Development of Real-Time Multiplex Nucleic Acid Sequence-Based ...

JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2008, p. 185–191 0095-1137/08/$08.00⫹0 doi:10.1128/JCM.00447-07 Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Vol. 46, No. 1

Development of Real-Time Multiplex Nucleic Acid Sequence-Based Amplification for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in Respiratory Specimens䌤 K. Loens,1* T. Beck,1 D. Ursi,1 M. Overdijk,2 P. Sillekens,2 H. Goossens,1 and M. Ieven1 Department of Medical Microbiology, University of Antwerp, Wilrijk, Belgium,1 and bioMe´rieux, Boxtel, The Netherlands2 Received 27 February 2007/Returned for modification 21 June 2007/Accepted 5 November 2007

Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMe´rieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run. potential contamination between samples. Real-time singletarget (mono) NASBA has been successfully used for the identification of West Nile and St. Louis encephalitis viruses (14), human immunodeficiency virus type 1 (8), M. pneumoniae (18), and Clavibacter michiganensis subsp. sepedonicus (30). Multiplex formats might solve the practical shortcoming of detecting only one agent at a time. Detection of multiple pathogens simultaneously would be economical for small volume samples and would reduce costs. Multiplex real-time NASBA has already been successfully applied for the detection of potato leafroll virus and potato virus Y in potato tubers (13), for simultaneous detection and typing of potato virus Y isolates (26), and for monitoring expression dynamics of human cytomegalovirus-encoded IE1 and pp67 RNA (9). The aim of this study was to develop a real-time multiplex NASBA assay for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens based on the amplification of a 16S rRNA target sequence using the NucliSens Basic Kit (17) and to compare the results with those of real-time NASBA.

Community-acquired pneumonia is associated with significant morbidity, mortality, and utilization of health service resources. No etiologic agent can be identified in ⬎35% of cases of community-acquired pneumonia when using conventional diagnostic methods such as culture and serology (21, 22, 31). Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella pneumophila may be responsible for 15 to 50% of community-acquired pneumonia (23, 25). Differentiation of infections due to Streptococcus pneumoniae from those due to M. pneumoniae, C. pneumoniae, or L. pneumophila as well as those due to viruses is essential to avoid inappropriate use of antibiotics. Culture and serological confirmation of the diagnosis of infections due to M. pneumoniae, C. pneumoniae, and L. pneumophila is difficult and may require several weeks. Therefore, nucleic acid amplification techniques are of considerable interest. PCR was shown to be significantly more sensitive than culture for the detection of M. pneumoniae (1, 33), C. pneumoniae (4, 32), and L. pneumophila (2, 11). Nucleic acid sequence-based amplification (NASBA; bioMe´rieux, Boxtel, The Netherlands) targeted at RNA has been adapted to the real-time format using DNA hybridization probes that fluoresce upon hybridization (15). The whole process of amplification and detection runs in a fluorescent reader. Real-time assays enable one-tube assays suitable for highthroughput applications, reducing the assay time and limiting

MATERIALS AND METHODS Bacterial strains. Bacterial strains, used to test the specificity of the NASBA primers, are presented in Table 1. Mycoplasma strains were cultured in homemade spiroplasma (SP4) medium (28) without thallium acetate and supplemented with amphotericin B (0.5 mg/ ml), polymyxin B (500 U/ml), glucose (0.5%), and arginine (0.25%) or urea (0.5%) depending on the nutritional needs of the species. M. pneumoniae strain ATCC 29085 was quantified by incubation of four replicates of 10-fold dilutions of a suspension in SP4 medium at 37°C. The cultures were examined weekly for color change from red to yellow during 2 months. The titer was expressed in color-changing units (CCU) per ml (3). C. pneumoniae (ATCC VR-1355) was grown in HEp-2 cells. After inoculation,

* Corresponding author. Mailing address: Department of Medical Microbiology, University of Antwerp, Universiteitsplein 1 S009a, B-2610 Wilrijk, Belgium. Phone: 32-3-820-24-18. Fax: 32-3-820-27-52. E-mail: [email protected]. 䌤 Published ahead of print on 21 November 2007. 185

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TABLE 1. Bacterial species and strains used to test the specificity of the assay No.

Strain/source

Species

Origina

1 2 3 4

ATCC VR-1355 Clinical isolate Kiq10 ATCC 29085 (PI1428) ATCC 15492 (MAC) NC10117 NC10111 ATCC 33530 (G-37) NC10112 NC10136 NC10113 NC11702 CDCBC1636 CDCBC1637 Clinical isolate Strain Los Angeles Strain Dallas Clinical isolate Strain Concor Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate Clinical isolate

Chlamydophila pneumoniae Chlamydia psittaci Chlamydia trachomatis Mycoplasma pneumoniae type 1 Mycoplasma pneumoniae type 2 Mycoplasma fermentans Mycoplasma hominis Mycoplasma genitalium

ATCC UZA ITG ATCC

Mycoplasma orale Mycoplasma buccale Mycoplasma salivarium Mycoplasma pirum Legionella pneumophila Legionella pneumophila Legionella pneumophila Legionella pneumophila

NCTC NCTC NCTC NCTC UZA UZA SEH UZA

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

1 2 3 4

Legionella pneumophila 5 Legionella pneumophila 6 Legionella pneumophila 8 Legionella pneumophila 10 Legionella longbeachae 4a Legionella longbeachae 4b Legionella bozemanii Legionella micdadei Ureaplasma urealyticum Moraxella catarrhalis Haemophilus influenzae Streptococcus pneumoniae Streptococcus pyogenes Streptococcus viridans Staphylococcus aureus Klebsiella pneumoniae Escherichia coli Neisseria meningitidis Pseudomonas aeruginosa

ATCC NTCC NCTC ATCC

UZA UZA UZA SEH UZA UZA SEH SEH UZA UZA UZA UZA UZA UZA UZA UZA UZA UZA UZA

a ATCC, American Type Culture Collection, Manassas, VA; ITG, Instituut voor Tropische Geneeskunde, Antwerp, Belgium; NCTC, National Collection of Type Cultures, Central Public Health Laboratory, London, United Kingdom; SEH, Sint Elisabeth Hospital, Tilburg, The Netherlands; UZA, Universitair Ziekenhuis Antwerpen, Edegem, Belgium.

the cell cultures were centrifuged at 3,500 rpm and 25°C for 60 min and subsequently incubated at 37°C for 1 h. Then the medium was aspirated and cell cultures were incubated with fresh medium containing cycloheximide (1 mg/ liter). After 3 days, the shell vials were aspirated and fixed with 96% ethanol. The fixed monolayers were rinsed with phosphate-buffered saline and stained by the fluorescent antibody technique with C. pneumoniae-specific mouse monoclonal antibodies (Dako A/S, Glostrup, Denmark). Rabbit anti-mouse immunoglobulin labeled with fluorescein isothiocyanate (Dako A/S) was used as a conjugate. C. pneumoniae (ATCC VR-1355) was quantified by incubation of five replicates of 10-fold dilutions on HEp-2 cells. Inclusion-forming units (IFU) were counted 72 h after infection by immunofluorescence microscopy. The titer was expressed in IFU/ml. Legionella strains were grown on buffered charcoal-yeast extract (Oxoid Ltd., Belgium) agar plates at 37°C for 48 to 72 h and quantified by incubation of four replicates of 10-fold dilutions on buffered charcoal-yeast extract agar. The titer was expressed as CFU/ml. The other organisms were cultivated on standard media supporting optimal growth. The clinical isolates were identified by standard methods. Respiratory specimens. Throat swabs, bronchoalveolar lavage (BAL) samples, nasopharyngeal aspirates (NPA), sputum, and bronchus aspirates (BA) from the Microbiology Laboratory of the University Hospital of Antwerp that tested

TABLE 2. Overview of test results for all Legionella-positive specimens Specimen type and no.

Real-time NASBA PCR

Culture Mono

Multiplex

Urinary antigen

IgM

IgGa

Lung 1 2 3 4 5

⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ND ND ND

NDc ND ND ND ND

ND ND ND ND ND

Sputum 6 7 8 9

⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫺

⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫺

NDb ⫹ ⫺ NDb

Water 10 11 12 13 14

⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫹ ⫹

⫹ ⫹ ⫹ ⫹ ⫹

NAd NA NA NA NA

NA NA NA NA NA

NA NA NA NA NA

a

Seroconversion or significant rise in titer. Only an acute-phase serum sample was available. c ND, not done. d NA, not applicable. b

negative for M. pneumoniae, C. pneumoniae, and L. pneumophila by PCR (12, 29) were spiked with dilutions of reference strains for sensitivity experiments. Archived specimens positive by PCR for one of the three organisms included three C. pneumoniae-positive nasopharyngeal aspirates, five L. pneumophilapositive lung biopsy specimens (Table 2), four sputum specimens, and five water samples. Fifty-one respiratory specimens were collected from 33 patients found to be M. pneumoniae positive by PCR (12). Forty of these specimens were M. pneumoniae PCR positive (Table 3). One hundred forty-nine specimens (44 sputum specimens, 36 throat swabs, 25 NPA, 22 BAs, 19 BAL samples, one gargle specimen, one lung biopsy specimen, and one pleural fluid) previously found negative for M. pneumoniae, C. pneumoniae, and L. pneumophila by PCR were also tested as negative-control specimens. Nucleic acid extraction. All respiratory specimens were protease treated before extraction (16). Nucleic acids were extracted as described by Boom et al. (5) using the NucliSens Basic Kit module (bioMe´rieux, Boxtel, The Netherlands). Briefly, 100 ␮l of protease-treated clinical specimens (16) or aliquots of a bacterial culture were added to a guanidinium thiocyanate lysis solution, pH 6.2, and mixed vigorously for rapid lysis. Fifty microliters of activated silica was added. The nucleic acid-silica complex was washed twice with guanidinium thiocyanate washing solution, twice with 70% (vol/vol) ethanol, and once with acetone. After being dried at 56°C, nucleic acids were eluted from the silica using 50 ␮l elution buffer and stored at ⫺80°C. Each nucleic acid extract was amplified both by real-time mono NASBA and by the real-time multiplex NASBA. Real-time NASBA. Real-time mono NASBA reactions were performed using the NucliSens Basic Kit amplification module (bioMe´rieux) as described previously (18, 19, 20). In negative-control reactions, target nucleic acid was replaced by RNase-/DNase-free water. Amplification reaction mixtures were incubated in a fluorescent reader, the NucliSens EasyQ Analyzer (bioMe´rieux), and results were calculated with the Ascent software (bioMe´rieux). NASBA with U1A, a low-abundance mRNA derived from a human cellular housekeeping gene, encoding the A protein present in the human U1 (U1A) small nuclear ribonucleoprotein (snRNP) particle (24)—to verify the presence of nucleic acid after nucleic acid extraction of specimens—was performed using the NucliSens Basic Kit amplification module (bioMe´rieux) on all nucleic acid extracts according to the instructions of the manufacturer in a separate reaction tube. For real-time multiplex NASBA, besides the species-specific P1 primers described previously (18, 19, 20), a generic P2 primer, 5⬘-GATGCAAGGTCGCA TATGAGAATTTGATCCTGGCTCAG-3⬘, was designed. The three P1 primers and this generic P2 primer were used in the real-time multiplex NASBA reaction. The following molecular beacons for real-time detection were used: 5⬘–6-

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DETECTION OF REAL-TIME MULTIPLEX NASBA

TABLE 3. Overview of test results for all M. pneumoniaepositive patients Patient no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15a 16 17 18 19 20 21a 22

a

23

a

24 25 26 27 28a 29 30a 31 32 33a

Specimen

BAL BA NPA NPA Sputum NPA NPA BAL BAL TSf Sputum BAL BAL NPA NPA TS Sputum NPA TS TS Gargle Sputum 1 Sputum 2 Sputum TS Gargle TS TS Gargle TS Sputum Sputum TS Sputum TS TS Sputum TS Sputum Sputum TS TS Gargle TS Sputum Sputum TS Sputum TS Sputum TS

PCR

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹

Realtime mono NASBA

Realtime multiplex NASBA

U1A resultb

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

9.61 11.00 9.76 14.30 11.27 9.00 10.55 11.46 10.27 7.28 9.78 14.58 15.94 11.50 11.78 9.89 12.68 11.86 12.30 4.07 3.04 13.44 7.83 13.53 1.34 8.69 1.56 3.46 6.24 11.50 13.25 1.34 8.29 1.72 4.58 6.50 9.71 5.58 12.52 7.82 11.31 10.02 6.16 1.38 11.09 11.59 9.99 13.50 8.04 1.58 1.67

IgM

IgGc

TABLE 4. Sensitivities of the real-time multiplex NASBA assay in comparison with the mono real-time NASBA assays in the amplification reaction and the extraction % Sensitivity

Culture No. of in vitro-generated RNA molecules

NAd NA NA NA NA NA NA NA NA ⫹

NA NA NA NA NA NA NA NA NA ⫹

NA NA NA NA ⫹

NA NA NA NA ⫹

⫺ ⫺ ⫹

ND ⫹ ⫹

NA

NA

⫹

⫹

NA

NA

⫺

ND

⫺

ND

⫹

ND

⫺

⫺

⫹

⫺

⫹

⫺

⫺

⫺

⫺

ND

⫺

⫺

⫺

ND

NA

NA

NA ⫺

NA ND

187

NDe ND ND ND ND ND ND ND ND ⫺ ⫹ ND ND ND ND ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ ND ND ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ND ⫺ ⫺ ⫺ ND ⫺ ND ⫺ ND ⫺ ND ⫺ ND ⫺ ⫺

a

Only an acute-phase serum sample was available. U1A positive result above 2.00. Seroconversion or significant rise in titer. d NA, not available. e ND, not done. f TS, throat swab. b c

carboxyfluorescein–CCATGGGTTGAAAGACTAGCTAATACCATGG-Dabcyl3⬘ for the detection of M. pneumoniae, 5⬘-ROX-CCGATCGTGTAGTGTAATTA GGCATCTAATATCGATCGG-Dabcyl-3⬘ for the detection of C. pneumoniae, and 5⬘-Cy5-CCGAGCTGAGTAACGCGTAGGAATATGCTCGG-Dabcyl-3⬘ for the detection of Legionella spp. The final concentration of each primer in the amplification reaction mixture was 0.2 ␮M. To determine the cutoff for real-time mono and multiplex NASBA detection, the results of the 142 different individual truly negative samples were measured. The mean was calculated. A sample was considered to be M. pneumoniae, C. pneumoniae, and/or Legionella positive when the signal was above the mean of the negative samples plus 20%. PCR. M. pneumoniae (12) and C. pneumoniae (29) PCR targeting the P1 cytadhesin gene and the PstI fragment, respectively, were done as described previously after nucleic acid extraction using the QiaAmp DNA blood minikit (Qiagen, Hilden, Germany). L. pneumophila real-time PCR was targeted at the mip gene. Four microliters of the nucleic acid extracts was used in a 20-␮l

L. pneumophila

M. pneumoniae

C. pneumoniae

Mono Multiplex Mono Multiplex Mono Multiplex Amplification (n ⫽ 15–20) 10 50 100 1,000 1,0000

31 70 91 100 100

23 57 82 100 100

33 82 89 100 100

7 50 79 100 100

80 100 100 100 100

65 100 100 100 100

Extraction (n ⫽ 10–12) 500 1,000 5,000 10,000 50,000

80 50 100 100 100

60 30 70 91 100

20 40 80 91 100

20 37 60 72 100

100 100 100 100 100

30 45 100 100 100

real-time PCR mixture using the LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). Ten picomoles of primers MIP1 (5⬘-CAACCGATGCCACAT CATTA-3⬘) and MIP2 (5⬘-TAGCCATTGCTTCCGGATTA-3⬘) and 4 pmol of probes MIPFL (5⬘-GCCTTGATTTTTAAAATTCTTCCCAA-FLU-3⬘) and MIPLC (5⬘-LCRed640-TCGGCACCAATGCTATAAGACAACT-3⬘) were used in combination with the LightCycler Faststart DNA hybridization probe kit (Roche Diagnostics) according to the instructions of the manufacturer. DNA was amplified by one incubation step at 94°C for 60 s, 45 cycles at 95°C for 10 seconds, 60°C for 10 s, and 72°C for 20 s each and one final incubation step at 40°C for 30 s. Sample preparation, setup of the reactions, and product analysis were done in separate rooms. As a control, negative samples were processed simultaneously. Sensitivity study. For the generation of wild-type RNA, cDNA from part of the 16S rRNA from each organism, obtained by PCR using adapted versions of the NASBA primers, containing an EcoRI site and a Csp45I site, was inserted into plasmid pG3O, a modified pGEM vector. The plasmids were transfected in Escherichia coli DH5␣ and used for large-scale generation of runoff transcripts after linearization with BamHI (Pharmacia Biotech). In vitro RNA was generated from these constructs with T7 RNA polymerase (Pharmacia Biotech) as described previously (16, 19, 20). The RNA was quantified by spectroscopy, and from the optical density the number of molecules was calculated. The analytical sensitivity of the multiplex real-time NASBA was studied on 10-fold dilutions in water of wild-type in vitro-generated RNA as single targets or as combinations and by using 10-fold dilutions of M. pneumoniae, C. pneumoniae, and Legionella spp. as single targets in lysis buffer (Tables 4 and 5). The sensitivity was also studied using 10-fold dilutions of cultured M. pneumoniae, C. pneumoniae, and L. pneumophila serotype 1 added as a single target to protease-treated samples (16) of each respiratory specimen pool. The 95% hit rate for in vitro-generated RNA was calculated by SAS version 6.12 software (SAS, Cary, NC). Reproducibility. The intrarun and interrun variations in multiplex real-time NASBA were estimated by running samples containing 500 or 5,000 CCU/100 ␮l

TABLE 5. Sensitivities of real-time multiplex NASBA assays when tested with multiple target in vitro-generated RNA molecules No. of in vitro-generated RNA molecules in the extraction (L. pneumophila/ M. pneumoniae/ C. pneumoniae)

33/33/33 330/330/330 3,300/3,300/3,300 330/3,300/3,300 3,300/330/3,300 3,300/3,300/330

No. of samples positive/no. tested by real-time multiplex NASBA for: M. pneumoniae

C. pneumoniae

L. pneumophila

1/2 2/2 2/2 2/2 2/2 2/2

2/2 2/2 2/2 2/2 2/2 2/2

1/2 2/2 2/2 0/2 2/2 2/2

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of M. pneumoniae; 0.1, 1, 10, or 100 IFU/100 ␮l of C. pneumoniae; and 100 or 1,000 CFU/100 ␮l of L. pneumophila serotype 1 added as single targets in duplicate to one BAL pool to determine the intrarun variation and to two BAL pools to determine the interrun variation. Five replicates of each nucleic acid extract were analyzed, respectively. The calculations on the final fluorescent value were done by using Microsoft Excel software. Negative controls were coanalyzed within each run. Serology and Legionella urinary antigen test. Serology and urinary antigen tests were performed upon physician request. If available, paired sera were tested. For the detection of M. pneumoniae antibodies, an immunoglobulin M (IgM) and an IgG enzyme immunoassay (Anilabsystems, Helsinki, Finland) were performed. An acute infection was defined when at least a 1.5-fold IgG enzyme immunoassay unit increase with paired sera, assayed in the same run, was obtained. With enzyme immunoassay unit values above 130, a 1.3-fold increase in the same run was indicative of a significant rise in antibodies. IgM was considered positive when the signal/cutoff unit ratios were above 1.1. C. pneumoniae-specific IgM and IgG antibodies were detected by the Anilabsystems enzyme immunoassay. The same definition of an acute infection as described above was used. A patient was considered positive for Legionella when ⬎70 U/ml and/or ⬎140 U/ml was measured for IgG and IgM, respectively, by using the Serion enzymelinked immunosorbent assay classic (Virion/Serion, Wu ¨rzburg, Germany) IgM and IgG test. The Binax Now urinary antigen test was used according to the instructions of the manufacturer. Statistical analysis. The ␹2 test was used for statistical analysis.

RESULTS Specificity of the 16S rRNA multiplex NASBA primers. Using real-time multiplex NASBA, positive results were obtained with nucleic acid extracts from M. pneumoniae, C. pneumoniae, and all Legionella spp. and serotypes but with none of the other organisms listed in Table 1. Sensitivity of the 16S rRNA multiplex NASBA. (i) Single target. The 95% hit rate for the analytical sensitivity of the 16S rRNA NASBA primers tested on dilutions of in vitro-generated wild-type RNA was 33 molecules, 153 molecules, and 169 molecules of C. pneumoniae, M. pneumoniae, and L. pneumophila in vitro-generated RNA, respectively, when immediately added to the amplification reaction mixtures. When extraction was done prior to the amplification, i.e., the isolation of in vitro-generated RNA from lysis buffer, 3,185 molecules, 18,489 molecules, and 16,697 molecules of C. pneumoniae, M. pneumoniae, and L. pneumophila, respectively, were needed in the extraction for a 95% hit rate in the amplification reaction. However, it should be mentioned that only 10% of the extracted nucleic acid is used in the amplification reaction. The results for the individual reactions are shown in Table 4. When lysis buffer was spiked with 5 CCU/100 ␮l of M. pneumoniae, four/four and two/four samples were found positive by real-time mono and multiplex NASBA, respectively. With an input of 0.1 IFU of C. pneumoniae/100 ␮l, four/four and three/four samples were positive by real-time mono and multiplex NASBA, respectively. Real-time mono and multiplex NASBA detected 0.1 CFU of L. pneumophila serotype 1 in 100 ␮l lysis buffer in four/four and two/four aliquots, respectively. When real-time mono and multiplex NASBAs were applied to the same nucleic acid extract from L. pneumophila serotypes 2, 3, 4, 5, 6, and 10 as well as to Legionella longbeachae 4a and Legionella micdadei, the difference in sensitivity between the real-time mono and multiplex NASBAs was 1 log with the former being the more sensitive assay. For L. longbeachae 4b and Legionella bozemanii the difference in sensitivity was 2 logs. The sensitivities on spiked respiratory specimens with M.

pneumoniae, C. pneumoniae, and L. pneumophila as a single target are shown in Table 6. (ii) Multiple targets. With inputs of 330 or 3,300 molecules of in vitro-generated RNA of each organism, all three organisms could be detected by the real-time multiplex NASBA. With an input of 33 molecules of in vitro-generated RNA of each organism, a positive result was always obtained for C. pneumoniae whereas the assay failed to detect Legionella and M. pneumoniae in some but not all samples (Table 5). In a mixture of 3,300 molecules of both M. pneumoniae and C. pneumoniae in vitro-generated RNA and 330 molecules of Legionella in vitro-generated RNA, an input of 330 molecules of Legionella in vitro-generated RNA was not detected by the real-time multiplex NASBA. (iii) Negative specimens. Out of 149 M. pneumoniae, C. pneumoniae, and L. pneumophila PCR-negative respiratory specimens, three, two, and two specimens were positive by real-time mono NASBA for M. pneumoniae, C. pneumoniae, and Legionella, respectively. One out of these three M. pneumoniae-positive specimens was also positive by real-time multiplex NASBA. Two specimens originated from the same patient. Reproducibility of the real-time multiplex NASBA. The intrarun variability coefficients for the detection of 500 and 5,000 CCU of M. pneumoniae were 13.5 and 13.5, respectively, whereas the interrun variation coefficients for the same inputs were 16.7 and 16.8, respectively. The intrarun variability coefficients for the detection of 0.1, 1, 10, and 100 IFU of C. pneumoniae were 13.3, 10.3, 10.7, and 13.0, respectively, by real-time multiplex NASBA. The interrun variation coefficients for the same inputs were 20.8, 11.0, 13.7, and 15.4, respectively. For L. pneumophila serotype 1, the intrarun variability coefficients were 13.4 and 13.1 for inputs of 100 and 1,000 CFU, respectively. The interrun variation coefficients for the same inputs were 10.6 and 11.6, respectively. Archived specimens from PCR-positive patients. From the 51 archived respiratory specimens collected from 33 patients who had at least one specimen positive for M. pneumoniae by PCR, 40 were PCR positive. Twenty-one specimens were M. pneumoniae positive by the three detection procedures: PCR, real-time mono NASBA, and multiplex NASBA. Four specimens were M. pneumoniae positive by PCR and real-time mono NASBA. Four M. pneumoniae PCR-negative specimens were positive by real-time mono NASBA only. Two of these were also positive in the multiplex assay. Seven specimens were M. pneumoniae negative by all three techniques (Table 3). Fifteen M. pneumoniae PCR-positive specimens were negative by both real-time mono and multiplex NASBAs. However, seven of these specimens were U1A negative and thus contained no RNA (samples 19, 20, 23, 24, 29, 32, and 33 [Table 3]). For three patients (patients 23, 25, and 26 [Table 3]), the PCR result was confirmed by a positive IgM result; one of these patients (patient 23 [Table 3]) had a second specimen positive for M. pneumoniae, by both real-time mono and multiplex NASBAs. For patients 19, 20, and 22, the PCR result was confirmed by a second specimen positive for M. pneumoniae by real-time NASBA, and for patient 24 the result was confirmed by culture (Table 3). The M. pneumoniae PCR-positive results for the specimens from the remaining four patients (patients

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TABLE 6. Sensitivities of real-time mono and multiplex NASBA assays when tested with a single target (M. pneumoniae, C. pneumoniae, or L. pneumophila) Species (type of unit) and test group Lysis buffer (negative control or no. of units in nucleic Mono Multiplex acid extraction)

No. of samples positive/no. testeda Throat swab

BAL

NPA

BA

Sputum

Total

Mono

Multiplex

Mono

Multiplex

Mono

Multiplex

Mono

Multiplex

Mono

Multiplex

Mono

Multiplex

M. pneumoniae (CCU) Negative control 5 50 500 5,000

0/4 4/4 4/4 4/4 ND

0/4 2/4 4/4 4/4 ND

0/4 2/4 3/4 4/4 ND

0/4 2/4 3/4 4/4 ND

0/4 2/4 3/4 4/4 ND

0/4 2/4 2/4 4/4 ND

0/4 2/4 3/4 4/4 ND

0/4 2/4 3/4 4/4 ND

0/4 ND 1/4 2/4 4/4

0/4 ND 1/4 2/4 4/4

0/4 ND 4/6 4/6 4/6

0/4 ND 3/6 4/6 5/6

0/24 10/16 18/26 22/26 8/10

0/24 8/16 16/26 22/26 9/10

C. pneumoniae (IFU) Negative control 0.1 1 10 100

0/4 4/4 4/4 4/4 4/4

0/4 3/4 4/4 4/4 4/4

0/4 4/4 4/4 4/4 4/4

0/4 4/4 4/4 4/4 4/4

0/4 4/4 4/4 4/4 4/4

0/4 3/4 4/4 4/4 4/4

0/4 4/4 4/4 4/4 4/4

0/4 3/4 4/4 4/4 4/4

0/4 0/4 0/4 0/4 0/4

0/4 0/4 0/4 0/4 0/4

0/4 0/4 0/4 4/4 4/4

0/4 0/4 0/4 4/4 4/4

0/24 16/24 16/24 20/24 20/24

0/24 13/24 16/24 20/24 20/24

L. pneumophila (CFU) Negative control 0.1 1 10 100 1,000 10,000

0/4 4/4 4/4 4/4 4/4 4/4 ND

0/4 2/4 4/4 4/4 4/4 4/4 ND

0/4 0/4 2/4 1/4 3/4 4/4 ND

0/4 0/4 1/4 3/4 4/4 4/4 ND

0/4 0/4 1/4 3/4 3/4 4/4 ND

0/4 0/4 0/4 1/4 4/4 4/4 ND

0/4 0/4 3/4 3/4 2/4 4/4 ND

0/4 0/4 0/4 1/4 2/4 4/4 ND

0/8 ND 0/8 3/8 4/8 6/8 8/8

0/8 ND 0/8 1/8 3/8 5/8 6/8

0/8 ND 1/8 2/8 4/8 4/8 7/8

0/8 ND 0/8 0/8 3/8 4/8 7/8

0/24 4/16 11/32 16/32 20/32 26/32 15/16

0/24 2/16 5/32 10/32 20/32 25/32 13/16

a

Boldface values represent differences in numbers of samples with correct results between real-time mono and multiplex NASBA. ND, not done.

27, 28, 30, and 31 [Table 3]) and from three patients with a negative U1A result (patients 29, 32, and 33 [Table 3]) could not be confirmed by serology or culture. Regarding C. pneumoniae-positive specimens, all three archived C. pneumoniae PCR-positive NPA were found positive in the NASBA assays. Of L. pneumophila-positive specimens, five/five lung biopsy specimens and five/five water samples were positive in the three assays, and the four sputum specimens were positive by PCR and real-time mono NASBA (Table 2). Statistical analysis. Statistical analysis using the ␹2 test was done for each organism on the total number of samples, spiked and real clinical samples, tested for each organism. The differences between real-time mono and multiplex NASBA were not significant for C. pneumoniae and M. pneumoniae (P ⫽ 0.11 and P ⫽ 0.24, respectively). A significant difference was observed for L. pneumophila-positive specimens (P ⫽ 0.04). DISCUSSION The aim of this study was to develop a real-time multiplex NASBA assay for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens based on 16S rRNA. The P1 primers and the beacons used were described previously (18, 19, 20). To reach a satisfactory sensitivity for all three pathogens, a generic P2 primer was designed to allow the simultaneous amplification of M. pneumoniae, C. pneumoniae, and as many Legionella spp. and serogroups as

possible. The specificity of the test was maintained in the multiplex format. The sensitivity of the real-time multiplex NASBA is between 5 and 50 CCU, 0.1 and 1 IFU, and 1 and 10 CFU of M. pneumoniae, C. pneumoniae, and L. pneumophila serotype 1 per 100 ␮l, respectively. The only significant difference between the real-time mono and multiplex NASBA reactions is for Legionella species. However, there is a tendency in all other instances for the multiplex format to be consistently (though not significantly) less sensitive than the monoplex reaction. All three individual primer pairs used in the real-time mono NASBAs were optimized for the absence of any cross-reactivity among the pathogens and were combined in a multiplex NASBA together with the differently colored molecular beacons. Unfortunately, a substantial decrease in sensitivity was observed for the detection of Mycoplasma pneumoniae and the Legionella species in this multiplex assay. This appeared to be due to primer interferences in the complex cocktail of P1 and P2 primers (results not shown). From the beginning this was known as a possible threat to NASBA multiplex assays, and therefore, the original P2 primers were chosen in the same segment of the 16S rRNA gene sequence and, as such, were combined into a single generic P2 primer for all three targets. Although several attempts were necessary, finally a generic P2 primer could be designed that abolished the observed primer interferences and revealed good sensitivity for all three targets when using in vitro-generated RNA. The spiking experiments with in vitro-generated RNA from multiple targets in one tube showed that double infections

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might not be detected by the real-time multiplex NASBA. Since the number of organisms present in clinical specimens of patients is not known, it is impossible to judge the clinical value of the NASBA reactions studied on the basis of the in vitro sensitivities presented. Until now, in the literature, M. pneumoniae and C. pneumoniae double infections have been seldom reported and if reported then they were detected by means of serology, which has well-known specificity and sensitivity problems for the detection of both organisms. Although it is very unlikely that double infections will occur, the possibility should be taken into account. Comparison between mono and multiplex assays has rarely been performed (6, 7, 9, 10, 27, 34). Greijer et al. reported a somewhat lower sensitivity of a real-time multiplex NASBA than of the mono NASBAs for the quantification of human cytomegalovirus IE1 mRNA (9). Corsaro et al. detected 2 CFU of M. pneumoniae in clinical samples by both duplex PCR and mono PCR (7). In the study by Welti et al. (34) there was no significant difference in sensitivity between the multiplex and monoplex PCR assays when tested on dilutions of DNA of C. pneumoniae, L. pneumophila, and M. pneumoniae cloned in plasmids. However, Tong et al. (27) found that the sensitivity of three PCRs applied in a triplex format decreased by about 1 log compared with the individual tests. In this study the lower sensitivity of the real-time multiplex NASBA for the detection of M. pneumoniae and Legionella species on spiked respiratory samples was confirmed when both real-time mono and multiplex NASBA were applied to dilutions of the wild-type in vitro-generated RNA and to archived L. pneumophila-positive specimens: none of the four L. pneumophila-positive sputum samples was positive by the multiplex assay. However, it is not clear if this is due to a low number of bacteria present in the samples or to a higher sensitivity of the real-time multiplex NASBA to inhibition. For C. pneumoniae, only three PCR-positive specimens could be tested. From the 51 specimens from 33 M pneumoniae PCR-positive patients, 40 were PCR positive upon reextraction-amplification. Within this group 15 specimens were negative in multiplex as well as in mono NASBA reactions, while four PCRnegative specimens were NASBA positive, two of them also in the multiplex format. The differences between the PCR and the NASBA results are significant (P ⫽ 0.01 and P ⫽ 0.05, respectively), but there is no difference between the two NASBA reactions (P ⫽ 0.2). For seven specimens the negative NASBA result is due to RNA degradation as shown by a negative U1A mRNA NASBA. In five cases (patients 22, 23, 24, 25, and 26 [Table 3]), the PCR result was confirmed either by a positive IgM result or by a positive NASBA result on a second specimen from the same patient or by culture. For the remaining patients (patients 27, 28, 29, 30, 31, 32, and 33 [Table 3]) the discordant amplification results for M. pneumoniae could not be resolved by serology or culture. Reextraction and reamplification confirmed all PCR- and NASBApositive results, classifying the results of the latter patients as truly M. pneumoniae positive. The archived nature of the specimens used in this study may be responsible for the negative results in some of the repeat PCRs and for some contradictions between PCR and real-time NASBA results. NASBA is inherently more sensitive to storage conditions than PCR, since

J. CLIN. MICROBIOL.

RNA is more easily degraded than DNA. A prospective study whereby specimens are put in lysis buffer immediately after production might avoid such contradictions. We conclude that the proposed real-time multiplex NASBA assay, although marginally less sensitive than the real-time mono NASBA assay (except for Legionella spp., where there is a significant difference between real-time mono and multiplex NASBA), is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run. A large number of clinical specimens from patients with community-acquired pneumonia should be analyzed for further evaluation of the assay. ACKNOWLEDGMENT This study was supported by European Commission (Framework V) grant no. QLK2-CT-2000-00294. REFERENCES 1. Abele-Horn, M., U. Busch, H. Nitschko, E. Jacobs, R. Bax, F. Pfaff, B. Schaffer, and J. Heesemann. 1998. Molecular approaches to diagnosis of pulmonary diseases due to Mycoplasma pneumoniae. J. Clin. Microbiol. 36: 548–551. 2. Bernander, S., H.-S. Hanson, B. Johansson, and L.-V. von Stedingk. 1997. A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens. Clin. Microbiol. Infect. 3:95–101. 3. Bernet, C., M. Garret, B. de Barbeyrac, C. Be´be´ar, and J. Bonnet. 1989. Detection of Mycoplasma pneumoniae by using the polymerase chain reaction. J. Clin. Microbiol. 27:2492–2496. 4. Boman, J., A. Allard, K. Persson, M. Lundborg, P. Juto, and G. Wadell. 1997. Rapid diagnosis of respiratory Chlamydia pneumoniae infection by nested touchdown polymerase chain reaction compared with culture and antigen detection by EIA. J. Infect. Dis. 175:1523–1526. 5. Boom, R., C. J. A. Sol, M. M. M. Salimans, C. L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495–503. 6. Cadieux, N., P. Lebek, and R. Brousseau. 1993. Use of a triplex polymerase chain reaction for the detection and differentiation of Mycoplasma pneumoniae and Mycoplasma genitalium in the presence of human DNA. J. Gen. Microbiol. 139:2431–2437. 7. Corsaro, D., M. Valassina, D. Venditti, V. Venard, A. Le Faou, and P. E. Valensin. 1999. Multiplex PCR for rapid and differential diagnosis of Mycoplasma pneumoniae and Chlamydia pneumoniae in respiratory infections. Diagn. Microbiol. Infect. Dis. 35:105–108. 8. de Baar, M. P., M. W. Van Dooren, E. De Rooij, M. Bakker, B. Van Gemen, J. Goudsmit, and A. De Ronde. 2001. Single rapid real-time monitored isothermal RNA amplification assay for quantification of human immunodeficiency virus type 1 isolates from groups M, N, and O. J. Clin. Microbiol. 39:1378–1384. 9. Greijer, A. E., H. M. A. Adriaanse, C. A. J. Dekkers, and J. M. Middeldorp. 2002. Multiplex real-time NASBA for monitoring expression dynamics of human cytomegalovirus encoded IE1 and pp67 RNA. J. Clin. Virol. 24:57– 66. 10. Gro ¨ndahl, B., W. Puppe, A. Hoppe, I. Ku ¨hne, J. A. I. Weigl, and H.-J. Schmitt. 1999. Rapid identification of nine microorganisms causing acute respiratory tract infections by single tube multiplex reverse transcription PCR: feasibility study. J. Clin. Microbiol. 37:1–7. 11. Helbig, J. H., T. Engelsta ¨dter, M. Maiwald, S. A. Uldum, W. Witzleb, and P. C. Lu ¨ck. 1999. Diagnostic relevance of the detection of Legionella DNA in urine samples by the polymerase chain reaction. Eur. J. Clin. Microbiol. Infect. Dis. 18:716–722. 12. Ieven, M., D. Ursi, H. Van Bever, W. Quint, H. G. M. Niesters, and H. Goossens. 1996. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J. Infect. Dis. 173:1445–1452. 13. Klerks, M. M., G. O. M. Leone, M. Verbeek, J. F. J. M. van den Heuvel, and C. D. Schoen. 2001. Development of a multiplex AmpliDet RNA for the simultaneous detection of potato leafroll virus and potato virus Y in potato tubers. J. Virol. Methods 93:115–125. 14. Lanciotti, R. S., and A. J. Kerst. 2001. Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses. J. Clin. Microbiol. 39:4506–4513. 15. Leone, G., H. van Schijndel, B. van Gemen, F. R. Kramer, and C. D. Schoen. 1998. Molecular beacon probes combined with amplification by NASBA enable homogeneous, real-time detection of RNA. Nucleic Acids Res. 26: 2150–2155.

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