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: peter.korosec@klinika-golnik.si

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

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 10

/27/

14Fo

r pe

rson

al u

se o

nly.

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

926 Case Reports

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 10

/27/

14Fo

r pe

rson

al u

se o

nly.

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

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 10

/27/

14Fo

r pe

rson

al u

se o

nly.

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.

References

[1] Fields BS, Benson RF, Besser RE. Legionella and Legion-

naires’ disease: 25 y of investigation. Clin Microbiol Rev

2002;/15:/506�26.

[2] Murdoch DR. Diagnosis of Legionella infection. Clin Infect

Dis 2003;/36:/64�9.

[3] Jaulhac B, Nowicki M, Bornstein N, Meunier O, Prevost G,

Piemont Y, et al. Detection of Legionella spp.in bronchoal-

veolar lavage fluids by DNA amplification. J Clin Microbiol

1992;/30:/920�4.

[4] Jonas D, Rosenbaum A, Weyrich S, Bhakdi S. Enzyme-

linked immunoassay for detection of PCR-amplified DNA of

legionellae in bronchoalveolar fluid. J Clin Microbiol 1995;/

33:/1247�52.

[5] Cloud JL, Carroll KC, Pixton P, Erali M, Hillyard DR.

Detection of Legionella species in respiratory specimens

using PCR with sequencing confirmation. J Clin Microbiol

2000;/38:/1709�12.

[6] Rantakokko-Jalava K, Jalava J. Development of conventional

and real-time PCR assays for detection of Legionella DNA in

respiratory specimens. J Clin Microbiol 2001;/39:/2904�10.

[7] Reischl U, Linde HJ, Lehn N, Landt O, Barratt K, Well-

inghausen N. Direct detection and differentiation of Legio-

nella spp. and Legionella pneumophila in clinical specimens

by dual-colour real-time PCR and melting curve analysis. J

Clin Microbiol 2002;/40:/3814�7.

[8] Templeton KE, Scheltinga SA, Sillekens P, Crielaard JW, van

Dam AP, Goossens H, et al. Development and clinical

evaluation of an internally controlled, single-tube multiplex

real-time PCR assay for detection of Legionella pneumophila

and other Legionella species. J Clin Microbiol 2003;/41:/

4016�21.

[9] Haufroid V, Clippe A, Knoops B, Bernard A, Lison D.

Genotyping in urine: an interesting tool for epidemiological

studies. Clin Chem 1998;/44:/2210�1.

[10] Lindsay DS, Abraham WH, Findlay W, Christie P, Johnston

F, Edwards GF. Laboratory diagnosis of Legionnaires’

disease due to Legionella pneumophila serogroup 1: com-

parison of phenotypic and genotypic methods. J Med

Microbiol 2004;/53:/183�7.

[11] Ramirez JA, Ahkee S, Tolentino A, Miller RD, Summersgill

JT. Diagnosis of Legionella pneumophila, Mycoplasma

pneumoniae, or Chlamydia pneumoniae lower respiratory

infection using polymerase chain reaction on a single throat

swab specimen. Diagn Microbiol Infect Dis 1996;/24:/7�14.

[12] Murdoch DR, Walford EJ, Jennings LC, Light GJ, Schous-

boe MI, Chereshsky AY, et al. Use of the polymerase chain

reaction to detect Legionella DNA in urine and serum

samples from patients with pneumonia. Clin Infect Dis

1996;/23:/475�80.

[13] Helbig JH, Engelstadter T, Maiwald M, Uldum SA, Witzleb

W, Luck PC. Diagnostic relevance of the detection of

Legionella DNA in urine samples by polymerase chain

reaction. Eur J Clin Microbiol Infect Dis 1999;/18:/716�22.

[14] Murdoch DR, Jennings LC, Light GJ, Chambers ST.

Detection of Legionella DNA in guinea pig peripheral

leukocytes, urine and plasma by polymerase chain reaction.

Eur J Clin Microbiol Infect Dis 1999;/18:/445�7.

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.

928 Case Reports

Scan

d J

Infe

ct D

is D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 10

/27/

14Fo

r pe

rson

al u

se o

nly.