Skip to main page content

• HOME
• Current Issue
• Archive
• Alerts
• About ASM
• Contact us
• Tech Support
• Journals.ASM.org

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ginevra, C. Articles by Molmeret, M. Search for Related Content PubMed PubMed Citation Articles by Ginevra, C. Articles by Molmeret, M.

 Previous Article  |  Next Article 

Journal of Clinical Microbiology, April 2009, p. 981-987, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02071-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease

Christophe Ginevra,^1,2,3,4 Marie Lopez,^3,4 Françoise Forey,^3,4 Monique Reyrolle,^3,4 Helene Meugnier,^3,4 François Vandenesch,^1,2,3,4 Jerome Etienne,^1,2,3,4 Sophie Jarraud,^1,2,3,4 and Maëlle Molmeret^1,2,3,4*

Université de Lyon, Lyon, France,¹ INSERM, U851, 21 Avenue Tony Garnier, Lyon, F-69007, France,² Université Lyon 1, IFR128, Lyon, France,³ Hospices Civils de Lyon, Faculté de Médecine Laënnec, Lyon, France⁴

Received 27 October 2008/ Returned for modification 22 December 2008/ Accepted 11 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sequence-based typing (SBT) is a powerful method based on the sequencing of seven genes of Legionella pneumophila isolates. SBT performed directly on clinical samples has been used only in a limited number of cases. In our study, its efficiency was tested with 63 legionellosis respiratory samples. Sixty-three clinical samples, which included 23 samples from sporadic cases and 40 collected during four French outbreaks, confirmed by culture or urinary antigen testing and all positive by L. pneumophila quantitative PCR were subtyped by SBT according to the European Working Group for Legionella Infections standard scheme. Only 28.6% of the samples provided nucleotide sequences by SBT. Nested-PCR-based SBT (NPSBT) applied to the same respiratory samples was thus evaluated with new PCR primers surrounding the first set of primers used for the SBT. Sequencing results were obtained with 90.5% of the samples. Complete allelic profiles (seven genes sequenced) were obtained for 3.2% versus 53.9% of the samples by SBT and NPSBT, respectively. More importantly, of the 28 culture-negative samples, only 4 did not give any sequencing results. Taken together, NPSBT applied directly to clinical specimens significantly improved epidemiological typing compared to the initial SBT, in particular when no isolates are available.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Legionellae are gram-negative bacteria that can cause sporadic cases and outbreaks of pneumonia when water droplets are inhaled from a variety of sources (8). Although 20 species of Legionella are documented as human pathogens (8), up to 90% of clinical cases are caused by Legionella pneumophila and about 84% are due to serogroup 1 strains (7, 38). The disease can be fatal unless diagnosed early and treated with specific antibiotics (12). Clinical signs are not specific, which makes prompt and accurate laboratory diagnosis crucial. Confirmed diagnostic methods include culture, direct fluorescent-antibody screening, serology, and urinary antigen testing (34). However, some of these tests are too slow for clinical use and others are inadequately sensitive or specific (5). Real-time PCR performed with clinical samples has been positively evaluated in different laboratories for the diagnosis of Legionnaires' disease, suggesting that its addition to confirmed diagnostic methods such as culture and urinary antigen testing could significantly help in the early detection of additional patients with suspected legionellosis (6, 16, 18, 21, 29, 30, 32, 35, 37).

Similarly, until recently, subtyping methods required an isolate. To subtype L. pneumophila strains, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism analysis are considered the most discriminatory methods (10, 11). However, in the past few years, epidemiological techniques have been developed to be used directly with clinical samples, independently of strain isolation, by PCR-based typing methods such as multiple-locus variable-number tandem-repeat (VNTR) and sequence-based typing (SBT) (13, 14, 25, 27, 33). Multiple-locus VNTR assays are based on the separation and sizing of amplified short to long tandem repeated sequences or microsatellites (up to 9 bp) and minisatellites (more than 9 bp) spread throughout the bacterial genome (28). This technique has an index of discrimination (28) similar to that of SBT. SBT is a powerful method based on the sequencing of seven gene loci (13, 31) that is already being recognized as the new EWGLI (European Working Group for Legionella Infections) "gold standard" tool for L. pneumophila typing and for which an important database of sequence types (STs) is available at http://www.ewgli.org. SBT produces robust data that can be easily exchanged between laboratories (4, 33). While culture is a prerequisite for PFGE- and amplified fragment length polymorphism analysis-based studies, a PCR-based typing method such as SBT can be performed directly with clinical samples (9, 24). SBT was the sole Legionella typing method to be applied directly to respiratory specimens and was only described in a limited number of legionellosis cases (9, 24). During three separate investigations of legionellosis outbreaks, Fry et al. obtained SBT results directly from seven respiratory samples, three of which were negative by culture (9). More recently, Lück et al. applied this culture-independent method to detect the source of infection in a case of hospital-acquired legionellosis (24).

In this study, 63 clinical samples from patients diagnosed with Legionnaires' disease by culture and/or urinary antigen testing and that were all positive for L. pneumophila by quantitative PCR (qPCR) were evaluated by sequence-based typing methods. We found that the efficiency of SBT, when performed directly with the clinical samples, was very poor. We therefore developed nested-PCR-based SBT (NPSBT) and evaluated its performance when applied directly to the same respiratory samples. We showed that NPSBT significantly improved epidemiological typing compared to the initial SBT when applied directly to clinical samples. NPSBT gives rapid and robust discriminatory epidemiological data, in particular when no isolates are available.

(Preliminary results of this study were presented at the EWGLI Meeting in Madrid, Spain, 11 to 13 May 2008).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Clinical specimens. We tested 63 samples of L. pneumophila serogroup 1 from patients with legionellosis confirmed either by culture (35 samples) or by urinary antigen testing (49 samples; Binax NOW Immunochromatographic Test; Oxoid, Dardilly, France). The specimens comprised 22 sputum samples, 28 tracheal aspirates, and 13 bronchoalveolar lavage (BAL) fluid samples and were obtained from 23 laboratories throughout France between February 2003 and December 2006. They were stored at –20°C.

Twenty-three samples corresponded to unrelated, sporadic cases. The other 40 samples were collected during four French outbreaks of Legionnaires' disease caused by L. pneumophila serogroup 1. Thirty-two samples were from the Lens outbreak of 86 cases that occurred from November 2003 to January 2004 and were linked to industrial cooling towers (26), three samples were from the Austerlitz outbreak of 26 cases that occurred between late July and early September 2006 and were also linked to a cooling tower, three samples were from the Sarrebourg outbreak of 12 cases in September 2006 (tentatively linked to a whirlpool spa display) (1), and two samples were from the Rueil-Malmaison outbreak of 12 cases in October and November 2006, also linked to a cooling tower.

DNA extraction. DNA was extracted with the automated MagNA Pure Compact System (Roche Diagnostics) and Nucleic Acid Isolation Kit I. Prior to automated extraction, samples were liquefied as follows: 22 µl of dithiothreitol was added to 200 µl of sample and incubated for 10 min at 37°C; 180 µl of bacteria lysis buffer and 20 µl of proteinase K (20 mg/ml) were then added to 200 µl of the liquefied sample and incubated for 10 min at 65°C. We used the manufacturer's DNA extraction protocol with an elution volume of 100 µl. Adequate extraction of all PCR-negative specimens was verified by amplifying part of the human β-globin gene with an in-house real-time PCR assay and using SYBR green I-based detection.

Primers and probes for real-time PCR. The oligonucleotide primers used are described elsewhere (19, 20, 36). The genus-specific PCR primers amplified a 386-bp portion of the 16S rRNA gene (base 451 to base 837) of L. pneumophila ATCC 33152. L. pneumophila-specific PCR amplified a 186-bp fragment of the mip gene. Amplification was detected with the LightCycler DNA Master Hybridization Probes kit as recommended by the manufacturer (Roche). Briefly, two different oligonucleotide probes specific to an internal sequence of the amplicon were used. One probe is labeled at the 5' end with the LightCycler Red 640 fluorophore, and the other probe is labeled at the 3' end with fluorescein. The specificity of the 16S rRNA and of the mip primers has been reported by Wellinghausen et al. (36) and Joly et al. (19).

The presence of PCR inhibitors was detected by including the internal inhibitor control (CI Duo) described by Joly et al. (19) in the PCR mixture and by using a dual-color hybridization probe assay.

PFGE subtyping. Culture-positive samples have been subtyped by a PFGE method routinely used in the laboratory as described previously (23, 26).

Genomic DNA was prepared as previously described, with some modifications. Briefly, L. pneumophila was treated with proteinase K (50 µg/ml) in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) for 24 h at 55°C and digested with 20 IU of SfiI restriction enzyme (Roche Diagnostic, Meylan, France) for 16 h at 50°C. Fragments of DNA were separated in a 0.8% agarose gel prepared and run in 0.5x Tris-borate-EDTA buffer (pH 8.3) in a contour-clamped homogeneous electric field apparatus (CHEF DRII system; Bio-Rad, Ivry sur Seine, France) with a constant voltage of 150 V. Runs were carried out with a constant pulse time (25 s) at 10°C for 11 h and increasing pulse times (35 to 60 s) at 10°C for 11 h. PFGE patterns were analyzed with GelComparII software (Applied Maths, Kortrijk, Belgium).

SBT and NPSBT. Samples that were positive by real-time PCR were processed for SBT with the EWGLI standard scheme (13). Briefly, 5 µl of extracted DNA was amplified with the EWGLI primers under the following conditions: 35 cycles of denaturation for 30 s at 94°C; annealing for 30 s at 50°C (for flaA, mompS, and proA), 55°C (for pilE), 60°C (for mip), or 62°C (for asd); and elongation for 30 s at 72°C. This technique was also applied to the isolates when available. Seven gene targets were used, namely, the usual six genes (asd, flaA, mip, mompS, pilE, and proA) (13) and the new gene target, neuA, recently published by Ratzow et al. (31). Oligonucleotide primers targeting regions of each gene were designed, yielding 245- to 648-bp products (primers for the second PCR described in Table 1). For the NPSBT, primers outside the first set of primers have been designed (primers for the first PCR described in Table 1) in a way that the annealing temperature equals 55°C. Then, 1 µl of the PCR product was used for the second PCR set according to the EWGLI standard scheme. Eight microliters of PCR product was electrophoresed through agarose gel containing ethidium bromide in TBE buffer at 135 V for 35 min. DNA fragment sizes were determined by using DNA molecular weight markers. Because of the high sensitivity of nested PCR, precautions must be taken to exclude the possibility of contamination of reaction tubes with previously amplified products. Aliquoting of samples, preparation of reagents, processing of samples, and nested PCR were performed in safety cabinets located in separated laboratories, all away from the area where amplified products were analyzed. Each cabinet was equipped with an independent batch of reagents, micropipette sets, sterile reagent tubes, and filtered pipette tips.

View this table: [in this window] [in a new window]

TABLE 1. Primers for amplification of L. pneumophila DNA by nested PCRa

Sequencing. The PCR products from either SBT or NPSBT were then sent to GATC Biotech (GATC Biotech, Constance, Germany) for purification and sequencing. The resulting consensus sequences of each gene locus were trimmed and compared to the online SBT database (version 3.0; http://www.ewgli.org), allowing assignment of the seven ordered alleles, flaA, pilE, asd, mip, mompS, proA, and neuA, as described by Gaia et al. and Ratzow et al. (13, 31), represented as an ST, e.g., ST1, or the allelic profile of the ordered string of allele numbers separated by commas, e.g., 1,4,3,1,1,1,1.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
SBT performance. In this study, we evaluated the subtyping of L. pneumophila by SBT-based methods with 63 respiratory samples from patients with Legionnaires' disease who were diagnosed by culture or/and urinary antigen testing and were all qPCR positive for Legionella and L. pneumophila. Among these 63 clinical samples, 28 samples from patients that were urinary antigen positive were negative by culture.

When the SBT method was directly applied to the 63 PCR-positive specimens, nucleotide sequence data were obtained for 18 (28.6%) of them (Table 2). None of them were culture negative. We obtained complete sequencing data (all seven alleles sequenced) for only 2 samples (3.2%); we also obtained almost 21% interpretable data (at least five alleles sequenced) from the 63 clinical samples tested (Table 2).

View this table: [in this window] [in a new window]

TABLE 2. SBT and NPSBT results obtained with 63 respiratory samples from patients with Legionnaires' disease

The flaA gene was the least able to provide nucleotide sequences, as only three sequences were obtained. This was followed by the mip and neuA genes, with nine sequenced alleles. The best results were obtained with the pilE gene and the asd, mompS, and proA genes which provided 15 and 17 sequenced alleles, respectively. Thus, the discriminative power of the flaA gene is the lowest of the seven alleles tested.

The isolates of culture-positive samples were subjected to SBT to ensure that the allelic profiles obtained from isolates were the same as those obtained directly from the clinical samples. In all of the cases in which sequencing data were obtained from clinical samples (14 samples), their allelic profile matched the one obtained from the corresponding isolate. All of the isolates had also been subjected to PFGE subtyping (data not shown). By SBT, the samples were clustered either according to the PFGE subtyping analysis when the isolates were recovered or to their historical and clinical background (Table 2). For instance, four samples from patients infected during the Lens outbreak (26) that provided partial or complete sequencing results, were correctly clustered within this outbreak group. However, out of the 22 sporadic cases, 15 samples associated with Legionella isolates (culture-positive samples), which had different PFGE patterns, showed identical SBT types, suggesting that SBT had a lower discriminatory power than PFGE (2). Taken together, these results obtained by SBT showed poor efficiency in providing nucleotide sequences, in particular when no isolates are available.

NPSBT performance. In order to increase the performance of the SBT, in particular when performed directly with clinical samples, we designed primers outside the ones initially chosen for the amplification of the seven loci used. The outside primers were used in the first round of PCR, and the initial primers were used in the second round of PCR. After the two rounds of amplification, the same sequencing primers were used as for the initial SBT. For the allele number assignment, sequences were trimmed similarly to the ones obtained with the initial SBT. The same 63 respiratory samples were used to assess the performance of NPSBT compared to that of SBT.

By NPBST, 57 out of the 63 samples provided sequencing data (90.5%) (Table 2). Thirty-four (53.9%) complete sequencing data (all seven alleles sequenced) and 50 (79.3%) interpretable data (at least five alleles sequenced) were obtained. Overall, the capacity to obtain complete sequencing data increased from 3.2% with SBT to 53.9% with NPSBT, representing an improvement of 16.4-fold.

Once again, the flaA gene was the least able to be amplified and sequenced with the new primers of the NPSBT; however, the overall performance was much better than with SBT, as 37 flaA sequences were provided, compared to only 3 with the initial SBT. The number of sequences obtained with the other genes and NPSBT primers ranged between 45 for the asd gene and 52 for the mip gene.

A good proportion of complete and partial sequencing data was obtained whether NPSBT was performed with sputa (72.7%), BAL fluids (76.9%), or tracheal aspirates (75%), suggesting that the sequencing results obtained with this technique were not related to the sample types.

More importantly, among the 28 samples that were negative by culture, only 4 did not provide any sequencing results (Table 2). Ten complete and 18 interpretable sequencing data with at least five alleles sequenced out of 28 samples were obtained.

As found with the SBT, some STs among the sporadic cases are identical despite the absence of epidemiological links between the patients and different PFGE patterns provided when isolates were available (Table 2 and data not shown). NPSBT allowed correct clustering of patients in the case of outbreak samples. Overall, NPSBT performed directly with clinical samples gives rapid and robust discriminatory epidemiological data. NPSBT significantly improves epidemiological typing compared to the initial SBT, in particular when no isolates are available.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This work provides a comprehensive study of the performance of SBT and its improved version based on a nested PCR called NPSBT performed directly with respiratory samples. The direct application of SBT to respiratory specimens has been previously described in a limited number of legionellosis cases (9, 24); in one study, only seven respiratory samples provided SBT results, three of which were negative by culture (9), and in another study, this method was used to detect the source of infection of only one hospital-acquired legionellosis case (24). In our study, 63 respiratory samples that were all positive for Legionella and L. pneumophila by qPCR were tested by SBT and provided only 3.2% of the complete sequencing data where the seven alleles were sequenced; none of them was culture negative. Improvement in the number of samples with seven loci sequenced by NPSBT, which represented 53.9% of the samples, of which 10 were culture negative, reached almost 17-fold compared to SBT.

The development of a nested-PCR-based technique has improved the sensitivity of SBT with seven newly designed sets of primers that are external to the seven existing ones that are already used for culture-based Legionella SBT. Such a system has already been developed for multilocus sequence typing of Neisseria meningitidis performed directly with clinical samples (3). The SBT database of EWGLI allows the assignment of the seven ordered alleles flaA, pilE, asd, mip, mompS, proA, and neuA as described by Gaia et al. and Ratzow et al. represented as an ST or an allelic profile of the ordered string of allele numbers (13, 31). Gaia et al. reported that maximal discrimination was obtained with the mompS gene, followed by asd, flaA, neuA, mip, pilE, and proA. In this study, the mompS allele of all 18 isolates providing sequences with SBT was indeed amplified 17 times, similar to the ads and proA alleles. With NPSBT, the mip allele was the most amplified (52 times out of 57 amplified alleles). The addition of neuA as a seventh allele did not add discriminatory power compared to the other genes in our study when using the SBT or NPSBT method (31). The flaA gene was the allele least able to be amplified and sequenced by both methods, even though the nested PCR significantly improves its yield. Redesigning primers for this locus might help in obtaining better results for its amplification and sequencing. Alternatively, changing target genes due to too high variability in nucleic acids for the flaA gene could be another way to increase SBT or NPSBT performance. This method can also be improved by using additional genes other than those already chosen, as has been previously tried (15). The detection limit of each gene amplification has not been determined, as it is most likely strain specific; indeed, several strain-dependent polymorphisms exist in the sequences targeted by the primers, resulting in mismatches during amplification and making the detection limit vary from one strain to another.

In the case of samples from sporadic cases, some of the STs determined by SBT and NPSBT were identical despite no evidence of epidemiological links between them (at least for the samples that were cultured and subtyped by PFGE), suggesting that SBT and NPSBT had a lower discriminatory power than PFGE with the selected genes (2). It is interesting that monoclonal antibody subtyping, which is performed with isolates as it has poor efficiency with clinical samples, enhances the discrimination of the SBT scheme (2). NPSBT nevertheless remains an excellent tool for L. pneumophila subtyping, in particular when no isolates are available. The initial SBT results and the NPSBT results clearly linked the patients to the outbreak when complete and partial STs were obtained. In the case of SBT, 4 STs allowed correct clustering to the Lens outbreak, whereas for the NPSBT, 32 STs were accordingly clustered within their own group of outbreak cases, which included 17 culture-negative samples. It is interesting that two outbreaks cases (from the Sarrebourg and Rueil-Malmaison outbreaks) showed the same ST (ST47), which corresponds to the endemic strain recently described, the Lorraine strain (17). This endemic strain has a high and increasing prevalence in clinical samples, although it is rarely detected in environmental samples, and is spread throughout France (17). In this case, isolates causing several epidemiologically unrelated cases of legionellosis have identical PFGE patterns and identical NPSBT profiles. In this study, one culture-positive sample belonging to each outbreak (Sb3 and RM1 samples in Table 2) was available and provided the same PFGE pattern as the Lorraine strain. This is confirmed by the profiles of the RM1 and Sb2 samples, which had six sequenced alleles corresponding to ST47 (with the allelic profile 5,10,22,15,6,2,6) and could not possibly be mixed up with another ST at the time we checked the online database. The only profile of those two outbreak cases that could be different from ST47 is the Sb1 profile, which could be mixed up with 5,10,3,15,6,2,6, where the differing number is in boldface (ST453). The PFGE pattern and historical background of Sb3, which could also have two other allelic profiles (5,10,22,15,6,10,6 [ST106] and 5,1,22,15,6,10,6 [ST109]), suggest that it most probably matches the Lorraine strain.

Possible explanations for the PCR-negative results include incorrect sampling and DNA degradation during storage at –20°C for several months. The impact of the interval between disease onset and respiratory sample collection is not clear. Some samples were collected several days after urinary antigen testing and therefore after the beginning of antibiotic treatment. Five samples with negative PCR results were obtained only 3 to 6 days after clinical disease onset. In contrast, four PCR-positive (and culture-negative) samples were collected 14 to 17 days after clinical disease onset, confirming that Legionella DNA can persist for long periods in some respiratory samples (21, 22). The somewhat modest success of SBT applied directly to respiratory specimens may also be due to nonspecific hybridization of contaminating DNA from the oropharyngeal flora. Alternatively, the PCR conditions (annealing temperature, cycle length, MgCl₂ concentration, etc.) may have been suboptimal for some of the seven target alleles. However, SBT has been performed, when possible, both with the isolate and directly with the respiratory sample, and the SBT profiles matched each other for the same legionellosis cases. This could also suggest that no variability in the sequences was created by this method. This hypothesis has been confirmed by the repeated sequencing of the seven loci of the Paris strain, which has been performed 10 times and provided the exact same sequence for each allele (data not shown).

Taken together, the results of this study show that SBT and, more importantly, its improved version, NPSBT, represent excellent tools for L. pneumophila subtyping, in particular when no isolates are available. Direct subtyping of clinical samples that have or have not been diagnosed by qPCR should be undertaken. This technique is being evaluated on a very large scale in our laboratory. The SBT-based subtyping technique should also be performed in a larger number of cases and eventually adapted for legionellosis cases caused by non-L. pneumophila strains.

 
We are grateful to the staff of the Legionella National Reference Center, particularly Nathalie Jacotin and Sami Slimani, for their technical assistance.

We have no commercial or other association that might pose a conflict of interest and received no funding for this research.

 
* Corresponding author. Mailing address: Université de Lyon, INSERM U851, Faculté de Médecine Laennec, 7 rue Guillaume Paradin, 69372 Lyon cedex 08, France. Phone: 33 (0) 478 77 86 57. Fax: 33 (0) 478 77 86 58. E-mail: maelle.molmeret{at}univ-lyon1.fr

Published ahead of print on 18 February 2009.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Alsibai, S., P. Bilo de Bernardi, C. Janin, D. Che, and J. V. Lee. 2006. Outbreak of legionellosis suspected to be related to a whirlpool spa display, September 2006, Lorquin, France. Eurosurveillance 11:E061012.3.
2
2. Amemura-Maekawa, J., F. Kura, B. Chang, and H. Watanabe. 2005. Legionella pneumophila serogroup 1 isolates from cooling towers in Japan form a distinct genetic cluster. Microbiol. Immunol. 49:1027-1033.[Medline]
3
3. Birtles, A., K. Hardy, S. J. Gray, S. Handford, E. B. Kaczmarski, V. Edwards-Jones, and A. J. Fox. 2005. Multilocus sequence typing of Neisseria meningitidis directly from clinical samples and application of the method to the investigation of meningococcal disease case clusters. J. Clin. Microbiol. 43:6007-6014.[Abstract/Free Full Text]
4
4. Cano, R., S. Jarraud, J. Pardos, C. Campese, and C. Pelaz. 2006. Legionnaires' disease and travel in Europe. Emerg. Infect. Dis. 12:1606-1607.[Medline]
5
5. Den Boer, J. W., and E. P. Yzerman. 2004. Diagnosis of Legionella infection in Legionnaires' disease. Eur. J. Clin. Microbiol. Infect. Dis. 23:871-878.[Medline]
6
6. Diederen, B. M., J. A. Kluytmans, C. M. Vandenbroucke-Grauls, and M. F. Peeters. 2008. Utility of real-time PCR for diagnosis of Legionnaires' disease in routine clinical practice. J. Clin. Microbiol. 46:671-677.[Abstract/Free Full Text]
7
7. Doleans, A., H. Aurell, M. Reyrolle, G. Lina, J. Freney, F. Vandenesch, J. Etienne, and S. Jarraud. 2004. Clinical and environmental distributions of Legionella strains in France are different. J. Clin. Microbiol. 42:458-460.[Abstract/Free Full Text]
8
8. Fields, B. S., R. F. Benson, and R. E. Besser. 2002. Legionella and Legionnaires' disease: 25 years of investigation. Clin. Microbiol. Rev. 15:506-526.[Abstract/Free Full Text]
9
9. Fry, N. K., B. Afshar, G. Wewalka, and T. G. Harrison. 2006. Epidemiological typing of Legionella pneumophila in the absence of isolates, p. 152-155. In N. P. Cianciotto, Y. A. Kwaik, and P. H. Edelstein (ed.), Legionella: state of the art 30 years after its recognition. ASM Press, Washington, DC.
10
10. Fry, N. K., S. Alexiou-Daniel, J. M. Bangsborg, S. Bernander, M. Castellani Pastoris, J. Etienne, B. Forsblom, V. Gaia, J. H. Helbig, D. Lindsay, P. Christian Luck, C. Pelaz, S. A. Uldum, and T. G. Harrison. 1999. A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study. Clin. Microbiol. Infect. 5:462-477.[Medline]
11
11. Fry, N. K., J. M. Bangsborg, A. Bergmans, S. Bernander, J. Etienne, L. Franzin, V. Gaia, P. Hasenberger, B. Baladron Jimenez, D. Jonas, D. Lindsay, S. Mentula, A. Papoutsi, M. Struelens, S. A. Uldum, P. Visca, W. Wannet, and T. G. Harrison. 2002. Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing using a standard protocol. Eur. J. Clin. Microbiol. Infect. Dis. 21:722-728.[CrossRef][Medline]
12
12. Gacouin, A., Y. Le Tulzo, S. Lavoue, C. Camus, J. Hoff, R. Bassen, C. Arvieux, C. Heurtin, and R. Thomas. 2002. Severe pneumonia due to Legionella pneumophila: prognostic factors, impact of delayed appropriate antimicrobial therapy. Intensive Care Med. 28:686-691.[CrossRef][Medline]
13
13. Gaia, V., N. K. Fry, B. Afshar, P. C. Luck, H. Meugnier, J. Etienne, R. Peduzzi, and T. G. Harrison. 2005. Consensus sequence-based scheme for epidemiological typing of clinical and environmental isolates of Legionella pneumophila. J. Clin. Microbiol. 43:2047-2052.[Abstract/Free Full Text]
14
14. Gaia, V., N. K. Fry, T. G. Harrison, and R. Peduzzi. 2003. Sequence-based typing of Legionella pneumophila serogroup 1 offers the potential for true portability in legionellosis outbreak investigation. J. Clin. Microbiol. 41:2932-2939.[Abstract/Free Full Text]
15
15. Gilmour, M. W., K. Bernard, D. M. Tracz, A. B. Olson, C. R. Corbett, T. Burdz, B. Ng, D. Wiebe, G. Broukhanski, P. Boleszczuk, P. Tang, F. Jamieson, G. Van Domselaar, F. A. Plummer, and J. D. Berry. 2007. Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada. J. Med. Microbiol. 56:336-341.[Abstract/Free Full Text]
16
16. Ginevra, C., C. Barranger, A. Ros, O. Mory, J. L. Stephan, F. Freymuth, M. Joannes, B. Pozzetto, and F. Grattard. 2005. Development and evaluation of Chlamylege, a new commercial test allowing simultaneous detection and identification of Legionella, Chlamydophila pneumoniae, and Mycoplasma pneumoniae in clinical respiratory specimens by multiplex PCR. J. Clin. Microbiol. 43:3247-3254.[Abstract/Free Full Text]
17
17. Ginevra, C., F. Forey, C. Campese, M. Reyrolle, D. Che, J. Etienne, and S. Jarraud. 2008. Lorraine strain of Legionella pneumophila serogroup 1, France. Emerg. Infect. Dis. 14:673-675.[CrossRef][Medline]
18
18. Hayden, R. T., J. R. Uhl, X. Qian, M. K. Hopkins, M. C. Aubry, A. H. Limper, R. V. Lloyd, and F. R. Cockerill. 2001. Direct detection of Legionella species from bronchoalveolar lavage and open lung biopsy specimens: comparison of LightCycler PCR, in situ hybridization, direct fluorescence antigen detection, and culture. J. Clin. Microbiol. 39:2618-2626.[Abstract/Free Full Text]
19
19. Joly, P., P. A. Falconnet, J. Andre, N. Weill, M. Reyrolle, F. Vandenesch, M. Maurin, J. Etienne, and S. Jarraud. 2006. Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl. Environ. Microbiol. 72:2801-2808.[Abstract/Free Full Text]
20
20. Jonas, D., A. Rosenbaum, S. Weyrich, and S. Bhakdi. 1995. Enzyme-linked immunoassay for detection of PCR-amplified DNA of legionellae in bronchoalveolar fluid. J. Clin. Microbiol. 33:1247-1252.[Abstract/Free Full Text]
21
21. Koide, M., F. Higa, M. Tateyama, I. Nakasone, N. Yamane, and J. Fujita. 2006. Detection of Legionella species in clinical samples: comparison of polymerase chain reaction and urinary antigen detection kits. Infection 34:264-268.[CrossRef][Medline]
22
22. Koide, M., F. Higa, M. Tateyama, H. Sakugawa, and A. Saito. 2004. Comparison of polymerase chain reaction and two urinary antigen detection kits for detecting Legionella in clinical samples. Eur. J. Clin. Microbiol. Infect. Dis. 23:221-223.[CrossRef][Medline]
23
23. Lawrence, C., M. Reyrolle, S. Dubrou, F. Forey, B. Decludt, C. Goulvestre, P. Matsiota-Bernard, J. Etienne, and C. Nauciel. 1999. Single clonal origin of a high proportion of Legionella pneumophila serogroup 1 isolates from patients and the environment in the area of Paris, France, over a 10-year period. J. Clin. Microbiol. 37:2652-2655.[Abstract/Free Full Text]
24
24. Lück, P. C., C. Ecker, U. Reischl, H. J. Linde, and R. Stempka. 2007. Culture-independent identification of the source of an infection by direct amplification and sequencing of Legionella pneumophila DNA from a clinical specimen. J. Clin. Microbiol. 45:3143-3144.[Free Full Text]
25
25. Nederbragt, A. J., A. Balasingham, R. Sirevag, H. Utkilen, K. S. Jakobsen, and M. J. Anderson-Glenna. 2008. Multiple-locus variable-number tandem repeat analysis of Legionella pneumophila using multi-colored capillary electrophoresis. J. Microbiol. Methods 73:111-117.[CrossRef][Medline]
26
26. Nguyen, T. M., D. Ilef, S. Jarraud, L. Rouil, C. Campese, D. Che, S. Haeghebaert, F. Ganiayre, F. Marcel, J. Etienne, and J. C. Desenclos. 2006. A community-wide outbreak of Legionnaires' disease linked to industrial cooling towers—how far can contaminated aerosols spread? J. Infect. Dis. 193:102-111.[CrossRef][Medline]
27
27. Pourcel, C., Y. Vidgop, F. Ramisse, G. Vergnaud, and C. Tram. 2003. Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping. J. Clin. Microbiol. 41:1819-1826.[Abstract/Free Full Text]
28
28. Pourcel, C., P. Visca, B. Afshar, S. D'Arezzo, G. Vergnaud, and N. K. Fry. 2007. Identification of variable-number tandem-repeat (VNTR) sequences in Legionella pneumophila and development of an optimized multiple-locus VNTR analysis typing scheme. J. Clin. Microbiol. 45:1190-1199.[Abstract/Free Full Text]
29
29. Raggam, R. B., E. Leitner, G. Muhlbauer, J. Berg, M. Stocher, A. J. Grisold, E. Marth, and H. H. Kessler. 2002. Qualitative detection of Legionella species in bronchoalveolar lavages and induced sputa by automated DNA extraction and real-time polymerase chain reaction. Med. Microbiol. Immunol. 191:119-125.[CrossRef][Medline]
30
30. Rantakokko-Jalava, K., and J. Jalava. 2001. Development of conventional and real-time PCR assays for detection of Legionella DNA in respiratory specimens. J. Clin. Microbiol. 39:2904-2910.[Abstract/Free Full Text]
31
31. Ratzow, S., V. Gaia, J. H. Helbig, N. K. Fry, and P. C. Luck. 2007. Addition of neuA, the gene encoding N-acylneuraminate cytidylyl transferase, increases the discriminatory ability of the consensus sequence-based scheme for typing Legionella pneumophila serogroup 1 strains. J. Clin. Microbiol. 45:1965-1968.[Abstract/Free Full Text]
32
32. Reischl, U., H. J. Linde, N. Lehn, O. Landt, K. Barratt, and N. Wellinghausen. 2002. Direct detection and differentiation of Legionella spp. and Legionella pneumophila in clinical specimens by dual-color real-time PCR and melting curve analysis. J. Clin. Microbiol. 40:3814-3817.[Abstract/Free Full Text]
33
33. Scaturro, M., M. Losardo, G. De Ponte, and M. L. Ricci. 2005. Comparison of three molecular methods used for subtyping of Legionella pneumophila strains isolated during an epidemic of legionellosis in Rome. J. Clin. Microbiol. 43:5348-5350.[Abstract/Free Full Text]
34
34. Stout, J. E., and V. L. Yu. 1997. Legionellosis. N. Engl. J. Med. 337:682-687.[Free Full Text]
35
35. Templeton, K. E., S. A. Scheltinga, P. Sillekens, J. W. Crielaard, A. P. van Dam, H. Goossens, and E. C. Claas. 2003. 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. 41:4016-4021.[Abstract/Free Full Text]
36
36. Wellinghausen, N., C. Frost, and R. Marre. 2001. Detection of legionellae in hospital water samples by quantitative real-time LightCycler PCR. Appl. Environ. Microbiol. 67:3985-3993.[Abstract/Free Full Text]
37
37. Wilson, D. A., B. Yen-Lieberman, U. Reischl, S. M. Gordon, and G. W. Procop. 2003. Detection of Legionella pneumophila by real-time PCR for the mip gene. J. Clin. Microbiol. 41:3327-3330.[Abstract/Free Full Text]
38
38. Yu, V. L., J. F. Plouffe, M. C. Pastoris, J. E. Stout, M. Schousboe, A. Widmer, J. Summersgill, T. File, C. M. Heath, D. L. Paterson, and A. Chereshsky. 2002. Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J. Infect. Dis. 186:127-128.[CrossRef][Medline]

---

Journal of Clinical Microbiology, April 2009, p. 981-987, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02071-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Coscolla, M., Gonzalez-Candelas, F. (2009). Direct Sequencing of Legionella pneumophila from Respiratory Samples for Sequence-Based Typing Analysis. J. Clin. Microbiol. 47: 2901-2905 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ginevra, C. Articles by Molmeret, M. Search for Related Content PubMed PubMed Citation Articles by Ginevra, C. Articles by Molmeret, M.