Toscana virus encephalitis in Southwest Germany: a retrospective study.

BACKGROUND: Toscana virus (TOSV) is an arthropod-borne virus transmitted by phlebotomine sandflies (Phlebotomus sp.) widespread throughout the Mediterranean having the potential to cause meningoencephalitis in humans. In Germany, the vectors of TOSV are introduced recently and become endemic especially in Southwestern Germany. As TOSV is not investigated regularly in patients with meningoencephalitis, cases of TOSV-neuroinvasive disease may remain mostly undetected. METHODS: We conducted a retrospective cohort study on patients with meningoencephalitis without identification of a causal pathogen from 2006 to 2016. Serologic assessment for anti-TOSV-IgG and IgM was performed on serum and CSF. Demographic, clinical and CSF data from TOSV-positive patients were compared to a cohort of patients with meningoencephalitis due to enterovirus. Informed consent was obtained from all included patients. RESULTS: We found 138 patients with meningoencephalitis without identified causal pathogen. From 98 of these patients CSF and serum was available for further testing. Additionally, we included 27 patients with meningoencephalitis due to enterovirus. We identified two patients with serological confirmed TOSV-neuroinvasive disease (TOSV-IgM and IgG positive, 2%) and two patients with possible TOSV-neuroinvasive disease (isolated TOSV-IgM positive, 2%). Overall, TOSV-neuroinvasive was detected in 4% of our cases with suspected viral meningoencephalitis. None of them had a history of recent travel to an endemic area. CONCLUSIONS: We found cases of TOSV-neuroinvasive disease in our German cohort of patients with meningoencephalitis. As no recent history of travel to an endemic area was reported, it remains probable that these cases resemble autochthonous infections, albeit we cannot draw conclusions regarding the origin of the respective vectors. TOSV could be considered in patients with meningoencephalitis in Germany.

Accurate detection of Zika virus IgG using a novel immune complex binding ELISA.

OBJECTIVES: Accurate serological assays are urgently needed to support public health responses to Zika virus (ZIKV) infection with its potential to cause foetal damage during pregnancy. Current flavivirus serology for ZIKV infections lacks specificity due to cross-reacting antibodies from closely related other flaviviruses. In this study, we evaluated novel serological tests for accurate ZIKV IgG detection. METHODS: Our ELISAs are based on immune complex binding. The high specificity is achieved by the simultaneous incubation of labelled ZIKV antigen and unlabelled flavivirus homolog protein competitors. Two assays were validated with a panel of 406 human samples from PCR-confirmed ZIKV patients collected in Brazil (n = 154), healthy blood donors and other infections from Brazil, Europe, Canada and Colombia (n = 252). RESULTS: The highest specificity (100% [252/252, 95% confidence interval (CI) 98.5-100.0]) was shown by the ZIKV ED3 ICB ELISA using the ED3 antigen of the ZIKV envelope. A similar test using the NS1 antigen (ZIKV NS1 ICB ELISA) was slightly less specific (92.1% [232/252, 95% CI 88.0-95.1]). The commercial Euroimmun ZIKV ELISA had a specificity of only 82.1% (207/252, 95% CI 76.8-86.7). Sensitivity was high (93-100%) from day 12 after onset of symptoms in all three tests. Seroprevalence of ZIKV IgG was analysed in 87 samples from Laos (Asia) confirming that the ED3 ELISA showed specific reactions in other populations. CONCLUSIONS: The novel ED3 ICB ELISA will be useful for ZIKV-specific IgG detection for seroepidemiological studies and serological diagnosis for case management in travellers and in countries where other flavivirus infections are co-circulating.

Geometric morphometric wing analysis represents a robust tool to identify female mosquitoes (Diptera: Culicidae) in Germany.

Accurate species identification is the prerequisite to assess the relevance of mosquito specimens, but is often hindered by missing or damaged morphological features. The present study analyses the applicability of wing geometric morphometrics as a low-cost and practical alternative to identify native mosquitoes in Germany. Wing pictures were collected for 502 female mosquitoes of five genera and 19 species from 80 sampling sites. The reliable species identification based on interspecific wing geometry of 18 landmarks per specimen was tested. Leave-one-out cross validation revealed an overall accuracy of 99% for the genus and 90% for the species identification. Misidentifications were mainly due to three pairings of Aedes species: Aedes annulipes vs. Aedes cantans, Aedes cinereus vs. Aedes rossicus and Aedes communis vs. Aedes punctor. Cytochrome oxidase subunit I (COI) gene region was sequenced to validate the morphological and morphometric identification. Similar to the results of the morphometric analysis, the same problematic three Aedes-pairs clustered, but most other species could be well separated. Overall, our study underpins that morphometric wing analysis is a robust tool for reliable mosquito identification, which reach the accuracy of COI barcoding.

Vector competence of field populations of the mosquito species Aedes japonicus japonicus and Culex pipiens from Switzerland for two West Nile virus strains.

Aedes (Hulecoeteomyia) japonicus japonicus (Diptera: Culicidae) (Theobald) is an invasive mosquito species in Central Europe, where it has colonized several areas. In this study, field-collected specimens of Ae. japonicus and Culex pipiens (Diptera: Culicidae) (Linnaeus) from Zürich (Switzerland) were orally exposed to two strains (NY99 and FIN) of the avian zoonotic pathogen West Nile virus (WNV) (family Flaviviridae, genus Flavivirus). Dissemination and transmission of the viruses after incubation for 12-15 days under a fluctuating Central European midsummer temperature regime (24 ± 7 °C) was investigated by detection of viral RNA in homogenates of pools of both head/thorax and saliva by reverse transcription real-time polymerase chain reaction (PCR). Culex pipiens was susceptible to WNV NY99 only, whereas both virus strains could be detected in Ae. japonicus, with the additional isolation of WNV NY99 in Vero cell culture from one saliva pool. Given the high abundances of Ae. japonicus in many newly colonized areas, its recently demonstrated broad host range, including mammalian and avian blood hosts, and its vector competence, this species is a potential key bridge vector of WNV in Central Europe.

Culex torrentium mosquitoes from Germany are negative for Wolbachia.

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high-throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia-induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.

Distribution of individual members of the mosquito Anopheles maculipennis complex in Germany identified by newly developed real-time PCR assays.

Owing to their role as vectors of malaria parasites, species of the Anopheles maculipennis complex (Diptera: Culicidae) Meigen were intensively studied in the past, but with the disappearance of malaria in Germany in the middle of the last century, the interest in this field of research declined. A comprehensive ecological analysis of the current species distribution for Germany is lacking. Between 2010 and 2013, a total of 1445 mosquitoes of the An. maculipennis complex were collected at 72 different sites in Germany. The samples comprise 722 single individuals as well as 723 individuals in 90 pools of up to 25 mosquitoes. All samples were analysed with newly developed species-specific qPCR assays for the identification of the four German species using nucleotide differences within the internal transcribed spacer 2 (ITS2) ribosomal DNA. All gathered data were used for species distribution modelling. The overall prevalence of An. messeae s.l. was highest with 98.89% of all pools; An. daciae with 6.93% of all individuals and An. messeae s.s. with 69.53%. The prevalence of the other two species was relatively low: An. maculipennis s.s. with 13.30% of all individuals (6.67% of all pools) and An. atroparvus with 1.80% of all individuals (1.11% of all pools).

Ross River virus disease in two Dutch travellers returning from Australia, February to April 2015.

We report two cases of Ross River virus (RRV) infection in Dutch travellers who visited Australia during February to April 2015. These cases coincided with the largest recorded outbreak of RRV disease in Australia since 1996. This report serves to create awareness among physicians to consider travel-related RRV disease in differential diagnosis of patients with fever, arthralgia and/or rash returning from the South Pacific area, and to promote awareness among professionals advising travellers to this region.

Zika virus infection in a traveller returning to Europe from Brazil, March 2015.

We report a case of laboratory-confirmed Zika virus infection imported into Europe from the Americas. The patient developed fever, rash, and oedema of hands and feet after returning to Italy from Brazil in late March 2015. The case highlights that, together with chikungunya virus and dengue virus, three major arboviruses are now co-circulating in Brazil. These arboviruses represent a burden for the healthcare systems in Brazil and other countries where competent mosquito vectors are present.

West Nile virus lineage 2 infection in a blood donor from Vienna, Austria, August 2014.

Eastern Austria is neighbouring regions with ongoing West Nile virus (WNV) transmissions. Three human WNV infections had been diagnosed during the past decade in Austria. The Austrian Red Cross Blood Service (ARC-BS) started a first voluntary screening for WNV in blood donors from Eastern Austria by Nucleic Acid Testing (NAT) in June 2014. This is also the most extensive WNV surveillance programme in humans in Austria so far. In August 2014, one autochthonous WNV infection was detected in a blood donor from Vienna. By now, one in 67,800 whole blood donations was found to be positive for WNV RNA.

Persisting arthralgia due to Mayaro virus infection in a traveler from Brazil: is there a risk for attendants to the 2014 FIFA World Cup?

The 2014 FIFA World Cup and the 2016 Olympic Games will attract large groups of visitors to Brazil. These visitors will be at risk for different arboviral infections, some of which not well known outside endemic areas. We report a case of a 52-year-old Dutch woman who presented with persistent arthralgia due to a Mayaro virus (MAYV) infection which she contracted in the Amazon basin in Brazil. MAYV is a mosquito-borne alphavirus which primarily circulates in humid tropical forests of South America. Infections are rarely reported in travelers and are characterized by an acute febrile illness which is often followed by a prolonged and sometimes incapacitating polyarthralgia. Both travelers and physicians should be aware of the risk of these arboviral infections and the importance of mosquito bite prevention should be stressed.

[Mosquitoes as vectors for exotic pathogens in Germany]. / Stechmücken als Überträger exotischer Krankheitserreger in Deutschland.

As a result of intensified globalization of international trade and of substantial travel activities, mosquito-borne exotic pathogens are becoming an increasing threat for Europe. In Germany some 50 different mosquito species are known, several of which have vector competence for pathogens. During the last few years a number of zoonotic arboviruses that are pathogenic for humans have been isolated from mosquitoes in Germany including Usutu, Sindbis and Batai viruses. In addition, filarial worms, such as Dirofilaria repens have been repeatedly detected in mosquitoes from the federal state of Brandenburg. Other pathogens, in particular West Nile virus, are expected to emerge sooner or later in Germany as the virus is already circulating in neighboring countries, e.g. France, Austria and the Czech Republic. In upcoming years the risk for arbovirus transmission might increase in Germany due to increased occurrence of new so-called "invasive" mosquito species, such as the Asian bush mosquito Ochlerotatus japonicus or the Asian tiger mosquito Aedes albopictus. These invasive species are characterized by high vector competence for a broad range of pathogens and a preference for human blood meals. For risk assessment, a number of mosquito and pathogen surveillance projects have been initiated in Germany during the last few years; however, mosquito control strategies and plans of action have to be developed and put into place to allow early and efficient action against possible vector-borne epidemics.

Autochthonous dengue virus infection in Japan imported into Germany, September 2013.

In September 2013, dengue virus (DENV) infection was diagnosed in a German traveller returning from Japan. DENV-specific IgM and IgG and DENV NS1 antigen were detected in the patient's blood, as were DENV serotype 2-specific antibodies. Public health authorities should be aware that autochthonous transmission of this emerging virus may occur in Japan. Our findings also highlight the importance of taking a full travel history, even from travellers not returning from tropical countries, to assess potential infection risks of patients.

Detection of Usutu virus infection in a healthy blood donor from south-west Germany, 2012.

From September 2011 until November 2012, 31 serum samples from German patients with clinically suspected acute Usutu virus (USUV) infections were tested for USUV-specific antibodies. All samples tested negative. In addition, 4,200 serum samples from healthy blood donors from south-west Germany were collected in January 2012 and also analysed for the presence of specific antibodies. One sample tested positive for USUV-IgG and -IgM. Thus, the seroprevalence of USUV antibodies in healthy blood donors from south-west Germany was low in January 2012.

Fatal course of an autochthonous hepatitis E virus infection in a patient with leukemia in Germany.

An acute infection with hepatitis E virus (HEV) genotype 3 subtype c was diagnosed in a patient with chronic lymphatic B-cell leukemia 6 weeks after the infusion of donor lymphocytes. Despite intensive care the patient died 39 days after admission due to pericardial effusion that was related to acute liver failure. We suggest that diagnostic procedures for detection of HEV infection should be seriously considered for the immunocompromised patient with elevated liver enzymes in the absence of a travel history to HEV endemic countries.

Fatal dengue hemorrhagic fever imported into Germany.

Dengue virus (DENV) is an arthropod-borne virus (family Flaviviridae) causing dengue fever or dengue hemorrhagic fever. Here, we report the first fatal DENV infection imported into Germany. A female traveler was hospitalized with fever and abdominal pain after returning from Ecuador. Due to a suspected acute acalculous cholecystitis, cholecystectomy was performed. After cholecystectomy, severe spontaneous bleeding from the abdominal wound occurred and the patient died. Postmortem analysis of transudate and tissue demonstrated a DENV secondary infection of the patient and a gallbladder wall thickening (GBWT) due to an extensive edema.