Leishmania infections in Austrian soldiers returning from military missions abroad: a cross-sectional study.

OBJECTIVES: The incidence of leishmaniasis is known to increase in conflict areas. The aims of this study were to determine the exposure to Leishmania species in Austrian soldiers returning from missions abroad and to assess possible risk factors. METHODS: A retrospective explorative cross-sectional serologic study was conducted in 225 healthy Austrian soldiers returning from UN or EU peacekeeping missions in Syria, Lebanon and Bosnia and Herzegovina (BIH). Sera were tested for anti-Leishmania antibodies using a commercial enzyme-linked immunosorbent assay. All positive individuals were screened for Leishmania DNA by PCR targeting the ITS1 region using EDTA blood samples. RESULTS: In total, 13.3% (30/225) of the individuals tested were either positive (8%, 18/225) or borderline (5.3%, 12/225) in the enzyme-linked immunosorbent assay, with the highest seroprevalence in soldiers returning from Syria (17.8%, 18/101; 12 positive, six borderline), second from Lebanon (11.1%, 7/63; four positive, three borderline) and lowest from BIH (8.2%, 5/61; two positive, three borderline). Ten soldiers returning from Syria and one from BIH were also positive for Leishmania DNA. Six of these were identified as Leishmania donovani/infantum complex, two as L. tropica and another three as mixed infections by DNA sequencing. Epidemiologic data were collected via a questionnaire, and seropositivity was correlated with a history of insect bites that took a long time to heal (odds ratio, 5.33; 95% confidence interval, 1.23-23.04; p 0.025). CONCLUSIONS: Although pretravel serologic data were not available in this study, the exposure of soldiers to Leishmania spp. during their missions can be assumed to be considerable. Because even asymptomatic infections may resurge in case of emerging immunodeficiencies, adequate prevention measures seem important.

In a trial of the use of miltefosine to treat HIV-related cryptosporidiosis in Zambian adults, extreme metabolic disturbances contribute to high mortality.

There is still no effective treatment for cryptosporidiosis even though the disease has a significant impact on HIV-infected adults and children. Following evidence of the drug's promising efficacy in vitro, a phase-1-phase-2 study of miltefosine (given at 2.5 mg/kg for 14 days, with the dose capped at 100 mg/day) was recently initiated among Zambian adults with HIV-related cryptosporidiosis. Seven patients were recruited before the trial was terminated prematurely because of lack of efficacy and the development of severe adverse events. The latter may have been entirely drug-related or the result of extreme metabolic abnormalities already present in the patients enrolled in the trial. In future trials of miltefosine, attention will have to be paid to the possibility of metabolic abnormalities in the subjects investigated.

Combination of cell culture and quantitative PCR for screening of drugs against Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic pathogen causing self-limiting diarrhea in immunocompetent patients. An assay combining cell culture and real time quantitative PCR (qPCR) is reported here to verify drug efficacy against C. parvum in vitro. The monolayers of Human ileocecal adenocarcinoma cells (HCT-8) were infected by sporozoites excysted directly on the cells and were incubated with monensin, halofuginone bromide and hexadecylphosphocholine until 45h post infection (p.i.). The genomic DNA was extracted at 3, 27 and 45h p.i. and subjected to qPCR targeting the 70kDa heat shock protein gene to quantify the development of C. parvum. The reliability of the method was validated by testing of monensin and halofuginone bromide, which are well known to be effective in vitro. With the dose dependency monensin and halofuginone showed a maximum inhibition of 98.15% and 98.05% at 0.144 and 25microM, respectively, compared with non-treated controls at the endpoint incubation, confirming previous reports. The reduction of the parasite DNA reproduction over 27h p.i. compared with 3h p.i. was found to be as 97-99% in 0.144microM monensin and 99% in 25microM halofuginone treated cells. The new antileishmanial compound hexadecylphosphocholine (24.5microM, Miltefosine) showed 78-98% inhibition at 45h p.i., however, the reproduction of parasite DNA was reduced to 96-98% over 27h p.i. The method has the potential to easily and reliably assess anticryptosporidial compounds in adequately equipped routine laboratories.

Evaluation of follow-up of therapy with fenbendazole incorporated into stabilized liposomes and immunomodulator glucan in mice infected with Toxocara canis larvae.

Anthelmintic activity of benzimidazole carbamate anthelmintics is low against dormant Toxocara canis larvae during late infections in paratenic hosts. The present study was conducted to examine the efficacy of pure fenbendazole, or drug incorporated into sterically stabilized liposomes (SL-FBZ) administered to T. canis-infected mice alone and after its co-administration with the immunomodulator (1-->3)-beta-D-glucan against larvae localized in muscles and brains. Therapy with either drug forms (in total 250 mg/kg in 10 doses) commenced on day 28 post-infection (p.i.) and the efficacy of treatment, examined on day 30 after the last dose of drug, was the highest in groups of mice treated with SL-FBZ in combination with glucan (89.5+/-5.8% in the muscles, 66.1+/-8.1% in brains). During 56 days of follow-up after termination of therapy, serum levels of anti-TES IgG antibodies, circulating IgG-TES immune complexes (CIC) as well as IgG antibodies to the most immunogenic part of recombinant myosin antigen of T. canis larvae were investigated. In contrast to anti-TES IgG antibodies, levels of CIC and anti-myosin antibodies were in the linear correlation with the efficacy of treatments beginning from day 38 post-therapy. We also showed that the serum levels of CIC as well as anti-myosin IgG antibodies seem to be the suitable serological markers for the monitoring of progress in larval destruction and TES resorption from the tissues.

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.

Characterisation and differentiation of pathogenic and non-pathogenic Acanthamoeba strains by their protein and antigen profiles.

Free-living amoebae of the genus Acanthamoeba are the causative agents of Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis. Acanthamoebae occur ubiquitously in the environment and are thus a constant cause of antigenic stimulation. In a previous study we have shown that compared to control sera, AK patients exhibit markedly lower immunoreactivities to whole cell antigen of Acanthamoeba spp. As the pathogenicity of acanthamoebae primarily relies on the excretion of proteins, it was the aim of the present study to investigate the immunoreactivity of metabolic antigen from different Acanthamoeba strains of varying pathogenicity. Three Acanthamoeba strains, one highly pathogenic, one non-pathogenic but thermophilic and one non-thermophilic non-pathogenic, were used for antigen extraction. The antigen was harvested before and after contact with human cells and all strains were tested with AK sera and with sera from healthy individuals. It was shown that the somatic protein profiles of the Acanthamoeba strains correlated to the morphological groups, and that within morphological group II-the group associated with AK-the profiles of the metabolic antigens correlated to strain pathogenicity. Moreover, it was shown that the control sera showed markedly higher immunoreactivities than the sera of the AK patients and that this immunoreactivity was generally higher to the non-pathogenic strains than to the pathogenic strain. Altogether our results once again raise the question of whether there is an immunological predisposition in AK. To our knowledge this is the first study on the immunoreactivity of metabolic antigen of acanthamoebae.

Anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities in correlation to strain pathogenicity.

Several representatives of the genus Acanthamoeba are known as causative agents of Acanthamoeba keratitis and granulomatous amoebic encephalitis. These occur predominantly in the immunocompromised host, but it is still unclear what primes the amoebae for pathogenicity. The aim of this study was to assess possible immunological differences between a highly pathogenic and a nonpathogenic Acanthamoeba strain. A total of 20 sera, including two sera of Acanthamoeba keratitis patients, were tested for anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities using immunoblotting. All sera were positive for Acanthamoeba, revealing two predominant bands at 29 kDa and at 47 kDa, respectively. Interestingly, IgG and particularly IgA immunoreactivity enabled a clear discrimination between the pathogenic and nonpathogenic strains. Moreover, compared to the control sera, the two sera of Acanthamoeba keratitis patients showed rather weak immunoreactivities and they lacked the 29 kDa and the 47 kDa band in the IgA immunoblot against the pathogenic strain. The results of our study support the assumption that immunological predisposition might also be of importance in Acanthamoeba keratitis.

Recombinant dissection of myosin heavy chain of Toxocara canis shows strong clustering of antigenic regions.

Myosins from nematode parasites elicit strong humoral and cellular immune responses and have been investigated as vaccine candidates. In this study we cloned and sequenced a cDNA coding for myosin heavy chain from Toxocara canis, a nematode parasite of canids which may also infect humans and cause various unspecific symptoms. To determine the major antigenic regions the myosin heavy chain was systematically dissected into ten overlapping recombinant fusion polypeptides which were purified by metal chelate chromatography. Single fragments were then tested for their IgG reactivity in sera from toxocarosis patients and healthy probands. Two regions, one region at the mid to carboxy-terminal end of the head domain and one region in the rod domain, were identified as major antigens, which in combination were positive with 86% of the sera. The other domains were less reactive. This shows that the patients' IgG reactivity was not directed evenly against all parts of the molecule, but was rather clustered in few regions.

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.

Discrimination between clinically relevant and nonrelevant Acanthamoeba strains isolated from contact lens- wearing keratitis patients in Austria.

Eighteen cases of Acanthamoeba-associated keratitis among contact lens wearers seen at the Department of Ophthalmology, Karl-Franzens-University, Graz, Austria, between 1996 and 1999 are reviewed. The amoebae were proven to be the causative agents in three patients. The aim of our study was to discriminate between clinically relevant and nonrelevant isolates and to assess the relatedness of the isolates to published strains. Altogether, 20 strains of free-living amoebae, including 15 Acanthamoeba strains, 3 Vahlkampfia strains, and 2 Hartmannella strains, were isolated from clinical specimens. The virulent Acanthamoeba strains were identified as A. polyphaga and two strains of A. hatchetti. To our knowledge this is the first determination of keratitis-causing Acanthamoeba strains in Austria. Clinically relevant isolates differed markedly from nonrelevant isolates with respect to their physiological properties. 18S ribosomal DNA sequence types were determined for the three physiologically most-divergent strains including one of the keratitis-causing strains. This highly virulent strain exhibited sequence type T6, a sequence type not previously associated with keratitis. Sequence data indicate that Acanthamoeba strains causing keratitis as well as nonpathogenic strains of Acanthamoeba in Austria are most closely related to published strains from other parts of the world. Moreover, the results of our study support the assumption that pathogenicity in Acanthamoeba is a distinct capability of certain strains and not dependent on appropriate conditions for the establishment of an infection.

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

Eleven Acanthamoeba isolates, obtained from Acanthamoeba 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 showed Acanthamoeba 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.

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. Subclass 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.

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 against Toxoplasma gondii. To examine the reliability of this ELISA using whole trophozoites produced in a serum-free 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 commercially 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, false-negative 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.