European Journal of Obstetrics & Gynecology and Reproductrve Bloloa, 32 (1989) 149-156 149

Elsevier

EUROBS 00781

Detection of Chlamydia trachomatis, Mycoplasma

hominis and Ureaplasma urealyticum in pregnant
Dutch women

H.J. Vonsee t, E.E. Stobbetingh 2, P.X.J.M. Bouckaert 3, J. de Haan ’

and C.P.A. van Boven 2
’ Department of Obstetrics and Gynaecology, University Hospital, and .’ Department of Medical
Microbiology, University of Limburg Maastricht, and ’ St. Elisabeth Kliniek, Heerlen, The Netherlands

Accepted for publication 10 November 1988

Summary

A prospective study was performed to determine the prevalence of endocervical

infection by Chlamydia trachomatis and vaginal colonization by Mycoplasma hominis
and Ureaplasma urealyticum in pregnant women seeking routine obstetrical care in
two clinics in the southern part of the Netherlands. C. trachomatis was detected
using the direct immunofluorescence staining technique. For the genital myco-
plasmata, generally accepted culture methods were used.
Evaluable samples were obtained from 691 of 770 women in the first trimester of
pregnancy. C. trachomatis was detected in 2.3%, M. hominis in 5.2% and 0:
urealyticum in 23.9% of the women. The isolation percentages of C. trachomatis and
U. urealyticum were almost equally distributed in the different age groups. The
prevalence of all three micro-organisms did not seem to be related to parity.
Smoking and alcohol consumption seemed to influence the isolation rate of M.
hominis and U. urealyticum.

Genital mycoplasmata; Pregnancy; C. trachomatis

Introduction

C. trachomatis has been recognized as the most prevalent sexually transmitted

pathogen responsible for a large number of infections in men and women [1,2]. In

Correspondence: E.E. Stobberingh, Department of Medical Microbiology, University of Limburg. P.O.

Box 616, 6200 MD Maastricht, The Netherlands.

002%2243/89/$03.50 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

150

addition, neonatal infection by C. trachomatis acquired during birth, manifested by

respiratory tract infection and conjunctivitis, is well established and may occur in
5-35% of the children born through an infected birth canal [3]. The incidence of
neonatal chlamydial diseases depends directly on the prevalence of genital chlamy-
dial infections in their mothers, which in turn is supposed to vary with the
socioeconomic background of the maternal population. In addition to these neonatal
diseases, several authors have drawn attention to a possible association between
cervical chlamydial infections in pregnancy and prematurity and low birth weight
[4]. Most studies dealing with the prevalence of C. trachomatis have been performed
in patients attending clinics for sexually transmitted diseases. Depending on the
population studied, i.e., Caucasian, Asian, Black or Hispanic, the prevalence ranged
from 2% to 37% [3,5,6].
M. hominis and U. ureai’yticum can be isolated from the lower genital tract
mucosa of healthy, sexually experienced individuals. M. hominis has been found in
35% to 50% and U. urealyticum in 50 to 70% of vaginal samples of healthy
individuals [7,8]. However, carefully controlled cultural and serological studies
suggest that under certain circumstances, such as low antibody levels and prolonged
rupture of membranes before delivery, these opportunistic micro-organisms are
capable of invading the upper genito-urinary tract and are of aetiological signifi-
cance in salpingitis and complications of pregnancy [9,10]. M. hominis and U,
urealyticum have been isolated from amniotic fluid as early as 16 weeks of gestation
[9]. Blanc0 et al. [ll] found that M. hominis was isolated significantly more often in
the amniotic fluid from women with intra-amniotic infection than in the control
group (36% vs. 8%, p < 0.001). However, the isolation rate of U. urealyticum (50%)
was similar in both groups. Although in general both micro-organisms may be
considered as common inhabitants of the urogenital tracts of healthy people, a
recent study by Waites et al. [12], stressed the importance of both micro-organisms
in the development of chronic infection of the central nervous system in preterm
infants. Recently Cassell et al. [13] suggested a strong association between the
isolation of U. urealyticum from endotracheal aspirates and respiratory disease in
preterm infants of less than 1000 g. The increased susceptibility of these very low
birth weight infants to this micro-organism is probably due to a deficiency in the
lung defence mechanism. Like C. trachomatis, the genital mycoplasmata have also
been implicated in preterm labour and low birth weight. Hillier and co-workers [14]
have noted an association between bacterial vaginosis and preterm labour, and
among the various micro-organisms involved, ureaplasmata were associated inde-
pendently with preterm delivery. To what extent the association of the organisms is
a causal one, is unknown.
Screening for C. trachomatis and both mycoplasmata in pregnancy, and subse-
quent treatment with antibiotics to prevent adverse pregnancy outcomes or neonatal
sequelae, is time consuming and expensive. Whether it is worthwhile and cost
effective depends largely on the magnitude of adverse pregnancy outcomes due to
these infections and on their prevalence in the local population. Screening of
pregnant women is recommended in order to determine whether the prevalence is
high enough to justify routine antenatal testing and treatment in that particular
population. We performed a prospective, population-based study among pregnant
 151

women seeking routine obstetrical care at two prenatal clinics in the southern part
of the Netherlands to determine the prevalence of these micro-organisms. To our
knowledge, no data are available of the prevalence of these micro-organisms among
unselected pregnant women in this part of the Netherlands.

Materials and methods

Study population
A total of 770 pregnant Dutch women attending the prenatal clinic of the
University Hospital in Maastricht and the training hospital for midwives in Heerlen
were enrolled in the study. Both clinics are located in the Southern part of the
Netherlands and are 30 km from each other. The study was conducted between
September 1985 and June 1987.
Women eligible to participate in the study had a gestational age of less than 18
weeks. The majority of the patients (> 80%) were enrolled between the 8th and 12th
week of their pregnancy. The patients were enrolled in the investigation after
written informed consent had been obtained. The study population comprised about
60% of all women seeking initial prenatal care at the two clinics. Among the 40% of
women not enrolled, about 8% refused to participate in the study. The remaining
32% either presented later in pregnancy or were not included because of personal
constraints, not maternal characteristics.

Specimerr collection
At the initial antenatal visit in the first trimester of pregnancy samples were
taken from endocervix and vaginal wall by the attending gynaecologists. Endocervi-
cal specimens for C. trachomatis were obtained using the Syva collection kit.
whereas samples for M. hominis and U. urealyticum were taken from the cervical
mucus and the posterior fomix of the vagina using separate cotton-tipped wooden
swabs.
During speculum examination, after removal of excess cervical mucus. specimens
for C. trachomatis were collected by firmly rotating a swab against the wall of the
endocervix for at least 5 s. Immediately after collection, the swab was rolled over a
designated area on a glass microscope slide. The smears were air-dried, fixed with
acetone for 1 min, and sent to the laboratory. The slides were stored at - 20 o C
prior to staining within 3 days.
Specimens for A4. hominis and U. urealyticum were immediately placed in
separate small vials containing 1.5 ml of Mycoplasma medium base (pH 6.0) and
stored at 4” C until transport to the laboratory, were they were stored at -20” C
until processing (within 3 days).

Laboratory procedures
C. trachomatis was detected using a direct immunofluorescent (IF) staining
(Microtrak, Syva), performed according to the manufacturer’s instructions. The
slides were examined with an epifluorescence microscope (Zeiss) at a magnification
of x500. Positive determinations were confirmed by examinations at a magnifica-
tion of x 1000. Slides not showing cervical columnar epithelial cells were considered
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inadequate and were not evaluated further. Slides were scored as positive if 10 or
more apple-green fluorescein-stained elementary bodies were observed.
ikf. hominis and U. ureulyticum were cultured using standard procedures [7]. M.
hominis was identified by typical morphological features on subculture plates
(fried-egg appearance), whereas characteristic brown colonies, due to a brown
deposit on mangane-containing medium, were identified as U. urealyticum.

Results

Evaluable endocervical swabs for C. truchomatis were obtained from 691 out of
770 specimens collected. Non-evaluable specimens were mostly due to the absence
of columnar epithelial cells. In patients with specimens evaluable for C. trachomatis,

TABLE I

Characteristics of pregnant patients with evaluable specimens of C. rruchomatis, M. hominis and U.

urealyticum

Characteristics C. trachomaris M. hominis U. urealyticum

pos./total(%) pos./total(%) pos./total(%)

Age (year)
< 20 O/20 (0) o/21 (0) 2/21 (10)
20-25 4/125 (3) 7/119 (6) 40/119 (34)
25-30 6/305 (2) 15/301 (5) 71/302 (24)
30-35 5/183 (3) 8/185 (4) 36/185 (19)
35-40 l/55 (2) 6/59 (10) 15/59 (25)
> 40 O/3 (0) O/3 (0) l/3 (33)

Parity
0 8/237 (3) 16/235 (7) 61/236 (26)
1 6/305 (2) 3/306 (3) 60/306 (20)
2 2/113 (2) 9/110 (8) 30/110 (27)
>3 O/36 (0) l/37 (3) 15/37 (43)

Education level *
1 7/213 (3) 16/212 (7) 53/204 (26)
2 3/249 (1) 5/248 (2) 68/251 (27)
3 5/172 (3) 11/171 (6) 41/171 (24)
4 l/57 (2) 3/57 (5) 3/42 (7)

Smoking habits (cigarettes per day)

0 8/406 (2) 16/402 (4) 87/363 (24)
l-4 l/45 (2) l/47 (2) 14/47 (30)
25 51240 (2) 17/239 (7) 60/207 (29)

Alcohol consumption (glasses per week)

0 15/541(3) 27/532 (5) 128/492 (26)
1-4 l/123 (1) 5/128 (4) 25/119 (21)
25 0127 (0) 2/28 (8) 12/28 (44)

* 1, less than high school; 2, high school; 3, postgraduate course or university; 4, unknown.
Note: Not all data of women with evaluable specimens were known.
 153

the presence of M. hominis and U. urealyticum was investigated as well. Cultures for
A4. hominis and U. urealyticum were evaluable from 688 and 689 women, respec-
tively. The distribution of the specimens was almost equally divided over the two
sampling sites (Heerlen 370/770, Maastricht 400/770).

Population characteristics
Characteristics of the women who participated in the study are shown in Table I.
The greater part (44%) of the women were between 25-30 years of age upon
enrollment in the study. Women younger than 20 years accounted for a little over
3% of the study population. One third of the women were nulliparous. Of the 61%
of women who graduated from high school, 25% completed a postgraduate course or
university education. Almost 35% of the patients smoked more than five cigarettes a
day, while a minority (4%) consumed more than five glasses of alcohol per week
during pregnancy (Table I).
The majority of the participating women were married (98%), or had a steady
relationship with one sexual partner. Only one patient had no partner at the time of
the investigation.

Isolation rates
The overall recovery rates for the three micro-organisms tested were as follows:
C. trachomatis was found in 2.3% (16/691) of the specimens, M. hominis and U.
urealyticum in 5.2% (36/688) and 23.9% (165/689), respectively. It is interesting to
note that several patients yielded positive results for two of the three micro-organisms
studied (Table II). Of the C. trachomatis-positive samples, 8% were found to be
positive for M. hominis and 47% for U. urealyticum. The specimens positive for U.
urealyticum were in 2% also positive for C. trachomatis and in 9% for M. hominis.
The distribution of the positive specimens according to age, parity, education
level, smoking habits and alcohol consumption is also shown in Table I. Chlamydia-
and Ureaplasma-positive cases were almost equally distributed through the different
age groups between 20 and 40 years. Mycoplasma-positive specimens seemed to be
more frequently isolated from women in the 35-40 age group. The isolation rate of
either of the three micro-organisms tended to be independent of the parity and the
education level.
Isolation of M. hominis and U. urealyticum tended to be higher in women
consuming more than five glasses of alcohol per week.

TABLE I1

Recovery of more than one micro-organism in one patient

Micro-organism No. of
patients

C. trachomatis + M. hominis 3
C. trachomatis + U. urealyticum 3
M. hominis + U. urealyticum 23
154

Discussion

The rate of inadequate specimens for C. trachomatis was approx. lo%, which is
rather high. During pregnancy the cervix is very vulnerable. Rotating a swab against
the wall of the endocervix often induces cervical bleeding, hampering the investiga-
tor in properly obtaining columnar epithelial cells.
In this study the direct IF test was used for the detection of C. trachomatis.
Support for this choice was found in the literature [15-171 as well as from our own
experience. In a limited number of patients (n = 459) the direct IF test was
compared with cell culture. With both methods the same specimens (n = 7) were
found to be positive for C. trachomatis by using a cut-off of 10 apple-green
fluorescein stained particles.
The prevalence of C. trachomatis in the present study (2.3%) falls towards the
lower end of the range of isolation percentages found in the different studies [3,5,6].
In this study there was no inverse relationship between the isolation rate of C.
trachomatis and the age of the patient nor her education level. The percentages
found were comparable with the data found by Kenter et al. [18] and Philips et al.
[16] in similar population groups. Kenter et al. [18] found 2 patients (1.2%) to be
positive for C. trachomatis in a group of 172 patients referred to the out-patient
department of Obstetrics and Gynaecology of an Amsterdam general hospital. In a
hospital-bound practice in Boston, 26 out of 527 patients (4.9%) seeking routine
gynaecological care were found to be positive [16]. However, Berman et al. [19]
found a prevalence of 22% in a selected study population of 1204 Navajo-Indian
women.
The isolation percentages of M. hominis and U. urealyticum found in this study
were in general lower than those found by other investigators [6,20]. A possible
explanation for the divergence might be differences in patient population and
sampling sites. Gibbs et al. [20] isolated M. hominis and U. urealyticum from the
amniotic fluid of asymptomatic patients in 11.3% and 47.78, respectively, whereas
the corresponding isolation percentages from maternal blood samples were 0% and
18.2% respectively. The isolation rate did not differ significantly from the samples
taken from patients with an intra-amniotic infection, except for the isolation
percentage of M. hominis from amniotic fluid samples (11.3% vs. 40% in case of an
infection). Sweet et al. [6] isolated M. hominis (18.9%) and l_J.urealyticum (68.2%)
from the endocervix of pregnant women. In the present study samples were taken
from the cervical mucus and posterior fornix of the vagina of asymptomatic women
and were found to be positive in 5.2% and 23.9%, respectively. These figures were
higher than the 1.8% and 9.3% isolated from the placentas in a control group as
reported by Embree et al. [lo].
Based on the relatively low frequency of detection of C. trachomatis, it seems not
justified either to screen all pregnant women attending our clinics for the presence
of this micro-organism or to treat them all blindly as in the study of Schachter et al.
[21]. The prevalence of vaginal colonization by M. hominis and U. urealyticum in the
present study was 5.2% and 23.9%, respectively. The association between genital
mycoplasmata and impaired course and outcome of pregnancy has not been seen in
all populations. However, there seems no doubt that in a few of the populations
 155

studied, the association between low birth weight and mycoplasmal colonization.
particularly with ureaplasmata is a real phenomenon.
Further studies to analyze the effect of infection or colonization of any one of
these micro-organisms on the course and outcome of pregnancy in this population
will be necessary.

Acknowledgements

We express our gratitude to Jose Philips, Monique Wunderink, Agnes Gerritsen

and Ton Houben for their technical assistance. We thank Ton van den Bogaard for
his assistance in culturing the mycoplasmata.

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