A cross-sectional study on phlebotomine sand flies in relation to disease transmission in the Republic of Kosovo.

Sand flies (Diptera: Psychodidae: Phlebotominae) are blood-feeding insects that transmit the protozoan parasites Leishmania spp. and various arboviruses. The Balkan region, including the Republic of Kosovo, harbours a diverse sand fly fauna. Vector species of Leishmania infantum as well as phleboviruses are endemic; however, recent data are scarce. We performed a cross-sectional study to update the current sand fly distribution in Kosovo and assess biological as well as environmental factors associated with sand fly presence. CDC light trapping was conducted at 46 locations in 2022 and 2023, specifically targeting understudied regions in Kosovo. Individual morphological species identification was supported by molecular barcoding. The occurrence data of sand flies was used to create distribution maps and perform environmental analyses, taking elevation, wind speed and climate-related factors into account. In addition, PCR-based blood meal analysis and pathogen screening were conducted. Overall, 303 specimens of six sand fly species were trapped, predominated by Phlebotomus neglectus (97%). Barcodes from eight of nine known endemic sand fly species were obtained. Combining our data with previous surveys, we mapped the currently known sand fly distribution based on more than 4000 specimens at 177 data points, identifying Ph. neglectus and Ph. perfiliewi as the predominant species. Environmental analyses depicted two geographical groups of sand flies in Kosovo, with notable differences between the species. In total, 223 blood meals of five sand fly species were analysed. Of seven identified host species, the predominant blood meal source was observed to be cattle, but the DNA of dogs and humans, among others, was also detected. This study assessed biological as well as ecological factors of sand fly occurrence, which should help better understand and evaluate potential hot spots of disease transmission in Kosovo.

On predatory fungi feeding on free-living amoebae harbouring yeast-like endoparasites.

Amoebae of the genus Vannella isolated from an ornamental fish aquarium were found to be infected with fungi. Upon plate culture, amoeba-trapping hyphal filaments were developed, and the amoeba trophozoites were found to harbour yeast-like parasites in their cytoplasm. Transfection of hyphae to a laboratory strain of Vannella resulted in the formation of conidia indicating the possible presence of zygomycetes of the genus Acaulopage, while efforts to culture the endoparasite remained unsuccessful. Biomolecular analysis based on rDNA revealed the presence of two distinct types of fungi, confirming the filamentous form as Acaulopage sp. (Zoopagomycota, Zoopagales) and identifying the yeast-like endoparasite as Cladosporium sp. (Ascomycota, Cladosporiales). To our knowledge, this is the first report of amoebae infected with Cladosporium.

Detection of Putative Virulence Genes alr, goiB, and goiC in Mycoplasma hominis Isolates from Austrian Patients.

In Mycoplasma hominis, two genes (alr and goiB) have been found to be associated with the invasion of the amniotic cavity, and a single gene (goiC) to be associated with intra-amniotic infections and a high risk of preterm birth. The syntopic presence of Ureaplasma spp. in the same patient has been shown to correlate with the absence of goiC in M. hominis. The aim of our study was to investigate the presence of alr, goiB, and goiC genes in two groups of M. hominis isolates collected from symptomatic and asymptomatic male and non-pregnant female patients attending an Outpatients Centre. Group A consisted of 26 isolates from patients with only M. hominis confirmed; group B consisted of 24 isolates from patients with Ureaplasma spp. as the only co-infection. We extracted DNA from all M. hominis isolates and analysed the samples for the presence of alr, goiB, and goiC in a qPCR assay. Additionally, we determined their cytotoxicity against HeLa cells. We confirmed the presence of the alr gene in 85% of group A isolates and in 100% of group B isolates; goiB was detected in 46% of the samples in both groups, whereas goiC was found in 73% of group A and 79% of group B isolates, respectively. It was shown that co-colonisation with Ureaplasma spp. in the same patient had no effect on the presence of goiC in the respective M. hominis isolate. We did not observe any cytotoxic effect of the investigated isolates on human cells, regardless of the presence or absence of the investigated genes.

The Palearctic blackfly Simulium equinum (Diptera: Simuliidae) as a biting pest of captive nyala antelopes (Tragelaphus angasii).

Blackflies (Diptera: Simuliidae) are cosmopolitan nuisance pests of great economic importance as well as vectors of many pathogens. After reports of massive blackfly biting of captive nyala antelopes in the Vienna Zoo, Austria, this study aimed to identify the species causing multiple skin lesions on the antelope hosts. The Palearctic species Simulium equinum, belonging to the medically and veterinary important Wilhelmia subgenus, was identified as the most likely causative agent. Barcoding and maximum likelihood analysis supported morphological species identification and highlighted the complex phylogeny of the subgenus Wilhelmia. Our study gives first evidence of the multi-host feeding blackfly S. equinum in the Vienna Zoo, thereby raising the question whether other hosts could also be bitten on a regular basis. The preliminary results urge for further analysis of blackfly breeding sites as well as the clarification of the host spectrum to assess the medical and veterinary importance of blackflies in the Zoo.

Comparison of Permethrin-Based Treatment Strategies against Scabies in Infants and Young Children.

OBJECTIVE: To optimize the permethrin-based therapies for scabies infestations in infants and young children, the efficacies of 3 different regimens were evaluated. STUDY DESIGN: The retrospective analysis encompassed 85 infants and children aged <4 years with scabies. The children had received either topical permethrin 5% on the entire body on days 1/8, on days 1/8/15, on days 1/8/15 plus interim applications restricted to hands and feet on days 2/3/4//9/10/11, or alternative treatments. RESULTS: The intensified regimen, consisting of full-body permethrin applied on days 1/8/15 and hands/feet on days 2/3/4//9/10/11, resulted in cure of scabies in 73.5% of the cases. The cure rates were significantly greater compared with full-body permethrin given on days 1/8, which led to eradication in 44%, and were greater compared with the clearance in children who had received full-body permethrin on days 1/8/15 (53.8%) or alternative treatments (60%). For patients in whom permethrin had previously been applied, the intensified regimen resulted in eradication of scabies in 71.4% of the cases, compared with 30% and 55.6% after full-body permethrin on days 1/8 and 1/8/15, respectively. CONCLUSIONS: The intensified regimen of full-body permethrin plus interim applications on hands/feet, which aims at reducing the number of mites present on the frequently heavily infested palmoplantar sites in addition to the standard entire body application, appears efficacious in curing scabies in young children.

An unusual thioredoxin system in the facultative parasite Acanthamoeba castellanii.

The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii's response to oxidative stress. The role of the large TrxR, however, remains elusive.

Main Metabolites of Pseudomonas aeruginosa: A Study of Electrochemical Properties.

Pseudomonas aeruginosa is a ubiquitously distributed soil and water bacterium and is considered an opportunistic pathogen in hospitals. In cystic fibrosis patients, for example, infections with P. aeruginosa can be severe and often lead to chronic or even fatal pneumonia. Therefore, rapid detection and further identification are of major importance in hospital hygiene and infection control. This work shows the electrochemical properties of five P. aeruginosa key metabolites considering their potential use as specific signaling agents in an electrochemical sensor system. The pure solutes of pyocyanin (PYO), Pseudomonas quinolone signal (PQS), pyochelin (PCH), 2-heptyl-4-hydroxyquinoline (HHQ), and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) were analyzed by different electrochemical techniques (cyclic and square wave voltammetry) and measured using a Gamry Reference 600+ potentiostat. Screen-printed electrodes (DropSens DRP110; carbon working and counter, silver reference electrode) were used to determine signal specificities, detection limits, as well as pH dependencies of the substances. All of the compounds were electrochemically inducible with well-separated oxidation and/or reduction peaks at specific peak potentials relative to the reference electrode. Additionally, all analytes exhibited linear concentration dependency in ranges classically reported in the literature. The demonstration of these properties is a promising step toward direct multiplexed detection of P. aeruginosa in environmental and clinical samples and thus, can make a significant contribution to public health and safety.

Characterisation of Trichomonas vaginalis Isolates Collected from Patients in Vienna between 2019 and 2021.

Trichomonas vaginalis (TV) is the causative agent of trichomoniasis, the most common nonviral sexually transmitted disease. TV can carry symbionts such as Trichomonas vaginalis virus (TVV) or Mycoplasma hominis. Four distinct strains of TV are known: TVV1, TVV2, TVV3, and TVV4. The aim of the current study was to characterise TV isolates from Austrian patients for the presence of symbionts, and to determine their effect on metronidazole susceptibility and cytotoxicity against HeLa cells. We collected 82 TV isolates and detected presence of TVV (TVV1, TVV2, or TVV3) in 29 of them (35%); no TVV4 was detected. M. hominis was detected in vaginal/urethral swabs by culture in 37% of the TV-positive patients; M. hominis DNA was found in 28% of the TV isolates by PCR. In 15% of the patients, M. hominis was detected in the clinical samples as well as within the respective TV isolates. In 22% of the patients, M. hominis was detected by culture only. In 11 patients, M. hominis was detected only within the respective cultured TV isolates (13%), while the swab samples were negative for M. hominis. Our results provide a first insight into the distribution of symbionts in TV isolates from Austrian patients. We did not observe significant effects of the symbionts on metronidazole susceptibility, cytotoxicity, or severity of symptoms.

Genetic diversity of Fasciola hepatica in Austria.

The worldwide occurring common liver fluke Fasciola hepatica can infect humans and animals and leads to considerable illness and economic loss annually. The aim of this study was to determine the genetic diversity of F. hepatica in Austria. In total, 31 adult flukes isolated from cattle from various regions in Austria were investigated for their cytochrome oxidase subunit 1 (cox1) and nicotinamide dehydrogenase subunit 1 (nad1) gene sequences. It was shown that Austrian isolates of F. hepatica reveal extensive genetic diversity. To the best of our knowledge, these are the first data on the diversity of F. hepatica in Austria.

Untargeted metagenomics shows a reliable performance for synchronous detection of parasites.

Shotgun metagenomics with high-throughput sequencing (HTS) techniques is increasingly used for pathogen identification and characterization. While many studies apply targeted amplicon sequencing, here we used untargeted metagenomics to simultaneously identify protists and helminths in pre-diagnosed faecal and tissue samples. The approach starts from RNA and operates without an amplification step, therefore allowing the detection of all eukaryotes, including pathogens, since it circumvents the bias typically observed in amplicon-based HTS approaches. The generated metagenomics datasets were analysed using the RIEMS tool for initial taxonomic read assignment. Mapping analyses against ribosomal reference sequences were subsequently applied to extract 18S rRNA sequences abundantly present in the sequence datasets. The original diagnosis, which was based on microscopy and/or PCR, could be confirmed in nearly all cases using ribosomal RNA metagenomics. In addition to the pre-diagnosed taxa, we detected other intestinal eukaryotic parasites of uncertain pathogenicity (of the genera Dientamoeba, Entamoeba, Endolimax, Hymenolepis) that are often excluded from routine diagnostic protocols. The study clearly demonstrates the applicability of untargeted RNA metagenomics for the parallel detection of parasites.

Solving an old enigma: Morellospora saccamoebae gen. nov., sp. nov. (Rozellomycota), a Sphaerita-like parasite of free-living amoebae.

The Rozellomycota form a lineage basal or sister to the Fungi, ancestor of Microsporidia. Their biodiversity is very rich but remains poorly characterized. The few known species are all parasites, whether of water molds and algae (Rozella), crustaceans (Mitosporidium), or as endonuclear parasites of amoebae (Nucleophaga, Paramicrosporidium). Since the nineteenth century, intracytoplasmic parasites of various protozoa have been described as species of the same genus Sphaerita. However, it was later thought possible to separate these parasites into at least two distinct groups, those forming flagellated zoospores, prevalent in Euglena and other flagellates, and those forming immobile spores, found mainly in free-living and endozoic amoebae. Herein, we report the recovery of a strain of the free-living amoeba species Saccamoeba lacustris, naturally infected by an intracytoplasmic parasite, which under light microscope has a morphology consistent with that of Sphaerita. Biomolecular analyses were thus performed. Our results show that the intracytoplasmic parasite of Saccamoeba belongs to the same subgroup of Mitosporidium and that it forms a new genus within Rozellomycota, Morellospora, that corresponds to the former spore-forming Sphaerita-like parasites of amoebae.

Successful extraction and PCR amplification of Giardia DNA from formalin-fixed stool samples.

Extracting genomic DNA of pathogenic agents from formalin-fixed specimens is inherently difficult. Storage of samples in formalin results in nucleic acid cross-linking and DNA fragmentation. In this study, DNA was extracted from 45 Giardia-positive stool samples stored in formalin and subjected to PCR amplification targeting the triose phosphate isomerase (tpi), beta gardin (bg) and glutamate dehydrogenase (gdh) genes. Samples were rehydrated by using a descending alcohol series before DNA extraction using a commercial kit. This was followed by EDTA-mediated inhibition of DNase activity and prolonged treatment with proteinase K to digest contaminating proteins. DNA was amplified at rates of 64.4% (29/45) at the tpi, 40% (18/45) at the bg and 20% (9/45) at the gdh loci as seen on nested PCR. DNA quality was subsequently tested in a genotyping experiment which produced high-quality sequences at the tpi (41.2%; 12/29) bg (50%; 9/18), and gdh (22.2%; 2/9) loci and enabled differentiation of Giardia strains at the subtype level. The modified extraction protocol was effective at removing inhibitors and reversing cross-linking of DNA. However, PCR amplification was limited to short fragments of DNA which resulted in highest success rate on amplification of the shortest (334 bp) gene fragment tested.

Filling gaps in the microsporidian tree: rDNA phylogeny of Chytridiopsis typographi (Microsporidia: Chytridiopsida).

Microsporidia are intracellular eukaryotic parasites of animals, characterized by unusual morphological and genetic features. They can be divided in three main groups, the classical microsporidians presenting all the features of the phylum and two putative primitive groups, the chytridiopsids and metchnikovellids. Microsporidia originated from microsporidia-like organisms belonging to a lineage of chytrid-like endoparasites basal or sister to the Fungi. Genetic and genomic data are available for all members, except chytridiopsids. Herein, we filled this gap by obtaining the rDNA sequence (SSU-ITS-partial LSU) of Chytridiopsis typographi (Chytridiopsida), a parasite of bark beetles. Our rDNA molecular phylogenies indicate that Chytridiopsis branches earlier than metchnikovellids, commonly thought ancestral, forming the more basal lineage of the Microsporidia. Furthermore, our structural analyses showed that only classical microsporidians present 16S-like SSU rRNA and 5.8S/LSU rRNA gene fusion, whereas the standard eukaryote rRNA gene structure, although slightly reduced, is still preserved in the primitive microsporidians, including 18S-like SSU rRNA with conserved core helices, and ITS2-like separating 5.8S from LSU. Overall, our results are consistent with the scenario of an evolution from microsporidia-like rozellids to microsporidians, however suggesting for metchnikovellids a derived position, probably related to marine transition and adaptation to hyperparasitism. The genetic and genomic data of additional members of Chytridiopsida and Rozellomycota will be of great value, not only to resolve phylogenetic relationships but also to improve our understanding of the evolution of these fascinating organisms.

Leishmaniasis in Northern Syria during Civil War.

Since the onset of the ongoing civil war in Syria, the governmental surveillance system for leishmaniasis has lost access to provinces of northern Syria. The MENTOR Initiative, an international not-for-profit organization, was commissioned to implement an integrated leishmaniasis control program, providing an opportunity to reassess the epidemiology of leishmaniasis in northern Syria. Epidemiologic data and biologic samples for molecular species diagnostics were collected from collaborating local health centers. Incidence peaked in March 2015 at 7,743 estimated monthly cases. High levels of transmission were observed in traditional endemic regions but extended to previously hypoendemic regions, such as Al-Raqqa and Al-Hasakah. Incidence decreased to 3,209 in July 2015. Data indicate that the prewar trend of increasing incidence of cutaneous leishmaniasis accelerated during the beginning of armed conflict but declined after implementation of the comprehensive control program by the MENTOR Initiative. Molecular analysis revealed a spectrum of Leishmania species and sporadic cases of visceral leishmaniasis.

Molecular evidence for relapse of an imported Plasmodium ovale wallikeri infection.

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.

Identification of free-living amoebae isolated from tap water in Istanbul, Turkey.

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.

Mycoplasma hominis impacts gene expression in Trichomonas vaginalis.

In Europe, up to 90% of isolated Trichomonas vaginalis strains are naturally infected with Mycoplasma hominis, a facultative pathogen of the human genital tract. The consequences of this endosymbiosis are not yet well understood. The aim of the current study was to evaluate the impact of natural and artificial infections with M. hominis on the RNA expression levels of metronidazole susceptibility-associated genes of T. vaginalis. Three T. vaginalis strains (TVSS10-, TVSS25-, G3) without M. hominis, as well as the same strains naturally (TVSS10+, TVSS25+) and artificially (G3-MhSS25, TVSS25-MhSS25) infected with M. hominis, were investigated for their expression profiles of three genes associated with metronidazole resistance (ferredoxin, flavin reductase 1 and pyruvate:ferredoxin oxidoreductase). The minimal inhibitory concentrations (MICs) of metronidazole were evaluated for all combinations and the respective M. hominis-free T. vaginalis strains were used as controls. The sole presence of M. hominis led to a down-regulation of metronidazole susceptibility-associated genes in all T. vaginalis strains tested. Interestingly, the effect was more prominent in the artificial symbioses. Moreover, a twofold enhancement of metronidazole tolerability was observed in three infected T. vaginalis strains, compared to the respective strains without M. hominis. In conclusion, M. hominis had an impact on gene expression in all T. vaginalis strains and on metronidazole MIC in all but one strain tested.

New insights from molecular phylogenetics of amoebophagous fungi (Zoopagomycota, Zoopagales).

Amoebophagous fungi are represented in all fungal groups: Basidiomycota, Ascomycota, Zygomycota, and Chytridiomycota. The amoebophagous fungi, within the zygomycota (Zoopagales, Zoopagomycota), mainly affect naked amoebae as ectoparasites or endoparasites. It is rather difficult to isolate members of the Zoopagales, because of their parasitic lifestyle, and to bring them into culture. Consequently, gene sequences of this group are undersampled, and its species composition and phylogeny are relatively unknown. In the present study, we were able to isolate amoebophagous fungi together with their amoeba hosts from various habitats (moss, pond, bark, and soil). Altogether, four fungal strains belonging to the genera Acaulopage and Stylopage plus one unidentified isolate were detected. Sequences of the 18S rDNA and the complete ITS region and partial 28S (LSU) rDNA were generated. Subsequent phylogenetic analyses showed that all new isolates diverge at one branch together with two environmental clonal sequences within the Zoopagomycota. Here, we provide the first molecular characterization of the genus Stylopage. Stylopage is closely related to the genus Acaulopage. In addition, taxonomy and phylogeny of amoebophagous fungi and their ecological importance are reviewed based on new sequence data, which includes environmental clonal sequences.

Candidatus Dirofilaria hongkongensis as Causative Agent of Human Ocular Filariosis after Travel to India.

We report a human case of ocular Dirofilaria infection in a traveler returning to Austria from India. Analysis of mitochondrial sequences identified the worm as Candidatus Dirofilaria hongkongensis, a close relative of Dirofilaria repens, which was only recently described in Hong Kong and proposed as a new species.