The initial detection of Toscana virus in phlebotomine sandflies from Turkey.

Toscana virus (TOSV) is a prominent arthropod-borne viral agent of human central nervous system infections occurring in the Mediterranean region. The main transmission route to susceptible individuals involves sandflies as vectors. Despite several reports revealing widespread TOSV activity in Turkey, vectors remained unidentified. A sandfly field survey was carried out in five provinces in Central, Southeast and Mediterranean Anatolia in 2017 to identify TOSV and related sandfly-borne phleboviruses and Leishmania parasites, with evidence for circulation in the region. A total of 7136 sandfly specimens, collected via standard methods, were evaluated in 163 pools. TOSV was detected in 11 pools (6.7%), comprising Phlebotomus major sensu lato, Sergentomyia dentata and Phlebotomus papatasi species. TOSV partial L and S segment sequences were characterized, that phylogenetically clustered with local and global genotype A strains. An amino acid substitution outside the conserved motifs of the viral polymerase, also present in previous TOSV sequences in endemic regions, was observed. Leishmania tropica was detected in a single pool of Ph. sergentii (0.6%). This is the first report of TOSV in sandflies from Turkey, and this study further provides evidence for additional sandfly species with the potential to transmit TOSV.

Sampling strategies for phlebotomine sand flies (Diptera: Psychodidae) in Europe.

The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.

Ongoing activity of Toscana virus genotype A and West Nile virus lineage 1 strains in Turkey: a clinical and field survey.

Toscana virus (TOSV), West Nile virus (WNV) and tickborne encephalitis virus (TBEV) are among major viral pathogens causing febrile disease and meningitis/encephalitis. The impact of these viruses was investigated at a referral centre in Ankara Province, Central Anatolia in 2012, where previous reports suggested virus circulation but with scarce information on clinical cases and vector activity. Serum and/or cerebrospinal fluid samples from 94 individuals were evaluated, in addition to field-collected arthropod specimens that included 767 sandflies and 239 mosquitoes. Viral nucleic acids in clinical samples and arthropods were sought via specific and generic nested/real-time PCRs, and antibody responses in clinical samples were investigated via commercial indirect immunofluorescence tests (IIFTs) and virus neutralization. A WNV antigen assay was also employed for mosquitoes. WNV neuroinvasive disease has been identified in a 63-year-old male via RNA detection, and the WNV strain was characterized as lineage 1. TOSV infections were diagnosed in six individuals (6.3%) via RNA or IgM detection. Partial sequences in a 23-year-old female, presented with fever and transient pancytopenia, were characterized as TOSV genotype A. Febrile disease with arthralgia and/or peripheral cranial nerve involvement was noted in cases with TOSV infections. Previous WNV and TOSV exposures have been observed in 5.3% and 2.1% of the subjects, respectively. No confirmed TBEV exposure could be identified. Morphological identification of the field-collected mosquitoes revealed Culex pipiens sensu lato (74.4%), Anopheles maculipennis (20.9%), An. claviger (2.1%) and others. Sandfly species were determined as Phlebotomus papatasi (36.2%), P. halepensis (27.3%), P. major s. l. (19.3%), P. sergenti (8.9%), P. perfiliewi (4.4%), P. simici (2.6%) and others. Viral infections in arthropods could not be demonstrated. TOSV genotype A and WNV lineage 1 activity have been demonstrated as well as serologically proven exposure in patients. Presence of sandfly and mosquito species capable of virus transmission has also been revealed.

Emergence and co-infections of West Nile virus and Toscana virus in Eastern Thrace, Turkey.

The objective of this study was to identify the impact of West Nile virus (WNV) and Toscana virus (TOSV) in febrile diseases of unknown aetiology in Eastern Thrace, Turkey; this study was conducted during August-October 2012, and included 18 clinical cases and 296 blood donors for local serosurveillance. Antibodies were determined via commercial assays and further tested for specificity via neutralization assays (NA). Viral RNAs were sought via specific and/or generic primers. WNV infections were diagnosed in seven patients (38.8%), detected via RNA+IgM in four, RNA in one and IgM and low avidity IgG in two cases. The most common symptom was fever (>38°C), followed by headache, malaise/fatigue, myalgia/arthralgia, muscle stiffness/lower back pain, anorexia, nausea/vomiting, diarrhoea, supraorbital/retrobulbar pain and abdominal pain. Neurological symptoms were noted in one individual. WNV strains in RNA-detectable patients were characterized as lineage 1. TOSV RNA or IgM were identified in two individuals with confirmed WNV infections and in one patient without evidence of WNV exposure. The clinical and laboratory findings in individuals with WNV/TOSV co-infection were comparable to those in WNV-induced disease. The TOSV strain in the patient with detectable viral RNA was characterized as genotype A. In local blood donors, seroreactivity for specific WNV and TOSV immunoglobulins was observed in 1.7% (5/296) and 14.4% (26/180), respectively. These findings indicate the emergence of WNV and TOSV-associated diseases in Eastern Thrace. WNV/TOSV co-infections were documented for the first time.

Toscana virus (TOSV) exposure is confirmed in blood donors from Central, North and South/Southeast Anatolia, Turkey.

Toscana virus (TOSV), a sandfly fever virus serotype of medical and public health importance, is a major pathogen involved in aseptic meningtis occurring in Mediterranean countries and poses a threat to the residents as well as travellers. Limited data on TOSV activity are present from Turkey despite being located in the endemic zone. We aimed to identify TOSV exposure in 1115 healthy blood donors at the Hacettepe University Hospital Blood Bank in Ankara, Turkey, using commercial indirect fluorescence assays (IFAs) and virus neutralization test (VNT) for antibody detection and specificity confirmation. A total of 199 samples (17.8%) were positive for anti-TOSV that include IgG reactivity in 10.4%, IgM reactivity in 8.2% and IgM + IgG reactivity in 0.7% of the sera. Anti-TOSV specificity could be confirmed via VNT in 56% of the IgG- and 43.6% of the IgM-positive sera, making up a total of 58 samples (5.2%). Risk factors associated with TOSV IgG reactivity were male gender, residing in rural areas, frequent sighting of mosquitoes/sandflies and working outdoors. TOSV-specific antibody prevalence increased significantly with age. Evidence of exposure to other sandfly fever viruses was noted. These data reveal that mild or asymptomatic infections with TOSV are frequent in central and northern Anatolia. TOSV exposure has also been identified in residents of 9 provinces in southern/southeastern Anatolia for the first time.

Confirmed exposure to tick-borne encephalitis virus and probable human cases of tick-borne encephalitis in Central/Northern Anatolia, Turkey.

Tick-borne encephalitis virus (TBEV) is the aetiological agent of tick-borne encephalitis (TBE), a potentially fatal central nervous system infection of humans. TBE is endemic in many areas of Europe and Asia; however, very scarce data on TBEV activity are available from Turkey. We aimed to identify TBEV exposure in healthy blood donors and the impact of TBEV in central nervous system infections in Central/Northern Anatolia. Two-thousand four hundred and fifty four sera, collected from blood donors at Ankara, Konya, Eskisehir and Zonguldak branches of the Turkish Red Crescent Middle Anatolia Regional Blood Center, were analysed for TBEV serosurveillance. Paired serum and cerebrospinal fluid samples from 108 patients with the diagnosis of aseptic meningitis/encephalitis of unknown aetiology were also evaluated to identify TBE and neuroborreliosis cases. Commercial enzyme-linked immunosorbent assays and indirect immunofluorescence tests were employed for antibody detection. Forty-seven donor samples (1.9%) were reactive for TBEV IgG. In 25 persons with IgG reactivity (53.1%), risk factors for tick-borne infections were revealed. One sample from Zonguldak province (1/198; 0.5%) in the Black Sea region of Turkey was confirmed to possess neutralizing antibodies via plaque reduction neutralization test. TBEV IgM was detected in 9.2% (8/108) of the patients. IgM was accompanied by IgG reactivity in two persons where, in one, recent history of a tick bite was also identified. Intrathecal antibody production for TBEV could not be demonstrated. No evidence for Borrelia infections could be found. Confirmed exposure to TBEV and/or an antigenically similar tick-borne flavivirus is documented for the first time in blood donors in Zonguldak in Northern Anatolia. Probable cases of TBE have also been identified from Central Anatolia. The epidemiology of TBEV activity in Turkey needs to be assessed and benefits of vaccination for general population, risk groups or travellers must be considered.

Sandfly fever virus activity in central/northern Anatolia, Turkey: first report of Toscana virus infections.

Sandfly fever viruses (SFVs) cause febrile diseases as well as aseptic meningitis/encephalitis and include serotypes sandfly fever Sicilian virus (SFSV), sandfly fever Naples virus (SFNV) and Toscana virus (TOSV). Infections are endemic in the Mediterranean basin and data on SFV activity in Turkey are limited. In this study, sera from 1533 blood donors from the Ankara, Konya, Eskisehir and Zonguldak provinces of Turkey were evaluated for SFV exposure by indirect immunofluorescence test (IIFT) and confirmed by virus neutralization test (VNT). One hundred and two patients with central nervous system (CNS) infections of unknown aetiology were also tested via IIFT and real-time reverse-transcription PCR for SFV/TOSV. Rate of overall IgG reactivity in IIFT was 32.9% (505/1533) among blood donors. TOSV exposure was confirmed by VNT in all study regions. Exposure to the recently-identified serotype sandfly fever Turkish virus, as evaluated by VNT, was revealed in Konya and Ankara. SFNV exposure was identified in Konya and SFSV was observed to be present in all regions except Zonguldak. TOSV RNA was detected in 15.7% (16/102) and was accompanied by TOSV IgM in 25% (4/16) of the patients. Partial L and S sequences suggested that TOSV circulating in Turkey can be grouped into TOSV genotype A strains. Exposure to TOSV and other SFV serotypes was revealed in blood donors and CNS infections by TOSV were identified for the first time in Turkey. Infections are observed to be endemic in central Anatolia and should be considered as aetiologic agents in cases/outbreaks of fever and meningoencephalitis.

Lack of known hepatitis virus in hepatitis-associated aplastic anemia and outcome after bone marrow transplantation.

Viral infection has been shown to induce aplastic anemia, unidentified types of hepatitis being the most common cause for aplastic anemia-associated viral hepatitis. The survival rate for this group of patients after bone marrow transplantation with stem cells from an HLA-matched sibling is not well known. The aim of this study was to determine the prevalence of hepatitis G virus (HGV) and transfusion transmitted virus (TTV) infection in non-A, non-B, non-C hepatitis associated-aplastic anemia (HAAA) patients, and to define the role of bone marrow transplantation (BMT) as a therapeutic modality for this disease. Sixty-eight patients (43 males and 25 females) with aplastic anemia, underwent allogeneic BMT at the Hadassah University Hospital between 1981 and 1997. Onset of hepatitis was defined as jaundice and elevated alanine aminotransaminase (ALT) levels. Onset of aplastic anemia was defined as the first date on which varying degrees of pancytopenia occurred: hemoglobin level below 10 g/dl, WBC below 2 x 10(9)/l and low platelet count 10 x 10(10)/l. Serial serum samples from HAAA patients were assayed for virological and/or serological markers of hepatitis A, B, C, D, E, G viruses, TTV and parvovirus B19. Seventeen of the 68 patients with aplastic anemia (25%) suffered from hepatitis, 12 males and five females, ages 5 to 36 years. The mean interval between onset of hepatitis and first indication of aplastic anemia was 62 days (range 14-225 days). The development of aplastic anemia was unrelated to age, sex or severity of hepatitis. Ten of the 17 patients (59%) achieved complete ALT recovery prior to the diagnosis of aplastic anemia. Serum samples were available for 15 patients; none had evidence of acute or active hepatitis A, B, C, D, E, G and TTV virus infection at the time of diagnosis. Parvovirus B19 DNA sequences were not detectable in 10 of 12 tested cases; two positive results were detected in serum samples obtained after blood transfusion, making the analysis of these positive results difficult. All 17 patients underwent BMT. The mean post-BMT follow-up period was 38 months (range 1 day-123 months), five patients (30%) died 1 to 160 days post BMT, and 12 (70%) are alive 31 to 123 months after BMT. Relapsing hepatitis was not observed in any of the patients. In conclusion, HAAA is a disease of the young and the etiologic agent associated with HAAA remains unknown. HGV, TTV and parvovirus B19 sequences were not detected in any of the HAAA cases. The survival rate after BMT with stem cells from an HLA-matched sibling is similar to that for patients with non-hepatitis-associated aplastic anemia.