Andreas Obwaller

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.

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.

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.

Toxocara infestations in humans: symptomatic course of toxocarosis correlates significantly with levels of IgE/anti‐IgE immune complexes

Infestations of humans with the parasitic nematode T. canis are common in both developing and industrialized countries. Most infestations induce a clinically inapparent course of infection, however, severe clinical manifestations, i.e. visceral larva migrans (VLM) or ocular larva migrans (OLM) syndromes are observed. To find an explanation for the different courses of toxocarosis we examined several serological parameters: the expression of (i) specific IgE (Immunoblot, IB), (ii) specific IgG subclasses (IgG1–4, ELISA) and the formation of (iii) IgE/anti‐IgE immune complexes. Serum samples were obtained from persons with symptomatic (VLM, OLM) and asymptomatic course (AS) of the infestation. As antigen, T. canis excretory/secretory (TES) antigen from L3 larvae was used. Reactivity of IgE against SDS‐PAGE separated TES antigens was marginally higher in toxocarosis patients (35%) than in asymptomatics (24%), but without statistical significance. TES‐specific IgG (1–4), predominant subclass in all three groups was IgG1, followed by IgG2, IgG4 and IgG3. Subclasses IgG1, 2, 4 showed significant differences between patients with VLM associated symptoms and asymptomatic persons (P < 0.001) but not between patients with OLM associated symptoms and asymptomatics. Significantly elevated levels of IgE/anti‐IgE immune complexes were detected in sera of patients with symptomatic course of the disease, both VLM and OLM (P < 0.001). Whereas specific IgG may act via antibody dependent cell‐mediated cytotoxicity mechanisms, IgE/anti‐IgE immune complexes might possibly participate in VLM and OLM by inducing type III hypersensitivity.

Anti-Acanthamoeba efficacy and toxicity of miltefosine in an organotypic skin equivalent

OBJECTIVES Acanthamoebae can cause infections of several organs, including eye, skin, lung and brain. Except for Acanthamoeba keratitis, these infections are linked to immunodeficiency. Treatment is generally problematic, due to the lack of sufficiently effective and also easily manageable drugs. In a previous study we discovered that miltefosine (hexadecylphosphocholine) is highly active against Acanthamoeba spp. in vitro. The aim of the current study was to evaluate the suitability of miltefosine for the topical treatment of Acanthamoeba infections. METHODS Storage life and time dependency, susceptibilities of opportunistic bacterial and fungal pathogens, and synergistic and adverse effects of combinations with other anti-Acanthamoeba substances were determined. Moreover, an organotypic skin equivalent was adapted for investigating the penetration of acanthamoebae into the epidermis and the human tissue tolerability of miltefosine. RESULTS It was shown that miltefosine can be stored as a 2 mM stock solution and also as a 50 microM dilution over a period of 12 months at 4 degrees C without any loss of activity. Efficacies against staphylococci and Candida albicans were established. Acanthamoebae were able to penetrate the skin equivalent within 24 h. This penetration was prevented by treatment with miltefosine, while miltefosine treatment was well tolerated by the skin equivalent. CONCLUSIONS Miltefosine has been approved for oral and topical treatment of leishmaniasis and may also be a promising candidate for the topical treatment of Acanthamoeba infections.

Miltefosine and polyhexamethylene biguanide: a new drug combination for the treatment of Acanthamoeba keratitis

In this study, a series of compounds – miltefosine, polyhexamethylene biguanide, chlorhexidine and propamidine isethionate – and combinations of the latter three agents with miltefosine were prepared and used in a rat model for the topical treatment of Acanthamoeba keratitis.

Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp.

Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to find out, whether the 18S rDNA sequence based identification correlates to immunological differentiation. The protein and antigen profiles of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Affinity purified antibodies were used to further explore immunological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classification in the investigated strains.

An enzyme-linked immunosorbent assay with whole trophozoites of Toxoplasma gondii from serum-free tissue culture for detection of specific antibodies

This paper describes a new procedure of preparation of the antigen for an enzyme-linked immunosorbent assay (ELISA) for detection of antibodies againstToxoplasma gondii. To examine the reliability of this ELISA using whole trophozoites produced in a serumfree tissue culture as an antigen, 221 sera were tested comparatively in the new system (TTE, total trophozoites ELISA), in the indirect fluorescent antibody test (IFAT), and in a commeriially available ELISA using sonicated trophozoites as an antigen (STE, sonicated trophozoites ELISA). The ELISA with antigen lysate showed a good correlation with the IFAT; however, falsenegative results were sometimes obtained. The TTE was performed with all sera in two modifications: one test with an anti-IgG conjugate (G-TTE) and the other with an anti-Ig-G,-M,-A conjugate (GMA-TTE). In none of these TTE modifications were insensitivities observed; however, the G-TTE seems to offer a clearer differentiation between specifically reactive and nonreactive findings. The present study shows that the ELISA with whole trophozoites produced in serum-free tissue culture might be used as an alternative test to the IFAT. This test combines the advantages of the ELISA system with the sensitivity and specificity of the IFAT.

Association of autoantibodies against small nuclear ribonucleoproteins (snRNPs) with symptomatic Toxocara canis infestation

Several studies have demonstrated the occurrence of autoantibodies in the course of infestations with helminth parasites and a number of target proteins have been identified. Sera from patients suffering from toxocarosis, a disease caused by the parasitic roundworm Toxocara canis, and from healthy individuals were tested for autoantibodies by immunofluorescence and immunoblot assays using HEp‐2 cells as antigen. A considerable proportion of the sera from patients with toxocarosis‐associated symptoms were autoantibody‐positive, with a speckled staining pattern in the immunofluorescence test (62%) and with anti‐snRNP reactivity in the immunoblot assay (98%). In contrast, significantly fewer sera from asymptomatic individuals scored positive in these assays (18% in the immunofluorescence test, P < 0·005; 24% in the immunoblot, P < 0·005). Although the causative link between Toxocara infestation and the occurrence of autoantibodies is still unclear, our results show that increased amounts of autoantibodies are associated with clinical symptoms of inflammation. Thus a serum test for autoantibodies in toxocarosis patients might be a valuable gatekeeper assay for the decision for or against anti‐inflammatory treatment.

Activity of selected phytochemicals against Plasmodium falciparum

According to the WHO, in 2008, there were 247 million reported cases of malaria and nearly one million deaths from the disease. Parasite resistance against first-line drugs, including artemisinin and mefloquine, is increasing. In this study the plant-derived compounds aglafolin, rocaglamid, kokusaginine, arborine, arborinine and tuberostemonine were investigated for their anti-plasmodial activity in vitro. Fresh Plasmodium falciparum isolates were taken from patients in the area of Mae Sot, north-western Thailand in 2008 and the inhibition of schizont maturation was determined for the respective compounds. With inhibitory concentrations effecting 50%, 90% and 99% inhibition (IC(50), IC(90) and IC(99)) of 60.95 nM, 854.41 nM and 7351.49 nM, respectively, rocaglamid was the most active of the substances, closely followed by aglafoline with 53.49 nM, 864.55 nM and 8354.20 nM. The activity was significantly below that of artemisinin, but moderately higher than that of quinine. Arborine, arborinine, tuberostemonine and kokusaginine showed only marginal activity against P. falciparum characterized by IC(50) and IC(99) values higher than 350 nM and 180 μM, respectively, and regressions with relatively shallow slopes S>14.38. Analogues of rocaglamid and aglafoline merit further exploration of their anti-plasmodial activity.