Julia Walochnik

Correlations between morphological, molecular biological, and physiological characteristics in clinical and nonclinical isolates of Acanthamoeba spp.

ABSTRACT Eleven Acanthamoeba isolates, obtained fromAcanthamoeba keratitis patients, from contact lens cases of non-Acanthamoeba keratitis patients, from asymptomatic individuals, from necrotic tissue, and from tap water and two reference strains were investigated by morphological, molecular biological, and physiological means in order to discriminate clinically relevant and nonrelevant isolates. All clinically relevant isolates showedAcanthamoeba sp. group II morphology. 18S ribosomal DNA sequencing revealed sequence type T4 to be the most prevalent group among the isolates and also the group recruiting most of the pathogenic strains. Interestingly, within T4 the strains of no clinical relevance clustered together. Moreover, physiological properties appeared to be highly consistent with initial pathogenicity and with sequence clustering. Altogether, the results of our study indicate a correlation between the phylogenetic relationship and pathogenicity.

The identification of free-living environmental isolates of amoebae from Bulgaria

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named “6” (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)—all exhibiting a highly efficient proliferation in axenic cultures—were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.

An update on Acanthamoeba keratitis: diagnosis, pathogenesis and treatment

Free-living amoebae of the genus Acanthamoeba are causal agents of a severe sight-threatening infection of the cornea known as Acanthamoeba keratitis. Moreover, the number of reported cases worldwide is increasing year after year, mostly in contact lens wearers, although cases have also been reported in non-contact lens wearers. Interestingly, Acanthamoeba keratitis has remained significant, despite our advances in antimicrobial chemotherapy and supportive care. In part, this is due to an incomplete understanding of the pathogenesis and pathophysiology of the disease, diagnostic delays and problems associated with chemotherapeutic interventions. In view of the devastating nature of this disease, here we present our current understanding of Acanthamoeba keratitis and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be potential targets for improved diagnosis, therapeutic interventions and/or for the development of preventative measures. Novel molecular approaches such as proteomics, RNAi and a consensus in the diagnostic approaches for a suspected case of Acanthamoeba keratitis are proposed and reviewed based on data which have been compiled after years of working on this amoebic organism using many different techniques and listening to many experts in this field at conferences, workshops and international meetings. Altogether, this review may serve as the milestone for developing an effective solution for the prevention, control and treatment of Acanthamoeba infections.

Cytotoxic Activities of Alkylphosphocholines against Clinical Isolates of Acanthamoeba spp.

ABSTRACT Free-living amoebae of the genus Acanthamoeba are causing serious chronic conditions such as destructive keratitis in contact lens wearers or granulomatous amoebic encephalitis in individuals with compromised immune systems. Both are characterized by the lack of availability of sufficiently effective and uncomplicated, manageable treatments. Hexadecylphosphocholine (miltefosine) is licensed for use as a topical antineoplastic agent, but it is also active in vitro against several protozoan parasites, and it was applied very successfully for the treatment of human visceral leishmaniasis. The aim of our study was to evaluate the efficacy of hexadecylphosphocholine and other alkylphosphocholines (APCs) against Acanthamoeba spp. The in vitro activities of eight different APCs against three Acanthamoeba strains of various pathogenicities were determined. All substances showed at least amoebostatic effects, and some of them disrupted the amoebae, as shown by the release of cytoplasmic enzyme activity. Hexadecylphosphocholine exhibited the highest degree of cytotoxicity against trophozoites, resulting in complete cell death at a concentration as low as 40 μM, and also displayed significant cysticidal activity. Hexadecylphosphocholine may be a promising new candidate for the topical treatment of Acanthamoeba keratitis and, conceivably, even for the oral treatment of granulomatous amoebic encephalitis.

Successful Treatment of Disseminated Acanthamoeba sp. Infection with Miltefosine

We report on an HIV-negative but immunocompromised patient with disseminated acanthamoebiasis, granulomatous amoebic encephalitis, and underlying miliary tuberculosis and tuberculous meningitis. The patient responded favorably to treatment with miltefosine, an alkylphosphocholine. The patient remained well with no signs of infection 2 years after treatment cessation.

Echinococcus granulosus strain differentiation based on sequence heterogeneity in mitochondrial genes of cytochrome c oxidase-1 and NADH dehydrogenase-1

Genetic analyses of Echinococcus granulosus isolates from different intermediate host species have demonstrated substantial levels of variation for some genotype (strain) clusters. To determine the range of genetic variability within and between genotypes we amplified and cloned partial cox1 and nadh1 genes from 16 isolates of E. granulosus from 4 continents. Furthermore, we sequenced different clones from a PCR product to analyse the intra-individual genetic variance. The findings showed a moderate degree of variance within single isolates and a significant degree of variance between the cluster of genotypes G1-G3 (sheep, Tasmanian sheep and buffalo strain), genotypes G4 (horse strain) and G5 (cattle strain) and the cluster of the genotypes G6 (camel strain) and G7 (pig strain). The variance of up to 2.2% within genotypes was relatively low compared with that of 4.3-15.7% among genotypes. The present results indicate that a re-examination of the classification of 5 genotypes of Echinococcus is warranted. Hence, our data highly support a re-evaluation of the taxonomy of the clades G1-G3, G4, G5, G6/7 and G8 (cervid strain) within the genus Echinococcus.

Early diagnosis of amoebic keratitis due to a mixed infection with Acanthamoeba and Hartmannella

A mixed keratitis due to Acanthamoeba and Hartmannella species is reported. The patient was a soft contact lens wearer. Early diagnosis was achieved by polymerase chain reaction and culture. The pathogenic potential of the isolated amoebae was proven using cytotoxicity assays. The reported case underlines the difficulties in identifying a corneal amoebic infection. In our case, the early diagnosis of a mixed infection allowed a proper antiamoebic treatment in an early stage of infection. This may have been the reason of a successful outcome after therapy.

Microsporidia-like parasites of amoebae belong to the early fungal lineage Rozellomycota

Molecular phylogenies based on the small subunit ribosomal RNA gene (SSU or 18S ribosomal DNA (rDNA)) revealed recently the existence of a relatively large and widespread group of eukaryotes, branching at the base of the fungal tree. This group, comprising almost exclusively environmental clones, includes the endoparasitic chytrid Rozella as the unique known representative. Rozella emerged as the first fungal lineage in molecular phylogenies and as the sister group of the Microsporidia. Here we report rDNA molecular phylogenetic analyses of two endonuclear parasites of free-living naked amoebae having microsporidia-like ultrastructural features but belonging to the rozellids. Similar to microsporidia, these endoparasites form unflagellated walled spores and grow inside the host cells as unwalled nonphagotrophic meronts. Our endonuclear parasites are microsporidia-like rozellids, for which we propose the name Paramicrosporidium, appearing to be the until now lacking morphological missing link between Fungi and Microsporidia. These features contrast with the recent description of the rozellids as an intermediate wall-less lineage of organisms between protists and true Fungi. We thus reconsider the rozellid clade as the most basal fungal lineage, naming it Rozellomycota.

Babesia Species Occurring in Austrian Ixodes ricinus Ticks

ABSTRACT Babesiosis is a tick-transmitted disease of veterinary and medical importance. The first Austrian case of human babesiosis was recently recorded. In the current study, ticks at all life cycle stages (instars), including 853 Ixodes ricinus and 11 Haemaphysalis concinna ticks, from sampling sites throughout Austria were examined for the presence of Babesia spp. by using 18S rRNA gene PCR and sequencing, and the overall mean infection rate was 51.04%. The infection rates for sampling sites were highly variable, ranging from 0% to almost 100%. Different instars and different sexes were infected almost equally. Babesia isolates occurring in Austrian ticks were identified as Babesia divergens, Babesia divergens-like, and Babesia sp. strain DD by sequencing a fragment of the heat shock protein 70 gene and internal transcribed spacer regions 1 and 2. To our knowledge, this is the first investigation of Babesia spp. in Austrian ticks.

Early Diagnosis of Acanthamoeba Infection during Routine Cytological Examination of Cerebrospinal Fluid

ABSTRACT Early identification of Acanthamoeba in cerebrospinal fluid is mandatory to prevent fatal granulomatous amebic encephalitis. In the case presented here amebic trophozoites were detected in a routine cerebrospinal fluid sample. The antibiotic treatment and the apparently low virulence of this isolate were responsible for the benign progression of the infection.