The relevance of molecular genotyping to allocate cases in a suspected outbreak of Legionella pneumonia in patients with prolonged immunosuppressive therapy

Three cases of pneumonia caused by Legionella pneumophila serogroup 1 (Lp1) in immunosuppressed patients with repeated hospitalization were suspected as a healthcare-associated cluster. The environmental investigation did not reveal the presence of legionellae in the hospital patient rooms. Water samples collected from the homes of two patients were also negative for Legionella spp. In the absence of environmental strains potentially involved in the infections, we proceeded to genotype environmental Lp1 strains isolated in the hospital during routine water sampling during the decade 2009–2019 and recovered after long-term storage at −20 °C. These 'historical' strains exhibited a high grade of similarity and stability over time, regardless of the disinfection systems. The different molecular profiles shown among the clinical and environmental strains excluded a nosocomial outbreak. The study suggests that the application of molecular typing may be a useful tool to discriminate hospital vs community-acquired cases, mostly for severely immunosuppressed patients in whom the symptomatology could be insidious and the incubation period could be prolonged. Moreover, the genotyping allowed us to exclude any link between the cases. Keywords: Legionnaires' disease, Immunosuppressed patients, Sequence-based typing, Cluster, Environmental strains, Clinical strain

Improvement of Legionnaires' disease diagnosis using real-time PCR assay: a retrospective analysis, Italy, 2010 to 2015

AimTo evaluate real-time PCR as a diagnostic method for Legionnaires' disease (LD). Detection of Legionella DNA is among the laboratory criteria of a probable LD case, according to the European Centre for Disease Prevention and Control, although the utility and advantages, as compared to culture, are widely recognised.MethodsTwo independent laboratories, one using an in-house and the other a commercial real-time PCR assay, analysed 354 respiratory samples from 311 patients hospitalised with pneumonia between 2010-15. The real-time PCR reliability was compared with that of culture and urinary antigen tests (UAT). Concordance, specificity, sensitivity and positive and negative predictive values (PPV and NPV, respectively) were calculated.ResultsOverall PCR detected eight additional LD cases, six of which were due to Legionella pneumophila (Lp) non-serogroup 1. The two real-time PCR assays were concordant in 99.4% of the samples. Considering in-house real-time PCR as the reference method, specificity of culture and UAT was 100% and 97.9% (95% CI: 96.2-99.6), while the sensitivity was 63.6% (95%CI: 58.6-68.6) and 77.8% (95% CI: 72.9-82.7). PPV and NPV for culture were 100% and 93.7% (95% CI: 91.2-96.3). PPV and NPV for UAT were 87.5% (95% CI: 83.6-91.4) and 95.8% (95% CI: 93.5-98.2).ConclusionRegardless of the real-time PCR assay used, it was possible to diagnose LD cases with higher sensitivity than using culture or UAT. These data encourage the adoption of PCR as routine laboratory testing to diagnose LD and such methods should be eligible to define a confirmed LD case

Two Overlapping Clusters of Group B Streptococcus Late-onset Disease in a Neonatal Intensive Care Unit

OBJECTIVES: Current predominant routes of group B Streptococcus (GBS) transmission in preterm neonates admitted to neonatal intensive care unit (NICU) are poorly defined. We report 2 overlapping clusters of GBS late-onset disease (LOD) from June to September 2015 in an Italian NICU.METHODS: During the outbreak, possible sources of transmission (equipment, feeding bottles and breast pumps) were swabbed. Specimens from throat and rectum were collected on a weekly basis from all neonates admitted to NICU. Colonized or infected neonates had cohorting. Bacterial isolates were characterized by serologic and molecular typing methods.RESULTS: GBS was isolated in 2 full-term and 7 preterm neonates. Strains belonged to serotype III, with 3 different sequence types (ST17, ST182 and ST19). Full-term neonates were colonized with GBS strains unrelated to the clusters (ST182 and ST19). Two distinct ST17 strains caused 2 clusters in preterm neonates: a first cluster with 1 case of LOD and a second, larger cluster with 6 LOD in 5 neonates (one of them had recurrence). ST17 strains were isolated from vaginorectal and milk samples of 2 mothers. Two preterm neonates had no evidence of colonization for weeks, until they presented with LOD.CONCLUSIONS: Molecular analyses identified the presence of multiclonal GBS strains and 2 clusters of 7 cases of GBS-LOD. The dynamics of transmission of GBS within the NICU were complex. Breast milk was suspected to be one of the possible sources. In a research setting, the screening of GBS carrier mothers who deliver very preterm could contribute to the tracking of GBS transmission