correlation between presence of Megasphera spp. with Leptotrichia spp. and G. vaginalis in vaginal flora (r=0,367;p<0,01 and r=0,324;p<0,01). Finding A. vaginae
in vaginal flora closely correlated with the pathogenic findings of flora (r = 0,7, p <0,01) .
|Vaginal flora is delicate and dynamic system, with dominating inhabitant Lactobacillus species
in the majority of women.10,11,17,18
|This study included only small female population – first trimester pregnant women. In this study
was confirmed by species-specific 16S rDNA gene PCR that L. crispatus, L. inners and L.
jensenii are the most common species in Latvian pregnant women normal flora, that does not
differ from the Finnish and Japanese data.6,14,19 The incidence of L. gasseri in other similar
studies match our data.19,20
|L. jensenii detection in the normal flora group was higher while L. inners frequency did not differ
between both groups, which was also confirmed in other similar studies.14
|A. vaginae, G. vaginalis, Megasphera mainly prevailed in abnormal vaginal flora that did not
differ from the global data. Interesting was the fact that one pathogenic bacteria correlated with
each other, but there was no similar data in literature. 14, 21, 22 A. vaginae finding significantly
correlated with pathogenic flora that is important because the literature describes a
microorganism high resistance to metronidazole and susceptibility to clarithromycin. It means
that in bacterial vaginosis treatment the use of metronidazole alone, which is effective against G.
vaginalis, may not give the expected result.4,14
|Comparing Grams staining method with pH measurements we found moderate correlation, but
the vaginal pH measurements did not show all abnormal flora cases. However the pH test is
specific enough, but with a low sensitivity, because not in all cases of abnormal flora pH is
increased (≥ 4.5), so if only pH diagnostic method is used there may be many undiagnosed
abnormal vaginal microflora cases, this makes it necessary to supplement this method of
investigation with vaginal discharge microscopy.1,3,17,18 Microscopy data were also compared
with PCR. Although, the PCR can accurately identify the composition of bacteria in the vagina,
but it is expensive and it has some drawbacks. There is no precise criteria for interpreting the
normal or pathogenic, and using conventional polymerase chain method it can only prove the
presence of microorganism in the vagina, but not the number of colony forming units, while
Gram staining microscopy is simple vaginal flora diagnostic techniques that have established
criteria for diagnostic the pathology.1,3,17,18
|Unfortunately, in our country, such studies using PCR have not been performed so far. Our study
group was relatively small, and analyzed cases only in Riga, therefore there is no clear vision on
our region vaginal microflora nuances. During the study, we confronted with several problems
and one of them was number of participants, although patient involvement lasted 6 months, the
normal pH group included 45 of the expected 50 and a high pH group, only 20 of the 50 samples,
this can be explained by the small population of our country. Patient involvement will continue to reach the necessary number of respondents. The other problem of the study was the
impossibility of using real-time PCR, because of study costs.
|Overall this study gave a deeper insight into the occurring lactobacilli species in the Latvia. This
is the root of future studies that will allow us to gain a deeper understanding of the vaginal flora
in the Baltic region.
|The most often isolated lactic acid bacteria in the vagina of pregnant Latvian women are L.
crispatus, L. jensenii and L. inners less common are L. gasseri and L. plantaris. L. jensenii is
the most common isolated lactic acid producing bacteria in normal flora of pregnant Latvian
women. A. vaginae, G. vaginalis, Megasphera are the most common microbes found in
pathogenic flora of vagina. Grams’ staining method and vaginal pH measurements correlate with
each other, but the vaginal pH measurements does not show all abnormal flora cases. In order to
evaluate the vaginal environment for pregnant women, in addition to vaginal pH measurement is
necessary for additional vaginal microflora diagnostics. Although the PCR can accurately
identify the composition of bacteria in the vagina it is expensive, while the Grams staining
microscopy is a simple vaginal flora change diagnostic methods. In order to better conclude on
normal and abnormal vaginal microflora composition in Latvian women the further research are
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