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Journal of Clinical Microbiology, May 1998, p. 1185-1188, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Two-Step PCR-Based Assay for Identification of Bacterial Etiology
of Otitis Media with Effusion in Infected Lebanese Children
Ghassan M.
Matar,1,*
Nada
Sidani,1
Michel
Fayad,2 and
Usamah
Hadi3
Department of Microbiology and
Immunology,1
Department of
Pediatrics,2 and
Department of
Otorhinolaryngology/ Head & Neck Surgery,3
Faculty of Medicine, American University of Beirut, New York, New York
10022
Received 26 August 1997/Returned for modification 22 December
1997/Accepted 30 January 1998
 |
ABSTRACT |
We developed and evaluated a two-step PCR-based assay with
universal primers and genus- or species-specific primers for the detection of the most prevalent bacterial etiologies of otitis media
with effusion (OME) in children from Lebanese hospitals. These
etiologies included Haemophilus, Streptococcus,
and Moraxella (Branhamella)
catarrhalis, which were detected in middle-ear effusion (MEE) samples taken from children with OME. A total of 47 MEE samples
were aspirated from 36 patients during insertion of a tympanostomy tube
performed particularly for OME. The duration of effusion in all
patients was 
2 months. DNA was extracted from MEE samples, and PCR
was initially done with DNA extracts by using the universal primers
RW01 and DG74, which flank an ~370-bp fragment found in the 16S rRNA
gene of all bacterial species. For the identification of specific
bacteria, we used in three separate reaction mixtures the following
genus- or species-specific primers: (i) a
Haemophilus-specific probe (probe RDR125) as a primer along
with DG74, (ii) a Streptococcus-specific primer (primer
STR1; designed by us) along with DG74, and (iii) an M. catarrhalis-specific primer pair (primer pair MCA1-MCA2). Thirty-five MEE samples (74.5%) gave the expected 370-bp band, indicating the presence of bacterial DNA in the tested samples. Of the
35 PCR-positive samples tested, 33 (94.3%) were positive for
Haemophilus, 3 (8.6%) were positive for
Streptococcus, and 10 (28.6%) were positive for M. catarrhalis. Ten samples (28.6%) exhibited a mixed infection and
were positive for both Haemophilus and M. catarrhalis. Culture was simultaneously performed for all 47 MEE
samples. Ten of the 47 MEE samples (21.3%) exhibited bacterial growth.
These 10 were PCR positive for bacterial DNA. The remaining 25 PCR-positive samples were negative by culture, thus showing about 53%
discordance between PCR results and those of culture. The PCR assay
proved to be more sensitive than culture, more rapid, less cumbersome,
and more cost-effective than the available PCR-Southern hybridization-based assays.
| |
INTRODUCTION |
Otitis media with effusion (OME) is
a common disease of childhood characterized by the presence of fluid in
the middle-ear cavity behind an intact tympanic membrane, also known as
middle-ear effusion (MEE) (5). OME often follows an episode
of acute otitis media (AOM) (12), which is a highly frequent
childhood disease due to its association with upper respiratory tract
viral infections (4). In OME, symptoms of acute disease like
fever and ear pain are usually lacking, and the disease is often
noticed when the affected child displays a delay in language
acquisition or inattentiveness due to the conductive type of hearing
loss caused by fluid accumulation in the middle-ear cavity. In OME, the
effusion is produced by the inflamed mucosa lining the cavity, and
according to the duration of persistence of this effusion, OME may be
classified as acute when the effusion lasts for 
3 weeks, as subacute
when the effusion lasts between 3 weeks and 3 months, and as chronic
when the effusion persists for 3 months (10).
In the past, OME was thought to be an entirely inflammatory process and
the MEE produced was considered sterile. This belief held true until
1958, when Senturia et al. (11) were able to recover
bacteria from cultures of MEE samples. Since then, several studies in
various regions of the world have been conducted in order to reveal the
bacteriology of OME, and almost all of these studies relied upon
culturing of the MEE samples (1, 2, 15); in addition,
PCR-based assays attempting to detect bacterial DNA in MEE samples were
also recently developed (9, 14). However, most of the
available PCR-based assays often use Southern hybridization, which is
time-consuming, costly, and cumbersome due to the involvement of
multiple procedures. According to the findings of culture and
PCR-based assays, the most common bacteria found in MEE samples
are Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella
(Branhamella) catarrhalis (9, 16). This is in addition to other bacteria like Streptococcus
pyogenes, Staphylococcus, Pseudomonas
aeruginosa, and anaerobes like Prevotella and
Porphyromonas (2).
The present work aimed at (i) the use and evaluation of a two-step
PCR-based assay that initially uses the universal primers RW01 and DG74
to amplify a 370-bp sequence in the conserved 16S rRNA gene common to
all bacteria to demonstrate the presence of bacteria and, in a second
step, single genus- or species-specific primers along with DG74 for the
specific identification of the bacteria involved in OME; (ii)
evaluation of the utility of a probe (probe RDR125), developed by
Greisen et al. (3), as a primer along with DG74 for the PCR
detection of Haemophilus and the utility of primer STR1,
developed by us, for the detection of Streptococcus when
used with DG74; and (iii) determination of the bacterial etiology of
OME in Lebanese patients by using the two-step PCR-based assay and
culture, since no such studies have been conducted in Lebanon.
| |
MATERIALS AND METHODS |
Case definition.
MEE samples were aspirated from children
undergoing myringotomy with or without the insertion of a
tympanostomy tube for OME. For all patients considered, the effusion
(MEE) had persisted for at least 2 months prior to the date of
operation; i.e., all cases of OME were of the subacute (MEE duration, 3 weeks to 3 months) and chronic (MEE duration, 3 months) types. All
patients had been on antibiotic therapy for at least 2 weeks prior to
the operation date.
Source of MEE samples.
A total of 47 MEE samples were
collected between September 1996 and May 1997 from 36 Lebanese children
undergoing tympanostomy-tube placement for OME. The ages of the
children ranged from 2 to 10 years. MEE samples were provided by three
medical centers in Beirut, Lebanon.
Sample aspiration and culture.
The surgeon aspirated MEE
samples from the OME patients during tympanostomy-tube insertion. Each
MEE sample was cultured on chocolate agar, and the culture was
incubated in 5% CO2 at 37°C overnight. Positive cultures
were further identified by Gram staining and standard identification
assays.
DNA extraction.
DNA was extracted from MEE samples by the
method of Loutit and Tompkins (7), with some modifications.
The method uses 800 µg of proteinase K per ml and 5% Triton X-100.
PCR.
PCR was done with DNA extracts first by using the
universal primers RW01 (5'-AAC TGG AGG AAG GTG GGG AT-3')
and DG74 (5'-AGG AGG TGA TCC AAC CGC A-3') which flank
an ~370-bp region in the 16S rRNA gene. MEE samples showing the
370-bp amplicon with the universal primers were further assessed by
using genus- or species-specific primers for the identification of
Haemophilus, Streptococcus, and M. catarrhalis. For Haemophilus, the primer pair used was RDR125 (5'-GG AGT GGG TTG TAC CAG AAG TAG AT 3') and DG74;
the RDR125 primer initially used as a probe by Greisen et al.
(3) hybridizes with an internal sequence within the 370-bp
fragment and was used with DG74 as a primer to flank a 124-bp region.
The data for Haemophilus were further confirmed by using a
second pair of specific primers, F1 (5'-AAC TTT TGG CGG TTA CTC TG
3') and R1 (5'-CTA ACA CTG CAC GAC GGT TT-3'),
designed by Ueyama et al. (14). Primers F1 and R1
flank a 351-bp fragment within the P6-encoding gene (outer membrane
protein-encoding gene) of Haemophilus. The results obtained
with the primer-probe combination were compared with those obtained
with the F1-R1 primer pair, and the degree of concordance between the
two results was recorded. The specificities of all these primers were
assessed by testing them with DNA from other genera. For
Streptococcus, DG74 was used together with primer STR1,
whose sequence was designed by us (5'-AGT CGG TGA GGT AAC CGT
AAG-3'). Primer STR1 lies within the 370-bp sequence and flanks,
along with DG74, a region of 105 bp. The specificity of primer STR1 was
assessed by using it along with DG74 to amplify DNA from other genera.
The primers used for M. catarrhalis were those designed
by Post et al. (9), and the expected band size is about 140 bp. These primers are MCAT1 (5'-TTG GCT TGT GCT AAA ATA TC-3')
and MCAT2 (5'-GTC ATC GCT ATC ATT CAC CT-3'). They
were also used to amplify DNA from other genera to assess their
specificities.
PCR amplification was carried in 100-µl reaction mixtures each
containing 56.5 µl of sterile distilled water, 16 µl of
deoxyribonucleoside triphosphate (0.2 mM), 10 µl of 10× PCR buffer
(100 mM Tris-HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2), 1 µl of each one of the primer pairs to be used (0.3 µg/µl), 0.5 µl of Taq DNA polymerase (5 U/µl), and 15 µl of the
DNA extract. Amplification was carried in a minicycler (MJ Research,
Watertown, Mass.), and the PCR programs used were optimized for each
primer set. The PCR programs for all primer pairs except F1 and R1
consisted of a three-step cycle repeated 34 times: denaturation at
95°C for 1 min, annealing at 55°C for 1 min, and extension at
72°C for 1 min. This was followed by a final extension at 72°C for
10 min. For the F1-R1 primer pair, the same PCR conditions were used,
except that a primer annealing temperature of 60°C rather than one of
55°C was used. The amplicons were separated on 1.5% SeaKem agarose
(FMC BioProducts, Rockland, Maine) gels stained with ethidium bromide
at a final concentration of 0.5 µg/ml. The gels were then visualized
on a UV-light transilluminator (HaakeBuchler, Saddle Brook, N.J.) and photographed with Polaroid type 667 film (Polaroid Ltd., St. Albans, Hertford, United Kingdom).
| |
RESULTS |
Our data have shown that of the 47 MEE samples tested, 35 (74.5%)
gave a band of ~370 bp in tests with universal primers, and of these,
33 (94%) were positive for Haemophilus in tests with the
genus-specific primer RDR125 and the universal primer DG74. These
samples exhibited the expected 124-bp band. No DNA from other genera
was amplified by these primers. Three (8.5%) samples were positive for
Streptococcus in tests with the genus-specific primer STR1
and the universal primer DG74 and gave the expected 105-bp band. No DNA
from other genera was amplified with these primers. Ten (28.5%)
samples were positive for M. catarrhalis in tests
with the species-specific primers MCAT1 and MCAT2 and gave the
expected 140-bp band. No DNA from other genera was amplified. None of the MEE samples that were positive for Haemophilus
in tests with genus-specific primer RDR125 and universal primer DG74 were negative for this organism in tests with the genus-specific F1-R1
primer pair which amplified the expected 351-bp segment, thus showing a
100% concordance between the results obtained by PCR with
Haemophilus-specific primer RDR125 and universal primer DG74
and Haemophilus-specific primer pair F1-R1. Figures
1 and 2
show representative amplicons.
View larger version (84K):
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FIG. 1.
Agarose gels containing representative amplicons. (A)
Universal primers RW01 and DG74. Lanes 1 and 9, 100-bp ladder; lane 2, negative control for the extraction process; lane 3, negative control
for PCR; lane 4, positive control for PCR (Escherichia coli
ATCC 25922 DNA); lanes 5 to 7, three PCR-positive MEE samples,
respectively (370-bp band); lane 8, a PCR-negative MEE sample. (B)
Haemophilus-specific primer-probe pair RDR125 and DG74.
Lanes 1 and 9, 50-bp ladder; lane 2, empty; lane 3, negative control
for PCR; lane 4, positive control for PCR (H. influenzae
ATCC 49766 DNA); lane 5, PCR-positive MEE sample (124-bp band);
lanes 6 and 7, two PCR-negative MEE samples, respectively; lane 8, empty. (C) Haemophilus-specific F1-R1 primer pair. Lanes 1 and 9, 100-bp ladder; lane 2, empty; lane 3, negative control for PCR;
lane 4, positive control for PCR (H. influenzae ATCC 49766 DNA); lane 5, PCR-positive MEE sample (351-bp band); lanes 6 and 7, two PCR-negative MEE samples, respectively; lane 8, empty.
|
|
View larger version (74K):
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[in a new window]
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FIG. 2.
Agarose gels containing representative amplicons. (A)
Universal primers RW01 and DG74. Lanes 1 and 9, 100-bp ladder; lane 2, negative control for the extraction process; lane 3, negative control
for PCR; lane 4, positive control for PCR (E. coli ATCC
25922 DNA); lanes 5 to 7, three PCR-positive MEE samples, respectively
(370-bp band); lane 8, PCR-negative MEE sample. (B)
Streptococcus-specific STR1 and DG74 primer pair. Lanes 1 and 9, 50-bp ladder; lane 2, empty; lane 3, negative control for PCR;
lane 4, positive control for PCR (S. pneumoniae ATCC 49619 DNA); lanes 5 and 7, two PCR-negative MEE samples, respectively; lane
6, PCR-positive MEE sample (105-bp band); lane 8, empty. (C)
M. catarrhalis-specific MCAT1 and MCAT2 primer pair.
Lanes 1 and 9, 50-bp ladder; lane 2, empty; lane 3, negative control
for PCR; lane 4, positive control for PCR (M. catarrhalis DNA); lanes 5 and 6, two PCR-negative MEE samples,
respectively; lane 7, a PCR-positive MEE sample (140-bp band); lane
8, empty.
|
|
Only 10 of the 47 MEE samples were culture positive, with 9 samples
yielding H. influenzae and 2 samples yielding M. catarrhalis after performing the appropriate biochemical tests.
Twelve of 33 (36.4%) samples from OME patients had mixed infections: 1 (3%) sample was positive for all 3 bacteria, 9 (27.3%) samples were positive for Haemophilus and M. catarrhalis,
and 2 (6%) samples were positive for Haemophilus and
Streptococcus. Few cases of OME were due to bacteria other
than the three tested for (PCR positive with universal primers but PCR
negative with specific primers), while others were not due to bacterial
infection (PCR negative with universal primers) but were possibly due
to viral infections or other causes. Table
1 presents the correlation between the
culture and the PCR data.
| |
DISCUSSION |
In the present study, we used a two-step PCR-based assay in order
to detect Haemophilus, M. catarrhalis, and
Streptococcus in MEE samples aspirated from Lebanese
children with OME. The efficiency of the assay for the detection of
bacteria was evaluated by comparing the PCR results to those of
culture. In addition we evaluated the use of a
Haemophilus-specific probe as a primer in an attempt to
detect this organism by PCR alone without resorting to Southern
hybridization.
Seventy-four percent of the MEE samples tested were PCR positive in
tests with the universal primers. In addition, all except two of these
samples were PCR positive in tests with genus- or species-specific
primers. Only 21% of the samples yielded bacterial growth. This
denotes that there is a 53% discordance between the PCR and the
culture data. This observation correlates with the findings of Post et
al. (9), who found that about 48% of the MEE samples tested
were PCR positive for H. influenzae, M. catarrhalis, or S. pneumoniae but culture negative for
these organisms. This implies that PCR is more efficient than culture
in detecting bacteria in MEE samples. However, since PCR amplifies DNA
from viable or dead bacteria, residual DNA from a previous episode of
AOM may have been detected as well. However, a study conducted by Post et al. (8) with chinchilla models has shown that the
bacterial DNA in MEE samples disintegrates within 2 days following
bacterial death. This finding suggests that the bacterial DNA detected
by PCR in our MEE samples may be originating from viable bacteria involved in disease and not residual DNA persisting from a previous episode of AOM unless the bacteria died only 2 days before the date of
operation and sample aspiration. The fact that these bacteria, if they
were viable, were not recovered by culture may be accounted for by the
inherent limited sensitivity of culture assays in general and/or by the
low bacterial count (low numbers of CFU) found in MEE samples. The
presence of low bacterial counts on the order of 104 CFU/ml
and less in MEE samples aspirated from OME patients has already been
documented (13). Such low bacterial counts may in turn be
due to the bactericidal effect of the antibiotics that are usually
administered to patients prior to operation and to the fact that OME,
unlike AOM, is due to a low-grade subclinical infection rather than an
active one. According to Klimek et al. (6), amoxicillin
administered to patients with chronic OME reaches levels of 6.2 µg/ml
in MEE samples. Such high levels are bactericidal enough to lower the
number of viable CFU in MEE samples, especially given the fact that the
antibiotics were administered to the patients whom we considered for at
least 10 days.
The two-step PCR-based assay which uses in step 1 universal primers for
the detection of bacterial DNA and in step 2 genus- or species-specific
primers for the detection of specific bacterial DNA offered a better
approach for the identification of bacteria than existing PCR-Southern
hybridization combinations. This is due to the fact that if bacterial
DNA is present in a given MEE sample, then the first step would detect
this DNA, while the second step would specifically define its identity
within a shorter period of time (12 h) than PCR-Southern hybridization
(2 days). This approach provided a rapid, cost-effective, and less
cumbersome procedure for the detection of bacteria at the genus or
species level.
For the specific PCR detection of Haemophilus in MEE
samples, a sequence originally used as a
Haemophilus-specific probe by Greisen et al. (3)
was used as a Haemophilus-specific primer in the
two-step PCR-based assay. This probe (RDR125) was used along with
one of the universal primers (DG74) to amplify a 124-bp fragment within
the 370-bp sequence of Haemophilus. The primer was shown to
be genus specific since it did not amplify the DNA of other genera. The
high prevalence (94%) of Haemophilus in our samples
necessitated confirmatory testing by using
Haemophilus-specific primer pair F1-R1; these primers flank
a 351-bp region in the P6-encoding gene of Haemophilus. All
samples were positive. This 100% concordance in the PCR results
obtained with the combination of RDR125 and DG74 and those obtained
with the F1-R1 primer pair shows the utility of RDR125 as a primer for
the identification of Haemophilus. Streptococcus-specific
primer STR1, which we designed, was used with DG74 to amplify a 105-bp
fragment. The primer's sequence was found to be unique for the genus
since DNA from closely related genera as well as the genera tested in
this study was not amplified when the primer was used along with DG74.
Finally, the specific primers used by Post et al. (9) for
the detection of M. catarrhalis in the two-step PCR
generated a 140-bp amplicon. The specificities of these primers were
also assessed by testing them with the DNA of a number of other genera.
In conclusion, the two-step PCR-based assay proved to be more sensitive
than culture. In addition, it appeared to be more rapid,
cost-efficient, and less cumbersome than available PCR-Southern hybridization-based assays. The use of a
Haemophilus-specific probe as a primer proved to be
efficient and allowed us to avoid cumbersome blotting and hybridization
procedures. The data generated by the two-step PCR indicate that
Haemophilus is the most prevalent bacterium among the
bacteria for which we tested, followed by M. catarrhalis and Streptococcus. Mixed infection was also
observed. Future work attempting to detect these three bacteria as well as other bacteria in a larger, statistically significant number of MEE
samples is needed in order to determine the prevalence of the bacterial
etiology of OME in Lebanon.
| |
ACKNOWLEDGMENTS |
We thank the Lebanese National Council for Scientific Research
for financial support and the Hotel Dieu de France and Makassed hospitals for provision of some MEE samples.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, American University of Beirut, 850, 3rd Ave., New York, NY 10022. Phone: (212) 583-7600. Fax: (212) 583-7650. E-mail: gmatar{at}aub.edu.lb.
| |
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Journal of Clinical Microbiology, May 1998, p. 1185-1188, Vol. 36, No. 5
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
