Pediatric visceral leishmaniasis in Austria: diagnostic difficulties in a non-endemic region.

Visceral leishmaniasis is usually fatal if left untreated. In Europe it is mainly caused by Leishmania infantum which is endemic in the whole Mediterranean region. While visceral leishmaniasis classically affects children, adults increasingly suffer infections in regions which are known to be endemic for HIV. Nowadays up to 70% of the patients with visceral leishmaniasis in southern Europe are HIV-infected adults. The diagnosis is known to be especially difficult to establish in this group of patients because of a frequently atypical clinical presentation, but even in non-HIV-infected patients visceral leishmaniasis often represents a diagnostic challenge particularly when the patient is living in a non-endemic region. We report on four children with visceral leishmaniasis diagnosed at St. Anna Children's Hospital, Vienna, in the last decade. Diagnostic difficulties arose (1) from inexperience with this rare disease, (2) from a long incubation period (6 to 8 months) and (3) from a travel history apparently unsuspicious for the contraction of what is considered a 'tropical' disease. In one case, specific problems resulted (4) from clinical appearance and laboratory data mimicking hemophagocytic lymphohistiocytosis. Consequently even in regions where leishmaniasis is not endemic, diagnostic efforts should be undertaken to rule out this disease especially in patients with the presumptive diagnosis of hemophagocytic lymphohistiocytosis.

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.

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.

Interactions of "Limax amoebae" and gram-negative bacteria: experimental studies and review of current problems.

Free-living amoebae can harbour bacteria inside their cysts giving them a microhabitat and protecting them from disinfectants. The aim of this study was to evaluate the potential importance of "Limax amoebae" as vectors for environmental and nosocomial bacteria in a hospital. It was shown that free-living amoebae are ubiquitous in the investigated hospital, occur syntopically with facultative human pathogens (Comamonas acidovorans and Pseudomonas aeruginosa) and may serve as hosts not only for these but also for bacteria isolated from clinical specimens (Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa); temperature is apparently of crucial importance for the interactions between these microorganisms. Recent studies have shown that "Limax amoebae" apart from acting as protective hosts, may also play a role for the thermotolerance, invasiveness and antibiotic-resistance of bacteria. Considering also the reduced immune-status of many patients, this "symbiosis" of free-living amoebae and bacteria might still be of underestimated hospital-hygienic importance.

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.

Comparison of different serotests for specific Toxoplasma IgM-antibodies (ISAGA, SPIHA, IFAT) and detection of circulating antigen in two cases of laboratory acquired Toxoplasma infection.

Two symptomatic Toxoplasma infections of laboratory personnel have been serologically followed up for 5.5 and 10 months, respectively. Results obtained by commonly used test systems (indirect fluorescent antibody tests for IgG and IgM antibodies, complement fixation test) were compared with those of two recently developed and improved tests for IgM detection (immunosorbent agglutination assay [ISAGA] and solid-phase indirect haemadsorption assay [SPIHA] as well as with those of a test designed for the detection of circulating antigen (cag-ELISA).

Combined application of enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination test (IHA) as a useful tool for the diagnosis and post-operative surveillance of human alveolar and cystic echinococcosis.

A combined application of enzyme-linked immunosorbent assay (ELISA) and indirect haemagglutination test (IHA) for the diagnosis and post-operative surveillance of human alveolar and cystic echinococcosis is described. Since January 1985 each serum sample submitted for the detection of specific antibodies was examined with both an ELISA using Echinococcus multilocularis antigen (EmELISA) and an IHA using E. granulosus antigen (EgIHA). In the course of our study altogether 72 human cases of Echinococcus infections were diagnosed. All 16 cases of alveolar echinococcosis (= 100%) and 48 out of 56 cases of cystic echinococcosis (= 86%) were revealed as Echinococcus infection at least in one of the two tests. Although crude antigens were used in both, EmELISA and EgIHA, species-specific diagnosis was achieved in 57 (= 89%) of 64 cases of the infections with E. multilocularis or E. granulosus. The diagnostic value of EmELISA and EgIHA for the post-operative surveillance is demonstrated by the follow-up of the immune response of one case of alveolar and three cases of cystic echinococcosis.

Experimental studies on circulating antigen of Toxoplasma gondii in intermediate hosts: criteria for detection and structural properties.

The present study was performed to demonstrate circulating antigen (cag) of Toxoplasma gondii in the sera of orally and intraperitoneally infected rabbits and swine, to determine the time of their appearance after infection, and to characterize the antigenic components of the cag by means of affinity chromatography, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic blot onto nitrocellulose sheets. Cag, as detected by an indirect ELISA, was found in the sera of both rabbits and swine from week 5 to week 8 after infection. Electrophoretic separation of cag extracted from swine and human sera showed 5 and 8 distinct protein bands, respectively, the molecular weight of which ranged between 25 and 100 kD. After Western blot, 2 of the 5 protein bands of swine-cag (27 and 57 kD) and 3 of the 8 protein bands of human cag (27, 32, and 57 kD) reacted with the anti-Toxoplasma antibody used in the ELISA. On the basis of the data presented, the influence of the dose and mode of infection as well as that of the preparation method of antisera on cag detection is discussed.

A solid-phase indirect haemadsorption assay (SPIHA) for detection of immunoglobulin m antibodies to Toxoplasma gondii: application to diagnosis of acute acquired toxoplasmosis.

A solid-phase indirect haemadsorption assay (SPIHA) for the detection of immunoglobulin M (IgM) antibodies to Toxoplasma gondii is described. Polystyrol microtiter plates are coated with anti-human IgM (mu-chain-specific) antibodies and then sequentially allowed to react with patient's serum and sheep erythrocytes sensitized with soluble antigen of Toxoplasma gondii. A total of 111 sera were tested in fluorescent antibody test (FAT and IgM-FAT), complement fixation test (CFT), indirect haemagglutination assay (IHA), and in SPIHA. 47 sera were from individuals with a suspected or verified acute Toxoplasma infection. In most of these cases the SPIHA allowed a clear interpretation with respect to the status of the infection, even when the IgM-FAT was not conclusive. In contrast to IgM-FAT, rheumatoid factor or exceedingly high specific IgG antibodies did not interfere with results in SPIHA. A laboratory-acquired infection enabled us to demonstrate the course of immune response measured by SPIHA as well as by IgM-FAT, FAT, CFT, and IHA. The method proposed here is well appropriated to IgM detection, simple to perform, inexpensive, and thus representing an alternative to IgM-FAT, convenient for the routine laboratory.

[Immunoenzyme test with whole trophozoites of Toxoplasma gondii]. / Enzymimmuntest mit ganzen Trophozoiten von Toxoplasma gondii.

In a comparative study 151 human sera were tested for IgG and IgM antibodies against Toxoplasma gondii in the Fluorescent-Antibody-Test (FAT) as well as in the Enzyme-Linked Immunosorbent Assay (ELISA) using the same antigen (whole trophozoites of Toxoplasma gondii). A positive correlation was found between the results obtained in FAT and ELISA. The detection of specific IgG antibodies revealed a correlation of 100% and 97.5% in positive and negative sera, respectively. Only 8 out of 111 positive sera showed deviations of more than +/- 1 step within the titre ranges. While detecting specific IgM antibodies a highly significant correlation (99.3%) between FAT and ELISA was obtained for negative sera. 5 positive IgM sera showed titre end-points of 1:64 and 1 serum an end-point titre of 1:256 in FAT as well as in ELISA; however, a comparison was not done because of the small amount of positive IgM sera having been available.

Reduction of carrageenan- bradykinin- and histamine-induced acute inflammation by experimental eosinophilia in rats.

An examination was made of the effects of eosinophilia on acute inflammation. Sprague Dawley rats were infected with Trichinella spiralis larvae which resulted in a blood eosinophilia. Groups of rats with the induced eosinophilia, and untreated rats without the eosinophilia, were treated with carrageenan, bradykinin or histamine in the hind paw to induce local inflammation. Paw oedema induced by carrageenan as a measure of the inflammation was much reduced in those rats with an eosinophilia, and slightly but significantly reduced in the site treated with histamine or bradykinin. Though other anti-inflammatory factors may have participated, it is believed that the reduction in inflammation was due to the eosinophils. Pretreatment of the rat paws with an eosinophil chemotactic factor tetrapeptide (ECF-A) caused no chemotaxis and therefore no effect on histamine-induced oedema.

[Human fascioliasis in Austria (author's transl)]. / Menschliche Infektionen mit Fasciola hepatica in Osterreich.

Two cases of human fascioliasis in Austria (a 3-year-old boy and a 4-year-ond girl) are reported. A description is given of the clinical picture and the diagnostic procedures employed in the two cases. The importance of the serodiagnosis of fascioliasis is demonstrated, especially in the case of liver flukes; 4 different serological tests, partly of high sensitivity, were performed. After unsuccessful therapy with Resochin both children were treated with dehydroemetine; after a 4-week interval during which no eggs of Fasciola were found in several stool samples from either child, ova were yet again detected in the faces of the boy. The problems concerning the chemotherapy of fascioliasis are discussed.

[Serum immunofluorescence examination in loa ophthalmia (author's transl)]. / Serumimmunfluoreszen-Untersuchung bei Loa-Ophthalmie

Loaiasis is a chronic parasitic disease caused by the filaria Loa loa.As soon as the worm reaches the subconjunctival space local irration ensues. This so-called Loa ophthalmia necessitates immediate surgical intervention which can be performed under topical anesthesia, using the slit lamp. The indirect immunofluorescence technique represents a valuable diagnostic test,since it is usually difficult to demonstrate the microfilaria in the peripheral blood.

[The diagnostic significance of the indirect hemagglutination test for the surveillance of toxoplasmosis during pregnancy (author's transl)]. / Die diagnostische Bedeutung des indirekten Hämagglutinationstests für die Toxoplasmoseüberwachung während der Schwangerschaft.

In order to clarify the relevance of the indirect hemagglutination test (IHAT) for the surveillance of toxoplasmosis during pregnancy 2417 sera of pregnant women were tested for antibodies against Toxoplasma gondii in this test and in the fluorescent-antibody test (FAT). In addition 1319 of these sera were tested in the Sabin-Feldman test (SFT) adn 681 samples also in the complement fixation test (CFT). Significant correlations were found between the results obtained in the IHAT on one hand and in the FAT and SFT on the other hand: 95% of the sera which were negative in the FAT or SFT did not react in the IHAT as well, while most (but not all!) sera which were positive in the FAT or SFT gave positive results in the IHAT also. No correlations could, however, be found between titres obtained in the IHAT on one hand and in the CFT on the other hand. The suggestion recently published by several authors that it might be possible to decide from one single serum sample only whether or not a fresh infection had occurred by correlating FAT- (SFT-) and CFT-titres with IHAT-titres could not be confirmed. For a definite interpretation of serological results with respect to the risk of an infection of the fetus with Toxoplasma gondii it will still be necessary to test a second serum sample. As a screening method for detection of antibodies against Toxoplasma gondii -- without determination of the duration of infection -- the indirect hemagglutination test appears to be, however, a highly effective and economic method.

[Frequency and significance of parasitic infections in refugees from vietnam (author's transl)]. / Häufigkeit und Bedeutung parasitärer Infektionen bei vietnamesischen Flüchtlingen.

In the course of the medical care which is provided for the refugees from Southeast Asia 100 persons were tested for intestinal parasites (helminths and protozoa). In addition serum samples were obtained from 72 of these and tested in several tests (CFT, FAT, IHA and SFT) using antigens of Entamoeba histolytica, Leishmania donovani, Toxoplasma gondii, Schistosoma spp., Fasciola hepatica, Echinococcus spp., Trichinella spiralis and Litomosoides carinii. In 49 persons infections with one, two or even three species of intestinal helminths were detected (helminthic index: 63%). In detail the following infection-rates were found: Ascaris lumbricoides 41%, Trichuris trichiura 11%, Ancylostoma spp. 10% and Clonorchis sinensis 1%. Besides infections with apathogenic protozoa (Entamoeba coli, Entamoeba hartmani, Dientamoeba fragilis, Endolimax nana, Jodamoeba buetschlii and Blastocystis hominis) infections with Entamoeba histolytica were found in 3% and with Lamblia intestinalis in 4%. The results of the serological tests did not reveal any acute infection with any of the extraintestinal parasites mentioned above, although some of the low titres may reflect old infections. In particular it is surprising that none of the sera reacted against Toxoplasma gondii in dilutions higher than 1 : 16. The results are discussed with respect to risks for the health of the refugees themselves and of the indigenous population.