KAROLINSKA INSTITUTET Microbiology & Tumor Biology Center • MTC Clinical Microbiology - KS

- A. Diagnosis of Legionnaires´ Disease (LD)
Construction of a nested PCR for detection of Legionella pneumophila in respiratory tract specimens, based on primers from mip gene sequences (1).

Improvement of the PCR technique using newer methods and evaluation in a clinical setting together with PCR methods for detection of other bacterial agents like Mycoplasma pneumoniae and Chlamydia pneumoniae.

- B. Genotyping of L. pneumophila for epidemiological use.
Participation in an ongoing international collaboration for harmonisation of legionella genotyping methods initiated by the European Working Group on Legionella Infections (EWGLI). The group has found Amplified Fragment Length Polymorphism (AFLP) to be the method of choice (2,3) and work is now directed towards designating AFLP types.

In cooperation with Monica Lundholm, Ph.D. at the Department of Clinical Microbiology, University Hospital (Akademiska sjukhuset) in Uppsala, Pulsed Field Electrophoresis subsequent to macrorestriction (PFGE) and AFLP have been used to study legionella outbreaks at the hospital. Both genotyping methods were found to be discriminatory and reproducible (4). This work also included monoclonal (MAb) serotyping using the Dresden panel in cooperation with Juergen Helbig, Ph.D., at the Universitätsklinikum in Dresden.

Recently the mip sequence of strains from the Uppsala outbreaks have been studied regarding possible variations. Differences between the L. pneumophila serogroup 1 outbreak strain and other strains were found (5th International Conference on Legionella, Ulm, Germany, 2000).

- C. Other fields of interest.
Aerobic Actinomycetes - participation in an international study coordinated by Prof. Patrick Boiron, Lyon.

Identification of bacterial isolates from clinical specimens by sequencing methods.

Development of PCR methods for detection of non-legionella agents causing atypical pneumonia.

Selected references:

1. Bernander S, Hanson H-S, Johansson B, von Stedingk L-V. A nested polymerase chain reaction for detection of Legionella pneumophila in clinical specimens. Clin. Microbiol. Infect. (CMI) 1997; 3:95-101

2. Fry NK, Alexiou-Daniel S, Bangsborg JM, Bernander S, et al. A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study. Clin. Microbiol. Infect. (CMI) 1999; 5: 462-477.

3. Fry NK, Bangsborg JM, Bernander S, Etienne J, et al. Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis. Eur. J. Clin. Microbiol. Infect. Dis. 2000; 19: 773-780.

4. Bernander S, Jacobson K, Zettersten U, Helbig JH, Lundholm M. PFGE , AFLP and MAb subtyping of Legionella pneumophila sg 1. Clin. Microbiol. Infect. (CMI) 2000; 6 suppl. 1, 223.