Extension of the Legionella pneumophila sequence-based typing scheme to include strains carrying a variant of the N-acylneuraminate cytidylyltransferase gene

Sequence-based typing (SBT) combined with monoclonal antibody subgrouping of Legionella pneumophila isolates is at present considered to be the reference standard during epidemiological investigation of Legionnaires' disease outbreaks. In some isolates of L. pneumophila, the seventh allele of the standard SBT scheme, neuA, is not amplified, because a homologue that is refractory to amplification with the standard neuA primers is present. Consequently, a complete seven-allele profile, and hence a sequence type, cannot be obtained. Subsequently, primers were designed to amplify both neuA and the homologue, but these yielded suboptimal sequencing results. In this study, novel primers specific for the neuA homologue were designed and internationally validated by members of the ESCMID Study Group for Legionella Infections at national and regional Legionella reference laboratories with a modified version of the online L. pneumophila sequence quality tool. To date, the addition of the neuAh target to the SBT protocol has allowed full typing data to be obtained for 108 isolates of 11 different serogroups, namely 1, 2, 3, 4, 5, 6, 7, 8, 10, 13, and 14, which could not previously be typed with the standard SBT neuA primers. Further studies are necessary to determine why it is still not possible to obtain either a neuA or a neuAh allele from three serogroup 11 isolates.