Key Role of Sequencing to Trace Hepatitis A Viruses Circulating in Italy During a Large Multi-Country European Foodborne Outbreak in 2013

Abstract

Background: Foodborne Hepatitis A Virus (HAV) outbreaks are being recognized as an emerging public health problem in industrialized countries. In 2013 three foodborne HAV outbreaks occurred in Europe and one in USA. During the largest of the three European outbreaks, most cases occurred in Italy (>1,200 cases as of March 31, 2014). A national Task Force was established at the beginning of the outbreak by the Ministry of Health. Mixed frozen berries were early demonstrated to be the source of infection by the identity of viral sequences in patients and in food. In the present study the molecular characterization of HAV isolates from 355 Italian cases is reported.

Methods: Molecular characterization was carried out by PCR/sequencing (VP1/2A region), comparison with reference strains and phylogenetic analysis.

Results: A unique strain was responsible for most characterized cases (235/355, 66.1%). Molecular data had a key role in tracing this outbreak, allowing 110 out of the 235 outbreak cases (46.8%) to be recognized in absence of any other link. The data also showed background circulation of further unrelated strains, both autochthonous and travel related, whose sequence comparison highlighted minor outbreaks and small clusters, most of them unrecognized on the basis of epidemiological data. Phylogenetic analysis showed most isolates from travel related cases clustering with reference strains originating from the same geographical area of travel.

Conclusions: In conclusion, the study documents, in a real outbreak context, the crucial role of molecular analysis in investigating an old but re-emerging pathogen. Improving the molecular knowledge of HAV strains, both autochthonous and circulating in countries from which potentially contaminated foods are imported, will become increasingly important to control outbreaks by supporting trace back activities, aiming to identify the geographical source(s) of contaminated food, as well as public health interventions.

Fig 1. Genotype distribution of HAV sequences (n = 355) from cases with clinical onset between January 1, 2013 and February 28, 2014.

The fraction of genotype IA isolates with the mixed frozen berry “outbreak” sequence is reported in purple.

Fig 2. Distribution of HAV strains in the 8 Italian Regions for which enough sequences were available.

Fig 3. Temporal distribution of the strains, based on the date of symptom onset.

* the lower number of cases in August is likely the result of under-delivery of samples, because the trend of notified cases did not show a parallel decrease to a similar extent.

Fig 4. Sequence type (“outbreak” or “non-outbreak”) associated to the risk factors reported in questionnaires by interviewed patients.

Multiple risk factors could be reported by each patient. In the “well-water”, “shellfish” and “travels” (outside Italy) groups the proportion of patients also reporting “frozen berries” is highlighted (striped region).