Firat University Medical Faculty Otorhinolaryngology,
Department Elazig, Turkey, and *Firat University Medical Faculty
Microbiology and ClinicMicrobiology Department Elazig, Turkey.
Correspondence to: Erol Keles, Assistant Professor,
Firat Üniversitesi Firat, Tip Merkezi KBB Klinigi, 23119 ELAZIG, Turkey.
E- mail: [email protected]
Manuscript received: June 4, 2004, Initial review
completed: August 13, 2004;
Revision accepted: February 2, 2005.
Otitis media with effusion (OME) is a type of
middle ear inflammation characterized by the accumulation of fluid
behind the intact tympanic membrane without the presence of acute signs
or symptoms of an infection, and it is the most common cause of hearing
loss in childhood. Traditionally, middle ear effusions were considered
to be sterile and any type of growth was regarded as a contamination(1).
However, in recent years, around 50% of the middle ear effusions were
reported to have bacterial growth(1,2). In several studies, middle ear
fluid cultures of OME cases were found to be in correlation with the
major bacteria profile of the acute otitis media cases(2,3).
The role of Chlamydia as an etiological agent
in otitis media is being discussed since 1960. Some authors report
isolating Chlamydia trachomatis in middle ear accumulates of
patients with OME and AOM(4,5). However, this situation was not
confirmed by others(6,7). Similar controversies also exist for
Chlamydia pneumoniae. In few isolated cases of OME, Chlamydia
pneumoniae is reported to have been isolated in the cultures of
middle ear fluid(8,9).
Polymerase chain reaction is a technique which allows
for the amplification of limited quantities of the nucleic acid of the
microorganism that is being questioned and aids in its identification.
This method helps us to identify the bacterial DNA present in the middle
ear fluid(3).
In this study, we analyzed the middle ear aspirates
of OME cases for the presence of Chlamydia trachomatis and tried
to demonstrate a possible relationship between the presence of
Chlamydia trachomatis by PCR and the nature of the
effusion material.
Subjects and M
ethods
Seventy eight middle ear aspiration samples from 59
children between the ages of 2-14 who have undergone myringotomy under
general anesthesia in Firat University School of Medicine, Department of
ENT from April 2002 to May 2003 were included in the study. Presence of
the effusion for longer than three months was taken as a criterion for
the presence of OME. Patients with infections, sinusitis, diabetes
mellitus, and immune insufficiencies were excluded from the study.
Effusion samples were obtained from the patients
under general anesthesia by using Zeiss Opmi-1 operation microscope.
Outer ear canal was filled with 70% alcohol and aspiration was carried
out after waiting for one minute. Following the aspiration, the anterior
lower quadrant of the tympanic membrane was entered with a No. 6 cannula
(if ventilation tube was to be inserted) or myringotomy was performed
with an angled politzer tympanic perforator from the same quadrant.
Effusion from the middle ear was directly transferred into a collector
tube with a sterile single-use aspirator set (Xomed Surgical Products,
Jacksonville, Florida, USA). PCR method was utilized for identification
of DNA of Chlamydia trachomatis in middle ear effusion media.
Statistical analyses were made by using Student’s
t test, for samples by using SPSS 10.0 for Windows 98. P
value of <0.05 was accepted as being statistically significant.
Chlamydia trachomatis DNA Identification and PCR
Middle ear effusion samples were transferred into 1.5
mL centrifuge tubes. 500 µL K buffer (20 mM Tris pH 8.0, 150 mM NaCl, %
0.2 SDS and 10 mg/mL Pronase) containing dithiotretiol (DTT) at a
concentration of 40 mM were added to the tubes. After vortexing the
tubes for a minute, they were left on a rotating surface at 37ºC
overnight. Following this procedure, standard phenol-chloroform
extraction and ethanol precipitation was carried out for total DNA
isolation. Isolated DNA was diluted with 50 µL dH20 and digested with
RNase A at a concentration of 50 µg/ml for 30 minutes at 37ºC. The
samples were kept at –20ºC until performing PCR(10). In this study,
sterile dH20 and Chlamydia trachomatis (ATCC 434) species were
used as negative and positive controls, respectively, for both
extraction of DNA and PCR analysis. All procedures were applied for both
the samples and negative and positive controls. Ten times diluted
solutions of DNA extraction product obtained from Chlamydia
trachomatis were utilized for the specificity of the PCR method. In
our experimental procedure, the highest Chlamydia trachomatis DNA
value was found to be 8 fg (0.8 µg/mL or 3.35 bacteria per reaction). In
brief, the specificity and standardization of the PCR procedure employed
in this study was found to be 3.35 bacteria.
For the PCR stage, 50 µL of mixture was prepared. To
each tube 5 µL of 10X reaction buffer (10 mM Tris-HCI (pH 9.0), 50 mM
KCI and % 0.1 Triton X-100, Promega) 5 µL of 25 mM MgCl2, 4
µL of 2 mM deoxy-nucleotide triphosphate, 10 µL of viral DNA, 1 µL of 5
U/mL Taq polymerase and 1 µL each of 20 pmol concentrations of two
primers: primer 1 which is specific to MOMP gene region of C.
trachomatis (Ct.0005: 5’ GAT AGC GAG CAC AAA GAG AGC TAA 3’) and
primer 2 ( Ct. 06: 5’ TTC ACA TCT GTT TGC AAA ACA CGG TCG AAA ACA AAG
3’) and 23 µL of sterile dH2O were added(10). Following 30 cycles of one
minute at 94ºC, one minute at 55ºC and one minute at 72ºC, amplification
was terminated by waiting ten minutes at 72ºC. The obtained PCR products
were run on 3% agarose gel containing ethidium bromide and were
visualized under UV light and bands of 281 base pairs (BP) were observed
as expected.
Results
All patients were between the ages of 2-14 (mean 5.20
± 2.31); there were 22 girls and 37 boys, adding to a total of 59
children and 78 ears. The most common complaint as expressed by the
parents was hearing loss. Of 59 children, 44 had hearing loss, six had
ear pain and three had fullness of the ear upon admission. The remaining
six patients who did not report any complaints were identified during
routine ENT examinations.
Of the seventy-eight middle ear effusions, 26 (33.3%)
were serous, 31 were (39.7%) mucoid and 21 (26.9) were sero-mucoid in
character. Chlamydia trachomatis DNA genome was identified in
seven (8.9%) effusion samples. Of these, three were mucoid (42.8%),
three were serous (42.8) and one was sero-mucoid (14.2%) in character.
There was no correlation of statistical significance between the
character of the middle ear effusion and the presence of Chlamydia
trachomatis (p >0.05).
Discussion
Chlamydia is an obligatory intracellular parasite
of 0.2-1.5 µm diameter; depending on its developmental phase within the
cell it might present different appearances. Chlamydia trachomatis
is a specific human parasite and the causative agent for trachoma,
inclusion conjunctivitis, urethritis and proctitis etc.(11).
Identification of Chlamydia trachomatis in infants as a
respiratory pathogen has resulted in focusing the attention to the
possible relationship between other respiratory diseases of the
childhood and Chlamydia trachomatis. If mother is infected with
Chlamydia trachomatis while giving birth, then the child acquires
the infection from the mother. The transmission rate from an infected
mother is around 50%. In 30-40% of these children, nasopharyngeal
infections develop and only 10% develop pneumonia afterwards.
The data pertaining to the role of Chlamydia
trachomatis in infants with otitis media was first collected by
Tipple, et al.(5) by isolating Chlamydia trachomatis in
middle ear effusions of few children with Chlamydial pneumonia. However,
we are not certain whether these children have AOM or not. Schachter,
et al.(12) reported that children born to Chlamydia positive
mothers did not demonstrate any significant difference in the incidence
of AOM when compared to controls. They thus concluded that AOM was not a
common complication of infantile chlamydial infection.
The possible relationship between OME and
Chlamydia trachomatis has been the topic of many research studies.
Of 44 patients with OME, Zhang, et al.(13) identified
Chlamydia trachomatis DNA in middle ear aspirates of 14 patients
with PCR technique. Zhang and colleagues(13) reported that in 14 out of
44 EOM patients, DNA of Chlamydia trachomatis examined by PCR in
the middle ear aspiration samples was positive. In other studies, no
relationship was reported between OME and Chlamydia trachomatis.
Hammerschlag, et al.(7) stated that they could not grow
Chlamydia trachomatis in any of the middle ear aspirates obtained
from 68 children with OME. In our study of the 78 middle ear effusion
samples obtained, we were able to identify Chlamydia trachomatis
DNA genome in seven samples (8.9%).
Hammerschlag, et al.(7) reported that
Chlamydia trachomatis related pneumonia would be a very rare
instance in infants above six months of age. In our study, the average
age of the patients was 5.20 ± 2.31years. We think that other than
transmission through infected mothers, insects and non-chlorinated
swimming pools might be other possible routes. Also, a postoperative
specific treatment was applied to the patients detected DNA genome of
Chlamydia trachomatis in their efusion liquid. No recurrence of
effusion was found while following the patients detected positively DNA
genome of Chlamydia trachomatis in an avarage period of 10 months.
Nowadays, there are studies comparing the character of the effusion and
the growth rates of the bacteria in the culture. In such studies,
bacterial growth rates of 29-80% are reported for purulent effusions and
25-43% for mucoid effusions(14).
The difficulty in obtaining material for culture in
cases of OME brings about the possibility of empirical treatment. In
empirical treatment, the important thing is to know the underlying
microbiology of the pathology. Concerning the involved microorganisms (H.
influenzae, S. pneumonia, B. catarrhalis) OME has similarities with
acute otitis media(3,15). Thus, most of OME patients have at least used
ampicillin group of anti-biotics once. Ampicillin has limited in
vitro effects against Chlamydia trachomatis.
As PCR detects DNA from both live and dead bacteria,
it can also detect DNA from a previous attack of AOM. Yet, in a research
conducted on chinchilla models Post, et al.(3) have demonstrated
bacterial DNA being eliminated from middle ear effusion samples two days
after bacterial death. These findings demonstrate that the bacterial DNA
that we have detected in middle ear effusion samples were probably
originating from live bacteria and was not due to a previous attack of
AOM. If these bacteria are alive, the reason behind their not being
grown in culture media is the low number of colony forming units (low
CFU). In middle ear effusion samples obtained from patients with OME,
the measurement of bacteria were reported as 104 CFU or below(16). This
number might be due to the bactericidal effects of the antibiotics used
prior to surgery. Moreover, in contrast to AOM, OME has a subclinical
presentation.
Klimek, et al.(17) demonstrated that in
patients with chronic OME, administration of amoxicillin resulted in
levels of 6.2 µg/mL in middle ear effusion samples. Imagining that the
antibiotics should at least be administered for a minimum of ten days,
they concluded that this high level of antibiotics resulted in lowered
number of live bacteria. We as well think that the reason behind not
being able to grow Chlamydia trachomatis in middle ear aspirate
cultures, is the low number of live bacteria due to the utilized
antibiotics.
The most important obstacle in detecting microbial
DNA in body fluids is the presence of heme or urea which both act like
polymerase inhibitors(18). This might cause false negative results.
Isolation of DNA from non-purified samples might eliminate these
polymerase inhibitors. However, a small part of target DNA might be lost
during this procedure(19). In our study, we did all our PCR assays after
the isolation of DNA in order to minimize the number of false
negativities.
Contributors: EK was responsible for planning and
design of the study, partially analysing the data, writing and editing
of the manuscript. YB and AO studies Chlamydia trachomatis in middle ear
fluid in otitis media with effusion. IK was responsible for planning and
design of the study. TK was responsible for planning and/or design of
the study, writing and/or editing of the manuscript. SY was responsible
for planning and/or design of the study, and HCA was involved in writing
and editing of the manuscript.
Funding: None.
Competing interests: None stated.
|
Key Messages |
• Identification of Chlamydia trachomatis in 8.9% of the
middle ear aspirate samples demonstrates that this microorganism
should be taken into consideration in the treatment of Otitis
media with effusion.
|
