EPIDEMIOLOGIA DELLA LISTERIOSI INVASIVA IN REGIONE LOMBARDIA (2006-2014): IDENTIFICAZIONE DEI GENOTIPI EMERGENTI

Listeriosis is a foodborne invasive disease that is of great interest to the field of public health due to the severity of clinical manifestations in patients at risk, like immunocompromised individuals and pregnant women. The incidence reported by the passive surveillance system in industrialized countries is less than 1 per 100,000 inhabitants, but under-diagnosis or under-reporting factors can affect these data and they are not easily quantifiable. The Italian listeriosis incidence is much lower (0.19-0.22 per 100,000 inhabitants) than the European incidence (0.33–0.44 per 100,000 inhabitants). In Lombardy, the national mandatory notification system has been implemented since 2006 with the regional electronic database (MAINF) and a Laboratory-based Surveillance System (LabSS). Considering that it is difficult to conduct epidemiological investigations of listeriosis and estimates may be biased downward, the present analysis aims to i) better understand the epidemiology of listeriosis in Lombardy, ii) characterise the Listeria monocytogens isolates by advanced molecular analysis in order to detect emerging strains and possible epidemic clusters. All cases of listeriosis in persons resident in the Lombardy Region and detected between 2006 and 2014 in at least one of two informative sources (MAINF and LabSS) were included. All human L. monocytogenes isolates collected by LabSS from 2006 to 2014 were studied. All isolates were serotyped by using antisera against O and H antigens. PFGE was performed according to the PulseNet protocol (Graves L.M. et al., 2001) with the AscI and ApaI enzymes. After AscI ans ApaI digestion, similarity clustering analysis was performed with BioNumerics software (version 5.1; Applied Maths, Saint-Martins-Latem, Belgium). Sequence types (STs) were identified by PCR amplification of seven housekeeping genes (Salcedo C. et al., 2003; Ragon et al., 2008). Alleles and STs were assigned by submitting the DNA sequences to the Listeria MLST database at the Pasteur Institute, France (http://www.pasteur.fr/mlst). Virulence types (VTs) were indentified by PCR amplification of six virulence genes (Chen Y et al., 2007; Knabel SJ et al., 2012). Virulence gene sequences were compared to available data obtained from isolates representing major human listeriosis outbreaks worldwide, and other unrelated isolates (https://sites.google.com/site/mvlstdatabase/home). Based on MAINF notifications and LabSS records, 580 cases of invasive listeriosis were observed in the nine year period under study (2006-2014). The mean annual incidence of listeriosis based on MAINF notifications was 0.56 per 100,000 inhabitants but it rose to 0.67 per 100,000 inhabitants once the cases detected only by the LabSS system and “lost” by the MAINF system were taken into account. The average incidence is high with a peak in 2011 (0.96 per 100,000 inhabitants), and in the Sondrio, Bergamo and Lecco provinces (1.11, 1.02, 1,00 per 100,000 inabitants, respectively). The “lost” cases, computed as the difference between the estimated cases and observed cases on estimated cases, was found to be 21% with significant differences by year and province. We found that the under-reporting factor affects all invasive listeriosis cases, while the under-diagnosis factor is more associated with pregnancy-related cases (6.7% of total cases). We hypothesize that the higher listeriosis incidence reported in Lombardy relative to the rest of the country as well as the upward trend in listeriosis incidence are attributable not only to the increased surveillance sensitivity, but also reflect a real increased incidence associated with the presence of people at risk for underlying disease conditions and/or social disadvantage. Regarding the microbiological and molecular characterizations, the predominant serotypes were 1/2a (60.9%), followed by 4b (26.3%) and 1/2b (10.4%). Multi-Locus Sequence Typing (MLST) revealed 52 Sequence Types (STs) were identified, of which the 6 most common STs were ST38 (14.9%), ST1 (12.8%), ST8 (9.3%), ST155 (7.6%), ST2 (5.9%) and ST3 (5.9%), accounting for 56.4% of the isolates. The distribution of ST38 and PFGE analysis led us to hypothesize the likely occurrence of an outbreak which had gone undetected by local health authorities during the period 2006-2014. In particular, identification of ST38 isolates began in 2006 (n=2), peaked in the period 2009-2011 (n=36) and continued in next years with sporadic cases (2013, n=1; 2014, n=2). The Multi-Virulence-Locus Sequence Typing (MVLST) revealed that the ST38 isolates split into two Virulence Type (VT) with different spatio-temporal distributions: VT80 and VT104. For these cases, an epidemiological investigation had been implemented and for only one case (ST38/VT104), the implicated food (smeared cheese) was recognized and confirmed by MVLST. This result represents an outstanding goal, as it is the first Outbreak Clone detected in Italy. Moreover, this study identified the prominent STs circulating in our regional area and contributed to our understanding of the genetic heterogeneity of L. monocytogenes. Monitoring STs can provide essential information for preventing listeriosis and supporting investigations on source identification and transmission routes, which, due to the peculiar epidemiological and clinical characteristics of this disease, are often very challenging. We propose that the integrated surveillance system in the Lombardy region is a highly acceptable model for the rest of the country, where the eating habits and the risk of exposure are more diversified.