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.

INSECT PHYLOGENOMICS. Response to Comment on "Phylogenomics resolves the timing and pattern of insect evolution".

Tong et al. comment on the accuracy of the dating analysis presented in our work on the phylogeny of insects and provide a reanalysis of our data. They replace log-normal priors with uniform priors and add a "roachoid" fossil as a calibration point. Although the reanalysis provides an interesting alternative viewpoint, we maintain that our choices were appropriate.

In vitro activity of hexadecylphosphocholine (miltefosine) against metronidazole-resistant and -susceptible strains of Trichomonas vaginalis.

OBJECTIVES: Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted disease with worldwide significance. Trichomoniasis can be treated with metronidazole; however, resistant strains of T. vaginalis have been isolated and there is a lack of useful alternative drugs. The aim of the present study was to examine the activity of hexadecylphosphocholine (HePC; miltefosine), a membrane-active alkylphospholipid, that is licensed as an antileishmanial agent against T. vaginalis. METHODS: The efficacy of HePC after 30 min, 1 h, 16 h and 24 h against four different T. vaginalis strains (with varying resistance to metronidazole) was evaluated. RESULTS: It was shown that all isolates, including the metronidazole-resistant strains, were susceptible to HePC, with EC50s of between 8 and 40 microM and EC90s of between 8 and 80 microM depending on time and on the medium used for the experiments. Treatment of trichomonads with HePC resulted in rounding up and, at concentrations of >or=40 microM, in subsequent total lysis of the organisms. CONCLUSIONS: HePC may be a promising new candidate for the treatment of trichomoniasis.

One- and two-step hydrogen peroxide contact lens disinfection solutions against Acanthamoeba: how effective are they?

PURPOSE: Effective contact lens disinfection solutions are important to keep the storage case free of acanthamoebae and thus prevent an infection of the eye. The aim of our study was to evaluate the effectivity of two new one-step hydrogen peroxide disinfecting solutions against Acanthamoeba spp. and compare it to the effectivity of other commercially available systems. METHODS: Nine one-step 3% hydrogen peroxide systems including the new systems Silver Sept (platinum and silver disk for intensifying disinfection) and Blue Vision (newly composed catalytic tablet) and 2 two-step systems (0.6 and 3.0% H(2)O(2)) were tested for their effectivity against cysts of two Acanthamoeba keratitis isolates at different concentrations. RESULTS: After a soaking time of 8 h (overnight soaking of contact lenses) the 2 two-step systems completely destroyed the cysts of both Acanthamoeba strains, even at the highest concentration of cysts tested. The nine tested one-step systems showed weaker effects. The new Blue Vision system was able to eradicate the cysts of one strain at the low concentration of cysts. CONCLUSIONS: One-step hydrogen peroxide systems do not have sufficient effects on Acanthamoeba cysts and therefore may not protect the contact lens user from a possible infection of the eye. Further development of tablets like the ones used in the Blue Vision system may result in better cysticidal effects for one-step systems.

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.

Analysis of the sensitization profile towards allergens in central Africa.

BACKGROUND: Almost no information is available regarding the prevalence of IgE-mediated allergies and the disease-eliciting allergens in tropical Africa. OBJECTIVE: To study IgE-mediated allergies and the allergen profile in allergic patients from Zimbabwe. METHODS: The frequency of sensitization to common environmental allergen sources was determined by skin prick testing in 650 allergic patients from Zimbabwe. Fifty representative sera were analysed for IgE reactivity to 20 respiratory and 20 food allergen extracts by multiallergen extract testing. The IgE reactivity profiles to recombinant pollen and mite allergens were compared between grass pollen- and mite-sensitized patients from Zimbabwe and central Europe. Sera from grass pollen-allergic patients were also analysed for IgE reactivity to nitrocellulose-blotted natural timothy grass and Bermuda grass pollen allergens. RESULTS: IgE-mediated allergies were found to be common in Zimbabwe. Similar to the situation in central Europe, mites and grass pollens represented the most prevalent allergen sources. However, the IgE reactivity profiles determined with single recombinant pollen and mite allergens revealed interesting differences between the European and African patients, which most likely reflect the local allergen exposure. CONCLUSIONS: The striking differences regarding sensitization to grass pollen and mite allergens between African and European patients revealed by recombinant allergen-based testing emphasize the need for component-resolved allergy testing to optimize allergy prevention and therapy in different populations.

Viability of Acanthamoeba after exposure to a multipurpose disinfecting contact lens solution and two hydrogen peroxide systems.

BACKGROUND/AIM: Contact lens cases contaminated with Acanthamoeba are a major risk factor for an infection of the eye. In this study the anti-Acanthamoeba activity of three different contact lens storage solutions was tested. METHODS: A new multipurpose contact lens storage solution (Meni Care Plus) and a two step (Titmus H(2)O(2)) and one step (Oxysept Comfort) hydrogen peroxide system were tested for their effects on trophozoites and cysts of three different Acanthamoeba species: A castellanii, A hatchetti, and A lenticulata. RESULTS: After a soaking time of 8 hours (overnight soaking of contact lenses) the Titmus H(2)O(2) 0.6% solution showed very good amoebicidal effects, while Oxysept Comfort 3% H(2)O(2) could not effectively destroy the cysts of any of the three tested species. Viable cysts of the species A lenticulata and A hatchetti were still present after exposure to Meni Care Plus (0.0005% PHMB) for 8 hours. CONCLUSION: Not all of the three tested contact lens storage solutions have sufficient amoebicidal effects. The two step peroxide system Titmus H(2)O(2) is a very effective disinfectant contact lens solution in order to avoid a possible Acanthamoeba infection of the eye.

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.

Microwave treatment of contact lens cases contaminated with acanthamoeba.

PURPOSE: Microbially contaminated contact lens cases are a predisposing risk factor for Acanthamoeba keratitis. Several findings have shown that microwave irradiation kills the six Food and Drug Administration test challenge microorganisms. We aimed to determine what effect microwave irradiation has on Acanthamoeba trophozoites and cysts. METHODS: Different types of contact lens cases were contaminated with trophozoites and cysts of three different Acanthamoeba species (A. comandoni, A. castellanii, A. hatchetti) and were exposed to microwave irradiation for 3, 5, and 8 minutes, respectively. RESULTS: Trophozoites, as well as cysts of the different Acanthamoeba strains, were effectively killed, even by only 3 minutes of microwave irradiation, and there were no negative effects of irradiation on the contact lens cases themselves. CONCLUSION: We demonstrate that microwave treatment is a very effective, easy, and cheap method to keep contact lens cases free of Acanthamoeba, thus considerably reducing the risk of an 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.

Radical surgical therapy of abdominal cystic hydatid disease: factors of recurrence.

A series of 74 consecutive patients (48 women, 26 men) were operated for abdominal hydatid disease between June 1949 and December 1995. The patients ranged in age from 15 to 81 years (median 49 years). In 69 cases only the liver was affected; two patients had concomitant extrahepatic disease (one spleen, one spleen and lung), and 3 had cysts in the spleen only. Cysts were multiple in 11 patients and calcified in 24. Conservative surgical procedures were used for 22 cysts in 20 patients [open partial (n = 3), open total (n = 6), closed total cystectomy (n = 9), marsupialization (n = 2), drainage (n = 2)] and radical surgical procedures for 72 cysts in 54 patients [pericystectomy (n = 41), wedge liver resection or hemihepatectomy (n = 25), splenectomy (n = 5), radical resection of a lung cyst (n = 1)]. Altogether 37 patients (50%) were given perioperative antihelmintic chemotherapy with mebendazole (18 patients) or albendazole (19 patients). Operative mortality rates were 5.0% after conservative surgery and 1.8% after radical surgery. Morbidity rates were 25.0% following conservative surgery and 24.1% following radical surgery. Antihelmintic therapy was well tolerated by all but five patients. All side effects were entirely reversible. Among the 74 patients, 60 (81.0%) were available for long-term follow-up (median 7.2 years; range 2.0-47.0 years). Recurrence of disease was seen in 9 of 60 patients at an interval of 3 months to 20 years from the first operation. The rate of recurrence was significantly lower after radical surgical procedures (p = 0.03) and after closed removal of the cyst (p = 0.04).

[Toxocara and bronchial hyperreactivity--results of a seroprevalence study]. / Toxocara und bronchiale Hyperreaktivität--Ergebnisse einer Seroprävalenz-Studie.

For more than 30 years the ascarid Toxocara canis, a parasite of the dog, has been considered a possible cause of allergic-pulmonal irritations (i.e. asthma bronchial) in man. According to a British study thousands of people are presumably suffering from asthma bronchial as a consequence of Toxocara infestations. The aim of our recent study was the assessment of the Toxocara seroprevalence in patients of varying bronchial reactivity and status of atopy suffering from respiratory disturbances. 191 serum samples from 59 male (mean age: 31.7 years) and 132 female patients (mean age: 36.6 years) with varying atopy status and degree of bronchial reactivity, living in or near Vienna, were examined for specific IgG antibodies against excretory-secretory (E/S) Toxocara canis antigen with enzyme-linked immunosorbent assay (TES-ELISA) and Western blot (TES-WB). In total a Toxocara seroprevalence of 9.4% could be assessed among these patients. 10% of the patients with and 7.8% of the patients without bronchial hyperreactivity were Toxocara-positive. Atopic patients were serologically positive in 7.1% of the cases tested whereas non-atopics showed an antibody prevalence of 14.3%. A comparison of Toxocara seroprevalence assessed within the recent study and in an earlier study among healthy pregnant women in Vienna did not show significant differences. The results of this study carried out in Vienna indicate that patients with bronchial hyper-reactivity or atopy show no higher seroprevalence than the normal population.

Clinical and diagnostic relevance of the Toxoplasma IgG avidity test in the serological surveillance of pregnant women in Austria.

For evaluation of the medical relevance of a Toxoplasma IgG avidity test within the Austrian program for screening of pregnant women, 23 sera from women with seroconversions (group 1) and with proven latent Toxoplasma infections (group 3), respectively, as well as 92 sera from women suspected of having a primary infection (group 2) were tested by the indirect immunofluorescence test (IFAT), Sabin-Feldman's dye test (SFT), IgM enzyme-linked immunofluorescence assay (ELFA-IgM), IgA microparticle enzyme immunoassay, and the IgG avidity test. Group 1 sera (seroconversions) revealed a median avidity index (AI) of 0.25, whereas the median AI of group 3 sera (latent infections) was 0.66. In 31 (33.7%) of 92 cases suspected of involving a primary Toxoplasma infection, low (<0.41) or borderline AIs (0.41-0.50) were assessed, and in 61 cases (66.3%) the AIs exceeded 0.50. Finally, a recent infection could be excluded due to the results of the IgG avidity test in 59 cases; in at least 34 IgM-positive cases an unnecessary and, thus, unjustified treatment could be avoided.

The past and present role of the Sabin-Feldman dye test in the serodiagnosis of toxoplasmosis.

The dye test for the detection of Toxoplasma-specific antibodies was first described by Sabin and Feldman 50 years ago. The test is highly specific and sensitive and considerable information is available on the development and persistence of dye test antibodies after primary Toxoplasma infection. However, the test uses live Toxoplasma gondii and is now only employed in a few laboratories. It is still the reference method for the serodiagnosis of toxoplasmosis, and a multicentre study comparing dye test results between different laboratories was much needed. We report in this article the results of a multicentre evaluation of the test involving nineteen laboratories in eight countries. The study revealed overall satisfactory standardization between the laboratories, but there were differences in the test protocols, the use of reference/standard preparations and the interpretation of results. There is still no agreement on the level of dye test values which reflect infection with the parasite, and conversion from titres to international units (IUs) did not improve standardization. However, the results indicated that a value of > 4 IU or a titre of 1:16 met the definition of positivity of most participants. We recommend that the dye test be retained as a reference method and that interlaboratory standardization be improved by the use of a common protocol and the expression of results in titres.

Experimental investigations on the B and T cell immune response in primary alveolar echinococcosis.

Susceptibility/resistance of the intermediate host to alveolar echinococcosis (AE) seems to be based on hitherto unknown immunological mechanisms, possibly involving the activation of different CD4+ T cell immune responses (Th1/Th2). Mice of two strains previously characterized as 'susceptible' (C57BL/6 J) and 'resistant' (C57BL/10 J) to secondary AE were orally infected with eggs of Echinococcus multilocularis and the course of infection was analysed by macroscopical, pathohistological and immunohistochemical examinations of the lymphocytes and cytokines participating in the periparasitic granulomas and by serological examinations of cytokines and E. multilocularis-specific antibodies. Although differences in the extent of parasitic growth were seen between the two groups, the composition of the granulomas was quite similar with CD4+ cells being the dominant lymphocyte subpopulation, succeeded by B cells and CD8+ cells. Interferon (IFN)-gamma-, interleukin (IL)-2- and IL-4-expressing cells could not be detected in the lesions of the early phase of the infection, possibly indicating the host's immunosuppression, but were present at the end. IL-10 was the most prominent cytokine throughout the course of the disease. Serological analyses of the cytokine concentrations revealed small amounts at the beginning and high levels at the end of the infection. The pattern of cytokine response was similar for IL-4 in both strains, but different for IL-2 and IL-10 in the late phase, when the C57BL/10 J strain developed higher levels than the C57BL/6 J strain. Correspondingly only small amounts of immunoglobulin (Ig)M, IgG1, IgG2a and IgG3 could be detected at the beginning of disease, followed by higher levels at the end. The courses of antibody titres were similar in both groups except IgG3, which was more pronounced in the C57BL/10 J strain. Parasite-specific IgG2b could neither be detected in the C57BL/6 J nor in the C57BL/10 J strain by the test system used. The results of the study suggest both subsets of CD4+ T cells (Th1 and Th2) being involved in murine primary alveolar echinococcosis. A strict differentiation of mice in susceptible and resistant animals based on the activation of different CD4+ T cell immune responses (Th1 'resistant' and Th2 'susceptible') should be avoided.