[The effect of cyclosporine A on the development of Toxoplasma gondii in the muscles and brain. Ultrastructural study]. / Vliv cyklosporinu A na vývoj Toxoplasma gondii ve svalech a mozku. Ultrastrukturální studie.

Presented are histological and electron microscopic findings of alterations in the muscles and brains of mice infected with T. gondii and suppressed with CyA during infection. Cya in the acute phase of infection promoted parasitemia. In the chronic stage it modified the formation of clustered cysts. Following repeated infection active tachyzoites appeared in the repleted macrophage populations of the interstitium. Polymyositis with manifested muscle fiber atrophy and allergic toxoplasmic encephalitis in the acute phase of infection is caused by: 1. active penetration of tachyzoites into non-phagocytizing muscle and nerve cells, 2. toxins (necrotizing enzymes) released by the destroyed reticuloendothelial cells due to rapid multiplication of tachyzoites and pseudocysts, 3. immunologic processes affecting factors regulating the multiplication rate and subsequent transformation of tachyzoites into cystozoites, and vice versa. The altered tissue tropism during toxoplasmic infection, i.e. suppression of the granulating tissue and fibrosis, results in a necrotizing process induced by tachyzoites or in the rupture of cysts. (Fig. 8, Ref. 18.)

[Cryptosporidium species in persons with HIV infection]. / Cryptosporidium sp. u osob infikovaných virem HIV.

Oocysts of Cryptosporidium sp. were detected in the faeces of 65 subjects with the HIV virus, using the concentrating flotation method in a sugar solution and differential staining with methyl violet. In positive cases (a total of 16 subjects) continuous as well as intermittent excretion of oocysts in faeces was recorded, the longest excretion period being up to 15 months. The presented findings of oocysts confirm that Cryptosporidium sp. is a common intestinal pathogenic organism in the investigated group of subjects, and with regard to its pathogenity and opportunistic character they must be subjected to regular parasitological examinations.

[Significance of the complement fixation reaction in the diagnosis of the acute phase of lymphatic toxoplasmosis]. / Význam komplement-fixacní reakce pro diagnostiku akutní fáze uzlinové formy toxoplazmózy.

In a group of 300 sera of subjects with clinically suspect nodular toxoplasmosis the authors tested by the IgM ELISA reaction in the reverse order the importance of the complement-fixation reaction for assessment of the acute stage of the infection. In view of the revealed prolonged detection of IgM antibodies the authors used quantitative evaluation of the reaction; they consider based on a probability pattern of antibody response coefficients of 10 and above important for differentiation of a recent infection. For the suggested method of evaluation it was confirmed that low CFR titres (1:8, 1:16, 1:32) imply usually (89.5%) latent infection, titres of 1:64 chronic (42.8%) and latent infection (57.2%); in no instance onset of the disease was involved. From the titre of 1:128 onwards there is a proportional increase of the probable detection of acute infections (titre 1:1024 as much as 72.7%. However, even high titres are not unequivocal evidence of recent infection. In view of the equivocal interpretation of results of the CFR the authors recommend for assessment of the acute stage of the nodular form of toxoplasmosis examination of the suspect subject by the CFR and in case of a medium or high level assessment of IgM by the ELISA method focused on the level and dynamics of the coefficient.

Toxoplasmic antibodies in sera of HIV-infected persons.

The sera of 67 HIV-infected persons without clinical signs of Toxoplasma gondii infection and sera of 777 immunocompetent persons from three distinct regions of Czechoslovakia were examined for the presence of toxoplasmic antibodies using the complement-fixation test (CFT). Additionally Toxoplasma positive HIV+ individuals were re-examined for the presence of IgG and IgM toxoplasmic antibodies by ELISA methods. Results show that overall prevalence of toxoplasmic antibodies is not significantly greater in HIV-positive subjects (29.8%) than in the general population (26.1%). Similarities between these two tested groups were also documented by a close correlation of their geometrical means of titres (13.9 versus 14.5). All 20 HIV-infected patients who were positive in CFT were positive in ELISA IgG reaction, and none in ELISA IgM reaction. The detected antibody levels were suggestive of a latent Toxoplasma infection only. But because of the risk of the infection reactivation all of these patients should be attended to on a systematic basis.

[Induction of experimental pneumocystosis, detection and primary isolation of Pneumocystis carinii]. / Indukce experimentální pneumocystózy, detecke a primární izolace Pnemocystis carinii.

The described model of experimental pneumocystosis is based on the induction of natural latent infection of P. carinii in Wistar strain laboratory rats. As to pharmacological inducing agents immunosuppresive preparations such as Hydrocortisone sol. inj. (Spofa), Cyclosporin A (Merck) and Dexamethasone (Spofa) were used whereby the latter was effective in up 50%. As to non-pharmacological inducing agents, the authors used in combination with the tested inducers a low protein diet (less than 8% protein in the diet); for suppression of associated bacterial contamination tetracycline was added to drinking water. From the infected lungs by two different methods two types of antigens were prepared 1) PCL antigen (Pneumocystis carinii lavage antigen) isolated by rinsing of the lungs and 2) PCWD antigen (P. carinii whole digest antigen) isolated by digestion of the lungs with collagenase and trypsin. Cysts of P. carinii were detected by staining according to Giemsa (staining of internal structures of nuclear cysts) and by modified staining with toluidine blue O (staining of the cyst wall). For isolation of the antigen and for detection of cysts a combination of the two described methods seems to be best.

[Interpretation of antitoxoplasma antibody findings]. / Interpretace nálezu antitoxoplazmických protilátek.

Between reactions assessing one class of immunoglobulins and reactions detecting all serum immunoglobulins comprehensively such as CFT, there is, as might be expected, a very poor quantitative correlation and thus in individual sera the result of one reaction cannot be reliably added to the result of another (CFT:ELISA/IgG). Even the correlation between reactions focused on the same Ig class (ELISA/IgG: IFT/IgG) is no better. Among reactions assessing specific IgM we consider ELISA/IgM better than IFT/IgM because there is not the risk of false negativity caused by concurrence of IgG. A combination of ELISA/IgG and ELISA/IgM gives good results as a statistical group: the distribution of results revealed agglomerations of sera corresponding to the assumed age of the infection derived from the generally accepted pattern of antibody formation. The applicability of the combination of these two reactions alone for evaluation of individual sera is a promising procedure but awaits further confirmation. Long-term investigations revealed within two years after infection a marked decline of CFT antibodies in the majority of cases but it was not sufficiently clear in ELISA/IgG. Despite the technical advantages of ELISA reactions, elimination af the CFT reaction is not foreseen in the near future. As the minimal combination of methods which provides adequate information we may consider at the present time CFT for assessment of total antibodies and ELISA/IgM for more marked differentiation of the acute stage. Evaluation of the lowest CFR titres considered hitherto as "practically negative" must be obviously revised in subjects with immunosuppression and organ donors for transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of cortisone on Toxoplasma gondii infection studied by electron microscopy.

The ultrastructural features of cortisone effect on the early development and formation of Toxoplasma gondii tissue cysts were examined in the brains of experimental mice on days 8-47 post inoculation. The cortisone was injected subcutaneously into mice twice a week: 1) at the early acute phase, 5 days post infection, and 2) at the transition phase of development, 12 days post infection. The disturbed regulation of the host-parasite relationship due to the effect of cortisone showed itself in an increase of T. gondii tachyzoites, cystozoites, and cysts not only by intracellular but also by as yet not described extracellular division. The persisting parasitaemia induced by tachyzoites and the earlier development and differentiation of cysts resulted in the cyst rupture releasing a highly resistant antigen of cystozoite type. The toxoplasmic encephalitis had a lethal effect in all mice. The tachyzoites penetrated into many types of cells, but a predilection of development into cysts was detected in neurons and astrocytes. According to the character of host cells, two types of cysts arose: less resistant electron-transparent cysts in neurons often rupturing or budding and more resistant electron-dense cysts in astrocytes.

[Determination of antitoxoplasma IgM antibodies using the ELISA method]. / Stanovení antitoxoplazmických protilátek IgM metodou ELISA.

The described ELISA method was developed for the examination of antitoxoplasmatic IgM. It is a double, i.e. 4-layer sandwich with the following layers: 1. anti. IgM antibody, 2. the examined serum in a uniform dilution of 1:100, 3. antigen + antitoxoplasmatic conjugate, 4. combined in a mixture prepared in advance. Thus the reaction is shorter but also more sensitive. The components in all three steps are combined for one hour at 37 degrees C. The entire process takes four hours. With regard to the course of the final answer in this type of ELISA reaction it is better to express the results by the level of the attained plateau of optic density than by a titre. The sera are thus examined in a uniform dilution of 1:100 and the results are expressed by a coefficient, the ratio of optic density of the examined serum to the optic density of the pooled negative control serum. By this evaluation the deviation of the mean is reduced to one half, i.e. V = 12.6%. The coefficient does not depend on the contents of specific IgM in a linear fashion, but an antibody change of 1:2 changes it about 1.55 times.

[Determination of antitoxoplasma IgG antibodies using the ELISA method]. / Stanovení antitoxoplazmických protilátek IgG metodou ELISA.

The described ELISA reaction is suggested as a method for examination of antitoxoplasmatic IgG antibodies. It is a simple, i.e. three-layer sandwich with the following layers: 1. antigen 2. the examined serum either in serial dilution or single dilution 1:300 and 3. a peroxidase anti-IgG conjugate with a final conclusive step, i.e. a substrate solution with OPD. Sera and conjugate are allowed to bind for 30 mins. at 37 degrees C and the substrate solution with OPD is allowed to act for 15 mins., also at 37 degrees C, and thus the entire reaction lasts about two and half hours. The authors describe alternative procedures with standard serum of a known number of international units (I.U.) or without it, evaluated optically or by a photometer. In the version with standard serum evaluated optically we compare the different localization of equally stained wells in the series of serially diluted (4n) examined and standard serum and thus we assess, based on the different dilutions the number of I.U. in the examined serum. If we compare the sera with the serum standard by means of a photometer, we examine them in a single dilution of 1:300 and compare the resulting optical density (OD) with the calibration curve made with four dilutions of the standard. The calibration curve is plotted on a scale which straightens it approximately at an angle close to the optimal 45 degrees and which makes it possible to take readings of interpolated international units.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immunoenzyme analysis with corpuscular antigen in the serology of leishmaniasis]. / Immunofermentnyi analiz s korpuskuliarnym antigenom v serologii leishmaniozov.

The authors propose polystyrol carrier enzyme-linked immunoassay using corpuscular Leishmania antigens (intact cells of Leishmania promastigotes)--C-ELISA. The standard lyophilic Leishmania antigen for ELISA may be used as an antigen in indirect immunofluorescence reaction. C-ELISA allowed the authors to determine clear antigenic differences between the mammalian leishmaniasis causative agents (subgen. Leishmania) and the reptilian ones (L. gymnodactyli--subgen. Sauroleishmania). Different levels of the antigenic relationship were demonstrated between separate species within the mammalian Leishmania serogroup. The method shows promise for elaboration of the specific test systems in order to differentially diagnose various nosologic entities of leishmaniasis, including ones in the mixed natural foci.

[Detection of Toxoplasma antibodies using the c-ELISA method]. / Detekce toxoplazmických protilátek metodou c-ELISA.

Sensitive and high capacity ELISA techniques introduced for the detection of toxoplasmatic antibodies so far do not correlate very well with standard serological examination systems used in common diagnosis of Toxoplasmosis. The most frequently used method in the CSSR - SFT using tween-ether soluble antigen extract, and displaying very satisfactory agreement with the Sabin-Feldman test, was compared with the system of direct ELISA, using the same antigen extract and the described c-ELISA method. The latter method uses for antibody detection whole non-disintegrated formol-treated trophozoites of Toxoplasma gondii and thus in the reaction in particular membrane antigens, incl. latent ones, are involved. Parallel examination of 160 sera of subjects with clinical suspicion of toxoplasma infection by the standard CFT, method and methods ELISA and c-ELISA revealed a better correlation between CFT and c-ELISA (r = 0.8031) than between CFT and ELISA with soluble antigen (r = 0.6016). Other positive specific features of the c-ELISA method are the use of commercially produced antigen for the immunofluorescence test, considerable elimination of selective adsorption of the polystyrene carrier and the perspective of the long-term preservation of plates with the fixed antigen.

[The importance of the microprecipitation reaction in agar in the diagnosis of toxoplasmosis]. / Význam mikroprecipitacní reakce v agaru pro diagnostiku toxoplazmózy.

The authors examined 56 pigs by an isolation experiment for Toxoplasma gondii and simultaneously by the Sabin-Feldman reaction, microprecipitation reaction in agar (MPA), quantitative microprecipitation reaction in agar (MPAK), reaction with complement-fixing and indirect haemagglutination reaction with toxoplasma antigen. In nine pigs with positive isolation finding, the Sabin-Feldman reaction and quantitative microprecipitation reaction in agar was positive in eight cases. In cases of negative Sabin-Feldman reaction, the quantitative microprecipitation reaction was always negative. The quantitative microprecipitation reaction in agar is therefore a suitable indicator of the success of isolation experiments. For a simplicity of the test it is suitable for immunological diagnostics of toxoplasmosis in pigs in common practice.

To the problem of toxoplasmosis in Vietnam.

The number of 259 human seria was examined for Toxoplasma gondii antibodies in 1984. Under survey were 140 persons from Duyen Thai village situated in a suburban agricultural area of Hanoi, and 119 persons from the villages of Mai Chau district in a mountainous valley near Laos border. A microreaction of complement fixation had shown higher titres in Mai Chau (24.3%) in comparison with 15.7% in Duyen Thai, using 1:16 and higher titres as significant reaction. Age distribution of antibodies revealed early infection in the childhood, as 50% of children had significant reactions in Mai Chau and 27.6% in Duyen Thai. Lately the level of antibodies was decreasing to a minimum (8%) in Duyen Thai in the age group of 30-49 years.

First experience with ELISA serosurvey for tick-borne borreliosis (Lyme disease) in Czechoslovakia.

Antibodies to Borrelia burgdorferi were searched for in the sera from two groups of hospital patients and one group of healthy agricultural workers. The antibody response was measured by the authors' own modification of the ELISA method employing the antigen prepared from Borrelia recurrentis strain propagated in laboratory mice. Positive antibody titres to Borrelia were demonstrated in 5 out of the 44 patients admitted to the Infectious Diseases Clinic, in 8 out of the 32 patients from the Neurologic Clinic and in 16 out of the 52 healthy agricultural workers living in areas infested with ticks. These findings are interpreted as showing the topicality of the Lyme disease problem in Czechoslovakia.