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Journal of Clinical Microbiology, September 2007, p. 3143-3144, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00685-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Culture-Independent Identification of the Source of an Infection by Direct Amplification and Sequencing of Legionella pneumophila DNA from a Clinical Specimen{triangledown}


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LETTER
 
Legionella spp. are a common cause of community-acquired and nosocomial pneumonia (1). So far, cultured legionellae are required for epidemiological typing of isolated strains to detect the source of the infection (1, 2, 3). However, due to the fastidious nature of these bacteria, culture techniques show a sensitivity of only 30 to 60% and require 3 to 7 days to grow visible colonies. Recently, a sequence-based typing (SBT) scheme of Legionella pneumophila that uses sequences from seven genes was described, and this is now considered the gold standard for epidemiological typing (2, 5). The discriminatory power of this typing approach was determined to be very high (2, 5). We have applied this culture-independent method to detect the source of infection in a case of nosocomial pneumonia.

A 49-year-old female patient suffering from a low-malignant B-cell lymphoma was admitted for chemotherapy. She was discharged after 14 days of hospitalization but was readmitted 11 days later. Her body temperature was elevated to 39.5°C, and a chest X ray revealed bilateral pulmonary infiltrates.

L. pneumophila was isolated from bronchial secretion and serotyped as serogroup 1 by use of latex reagents from Oxoid (Wesel, Germany). Unfortunately, the strain was not preserved and so further subtyping could not be performed. In parallel, DNA was extracted from this sample and found to be positive by PCR (6). As the question of whether the infection was acquired during the hospitalization period arose, this DNA preparation was used to amplify and sequence fragments of 360 to 500 bp from seven Legionella genes: flaA, pilE, asd, mip, mompS, proA, and neuA (2, 5). The sequences were analyzed, and the SBT type was determined. An SBT type comprises a string of the individual allele numbers separated by commas (2). Currently, the determination of the SBT type can be performed by using the online tool on the website of the European Working Group on Legionella Infections (www.ewgli.org). DNA sequences of the seven genes obtained by direct amplification from clinical samples revealed the SBT profile 2,10,3,10,9,4,11.

Six days after the onset of infection, an epidemiological investigation that included culturing environmental samples from the water supplies in the hospital and the patient's home was started (4). In the latter samples, no legionellae were detected. The Legionella count in the hospital water was less than 100 CFU (range from 1 to 76) per 100 ml (L. pneumophila serogroup 1) in 14 of 15 water samples. Only one sample contained 1,300 CFU per 100 ml. The detection limit of our culture method is 1 CFU per 100 ml (4). In general, this concentration is considered to be relatively safe. Although we do not know the exact concentration of legionellae at the presumed time of infection, our observation confirms that low levels of Legionella might present a risk for immunologically incompetent patients. Altogether, seven isolates belonging to serogroup 1 obtained from different water outlets in the hospital, including the patient's room, were forwarded to the National Reference Laboratory for Legionella. Six isolates belonged to monoclonal subgroup Bellingham and one to monoclonal subgroup OLDA (3). Although monoclonal subgrouping was performed on only seven isolates, it is safe to assume that the disease-causing strain represents the majority in the hospital water supply. Neither strain reacted with our monoclonal antibody (MAb) 3-1. Such strains are considered to be less virulent than MAb 3-1-positive strains (3). However, as shown in the present study, such strains can cause disease in immunocompromised persons (3). One strain of each MAb type was analyzed by SBT. The resulting SBT profiles were 2,10,3,10,9,4,11 for the Bellingham subtype strain and 7,6,17,10,9,4,11 for the OLDA strain. The typing results confirm the hospital water supply as the probable source of the infection. This is remarkable since the incubation time, i.e., the time between discharge and readmittance, was more than 10 days. The profile of the patient strain was not found among 187 L. pneumophila serogroup 1 strains isolated in Germany but in one strain isolated in Hokkaido, Japan, in 1986. The OLDA strain SBT profile was not found in the database. This observation underlines the excellent discriminatory power of the SBT method. In previous studies, it was shown that monoclonal subgrouping further increased the index of discrimination (2, 5). However, this applies mostly to common SBTs like 1,4,3,1,1,1 (2, 5). We could not perform monoclonal subgrouping on the clinical isolate. Because the disease-causing strain is rarely found in unrelated strains, it is safe to assume that in our case the monoclonal subgrouping would not give additional information.

The case presented here demonstrates that sequence data alone were sufficient to detect not only the infectious agent causing the pneumonia but also the source of infection. The most striking advantage of this DNA-based approach is that it can be applied in cases where no isolates are available either from the patient or from the environmental source, e.g., after hyperchlorination of the water supply. Furthermore, time might be saved both to diagnose legionellosis and to rapidly identify the source of the infection. The whole procedure could be performed within 24 h. Nucleotide sequence data could easily and reliably be compared with data obtained in other studies and are interchangeable with other laboratories (2). In this way, the rapid identification of the source of the infection may help to prevent further infections.


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ACKNOWLEDGMENTS
 
We are grateful to Jutta Paasche and Kerstin Seeliger for technical assistance.

This study was supported by the Federal Ministry of Education and Research of Germany, Network of Competence in Medicine CAPNETZ.


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FOOTNOTES
 
{triangledown} Published ahead of print on 25 July 2007. Back


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REFERENCES
 
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  1. 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. C. Lück, C. Pelaz, S. A. Uldum, and T. G. Harrison. 1999. A multicenter evaluation of genotyping methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results from a pan-European study. Clin. Microbiol. Infect. 5:462-477.[Medline]
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  2. Gaia, V., N. K. Fry, B. Afshar, P. C. Lück, 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]
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  3. Helbig, J. H., S. Bernander, P. Castellani, J. Etienne, V. Gaia, S. Lauwers, D. Lindsay, P. C. Lück, T. Marques, S. Mentula, M. F. Peeters, C. Pelaz, M. Struelens, S. A. Uldum, G. Wewalka, and T. G. Harrison. 2002. Pan-European study on culture-proven Legionnaires’ disease: distribution of Legionella pneumophila serogroups and monoclonal subgroups. Eur. J. Clin. Microbiol. Infect. Dis. 21:710-716.[CrossRef][Medline]
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  4. International Organization for Standardization. 2004. Water quality—detection and enumeration of Legionella. ISO 11731-2. International Organization for Standardization, Geneva, Switzerland.
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  5. Ratzow, S., V. Gaia, J. H. Helbig, N. K. Fry, and P. C. Lück. 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]
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  6. 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]
Paul Christian Lück*
Institute of Medical Microbiology and Hygiene
National Reference Laboratory for Legionella
Fiedlerstrasse 42
D-01307 Dresden, Germany

Christa Ecker
Landesamt für Soziales, Gesundheit und Verbraucherschutz
Saarbrücken, Germany

Udo Reischl
Hans-Jörg Linde
Institute of Medical Microbiology and Hygiene
University of Regensburg
Regensburg, Germany

Roland Stempka
Institute of Medical Microbiology and Hygiene
Saarland University Hospital
Homburg/Saar, Germany

* E-mail: Christian.Lueck{at}tu-dresden.de

Journal of Clinical Microbiology, September 2007, p. 3143-3144, Vol. 45, No. 9
0095-1137/07/$08.00+0     doi:10.1128/JCM.00685-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Thurmer, A., Helbig, J. H., Jacobs, E., Luck, P. C. (2009). PCR-based 'serotyping' of Legionella pneumophila. J Med Microbiol 58: 588-595 [Abstract] [Full Text]  
  • Ginevra, C., Lopez, M., Forey, F., Reyrolle, M., Meugnier, H., Vandenesch, F., Etienne, J., Jarraud, S., Molmeret, M. (2009). Evaluation of a Nested-PCR-Derived Sequence-Based Typing Method Applied Directly to Respiratory Samples from Patients with Legionnaires' Disease. J. Clin. Microbiol. 47: 981-987 [Abstract] [Full Text]  

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