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Background: Malaria caused by Plasmodium ovale spp. has been neglected by and large from research and has received only little scientific attention during the past decades. Ovale malaria is considered to feature relapses by liver hypnozoites although scientific evidence for this paradigm is scarce. Case presentation: Here, the case of a 16-year-old male, who presented with fevers to the outpatient department in Vienna, Austria, after travelling to Uganda and Papua New Guinea is described. Infection with Plasmodium malariae was diagnosed by microscopy and the patient was treated accordingly with a full course of supervised artemether-lumefantrine. He was discharged in good clinical condition with a negative blood smear. One month after initial diagnosis, he returned complaining of fever. Thick blood smear was positive again for malaria parasites, which were confirmed as P. ovale wallikeri by PCR. Retrospective analysis revealed the identical Plasmodium spp. in the initial blood samples. Molecular analysis of various gene loci (nuclear porbp2, 18S rRNA and potra genes) gave identical results providing further evidence for relapse by an identical parasite genotype. Consecutively, the patient was retreated with artemether-lumefantrine and received a regimen of primaquine according to WHO guidelines. Conclusion: Conclusive evidence for relapses with P. ovale spp. is rare. The presented case provides convincing confirmation for the relapse paradigm based on re-appearing parasitaemia following supervised treatment in a non-endemic region with a parasite strain of identical genotype.

List of samples

Background: Bats are among the most widely distributed mammals worldwide and can represent hosts or reservoirs for a number of different pathogens. Bartonella spp. are opportunistic bacterial pathogens, which are transmitted by a large variety of arthropods. The aim of this study was to investigate the presence and host-associations of these Gram-negative bacteria in heart tissues of bats collected in four different countries from eastern and central Europe and to analyze their phylogenetic relationship with other bat-associated bartonellae. Results: The results of this study show for the first time the presence of Bartonella spp. DNA in heart tissues of bats from central and eastern Europe. The overall prevalence of the infection was 1.38%. Phylogenetic analysis identified four new Bartonella spp. sequences, which were closely related with other Bartonella previously isolated from bats in Europe and North America. Conclusions: The gltA sequences of Bartonella spp. showed considerable heterogeneity in the phylogenetic analysis resulting in six different clades. Our study demonstrated the presence of Bartonella spp. only in heart tissues of bats from Romania, with two new bat species recorded as hosts (Myotis cf. alcathoe and Pipistrellus pipistrellus).

Human malaria parasites have rarely been reported from free-ranging great apes. Our study confirms the presence of the human malaria parasite Plasmodium ovale wallikeri in western lowland gorillas and humans in Dzanga Sangha Protected Areas, Central African Republic, and discusses implications for malaria epidemiology.

Trypanosomatid flagellates have not been studied in Austria in any detail. In this study, specific nested PCR, targeted on the ribosomal small subunit, was used to determine the occurrence and diversity of trypanosomatids in wild-caught mosquitoes sampled across Eastern Austria in the years 2014−2015. We collected a total of 29,975 mosquitoes of 19 species divided in 1680 pools. Of these, 298 (17.7%), representing 12 different mosquito species, were positive for trypanosomatid DNA. In total, seven trypanosomatid spp. were identified (three Trypanosoma, three Crithidia and one Herpetomonas species), with the highest parasite species diversity found in the mosquito host Coquillettidia richiardii. The most frequent parasite species belonged to the mammalian Trypanosoma theileri/cervi species complex (found in 105 pools; 6.3%). The avian species T. culicavium (found in 69 pools; 4.1%) was only detected in mosquitoes of the genus Culex, which corresponds to their preference for avian hosts. Monoxenous trypanosomatids of the genus Crithidia and Herpetomonas were found in 20 (1.3%) mosquito pools. One third (n = 98) of the trypanosomatid positive mosquito pools carried more than one parasite species. This is the first large scale study of trypanosomatid parasites in Austrian mosquitoes and our results are valuable in providing an overview of the diversity of these parasites in Austria.

Overall trypanosomatid prevalence (calculated as a minimum infection rate, MIR), pool positivity, and parasite diversity found in mosquitoes sampled in Vienna and Eastern Austria (2014 and 2015). (DOCX)

Occurrence of trypanosomatid DNA in pools of mosquito species collected in Vienna and Eastern Austria (2014 and 2015). (DOCX)

Sampling sites for mosquitoes in Eastern Austria during the years 2013−2015. The close-up provides an overview of the city of Vienna where sampling sites were densest. Sites positive for trypanosomatid parasites are marked by stars, negative sites are marked by triangles. The map was constructed using our data and the software: ArcGIS 10.1 (ESRI, Redlands, CA, USA, https://www.esri.com). (TIF)

Information on mosquito-borne filarioid helminths in Austria is scarce, but recent discoveries of Dirofilaria repens indicate autochthonous distribution of this parasite in Eastern Austria. In the current xenomonitoring study, more than 48,000 mosquitoes were collected in Eastern Austria between 2013 and 2015, using different sampling techniques and storage conditions, and were analysed in pools with molecular tools for the presence of filarioid helminth DNA. Overall, DNA of D. repens , Setaria tundra , and two unknown filarioid helminths were documented in twenty mosquito pools within the mitochondrial cox1 gene (barcode region). These results indicate that S. tundra , with roe deer as definite hosts, is common in Eastern Austria, with most occurrences in floodplain mosquitoes (e.g., Aedes vexans ). Moreover, DNA of D. repens was found in an Anopheles plumbeus mosquito close to the Slovakian border, indicating that D. repens is endemic in low prevalence in Eastern Austria. This study shows that xenomonitoring is an adequate tool to analyse the presence of filarioid helminths, but results are influenced by mosquito sampling techniques, storage conditions, and molecular protocols.

Background Vector-pathogen dynamics are controlled by fluctuations of potential vector communities, such as the Culicidae. Assessment of mosquito community diversity and, in particular, identification of environmental parameters shaping these communities is therefore of key importance for the design of adequate surveillance approaches. In this study, we assess effects of climatic parameters and habitat structure on mosquito communities in eastern Austria to deliver these highly relevant baseline data. Methods Female mosquitoes were sampled twice a month from April to October 2014 and 2015 at 35 permanent and 23 non-permanent trapping sites using carbon dioxide-baited traps. Differences in spatial and seasonal abundance patterns of Culicidae taxa were identified using likelihood ratio tests; possible effects of environmental parameters on seasonal and spatial mosquito distribution were analysed using multivariate statistical methods. We assessed community responses to environmental parameters based on 14-day-average values that affect ontogenesis. ResultsAltogether 29,734 female mosquitoes were collected, and 21 of 42 native as well as two of four non-native mosquito species were reconfirmed in eastern Austria. Statistical analyses revealed significant differences in mosquito abundance between sampling years and provinces. Incidence and abundance patterns were found to be linked to 14-day mean sunshine duration, humidity, water–level maxima and the amount of precipitation. However, land cover classes were found to be the most important factor, effectively assigning both indigenous and non-native mosquito species to various communities, which responded differentially to environmental variables. Conclusions These findings thus underline the significance of non-climatic variables for future mosquito prediction models and the necessity to consider these in mosquito surveillance programmes.

Background Mosquitoes are arthropods of major importance to animal and human health because they are able to transmit pathogenic agents such as filarioids (Spirurida), vector-borne nematodes, which reside in the tissues of vertebrates. In Europe, recent research has mostly focused on mosquito-borne zoonotic species, while others remain neglected. Mosquitoes are also vectors of avian malaria, which has an almost worldwide distribution, and is caused by several Plasmodium species and lineages, the most common being P. relictum. The Danube Delta region of Romania is one of the most important stopover sites for migratory birds. The local mosquito fauna is diverse and well represented, while filarial infections are known to be endemic in domestic dogs in this area. The aim of the present study was thus to assess the potential vector capacity for various filarial helminths and avian malaria of mosquitoes trapped in the Danube Delta. Methods In July 2015, mosquitoes were collected at seven sites located in and around a rural locality in the Danube Delta region of Romania, using CO2-baited traps and hand aspirators. Additionally, a trap was placed next to a microfilaremic dog co-infected with Dirofilaria repens and D. immitis. All randomly trapped mosquitoes were identified to the species level and pooled according to date, sampling site, and taxon. Three hundred individual mosquitoes sampled next to the microfilaremic dog were processed individually and divided into abdomen and thorax/head. Following DNA extraction, all samples were screened for the presence of DNA of filarioid helminths and avian malaria agents by PCR techniques. Results All 284 pools (a total of 5855 mosquitoes) were negative for filarioid DNA. One pool of Culex modestus mosquitoes was positive for Plasmodium sp. lineage Donana03. In the individually extracted mosquitoes, one abdomen of Aedes vexans was positive for D. repens DNA, one thorax/head of Ae. vexans was positive for DNA of Setaria labiatopapillosa, and two thorax/head of Cx. pipiens f. pipiens were positive for P. relictum lineage pSGS1. Conclusion The present study suggests the vector competence of Cx. modestus and Cx. pipiens for avian Plasmodium including pathogenic species P. relictum and Ae. vexans for mammalian filarioids. Moreover, it indicates the role of Cx. pipiens f. pipiens as a potential natural vector of P. relictum lineage pSGS1 in nature.

Background Insect vectors, namely mosquitoes (Diptera: Culicidae), are compulsory for malaria parasites (Plasmodium spp.) to complete their life cycle. Despite this, little is known about vector competence of different mosquito species for the transmission of avian malaria parasites. Methods In this study, nested PCR was used to determine Plasmodium spp. occurrence in pools of whole individuals, as well as the diversity of mitochondrial cytochrome b gene sequences in wild-caught mosquitoes sampled across Eastern Austria in 2013–2015. Results A total of 45,749 mosquitoes in 2628 pools were collected, of which 169 pools (6.43%) comprising 9 mosquito species were positive for avian Plasmodium, with the majority of positives in mosquitoes of Culex pipiens s.l./Culex torrentium. Six different avian Plasmodium lineages were found, the most common were Plasmodium vaughani SYAT05, Plasmodium sp. Linn1 and Plasmodium relictum SGS1. In 2014, mosquitoes of the Culex pipiens complex were genetically identified and Culex pipiens f. pipiens presented with the highest number of avian Plasmodium positives (n = 37; 16.74%). Despite this, the minimum infection rate (MIR) was highest in Culex torrentium (5.36%) and Culex pipiens f. pipiens/f. molestus hybrids (5.26%). During 2014 and 2015, seasonal and annual changes in Plasmodium lineage distribution were also observed. In both years P. vaughani SYAT05 dominated at the beginning of the sampling period to be replaced later in the year by P. relictum SGS1 (2014) and Plasmodium sp. Linn1 (2015). Conclusions This is the first large-scale study of avian Plasmodium parasites in Austrian mosquitoes. These results are of special interest, because molecular identification of the taxa of the Cx. pipiens complex and Cx. torrentium enabled the determination of Plasmodium prevalence in the different mosquito taxa and hybrids of this complex. Since pools of whole insects were used, it is not possible to assert any vector competence in any of the examined mosquitoes, but the results are nonetheless valuable in providing an overview of avian Plasmodium species and lineages present in Austria.

Background Babesia spp. are hemoparasites which infect the red blood cells of a large variety of mammals. In bats, the only known species of the genus is Babesia vesperuginis. However, except a few old reports, the host range and geographical distribution of this bat parasite have been poorly studied. This study aimed to investigate the presence of piroplasms in tissues of bats collected in four different countries from eastern and central Europe: Austria, Czech Republic, Hungary and Romania. Methods A total of 461 bat carcasses (24 species) were collected between 2001 and 2016 from caves, mines and buildings. PCR was performed using specific primers targeting a portion of the 18S rDNA nuclear gene and cytochrome c oxidase subunit 1 mitochondrial gene, followed by sequencing. Results The results of this study show for the first time the presence of B. vesperuginis in bats in central and eastern Europe. The phylogenetic analysis of the 18S rDNA nuclear gene revealed no variability between the sequences and the phylogenetic analysis of the cox1 mitochondrial gene proved that B. vesperuginis could be divided into two subclades. Conclusion Our study showed a broad geographical distribution of B. vesperuginis in European bats, reporting its presence in five new host species (M. cf. alcathoe, M. bechsteinii, M. myotis, Pi. nathusii and V. murinus) and three new countries. Electronic supplementary material The online version of this article (10.1186/s13071-017-2536-3) contains supplementary material, which is available to authorized users.

In the framework of the biodiversity initiative and barcoding project “Austrian Barcode of Life” (ABOL) post mortem examinations of the gastro-intestinal tracts of different species of wild birds were carried out and several adult helminths were retrieved. In the gizzard of two barn owls (Tyto alba) and one common kestrel (Falco tinnuculus) acuariid nematodes belonging to the species Synhimantus (Synhimantus) laticeps (Rudolphi, 1819) were discovered. This report illustrates the identification of this parasitic nematode by morphometric comparison and scanning electron microscopic photographs. Furthermore, genetic identification of individual parasites based on a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear 18S ribosomal RNA gene was carried out. This report constitutes the first COI-based DNA barcoding of S. (S.) laticeps and its first record in the barn owl (Tyto alba) in Austria.

Red fox (Vulpes vulpes) is the most abundant wild canid species in Austria, and it is a well-known carrier of many pathogens of medical and veterinary concern. The main aim of the present study was to investigate the occurrence and diversity of protozoan, bacterial and filarial parasites transmitted by blood-feeding arthropods in a red fox population in western Austria. Blood (n = 351) and spleen (n = 506) samples from foxes were examined by PCR and sequencing and the following pathogens were identified: Babesia canis, Babesia cf. microti (syn. Theileria annae), Hepatozoon canis, Anaplasma phagocytophilum, Candidatus Neoehrlichia sp. and Bartonella rochalimae. Blood was shown to be more suitable for detection of Babesia cf. microti, whilst the spleen tissue was better for detection of H. canis than blood. Moreover, extremely low genetic variability of H. canis and its relatively low prevalence rate observed in this study may suggest that the parasite has only recently been introduced in the sampled area. Furthermore, the data presented here demonstrates, for the first time, the possible vertical transmission of H. canis from an infected vixen to the offspring, and this could explain the very high prevalence in areas considered free of its main tick vector(s).

During a three-year mosquito monitoring from 2014 to 2016, the strictly ornithophilic, originally Mediterranean species Orthopodomyia pulcripalpis (Rondani, 1872) was collected as single specimen for the first time in Austria in the district of Penzing in Vienna. Morphological species determination was confirmed by analysis of the mitochondrial cytochrome c oxidase subunit I gene. We thus not only confirm the existence of another mosquito species in Austria, but also add a new genus to the Austrian Culicidae taxa list.

Vector-borne diseases (VBD) are of major importance to human and animal health. In recent years, VBD have been emerging or re-emerging in many geographical areas, alarming new disease threats and economic losses. The precise diagnosis of many of these diseases still remains a major challenge because of the lack of comprehensive data available on accurate and reliable diagnostic methods. Here, we conducted a systematic and in-depth review of the former, current, and upcoming techniques employed for the diagnosis of VBD.

Background: Although camels represent a valuable source of food, wool and hide in many countries, in-depth information about their vector-borne pathogens is scarce compared to other animals. The aim of the current study was to characterize vector-borne protozoa in the blood of dromedaries from Iran by molecular tools. Methods: From June to July 2014, 200 peripheral blood samples were collected from asymptomatic one-humped camels in two provinces of Kerman and Sistan-va-Baloochestan in central and southeastern Iran. Microscopic examination was performed on Giemsa-stained blood smears, and drops of blood were spotted on Whatman FTA® cards for further analyses. Genomic DNA was extracted from the cards, and PCR was carried out for the detection of piroplasms and trypanosomes, followed by sequence analysis of positive samples. Results: One sample was positive Trypanosoma spp. trypomastigotes in light microscopy. PCR results revealed one positive sample each with Theileria annulata and Trypanosoma evansi. Conclusion: Camels were identified as hosts for bovine Mediterranean theileriosis in the investigated area. The presence of Tr. evansi, the causative agent of surra disease, was also confirmed in camels of Iran. Further studies are recommended in order to investigate their impact on the health and productivity of camels and other livestock in this region.

Nasopharyngeal myiases are caused by larvae of bot flies (Diptera: Oestridae), which have evolved a high specificity for their hosts. Bot flies (n = 916) were collected from 137 (57.6 %) out of 238 red deer (Cervus elaphus) hunted in Vorarlberg and Tyrol (Western Austria). After being stored in 75 % ethanol, larvae were identified to species level and developmental stage using morphological and morphometric keys. Larvae were also molecularly characterized by polymerase chain reaction (PCR) amplification and partial sequencing of the mitochondrial cytochrome oxidase subunit I gene. Morphological and molecular analysis allowed identification of larvae as Cephenemyia auribarbis and Pharyngomyia picta. Genetic variations were also examined within the specimens collected in both geographical locations.

Background Habitat types can affect vector and pathogen distribution and transmission dynamics. The prevalence and genetic diversity of Plasmodium spp. in two eastern chimpanzee populations—Kalinzu Forest Reserve, Uganda and Issa Valley, Tanzania—inhabiting different habitat types was investigated. As a follow up study the effect of host sex and age on infections patterns in Kalinzu Forest Reserve chimpanzees was determined. Methods Molecular methods were employed to detect Plasmodium DNA from faecal samples collected from savanna-woodland (Issa Valley) and forest (Kalinzu Forest Reserve) chimpanzee populations. ResultsBased on a Cytochrome-b PCR assay, 32 out of 160 Kalinzu chimpanzee faecal samples were positive for Plasmodium DNA, whilst no positive sample was detected in 171 Issa Valley chimpanzee faecal samples. Sequence analysis revealed that previously known Laverania species (Plasmodium reichenowi, Plasmodium billbrayi and Plasmodium billcollinsi) are circulating in the Kalinzu chimpanzees. A significantly higher proportion of young individuals were tested positive for infections, and switching of Plasmodium spp. was reported in one individual. Amongst the positive individuals sampled more than once, the success of amplification of Plasmodium DNA from faeces varied over sampling time. Conclusion The study showed marked differences in the prevalence of malaria parasites among free ranging chimpanzee populations living in different habitats. In addition, a clear pattern of Plasmodium infections with respect to host age was found. The results presented in this study contribute to understanding the ecological aspects underlying the malaria infections in the wild. Nevertheless, integrative long-term studies on vector abundance, Plasmodium diversity during different seasons between sites would provide more insight on the occurrence, distribution and ecology of these pathogens.

Wild ungulates may act as reservoirs of various vector borne pathogens that can infect humans and domestic animals. In the present study, blood samples from 196 red deer (Cervus elaphus) from Western Austria (Vorarlberg, Tyrol and Salzburg) were collected on filter paper and tested for Anaplasmataceae, Piroplasmida, Rickettsia and filarioid helminths using molecular tools. Babesia divergens was detected in ten (5.1%) and Anaplasma phagocytophilum in three (1.5%) of the 196 samples. Filarioid helminths, Rickettsia spp. and Theileria spp. were not detected. These findings indicate that red deer may serve as reservoirs of Babesia divergens and Anaplasma phagocytophilum in Western Austria. Further investigations are needed to assess the presence of these pathogens in ticks in this geographical region, and the significance of these pathogens in both animals and humans.

Background Recent gains in reducing the global burden of malaria are threatened by the emergence of Plasmodium falciparum resistance to artemisinins. The discovery that mutations in portions of a P. falciparum gene encoding kelch (K13)–propeller domains are the major determinant of resistance has provided opportunities for monitoring such resistance on a global scale. Methods We analyzed the K13-propeller sequence polymorphism in 14,037 samples collected in 59 countries in which malaria is endemic. Most of the samples (84.5%) were obtained from patients who were treated at sentinel sites used for nationwide surveillance of antimalarial resistance. We evaluated the emergence and dissemination of mutations by haplotyping neighboring loci. Results We identified 108 nonsynonymous K13 mutations, which showed marked geographic disparity in their frequency and distribution. In Asia, 36.5% of the K13 mutations were distributed within two areas — one in Cambodia, Vietnam, and Laos and the other in western Thailand, Myanmar, and China — with no overlap. In Africa, we observed a broad array of rare nonsynonymous mutations that were not associated with delayed parasite clearance. The gene-edited Dd2 transgenic line with the A578S mutation, which expresses the most frequently observed African allele, was found to be susceptible to artemisinin in vitro on a ring-stage survival assay. Conclusions No evidence of artemisinin resistance was found outside Southeast Asia and China, where resistance-associated K13 mutations were confined. The common African A578S allele was not associated with clinical or in vitro resistance to artemisinin, and many African mutations appear to be neutral. (Funded by Institut Pasteur Paris and others.)

Eucoleus boehmi (syn. Capillaria boehmi) is a canine trichuroid nematode affecting the upper respiratory airways (i.e., nasal cavity and paranasal sinuses) of dogs, foxes, and wolves. In the past few years, reports in dogs and wild canids have increased from across Europe, but data on its occurrence and distribution in Austria is scanty. A total of 47 red foxes (Vulpes vulpes) from the two westernmost provinces (Tyrol and Vorarlberg) of Austria were therefore examined for the presence of E. boehmi at necropsy. Eggs and adult nematodes were identified morphologically and molecularly (cox1) as E. boehmi. These nematodes were found in 26 (78.8 %) and 13 (92.9 %) foxes from Tyrol and Vorarlberg, respectively, with an overall prevalence of 83.0 % (39/47). The prevalence rate of infection recorded in this study is among the highest in Europe. These results suggest that foxes may represent an important source of infection for dogs and other canids, but further studies are needed to elucidate the transmission dynamics.

Background: Dirofilaria repens and D. immitis are filarioid helminths with domestic and wild canids as main hosts and mosquitoes as vectors. Both species are known to cause zoonotic diseases, primarily pulmonary (D. immitis), ocular (D. repens), and subcutaneous (D. repens) dirofilariosis. Both D. immitis and D. repens are known as invasive species, and their distribution seems associated with climate change. Until very recently, both species were known to be nonendemic in Austria. Methodology and principal findings: Metadata on introduced and possibly autochthonous cases of infection with Dirofilaria sp. in dogs and humans in Austria are analysed, together with analyses of mosquito populations from Austria in ongoing studies. In Austria, most cases of Dirofilaria sp. in humans (30 cases of D. repens-six ocular and 24 subcutaneous) and dogs (approximately 50 cases-both D. immitis and D. repens) were most likely imported. However, occasionally infections with D. repens were discussed to be autochthonous (one human case and seven in dogs). The introduction of D. repens to Austria was confirmed very recently, as the parasite was detected in Burgenland (eastern Austria) for the first time in mosquito vectors during a surveillance program. For D. immitis, this could not be confirmed yet, but data from Germany suggest that the successful establishment of this nematode species in Austria is a credible scenario for the near future. Conclusions: The first findings of D. repens in mosquito vectors indicate that D. repens presumably invaded in eastern Austria. Climate analyses from central Europe indicate that D. immitis also has the capacity to establish itself in the lowland regions of Austria, given that both canid and culicid hosts are present.

Background Culex pipiens complex taxa differ in behaviour, ecophysiology and epidemiologic importance. Despite their epidemiologic significance, information on genetic diversity, occurrence and seasonal and spatial distribution patterns of the Cx. pipiens complex is still insufficient. Assessment of seasonal and spatial distribution patterns of Culex pipiens forms and their congener Cx. torrentium is crucial for the understanding of their vector–pathogen dynamics. Methods Female mosquitoes were trapped from April–October 2014 twice a month for a 24-h time period with BG-sentinel traps at 24 sampling sites in eastern Austria, using carbon dioxide as attractant. Ecological forms of Cx. pipiens s.l. and their hybrids were differentiated using the CQ11 locus, and Cx. pipiens forms and their congener Cx. torrentium using the ACE-2 gene. Differential exploitation of ecological niches by Cx. pipiens forms and Cx. torrentium was analysed using likelihood ratio tests. Possible effects of environmental parameters on these taxa were tested using PERMANOVA based on distance matrices and, if significant, were modelled in nMDS ordination space to estimate non-linear relationships. Results For this study, 1476 Culex spp. were sampled. Culex pipiens f. pipiens representing 87.33 % of the total catch was most abundant, followed by hybrids of both forms (5.62 %), Cx. torrentium (3.79 %) and Cx. pipiens f. molestus (3.25 %). Differences in proportional abundances were found between land cover classes. Ecological parameters affecting seasonal and spatial distribution of these taxa in eastern Austria are precipitation duration, air temperature, sunlight and the interaction term of precipitation amount and the Danube water level, which can be interpreted as a proxy for breeding habitat availability. Conclusions The Cx. pipiens complex of eastern Austria comprises both ecologically different forms, the mainly ornithophilic form pipiens and the mainly mammalophilic and anthropophilic form molestus. Heterogeneous agricultural areas as areas of coexistence may serve as hybridization zones, resulting in potential bridge vectors between birds and humans. Occurrence, seasonal and spatial distribution patterns of the Cx. pipiens complex and Cx. torrentium and the presence of hybrids between both forms were quantified for the first time in Austria. These findings will improve the knowledge of their vector–pathogen dynamics in this country.

Despite the economic importance of camels, the parasites that affect them have not received adequate attention so far, and molecular studies are scarce compared to other livestock. In this study we characterized peripheral blood microfilariae in 200 healthy one-humped camels (Camelus dromedarius) from south-east Iran by microscopy and molecular tools to receive a more detailed insight into prevalence and species that affect them. Moreover, adult specimens of the filarial nematode Dipetalonema evansi were collected from the carcass of an infected animal. Microscopic examination was performed on Giemsa stained blood smears, and blood was also spotted on Whatman FTA® cards for DNA analysis. Genomic DNA was extracted, and PCR was carried out for the detection of filaroid helminths, followed by sequence analysis of positive samples. Four samples were positive for microfilariae by microscopy, while 16 animals (8%) were positive by PCR. Sequence analysis revealed D. evansi in all cases. Phylogenetic analysis of a cytochrome C oxidase subunit I (COI) sequence of filaroid nematodes showed that most species in a single genus cluster in the same clade; however D. evansi and D. gracile are not monophyletic and branch rather at the base of the tree. Further studies on the life cycle of D. evansi, specifically the identification of intermediate host(s) have become feasible with the provision of the first specific COI sequences in this study.

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.

In Bosnia and Herzegovina, the tick fauna is very diverse, but data on the occurrence of zoonotic tick-borne bacteria is 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%) out 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 Glamoč Municipality (southwestern 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. holarctica (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%). The present 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.

Background Candidatus Neoehrlichia came under the focus of recent research in terms of human and pet relevance. Candidatus Neoehrlichia mikurensis seems to be relatively abundant in animals and humans from Central European countries, whereas Candidatus Neoehrlichia lotoris was found solely in raccoons from the USA. Findings Spleen samples from a total of 164 red foxes, originating from two western provinces in Austria (Tyrol and Vorarlberg), were collected and examined for the presence of tick-borne bacteria of the family Anaplasmataceae by PCR and sequencing. In a fox sample originating from Vorarlberg Candidatus Neoehrlichia sp. was found, which is genetically (16S rRNA, groEL) closely related to Candidatus Neoehrlichia lotoris but clearly distinct from Candidatus Neoehrlichia mikurensis. Conclusions The present study revealed, for the first time, the occurrence of Candidatus Neoehrlichia sp. in a red fox worldwide. A continuing screening of wild carnivores, especially foxes, and ticks for this potential pathogen is required to evaluate the actual occurrence and distribution of these bacteria. Further research is needed to elucidate the relationships of Neoehrlichia, as well as their reservoir and impact on wildlife, pets and humans.

The present study focuses on development of phytochemical methods for quality assessment of two West-African Cochlospermum species (Cochlospermum planchonii and Cochlospermum tinctorium) traditionally used for malaria treatment in Burkina Faso. Antimalarial activity of preparations from dried rhizomes (decoction) was tested against the chloroquine-sensitive Plasmodium strain 3D7 using the histidine-rich protein II (HRP2) drug susceptibility assay and compared with extract preparations using organic solvents of different polarity. Two main apocarotenoids were isolated from rhizomes of C. planchonii and unambiguously identified as dihydrocochloxanthine and cochloxanthine by spectroscopic methods. Comparative HPLC analyses of thirty-nine (39) samples from markets and from collections in natural habitats of both species showed a high variability in the accumulation of cochloxanthines and related carotenoids which were proven to be characteristic for rhizomes of both species and generally absent in leaves. Furthermore, content of total phenolics and antioxidant activities (DPPH and FRAP) as well as haemolytic activity of various extracts was tested. The HPLC method presented here was validated and provides a good separation of both compounds including 10 minor carotenoids. Extracts from both species and pure cochloxanthine offered pronounced antioxidant activities and weak haemolytic activity while, in contrast, dihydrocochloxanthine had a strong haemolytic effect at the highest concentration analysed. However, cochloxanthine as well as dihydrocochloxanthine showed erythroprotective effects against the haemolytic activity of the reference saponin. Moderate antiplasmodial activity between 16 and 63μg/ml were observed with all tested extracts, and lower IC50 values were obtained with pure dihydrocochloxanthine (IC50=6.9μg/ml), cochloxanthine (IC50=6.8μg/ml), the DCM fraction (IC50=2.4μg/ml) and the ethyl acetate fraction (IC50=11.5μg/ml) derived from a methanolic extract of C. planchonii. This study shows a major variability of carotenoid content and antiplasmodial activity of both C. planchonii and C. tinctorium. The high haemolytic activity of dihydrocochloxanthine (at 100μg/ml) should be considered as a selection criterion for choosing species phenotypes for treatment.

Plasmodium falciparum accounts for approximately 60% of malaria cases in Ethiopia and artemether-lumefantrine has been used as a first-line treatment for uncomplicated P. falciparum malaria since 2004. The aim of this study was to assess the therapeutic efficacy of artemether-lumefantrine (AL) for the treatment of uncomplicated P. falciparum malaria in north-western Ethiopia. A 28-day one-arm, prospective evaluation of the clinical and parasitological response to the first-line treatment for uncomplicated P. falciparum malaria was conducted in Enfranze Health Centre in accordance with the 2009 WHO efficacy study guidelines. Patients were treated with a 3-day course of AL and clinical and parasitological parameters were monitored over a 28-day follow-up. All data from recruited patients were imported into an electronic data base and Kaplan-Meier survival analysis was used for analysing primary [early treatment failures (ETF), late clinical failure (LCF), late parasitological failures (LPF), and adequate clinical and parasitological response (ACPR)] and secondary (PCT, GCT and FCT) outcomes. Eighty patients were enrolled and all of them completed the 28-day follow-up period. The PCR-corrected cure rate was 95.0% (95% CI 87.0-98.4%) and there were two ETF, one LCF and three LPF. Two of the LPF were classified as re infections by PCR. Seventy three point seven five percent, 91.25 and 95% of patients had cleared their parasitaemia by days 1, 2, and 3, respectively, and 75, 91.25 and 96.25% of patients had cleared their fever by days 1, 2, and 3. All patients completely cleared their gametocytes by day 7. The relatively high cure rate, low proportion of patients still positive on day 3 as well as parasite clearance times in this study would indicate no imminent threat of artemisinin resistance development in the region. However, the threat of spreading or de novo development of artemisinin resistance warrants regular monitoring of drug efficacy throughout the region.

As a consequence of climate change and globalization alien mosquito species and also mosquito-borne pathogens (e. g. Dirofilaria) have been imported or immigrated into areas of Central Europe. Currently, 46 different mosquito species have been described from Austria, and four of those are alien species. These include the Tiger mosquito (Aedes albopictus) as a potential vector of dengue virus, and the Asian Bush mosquito (Ochlerotatus japonicus japonicus). Beside the increase of mosquito species also an increasing number of cases of dirofilariosis (caused by the filarioid nematodes Dirofilaria repens or D. immitis) were observed in the last years. First autochthonous findings of D. repens in humans and dogs had been discussed previously. However, only recently the detection of D. repens in mosquitoes confirmed its autochthonous presence in Austria. This highlights the increasing importance of monitoring of indigenous as well as alien mosquito species for the surveillance of mosquito-borne pathogens.

Domestic cats can be infected with a variety of enteric protozoa. Genotyping of protozoan species, especially Giardia as the most common, can improve assessment of their relevance as zoonotic agents. For an overview on the occurrence of feline enteric protozoa, 298 faecal samples of cats from private households, catteries and animal shelters in Austria were collected. All samples were examined by flotation and using a rapid test for Giardia (FASTest®). For the detection of Tritrichomonas blagburni, freshly voided faeces (n = 40) were processed using a commercial culturing system (InPouch™ TF-Feline). Genotyping was done at the β-giardin gene loci (each sample) and triosephosphate isomerase gene loci (positive samples) for Giardia and at the 18S rRNA gene (positive samples) for Cryptosporidium. Thirty-seven samples (12.4 %) were positive for Giardia by flotation and/or using a rapid test. Cryptosporidium was present in 1.7 %, Cystoisospora in 4.0 %, Sarcocystis in 0.3 % and T. blagburni in 2.5 % of the samples. Genotyping revealed Giardia cati, the potentially zoonotic Giardia duodenalis and Cryptosporidium felis. Most of the infected cats had no diarrhoea. Cats from shelters were significantly more often infected than owned cats (p = 0.01). When comparing Giardia detection methods, the rapid test had a higher sensitivity than flotation. Polymerase chain reaction (PCR) results were mostly independent from the other two tests.

The trematode This is a report of coinfection of both the trematode and the nematode, in a polecat from Lower Austria, as well as the first attempt to barcode

Background Red foxes (Vulpes vulpes) have recently been recognized as potential reservoirs of several vector-borne pathogens and a source of infection for domestic dogs and humans, mostly due to their close vicinity to urban areas and frequent exposure to different arthropod vectors. The aim of this study was to investigate the presence and distribution of Babesia spp., Hepatozoon canis, Anaplasma spp., Bartonella spp., `Candidatus Neoehrlichia mikurensis', Ehrlichia canis, Rickettsia spp. and blood filaroid nematodes in free-ranging red foxes from Bosnia and Herzegovina. Methods Spleen samples from a total of 119 red foxes, shot during the hunting season between October 2013 and April 2014 throughout Bosnia and Herzegovina, were examined for the presence of blood vector-borne pathogens by conventional PCRs and sequencing. Results In the present study, three species of apicomplexan parasites were molecularly identified in 73 red foxes from the entire sample area, with an overall prevalence of 60.8%. The DNA of B. canis, B. cf. microti and H. canis was found in 1 (0.8%), 38 (31.9%) and 46 (38.6%) spleen samples, respectively. In 11 samples (9.2%) co-infections with B. cf. microti and H. canis were detected and one fox harboured all three parasites (0.8%). There were no statistically significant differences between geographical region, sex or age of the host in the infection prevalence of B. cf. microti, although females (52.9%; 18/34) were significantly more infected with H. canis than males (32.9%; 28/85). The presence of vector-borne bacteria and filaroid nematodes was not detected in our study. Conclusion This is the first report of B. canis, B. cf. microti and H. canis parasites in foxes from Bosnia and Herzegovina and the data presented here provide a first insight into the distribution of these pathogens among the red fox population. Moreover, the relatively high prevalence of B. cf. microti and H. canis reinforces the assumption that this wild canid species might be a possible reservoir and source of infection for domestic dogs.

Background Both Dirofilaria repens and recently D. immitis are known to be endemic in Hungary. As one of several recent cases, the fatal case of a dog infested with D. immitis in Szeged, Southern Hungary, received attention from the media. Hence it was decided to catch mosquitoes in the garden where the dog lived to screen for filarioid helminths and Plasmodium spp. using molecular tools.Methods Mosquitoes were caught in Szeged, in the garden where the infected dog was kept, in July 2013 with M-360 electric mosquito traps and were stored in ethanol until further procedure. Female mosquitoes were classified to genus level by morphology. Each mosquito was homogenized and analyzed for filarioid helminths and avian malaria using standardized PCR techniques. Positive mosquito samples were further identified to species level by comparing a section of the mitochondrial COI gene to GenBank® entries.ResultsIn this study, 267 blood-fed mosquitoes were caught in July 2013 in Szeged. Subsequent molecular screening revealed that not only D. immitis was present in the analyzed specimens but also DNA of D. repens, Setaria tundra and Plasmodium spp. was confirmed.Conclusions The analysis of blood-fed mosquitoes for the diagnosis of Dirofilaria spp. and other mosquito-borne pathogens seems to be an adequate technique to evaluate if filarioid helminths are present in a certain area. Usually only unfed female mosquitoes are analyzed for epidemiological studies. However, blood-fed mosquitoes can only be used for screening if a pathogen is present because the role of the mosquito as vector cannot be classified (blood of bitten host). Furthermore, Setaria tundra was confirmed for the first time in Hungary.

The term helminth summarizes numerous groups of metazoan parasites which are phylogenetically unrelated– Nematoda, Plathelminthes (Trematoda, Cestoda) and Acanthocephala. Most helminths lack discriminating features at least in some stages and are therefore difficult to determine to species level. Although many of these parasites are of veterinary and/or medical importance, DNA barcode sequences are still missing for most of them. Parasite DNA-barcodes enable the identification especially of specimen for diagnostic purposes that cannot be determined otherwise. Due to the phylogenetic distance between the taxa, however, group group-specific primers are required for barcoding within this mixed group of animals, which can pose a technical challenge. To highlight the problems parasitologists are faced with, the case of a filarioid helminth in the skin is described. Filarioid specimens were found in subcutaneous nodules in a red deer (Cervus elaphus) from Tyrol and barcoded. However, sequence analysis of several additional genes was necessary to confirm the worm as Onchocerca jakutensis, due to the lack of available DNA-barcodes. In the meantime, barcodes for this species are available and can be used to detect infections especially in aberrant hosts or locations.

Austria's mammalian wildlife comprises a large variety of species, acting and interacting in different ways as reservoir and intermediate and definitive hosts for different pathogens that can be transmitted to pets and/or humans. Foxes and other wild canids are responsible for maintaining zoonotic agents, e.g. Echinococcus multilocularis, as well as pet-relevant pathogens, e.g. Hepatozoon canis. Together with the canids, and less commonly felids, rodents play a major role as intermediate and paratenic hosts. They carry viruses such as tick-borne encephalitis virus (TBEV), bacteria including Borrelia spp., protozoa such as Toxoplasma gondii, and helminths such as Toxocara canis. The role of wild ungulates, especially ruminants, as reservoirs for zoonotic disease on the other hand seems to be negligible, although the deer filaroid Onchocerca jakutensis has been described to infect humans. Deer may also harbour certain Anaplasma phagocytophilum strains with so far unclear potential to infect humans. The major role of deer as reservoirs is for ticks, mainly adults, thus maintaining the life cycle of these vectors and their distribution. Wild boar seem to be an exception among the ungulates as, in their interaction with the fox, they can introduce food-borne zoonotic agents such as Trichinella britovi and Alaria alata into the human food chain.

Mosquitoes (Diptera: Culicidae) are important vectors for a wide range of pathogenic organisms. As large parts of the human population in developed countries live in cities, the occurrence of vector-borne diseases in urban areas is of particular interest for epidemiologists and public health authorities. In this study, we investigated the mosquito occurrence in the city of Vienna, Austria, in order to estimate the risk of transmission of mosquito-borne diseases. Mosquitoes were captured using different sampling techniques at 17 sites in the city of Vienna. Species belonging to the Culex pipiens complex (78.8 %) were most abundant, followed by Coquillettidia richiardii (10.2 %), Anopheles plumbeus (5.4 %), Aedes vexans (3.8 %), and Ochlerotatus sticticus (0.7 %). Individuals of the Cx. pipiens complex were found at 80.2 % of the trap sites, while 58.8 % of the trap sites were positive for Cq. richiardii and Ae. vexans. Oc. sticticus was captured at 35.3 % of the sites, and An. plumbeus only at 23.5 % of the trap sites. Cx. pipiens complex is known to be a potent vector and pathogens like West Nile virus (WNV), Usutu virus (USUV), Tahyna virus (TAHV), Sindbis virus (SINV), Plasmodium sp., and Dirofilaria repens can be transmitted by this species. Cq. richiardii is a known vector species for Batai virus (BATV), SINV, TAHV, and WNV, while Ae. vexans can transmit TAHV, USUV, WNV, and Dirofilaria repens. An. plumbeus and Oc. sticticus seem to play only a minor role in the transmission of vector-borne diseases in Vienna. WNV, which is already wide-spread in Europe, is likely to be the highest threat in Vienna as it can be transmitted by several of the most common species, has already been shown to pose a higher risk in cities, and has the possibility to cause severe illness.

During a two-year mosquito monitoring program in Danubean wetlands (Lower Austria) from 2012 to 2013 the newly introduced culicid species Culiseta (Allotheobaldia) longiareolata (Macquart 1838) was detected. This species invaded southern Austria from Italy and Slovenia in 2012 and is now reconfirmed in an artificial water storage container in October and November 2013 in northeastern Austria (Orth an der Donau, Lower Austria) at 150 m.a.s.l., N48°8’40.68’’, E16°41’57.85’’. A total of 100 larvae, three pupae and 37 adults (14 males and 23 females) were sampled. Species were determined morphologically and genetically by barcode methods. Biometrical data such as larval head capsule widths and body lengths were measured in order to define the four larval instars for lifecycle reconstruction. This species had one generation emerging in October/November 2013. Despite intensive monitoring, development stages of Cs. longiareolata were exclusively found in artificial, but not in natural breeding habitats.

Background The red fox (Vulpes vulpes) is a widespread species, harbouring many pathogens relevant for humans and pets. Indeed, Anaplasma spp., Ehrlichia canis and Rickettsia spp. among the bacteria and Hepatozoon canis as well as Babesia sp. among the parasites have been the focus of several studies.FindingsIn a cohort of 36 foxes shot on one day in the north-eastern part of Austria, Babesia microti-like pathogens were found in 50%, while H. canis was detected in 58.3% of the samples. The spleen was more useful for detection of H. canis, whereas B. microti-like parasites were more frequently found in the blood. Bacteria could not be confirmed in any of the cases to demonstrate the occurrence of such tick-borne pathogens using PCR and sequencing on blood and spleen samples.Conclusions The occurrence of B. microti-like and H. canis parasites raised many questions, because these infections have never been found autochthonously in dogs. Furthermore in the case of H. canis the main vector tick, Rhipicephalus sanguineus, is absent in the sampling area, leaving space for further hypotheses for transmission such as vertical transmission, transmission via ingestion of prey animals or other vector ticks. Further studies are needed to evaluate the risks for pets in this area. PCRs delivered differing results with the different tissues, suggesting the use of both spleen and blood to obtain an integral result.

Background: The emergence of artemisinin-resistant Plasmodium falciparum in Southeast Asia threatens malaria treatment efficacy. Mutations in a kelch protein encoded on P. falciparum chromosome 13 (K13) have been associated with resistance in vitro and in field samples from Cambodia. Methods: P. falciparum infections from artesunate efficacy trials in Bangladesh, Cambodia, Laos, Myanmar, and Vietnam were genotyped at 33 716 genome-wide single-nucleotide polymorphisms (SNPs). Linear mixed models were used to test associations between parasite genotypes and parasite clearance half-lives following artesunate treatment. K13 mutations were tested for association with artemisinin resistance, and extended haplotypes on chromosome 13 were examined to determine whether mutations arose focally and spread or whether they emerged independently. Results: The presence of nonreference K13 alleles was associated with prolonged parasite clearance half-life (P = 1.97 × 10(-12)). Parasites with a mutation in any of the K13 kelch domains displayed longer parasite clearance half-lives than parasites with wild-type alleles. Haplotype analysis revealed both population-specific emergence of mutations and independent emergence of the same mutation in different geographic areas. Conclusions: K13 appears to be a major determinant of artemisinin resistance throughout Southeast Asia. While we found some evidence of spreading resistance, there was no evidence of resistance moving westward from Cambodia into Myanmar.

Background Spreading resistance of Plasmodium falciparum to existing drugs calls for the search for novel anti-malarial drugs and combinations for the treatment of falciparum malaria. Methods In vitro and ex vivo investigations were conducted with fresh P. falciparum field isolates and culture-adapted P. falciparum clones to evaluate the anti-malarial potential of mirincamycin, a lincosamide, alone and in combination with tafenoquine (TQ), dihydroartemisinin (DHA), and chloroquine (CQ). All samples were tested in a histidine-rich protein 2 (HRP2) drug susceptibility assay. Results Interaction analysis showed additive to synergistic interaction profiles with these potential partner drugs, with an overall geometric mean fractional inhibitory concentration at 50% inhibition (FIC50) of 0.78, 0.80 and 0.80 for mirincamycin with TQ, DHA, and CQ, respectively. Antagonism was not found in any of the tested field isolates or clones. The strongest tendency toward synergy (i.e. the lowest FIC) was seen with a combination ratio of 1:0.27 to 1:7.2 (mean 1:2.7) for the combination with tafenoquine. The optimal combination ratios for DHA and CQ were 1:444.4 to 1:36,000 (mean 1:10,755.5) and 1:2.7 to 1:216 (mean 1:64.5), respectively. No evidence of an activity correlation (i.e. potential cross-resistance) with DHA, mefloquine, quinine or chloroquine was seen whereas a significant correlation with the activity of clindamycin and azithromycin was detected. Conclusions Mirincamycin combinations may be promising candidates for further clinical investigations in the therapy and prophylaxis of multidrug-resistant falciparum malaria or in combination with 4 or 8-aminoquinolines for the treatment and relapse prevention of vivax malaria.

Background In Europe animal and human infections due to Dirofilaria repens are increasing. Findings In a nationwide screening for filarioid parasites in Austria, 7,632 mosquitoes were collected from June till October 2012 and divided into 437 pools according to same trapping date and sight and mosquito species. For the molecular detection, a real-time PCR approach was followed by conventional PCR. D. repens was detected in the villages Moerbisch and Rust, Burgenland in one Anopheles maculipennis group and one Anopheles algeriensis species pool, respectively. Conclusions The geographical distribution of the two positive pools points to the invasion of D. repens from Eastern neighboring countries. The finding of D. repens in mosquito vectors suggests the occurrence of the causative agent for cutaneous dirofilariosis in Austria.

Background: Malaria remains one of the leading communicable diseases in Ethiopia. Early diagnosis combined with prompt treatment is one of the main strategies for malaria prevention and control. Despite its limitation, Giemsa microscopy is still considered to be the gold standard for malaria diagnosis. This study aimed to compare the performance of Giemsa microscopy with nested polymerase chain reaction (nPCR) for the diagnosis of malaria in north-west Ethiopia. Methods: A cross sectional study was conducted in public health facilities in North Gondar, from March 2013 to April 2013. A total number of 297 subjects with suspected malaria were enrolled in the study. Finger-prick blood samples were collected and examined for Plasmodium parasites using Giemsa microscopy and standard nPCR. Results: Among the study participants, 61.6% (183/297) patients tested positive for malaria by Giemsa microscopy of which, 72.1% (132/183) and 27.9% (51/183) were diagnosed as Plasmodium falciparum and Plasmodium vivax, respectively. By nPCR, 73.1% (217/297) were malaria-positive. Among microscopy-negative samples, 13.1% (39/297) samples turned malaria-positive in nPCR. In nPCR, the rate of mixed Plasmodium infections was 4.7% (14/297) and 3.03% (9/297) were positive for Plasmodium ovale. Using nPCR as reference the sensitivity, specificity, positive predictive and negative predictive values of Giemsa microscopy were 82.0%, 93.8%, 97.3% and 65.8%, respectively, with a good agreement (κ = 0.668) to nested PCR. The sensitivity and specificity of Giemsa microscopy in identifyingP. falciparium infections were 74.0% and 87.4% and 63.2% and 96.5% for P. vivax infections, respectively. Conclusion: Although Giemsa microscopy remains the gold standard for malaria diagnosis in resource-limited environments, its sensitivity and specificity as compared to nPCR is limited suggesting exploration of novel rapid and simplified molecular techniques for malaria-endemic countries. A high rate of misclassification and misidentification highlights the importance of adequate training for staff involved in malaria diagnosis.

Although the prevalence of malaria remains high in parts of Bangladesh, there continues to be a substantial shortage of information regarding the less common malaria parasites such as Plasmodium malariae or Plasmodium knowlesi. Recent studies indicate that P. malariae may be extremely rare, and so far, there are no data on the presence (or absence) of P. knowlesi in southeastern Bangladesh. Genus- and species-specific nested polymerase chain reaction (PCR) analysis of the small subunit ribosomal RNA gene was performed to assess the presence and prevalence of P. malariae and P. knowlesi in 2,246 samples originating from asymptomatic and febrile participants of a cross-sectional and a febrile illnesses study in the Chittagong Hill Tracts in southeastern Bangladesh. P. malariae was detected in 60 samples (2.7 %) corresponding to 8 % of the 746 samples giving positive PCR results for Plasmodium sp., mainly because of the high prevalence (9.5 %) among asymptomatic study participants testing positive for malaria. Symptomatic cases were more common (4.3 % of all symptomatic malaria cases) during the dry season. Parasitemias were low (1,120–2,560/μl in symptomatic and 120–520/μl in asymptomatic carriers). Symptomatic patients presented mild to moderate symptoms like fever, chills, headache, dizziness, fatigue and myalgia. Although both the intermediate as well as the definite host are known to be endemic in southeastern Bangladesh, no evidence for the presence of P. knowlesi was found. We conclude that the role of P. malariae is highly underestimated in rural Bangladesh with major implications for malaria control and elimination strategies.

In malaria-endemic regions any febrile case is likely to be classified as malaria based on presumptive diagnosis largely caused by a lack of diagnostic resources. A district-wide prevalence study assessing etiologies of fever in 659 patients recruited in rural and semi-urban areas of Bandarban district in southeastern Bangladesh revealed high proportions of seropositivity for selected infectious diseases (leptospirosis, typhoid fever) potentially being misdiagnosed as malaria because of similarities in the clinical presentation. In an area with point prevalences of more than 40% for malaria among fever cases, even higher seroprevalence rates of leptospirosis and typhoid fever provide evidence of a major persistent reservoir of these pathogens.

WHO has reported that RDT and microscopy-confirmed malaria cases have declined in recent years. However, it is still unclear if this reflects a real decrease in incidence in Bangladesh, as particularly the hilly and forested areas of the Chittagong Hill Tract (CHT) Districts report more than 80% of all cases and deaths. PCR-confirmed surveillance and epidemiological data on malaria from the CHT are limited; existing data report Plasmodium falciparum and Plasmodium vivax as the dominant species. A cross-sectional survey was conducted in the District of Bandarban, the southernmost of the three Hill Tracts Districts, to collect district-wide malaria prevalence data from one of the regions with the highest malaria endemicity in Bangladesh. A multistage cluster sampling technique was used to collect blood samples from febrile and afebrile participants and malaria microscopy and standardized nested PCR for diagnosis were performed. Demographic data, vital signs and splenomegaly were recorded. Malaria prevalence across all subdistricts in the monsoon season was 30.7% (95% CI: 28.3-33.2) and 14.2% (95% CI: 12.5-16.2) by PCR and microscopy, respectively. Plasmodium falciparum mono-infections accounted for 58.9%, P. vivax mono-infections for 13.6%, Plasmodium malariae for 1.8%, and Plasmodium ovale for 1.4% of all positive cases. In 24.4% of all cases mixed infections were identified by PCR. The proportion of asymptomatic infections among PCR-confirmed cases was 77.0%, oligosymptomatic and symptomatic cases accounted for only 19.8 and 3.2%, respectively. Significantly (p < 0.01) more asymptomatic cases were recorded among participants older than 15 years as compared to younger participants, whereas prevalence and parasite density were significantly (p < 0.01) higher in patients younger than 15 years. Spleen rate and malaria prevalence in two to nine year olds were 18.6 and 34.6%, respectively. No significant difference in malaria prevalence and parasite density was observed between dry and rainy season. A large proportion of asymptomatic plasmodial infections was found which likely act as a reservoir of transmission. This has major implications for ongoing malaria control programmes that are based on the treatment of symptomatic patients. These findings highlight the need for new intervention strategies targeting asymptomatic carriers.

Calodium hepaticum (syn. Capillaria hepatica) is a globally distributed zoonotic nematode with low host specificity and a high affinity to the liver. Although murid rodents are the main definite hosts, various other mammals can be affected with hepatic capillariasis: non-murid rodents, Insectivora, Chiroptera, Lagomorpha, Artiodactyla, Perissodactyla, Hyracoidea, Marsupialia, Carnivora, and Primates. Overall, more than 180 mammalian species (including humans) are known as suitable hosts of this pathogen. This review gives an overview of the distribution and host spectrum of C. hepaticum in non-Muroidean mammals in wildlife and zoos as well as in domesticated and laboratory animals. Furthermore, the role of spurious infections in animals and the dissemination of C. hepaticum by mammalian and non-mammalian animals are summarized.

Calodium hepaticum (syn. Capillaria hepatica) is a worldwide-distributed species of zoonotic nematodes with a high affinity to the liver. Several rodent species of the super-family Muroidea serve as main hosts for this pathogen. C. hepaticum has been found in Muroidean hosts in more than 60 countries in Europe; North, Central, and South America; Asia; Africa; and Oceania. C. hepaticum was documented in more than 90 Muroidean rodent species (Murinae, Deomyinae, Arvicolinae, Neotominae, Cricetinae, Sigmodontinae, Gerbillinae, and Cricetomyinae). Globally, the Norway rat (Rattus norvegicus) seems to be the main host species for this nematode. However, locally high prevalences (above 50 %) have also been observed in several other synanthropic (commensal and non-commensal) Muroidea species (e.g., Rattus tanezumi, Ondatra zibethicus, Apodemus sylvaticus). This review gives an overview of the distribution and host spectrum of C. hepaticum in Muroidea host species.

A novel direct PCR assay for the detection of Dirofilaria spp. from EDTA blood, Knott test, FTA cards, adult filarial worms, skin nodules and Dirofilaria spp.-infected mosquitoes was tested. Larval and adult DNA of Dirofilaria spp. from FTA cards, from the mosquito vector and from worm fragments without prior DNA extraction was successfully obtained. As little as 3.11 larvae/100 μl blood on FTA cards could be detected. Thus, direct PCR is capable of directly detecting first larval stages in the blood, third larval stages in the mosquito vector and pieces of mature stages of Dirofilaria spp. The assay is a rapid, sensitive and cost-effective alternative to standard PCR.

Recent molecular studies indicate that Plasmodium ovale malaria is caused by two closely related species of protozoan parasites, thereby imposing new challenges for detection and species differentiation. This minireview explores the potential value of innovative methods for the molecular diagnosis of malaria with a strong emphasis on the discrimination and genotyping of P. ovale wallikeri and P. ovale curtisi as well as tools for the simultaneous detection of P. ovale sp. An update for the widely used NP-1993 to NP-2005 (SSU rRNA) protocols for all human malaria parasites is discussed.

In Ethiopia Plasmodium falciparum and Plasmodium vivax are the dominant species accounting for roughly 60 and 40% of malaria cases, respectively. Recently a major shift from P. falciparum to P. vivax has been observed in various parts of the country but the epidemiology of the other human malaria species, Plasmodium ovale spp. and Plasmodium malariae remains poorly understood. The aim of this study was to assess P. ovale curtisi and wallikeri infection in north-west Ethiopia by using microscopy and nested PCR. A health institution-based survey using non-probability sampling techniques was conducted at Maksegnet, Enfranze and Kola Diba health centres and Metema hospital in North Gondar. Three-hundred patients with signs and symptoms consistent with malaria were included in this study and capillary blood was collected for microscopic examination and molecular analysis of Plasmodium species. Samples were collected on Whatman 903 filter papers, stored in small plastic bags with desiccant and transported to Vienna (Austria) for molecular analysis. Data from study participants were entered and analysed by SPSS 20 software. Out of 300 study participants (167 males and 133 females), 184 samples were classified positive for malaria (133 P. falciparum and 51 P. vivax) by microscopy. By species-specific PCR 233 Plasmodium spp (95% CI: 72.6-82) were detected and the majority 155 (66.5%, 95% CI: 60.2-72.3) were P. falciparum followed by P. vivax 69 (29.6%, 95% CI; 24.1-35.8) and 9 (3.9%, 95% CI: 2-7.2) samples were positive for P. ovale. Seven of P. ovale parasites were confirmed as P. ovale wallikeri and two were confirmed as P. ovale curtisi. None of the samples tested positive for P. malariae. During microscopic examination there were high (16.3%) false negative reports and all mixed infections and P. ovale cases were missed or misclassified. This study indicates that P. ovale malaria is under-reported in Ethiopia and provides the first known evidence of the sympatric distribution of indigenous P. ovale wallikeri and P. ovale curtisi in Ethiopia. Therefore, further studies assessing the prevalence of the rare species P. ovale and P. malariae are urgently needed to better understand the species distribution and to adapt malaria control strategies.

Theileria spp. are intracellular protozoa transmitted by ixodid ticks. T. parva and T. annulata are highly pathogenic and responsible for serious disease in domestic ruminants in tropical and subtropical countries. However, asymptomatic findings of Theileria sp. in wild ungulates lead to the suggestion that wild ruminants play a role as reservoirs for these piroplasms. In a game enclosure in Eastern Austria (Federal county of Burgenland), piroplasms were detected with molecular analysis in blood samples of all 80 examined asymptomatic red deer (Cervus elaphus). Furthermore, piroplasms were detected in four out of 12 questing nymphs of Haemaphysalis concinna. In 32 Ixodes ticks sampled on-site, no Theileria DNA was detected. Sequence analysis identified these samples from both red deer and ticks as Theileria sp. ZS TO4. Our findings indicate that farmed red deer serve as asymptomatic carriers and adapted intermediate hosts of Theileria sp. in Central Europe and H. concinna was identified as a possible vector species of Theileria sp. ZS TO4.

Dirofilaria immitis is a parasite of domestic and wild canids and felids in tropical, subtropical and temperate regions throughout the world. The canine heartworm (D. immitis) is the causative agent of canine and feline cardiopulmonary dirofilariasis. This parasite is known to cause a zoonotic disease, namely human pulmonary dirofilariasis. D. immitis is known to be endemic in several South and Southeast Asian countries (e.g. India and Malaysia), but there has previously been no information about the presence of this pathogen in Bangladesh. We present a case of canine dirofilariasis caused by D. immitis in rural southeastern Bangladesh. A male filaroid nematode (95 mm in length and 1.94 mm in width) was identified in the heart of a dog. Species classification was performed by microscopy and molecular tools. Sequence analysis revealed a 100 % identity within the mitochondrial cytochrome c oxidase I (CO1) gene to two Chinese and one Australian D. immitis samples. Usually, dogs stay outside overnight with a high risk to get infected with D. immitis via nocturnal mosquito vectors, which may lead to high prevalences of this pathogen in the canine population and thus increase the risk of human infections with this neglected parasitic disease.

Recent reports indicate that first cases of genuine artemisinin resistance have already emerged along the Thai-Cambodian border. The main objective of this trial was to track the potential emergence of artemisinin resistance in Bangladesh, which in terms of drug resistance forms a gateway to the Indian subcontinent. We conducted an open-label, randomized, controlled 42-day clinical trial in Southeastern Bangladesh to investigate the potential spread of clinical artemisinin resistance from Southeast Asia. A total of 126 uncomplicated falciparum malaria patients were randomized to one of 3 treatment arms (artesunate monotherapy with 2 or 4 mg/kg/day once daily or quinine plus doxycycline TID for 7 days). Only cases fulfilling a stringent set of criteria were considered as being artemisinin-resistant. The 28-day and 42-day cure rates in the artesunate monotherapy (2 and 4 mg/kg) and quinine/doxycyline arms were 97.8% (95% confidence interval, CI: 87.8-99.8%), 100% (95% CI: 91.1-100%), and 100% (95% CI: 83.4-100%), respectively. One case of re-infection was seen in the artesunate high dose arm, and a single case of recrudescence was observed in the low dose group on day 26. No differences in median parasite and fever clearance times were found between the 2 artesunate arms (29.8 h and 17.9 h vs. 29.5 h and 19.1 h). Not a single case fulfilled our criteria of artemisinin resistance. Parasite clearance times were considerably shorter and ex vivo results indicate significantly higher susceptibility (50% inhibitory concentration for dihydroartemisinin was 1.10 nM; 95% CI: 0.95-1.28 nM) to artemisinins as compared to SE-Asia. There is currently no indication that artemisinin resistance has reached Bangladesh. However, the fact that resistance has recently been reported from nearby Myanmar indicates an urgent need for close monitoring of artemisinin resistance in the region. ClinicalTrials.gov NCT00639873.

CONSORT Checklist. (DOC)

Trial Protocol. (PDF)

The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.

Rodents are a key mammalian group highly successful in adapting to a variety of environments throughout the world and play an important role in many zoonotic cycles. Within this project, the gastrointestinal and extraintestinal parasite fauna of 76 rodents (Muroidea and Sciuridae) was determined in the District of Bandarban (Chittagong Hill Tracts) in Southeastern Bangladesh. Gastrointestinal and extraintestinal parasites were examined with macro- and microscopical tools (e.g. Ziehl-Neelsen Staining) at a field site in Bandarban. A wide variety of parasites were found in rodent hosts, including protozoa—Giardia sp. (n = 8), Cryptosporidium sp. (n = 1), Entamoeba sp. (n = 8), Trichomonadida (n = 4), Isospora sp. (n = 1), trematodes (Echinostoma sp.; n = 3), cestodes—Hymenolepis nana (n = 1), Hymenolepis diminuta (n = 3), Hymenolepis sp. (n = 2), Taenia taeniaeformis-Larven (n = 4), Catenotaenia sp. (n = 1), Taenia sp. (n = 1), nematodes—Heterakis spumosa (n = 4), Heterakis sp. (n = 1), Aspiculuris tetraptera (n = 2), Capillaria hepatica (n = 2), Capillaria sp. (n = 3), Syphacia sp. (n = 2), Strongyloides sp. (n = 10), Trichostrongylus sp. (n = 2) and Trichuris sp. (n = 1)—and acanthocephalans (Moniliformis moniliformis; n = 2). Several of the examined parasites are of zoonotic importance via direct or indirect transmission (e.g. C. hepatica) and may affect humans.

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.

The primers traditionally used to detect Plasmodium ovale infections are known for not binding all P. ovale parasites within the small-subunit rRNA gene when used alone. We describe a simple, cost- and time-efficient multiplex nested PCR and a nested PCR using a novel set of primers for the simultaneous detection of P. ovale curtisi and P. ovale wallikeri.

Taenia taeniaeformis and Taenia crassiceps are cestodes with voles as intermediate hosts and Felidae, Canidae and Mustelidae as definitive hosts. To evaluate the influence of T. taeniaeformis metacestodes on voles in Vorarlberg (Western Austria), a helminthological survey was performed on 318 common voles (Microtus arvalis) and 93 water voles (Arvicola terrestris). Furthermore the metacestodes themselves were analysed by morphometric methods. Our results demonstrate that both T. taeniaeformis and T. crassiceps are endemic in Vorarlberg, and that there is a significant difference between those infected with larvae of T. taeniaeformis and uninfected voles regarding body weight, but not sex or body length.

In spite of the high prevalence of malaria in Bangladesh and other southern Asian countries, there remains a substantial shortage of knowledge about the less common human malaria parasites. Recent studies indicate that Plasmodium ovale is made up of two species, namely Plasmodium ovale wallikeri and Plasmodium ovale curtisi. Genus- and species-specific nested PCR analyses of the ssrRNA gene was used to detect P. ovale infections among 2,246 diagnostic samples. Plasmodium ovale infections were further differentiated by nested PCR of the potra gene and multilocus sequence analysis of the cox1, porbp2 and the ssrRNA genes. Both P. ovale curtisi and P. ovale wallikeri occur sympatrically in the Chittagong Hill Tracts, Bangladesh and all patients presented with a mild or asymptomatic symptom complex at the time of diagnosis. The pathogens can be differentiated by nested PCRs targeting the ssrRNA and potra genes, and display dimorphism in multilocus sequence analyses. We believe that we report the first evidence of sympatric P. ovale curtisi and P. ovale wallikeri in southern Asia within a relatively confined study area of less than 5,000 km(2). High rates of mixed infections, the emergence of "new" human malaria parasite species and the evidence of zoonotic capability call for optimised diagnostic strategies for a new era of eradication.

A large variety of ectoparasites parasitizing on livestock, dogs, and rodents are documented throughout the world, of which several are proven vectors for major (including zoonotic) diseases affecting humans and/or livestock. However, there remains a significant lack of knowledge in regard to the ectoparasite fauna in remote regions of the developing world, such as southeastern Bangladesh, and an urgent need to investigate this fauna to improve diagnostic options. In the course of the present study, more than 5,300 ectoparasites were collected by flag dragging and handpicking of livestock, dogs, and rodents in the District of Bandarban (Chittagong Hill Tracts) in southeastern Bangladesh. Three tick species were identified: Haemaphysalis bispinosa (flagging, cattle, goats, and dogs), Rhipicephalus microplus (cattle, goats), and Rhipicephalus sanguineus (dogs, goats, and flagging). H. bispinosa was the dominant tick species on mammalian hosts as well as on vegetation. Furthermore, Ctenocephalides canis (dogs, goats) and Linognatus sp. (goat) were found. Overall, 73 rodents of eight different species (e.g., Mus musculus, Rattus sikkimensis, Bandicota bengalensis, and Niviventer sp.) hosted a variety of ectoparasites such as mites (Laelaps nuttali, Laelaps echidninus, Lyponissoides sp. and Ornithonyssus bacoti), fleas (Xenopsylla cheopis), and one myiasis-causing dipteran species. Monitoring the ectoparasite burden of livestock and other mammals is urgently needed in order to control ectoparasites associated with social and economic burden (e.g., reduced milk production, weight loss). Several zoonotic diseases can be transmitted by ectoparasites in this area, where the majority of the population live in basic housing conditions and in direct contact with livestock, dogs, and rodents.

Malaria is still a major threat in many parts of the world with resistance spreading to almost all classes of antimalarials. The limited arsenal of available antimalarial drugs emphasizes the urgent need for novel antimalarial compounds. Owing to the fact that novel leads from nature have traditionally played a pivotal role in the development of various classes of antimalarials, we investigated a set of eight naturally occurring dietary flavonoids and their analogues for their antiplasmodial activity on clinical field isolates in southeastern Bangladesh and culture-adapted chloroquine-sensitive and chloroquine-resistant parasite clones. Except for taxifolin, all the other flavonoids had 50% inhibitory concentrations below 14 μM, both in the field and laboratory-adapted parasites. Neither of the flavonoids showed any activity correlation with chloroquine. The quercetin analogue rutin (7.10 ± 10.32 μM) was the most active substance in field isolates as well as laboratory-adapted cultures (3.53 ± 13.34 μM in 3D7 and 10.38 ± 15.08 μM in K1), providing the first evidence of its activity against Plasmodium falciparum parasites. Thus, our results provide important evidence of the antimalarial activity of flavonoids in traditional use and thus warrant further investigation of these compounds as potential antiplasmodial agents.

Capillaria hepatica (syn. for Calodium hepaticum) is a zoonotic nematode parasitizing in the livers of rodents as main hosts and in numerous other mammals including humans. It is the causative agent of the rare conditions of hepatic capillariosis and spurious C. hepatica infections in humans. In this review, 163 reported cases of infestations with this parasite (72 reports of hepatic capillariosis, 13 serologically confirmed infestations and 78 observations of spurious infections) are summarized with an overview on the distribution, symptoms, pathology, diagnosis, serology and therapy of this rare human pathogen.