High impact of molecular surveillance on hepatitis A outbreak case detection in Sweden: a retrospective study, 2009 to 2018.

BackgroundSwedish hepatitis A surveillance includes sequence-based typing, but its contribution to outbreak detection in relation to epidemiological investigations has not been fully evaluated.AimTo evaluate the role of sequence-based typing in hepatitis A outbreak detection and to describe the hepatitis A epidemiology in Sweden to improve surveillance.MethodsWe retrospectively investigated hepatitis A virus sequences of 447 cases notified in Sweden 2009-18. We performed a phylogenetic analysis of evolutionary distances to identify cases with similar virus sequences (≥ 459/460 identical nt in the VP1/P2A junction). Unique sequences, dyads and sequence-based clusters (SBCs) were identified. We linked non-sequenced cases by epidemiological information and retrospectively assessed the value of typing for outbreak identification.ResultsFifty-five percent (n = 542/990) of the notified hepatitis A cases were referred to the Public Health Agency of Sweden for typing and 447 (45%) were sequenced successfully. Subgenotypes included IA (42.5%, n = 190), IB (42.7%, n = 191) and IIIA (14.8%, n = 66). Phylogenetic analysis identified 154 unique sequences, 33 dyads (66 cases) and 34 SBCs (227 cases). The combination of molecular and epidemiological data revealed 23 potential outbreaks comprising 201 cases. Cases were linked by sequence (59%, n = 118), epidemiological data (11%, n = 23) or both (30%, n = 60). Typing was needed to identify 15 of 23 potential outbreak signals.ConclusionSequence-based typing contributed substantially to detecting clustering cases and identifying outbreaks in Sweden. The results show routine sequence-based typing detects outbreaks, promotes timely outbreak investigations and facilitates international collaboration.

Outbreak of unusual H2S-negative monophasic Salmonella Typhimurium strain likely associated with small tomatoes, Sweden, August to October 2019.

Sweden is investigating an outbreak of monophasic Salmonella Typhimurium. Eighty-two nationally-distributed cases have been confirmed, with date of symptom onset between 28 August and 29 October. Cases were 51 years of age on average (range: 0-89) and the majority of cases were female (62%). A case-control study was conducted and suggested small tomatoes as source of the outbreak (adjusted odds ratio (OR): 10.8, 95% confidence interval (CI): 4.15-112.68, p value < 0.001), and a trace-back investigation led to a single, non-Swedish producer in Europe. Both the Salmonella strain and the source of the outbreak are rarely encountered in Europe. Results from investigation at the producer are pending.

Standardising surveillance of hepatitis E virus infection in the EU/EEA: A review of national practices and suggestions for the way forward.

BACKGROUND: Hepatitis E virus (HEV) infection is not notifiable at EU/EEA level, therefore surveillance relies on national policies only. Between 2005 and 2015, more than 20,000 cases were reported in EU/EEA countries. HEV testing is established in 26 countries and 19 countries sequence HEV viruses. OBJECTIVE AND STUDY DESIGN: WHO's European Action plan for viral hepatitis recommends harmonised surveillance objectives and case definitions. ECDC's HEV expert group developed minimal and optimal criteria for national hepatitis E surveillance to support EU/EEA countries in enhancing their capacity and to harmonise methods. RESULTS: The experts agreed that the primary objectives of national surveillance for HEV infections should focus on the basic epidemiology of the disease: to monitor the incidence of acute cases and chronic infections. The secondary objectives should be to describe viral phylotypes or subtypes and to identify potential clusters/outbreaks and possible routes of transmission. Seventeen of 20 countries with existing surveillance systems collect the minimal data set required to describe the epidemiology of acute cases. Eleven countries test for chronic infections. Twelve countries collect data to identify potential clusters/outbreaks and information on possible routes of transmission. DISCUSSION: Overall, the majority of EU/EEA countries collect the suggested data and meet the outlined requirements to confirm an acute case.

Improving preparedness to respond to cross-border hepatitis A outbreaks in the European Union/European Economic Area: towards comparable sequencing of hepatitis A virus.

IntroductionSequence-based typing of hepatitis A virus (HAV) is important for outbreak detection, investigation and surveillance. In 2013, sequencing was central to resolving a large European Union (EU)-wide outbreak related to frozen berries. However, as the sequenced HAV genome regions were only partly comparable between countries, results were not always conclusive.AimThe objective was to gather information on HAV surveillance and sequencing in EU/European Economic Area (EEA) countries to find ways to harmonise their procedures, for improvement of cross-border outbreak responses.MethodsIn 2014, the European Centre for Disease Prevention and Control (ECDC) conducted a survey on HAV surveillance practices in EU/EEA countries. The survey enquired whether a referral system for confirming primary diagnostics of hepatitis A existed as well as a central collection/storage of hepatitis A cases' samples for typing. Questions on HAV sequencing procedures were also asked. Based on the results, an expert consultation proposed harmonised procedures for cross-border outbreak response, in particular regarding sequencing. In 2016, a follow-up survey assessed uptake of suggested methods.ResultsOf 31 EU/EEA countries, 23 (2014) and 27 (2016) participated. Numbers of countries with central collection and storage of HAV positive samples and of those performing sequencing increased from 12 to 15 and 12 to 14 respectively in 2016, with all countries typing an overlapping fragment of 218 nt. However, variation existed in the sequenced genomic regions and their lengths.ConclusionsWhile HAV sequences in EU/EEA countries are comparable for surveillance, collaboration in sharing and comparing these can be further strengthened.

Hepatitis A outbreak linked to imported frozen strawberries by sequencing, Sweden and Austria, June to September 2018.

Between June-September 2018, 20 hepatitis A cases were notified in six counties in Sweden. Combined epidemiological and microbiological investigations identified imported frozen strawberries produced in Poland as the source of the outbreak. Sequence analysis confirmed the outbreak strain IB in the strawberries with 100 % identity and the respective batch was withdrawn. Sharing the sequence information internationally led to the identification of 14 additional cases in Austria, linked to strawberries from the same producer.

Hepatitis A outbreak disproportionately affecting men who have sex with men (MSM) in the European Union and European Economic Area, June 2016 to May 2017.

Between 1 June 2016 and 31 May 2017, 17 European Union (EU) and European Economic Area countries reported 4,096 cases associated with a multi-country hepatitis A (HA) outbreak. Molecular analysis identified three co-circulating hepatitis A virus (HAV) strains of genotype IA: VRD_521_2016, V16-25801 and RIVM-HAV16-090. We categorised cases as confirmed, probable or possible, according to the EU outbreak case definitions. Confirmed cases were infected with one of the three outbreak strains. We investigated case characteristics and strain-specific risk factors for transmission. A total of 1,400 (34%) cases were confirmed; VRD_521_2016 and RIVM-HAV16-090 accounted for 92% of these. Among confirmed cases with available epidemiological data, 92% (361/393) were unvaccinated, 43% (83/195) travelled to Spain during the incubation period and 84% (565/676) identified as men who have sex with men (MSM). Results depict an HA outbreak of multiple HAV strains, within a cross-European population, that was particularly driven by transmission between non-immune MSM engaging in high-risk sexual behaviour. The most effective preventive measure to curb this outbreak is HAV vaccination of MSM, supplemented by primary prevention campaigns that target the MSM population and promote protective sexual behaviour.

Two concurrent outbreaks of hepatitis A highlight the risk of infection for non-immune travellers to Morocco, January to June 2018.

From January to June 2018, two ongoing hepatitis A outbreaks affected travellers returning from Morocco and cases in Europe without travel history, resulting in 163 patients in eight European countries. Most interviewed travel-related cases were unaware of the hepatitis A risk in Morocco. Molecular analysis revealed two distinct hepatitis A virus (HAV) strains (subgenotype IA DK2018_231; subgenotype IB V18-16428). Vaccination recommendations should be emphasised to increase awareness among non-immune travellers to Morocco and HAV-endemic countries.

A nationwide outbreak of listeriosis associated with cold-cuts, Sweden 2013-2014.

In January 2014, the Public Health Agency of Sweden noticed an increase in listeriosis cases. Isolates from 10 cases had identical pulsed field gel electrophoresis (PFGE) profiles, suggesting a common source. We investigated the outbreak to identify the source and stop transmission. We looked for cases in 2013-2014 and also compared cases notified after February 2014 to randomly selected controls. We surveyed food items consumed two weeks prior to symptom onset. Listeria monocytogenes isolates found by food producers were PFGE-typed. Patient and food isolates with the outbreak PFGE profile were whole-genome sequenced and 51 cases with identical PFGE profile were identified; 12/20 cases and 108/186 controls responded to the survey. All cases were exposed to cold-cuts, compared with 72% of controls (p = 0.034). Five isolates of L. monocytogenes with the outbreak PFGE profile were found in cold-cuts from a food producer which stopped production in February 2014, but cases appeared until October 2014. Whole-genome sequencing showed that cold-cut and patient isolates differed by eight single nucleotide polymorphisms. Three patient isolates differed more and were probably not part of the outbreak. Epidemiological and microbiological results indicated cold-cuts as a possible source of the outbreak.

Hepatitis E virus: Assessment of the epidemiological situation in humans in Europe, 2014/15.

BACKGROUND: Hepatitis E virus (HEV) is endemic in EU/EEA countries, but the understanding of the burden of the infection in humans is inconsistent as the disease is not under EU surveillance but subject to national policies. STUDY: Countries were asked to nominate experts and to complete a standardised questionnaire about the epidemiological situation and surveillance of HEV in their respective EU/EEA country. This study reviewed surveillance systems for human cases of HEV in EU/EEA countries and nominated experts assessed the epidemiology in particular examining the recent increase in the number of autochthonous cases. RESULTS: Surveillance systems and case definitions across EU/EEA countries were shown to be highly variable and testing algorithms were unreliable. Large increases of autochthonous cases were reported from Western EU/EEA countries with lower case numbers seen in Northern and Southern European countries. Lack of clinical awareness and variability in testing strategies might account for the observed differences in hepatitis E incidence across EU/EEA countries. Infections were predominantly caused by HEV genotype 3, the most prevalent virus type in the animal reservoirs. CONCLUSION: Discussions from the expert group supported joint working across countries to better monitor the epidemiology and possible changes in risk of virus acquisition at a European level. There was agreement to share surveillance strategies and algorithms but also importantly the collation of HEV data from human and animal populations. These data collected at a European level would serve the 'One Health' approach to better informing on human exposure to HEV.

Molecular epidemiological studies show that hepatitis A virus is endemic among active homosexual men in Europe.

Large outbreaks of hepatitis A have occurred in Denmark, Germany, the Netherlands, Norway, Spain, Sweden, and the United Kingdom during the period 1997-2005 affecting homosexual men. A collaborative study was undertaken between these countries to determine if the strains involved in these hepatitis A outbreaks were related genetically. The N-terminal region of VP1 and the VP1/P2A region of the strains were sequenced and compared. The majority of the strains found among homosexual men from the different European countries formed a closely related cluster, named MSM1, belonging to genotype IA. Different HAV strains circulated among other risk groups in these countries during the same period, indicating that specific strains were circulating among homosexual men exclusively. Similar strains found among homosexual men from 1997 to 2005 indicate that these HAV strains have been circulating among homosexual men for a long time. The homosexual communities are probably too small within the individual countries to maintain HAV in their population over time, whereas the homosexual communities across Europe are probably sufficiently large to sustain continued circulation of homologous HAV strains for years resulting in an endemic situation among homosexual men.

Kluyveromyces lactis SSO1 and SEB1 genes are functional in Saccharomyces cerevisiae and enhance production of secreted proteins when overexpressed.

The SEB1/SBH1 and the SSO genes encode components of the protein secretory machinery functioning at the opposite ends, ER translocation and exocytosis, respectively, of the secretory pathway in Saccharomyces cerevisiae. Overexpression of these genes can rescue temperature-sensitive (ts) growth defect of many sec mutants impaired in protein secretion. Furthermore, their overexpression in wild-type yeast enhances production of secreted proteins in S. cerevisiae, which suggests that they may be rate-limiting factors in this process. Here we report isolation of Kluyveromyces lactis homologues of these genes. KlSSO1 and KlSEB1 were isolated as clones capable of rescuing growth of ts sso2-1 and seb1Delta seb2Delta sem1Delta strains, respectively, at the restrictive temperature. The encoded Kluyveromyces proteins are up to 70% identical with the S. cerevisiae homologues at the amino acid level and can functionally replace them. Interestingly, KlSSO1 and KlSEB1 show similar enhancing effect on production of a secreted protein as the SSO and SEB1 genes of S. cerevisiae when overexpressed. In accordance with the high homology level of the secretory pathway proteins in different yeast species, the polyclonal antibodies raised against S. cerevisiae Seb1p, Sso2p and Sec4p can detect homologous proteins in cell lysates of K. lactis and Pichia pastoris, the latter also in Candida utilis. The GenBank Accession Nos are AF307983 (K. lactis SSO1) and AF318314 (K. lactis SEB1).

Identification of the first fungal NADP-GAPDH from Kluyveromyces lactis.

Deletion of the phosphoglucose isomerase gene, PGI1, in Saccharomyces cerevisiae leads to a phenotype for which glucose is toxic. This is related to overproduction of NADPH through the oxidative part of the pentose phosphate pathway and the incompetence of S. cerevisiae to deal with this overproduction. A similar deletion (rag2) in Kluyveromyces lactis does not lead to such a phenotype. We transformed a genomic library of K. lactis in a yeast vector to a S. cerevisiae strain with a pgi1 deletion and screened for growth on glucose. We found a gene (GDP1) which encodes a phosphorylating glyceraldehyde-3-phosphate dehydrogenase, NADP-GAPDH (EC 1.2.1.13), that accepts both NADP and NAD. This is the first report of a eukaryotic, nonplant, NADP-linked GAPDH. Presumably, operation of this enzyme in the reverse direction enabled the transformed S. cerevisiae pgi1 deletion mutant to reoxidize the excess NADPH produced when glucose catabolism was forced through the pentose pathway. On the other hand, transcription of the gene in K. lactis was upregulated during growth on D-xylose, which suggests that in K. lactis the enzyme is involved in regeneration of NADPH needed for xylose assimilation, but transcription was not detected in a rag2 mutant grown on glucose. The presence of an asparagine (Asn46 in NADP-GAPDH) instead of the conserved aspartate found in related but NAD-specific enzymes may explain the ability of NADP-GAPDH to work with NADP as well as NAD.

[Results of the hepatitis A epidemic in Blekinge. Transmission links surveyed with the help of sequence analysis of viral RNA]. / Facit för hepatit A-epidemi i Blekinge. Smittsambanden kartlades med hjälp av sekvensanalys av viralt RNA.

In 1994-1995 there was an epidemic of hepatitis A (HAV) among drug abusers in the county of Blekinge, Sweden. There were also a number of other cases in which the epidemiological links were unclear. In order to uncover possible links between unclear cases retrospective sequencing was carried out of the VP1 region of HAV-RNA from patient sera. It was found that the epidemic among drug abusers spread beyond their closest contacts, revealing links that would otherwise not have been suspected. The extent of the epidemic also demonstrated the need to vaccinate injecting drug users against hepatitis A.

Characterization of temperature-sensitive mutations in the yeast syntaxin 1 homologues Sso1p and Sso2p, and evidence of a distinct function for Sso1p in sporulation.

The duplicated genes SSO1 and SSO2 encode yeast homologues of syntaxin 1 and perform an essential function during fusion of secretory vesicles at the plasma membrane. We have used in vitro mutagenesis to obtain a temperature-sensitive SSO2 allele, sso2-1, in which a conserved arginine has been changed to a lysine. A yeast strain that lacks SSO1 and carries the sso2-1 allele ceases growth and accumulates secretory vesicles at the restrictive temperature. Interestingly, the strain also has a pronounced phenotype at the permissive temperature, causing a defect in bud neck closure that prevents separation of mother and daughter cells. The same mutation was introduced into SSO1, producing the sso1-1 allele, which also has a temperature-sensitive phenotype, although less pronounced than sso2-1. A screen for high copy number suppressors of sso2-1 yielded three genes that are involved in the terminal step of secretion: SNC1, SNC2 and SEC9. The sso1-1 mutation interacts synthetically with a disruption of the MSO1 gene, which encodes a Sec1p interacting protein. Interestingly, we further found that both MSO1 and SSO1, but not SSO2, are required for sporulation. This difference is not due to differential expression, since SSO2 expressed from the SSO1 promoter failed to restore sporulation. We conclude that a functional difference exists between the Sso1 and Sso2 proteins, with the former being specifically required during sporulation.