Development of Real-Time Multiplex NASBA® for the Detection of ...

JCM Accepts, published online ahead of print on 21 November 2007 J. Clin. Microbiol. doi:10.1128/JCM.00447-07 Copyright © 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Development of Real-Time Multiplex NASBA® for the Detection of Mycoplasma pneumoniae,

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Chlamydophila pneumoniae and Legionella spp. in Respiratory Specimens 5

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K. Loens1, T. Beck1, D. Ursi1, M. Overdijk2, P. Sillekens2, H. Goossens1 and M. Ieven1 Submitted to: The Journal of Clinical Microbiology

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Correspondent footnote: Department of Medical Microbiology, University of Antwerp, Universiteitsplein 1 S009a, B-2610 Wilrijk, Belgium. Phone: 32-3-820-24-18. Fax: 32-310

820-27-52. E-mail: [email protected]

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Abstract 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 (bioMérieux, 15

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Boxtel, The Netherlands).

Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae and L. pneumophila 16S rRNA. For real-time detection, molecular beacons were used.

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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, 20

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

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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 25

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diminished compared to the results obtained with the 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, and C. pneumoniae and Legionella spp. in respiratory specimens, regarding handling, speed and number of samples that can be analyzed in a single run.

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1.1 Introduction

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 CAP when using conventional diagnostic methods such as culture and serology (21, 22, 31). M. pneumoniae, C. pneumoniae and L. pneumophila may be responsible for 35

15-50% of CAP (23, 25). Differentiation of infections due to S. 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.

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Culture and serological confirmation of the diagnosis of infections due to M. pneumoniae, C. pneumoniae and L. pneumophila is difficult and may require several 40

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).

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Nucleic acid sequence-based amplification (NASBA, bioMérieux, Boxtel, The Netherlands) targeted at RNA has been adapted to the real-time format using DNA 45

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

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assays suitable for high-throughput applications, reducing the assay time and limiting potential contamination between samples. Real-time single-target (mono) NASBA has been successfully used for the identification of West Nile and St. Louis encephalitis 50

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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

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volume samples and reduce costs. Multiplex real-time NASBA has already been 55

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 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 60

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.

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1.2 Materials and Methods Bacterial strains 65

Bacterial strains, used to test the specificity of the NASBA® primers, are presented in

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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 70

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nutritional needs of the species.

M. pneumoniae strain ATCC 29085 was quantified by incubation of 4 replicates of 10fold dilutions of a suspension in SP4 medium at 37°C. The cultures were examined

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weekly for color change from red to yellow during 2 months. The titer was expressed in color changing units (CCU) per ml (3). 75

C. pneumoniae (ATCC VR-1355) was grown in Hep-2 cells. After inoculation, the cell

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cultures were centrifuged at 3500 rpm and 25°C for 60 min and subsequently incubated at 37°C for 1h. Then the medium was aspirated and cell cultures were incubated with fresh medium containing cycloheximide (1mg/l). After 3 days, the shell vials were aspirated and fixed with 96% ethanol. The fixed monolayers were rinsed with phosphate buffered 80

saline and stained with fluorescent antibody technique with C. pneumoniae specific mouse monoclonal antibodies (Dako A/S, Glostrup, Denmark). Rabbit anti-mouse immunoglobulin labeled with fluoresceine isothiocyanate (Dako A/S) was used as a conjugate. C. pneumoniae (ATCC VR-1355) was quantified by incubation of 5 replicates of 10-fold

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dilutions on Hep-2 cells. Inclusion forming units (IFU) were counted 72h after infection by immunofluorescence microscopy. The titer was expressed in IFU/ml. Legionella strains were grown on buffered charcoal-yeast (Oxoid Ltd., Belgium) agar plates at 37°C for 48-72h and quantified by incubation of 4 replicates of 10-fold dilutions on buffered charcoal-yeast agar. The titer was expressed as CFU/ml.

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The other organisms were cultivated on standard media supporting optimal growth. The clinical isolates were identified by standard methods. 4

Respiratory specimens Throat swabs, broncho-alveolar lavages (BAL), nasopharyngeal aspirates (NPA), sputum and bronchus aspirates (BA) from the Microbiology Laboratory of the University 95

Hospital of Antwerp and tested negative for M. pneumoniae, C. pneumoniae and L. pneumophila by PCR (12, 29) were spiked with dilutions of reference strains for

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sensitivity experiments.

Archived specimens positive by PCR for one of the three organisms: 3 C. pneumoniae positive nasopharyngeal aspirates, 5 L. pneumophila positive lung biopsy specimens 100

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(Table 2b), 4 sputum specimens and 5 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 2).

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One hundred forty nine specimens (44 sputum specimens, 36 throat swabs, 25 NPA, 22 BA, 19 BAL, 1 gargle specimen, 1 lung biopsy specimen and 1 pleural fluid) previously 105

found negative for M. pneumoniae, C. pneumoniae and L. pneumophila by PCR were also tested as negative control specimens.

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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 110

(bioMérieux, Boxtel, The Netherlands).

Briefly, 100 µl of protease treated clinical

specimens, (16) or aliquots of a bacterial culture, were added to a guanidinium thiocyanate (GuSCN) lysis solution, pH 6.2 and mixed vigorously for rapid lysis. Fifty µl of activated silica were added. The nucleic acid-silica complex was washed twice with GuSCN washing solution, twice with 70% (v/v) ethanol and once with acetone. After 115

drying at 56°C, nucleic acids were eluted from the silica using 50µl elution buffer and stored at -80°C. Each nucleic acid extract was amplified by both real-time mono NASBA and by the real-time multiplex NASBA.

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Real-time NASBA 120

Real-time mono NASBA reactions were performed using the NucliSens Basic Kit amplification module (bioMérieux) as described previously (18, 19, 20). In negative control reactions, target nucleic acid was replaced by RNase-/DNase-free water. Amplification reactions were incubated in a fluorescent reader, the NucliSens EasyQ

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Analyzer (bioMérieux) and results calculated with the Ascent software (bioMérieux). 125

U1A, a low abundance mRNA derived from a human cellular house keeping gene, encoding the ‘A’ protein present in the human U1 (U1A) small nuclear ribonucleoprotein

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(snRNP) particle, (23) NASBA -to verify the presence of nucleic acid after nucleic acid

extraction of specimens- was performed using the NucliSens Basic Kit® amplification

module (bioMérieux) on all nucleic acid extracts according to the instructions of the 130

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manufacturer in a separate reaction tube.

Real-time multiplex NASBA: Besides the species specific P1 primers described previously (18,

19,

20),

a generic P2

primer,

5'-GATGCAAGGTCGCATA

TGAGAATTTGA TCCTGGCTCAG-3' was designed. The three P1 primers and this

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generic P2 primer were used in the real-time multiplex NASBA reaction. 135

The following molecular beacons for real-time detection were used: 5’FAMCCATGGGTTGAAAGACTAGCTAATACCATGG-Dabcyl3’ for the detection of M. pneumoniae, 5’ROX-CCGATCGTGTAGTG TAATTAGGCATCTAATATCGATCGGDabcyl3’

for

the

detection

of

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pneumoniae,

and

5’

Cy5-

CCGAGCTGAGTAACGCGTAGGAATATGCTCGG-Dabcyl 3’ for the detection of 140

Legionella spp. The final concentration of each primer in the amplification reaction was 0.2µM. To determine the cut off 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

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Legionella positive when the signal was above the mean of the negative samples plus 20%.

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PCR: M. pneumoniae (12) and C. pneumoniae PCR (29) targeting the P1 cytadhesin gene and the PstI fragment, respectively, were done as described previously after nucleic acid 150

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

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acid extracts were used in a 20 µl real-time PCR reaction using the LightCycler (Roche Diagnostics

GmbH,

Mannheim,

Germany).

Ten

pmol

of

primers

MIP1

(5'CAACCGATGCCACATCATTA3') and MIP2 (5' TAGCCATTGCTTCCGGATTA3'), 155

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4 pmol of probes MIPFL (5'GCCTTGATTTTTAAAATTCTTCCCAA FLU3') and MIPLC (5' LCRed640TCGGCACCAATGCTATAAGACAACT3') were used in

combination with the LightCycler Faststart DNA hybridization probes kit (Roche

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Diagnostics) according to the instructions of the manufacturer. DNA was amplified by 1 incubation step at 94°C for 60 sec, 45 cycles at 95°C for 10 seconds, 60°C for 10 sec and 160

72°C for 20 sec each and 1 final incubation step at 40°C for 30 sec. Sample preparation, set up of the reactions and product-analysis were done in separate rooms. As a control,

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negative samples were processed simultaneously.

Sensitivity study

For the generation of wild type (WT) RNA, cDNA from part of the 16S rRNA from each 165

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 DH5α 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

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Biotech) as described previously (16, 19, 20) The RNA was quantified by spectroscopy and from the OD 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

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combinations and by using 10-fold dilutions of M. pneumoniae, C. pneumoniae and Legionella spp. as single targets in lysis buffer (Tables 4a, 4b 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.

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The 95% hitrate for in vitro generated RNA was calculated by SAS version 6.12 software (SAS, Cary, USA).

Reproducibility

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The intrarun and interrun variations in multiplex real-time NASBA were estimated by running samples containing 500 or 5000 CCU/100 µl of M. pneumoniae; 0.1, 1, 10 or 100 185

IFU/100 µl of C. pneumoniae and of 100 or 1000 CFU/100 µl of L. pneumophila

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serotype 1 added as single targets in duplicate to 1 BAL pool to determine the intrarun variation and to 2 BAL pools to determine the interrun variation. Five replicates of each nucleic acid extract were analysed, respectively. The calculations on the final fluorescent

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value were done by using the Microsoft Excel software. Negative controls were co190

analyzed within each run.

Serology and Legionella urinary antigen test Serology and urinary antigen tests (uAg) were performed on physician's request. If available, paired sera were tested. For the detection of M. pneumoniae antibodies, an IgM and an IgG enzyme immunoassay (Anilabsystems, Helsinki, Finland) were performed. 195

An acute infection was defined when at least a 1.5-fold IgG EIU increase with paired sera, assayed in the same run was obtained. With EIU 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/cut-off units were above 1.1. C. pneumoniae specific IgM and IgG antibodies were detected by the Anilabsystems

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EIA. The same definition of an acute infection as described above was used. A patient was considered positive for Legionella when >70U/ml and/or >140 U/ml were measured for IgG and IgM, respectively, by using the Serion ELISA classic

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(Virion/Serion, Würzburg, Germany) IgM and IgG test. The BINAX NOW urinary antigen test was used according to the instructions of the manufacturer. 205

Statistical analysis: The Х2-test was used for statistical analysis.

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Results

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Specificity of the 16S rRNA multiplex NASBA primers

Using real-time multiplex NASBA, positive results were obtained with nucleic acid 210

extracts from M. pneumoniae, C. pneumoniae, and all Legionella spp and serotypes but

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with none of the other organisms listed in Table 1.

Sensitivity of the 16S rRNA multiplex NASBA

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Single target:

The 95% hit-rate for the analytical sensitivity of the 16S rRNA NASBA primers tested on 215

dilutions of in vitro generated WT 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 reactions. When extraction was done prior to the amplification, i.e. the isolation of in vitro generated RNA from lysis buffer, 3185 molecules, 18489 molecules and 16697 molecules of C.

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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 Tables 4a and 4b. When lysis buffer was spiked with 5 CCU/100 µl of M. pneumoniae, 4/4 and 2/4 samples

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were found positive by real-time mono and multiplex NASBA, respectively. With an input of 0.1 IFU/100µl, of C. pneumoniae 4/4 and 3/4 samples were positive by real-time mono and multiplex NASBA respectively. Real-time mono and multiplex NASBA

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detected 0.1 CFU of L. pneumophila serotype 1 in 100µl lysis buffer in 4/4 and 2/4 aliquots, respectively. When real-time mono and multiplex NASBA were applied to the 230

same nucleic acid extract from L. pneumophila serotypes 2, 3, 4, 5, 6, and 10 as well as to L. longbeachae 4a, and L. micdadei spp the difference in sensitivity between the real-time mono and multiplex NASBA was 1 log with the former being the more sensitive assay.

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For L. longbeachae 4b and L. bozemanii the difference in sensitivity was 2 logs. The sensitivity on spiked respiratory specimens with M. pneumoniae, C. pneumoniae and L. 235

pneumophila as a single target are shown in Tables 3A, 3B and 3C, respectively.

Multiple targets:

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With inputs of 330 or 3300 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

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33 molecules of in vitro generated RNA of each organism, a positive result was always 240

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 3300 molecules of both M. pneumoniae and C. pneumoniae in vitro

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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 245

multiplex NASBA.

Negative specimens:

Out of 149 M. pneumoniae, C. pneumoniae and L. pneumophila PCR negative respiratory specimens, 3, 2 and 2 specimens were positive by real-time mono NASBA for M. pneumoniae, C. pneumoniae, and Legionella, respectively. One out of these three M. 250

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 5000 CCU of M. pneumoniae were 13.5 and 13.5 whereas the interrun variation coefficients for the same 255

inputs were 16.7 and 16.8.

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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, 13.0 by real-time multiplex NASBA. The interrun variation coefficients for the same inputs were 20.8, 11.0, 13.7, and 15.4. For L. pneumophila serotype 1, the intrarun variability coefficients were 13.4 and 13.1 for 260

an input of 100 and 1000 CFU respectively. The interrun variation coefficients for the

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same inputs were 10.6 and 11.6.

Archived specimens from PCR positive patients:

From the 51 archived respiratory specimens collected from 33 patients who had at least

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one specimen positive for M pneumoniae by PCR, 40 were PCR positive. Twenty-one 265

specimens were M. pneumoniae positive by the three detection procedures: PCR, realtime mono and multiplex NASBA. Four specimens were M. pneumoniae positive by PCR

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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 270

(Table 2). Fifteen M. pneumoniae PCR positive specimens were negative by both real-

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time mono and multiplex NASBA. However seven of these specimens were U1A negative and thus contained no RNA (nos. 19, 20, 23, 24, 29, 32, and 33. Table 2). For 3 patients (nrs. 23, 25, and 26. Table 2), the PCR-result was confirmed by a positive IgM result, one of whom (no. 23. Table 2) had a second specimen positive for M. pneumoniae, 275

by both real-time mono and multiplex NASBA. For patients no. 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 by culture, (Table 2). The M. pneumoniae PCR positive results on the specimens from the remaining 4 patients (nos. 27, 28, 30, and 31. Table 2) and from 3 patients with a negative U1A result (29, 32, and 33. Table 2) could not be

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confirmed by serology or culture. C. pneumoniae positive specimens: all 3 archived C. pneumoniae PCR positive nasopharyngeal aspirates were found positive in the NASBA assays. L. pneumophila positive specimens: 5/5 lung biopsy specimens and 5/5 water samples were positive in the three assays, the 4 sputum specimens were positive by PCR and real-

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time mono NASBA (Table 2b).

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Statistical analysis: Statistical analysis using the X2-test was done for each organisms 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 290

and M. pneumoniae, p=0.11 and p= 0.24 respectively. A significant difference was

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observed for L. pneumophila positive specimens, p=0.04.

1.3 DISCUSSION

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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 295

based on 16S rRNA. The P1 primers and the beacons used were described previously (18,

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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 as many serogroups as possible. The specificity of the test was maintained in the multiplex format. 300

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The sensitivity of the real-time multiplex NASBA is between 5-50 CCU, 0.1-1 IFU and

1-10 CFU/100µl of M. pneumoniae, C. pneumoniae, and L. pneumophila serotype 1 per 100µl, 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, (be it not significantly), less 305

sensitive than the monoplex reaction. All 3 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

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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 for NASBA multiplex assays and, therefore, the original P2 primers were chosen in the same segment of the 16S ribosomal RNA gene sequence and, as such, were combined into a single generic P2 primer for all three targets. Although several attempts

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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 1 tube showed that double infections might not be detected by the real-time multiplex NASBA.

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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 seldomly reported and if reported then they

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were detected by means of serology which has well known specificity and sensitivity 325

problems for the detection of both organisms. Although it is very unlikely that double infections will occur the possibility should be taken into account.

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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 compared with the mono NASBAs, for the quantification of HCMV IE1 mRNA 330

(9). Corsaro et al. detected 2 CFU of M. pneumoniae in clinical samples by both duplex

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PCR as well as mono PCR (7). In the study of 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 335

triplex format decreased by about 1 log as 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 in wild type in vitro generated RNA and to archived L. pneumophila positive specimens:

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none of the 4 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

345

positive upon re-extraction-amplification. Within this group 15 specimens were negative

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in multiplex as well as in mono NASBA reactions, while 4 PCR negative specimens were NASBA positive 2 of which 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 7 specimens the negative 350

NASBA result is due to RNA degradation as shown by a negative U1A mRNA NASBA.

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In five cases (nos. 22, 23, 24, 25 and 26. Table 2), 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 (nrs. 27, 28, 29, 30, 31, 32 and 33.

Table 2) the discordant amplification results for M. pneumoniae could not be resolved by 355

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serology or culture. Re-extraction and re-amplification confirmed all PCR and NASBA

positive results, classifying the results of the latter patients as true M. pneumoniae positive. The archived nature of the specimens used in this study may be responsible for

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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 360

than PCR, since RNA is more easily degraded than DNA. A prospective study whereby specimens are put in lysis buffer immediately after production might avoid such

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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 365

there is a significant difference between real-time mono and multiplex NASBA, is a promising tool for the detection of M. pneumoniae, and 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.

1.4 Acknowledgements

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This study was supported by European Commission (Framework V) grant no QLK2-CT2000-00294.

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17

Table 1. Bacterial species and strains used to test the specificity of the assay

480

NO..

Strain / Source

Species

Origin

1 2 3 4 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

ATCC VR-1355 Clinical isolate Kiq10 ATCC 29085 (PI1428) ATCC 15492 (MAC) NC 10117 NC10111 ATCC 33530 (G-37) NC10112 NC10136 NC10113 NC11702 CDC BC 1636 CDC BC 1637 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 Mycoplasma orale Mycoplasma buccale Mycoplasma salivarium Mycoplasma pirum Legionella pneumophila 1 Legionella pneumophila 2 Legionella pneumophila 3 Legionella pneumophila 4 Legionella pneumophila 5 Legionella pneumophila 6 Legionella pneumophila 8 Legionella pneumophila 10 Legionella. longbeachae 4a Legionella. longbeachae 4b Legionella bozemanni 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 UZA ITGa ATCCb ATCC NTCCc NCTC ATCC NCTC NCTC NCTC NCTC UZAd UZA SEHe UZA UZA UZA UZA SEH UZA UZA SEH SEH UZA UZA UZA UZA UZA UZA UZA UZA UZA UZA UZA

T P

E C

C A

a

D E

ITG: Instituut voor Tropische Geneeskunde, Antwerp, Belgium,

Type Culture Collection, Manassas, Va, USA,

c

b

ATCC: American

NCTC: National Collection of Type

Cultures, Central Public Health Laboratory, London, UK, d UZA: Universitair Ziekenhuis Antwerpen, Edegem, Belgium, e SEH: Sint Elisabeth Hospital, Tilburg, The Netherlands.

18

Table 2a. Overview of test results for all M. pneumoniae positive patients Patient Nr

Specimen

1 2 3 4 5 6

BAL BA NPA NPA Sputum NPA NPA BAL BAL TS 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 Sput TS Sputum Sputum TS TS Gargle TS Sputum Sputum TS Sputum TS Sputum TS

7 8 9 10 11 12 13 14 15a 16 17 18 19

PCR

Real-time mono NASBA

Real-time MX NASBA

U1A resultb

IgM

IgGc

culture

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + -

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

NA NA NA NA NA NA NA NA NA +

NA NA NA NA NA NA NA NA NA +

ND ND ND ND ND ND ND ND ND + ND ND ND ND + + ND ND + + ND ND ND ND ND ND -

E C

C A

20

21a 22a 23a 24 25 26 27 28a 29 30a 31 32 33a

485

a

D E

T P NA NA NA NA + +

NA NA NA NA +

ND + +

NA

NA

+

+

NA

NA

-

ND

-

ND

+

ND

-

-

+

-

+

-

-

-

-

ND

-

-

-

ND

NA

NA

NA -

NA ND

: only an acute serum sample available, b: U1A positive above 2.00, c: Seroconversion or significant rise in

titre BA: bronchus aspirate; BAL: bronchoalveolar lavage; NA: not available, ND: not done, NPA: nasopharyngeal aspirate; TS: throat swab

19

Table 2b: Overview of test results for all Legionella positive specimens Nr

1 2 3 4 5 6 7 8 9 10 11 12 13 14

490

a

Specimen

Lung Lung Lung Lung Lung Sputum Sputum Sputum Sputum Water Water Water Water Water

PCR

Cult.

+ + + + + + + + + + + + + +

+ + + + + + + + -

Real-time mono NASBA + + + + + + + + + + + + + +

Real-time MX NASBA + + + + + + + + + +

uAG

IgM

IgGb

+ + ND ND ND + + + + NA NA NA NA NA

ND ND ND ND ND + + + NA NA NA NA NA

ND ND ND ND ND NDa + NDa NA NA NA NA NA

T P

D E

: only an acute serum sample available, b: Seroconversion or significant rise in titre, NA: not applicable,

ND: not done

E C

C A

20

Table 3A. Sensitivity of real-time mono and multiplex NASBA assays when tested with a 495

single target: M. pneumoniae

LY mono LY multiplex TS mono TS multiplex BAL mono BAL multiplex NA mono NA multiplex BA mono BA multiplex SP mono SP multiplex Total mono Total multiplex

Negative control

5a

50 a

500 a

5000 a

0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/24 0/24

4/4 2/4 2/4 2/4 2/4 2/4 2/4 2/4 ND ND ND ND 10/16 8/16

4/4 4/4 3/4 3/4 3/4 2/4 3/4 3/4 1/4 1/4 4/6 3/6 18/26 16/26

4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 2/4 2/4 4/6 4/6 22/26 22/26

ND ND ND ND ND ND ND ND 4/4 4/4 4/6 5/6 8/10 9/10

T P

D E

BAL: bronchoalveolar lavage, BA: bronchus aspirate, CCU: colour changing units in the

E C

extraction, LY: lysis buffer, NA: nasopharyngeal aspirate, ND: not done, SP: sputum, TS: throat swab a

500

number of CCU in nucleic acid extraction

C A

Underlined numbers represent differences in number of samples with correct results between real-time mono and multiplex NASBA.

21

Table 3B. Sensitivity of real-time mono and multiplex NASBA assays when tested with a single target: C. pneumoniae

LY mono LY multiplex TS mono TS multiplex BAL mono BAL multiplex NA mono NA multiplex BA mono BA multiplex SP mono SP multiplex Total mono Total multiplex

Negative control

0.1 a

1a

10 a

100 a

0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/24 0/24

4/4 3/4 4/4 4/4 4/4 3/4 4/4 3/4 0/4 0/4 0/4 0/4 16/24 13/24

4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 0/4 0/4 0/4 0/4 16/24 16/24

4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 0/4 0/4 4/4 4/4 20/24 20/24

4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 0/4 0/4 4/4 4/4 20/24 20/24

T P

D E

BAL: bronchoalveolar lavage, BA: bronchus aspirate, IFU: inclusion forming units in the 505

extraction, LY: lysis buffer, NA: nasopharyngeal aspirate, ND: not done, SP: sputum, TS:

E C

throat swab a

number of IFU in nucleic acid extraction

Underlined numbers represent differences in number of samples with correct results

C A

between real-time mono and multiplex NASBA.

22

510

Table 3C. Sensitivity of real-time mono and multiplex NASBA assays when tested with a single target: L. pneumophila

LY mono LY multiplex TS mono TS multiplex BAL mono BAL multiplex NA mono NA multiplex BA mono BA multiplex SP mono SP multiplex Total mono Total multiplex

Negative control

0.1 a

1a

10 a

100 a

1000 a

10000 a

0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/8 0/8 0/8 0/8 0/24 0/24

4/4 2/4 0/4 0/4 0/4 0/4 0/4 0/4 ND ND ND ND 4/16 2/16

4/4 4/4 2/4 1/4 1/4 0/4 3/4 0/4 0/8 0/8 1/8 0/8 11/32 5/32

4/4 4/4 1/4 3/4 3/4 1/4 3/4 1/4 3/8 1/8 2/8 0/8 16/32 10/32

4/4 4/4 3/4 4/4 3/4 4/4 2/4 2/4 4/8 3/8 4/8 3/8 20/32 20/32

4/4 4/4 4/4 4/4 4/4 4/4 4/4 4/4 6/8 5/8 4/8 4/8 26/32 25/32

ND ND ND ND ND ND ND ND 8/8 6/8 7/8 7/8 15/16 13/16

T P

D E

BAL: bronchoalveolar lavage, BA: bronchus aspirate, CFU: colony forming units in the extraction, LY: lysis buffer, NA: nasopharyngeal aspirate, ND: not done, SP: sputum, TS:

E C

throat swab 515

a

number of CFU in nucleic acid extraction

Underlined numbers represent differences in number of samples with correct results

C A

between real-time mono and multiplex NASBA.

23

Table 4a: Sensitivity of the real-time multiplex NASBA in comparison with the mono real-time NASBA assays in the amplification reaction. Number of in vitro generated RNA molecules in the amplification (n=15-20) 10

50

100

1000

L. pneumophila mono

31%

70%

91%

100%

L. pneumophila MX

23%

57%

82%

100%

M. pneumoniae mono

33%

82%

89%

100%

M. pneumoniae MX

7%

50%

79%

100%

C. pneumoniae mono

80%

100%

100%

100%

C. pneumoniae MX

65%

100%

100%

100%

520

10000

T P

D E 100%

100%

100% 100% 100%

100%

E C

MX: multiplex

Table 4b: Sensitivity of the real-time multiplex NASBA in comparison with the mono

C A

real-time NASBA assays in the extraction. 525

Number of in vitro generated RNA molecules in the extraction (n=10-12) 500

1000

5000

10000

50000

L. pneumophila mono

80%

50%

100%

100%

100%

L. pneumophila MX

60%

30%

70%

91%

100%

M. pneumoniae mono

20%

40%

80%

91%

100%

M. pneumoniae MX

20%

37%

60%

72%

100%

C. pneumoniae mono

100%

100%

100%

100%

100%

C. pneumoniae MX

30%

45%

100%

100%

100%

MX: multiplex

24

Table 5. Sensitivity of real-time multiplex NASBA assays when tested with multiple target in vitro generated RNA molecules. 530 Nr of in vitro generated RNA molecules

Detection result positive by real-time multiplex NASBA for:

in the extraction M. pneumoniae

C. pneumoniae

L. pneumophila

33 Lpn + 33 Mpn + 33 Cpn

1/2

2/2

1/2

330 Lpn + 330 Mpn + 330 Cpn

2/2

2/2

3300 Lpn + 3300 Mpn + 3300 Cpn

2/2

2/2

330 Lpn + 3300 Mpn + 3300 Cpn

2/2

2/2

3300 Lpn + 330 Mpn + 3300 Cpn

2/2

3300 Lpn + 3300 Mpn + 330 Cpn

2/2

E C

Mpn: Mycoplasma pneumoniae in vitro generated RNA Cpn: Chlamydia pneumoniae in vitro generated RNA

C A

25

D E

T P

Lpn: Legionella pneumophila in vitro generated RNA

2/2

2/2

2/2

2/2 0/2

2/2 2/2