the influence of antimicrobial therapy on the sensitivity of legionella pcr
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The influence of antimicrobial therapy on the sensitivity ofLegionella PCR
PETER KOROSEC, MIRA SILAR, RENATO ERZEN & MITJA KOSNIK
From the University Clinic of Respiratory and Allergic Diseases, Golnik, Slovenia
AbstractThe aim of our study was to establish the sensitivity of Legionella DNA detection in lower respiratory tract samples in 3cases of Legionnaires’ disease after initiation of specific antibiotic therapy. The results showed that Legionella ampliconintensity was highest in the sputum or bronchial aspirates collected at or before the start of appropriate therapy anddecreased markedly within 3 days of therapy. PCR testing was negative within 4 to 6 days of therapy. These data suggest thatwithin a few days specific antimicrobial therapy induces a significant drop of bacterial concentration in respiratory secretionsreaching the detection limit of PCR assay. Respiratory samples for Legionella PCR should be obtained before or earlyafterinitiating antimicrobial therapy.
Introduction
The ability to diagnose Legionnaires’ disease, which
is a common cause of severe pneumonia and
requires specific antimicrobial therapy, is limited
by the non-specific nature of clinical features and
the shortcomings of the diagnostic tests [1]. De-
spite the obvious utility of Legionella culture and
serology, both tests either lack sensitivity or are
unable to provide results within a clinically useful
time [2]. Therefore, detection of Legionella DNA
by PCR plus urinary L. pneumophila antigen
testing is likely to be the best diagnostic tool for
detection of all species within a time frame that will
affect clinical management [2]. When testing lower
respiratory tract secretions, Legionella PCR has
repeatedly been shown to have sensitivity equal to
or greater than cultures [3�8]. However, there is a
lack of standardized PCR assays robust enough to
be used outside the setting of a research laboratory.
An important issue concerning standardization is
the influence of antimicrobial therapy on the quality
of respiratory samples, as Legionella bacteria may
survive poorly in respiratory secretions. This ques-
tion has been poorly evaluated; therefore, the aim
of our study was to establish the sensitivity of PCR
during the course of specific antibiotic therapy. For
this reason we followed up 3 cases of severe
pneumonia with PCR, urinary antigen detection
and serology.
Materials and methods
Study cases
Case 1. A 30-y-old male, smoker, was admitted 4 d
after returning from travel in the Middle East with
dry cough, chest pain, fever, chills and myalgias.
Before hospitalization he was treated with amoxycil-
lin-clavulanic acid for 2 d, but the fever persisted. At
admittance he was tachypnoic with 28 respirations
per min, febrile (39.98C), with inspiratory rales over
the left pulmonary base and non-homogenous in-
filtrate in the left lower lobe. Erythromycin 600 mg 4
times daily was introduced into therapy.
Case 2. A 63-y-old male, ex-smoker, was admitted
after 3 d of chills, fever, dyspnoea and productive
cough. He was tachypnoic, with 24 respirations per
min, febrile (38.58C), with inspiratory rales over the
pulmonary bases and a homogenous alveolar infil-
trate in the left lower lobe. He was treated with
intravenous penicillin 6M 4 times daily for 2 d. As
the fever persisted, penicillin was exchanged for
ceftriaxone 2 g daily and gentamicin 80 mg 3 times
daily for the next 2 d. After Legionella was con-
firmed by urinary antigen testing, intravenous levo-
floxacin 500mg was introduced into therapy.
Case 3. A 45-y-old male, heavy smoker was admitted
after 5 d of illness. He was febrile, had started to
Correspondence: P. Korosec, University Clinic of Respiratory and Allergic Diseases, Laboratory of Clinical Immunology and Molecular Genetics, Golnik 36,
4204 Golnik, Slovenia. Tel: �/386 4 2569 432. Fax: �/386 4 2569 143. E-mail: [email protected]
Case Reports 925
(Received 22 November 2005; accepted 3 January 2006)
ISSN 0036-5548 print/ISSN 1651-1980 online # 2006 Taylor & Francis
DOI: 10.1080/00365540600561777
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cough and had myalgias. At admittance he was
febrile (40.28C), tachypnoic with 32 respirations
per min, tachycardic 110/min, and dullness of
percussion and decreased breath sounds over the
right lungs were heard. Right-sided pleural effusion
and homogenous infiltrate in the right upper lobe
were seen on the chest X-ray. Empirical treatment
with amoxycillin-clavulanate 1.2 g 3 times daily was
started. After 2 d, owing to clinical deterioration and
after confirmation of legionellosis by PCR and
urinary antigen testing, treatment was switched to
intravenous levofloxacin 500 mg daily.
DNA extraction
Respiratory samples (sputum or bronchial aspirate)
were processed as previously described [6]. Briefly,
samples were diluted with phosphate-buffered saline
(PBS), vortexed until they were homogeneous and
centrifuged at 1800�/g for 5 min; the supernatant
was discarded and the pellet volume was brought up
to 0.5 ml with PBS. DNA extraction was performed
with the QIAamp DNA Mini Kit (Qiagen, Hilden,
Germany) according to the manufacturer’s instruc-
tions. To extract DNA from urine samples, the
procedure of the QIAamp Viral RNA kit (Qiagen)
was followed [9].
DNA amplification
DNA amplification was performed with PCR-based
OnarLP test (Minerva Biolabs, Berlin, Germany),
with primers that amplified the portion of the 16S
rRNA region specific for the Legionella spp. genome
(all clinically relevant species are detected, e.g. L.
pneumophila 1�15, L. longbeachae, L. dumoffii, L.
bozemanii and L. gormanii), with achieved sensitiv-
ity of 5 genome copies per PCR reaction (data not
shown). Briefly, 5 ul of extracted DNA was used in a
25 ul reaction mixture that included 10 mM Tris-
HCl (pH 8.5), 50 mM KCl, 4 mM MgCl2, 16S
rRNA primer set for 245 bp amplicon, nucleotides
mix, internal control (lyophilized plasmid DNA of
the HTLV-I tax with a specific primer set for 150 bp
amplicon) and 1 U of Taq DNA polymerase
(Minerva Biolabs). Positive control consisted of
DNA fragments of L. pneumophila genome pre-
pared by PCR (Minerva Biolabs) and negative
control was deionized nuclease-free water. Thermal
cycling was performed with a Primus 96 system
(MWG Biotech, Ebersberg, Germany). Cycling
conditions began with an initial incubation at 948Cfor 2 min followed by 35 cycles consisting of 948Cfor 30 s, 558C for 30 s, and 728C for 30 s and final
cool-down to 88C.
Amplicon detection
10 ul of the PCR amplicon was mixed with 10 ul
deionized nuclease-free water and the mixture was
electrophoresed on 2% agarose E-Gel with ethidium
bromide (Invitrogen, CA, USA). The gels were
viewed under UV light (Gel Doc 2000, Bio-Rad,
Hercules, CA, USA) and migration distance was
compared to that of PCR 100 bp Low Ladder (Sigma,
St. Louis, MO, USA) to determine the approximate
number of base pairs of the amplification product.
Urine antigen detection
Legionella antigen detection was performed by
Binax NOW Legionella Urinary Antigen Test (Bi-
nax, Portland, Maine) according to the manufac-
turer’s instructions.
Serological diagnosis
The presence of L. pneumophila 1�7 IgM and
IgG antibodies in serum samples was investigated
by Serion ELISA assay (Virion/Serion, Wurzburg,
Germany).
Results
A summary of PCR, urinary antigen and serological
diagnostic results are presented in Table 1A�C and
in Figure 1A�B. Specific antimicrobial treatment
was started on d 1 in case 1, on d 4 in case 2, and on
d 3 of hospitalization in case 3. In patient 1,
Legionella DNA was detected in sputum samples
obtained within the first 3 d. However, in sputum d
5 it was negative. In the urine samples PCR was
negative. Urine antigen was positive during the first
2 d. Seroconversion to positive IgM and IgG was
detected within 7 d. In patient 2, Legionella PCR
was positive in the urine but not the sputum sample
of d 4, positive in sputum of d 7 and negative in the
urine and sputum of d 10 and 14. Urine antigen was
positive in all samples. Seroconversion to positive
IgM was detected within 10 d. In patient 3, PCR was
positive in the first 3 sputum samples from d 3 to 5,
but negative in the sample of d 6. Legionella DNA
was not detected in the urine samples. Urinary
antigen testing was positive in the first 2 samples.
A quantitative rise in antibody IgM and IgG
response was detected from d 3 to 9, but not to
positive seroconversion.
Discussion
The performance of Legionella DNA detection in
routine practice is connected with standardized
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clinical samples, and we demonstrated that specific
antimicrobial therapy could have a major influence
on the sensitivities of PCR in lower respiratory tract
samples. We showed that Legionella amplicon
intensity was highest in samples collected at or
before the start of antibiotic therapy. Thereafter,
amplicon intensity decreased within 3 d of therapy
and was negative within 4 to 6 d of therapy. These
data suggest that a few d of therapy induce a
significant drop of bacterial concentration in re-
spiratory secretions reaching the detection limit of
the PCR assay.
In all 3 cases PCR permitted early diagnosis of
Legionnaires’ disease and initiation of appropriate
therapy. In patients 1 and 3, PCR was more sensitive
than urinary antigen detection, as on d 3 (case 1) or
5 (case 3) of hospitalization, PCR testing was still
positive while antigen testing was already negative. A
urinary antigen peak is often observed at 5 to 10 d
after onset of disease symptoms [10]. Nevertheless,
in patient 2 the first sputum sample showed negative
results, suggesting that PCR should not be used as
the sole diagnostic tool. Legionella nucleic acid
amplification was carried out prospectively and in
the same way as routinely performed.
Sputum is considered the specimen of choice for
Legionella DNA detection [2]. However, many
patients produce few or non-purulent samples. In
those patients, throat swabs [11], urine [12�14],
serum [10,12] or peripheral leukocytes [14] may
also be suitable for PCR testing. For that reason we
additionally tested all urine samples, yet with
Table 1. Follow-up of Legionella PCR, urinary antigen detection and serology in pneumonia patient 1 (A), 2 (B) and 3 (C).
1A
Hospitalization
(after symptom onset)
d 1
(3 d)
d 2
(5 d)
d 3
(6 d)
d 5
(8 d)
d 7
(10 d)
d 32a
(35 d)
Antibiotic therapy Erythromycin (600 mg 4�/daily)
PCR in sputum Positive Positive Positive Negative
PCR in urine Negative Negative Negative
Urine antigen Positive Positive Negative
IgMb Neg./10 Pos./224 Neg./67
IgGb Neg./5 Pos./316 Pos./179
1B
Hospitalization (after
symptom onset)
d 1
(3 d)
d 2
(5 d)
d 4
(7 d)
d 7
(10 d)
d 10
(13 d)
d 14
(17 d)
d 31a
(34 d)
d 66a
(69 d)
Antibiotic therapy Penicillin
(6M 4�/daily)
Ceftriaxon (2 g)
Gentamicin
(80 mg 3�/daily)
Levofloxacin
(500 mg)
PCR in sputum Negative Positive Negative Negative
PCR in urine Positive Negative Negative Negative
Urine antigen Positive Positive Positive Positive
IgMb Neg./6 Pos./142 Pos./456 Pos./438 Pos./378
IgGb Neg./11 Pos./51 Pos./116 Pos./96 Pos./297
1C
Hospitalization
(after symptom onset)
d 1
(5 d)
d 3
(8 d)
d 4
(9 d)
d 5
(10 d)
d 6
(11 d)
d 9
(14 d)
Antibiotic therapy Amoxycillin Clavulanate
(1.2 g 3�/daily)
Levofloxacin
(500 mg)
PCR in aspirate Positive Positive Positive Negative
PCR in urine Negative Negative
Urine antigen Positive Positivea Negative
IgMb Neg./7 Neg./55
IgGb Neg./13 Neg./29
a 1A-B convalescent-phase serum sample, 1C weakly positive sample.b Quantitative IgM/IgG ELISA; IgM positive results: �/120 U/ml; IgG positive results: �/50 U/ml.
Case Reports 927
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disappointing results, although previous studies
have shown moderate [14] or good [12,13] sensi-
tivities. Urinary inhibitors could not influence these
results, as internal control was constantly amplified.
Perhaps the reason was the difference in Legionella
DNA urine fragment isolation [12�14] and/or
DNAses in the urine, or that we used primers
that amplified a larger segment (16S rRNA; 245bp)
than in previous studies (5S rRNA; 104 or 108bp)
[12�14].
In all 3 patients the first serological samples
were negative, and seroconversion was observed
within 9 to 14 d after symptoms onset. Thus,
serological testing was simply a valuable epidemio-
logical tool.
In conclusion, antimicrobial therapy can signifi-
cantly influence the sensitivity of Legionella DNA
detection in respiratory samples. Consequently, it
would be important to consider the possible influ-
ence of antimicrobial therapy on sensitivities of
Legionella culture and/or direct fluorescent antibody
staining.
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Figure 1. Detection of 16S rRNA-gene (245 bp amplicon)
specific for Legionella spp. in sputum of patient 1 (A) and
bronchial aspirate of patient 3 (B). Internal control amplicon
has 150 bp, sample numbers represent d of hospitalization, (�/)
positive control, (�/) negative control. Last line represents 100 bp
DNA ladder.
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