Investigation of Three Different UV-C Irradiation Schemes for Bacterial Decontamination of FFP2 Masks to Make Them Reusable.

The effect of filtering face piece grade 2 (FFP2) masks for infection prevention is essential in health care systems; however, it depends on supply chains. Efficient methods to reprocess FFP2 masks may be needed in disasters. Therefore, different UV-C irradiation schemes for bacterial decontamination of used FFP2 masks were investigated.Seventy-eight masks were irradiated with UV light for durations between 3 and 120 seconds and subsequently analyzed for the presence of viable bacteria on the inside. Ten masks served as the control group. Irradiation on the inside of the masks reduced bacteria in proportion to the dose, with an almost complete decontamination after 30 seconds. Outside irradiation reduced the quantity of colonies without time-dependent effects. Both sides of irradiation for a cumulated 30 seconds or more showed almost complete decontamination.Overall, this study suggests that standardized UV irradiation schemes with treatment to both sides might be an efficient and effective method for FFP2 mask decontamination in times of insufficient supplies.

Induction of Programmed Cell Death in Acanthamoeba culbertsoni by the Repurposed Compound Nitroxoline.

Acanthamoeba is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies. Despite advances in Acanthamoeba therapy, the management of patients with Acanthamoeba infections remains a challenge for health services. Therefore, there is a need to search for new active substances against Acanthamoebae. In the present study, we evaluated the amoebicidal activity of nitroxoline against the trophozoite and cyst stages of six different strains of Acanthamoeba. The strain A. griffini showed the lowest IC50 value in the trophozoite stage (0.69 ± 0.01 µM), while the strain A. castellanii L-10 showed the lowest IC50 value in the cyst stage (0.11 ± 0.03 µM). In addition, nitroxoline induced in treated trophozoites of A. culbertsoni features compatibles with apoptosis and autophagy pathways, including chromatin condensation, mitochondrial malfunction, oxidative stress, changes in cell permeability and the formation of autophagic vacuoles. Furthermore, proteomic analysis of the effect of nitroxoline on trophozoites revealed that this antibiotic induced the overexpression and the downregulation of proteins involved in the apoptotic process and in metabolic and biosynthesis pathways.

Repurposing of Nitroxoline as an Alternative Primary Amoebic Meningoencephalitis Treatment.

Among the pathogenic free-living amoebae (FLA), Naegleria fowleri is the etiological agent of a fatal disease known as primary amoebic meningoencephalitis (PAM). Once infection begins, the lesions generated in the central nervous system (CNS) result in the onset of symptoms leading to death in a short period of time. Currently, there is no standardized treatment against the infection, which, due to the high virulence of the parasite, results in a high case fatality rate (>97%). Therefore, it is essential to search for new therapeutic sources that can generate a rapid elimination of the parasite. In recent years, there have already been several successful examples of drug repurposing, such as Nitroxoline, for which, in addition to its known bioactive properties, anti-Balamuthia activity has recently been described. Following this approach, the anti-Naegleria activity of Nitroxoline was tested. Nitroxoline displayed low micromolar activity against two different strains of N. fowleri trophozoites (IC50 values of 1.63 ± 0.37 µM and 1.17 ± 0.21 µM) and against cyst stages (IC50 of 1.26 ± 0.42 µM). The potent anti-parasitic activity compared to the toxicity produced (selectivity index of 3.78 and 5.25, respectively) in murine macrophages and human cell lines (reported in previous studies), together with the induction of programmed cell death (PCD)-related events in N. fowleri make Nitroxoline a great candidate for an alternative PAM treatment.

Molecular detection of Leishmania (Sauroleishmania) adleri (Trypanosomatida: Trypanosomatidae) in Sergentomyia sp. sand flies (Diptera: Psychodidae) in Mali and Niger.

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sauroleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies.

Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of Leishmania spp. in Human Serum.

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.

Acanthamoeba species isolated from Philippine freshwater systems: epidemiological and molecular aspects.

Free-living amoeba (FLA) research in the Philippines is still in its infancy but has, by far, demonstrated the presence of potentially pathogenic species. Acanthamoeba may cause sight-threatening and central nervous system infections to humans, yet its epidemiologic distribution from local environmental sources is yet to be defined. The present study aimed to provide a baseline epidemiologic distribution of Acanthamoeba spp. in freshwater systems in the Philippines and establish potential pathogenicity of isolates through thermo-tolerance assay. A total of 63 water samples were collected from 13 freshwater systems all over the Philippine archipelago. The low-volume (50 ml) water samples were processed and cultured on non-nutrient agar lawned with Escherichia coli and observed for amoebic growth using light microscopy. Amoebic culture demonstrated 14.28% (9/63) positivity while further molecular testing of culture-positive plates using Acanthamoeba-specific primers demonstrated 100% (9/9) confirmation of Acanthamoeba species. Genotyping of Acanthamoeba isolates revealed T1, T3, T4, T5, T7, T11, and T15 genotypes. Thermo-tolerance assay demonstrated that T5 and T7 genotypes were potentially pathogenic strains. The evidence of environmental distribution of Acanthamoeba spp. in the freshwater systems in the Philippines and thermo-tolerance profile of isolates are significant aspects of amoeba study in public health and calls for initiatives in the dissemination of relevant information and the expansion of knowledge, awareness, and policies on pathogenic waterborne amoeba to mitigate, prevent, detect, and report cases of human infections.

Molecular phylogeny of Megalobatrachonema (Nematoda: Ascaridida), with description of a new species based on morphological and molecular evidence.

Species of MegalobatrachonemaYamaguti, 1941 (Ascaridida: Cosmocercoidea) are important nematode parasites in amphibians and reptiles. However, the phylogenetic relationship of its included two subgenera Megalobatrachonema and Chabaudgolvania remains unclear. In the present study, a new species of Megalobatrachonema, M. (Chabaudgolvania) wangi sp. nov., was described based on the specimens collected from the lesser spiny frog Quasipaa exilispinosa (Liu & Hu) (Amphibia: Anura) in China. The ribosomal [large ribosomal DNA (28S) and internal transcribed spacer (ITS1-5.8S-ITS2)] and mitochondrial [12S small subunit ribosomal DNA and cytochrome c oxidase subunit 1 (cox1)] target regions of the new species and M. (Chabaudgolvania) terdentatum, together with the 12S region of M. (Megalobatrachonema) hainanensis, were amplified and sequenced for molecular identification and phylogeny. Moreover, in order to clarify the systematic position of the new species and the phylogenetic relationship of the two subgenera Megalobatrachonema and Chabaudgolvania, phylogenetic analyses based on 28S + ITS1-5.8S-ITS2 + 12S sequence data were performed using maximum likelihood (ML) inference and Bayesian inference (BI). The molecular phylogenetic results conflicted with the current classification and challenged the validity of the subgenus Chabaudgolvania, that should be a synonym of the subgenus Megalobatrachonema. The presence or absence of valves in the oesophageal bulb as a key criterion for delimitation of the two subgenera Megalobatrachonema and Chabaudgolvania seems to be unreliable.

Vermamoeba vermiformis as etiological agent of a painful ulcer close to the eye.

In the present article, we report on the identification of Vermamoeba (Hartmannella) vermiformis as the etiological agent of a tissue infection close to the eye of a female patient. Laboratory examination revealed no involvement of any pathogenic bacteria or fungi in the tissue infection. V. vermiformis was identified by cultivation and morphology of trophozoites and cysts as well as phylogenetic analysis of nuclear 18S rDNA. The lesion improved in the course of 4 weeks by application of zinc paste.

Lyophilisation as a simple and safe method for long-term storage of free-living amoebae at ambient temperature.

Free-living amoebae (FLA) are protozoa ubiquitously found in nature. As some species or strains of these FLA are pathogenic for humans and animals, they represent objects of medical and parasitological research worldwide. Storage of valuable FLA strains in laboratories is often time- and energy-consuming and expensive. The shipment of such strains as frozen stocks is cumbersome and challenging in terms of cooling requirements as well as of transport regulations. To overcome these difficulties and challenges in maintenance and transport, we present a new method to generate lyophilised samples of non-cyst-forming FLA (Ripella (Vannella) spp.) and cyst-forming FLA (Acanthamoeba spp.) strains which guarantees a simple mechanism for long-term storage at ambient temperature, as well as easy handling and/or shipment. The survival rate of all FLA lyophilisates after short-term storage (2 months) was comparable to the survival rate of freeze cultures of the respective strains. Furthermore, the viability of Acanthamoeba spp. cysts after storage for 29 months was 20 to 40% following lyophilisation and rehydration, with strain variation.

Failure of molecular diagnostics of a keratitis-inducing Acanthamoeba strain.

An otherwise healthy 49-year-old female patient presented at the local hospital with severe keratitis in both inflamed eyes. She was a contact lens wearer and had no history of a corneal trauma. In our laboratory for medical parasitology Acanthamoebae were detected microscopically from the cornea scraping and from the fluid of the contact lens storage case after xenical culture and showed the typical cyst morphology of Acanthamoebae group II. The diagnosis of "Acanthamoeba keratitis" was established and successful therapy was provided. While the morphological microscopic method led to the correct diagnosis in this case, an in-house multiplex qPCR and a commercial qPCR showed false negative results regarding Acanthamoeba sp. The subsequent sequencing revealed the Acanthamoeba genotype T4. In the present case report, the inability to detect Acanthamoebae using qPCR only is presented. Therefore, we recommend the utilization of combined different assays for optimal diagnostic purposes.

Free-Living Amoebae as Hosts for and Vectors of Intracellular Microorganisms with Public Health Significance.

Free-living amoebae (FLA) are parasites within both humans and animals causing a wide range of symptoms and act as hosts of, and vehicles for phylogenetically diverse microorganisms, called endocytobionts. The interaction of the FLA with sympatric microorganisms leads to an exceptional diversity within FLA. Some of these bacteria, viruses, and even eukaryotes, can live and replicate intracellularly within the FLA. This relationship provides protection to the microorganisms from external interventions and a dispersal mechanism across various habitats. Among those intracellularly-replicating or -residing organisms there are obligate and facultative pathogenic microorganisms affecting the health of humans or animals and are therefore of interest to Public Health Authorities. Mimiviruses, Pandoraviruses, and Pithoviruses are examples for interesting viral endocytobionts within FLA. Future research is expected to reveal further endocytobionts within free-living amoebae and other protozoa through co-cultivation studies, genomic, transcriptomic, and proteomic analyses.

Detection of Rickettsia helvetica in Ixodes ricinus infesting wild and domestic animals and in a botfly larva (Cephenemyia stimulator) infesting roe deer in Germany.

Ixodes ricinus is a well-known vector of different human pathogens including Rickettsia helvetica. The role of wild mammals in the distribution and probable maintenance of Rickettsia in nature is still to be determined. We therefore investigated various parasites from different wild mammals as well as companion animals for the presence of Rickettsia. A total of 606 I. ricinus, 38 Cephenemyia stimulator (botfly larvae), one Dermacentor reticulatus, 24 Haematopinus suis (hog lice) and 30 Lipoptena cervi (deer flies) were collected from free-ranging animals during seasonal hunting, and from companion animals. Sample sites included hunting leases at three main sampling areas and five additional areas in West and Central Germany. All collected parasites were screened for Rickettsia spp. and I. ricinus were investigated for tick-borne encephalitis virus (TBEV) in addition. While no TBEV was detected, the minimum infection rate (MIR) of I. ricinus with Rickettsia was 4.1% referring to all sampling sites and up to 6.9% at the main sampling site in Koblenz area. Sequencing of a fragment of the ompB gene identified R. helvetica. Approximately one third (29.5%) of the animals carried Rickettsia-positive ticks and the MIR in ticks infesting wild mammals ranged from 4.1% (roe deer) to 9.5%. These data affirm the widespread distribution of R. helvetica in Germany. One botfly larva from roe deer also harboured R. helvetica. Botfly larvae are obligate parasites of the nasal cavity, pharynx and throat of cervids and feed on cell fragments and blood. Based on this one might hypothesise that R. helvetica likely induces rickettsemia in cervids thus possibly contributing to maintenance and distribution of this rickettsia in the field.

Detection of Balamuthia mandrillaris DNA in the storage case of contact lenses in Germany.

Acanthamoeba spp. are frequently the etiological agents of a severe form of sight-threatening keratitis, called Acanthamoeba keratitis. The contact lens storage solution of a patient with keratitis of unknown genesis was screened using our diagnostic tools to detect potentially pathogenic free-living amoebae (FLA). Culture methods and a triplex quantitative real-time polymerase chain reaction (qPCR) targeting Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris were used in context of this routine screening. While no amoebae were detected by culture, qPCR specifically detected DNA of B. mandrillaris. This FLA is known as the etiological agent of a fatal form of encephalitis in humans and other mammals, Balamuthia amoebic encephalitis (BAE). A fragment of the 18S rDNA gene was amplified from the sample and showed 99 % sequence identity to B. mandrillaris sequences from GenBank. To the best of our knowledge, this is the first report of B. mandrillaris found in association with contact lenses. Although no viable amoeba was obtained by culturing efforts, the verification of B. mandrillaris DNA in the contact lens storage solution demonstrates how easily this pathogen might come into close contact with humans.

Viruses in close associations with free-living amoebae.

As both groups of organisms, free-living amoebae (FLA) and viruses, can be found in aquatic environments side by side, it appears obvious that there are multiple interactions with respect to host-endocytobiont relationships. Several relationships between viruses and protozoan hosts are described and it was the discovery of the so called "giant viruses," associated with amoebae, which gave another dimension to these interactions. Mimiviruses, Pandoraviruses and Pithoviruses are examples for interesting viral endocytobionts within FLA. In the Mimivirus viral factories, viral DNA undergoes replication and transcription, and the DNA is prepared to be packed in procapsids. Theses Mimivirus factories can be considered as efficient "production lines" where, at any given moment, all stages of viral generation including membrane biogenesis, capsid assembly and genome encapsidation, are occurring concomitantly. There are some hints that similar replication factories are involved as well during the Pandoravirus development. Some scientists favour the assumption that the giant viruses have received many of their genes from their hosts or from sympatric occurring endocytobionts via lateral gene transfer. This hypothesis would mean that this type of transfer has been an important process in the evolution of genomes in the context of the intracellular parasitic or endocytobiotic lifestyle. In turn, that would migitate against hypothesizing development of a new branch in the tree of life. Based on the described scenarios to explain the presence of genes related to translation, it is also possible that earlier ancestors of today's DNA viruses were involved in the origin of eukaryotes. That possibly could in turn support the idea that cellular organisms could have evolved from viruses with growing autarkic properties. In future we expect the discovery of further (giant) viruses within free-living amoebae and other protozoa through genomic, transcriptomic and proteomic analyses.

Rhinosporidiosis in African reed frogs Hyperolius spp. caused by a new species of Rhinosporidium.

We report the identification of a new Rhinosporidium species (Dermocystida, Mesomycetozoea) infecting amphibian hosts, while showing a species specificity for African reed frogs of the genus Hyperolius. Large dermal cysts (sporangia) of R. rwandae sp. nov. were observed in 18% of H. lateralis and similar cysts in 0.7% of H. viridiflavus surveyed. Fully developed R. rwandae cysts are about 500 to 600 µm in diameter and sealed from the frog tissue by a thick chitinous wall. Some cysts were filled with numerous round-oval basophilic microspores of 8 to 12 µm diameter. With the exception of legs, nodules were visible over the complete torso surface including the vocal sac of males, but the most affected skin region was the area around the cloaca. Behavior, condition, and lifespan of infected frogs do not seem to be distinct from that of healthy individuals. The mode of infection remains unknown, but we hypothesize that the infectious life stage reaches the dermis via the intraepidermal ducts of the skin glands. Molecular evidence places the new frog pathogen as a sister species of the human pathogen R. seeberi.

Welcome to pandoraviruses at the 'Fourth TRUC' club.

Nucleocytoplasmic large DNA viruses, or representatives of the proposed order Megavirales, belong to families of giant viruses that infect a broad range of eukaryotic hosts. Megaviruses have been previously described to comprise a fourth monophylogenetic TRUC (things resisting uncompleted classification) together with cellular domains in the universal tree of life. Recently described pandoraviruses have large (1.9-2.5 MB) and highly divergent genomes. In the present study, we updated the classification of pandoraviruses and other reported giant viruses. Phylogenetic trees were constructed based on six informational genes. Hierarchical clustering was performed based on a set of informational genes from Megavirales members and cellular organisms. Homologous sequences were selected from cellular organisms using TimeTree software, comprising comprehensive, and representative sets of members from Bacteria, Archaea, and Eukarya. Phylogenetic analyses based on three conserved core genes clustered pandoraviruses with phycodnaviruses, exhibiting their close relatedness. Additionally, hierarchical clustering analyses based on informational genes grouped pandoraviruses with Megavirales members as a super group distinct from cellular organisms. Thus, the analyses based on core conserved genes revealed that pandoraviruses are new genuine members of the 'Fourth TRUC' club, encompassing distinct life forms compared with cellular organisms.

Article for the "Free-living amoebae special issue": Isolation and characterisation of various amoebophagous fungi and evaluation of their prey spectrum.

This article gives an overview on the isolation and characterisation of endoparasitic fungi invading free-living amoebae (FLA), including the ones forming thalli inside their hosts such as Cochlonema euryblastum and also the predatory fungi which capture amoebae by adhesive hyphae. Acaulopage spp. and Stylopage spp. trap, intrude, and exploit amoebal trophozoites. Previous phylogenetic studies proved Cochlonema to be a member of the Zoopagales. The genetic investigation of Acaulopage tetraceros demonstrated its close relationship to Cochlonema. Co-cultivation of A. tetraceros with a number of FLA revealed a great prey spectrum of this amoebophageous fungus. In addition it was shown that solitary amoebal stages of slime moulds such as Dictyostelium sp. and Physarum sp. are also suited as welcome prey amoebae.

Some secrets are revealed: parasitic keratitis amoebae as vectors of the scarcely described pandoraviruses to humans.

In this article, the results of a long effort to derive valuable phylogenetic data about an extraordinary spore-like infectious particle (endocytobiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens and the inflamed eye of a patient with keratitis are presented. The development of these endocytobionts has already been demonstrated with electron microscopic photo sequences, leading to a relevant model of its development presented here. The molecular biological investigation following the discovery of two other Pandoravirus species within aquatic sediments in 2013 led to the taxonomic affiliation of our endocytobiont with the genus Pandoravirus. A range of endocytobionts (intracellular biofilms) have been found in recent years, among which are several viruses which obligatorily proliferate within free-living amoebae. In human medicine, foreign objects which are placed in or on humans cause problems with microorganisms in biofilms. Contact lenses are especially important, because they are known as a source of a rapid formation of biofilm. These were the first Pandoraviruses described, and because this is additionally the first documented association with humans, we have clearly demonstrated how easily such (viral) endocytobionts can be transferred to humans. This case counts as an example of parasites acting as vectors of phylogenetically different microorganisms especially when living sympatric within their biocoenosis of biofilms. As the third part of the "Pandoravirus trilogy", it finally reveals the phylogenetic nature of these "extraordinary endocytobionts" within Acanthamoebae.

First report of an Acanthamoeba genotype T13 isolate as etiological agent of a keratitis in humans.

Several strains of free-living amoebae (FLA) belonging to the genus Acanthamoeba are able to cause a painful sight-threatening disease of the cornea designated as Acanthamoeba keratitis (AK). In this case report, a 22-year-old woman, wearer of soft contact lenses, was treated after the initial examination, and follow-up laboratory results led to the diagnosis of Acanthamoeba keratitis. The patient recovered under the targeted therapy, demonstrating that the acanthamoebae were the etiological agents of the keratitis in this case. The acanthamoebae belonged morphologically to group II. Genotyping of the causative Acanthamoeba strain based on sequences of the PCR amplimer ASA.S1 amplified from 18S ribosomal DNA by using the genus-specific primers JDP1 and JDP2 followed. The phylogenetic comparison of ASA.S1 confirmed that the isolated Acanthamoeba strain is closely related to genotype T13 supported by pairwise sequence identities of 97.1-98.0% and bootstrap support of 980 replicates with reference sequences of genotype T13. These results regarding the Acanthamoeba keratitis-causing isolate KaBo expands the number of known pathogenic genotypes to 12. To our knowledge, this is the first report of a T13 Acanthamoeba genotype being associated with keratitis in humans.