Comparison of Legionella pneumophila isolates by arbitrarily primed PCR and pulsed-field gel electrophoresis: analysis from seven epidemic investigations

Pruckler, Janet M.; Mermel, Leonard A.; Benson, Robert F.; Giorgio, Christina; Cassiday, Pamela K.; Breiman, Robert F.; Whitney, Cynthia G.; Fields, Barry S.

doi:10.1128/jcm.33.11.2872-2875.1995

Cited by 72 publications

(44 citation statements)

References 23 publications

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“…Of the many molecular typing methods applied to L. pneumophila, PFGE is probably the most widely used and is generally considered to be highly discriminatory [16,18,19,43]. Again, the results of this study confirm this view.…”

Section: Discussionsupporting

confidence: 79%

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study

Fry

Alexiou-Daniel

Bangsborg

et al. 1999

Clinical Microbiology and Infection

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OBJECTIVES: To compare genotypic methods for epidemiologic typing of Legionella pneumophila serogroup (sg) 1, in order to determine the best available method within Europe for implementation and standardization by members of the European Working Group on Legionella Infections. METHODS: Coded isolates (114) of L. pneumophila sg 1 comprising one epidemiologically 'unrelated' (79) and one 'related' panel of isolates (35) were sent to 12 laboratories in 11 European countries. Analysis was undertaken in each laboratory using one or more of the following methods: ribotyping, restriction fragment length polymorphism analysis, restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), PCR using arbitrary/repeat sequence primers (AP-, AP/rep-PCR), and amplified fragment length polymorphism (AFLP) analysis. Results were analyzed visually or using gel analysis software. Each method was assessed for its: index of discrimination (D), epidemiologic concordance (E), speed of application and ease of use. In addition, phenotypic analysis was performed in two laboratories using monoclonal antibodies (mAbs). RESULTS: The D of each of the genotypic methods ranged from 0.840 for ribotyping to 0.990 for PFGE using Sfil: E ranged from 0.06 for AP- and AP/rep-PCR to 1.00 for ribotyping using Pstl/EcoRI and AFLP: in general, E was inversely related to D. Although offering only limited discrimination (D=0.838), mAb typing was both rapid and highly epidemiologically concordant (E=1.00). CONCLUSIONS: Two methods, PFGE using Sfil and AFLP, were selected for further study. AFLP is rapid and highly epidemiologically concordant (E=1.00), but is not highly discriminatory. This method will be developed as a rapid screening tool. PFGE using Sfil is highly discriminatory but, in the present study, yielded low values of E (0.12-0.71). Attempts will be made to rigorously standardize this method for use as the reference method. Primary screening of isolates by mAb subgrouping is recommended.

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Section: Discussionsupporting

confidence: 79%

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study

Fry

Alexiou-Daniel

Bangsborg

et al. 1999

Clinical Microbiology and Infection

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“…Therefore, multiple, simple PCR-based approaches for the typing of Legionella organisms have been proposed, such as AP-PCR (2,9,10,22) or rep-PCR (7,31).…”

Section: Discussionmentioning

confidence: 99%

“…The AP-PCR was essentially performed as described previously (9,22). The M13 primer (5Ј TTA TGT AAA ACG ACG GCC AGT 3Ј) used for typing was fluorescently labelled with Cy-5 during manufacture (Pharmacia Biotech, Freiburg, Germany).…”

Section: Methodsmentioning

confidence: 99%

Comparative Evaluation of Three Different Genotyping Methods for Investigation of Nosocomial Outbreaks of Legionnaires' Disease in Hospitals

Jonas

Meyer

Matthes

et al. 2000

J. Clin. Microbiol.

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The increased incidence of nosocomial Legionnaires' disease in two hospitals prompted investigation of possible environmental sources. In the search for an effective DNA-typing technique for use in hospital epidemiology, the performance and convenience of three methods-SfiI macrorestriction analysis (MRA), amplified fragment length polymorphism (AFLP), and arbitrarily primed PCR (AP-PCR)-were compared. Twenty-nine outbreak-associated and eight nonassociated strains of Legionella pneumophila with 13 MRA types and subtypes were investigated. These strains comprised isolates from bronchoalveolar lavages, from environmental, patient-related sources, and type strains. All three typing methods detected one predominant genotype associated with the outbreaks in both hospitals. All of them correctly assigned epidemiologically associated, environmental isolates to their respective patient specimens. AP-PCR was the least discriminating and least reproducible technique. In contrast, AFLP was demonstrated as being the method with the best interassay reproducibility (90%) and concordance (94%) in comparison to the genotyping standard of MRA and the epidemiological data. Analysis of AFLP fragments revealed 12 different types and subtypes. Because of its simplicity and reproducibility, AFLP proved to be the most effective technique in outbreak investigation.

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“…Genomic investigation. PFGE analysis was done as described in Pruckler (25), with slight modifications. Briefly, cultures grown for 72 hr on buffered charcoalyeast extract agar were suspended in TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0]) with turbidity adjusted to 25 (Vitek special DR100 colorimeter).…”

Section: Methodsmentioning

confidence: 99%

A Legionellosis Case Due to Contaminated Spa Water and Confirmed by Genomic Identification in Taiwan

Tseng

Tzeng

et al. 2006

Microbiology and Immunology

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Legionellosis (LG, infection by members of the genus Legionella) can range from mild respiratory illness to acute life-threatening pneumonia. The majority of LG cases are caused by Legionella pneumophila (LP), particularly serogroup 1 (18). Since the first outbreak in Philadelphia in 1976 (12), LP has been recognized as an important etiological agent of hospital-and community-acquired pneumonia. This microbe can survive in a wide range of temperature (5-65 C) and pH (5.5-9.5), particularly in warm and damp environments of 35-45 C which is their favorable growth temperature range. Because of their high survival rate in a thermal and wet environment, which happens to be the atmosphere regularly established in a whirlpool spa, numerous outbreaks of LG have been traced to the spa water as the source of their causative agents (2,17,20,21,23,31).Tracing the source of LG was often determined by linking environmental isolates to clinical isolates by various molecular subtyping methods, of which at least 7 kinds have been reported (15). Among them, amplified fragment length polymorphism (AFLP) and pulsefield gel electrophoresis (PFGE) were two methods most often used and highly recommended (3, 13).

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Comparison of Legionella pneumophila isolates by arbitrarily primed PCR and pulsed-field gel electrophoresis: analysis from seven epidemic investigations

Cited by 72 publications

References 23 publications

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study

A multicenter evaluation of genotypic methods for the epidemiologic typing of Legionella pneumophila serogroup 1: results of a pan-European study

Comparative Evaluation of Three Different Genotyping Methods for Investigation of Nosocomial Outbreaks of Legionnaires' Disease in Hospitals

A Legionellosis Case Due to Contaminated Spa Water and Confirmed by Genomic Identification in Taiwan

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