Parasites and their impact on thick-shelled river mussels Unio crassus from two populations in Luxembourg.

The thick-shelled river mussel Unio crassus Philipsson, 1788 is a species native to many European habitats, with declining populations. The impact of parasite communities on health status of this species is poorly understood. In this study, parasites of 30 U. crassus specimens from the Our and Sauer Rivers in Luxembourg were identified morphologically and, in some cases, using molecular genetic methods. The findings were correlated to selected parameters (total length, visceral weight, shell lesions, gonadal stage). The 2 populations did not differ in shell length, visceral weight, number of males and females, gonadal scoring, shell lesions, and the occurrence of glochidia. The prevalence and infestation intensities of detected Trichodina sp., Conchophthirus sp., and freshwater mite larvae did not differ between the 2 populations, whereas the prevalence and infestation intensities of mite eggs, nymphs, and adults were significantly higher in the Sauer River. Rhipidocotyle campanula and European bitterling Rhodeus amarus larvae were only present in the Sauer. Histopathology revealed the destruction of the gonads by R. campanula and tissue damage by the mites. The only significant correlation of the selected parameters was a positive correlation between R. amarus occurrence and total length as well as a negative correlation between R. amarus occurrence and gonadal stage. In the Sauer River, 2 mussels were found to be hermaphrodites.

Viral metagenomics reveals the presence of novel Zika virus variants in Aedes mosquitoes from Barbados.

BACKGROUND: The Zika virus (ZIKV) epidemic of 2015/2016 spread throughout numerous countries. It emerged in mainland Latin America and spread to neighboring islands, including the Caribbean island of Barbados. Recent studies have indicated that the virus must have already been circulating in local mosquito populations in Brazil for almost 2 years before it was identified by the World Health Organization in 2015. Metagenomic detection assays have the potential to detect emerging pathogens without prior knowledge of their genomic nucleic acid sequence. Yet their applicability as vector surveillance tools has been widely limited by the complexity of DNA populations from field-collected mosquito preparations. The aim of this study was to investigate local vector biology and characterize metagenomic arbovirus diversity in Aedes mosquitoes during the ongoing 2015/2016 ZIKV epidemic. METHODS: We performed a short-term vector screening study on the island of Barbados during the ongoing 2015/2016 ZIKV epidemic, where we sampled local Aedes mosquitoes. We reanalyzed mosquito viral microbiome data derived from standard Illumina MiSeq sequencing to detect arbovirus sequences. Additionally, we employed deep sequencing techniques (Illumina HiSeq) and designed a novel bait capture enrichment assay to increase sequencing efficiency for arbovirus sequences from complex DNA samples. RESULTS: We found that Aedes aegypti seemed to be the most likely vector of ZIKV, although it prevailed at a low density during the observed time period. The number of detected viruses increased with sequencing depth. Arbovirus sequence enrichment of metagenomic DNA preparations allowed the detection of arbovirus sequences of two different ZIKV genotypes, including a novel one. To our knowledge, this is the first report of the S3116W mutation in the NS5 gene region of ZIKV polyprotein. CONCLUSIONS: The metagenomic arbovirus detection approach presented here may serve as a useful tool for the identification of epidemic-causing arboviruses with the additional benefit of enabling the collection of phylogenetic information on the source. Apart from detecting more than 88 viruses using this approach, we also found evidence of novel ZIKV variants circulating in the local mosquito population during the observed time period.

First Molecular Evidence of Zoonotic Bacteria in Ticks in Bosnia and Herzegovina.

In Bosnia and Herzegovina, the tick fauna is very diverse, but data on the occurrence of zoonotic tick-borne bacteria are lacking. Thus, the aim of this study was to investigate the presence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, 'Candidatus Neoehrlichia mikurensis', spotted fever group (SFG) rickettsiae and Francisella tularensis in questing ticks. In 19 (21.8%) of 87 ticks (Ixodes ricinus, n = 30; Dermacentor reticulatus, n = 54; D. marginatus, n = 3) collected by flagging the vegetation at the collection site in the Glamoc Municipality (south-western Bosnia and Herzegovina), Rickettsia monacensis (1.1%), R. helvetica (5.7%), R. raoultii (5.7%), R. slovaca (8.0%), A. phagocytophilum (1.1%) and F. tularensis subsp. holartica (1.1%) were detected and identified by molecular methods. None of the tested ticks were positive for B. burgdorferi s.l. and 'Candidatus N. mikurensis', and co-infection of R. slovaca and F. tularensis subsp. holarctica was detected in only one D. marginatus (1.1%). This study reports the occurrence of emerging zoonotic bacteria in ticks from Bosnia and Herzegovina for the first time, indicating a public health threat to humans. Therefore, physicians and practitioners should be aware of the presence of these tick-borne bacteria, especially when they are faced with acute febrile illnesses after tick exposure.

Direct PCR of indigenous and invasive mosquito species: a time- and cost-effective technique of mosquito barcoding.

Millions of people die each year as a result of pathogens transmitted by mosquitoes. However, the morphological identification of mosquito species can be difficult even for experts. The identification of morphologically indistinguishable species, such as members of the Anopheles maculipennis complex (Diptera: Culicidae), and possible hybrids, such as Culex pipiens pipiens/Culex pipiens molestus (Diptera: Culicidae), presents a major problem. In addition, the detection and discrimination of newly introduced species can be challenging, particularly to researchers without previous experience. Because of their medical importance, the clear identification of all relevant mosquito species is essential. Using the direct polymerase chain reaction (PCR) method described here, DNA amplification without prior DNA extraction is possible and thus species identification after sequencing can be achieved. Different amounts of tissue (leg, head; larvae or adult) as well as different storage conditions (dry, ethanol, -20 and -80 °C) and storage times were successfully applied and showed positive results after amplification and gel electrophoresis. Overall, 28 different indigenous and non-indigenous mosquito species were analysed using a gene fragment of the COX1 gene for species differentiation and identification by sequencing this 658-bp fragment. Compared with standard PCR, this method is time- and cost-effective and could thus improve existing surveillance and control programmes.

The role of CD4(+) T cells in BKV-specific T cell immunity.

Reactivation of polyomavirus BK (BKV) infection represents a severe complication in kidney transplant (KTX) patients. We previously reported an association between a declining BK viral load and the reconstitution of CD4(+) T cell BKV-specific immunity in patients following kidney transplantation. However, the specific contribution of CD4(+) T cells in the regulation of BKV-replication is unknown. Nevertheless, in vitro enrichment of BKV-specific T cells and subsequent adoptive T cell transfer may improve the restoration of immune competence in KTX patients with BKV infection. To date, strategies to capture human BKV-specific T cells with the ensuing expansion to clinically useful numbers are lacking. Here, we demonstrated a comprehensive flow cytometric analysis of the BKV-specific T cell response that permits access to the majority of T cells specific for immunodominant BKV antigens. A full-spectrum evaluation of the BKV-specific T cell response was performed by stimulating peripheral blood mononuclear cells (PBMC) with a mixture of BKV immunodominant peptide pools at varying concentrations and measuring activation marker expression and cytokine secretion. We also examined the effects of co-stimulation and PBMC resting time prior to activation. We defined the narrow range of stimulation conditions that permit the capture and expansion of functional BKV-specific T cell lines. The generated BKV-specific T cell lines showed the highest specificity and functionality when the T cells were captured according to IFNγ-secretion. This study highlights the multifunctional and cytolytic BKV-specific CD4(+) T cells as a dominant population within the generated T cell product. This method offers a novel approach for the generation of BKV-specific T cell lines for adoptive immunotherapy and underscores the critical role of CD4(+) T cells in the clearance of BKV.

Characterization and epidemiology of influenza viruses in patients seeking treatment for influenza-like illnesses in rural Bangladesh.

CONTEXT: Infections caused by influenza viruses are a major health burden, both in developed and developing countries worldwide. Nevertheless, the overwhelming majority of influenza reports originate from industrialized countries in northern and southern temperate zones. AIMS: The aim of this study was to determine the epidemiology of influenza viruses in patients seeking treatment for acute febrile illnesses in rural Bangladesh. SETTINGS AND DESIGN: As part of our research on the causes of febrile illnesses in rural Bangladesh, nasopharyngeal swabs from patients with signs and symptoms consistent with influenza were collected from 2008 onwards. MATERIALS AND METHODS: Viral infection was established using two independent rapid diagnostic tests (RDTs) and later confirmed by RT-PCR. RESULTS: A total of 314 fever cases were enrolled in a survey of febrile illnesses carried out in Bandarban District in southeastern Bangladesh, out of whom 38 (12.1%) tested positive by RDT. Molecular subtyping showed that seasonal H3 strains (N=22; 7.0%) as well as the new H1N1v pandemic influenza subtype (N=13; 4.1%) had been circulating at the time of our investigations resulting in a PCR-adjusted positivity rate of 11.1% (95% CI 8.0 - 15.3). The positive predictive values for the RDTs used were 90.9% and 94.4%, respectively. CONCLUSIONS: This study provides a first insight into influenza epidemics in one of the most remote parts of Asia. Our findings suggest that respiratory illnesses due to influenza viruses are underreported in areas with limited access to health care and show a distinct seasonality also in rural areas of tropical countries.

Antimalarial activity of tigecycline, a novel glycylcycline antibiotic.

Tigecycline is a novel glycylcycline antibiotic with a broad antibacterial spectrum. Tigecycline was tested with 66 clinical isolates of Plasmodium falciparum from Bangladesh using the histidine-rich protein 2 in vitro drug susceptibility assay. The 50% and 90% inhibitory concentrations of tigecycline were 699 (95% confidence interval, 496 to 986) and 5,905 nM (4,344 to 8,028). Tigecycline shows no activity correlation with traditional antimalarials and has substantial antimalarial activity on its own.