Methods of assessing virulence of Legionella pneumophila, the etiologic agent of Legionnaires disease, include the infection of guinea pigs, fertile chicken eggs, and mammalian and protozoan cell cultures. Guinea pig assays, in particular, are expensive, laborious, or unsuitable for routine screening of Legionella isolates. We have developed a virulence assay that requires the enumeration of viruslike plaques which are the result of virulent L. pneumophila infecting mouse L929 cells. Each plaque is the consequence of the initial infection of an L cell with a single bacterium. A nonvirulent mutant derived from the serial passage of virulent L. pneumophila on Mueller-Hinton agar fails to survive within L cells and consequently fails to produce plaques.
Samples of sputum were examined microscopically to determine their suitability for routine culture. When the number of squamous epithelial cells per field was less than 10, the number of bacterial species generally fell within the range of one to four. Squamous epithelial cells were not always a true indication because some unmarked transtracheal specimens showing more than 10 squamous epithelial cells also gave a range of isolation falling between one and four. When the presence of 25 or more polymorphs was used as the parameter, the number of bacterial isolates generally fell within the range of one to three, but this resulted in positive overbiasing with consequent rejection of valid specimens. Later it was found that when a differential system using both polymorphonuclear cells and squamous epithelial cells was applied, a significant number of specimens could be salvaged which would otherwise have been discarded.
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