Zoonotic and arthropod-borne diseases

Neurotropic arboviruses are increasingly recognised as causative agents of neurological disease in Europe but underdiagnosis is still suspected. Capability for accurate diagnosis is a prerequisite for adequate clinical and public health response.

To improve diagnostic capability in EVD-LabNet laboratories, we organised an external quality assessment (EQA) focusing on molecular detection of Toscana (TOSV), Usutu (USUV), West Nile (WNV) and tick-borne encephalitis viruses (TBEV).

Sixty-nine laboratories were invited. The EQA panel included two WNV RNA-positive samples (lineages 1 and 2), two TOSV RNA-positive samples (lineages A and B), one TBEV RNA-positive sample (Western subtype), one USUV RNA-positive sample and four negative samples. The EQA focused on overall capability rather than sensitivity of the used techniques. Only detection of one, clinically relevant, concentration per virus species and lineage was assessed.

The final EQA analysis included 51 laboratories from 35 countries; 44 of these laboratories were from 28 of 31 countries in the European Union/European Economic Area (EU/EEA). USUV diagnostic capability was lowest (28 laboratories in 18 countries), WNV detection capacity was highest (48 laboratories in 32 countries). Twenty-five laboratories were able to test the whole EQA panel, of which only 11 provided completely correct results. The highest scores were observed for WNV and TOSV (92%), followed by TBEV (86%) and USUV (75%).

We observed wide variety in extraction methods and RT-PCR tests, showing a profound absence of standardisation across European laboratories. Overall, the results were not satisfactory; capacity and capability need to be improved in 40 laboratories.

During the 2018 WNV transmission season, similarly to other endemic areas in Europe, a large number of human West Nile virus (WNV) infections were reported in Hungary.

We summarise the epidemiological and laboratory findings of the 2018 transmission season and expand experiences in flavivirus differential diagnostics.

Every patient with clinical suspicion of acute WNV infection was in parallel tested for WNV, tick-borne encephalitis virus and Usutu virus (USUV) by serological methods. Sera, whole blood and urine samples were also tested for the presence of viral nucleic acid.

Until the end of December 2018, 215 locally acquired and 10 imported human WNV infections were notified in Hungary. All reported cases were symptomatic; most of them exhibited neurological symptoms. In a large proportion of tested individuals, whole blood was the most appropriate sample type for viral nucleic acid detection, but because whole blood samples were not always available, testing of urine samples also extended diagnostic possibilities. In addition, the first human USUV infection was confirmed in 2018 in a patient with aseptic meningitis. Serological cross-reactions with WNV in different serological assays were experienced, but subsequent molecular biological testing and sequence analysis identified Europe lineage 2 USUV infection.

Careful interpretation and simultaneous application of different laboratory methods are necessary to avoid misdiagnosis of human USUV cases. Expansion of the laboratory-confirmed case definition criteria for detection of viral RNA in any clinical specimens to include urine samples could increase diagnostic sensitivity.

In 2016, an uncommon outbreak of oropharyngeal tularaemia involving six human cases occurred in Germany, caused by drinking contaminated fresh must after a grape harvest.

We describe the details of laboratory investigations leading to identification of the outbreak strain, its characterisation by next generation sequencing (NGS) and the finding of the possible source of contamination.

We incubated wine samples in different media and on agar plates. NGS was performed on DNA isolated from young wine, sweet reserve and an outbreak case’s lymph node. A draft genome of the outbreak strain was generated. Vertebrate-specific PCRs using primers targeting the mitochondrial cytochrome b gene and product analyses by blast search were used to identify the putative source of must contamination.

No bacterial isolate could be obtained. Analysis of the draft genome sequence obtained from the sweet reserve attributed this sequence to Francisella tularensis subsp. holarctica, belonging to the B.12/B.34 phylogenetic clade (erythromycin-resistant biovar II). In addition, the DNA sequence obtained from the case’s isolate supported our hypothesis that infection was caused by drinking contaminated must. The vertebrate-specific cytochrome b sequence derived from the young wine and the sweet reserve could be assigned to Apodemus sylvaticus (wood mouse), suggesting that a wood mouse infected with F. tularensis may have contaminated the must.

The discovered source of infection and the transmission scenario of F. tularensis in this outbreak have not been observed previously and suggest the need for additional hygienic precautionary measures when processing and consuming freshly pressed must.